Regulatory nucleic acid molecules for enhancing gene expression in plants

Abstract

The current disclosure generally relates to the technical field of plant molecular biology and provides methods for the production of high expressing promoters and the production of plants with enhanced expression of nucleic acids wherein one or more nucleic acid expression enhancing nucleic acid (NEENA) is functionally linked to said promoters and / or introduced into plants.

Description

BASF Agricultural Solutions US LLC 231727WO01REGULATORY NUCLEIC ACID MOLECULES FOR ENHANCING GENE EXPRESSION IN PLANTSField of the invention

[0001] The present invention generally relates to the technical field of plant molecular biology and provides methods for production of high expressing promoters and the production of plants with enhanced expression of nucleic acids wherein nucleic acid expression enhancing nucleic acids (NEENAs) are functionally linked to said promoters and / or introduced into plants.

[0002] Expression levels of transgenes in plants are strongly affected by various external and internal factors resulting in a variable and unpredictable level of transgene expression. Often a high number of transformants must be produced and analyzed to identify lines with desirable expression strength. As transformation and screening of lines with desirable expression strength is costly and labor intensive there is a need for high expression of one or more transgenes in a plant. This problem is especially pronounced, when several genes must be coordinately expressed in a transgenic plant to achieve a specific effect as a plant has to be identified in which each gene is strongly expressed. Alternatively, the endogenous gene expression level of a target gene is often lower than needed in order to obtain the desired effect. Strong promoters can partially overcome these challenges. Unfortunately, the availability of suitable promoters showing strong expression with the desired specificity is limited. The identification and characterization of new promoters with the respective specificity and strength is, however, a time-consuming process.

[0003] In addition to promoters, additional genetic elements and / or nucleic acid motifs that positively affect gene expression can be used to regulate the expression levels of target genes. In particular, enhancers are cis-regulatory DNA elements that regulate transcription programs for example by recruiting transcription factors and directing them to the promoters of target genes in a cell-type / tissue-specific manner. The expression of a gene can be regulated by one or multiple enhancers (Marand et al 2017; Biochimica and Biophysica Acta 1860(131-139). Enhancers may be located upstream or downstream of the transcription start site of a certain expressed nucleic acid, may be found within introns and may function at positions 5000 or more nucleotides away from the respective promoter. The unpredictable positions of enhancers, relative to their cognate promoters, has resulted in the identification of only a limited number of enhancers in plant species. WO 2024 / 08357, WO 2021 / 048316 Al, WO 2021 / 069387 Al, WO 2021 / 110582 Al and WO 2020 / 229241 Al describe the identification of a number of enhancers in plants. While the insertion of strong enhancers in promoters, untranslated regions or introns of endogenous genes using genome editing techniques could allow to significantly increase the expression levels of endogenous genes, this approach is currently impeded by the limited number of enhancers identified in plant species conferring the desired levels of expression.BASF Agricultural Solutions US LLC 231727WO01

[0004] There remains a need for new gene regulatory elements that can drive strong expression of transgenes as well as of endogenous genes in plants.

[0005] Nucleic acid molecules enhancing expression of functionally linked nucleic acids are in the present disclosure described as "nucleic acid expression enhancing nucleic acids" (NEENA).

[0006] In one aspect, an isolated NEENA molecule is provided selected from the group consisting of: i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement, ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-15 or their complement, iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement, and iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii), wherein said NEENA molecule has expression enhancing activity.

[0007] In another aspect, a recombinant nucleic acid sequence for regulating expression of a polynucleotide of interest is provided, comprising a nucleic acid having promoter activity functionally linked to one or more NEENA molecules as defined in the previous paragraph, wherein said NEENA molecule is heterologous to the nucleic acid having promoter activity. The inventors have found that operably linking at least one NEENA molecule as described herein to a promoter, results in an enhanced expression derived from that promoter as compared to a promoter lacking said NEENA molecule.

[0008] In another aspect, a method is provided for enhancing expression derived from a plant promoter, comprising functionally linking to the promoter one or more heterologous NEENA molecules as defined above.

[0009] In another aspect, a method is provided for producing a plant, plant part or plant cell with enhanced expression of one or more nucleic acid molecules , as compared to a respective control plant, plant part or plant cell, comprising the steps of: a) introducing into the plant, plant part or plant cell one or more heterologous NEENA molecule as defined above, and b) functionally linking the one or more NEENA molecule to a promoter and to a nucleic acid molecule being under the control of said promoter, wherein the NEENA molecule is heterologous to that promoter.

[0010] In another aspect, a recombinant expression construct is provided comprising one or more NEENA molecule functionally linked to a promoter and one or more expressed nucleic acid molecules, whereinBASF Agricultural Solutions US LLC 231727WO01 the one or more NEENA molecule is heterologous to the promoter and comprises a nucleic acid as defined above. In another or further aspect, a recombinant expression vector is provided comprising one or more such recombinant expression constructs. In yet another or further aspect, a transgenic cell plant or plant part is provided comprising a recombinant expression construct as described above, a recombinant expression vector as described above, or one or more heterologous NEENA molecules wherein the one or more NEENA molecule comprises a nucleic acid as described above. In yet another or further aspect, a transgenic cell culture, transgenic seed or propagation material is provided herein derived from the transgenic cell or transgenic plant or transgenic plant part described above, comprising a recombinant expression construct, a recombinant expression vector or one or more heterologous NEENA molecules as described above.

[0011] In yet another aspect, provided herein is the use of the above described NEENA molecule, recombinant construct or recombinant vector for enhancing expression in plants or in plant parts as well as the use of the above described transgenic cell culture, transgenic seed, transgenic plant, transgenic plant part or propagation material derived thereof for the production of foodstuffs, animal feeds, seeds, pharmaceuticals or fine chemicals.Brief description of the figures

[0012] Figure 1: Cloning strategy of the MPRA expression library and vector configuration to test individual candidate enhancers.

[0013] Figure 2: Impact of candidate NEENA molecules on the activity of a CaMV 35S enhancer-minimal promoter combination in transiently transformed canola mesophyll protoplasts.

[0014] Figure 3: Impact of the EN-35S2-1.1 sequence on the activity of the Pbdc7 promoter in transiently transformed soybean protoplasts.

[0015] Figure 4: Impact of candidate NEENA molecules on the activity of the Pbdc7 promoter in transiently transformed canola protoplasts.

[0016] Figure 5: Impact of candidate NEENA molecules on the activity of the Pbdc7 promoter in transiently transformed soybean protoplasts.

[0017] Figure 6: Impact of candidate NEENA molecules and introns on the activity of the Pbdc7 promoter in transiently transformed canola and soybean protoplasts.

[0018] Figure 7: Comparison of the activity of the PubilOAt promoter and a Pbdc7 promoter functionally linked to candidate NEENA SOY-1 and intron NEENAc5 in transiently transformed canola protoplasts.

[0019] Figure 8: Impact of the EN-35S2-1.1 sequence on the activity of the Pbdcl6 promoter in transiently transformed soybean protoplasts.

[0020] Figure 9: Impact of candidate NEENA molecules on the activity of the Pbdcl6 promoter in transiently transformed canola protoplasts.BASF Agricultural Solutions US LLC 231727WO01

[0021] Figure 10: Impact of candidate NEENA molecules on the activity of the Pbdcl6 promoter in transiently transformed soybean protoplasts.

[0022] Figure 11: Impact of the EN-35S2-1.1 sequence on the activity of the GmUbi9 promoter in transiently transformed canola protoplasts.

[0023] Figure 12: Impact of the EN-35S2-1.1 sequence on the activity of the GmUbi2 promoter in transiently transformed canola protoplasts.

[0024] Figure 13: Impact of the EN-35S2-1.1 sequence on the activity of the GmUbi7 promoter in transiently transformed canola protoplasts.

[0025] Figure 14: Impact of the EN-35S2-1.1 sequence on the activity of the pCH3 promoter in transiently transformed canola protoplasts.

[0026] Figure 15: Impact of the EN-35S2-1.1 sequence on the activity of the pCH5 promoter in transiently transformed canola protoplasts.

[0027] Figure 16: Impact of the EN-35S2-1.1 sequence on the activity of the pNelF-4A10 promoter in transiently transformed canola protoplasts.

[0028] Figure 17: Impact of the EN-35S2-1.1 sequence on the activity of the pCYPl promoter in transiently transformed canola protoplasts.

[0029] Figure 18: Impact of candidate NEENA sequences on the activity of the soybean GmUbi9 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0030] Figure 19: Impact of candidate NEENA sequences on the activity of the soybean GmUbi2 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0031] Figure 20: Impact of candidate NEENA sequences on the activity of the soybean GmUbi7 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0032] Figure 21: Impact of candidate NEENA sequences on the activity of the Arabidopsis pCH3 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0033] Figure 22: Impact of candidate NEENA sequences on the activity of the Arabidopsis pCH5 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0034] Figure 23: Impact of candidate NEENA sequences on the activity of the tobacco NelF-4A10 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0035] Figure 24: Impact of candidate NEENA sequences on the activity of the soybean CYP1 promoter in transiently transformed canola (A) and soybean (B) protoplasts.

[0036] Figure 25: Nucleotide sequence of candidate NEENA molecules with SEQ ID NOs: 1-9 (A) and SEQ ID NOs: 10-15 (B).

[0037] Figure 26: Graph showing expression data (RNA-seq) for the native soybean gene Glyma.llG098700 from which pCYPl promoter (SEQ ID NO: 142) is derived, as described in Examples 19 and 20.BASF Agricultural Solutions US LLC 231727WO01

[0038] Figure 27: Impact of candidate NEENA sequence SOY-3 and the NEENAc5 intron on the activity of the soybean Pbdc7 promoter in stably transformed soybean plants.

[0039] Figure 28: Impact of candidate NEENA sequence SOY-3 and the NEENAcS intron on the activity of the soybean Pbdc7 promoter in the leaves (A) , roots (B) or stems (C) in stably transformed soybean plants.

[0040] Figure 29: Impact of candidate NEENA sequences on the activity of promoters in stably transformed soybean plants.Detailed description

[0041] The present invention concerns methods to enhance expression of nucleic acids derived from a promoter in plants by operably linking at least one nucleic acid expression enhancing nucleic acid (NEENA) molecule to the respective promoter. The inventors have found that operably linking at least one NEENA molecule as described hereunder to a promoter, results in an enhanced expression derived from that promoter as compared to a promoter lacking said NEENA molecule.

[0042] It is to be understood that this invention is not limited to the particular methodology or protocols. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms "a", "and" and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art. The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%, preferably 10%, more preferably 5%, even more preferably 2%, most preferably 1% up or down (higher or lower). As used herein, the word "or" means any one member of a particular list and includes any combination of members of that list. The words "comprise," "comprising," "include," "including," and "includes" when used in this specification and in the following claims are intended to specify the presence of one or more stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.BASF Agricultural Solutions US LLC 231727WO01

[0043] The term "nucleic acid expression enhancing nucleic acid" or "NEENA" refers to a sequence and / or a nucleic acid molecule of a specific sequence having the intrinsic property to enhance expression of a nucleic acid under the control of a promoter to which the NEENA is functionally linked. Unlike promoter sequences, the NEENA as such is not able to drive expression. In order to fulfill the function of enhancing expression of a nucleic acid molecule functionally linked to the NEENA, the NEENA itself has to be functionally linked to a promoter. In distinction to enhancer sequences known in the art, the NEENA is acting in cis but not in trans and has to be located close to the transcription start site of the nucleic acid to be expressed.

[0044] In one aspect, the invention comprises an isolated "nucleic acid expression enhancing nucleic acid (NEENA)" molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement,(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-15 or their complement,(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement, and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii), wherein said NEENA molecule has expression enhancing activity.

[0045] The term "isolated" as used herein means that a material has been removed by the hand of man and exists apart from its original, native environment and is therefore not a product of nature. An isolated material or molecule (such as a DNA molecule or enzyme) may exist in a purified form or may exist in a non-native environment such as, for example, in a transgenic host cell. For example, a naturally occurring polynucleotide or polypeptide present in a living plant is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides could be part of a composition and would be isolated in that such a vector or composition is not part of its original environment. Preferably, the term "isolated" when used in relation to a nucleic acid molecule, as in "an isolated nucleic acid sequence" refers to a nucleic acid sequence that is identified and separated from at least one flanking nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated nucleic acid molecule is a nucleic acid molecule present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acid molecules are nucleic acid molecules such as DNA and RNA, which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighbouring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs, which encode a multitude of proteins. However, an isolated nucleicBASF Agricultural Solutions US LLC 231727WO01 acid sequence comprising, for example, one or more of SEQ ID NOs: 1-15 includes, by way of example, such nucleic acid sequences in cells which ordinarily contain said one or more of SEQ ID NOs: 1-15 where the nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be used to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be singlestranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded). As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences that are removed from their natural environment, isolated or separated. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. A purified nucleic acid sequence may be an isolated nucleic acid sequence.

[0046] In another aspect, the invention provides a recombinant nucleic acid sequence for regulating expression of a polynucleotide of interest comprising:- a nucleic acid molecule having promoter activity, and- one or more NEENA molecules as defined above in (i) to (iv) functionally linked to the nucleic acid molecule having promoter activity, wherein the NEENA molecule is heterologous to the nucleic acid having promoter activity.

[0047] In yet another aspect, the invention comprises a method for enhancing expression derived from a plant promoter, comprising functionally linking to the promoter one or more heterologous nucleic acid expression enhancing nucleic acid (NEENA) molecule, said NEENA molecule comprising:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or(ii) a nucleic acid having at least 90 % sequence identity to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of (i) to (iii).

[0048] The inventors have found that functionally linking one or more NEENA molecule to a promoter according to the method disclosed above results in an unexpected enhancement of the expression derived from that promoter. This method thus allows to efficiently enhance expression of a desired product when the expression driven by a desired promoter fails to reach the intended strength.BASF Agricultural Solutions US LLC 231727WO01

[0049] The term "expression" or "gene expression" includes the transcription of a specific gene or specific genes or specific genetic construct. The term "expression" or "gene expression", in particular, means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product. Yet, the term "expression" as used herein may also include the translation of process of an mRNA molecule where a polypeptide is formed. Thus, the term "expression" may include the transcription process alone, the translation process alone, or both processes combined.

[0050] "Enhance" or "increase" expression are used equivalently herein and mean that the level of expression of a nucleic acid molecule in a plant, part of a plant or plant cell after applying a method of the present invention is higher than its original level of expression in the plant, part of the plant or plant cell before applying the method or compared to a reference plant lacking a recombinant nucleic acid molecule of the invention. The original level of expression includes the absence of expression and immeasurable expression. For example, the reference plant is comprising the same construct which is only lacking the respective one or more NEENA molecule. As used herein, an "enhancement" or "increase" of the level of an agent such as a protein, mRNA or RNA means that the level is increased relative to a substantially identical plant, part of a plant or plant cell grown under substantially identical conditions, lacking a recombinant nucleic acid molecule of the invention, for example lacking the one or more NEENA molecule, the recombinant construct or recombinant vector of the invention. As used herein, "enhancement" or "increase" of the level of an agent, such as for example a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by the target gene and / or of the protein product encoded by it, means that the level is increased 50% or more, for example 75% or more, for example 100% or more, preferably 200% or more, more preferably 5 fold or more, even more preferably 10 fold or more, most preferably 20 fold or more for example 50 fold relative to a cell or organism lacking a NEENA molecule or recombinant expression construct of the invention.

[0051] The enhancement or increase can be determined by methods with which a person skilled in the art is familiar. Thus, the enhancement or increase of the nucleic acid or protein expression can be determined for example by an immunological detection of the protein. Moreover, techniques such as protein assay, fluorescence, Northern hybridization, nuclease protection assay, reverse transcription (quantitative RT-PCR), ELISA (enzyme-linked immuno-sorbent assay), Western blotting, radioimmunoassay (RIA) or other immunoassays or enzymatic assays and fluorescence-activated cell analysis (FACS) can be employed to measure a specific protein or RNA in a plant or plant cell. Depending on the type of the induced protein product, its' activity or the effect on the phenotype of the organism or the cell may also be determined. Methods for determining the protein quantity are known to the skilledBASF Agricultural Solutions US LLC 231727WO01 worker. Examples, which may be mentioned, are: the micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteau method (Lowry OH et al. (1951) J Biol Chem 193:265-275) or measuring the absorption of CBB G-250 (Bradford MM (1976) Analyt Biochem 72:248-254). As examples for quantifying the enhanced expression derived from a promoter, the detection of luciferase activity or GUS activity is described in the Examples below.

[0052] The terms "Nucleic Acids" and "Nucleotides" refer to naturally occurring or synthetic or artificial nucleic acid or nucleotides. The terms "nucleic acids" and "nucleotides" comprise deoxyribonucleotides or ribonucleotides or any nucleotide analogue and polymers or hybrids thereof in either single- or doublestranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used inter-changeably herein with "gene", "cDNA, "mRNA", "oligonucleotide," and "polynucleotide". Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and / or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, and the like; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group selected from H, OR, R, a halogen, SH, SR, NH2, NHR, NR2, or CN. Short hairpin RNAs (shRNAs) also can comprise non-natural elements such as non-natural bases, e.g., ionosin and xanthine, non-natural sugars, e.g., 2'-methoxy ribose, or non-natural phosphodiester linkages, e.g., methylphosphonates, phosphorothioates and peptides.

[0053] The phrase "nucleic acid sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'- to the 3'-end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. "Nucleic acid sequence" also refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length. A "target region" of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A "coding region" of a nucleic acid is the portion of the nucleic acid, which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein.

[0054] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.BASF Agricultural Solutions US LLC 231727WO01

[0055] The terms "promoter", or "promoter sequence" are equivalents and as used herein, refer to a DNA sequence which, when operably linked to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into RNA. A "plant promoter" comprises promoters which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may also originate for example from viruses or microorganisms or it may be a synthetic promoter designed by man. Such plant promoters can for example be found in the following public databases GrassPROMDB (https: / / www.grassius.org / grasspromdb.php), PlantPromDB (http: / / www.softberry.com / ), PPDB (http: / / ppdb.agr.gifu-u.ac.jp). Promoters listed there may be addressed with the methods of the invention and are herewith included by reference. A promoter is located 5' (i.e., upstream), proximal to the transcriptional start site of a nucleotide sequence of interest whose transcription it controls and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. Said promoter comprises for example the at least 10 kb, for example 5 kb or 2 kb proximal to the transcription start site. It may also comprise the at least 1500 bp proximal to the transcriptional start site, preferably the at least 1000 bp, more preferably the at least 500 bp, even more preferably the at least 400 bp, the at least 300 bp, the at least 200 bp or the at least 100 bp, most preferably the at least 50 bp proximal to the transcription start site, for example, at least 25 bp. The promoter does not comprise exon and / or intron regions or 5' untranslated regions. The promoter may be heterologous or homologous to the respective plant.

[0056] The term "heterologous" (or exogenous or foreign or recombinant) polynucleotide refers to a polynucleotide that is not native to the host cell; or to a polynucleotide native to the host cell but including structural modifications, e.g., deletions, substitutions, and / or insertions as a result of manipulation of the DNA of the host cell by recombinant DNA techniques to alter the native polynucleotide; or to a polynucleotide native to the host cell whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter; or to a polynucleotide native to the host cell, but integrated not within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques. With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not occur naturally in the specific combination with each other. For example, a promoter operably linked to a heterologous nucleic acid sequence refers to a nucleic acid sequence from a species different from that from which the promoter was derived, or when it originates from the same species, to a nucleic acid sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).BASF Agricultural Solutions US LLC 231727WO01

[0057] The one or more NEENA molecule preferably are heterologous to the nucleic acid molecule which is under the control of the promoter to which the NEENA is functionally linked. The one or more NEENA molecule may be heterologous to the promoter to which it is functionally linked or it may be heterologous to both the promoter and the nucleic acid molecule under the control of said promoter.

[0058] Preferably, the term "heterologous" with respect to NEENA molecule, refers to a NEENA which is operably linked to, or is manipulated to become operably linked to, a nucleic acid molecule, for example a promoter or an additional NEENA molecule, to which it is not operably linked in nature. For example, a NEENA is in its natural environment functionally linked to its native promoter, whereas in the present invention it is linked to another promoter which might be derived from the same organism, a different organism or might be a synthetic promoter such as the SUPER-promoter. It may also mean that a NEENA according to the present invention is linked to its native promoter but the nucleic acid molecule under control of said promoter is heterologous to the promoter comprising its native NEENA. It is in addition to be understood that the promoter and / or the nucleic acid molecule under the control of said promoter functionally linked to one or more NEENA of the invention are heterologous to said NEENA as their sequence has been manipulated by for example mutation such as insertions, deletions and the forth so that the natural sequence of the promoter and / or the nucleic acid molecule under control of said promoter is modified and therefore have become heterologous to the NEENA of the invention. It may also be understood that a NEENA is heterologous to the nucleic acid to which it is functionally linked when the NEENA is functionally linked to its native promoter wherein the position of the NEENA in relation to said promoter is changed.

[0059] The term "specificity" when referring to a promoter means the pattern of expression conferred by the respective promoter. The specificity describes the tissues and / or developmental status of a plant, plant part or plant cell, in which the promoter is conferring expression of the nucleic acid molecule under the control of the respective promoter. Specificity of a promoter may also comprise the environmental conditions, under which the promoter may be activated or down-regulated such as induction or repression by biological or environmental stresses such as cold, drought, wounding or infection.

[0060] If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. Also, the promoter may be regulated in a tissue-specific or tissue preferred manner such that it is only or predominantly active in transcribing the associated nucleic acid molecule in a specific tissue type(s) such as leaves, roots or meristem.

[0061] The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in the relative absence of expression of the same nucleotide sequence of interest in a different type ofBASF Agricultural Solutions US LLC 231727WO01 tissue (e.g., roots). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected. The term "cell type specific" as applied to a promoter refers to a promoter, which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., GUS activity staining, GFP protein or immunohistochemical staining. The term "constitutive" when made in reference to a promoter or the expression derived from a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid molecule in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.) in at least one cell, tissue or organ, during most, but not necessarily all, phases of growth and development of a plant or a part of a plant. A "ubiquitous promoter" as used herein refers to a promoter that is active in substantially all tissues or cells of an organism.

[0062] In principle, any promoter is suitable for use in the methods provided herein. These promoters include but are not limited to tissue specific, organ specific, inducible, developmental specific and constitutive promoters.

[0063] The one or more NEENA may be functionally linked to any promoter and will enhance expression of the nucleic acid molecule under the control of said promoter. Constitutive promoters to be used in any method of the invention may be derived from plants, for example monocotyledonous or dicotyledonous plants, from bacteria and / or viruses or may be synthetic promoters. Constitutive promoters to be used are for example the Cassava vein mosaic virus-Promoter (Verdaguer B et al. (1996). PMB 31(6), 1129-39), the Subterrenean Clover Stunt Virus-Promoter (Boevink P, et al. (1995). Virology 207(2), 354-61), the A. thaliana histone 4A promoter in combination with the histone 3A intron (Chaboute et al. (1984). PMB 8(2), 179-91), the B. napus P450-dependent fatty acid omega-hydroxylase promoter (WO2016113333), the pActlOs promoter from rice (McElroy et al. (1990). Plant Cell 2(2), 163-71), the Pcllbi-Promoter from P. crispum (WO 2003102198), the ZmUbi-Promoter from Zea mays (Christensen et al (1992). Plant Mol Biol. 18(4), 675-89), AtNit-promoter from the A. thaliana gene At3g44310 encoding nitrilase 1, the 34S-BASF Agricultural Solutions US LLC 231727WO01 promoter from figwort mosaic virus (Sanger et al., 1990, PMB 14(3)), the 35S-promoter from Cauliflower mosaic virus (Odell et al (1985). Nature 313(6005), 810-2), the nos (Depicker et al (1982). J Mol Appl Genet. 1(6), 561-73) and ocs-promoter derived from Agrobacterium tumefaciens, the ScBV-promoter (US 5 994 123), the SUPER-promoter (Lee et al. 2007, Plant. Phys. 145), the AtFNR-promoter from the A. thaliana gene At5g66190 encoding the ferredoxin NADH reductase, the ptxA promoter from Pisum sativum (W02005085450), the AtTPT-promoter from the A. thaliana gene At5g46110 encoding the triose phosphate translocator, the bidirectional AtOASTL-promoter from the A. thaliana genes At4gl4880 and At4gl4890 , the PRO0194 promoter from the A. thaliana gene Atlgl3440 encoding the glyceraldehyde- 3-phosphate dehydrogenase, the PRO0162 promoter from the A. thaliana gene At3g52930 encoding the fructose-bis-phosphate aldolase, the promoter from A.thaliana ubiquitin-10 gene (pUbilOAt, Grefen et al, 2010, Plant J 64(2), 355-65), the pCH3 promoter of A. thaliana hydroxymethyltransferase 4 gene At4gl3930 or the pCH5 promoter of A. thaliana Arabinogalactan protein 5 gene At5gll740 (Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545), the AHAS-promoter (WO2008124495), the soybean promoters Pbdc7 (WO2014150449) and Pbdcl6 (WO2021183803), the soybean promoters of the GmUbi9 gene, the GmUbi2 gene and the GmUbi7 gene (Hernandez-Garcia et al. 2010, BMC Plant Biol. 10, 237), the tobacco elF4A-10 promoter (pNelF-4A10, Mandel et al. 1995, Plant Mol. Biol. 29, 995-1004), the CaffeoylCoA-MT promoter and the OsCP12 from rice (W02006084868) or the pGOS2 promoter from rice (de Pater et al. (1992). Plant J. 2(6), 837-44). In a preferred embodiment, the nucleic acid having promoter activity is selected from the group consisting of the soybean promoters Pbdc7, Pbdcl6, GmUbi9, GmUbi2, GmUbi7 and pCYPl, the tobacco promoter elF4A-10, the Arabidopsis promoters pCH3 and pCH5 and their functional variants having similar promoter activity such as for example, but not limited to, the Arabidopsis pCH5 promoter lacking the first 200 nt (SEQ ID NO: 120) and the Arabidopsis pCH3 promoter variant having a T-to-C mutation at nt 1590 (SEQ ID NO: 108).

[0064] Tissue or developmental specific or inducible promoters to be used in any method of the invention may be derived from plants, for example monocotyledonous or dicotyledonous plants, from bacteria and / or viruses or may be synthetic promoters. Tissue or developmental specific or inducible promoters to be used are for example the seed specific and / or seed-preferential promoters for example the High Molecular Weight Glutenin Bxl7 promoter from T. aestivum (Reddy P and Appels R (1993) Theor Appl Genet. 85(5), 616-24), High Molecular Weight Glutenin 1DX5 promoter from T. aestivum (Lamacchia et al. (2001) J Exp Bot. 52(355), 243-50), the plastidic AGPase promoter from T. aestivum (Thorneycroft et al. (2003) Plant Biotechnol J. 1(4), 259-70), the hordein Bl promoter from Hordeum vulgare (Brandt et al. (1985) Carlsberg Research Communications 50, 333), the SBP-promoter from Vicia faba (WQ2000026388), the Unknown Seed Protein-promoter (USP) from Vicia faba (WQ2003092362), the napin promoter from Brassica napus (EP0255378), the conlinin-promoter from Linum usitatissmum (WQ2001016340), the promoter from the A. thaliana gene At5g01670 encoding the peroxiredoxin likeBASF Agricultural Solutions US LLC 231727WO01 protein (W02006089950), the promoter of the peroxiredoxin like protein from Linum usitatissmum (W02006089950), the globulin like protein promoter from Brassica napus (Roh et al., 2014, Journal of the Korean Society for Applied Biological Chemistry 57(5)), the arcelin5-l promoter from Phaseolus vulgaris (WO 2012077020), the Zein promoter from Zea mays (Shepherd and Scott Biotechnol Appl Biochem. 2009, 52(3)), the globulin promoter from Zea mays (Mei et al., 2004, Maydica 49(4)), the pKG86 promoter from Zea mays (WO 2010122110), the leaf specific ST-LS1 promoter from Solanum tuberosum (Stockhaus et al (1989) EMBO J. 8(9), 2445-51), the leaf specific thioredoxin promoter from Oryza sativa (Fukuda et al. (2005) Plant Cell Physiol. 46(11), 1779-86), the root specific or root preferential promoters Pbtg-26D from G. hirsutum (WO2017 / 025282), PGL4 and 5 from Zea mays (EP1862473) or Pzrp2 from Zea mays (Held et al. (1997) PMG 35(3), 367-375), the inducible promoters Phprl from A. thaliana (Wang et al. (2009) Molecular Plant 2(1), 191-200), the rd29a promoter from A. thaliana (Yamaguchi-Shinozaki K and Shinozaki K (1994) Plant Cell 6(2), 251-64), the proteinase inhibitor promoter from Zea mays (Cordero et al (1994) Plant J. 6(2), 141-50), or the fiber specific or preferential promoters from G. hirsutum as described in W02012093032, US2013081154, W02004065571, W02008083969 or WO2012136788.

[0065] The inventors have found that functionally linking one or more NEENA molecule as described above to any type of promoter as described above results in an unexpected enhancement of the expression derived from that promoter, and that the degree of enhancement can be influenced by the use of specific NEENA-promoter combinations.

[0066] Accordingly, in a preferred embodiment, the soybean Pbdc7 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 5-7, 9-11, 15 or their complement, preferably any one of SEQ ID NOs: 1, 3, 5, 6 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 5-7, 9-11, 15 or their complement, preferably to any one of SEQ ID NOs: 1, 3, 5, 6 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 5-7, 9-11, 15 or their complement, preferably to any one of SEQ ID NOs: 1, 3, 5, 6 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0067] In another preferred embodiment, the soybean Pbdcl6 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 5-7, 9-11 or their complement, preferably any one of SEQ ID NOs: 1, 3, 5, 9 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 5-7, 9-11 or their complement, preferably to any one of SEQ ID NOs: 1, 3, 5, 9 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 5-7, 9-11 or theirBASF Agricultural Solutions US LLC 231727WO01 complement, preferably to any one of SEQ ID NOs: 1, 3, 5, 9 or their complement; and (iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0068] In another preferred embodiment, the soybean GmUbi9 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 5-6, 9 or their complement, preferably any one of SEQ ID NOs: 1-3, 6, 9 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 5-6, 9 or their complement, preferably to any one of SEQ ID NOs: 1-3, 6, 9 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 5-6, 9 or their complement, preferably to any one of SEQ ID NOs: 1-3, 6, 9 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0069] In another preferred embodiment, the soybean GmUbi2 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 5-7, 9, 10 or their complement, preferably any one of SEQ ID NOs: 1-3, 5, 9 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 5-7, 9, 10 or their complement, preferably to any one of SEQ ID NOs: 1-3, 5, 9 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 5-7, 9, 10 or their complement, preferably to any one of SEQ ID NOs: 1-3, 5, 9 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0070] In another preferred embodiment, the soybean GmUbi7 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1, 3, 5, 9 or their complement, preferably any one of SEQ ID NOs: 1, 3 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 9 or their complement, preferably to any one of SEQ ID NOs: 1, 3 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1, 3, 5, 9 or their complement, preferably to any one of SEQ ID NOs: 1, 3 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0071] In another preferred embodiment, the Arabidopsis pCH3 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 5-7, 9-11 or their complement, preferably any one of SEQ ID NOs: 1, 3, 5 or their complement;BASF Agricultural Solutions US LLC 231727WO01(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 5-7, 9-11 or their complement, preferably to any one of SEQ ID NOs: 1, 3, 5 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 5-7, 9-11 or their complement, preferably to any one of SEQ ID NOs: 1, 3, 5 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0072] In another preferred embodiment, the Arabidopsis pCH5 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 9 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 9 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 9 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0073] In another preferred embodiment, the tobacco NelF-4A10 promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1, 3, 5 or their complement, preferably to any one of SEQ ID NOs: 1, 3 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1, 3, 5 or their complement, preferably to any one of SEQ ID NOs: 1, 3 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1, 3, 5 or their complement, preferably to any one of SEQ ID NOs: 1, 3 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0074] In another preferred embodiment, the soybean pCYPl promoter is functionally linked to a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-3, 5, 9, 10 or their complement, preferably to any one of SEQ ID NOs: 1-3, 5 or their complement;(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-3, 5, 9, 10 or their complement, preferably to any one of SEQ ID NOs: 1-3, 5 or their complement;(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-3, 5, 9, 10 or their complement, preferably to any one of SEQ ID NOs: 1-3, 5 or their complement; and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0075] In another aspect, provided herein is an isolated nucleic acid having constitutive promoter activity ("CYP1 promoter" or "pCYPl") selected from the group consisting of a) SEQ ID NO: 142 or a functionalBASF Agricultural Solutions US LLC 231727WO01 fragment thereof; and b) a nucleic acid sequence having at least 90%, preferably 95%, more preferably 98%, most preferably 99% sequence identity to SEQ ID NO: 142, or a functional fragment thereof. In a further embodiment a recombinant nucleotide for regulating gene expression of a polynucleotide of interest is provided, comprising the isolated nucleotide having constitutive promoter activity selected from the group consisting of a) SEQ ID NO: 142 or a functional fragment thereof; and b) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 142, or a functional fragment thereof. In another further embodiment, a recombinant gene is provided comprising: the isolated nucleic acid having constitutive promoter activity or the recombinant nucleotide for regulating gene expression of a polynucleotide of interest comprising said isolated nucleic acid having constitutive promoter activity operably linked to a heterologous nucleic acid sequence. Said recombinant gene may further comprise a transcription termination and polyadenylation sequence, preferably a transcription termination and polyadenylation region functional in plant cells. The heterologous nucleic acid sequence may encode an expression product of interest, for example, but not limited to, an RNA molecule capable of modulating the expression of a gene or a protein. Said heterologous nucleic acid sequence may also be a NEENA molecule. In another or further aspect, an expression cassette for regulating constitutive expression of a polynucleotide of interest is provided, said expression cassette comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) SEQ ID NO: 142 or a functional fragment thereof; and b) a nucleic acid sequence having at least 90%, preferably 95%, more preferably 98%, most preferably 99% sequence identity to SEQ ID NO: 142, or a functional fragment thereof. Alternatively, said expression cassette may comprise a recombinant nucleotide or a recombinant gene comprising the CYP1 promoter as described above. In another aspect, a vector is provided herein, the vector comprising any one of the expression cassettes described above. In some embodiments, the vector can be an expression vector. In another aspect, a host cell is provided, comprising the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) SEQ ID NO: 142 or a functional fragment thereof; and b) a nucleic acid sequence having at least 90%, preferably 95%, more preferably 98%, most preferably 99% sequence identity to SEQ ID NO: 142, or a functional fragment thereof; or comprising any one of the expression cassettes or vectors described above. In another aspect, provided herein is a plant tissue, plant organ, plant part, plant, or seed comprising: (1) the isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) SEQ ID NO: 142 or a functional fragment thereof; and b) a nucleic acid sequence having at least 90%, preferably 95%, more preferably 98%, most preferably 99% sequence identity to SEQ ID NO: 142, or a functional fragment thereof; (2) the recombinant nucleotide for regulating gene expression of a polynucleotide of interest described above; or (3) any one of the expression cassettes or vectors described above.

[0076] In another aspect, a method is provided herein for producing a plant tissue, plant organ, plant part, plant, or seed, the method comprising: a) introducing or providing the nucleic acid havingBASF Agricultural Solutions US LLC 231727WO01 constitutive promoter activity, the recombinant nucleic acid, the expression cassette, or the vector described in the previous paragraph to a plant cell to create a modified plant cell; and b) regenerating the modified plant cell to form a plant tissue, plant organ, plant part, plant, or seed. In some embodiments, the method further comprises selecting said plant cell to form a plant tissue, plant organ, plant or seed for the presence of the isolated nucleic acids, the recombinant nucleotides , the expression cassettes, or the vectors described in the previous paragraph. In another aspect, provided herein is a method for expressing a polynucleotide of interest in a host cell comprising: (a) introducing any of the recombinant nucleotides described above, any of the expression cassettes described above, or any of the vectors described above into the host cell, and (b) expressing at least one polynucleotide of interest in said host cell. In some embodiments, the host cell can be a plant cell. In another aspect, provided herein is a method for effecting constitutive expression of a nucleic acid in a plant, comprising introducing any of the isolated nucleic acids having constitutive promoter activity described above, or any of the recombinant nucleotides described above, into the genome of a plant. In another aspect, provided herein is a use of the isolated nucleic acids, the recombinant nucleotides , the expression cassettes or the vectors described in the previous paragraph to regulate expression of an operably linked nucleic acid sequence in a plant or to identify other nucleic acids having constitutive promoter activity or having expression enhancing activity. In some embodiments, the plant tissue, plant organ, plant part, plant, or seed is dicotyledonous. In some embodiments, the plant tissue, plant organ, plant part, plant, or seed is a soybean or canola plant tissue, plant organ, plant part, plant, or seed. In another aspect, a method is provided herein for producing food, feed, or an industrial product comprising: a) obtaining the plant tissue, plant organ, plant part, plant, or seed comprising the isolated nucleic acid having constitutive promoter activity, the recombinant nucleotide for regulating expression of a polynucleotide of interest, any one of the expression cassettes or any one of the vectors described in the previous paragraph; and b) preparing the food, feed or industrial product from the plant tissue, plant organ, plant part, plant, or seed. In some embodiments of the method, the food or feed is meal, grain, starch, flour or protein. In some embodiments of the method, the industrial product is biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.

[0077] Embodiments presented herein with respect to the pCYPl promoter include:

[0078] I. An isolated nucleic acid having constitutive promoter activity selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 142 or a functional fragment thereof; and b) a nucleic acid comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 142, or a functional fragment thereof.

[0079] II. A recombinant nucleotide for regulating expression of a polynucleotide of interest, comprising the nucleic acid having constitutive promoter of embodiment I.BASF Agricultural Solutions US LLC 231727WO01

[0080] III. An expression cassette for regulating constitutive expression of a polynucleotide of interest, said expression cassette comprising the nucleic acid having constitutive promoter activity of embodiment I, or the recombinant nucleotide of embodiment II.

[0081] IV. A vector comprising the expression cassette of embodiment III, optionally the vector is an expression vector.

[0082] V. A host cell comprising the nucleic acid having constitutive promoter activity of embodiment I., the recombinant nucleotide of embodiment II., the expression cassette of embodiment III., or the vector of embodiment IV.

[0083] VI. A plant tissue, plant organ, plant part, plant cell, plant, or seed comprising the nucleic acid having constitutive promoter activity of embodiment I. or the recombinant nucleotide of embodiment II., the expression cassette of embodiment III., or the vector of embodiment IV.

[0084] VII. A method of producing a plant tissue, plant organ, plant part, plant cell, plant, or seed, comprising: introducing or providing the nucleic acid having constitutive promoter activity of embodiment I., the recombinant nucleic acid of embodiment II., the expression cassette of embodiment III., or the vector of embodiment IV. to a plant cell to create a modified plant cell; and regenerating the modified plant cell to form a plant tissue, plant organ, plant part, plant cell, plant, or seed.

[0085] VIII. A method for expressing a polynucleotide of interest in a host cell comprising: introducing the recombinant nucleotide of embodiment II., the expression cassette of embodiment III., or the vector of embodiment IV. into the host cell, and expressing at least one polynucleotide of interest in said host cell.

[0086] IX. A method of effecting constitutive expression of a nucleic acid, comprising introducing the nucleic acid having constitutive promoter activity of embodiment I., the recombinant nucleic acid of embodiment II., into the genome of a plant.

[0087] X. Use of the nucleic acid having constitutive promoter activity according to embodiment I. to regulate expression of an operably linked nucleic acid sequence in a plant.

[0088] XI. Use of the nucleic acid having constitutive promoter activity according to embodiment I. to identify other nucleic acids having constitutive promoter activity or having expression enhancing activity.

[0089] XII. A method of producing food, feed, or an industrial product comprising:BASF Agricultural Solutions US LLC 231727WO01 obtaining the plant tissue, plant organ, plant part, plant, plant cell or seed of embodiment VI. ; and preparing the food, feed or industrial product from that plant tissue, plant organ, plant part, plant, plant cell or seed.

[0090] Embodiment II. may further comprise one or more of the following characteristics in any combination:

[0091] Characteristic 1: wherein the nucleic acid having constitutive promoter activity is operably linked to a heterologous nucleic acid sequence.

[0092] Characteristic 2: further comprising a transcription termination and polyadenylation sequence, optionally wherein the transcription termination and polyadenylation region is functional in plants.

[0093] Characteristic 3: wherein the heterologous nucleic acid sequence may- encode an expression product of interest, which expression product of interest may optionally be an RNA molecule capable of modulating the expression of a gene or may be a protein such as for example a herbicide selectable marker, an insecticidal protein, an antibiotic resistance protein, a herbicide resistance protein, an insect resistance protein, a disease resistance protein, a herbicide tolerance protein, or- be a nucleic acid expression enhancing nucleic acid (NEENA) molecule having expression enhancing activity.

[0094] The host cell of embodiment V. or VIII. may be an E. coli cell, an Agrobacterium cell, an algal cell, a yeast cell or a plant cell.

[0095] The plant tissue, plant organ, plant part, plant cell, or seeds of embodiments VI. , VII. , IX. and X. may be a dicotyledonous plant tissue, plant organ, plant part, plant cell, or seed, preferably it is a soybean plant, plant tissue, plant organ, plant part, plant cell or seed or oilseed rape plant, plant tissue, plant organ, plant part, plant cell or seed.

[0096] The method of embodiment VII, may further comprises selecting the plant cell to form a plant tissue, plant organ, plant, plant cell or seed for the presence of the nucleic acid having constitutive promoter activity of embodiment I., the recombinant nucleic acid of embodiment II., the expression cassette of embodiment III., or the vector of embodiment IV.

[0097] Embodiment XII. may further comprise one or more of the following characteristics:

[0098] Characteristic 1: the food or feed may be oil, meal, grain, starch, flour or protein.BASF Agricultural Solutions US LLC 231727WO01

[0099] Characteristic 2: the industrial product is a biofuel, a fiber, an industrial chemical, a pharmaceutical or a nutraceutical.

[0100] In another aspect, provided herein is a hybrid promoter comprising a nucleic acid having promoter activity, and a NEENA molecule selected from the group consisting of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement,(ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-15 or their complement,(iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement, and(iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii), wherein said NEENA molecule has expression enhancing activity.

[0101] In an embodiment, the nucleic acid having promoter activity is a tissue specific, developmental specific or inducible promoter. In another embodiment, the nucleic acid having promoter activity is a consitutive promoter. In a further embodiment, the nucleic acid having consitutive promoter activity is selected from the group consisting of the soybean promoters Pbdc7, Pbdcl6, GmUbi9, GmUbi2, GmUbi7 and pCYPl, the tobacco promoter elF4A-10, the Arabidopsis promoters pCH3 and pCH5 and their functional variants having similar promoter activity such as for example, but not limited to, the Arabidopsis pCH5 promoter lacking the first 200 nt (SEQ ID NO: 120) and the Arabidopsis pCH3 promoter variant having a T-to-C mutation at nt 1590 (SEQ ID NO: 108).

[0102] The expression derived from promoters functionally linked to one or more NEENA molecule according to the current disclosure may be employed in any plant comprising for example moss, fern, gymnosperm or angiosperm, for example monocotyledonous or dicotyledonous plant. In an embodiment the promoter which is functionally linked to one or more NEENA molecule may be employed in monocotyledonous or dicotyledonous plants, preferably crop plant such as members of the Brassica family including mustard, cabbage, turnip and canola, corn, soy, cotton, potato, sugar beet, rice, wheat, sorghum, barley, musa, sugarcane, miscanthus and the like. In a preferred embodiment, said promoter which is functionally linked to the one or more NEENA may be employed in crop plants such as members of the Brassica family including mustard, cabbage, turnip and canola, corn, rice, wheat, soy, cotton or potato. In an especially preferred embodiment, said promoter is employed in dicotyledonous crop plants such as soy, canola, cotton or potato. In a most preferred embodiment, the promoter functionally linked to the NEENA may be employed in canola or soy.

[0103] A high expressing promoter as used in the application means for example a promoter which is functionally linked to one or more NEENA molecule causing enhanced expression of the promoter in aBASF Agricultural Solutions US LLC 231727WO01 plant, a plant part or a plant cell, wherein the accumulation of RNA or rate of synthesis of RNA derived from the nucleic acid molecule under the control of the respective promoter functionally linked to one or more NEENA molecule is higher, preferably significantly higher than the expression caused by the same promoter not functionally linked to said NEENA. Preferably the amount of RNA of the respective nucleic acid and / or the rate of RNA synthesis and / or the RNA stability in a plant is increased 25% or more, for example 50% or more, preferably 100% or more, more preferably 200% or more, more preferably 5 fold or more, even more preferably 10 fold or more, most preferably 20 fold or more for example 50 fold compared to a control plant, plant part or plant cell of same age grown under the same conditions comprising the same promoter the latter not being functionally linked to said NEENA.

[0104] When used herein, significantly higher refers to statistical significance. The skilled person is aware how to determine statistical significance for example by applying statistical tests, such as the t-test, to the respective data sets. An increase or decrease, for example in gene expression, is larger than the margin of error inherent to the measurement technique, and preferably constitutes an increase or decrease by 25% or more of the expression in the control cell or plant, for example 50% or more, preferably 100% or more, more preferably 200% or more, even more preferably 5 fold or more, even more preferably 10 fold or more, most preferably 20 fold or more for example 50 fold.

[0105] Methods for detecting expression conferred by a promoter are known in the art. For example, the promoter may be functionally linked to a marker gene such as P-Glucuronidase (GUS), green fluorescence protein (GFP) or luciferase (LUC) and the activity of the respective protein encoded by the respective marker gene may be determined in the plant, plant part or plant cell. Other methods include, but are not limited to, measuring the steady state level or synthesis rate of RNA of the nucleic acid molecule controlled by the promoter by methods known in the art such as Northern blot analysis, qPCR, run-on assays, microarray analysis, RNA sequencing or other methods described in the art.

[0106] The term "functional linkage" means that the described components are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences. Further, with respect to regulatory elements, is to be understood as meaning the sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator or a NEENA) in such a way that each of the regulatory elements can fulfil its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. As a synonym the wording "operable linkage" or "operably linked" may be used. The expression may result, depending on the arrangement of the nucleic acid sequences, in sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences suchBASF Agricultural Solutions US LLC 231727WO01 as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, more preferably less than 100 base pairs, most preferably less than 50 base pairs. In a preferred arrangement, the nucleic acid sequence to be transcribed is located behind the promoter in such a way that the transcription start is identical with the desired beginning of the RNA.

[0107] Functional linkage, and an expression construct, can be generated by means of customary recombination and cloning techniques described in the examples and also described in, for example, Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Gelvin et al. (Eds) (1990) Plant Molecular Biology Manual, Kluwer Academic Publisher, Dordrecht, The Netherlands. However, further sequences, which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. Similarly, a linker comprising non-coding nucleotides, herein referred to as "spacer" sequence may be positioned between two sequences of interest. The insertion of sequences may also lead to the expression of fusion proteins. Preferably, the expression construct, consisting of a linkage of a regulatory region for example a promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and can be inserted into a plant genome, for example by transformation.

[0108] A skilled person is aware of various methods for functionally linking two or more nucleic acid molecules. Such methods may encompass restriction / ligation, ligase independent cloning, recombineering, recombination or synthesis. Other methods may be employed to functionally link two or more nucleic acid molecules.

[0109] The term "sense" is understood to mean a nucleic acid molecule having a sequence which is complementary or identical to a target sequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of a given gene. The term "antisense" refers to a nucleotide sequence that is reverse complementary or inverted relative to its normal orientation for transcription or function and so expresses an RNA transcript that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene mRNA molecule or single stranded genomic DNA through Watson-Crick base pairing) or that is complementary to a target DNA molecule such as, for example genomic DNA present in the host cell.

[0110] In an embodiment the nucleic acids of (ii) have at least 90% sequence identity to any one of SEQ.ID NOs: 1-15 or their complement or reverse complement, preferably at least 92%, more preferably atBASF Agricultural Solutions US LLC 231727WO01 least 94%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 1-15 when sequence identity is determined over the complete length of the respective SEQ ID NOs: 1-15.

[0111] "Identity" when used in respect to the comparison of two or more nucleic acid molecules means that the sequences of said molecules share a certain degree of sequence similarity, the sequences being partially identical. Sequence identity usually is provided as "% sequence identity" or "% identity". To determine the percent-identity between two nucleic acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters for nucleic acid alignments (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL).

[0112] The following example is meant to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:Seq A: AAGATACTG length: 9 basesSeq B: GATCTGA length: 7 basesHence, the shorter sequence is sequence B.

[0113] Producing a pairwise global alignment which is showing both sequences over their complete lengths results in:Seq A: AAGATACTG-Seq B:

[0114] The "I" symbol in the alignment indicates identical residues (which means bases for DNA or amino acids for proteins). The number of identical residues is 6. The symbol in the alignment indicates gaps. The number of gaps introduced by alignment within the Seq B is 1. The number of gaps introduced by alignment at borders of Seq B is 2, and at borders of Seq A is 1. The alignment length showing the aligned sequences over their complete length is 10.

[0115] Producing a pairwise alignment which is showing the shorter sequence over its complete length according to the invention consequently results in:Seq A:Seq B:

[0116] Producing a pairwise alignment which is showing sequence A over its complete length according to the invention consequently results in:BASF Agricultural Solutions US LLC 231727WO01Seq A:Seq B:

[0117] Producing a pairwise alignment which is showing sequence B over its complete length according to the invention consequently results in:Seq A:Seq B:

[0118] The alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence). Accordingly, the alignment length showing Seq A over its complete length would be 9 (meaning Seq A is the sequence of the invention). Accordingly, the alignment length showing Seq B over its complete length would be 8 (meaning Seq B is the sequence of the invention).

[0119] In a second step, after aligning the two sequences, an identity value is determined from the alignment produced. Percent identity is then calculated by: %-identity = (identical residues / length of the alignment region which is showing the sequence of this invention over its complete length) *100. According to the example provided above, %-identity is: for Seq A being the sequence of the invention (6 / 9) * 100 = 66.7 %; for Seq B being the sequence of the invention (6 / 8) * 100 =75%. For nucleic acid sequences, to compare a sequence to any sequences of this invention (SEQ ID NOs: 1-15), the pairwise alignment shall be made over the complete length of the sequences of this invention.

[0120] "Complementary" or "complementarity" refers to two nucleotide sequences which comprise antiparallel nucleotide sequences capable of pairing with one another (by the base-pairing rules) upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences. For example, the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases are not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acid molecules is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid molecule strands has significant effects on the efficiency and strength of hybridization between nucleic acid molecule strands. A "complement" of a nucleic acid sequence as used herein refers to a nucleotide sequence whose nucleic acid molecules show total complementarity to the nucleic acid molecules of the nucleic acid sequence.

[0121] The term "antiparallel" refers to two nucleotide sequences paired through hydrogen bonds between complementary base residues with phosphodiester bonds running in the 5'-3' direction in one nucleotide sequence and in the 3'-5' direction in the other nucleotide sequence.BASF Agricultural Solutions US LLC 231727WO01

[0122] The term "hybridization" as defined herein is a process wherein substantially complementary nucleotide sequences anneal to each other. The term hybridization according to this invention means, that hybridization must occur over the complete length of the sequence of the invention. The hybridization process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridization process can also occur with one of the complementary nucleic acids immobilized to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridization process can furthermore occur with one of the complementary nucleic acids immobilized to a solid support such as a nitro-cellulose or nylon membrane or immobilized by e.g. photolithography to, for ex-ample, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridization to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and / or to remove hairpins or other secondary structures from single stranded nucleic acids.

[0123] In its broadest sense, the term "substantially complementary", when used herein with respect to a nucleotide sequence in relation to a reference or target nucleotide sequence, means a nucleotide sequence having a percentage of identity between the substantially complementary nucleotide sequence and the exact complementary sequence of said reference or target nucleotide sequence of at least 60%, more desirably at least 70%, more desirably at least 80% or 85%, preferably at least 90%, more preferably at least 93%, still more preferably at least 95% or 96%, yet still more preferably at least 97% or 98%, yet still more preferably at least 99% or most preferably 100% (the latter being equivalent to the term "identical" in this context). Sequence comparisons are carried out using default GAP analysis with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as defined above). A nucleotide sequence "substantially complementary" to a reference nucleotide sequence hybridizes to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions.

[0124] The term "stringency" refers to the conditions under which a hybridization takes place. The stringency of hybridization is influenced by conditions such as temperature, salt concentration, ionic strength and hybridization buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below Tm, and high stringency conditions are when the temperature is 10°C below Tm. High stringency hybridization conditions are typically used for isolating hybridizing sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore, medium stringency hybridizationBASF Agricultural Solutions US LLC 231727WO01 conditions may sometimes be needed to identify such nucleic acid molecules.

[0125] The "Tm" is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridizes to a perfectly matched probe. TheTm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. The maximum rate of hybridization is obtained from about 16°C up to 32°C below Tm. The presence of monovalent cations in the hybridization solution reduces the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridization to be performed at 30 to 45°C, though the rate of hybridization will be lowered. Base pair mismatches reduce the hybridization rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids: DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):Tm= 81.5°C + 16.6xlog[Na+]a+ 0.41x%[G / Cb] - 500x[Lc]1- 0.61x% formamideDNA-RNA or RNA-RNA hybrids:Tm= 79.8 + 18.5 (logl0[Na+]a) + 0.58 (%G / Cb) + 11.8 (%G / Cb)2- 820 / Lcoligo-DNA or oligo-RNAdhybrids:For <20 nucleotides: Tm= 2 ( / n)For 20-35 nucleotides: Tm= 22 + 1.46 ( / n)aor for other monovalent cation, but only accurate in the 0.01-0.4 M range.bonly accurate for %GC in the 30% to 75% range.cL = length of duplex in base pairs.dOligo, oligonucleotide; / n, effective length of primer = 2x(no. of G / C)+(no. of A / T).

[0126] Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. For non-related probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridization and which will either maintain or change the stringency conditions.

[0127] Besides the hybridization conditions, specificity of hybridization typically also depends on the function of post-hybridization washes. To remove background resulting from non-specific hybridization, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strengthBASF Agricultural Solutions US LLC 231727WO01 and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridization stringency. A positive hybridization gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.

[0128] For example, typical high stringency hybridization conditions for DNA hybrids longer than 50 nucleotides encompass hybridization at 65°C in lx SSC or at 42°C in lx SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC. Examples of medium stringency hybridization conditions for DNA hybrids longer than 50 nucleotides encompass hybridization at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC. The length of the hybrid is the anticipated length for the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. lxSSC is 0.15M NaCI and 15mM sodium citrate; the hybridization solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1.0% SDS, 100 pg / ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridization at 65°C in O.lx SSC comprising 0.1 SDS and optionally 5x Denhardt's reagent, 100 pg / ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by the washing at 65°C in 0.3x SSC.

[0129] In another embodiment, the nucleic acid of (iii) which is a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, is hybridizing to any one of SEQ ID NOs: 1-15 or their complement or reverse complement under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2 X SSC, 0.1% SDS at 50°C or 65°C, preferably 65°C. Preferably, the nucleic acid of (iii) is hybridizing to any one of SEQ ID NOs: 1-15 or their complement under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1% SDS at 50°C or 65°C, preferably 65°C. More preferably, the nucleic acid of (iii) is hybridizing to any one of SEQ ID NOs: 1-15 or their complement under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0,1 X SSC, 0.1% SDS at 50°C or 65°C, preferably 65°C.

[0130] For the purposes of defining the level of stringency, reference can be made to Sambrook and Russell (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, NY, or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates) and to Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology.BASF Agricultural Solutions US LLC 231727WO01

[0131] In another embodiment, the functional fragment of (iv) which is a functional fragment of 30 or more consecutive bases of:(i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or (ii) a nucleic acid having at least 90 % sequence identity to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or (iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement or reverse complement; comprises at least about 30, at least about 35, at least about 41, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140 consecutive bases of the original nucleic acid sequence.

[0132] "Fragment", or "subsequence" as used herein is a portion of a polynucleotide or an amino acid sequence. In some aspects, such fragments or subsequences retain or encode for at least one functional activity of the sequence to which it is related.

[0133] The term "functional fragment" refers to any nucleic acid sequence which comprises merely a part of the full-length nucleic acid sequence, but still has the same or similar activity and / or function. A "functional fragment" of a nucleic acid having expression enhancing activity denotes a nucleic acid comprising a stretch of the nucleic acid sequence of any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or of the nucleic acid having at least 95% sequence identity to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or of the nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement, which still exerts the desired function, i.e. which has 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, 85% or more or 90% or more, most preferred 95% or more of the expression enhancing activity as the corresponding original nucleic acid sequence. Functional fragments of nucleic acids having promoter activity or, of NEENA molecules, can be useful for finetuning expression levels to, in turn, obtain the desired effect of transgene expression in the plant, plant part, plant cell or seed.

[0134] In another aspect, the invention provides a method for producing a plant, plant part or plant cell with enhanced expression of one or more nucleic acid molecules, as compared to a respective control plant, plant part or plant cell, comprising the steps of: a) introducing into the plant, plant part or plant cell one or more heterologous NEENA molecule as defined above in (i) to (iv), and b) functionally linking said one or more NEENA molecule to a promoter and to a nucleic acid molecule being under the control of said promoter, wherein said NEENA molecule is heterologous to said promoter.

[0135] A "plant" is generally understood as meaning any eukaryotic single or multi-celled organism or a cell, tissue, organ, plant part or propagation material (such as seeds or fruit) of same which is capable of photosynthesis. Included for the purpose of the invention are all genera and species of higher and lowerBASF Agricultural Solutions US LLC 231727WO01 plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. The term includes whole plants, ancestors and progeny of the plants and plant parts, including shoots, stems, leaves, roots (including tubers), propagation material (such as seeds or microspores), flowers, seedlings and mature plants, tissues and organs (mature plants and seedlings) and protoplasts and their derived parts.

[0136] The term "plant" also refers to plant parts, plant cells, cell cultures such as suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores. Hence, when the term "plant" is used herein, the term is intended to encompass "a plant, plant part or plant cell". In a particular embodiment, the term "plant cell" is a non-propagating plant cell.

[0137] Mature plants refer to plants at any desired developmental stage beyond that of the seedling. Seedling refers to a young immature plant at an early developmental stage. Annual, biennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants.

[0138] The expression of genes is furthermore advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses); Pteridophytes such as ferns, horsetail and club mosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms), and Euglenophyceae. Preferred are plants which are used for food or feed purpose such as the families of the Leguminosae such as pea, alfalfa and soya; Gramineae such as rice, maize, wheat, barley, sorghum, millet, rye, triticale, or oats; the family of the Umbelliferae, especially the genus Daucus, very especially the species carota (carrot) and Apium, very especially the species Graveolens dulce (celery) and many others; the family of the Solanaceae, especially the genus Solanum, very especially the species lycopersicum (tomato) and the genus Solanum, very especially the species tuberosum (potato) and melongena (egg plant), and many others (such as tobacco); and the genus Capsicum, very especially the species annuum (peppers) and many others; the family of the Leguminosae, especially the genus Glycine, very especially the species max (soybean), alfalfa, pea, lucerne, beans or peanut and many others; and the family of the Cruciferae (Brassicacae), especially the genus Brassica, very especially the species napus (oil seed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and of the genus Arabidopsis, very especially the species thaliana and many others; the family of the Compositae, especially the genus Lactuca, very especially the species sativa (lettuce) and many others; the family of the Asteraceae such as sunflower, Tagetes, lettuce or Calendula and many other; the family of the Cucurbitaceae such as melon, pumpkin / squash or zucchini, and linseed. Further preferred are cotton,BASF Agricultural Solutions US LLC 231727WO01 sugar cane, hemp, flax, chillies, and the various tree, nut and wine species. Preferred crop plants are corn, wheat, rice, soy, rape seed, canola and potato.

[0139] A plant, plant part or plant cell exhibiting enhanced expression of a nucleic acid molecule as used herein refers to a plant having a higher, preferably statistically significant higher expression of a nucleic acid molecule compared to a control plant, plant part or plant cell grown under the same conditions without the respective one or more NEENAs functionally linked to the respective nucleic acid molecule. Such control plant, plant part or plant cell may be a wild-type or transgenic plant, plant part or plant cell wherein the promoter is not functionally linked to the one or more NEENA molecule. The term "wildtype", "natural" or "natural origin", as used herein, refers to an organism, polypeptide, or nucleic acid sequence which is naturally occurring or, in the case of a polypeptide or a nucleic acid, which is available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.

[0140] Producing a plant, plant part or plant cell, as used herein, comprises methods for stable transformation such as introducing a recombinant DNA construct into a plant, plant part or plant cell by means of Agrobacterium mediated transformation, protoplast transformation, particle bombardment or the like and optionally subsequent regeneration of a transgenic plant, plant part or plant cell. Producing a plant, plant part or plant cell, as used herein, also comprises methods for transient transformation of a plant, plant part or plant cell such as viral infection or Agrobacterium infiltration. A skilled person is aware of further methods for stable and / or transient transformation of a plant, plant part or plant cell.

[0141] Approaches such as breeding methods, protoplast fusion or recombination techniques using a donor DNA might also be employed for production of a plant of the invention and are covered herewith. For example, a single strand break (nick) or a double strand break may be introduced into the genome of a plant using recombinant technologies known in the art such as TALEN (WO12138939, WO12138927); Zink finger proteins (WO02057293, W005084190), homing endonucleases (WO11104382, WO14199358) or nucleic acid guided nucleases such as AGO, Cas9 or Casl2 (WO13141680, WO13176772, WO14093595, WO15157534 or WO16205711). Together with the introduction of such single or double-strand break inducing agents, one or more donor DNA (WO13176772, W014089290) may be introduced into the plant, plant part or plant cell comprising one or more NEENA molecule flanked by nucleic acid molecules comprising sequences essentially identical or essentially complementary to the regions adjacent to the nick or double strand break thereby facilitating homologous recombination and introducing the NEENA molecule into the genome of the plant, plant part or plant cell.

[0142] The methods of the invention may be applied to any plant or part of or cell of any plant, for example gymnosperm or angio-sperm, preferably angiosperm, for example dicotyledonous orBASF Agricultural Solutions US LLC 231727WO01 monocotyledonous plants, preferably dicotyledonous plants. Preferred monocotyledonous plants are for example corn, wheat, rice, barley, sorghum, musa, sugarcane, miscanthus and brachypodium, especially preferred monocotyledonous plants are corn, wheat and rice, most preferred is wheat. Preferred dicotyledonous plants are for example soy, rape seed, canola, linseed, cotton, potato, sugar beet, tagetes and Arabidopsis, especially preferred dicotyledonous plants are soy, rape seed, canola and potato.

[0143] In an embodiment of the invention, the methods as defined above comprise the steps of a) introducing one or more NEENA molecule comprising a nucleic acid molecule as described above in (i) to (iv) into a plant, a plant part or a plant cell, and b) integrating the NEENA molecule into the genome of the plant, plant part or plant cell whereby the NEENA molecule is functionally linked to an endogenous promoter, the NEENA molecule being heterologous to the promoter and, optionally, c) regenerating a plant or plant part comprising the one or more NEENA molecule functionally linked to the promoter from said plant, plant part or plant cell.

[0144] The one or more NEENA molecule may be introduced into the plant, plant part or plant cell by means of particle bombardment, protoplast electroporation, virus infection, Agrobacterium mediated trans-formation, CRISPR / Cas or any other approach known in the art. The NEENA molecule may be introduced integrated for example into a plasmid or viral DNA or viral RNA or a donor DNA in a CRISPR / Cas approach. The NEENA molecule may also be comprised on a BAC, YAC or artificial chromosome prior to introduction into the plant or part of the plant. Each NEENA molecule may also be introduced as a linear nucleic acid molecule comprising the respective NEENA sequence wherein additional sequences may be present adjacent to the NEENA sequence on the nucleic acid molecule. These sequences neighboring the NEENA sequences may be from about 20 bp, for example 20 bp to several hundred base pairs, for example 100 bp or more and may facilitate integration into the genome for example by homologous recombination. Any other method for genome integration may be employed, be it targeted integration approaches, such as homologous recombination or random integration approaches, such as illegitimate recombination.

[0145] The nucleic acid molecule to which the one or more NEENA molecule is functionally linked may be any nucleic acid, preferably any expressed nucleic acid molecule. The nucleic acid molecule may be a protein coding nucleic acid molecule or a non-coding molecule such as antisense RNA, rRNA, tRNA, miRNA, ta-siRNA, siRNA, dsRNA, snRNA, snoRNA or any other noncoding RNA known in the art. The nucleic acid molecule functionally linked to one or more NEENA molecule may be an endogenous or heterologous nucleic acid molecule.

[0146] An "endogenous" nucleotide sequence or nucleic acid molecule refers to a nucleotide sequence,BASF Agricultural Solutions US LLC 231727WO01 which is present in the genome of an untransformed plant cell. A "double-stranded RNA" molecule or "dsRNA" molecule comprises a sense RNA fragment of a nucleotide sequence and an antisense RNA fragment of the nucleotide sequence, which both comprise nucleotide sequences complementary to one another, thereby allowing the sense and antisense RNA fragments to pair and form a double-stranded RNA molecule. The term "non-coding" refers to nucleotide sequences or nucleic acid molecules that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited to introns, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions.

[0147] In a further embodiment of the methods of the invention the one or more NEENA molecule or NEENA sequence is integrated into the genome of a plant, plant part or plant cell by applying genome editing technologies.

[0148] "Genome editing", "genome engineering" or "gene editing" technologies are used herein interchangeably and refer to genetic engineering techniques allowing targeted modifications of the genetic material of a host cell or organism. Genome editing technologies may be used for the production of a plant, plant part or plant cell of the invention.

[0149] Genome editing technologies encompass technologies comprising the introduction of single or double strand breaks near to the position where a target molecule or sequence, such as for example a NEENA molecule, is to be integrated into the genome. These technologies are for example based on the use of targeted nucleases and a DNA repair template. Suitable targeted nucleases include for example nucleic acid guided nucleases such as AGO, Cas9 or Casl2 nucleases, TALENs, homing endonucleases and Zinc finger proteins. Suitable DNA repair templates comprise the desired molecule or sequence, such as for example a NEENA molecule, flanked at their 3' and 5' ends by sequences which are essentially identical or essentially complementary to the sequences upstream and / or downstream of the single or double strand break.

[0150] Further, the sequence of one or more NEENAs of the invention may be introduced into the genome and functionally linked to the respective heterologous promoter by introducing into the genome a series of point mutations using gene editing technologies encompassing, for example, the use of deaminases (WO 2000 / 058480 Al, WO 2018 / 027078 Al) and the like which may be directed to a specific region in the genome of a plant, plant part or plant cell by fusing the mutating polypeptide portion e.g. a deaminase or glycosidase to a DNA binding polypeptide such as, for example a TALEN, a Zinc finger protein, a homing endonuclease or an RNA guided nuclease, nickase or inactivated nuclease such as Cas9 or Casl2, as described in WO 2015 / 089406 Al, US 2017321210 Al, WO 2015 / 133554 Al or WO 2017 / 070632 A2). By application of these methods, the NEENA sequence is introduced into the genome without introduction of a heterologous molecule, the NEENA sequence replacing other sequences in theBASF Agricultural Solutions US LLC 231727WO01 genome. Such technologies are encompassed by the term "integrate", "integrating", "introduce" or "introducing" a NEENA sequence or a NEENA molecule into the genome and functionally linking such sequences and / or molecules to a heterologous promoter.

[0151] In a further preferred embodiment, the genome editing technology comprises the introduction of single or double strand breaks and the introduction of a DNA repair template comprising the NEENA molecule or sequence. Accordingly, single or double strand breaks are introduced near to the position where a NEENA molecule is to be integrated into the genome using targeted nucleases. In addition, a DNA repair template is introduced comprising said NEENA molecule and at its 3' and 5' end sequences essentially identical or complementary to the sequences upstream and downstream of the single or double strand break facilitating recombination at the position of the single or double strand break. Preferably, the essentially identical or essentially complementary sequences are each individually at least 1000 bases, at least 500 bases, at least 450 bases, at least 400 bases, at least 350 bases, at least 300 bases, at least 250 bases, at least 200 bases, at least 150 bases, at least 100 bases or at least 50 bases long. Preferably, the identity or complementarity of the sequences is at least 50%, at least 60%, at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98 or at least 99% identical or complementary to the respective genomic region with which they recombine.

[0152] In another or further preferred embodiment, the genome editing technology comprises introduction of point mutations in the genome of the plant, plant part or plant cell thereby introducing the sequence of a NEENA molecule in the genome. This can, for example, be achieved by introducing DNA binding proteins, for example Zinc finger proteins, TALE proteins or a nucleic acid guided nuclease, for example Cas9, Casl2 (Cpfl) or AGO functionally bound to a cytidine deaminase (W017070633) or adenine deaminase (W018027078).

[0153] In another embodiment of the invention, the methods described above comprise the steps of: a) providing to a plant, a plant part or a plant cell an expression construct comprising one or more NEENA molecules functionally linked to a promoter, the latter being heterologous to said one or more NEENA molecule, wherein the one or more NEENA molecule comprises a nucleic acid molecule as described above in (i) to (iv); and b) integrating the expression construct into the genome of a plant, plant part or plant cell; and, optionally, c) regenerating a plant or plant part comprising said one or more expression construct from said transformed plant, plant part or plant cell.

[0154] The one or more NEENA molecule may be heterologous to the nucleic acid molecule which is under the control of the promoter to which the NEENA is functionally linked or they may be heterologousBASF Agricultural Solutions US LLC 231727WO01 to both the promoter and the nucleic acid molecule under the control of that promoter.

[0155] The expression construct may be integrated into the genome of the respective plant with any method known in the art. The integration may be random or targeted using methods such as for example, but not limited to, particle bombardment or Agrobacterium mediated transformation or CRISPR / Cas applications. In a preferred embodiment, the integration is via targeted integration for example by homologous recombination. The latter method would allow integrating the expression construct comprising a high expression promoter functionally linked to one or more NEENA molecule into a favorable genome region. Favorable genome regions are for example genome regions known to comprise genes that are highly expressed for example in seeds and hence may increase expression derived from said expression construct compared to a genome region which shows no transcriptional activity.

[0156] In another preferred embodiment, the one or more NEENA molecule is functionally linked to the promoter close to the transcription start site of the nucleic acid molecule which is under control of that promoter. Close to the transcription start site, as meant herein, comprises functionally linking one or more NEENA molecule to a promoter 5000 bp or less, 4000 bp or less, 3000 or less, 2500 bp or less, preferably 2000 bp or less, more preferred 1500 bp or less, even more preferred 1000 bp or less and most preferred 500 bp or less away from the transcription start site of said nucleic acid molecule. It is to be understood that the NEENA molecule or sequence may be integrated upstream or downstream in the respective distance from the transcription start site of the respective promoter. Hence, the one or more NEENA molecule may be included in the primary transcript of the respective nucleic acid under control of the promoter to which the NEENA molecule is functionally linked, or it may be integrated within the promoter. If the NEENA molecule is integrated downstream of the transcription start site of the respective promoter, the integration site is preferably in the 5' UTR, the 3' UTR or in an intron of the nucleic acid the expression of which is under the control of that promoter. Preferably, the one or more NEENA molecule or sequence is integrated in the promoter, the 5' UTR or the first intron or, similarly, the NEENA molecule is replacing a part of the promoter, the 5'UTR or of the first intron. More preferably, the one or more NEENA molecule is integrated in the first intron of the respective nucleic acid under control of the promoter to which the NEENA molecule is functionally linked.

[0157] The term "intron" as used herein, refers to sections of DNA (intervening sequences) within a gene that do not encode part of the protein that the gene produces, and that is spliced out of the mRNA that is transcribed from the gene before it is exported from the cell nucleus. Intron sequence refers to the nucleic acid sequence of an intron. Thus, introns are those regions of DNA sequences that are transcribed along with the coding sequence (exons) but are removed during the formation of mature mRNA. Introns can be positioned within the actual coding region or in either the 5' or 3' untranslated leaders of the pre- mRNA (unspliced mRNA). Introns in the primary transcript are excised and the coding sequences areBASF Agricultural Solutions US LLC 231727WO01 simultaneously and precisely ligated to form the mature mRNA. The junctions of introns and exons form the splice site. The sequence of an intron begins with GU and ends with AG. Furthermore, in plants, two examples of AU-AC introns have been described: the fourteenth intron of the RecA-like protein gene and the seventh intron of the G5 gene from Arabidopsis thaliana are AT-AC introns. Pre-mRNAs containing introns have three short sequences that are, in addition to other sequences, essential for the intron to be accurately spliced. These sequences are the 5' splice-site, the 3' splice-site, and the branchpoint. mRNA splicing is the removal of intervening sequences (introns) present in primary mRNA transcripts and joining or ligation of exon sequences. This is also known as cis-splicing which joins two exons on the same RNA with the removal of the intervening sequence (intron). The functional elements of an intron are comprising sequences that are recognized and bound by the specific protein components of the spliceosome (e.g. splicing consensus sequences at the ends of introns). The interaction of the functional elements with the spliceosome results in the removal of the intron sequence from the premature mRNA and the rejoining of the exon sequences. The branchpoint sequence is important in splicing and splicesite selection in plants. The branchpoint sequence is usually located 10-60 nucleotides upstream of the 3' splice-site.

[0158] Alternatively, some introns have been recognized as genetic elements with a strong potential for improving gene expression. Although the mechanism is largely unknown, it has been shown that some introns positively affect the steady state amount of mature mRNA, possibly by enhanced transcriptional activity, improved mRNA maturation, enhanced nuclear mRNA export and / or improved translation initiation (e.g. Huang and Gorman, 1990; Le Hir et al., 2003; Nott et al., 2004). Since only selected introns were shown to increase expression, splicing as such is likely not accountable for the observed effects. The increase of gene expression observed upon functionally linking introns to promoters is called intron mediated enhancement of gene expression and has been shown in various monocotyledonous (e.g. Callis et al., 1987; Vasil et al., 1989; Bruce et al., 1990; Lu et al., 2008) and dicotyledonous plants (e.g. Chung et al., 2006; Kim et al., 2006; Rose et al., 2008).

[0159] In a further embodiment of the invention, the nucleic acid having promoter activity, to which the one or more heterologous NEENA molecule as defined above is functionally linked, is further functionally linked to an intron having expression enhancing activity. Preferably, the intron having expression enhancing activity is selected from the group comprising NEENAc5, NEENAC18 (SEQ ID NOs: 153, 155; WO 2011 / 023537 Al), NEENAssl3 (SEQ ID NO: 154; WO 2011 / 023800 Al) or intronl_h3At (Chaubet et al. 1992, J. Mol. Biol. 225, 569-574). In another preferred embodiment the nucleic acid having promoter activity is the soybean Pbdc7 promoter. In another, further preferred embodiment, the soybean Pbdc7 promoter is functionally linked to the NEENA molecule comprising the nucleic acid sequence of SEQ ID NO: 1 and to the NEENAc5 introns as defined above. In yet another, further preferred embodiment, theBASF Agricultural Solutions US LLC 231727WO01 soybean Pbdc7 promoter is functionally linked to the NEENA molecule comprising the nucleic acid sequence of SEQ ID NO: 3 and to the NEENAcl8 intron.

[0160] As used herein, the term "coding region", when used in reference to a structural gene, refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' -side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3' -side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA). In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5'- and 3' -end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions. These flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript. The 5' -flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' -flanking region may contain sequences which direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

[0161] The term "primary transcript" as used herein refers to a premature RNA transcript of a gene. A "primary transcript", for example, still comprises introns and / or is not yet comprising a polyA tail or a cap structure and / or is missing other modifications necessary for its correct function as a transcript such as, for example, trimming or editing.

[0162] In another aspect of the invention, when said one or more NEENA molecule is functionally linked to a promoter, the NEENA molecule may be inserted at a position between 50 and 150 bp, 150 and 250 bp, between 250 and 350 bp, between 350 and 450 bp, between 450 bp and 620 bp, between 620 and 720 bp, between 720 and 950 bp or between 950 and 1000 bp upstream of the transcriptional start site of a nucleic acid molecule the expression of which is under control of that promoter.

[0163] When said one or more NEENA molecule is functionally linked to the Pbdc7 promoter (WO2014150449), said one or more NEENA molecule is preferably inserted between nucleotide (nt) 402 and 403, nt 628 and 629, nt 827 and 828, nt 1052 and 1053, nt 1188 and 1189, nt 1300 and 1301, nt 1413 and 1414 or nt 1508 and 1509, more preferably between nt 1413 and 1414 or nt 1508 and 1509, most preferably between nt 1508 and 1509 of the Pbdc7 promoter (SEQ ID NO: 28).

[0164] When said one or more NEENA molecule is functionally linked to the Pbdcl6 promoter (WQ2021183803), said one or more NEENA molecule is preferably inserted between nt 66 and 67, nt 335 and 336, nt 493 and 494, nt 600 and 601, nt 695 and 696, nt 729 and 730, nt 767 and 768, nt 833 and 834, nt 857 and 858, nt 887 and 888 or nt 925 and 926, more preferably between nt 335 and 336, nt 493 and 494, nt 695 and 696, nt 729 and 730, nt 767 and 768, nt 833 and 834, nt 857 and 858, nt 887 and 888 orBASF Agricultural Solutions US LLC 231727WO01 nt 925 and 926, most preferably between nt 925 and 926 of the Pbdcl6 promoter (SEQ ID NO: 61).

[0165] When said one or more NEENA molecule is functionally linked to the GmUbi9 promoter (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237), said one or more NEENA molecule is preferably inserted between nt 1585 and 1586, nt 1632 and 1633, nt 1689 and 1690, nt 1740 and 1741, nt 1799 and 1800, nt 1830 and 1831, nt 1862 and 1863 or nt 1902 and 1903, more preferably between nt 1862 and 1863 or nt 1902 and 1903, most preferably between nt 1862 and 1863 of the GmUbi9 promoter (SEQ ID NO: 73).

[0166] When said one or more NEENA molecule is functionally linked to the GmUbi2 promoter (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237), said one or more NEENA molecule is preferably inserted between nt 918 and 919, nt 967 and 968, nt 1078 and 1079, nt 1138 and 1139, nt 1188 and 1189, nt 1228 and 1229, nt 1256 and 1257 or nt 1289 and 1290, more preferably between nt 1078 and 1079, nt 1188 and 1189, nt 1228 and 1229 or nt 1256 and 1257, most preferably between nt 1228 and 1229 of the GmUbi2 promoter (SEQ ID NO: 85).

[0167] When said one or more NEENA molecule is functionally linked to the GmUbi7 promoter (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237), said one or more NEENA molecule is preferably inserted between nt 897 and 898, nt 996 and 997, nt 1071 and 1072, nt 1140 and 1141, nt 1190 and 1191, nt 1221 and 1222, nt 1253 and 1254 or nt 1282 and 1283, more preferably between nt 1190 and 1191, nt 1253 and 1254 or nt 1282 and 1283, most preferably between nt 1282 and 18283 of the GmUbi7 promoter (SEQ ID NO: 96).

[0168] When said one or more NEENA molecule is functionally linked to the Arabidopsis pCH3 promoter (Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545), said one or more NEENA molecule is preferably inserted between nt 1470 and 1471, nt 1560 and 1561, nt 1607 and 1608, nt 1660 and 1661, nt 1709 and 1710, nt 1740 and 1741, nt 1775 and 1776, nt 1805 and 1806, more preferably between nt 1560 and 1561, nt 1607 and 1608, nt 1660 and 1661, nt 1709 and 1710, nt 1740 and 1741, nt 1775 and 1776, nt 1805 and 1806, most preferably between nt 1775 and 1776 of the pCH3 promoter (SEQ ID NO: 107).

[0169] When said one or more NEENA molecule is functionally linked to the Arabidopsis pCH5 promoter (Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545), said one or more NEENA molecule is preferably inserted between nt 1515 and 1516, nt 1572 and 1573, nt 1632 and 1633, nt 1695 and 1696, nt 1755 and 1756, nt 1796 and 1797, nt 1833 and 1834 or nt 1866 and 1867, most preferably between nt 1833 and 1834 of the pCH5 promoter (SEQ ID NO: 119).

[0170] When said one or more NEENA molecule is functionally linked to the tobacco NelF-4A10 promoter (Mandel et al. 1995, Plant Mol. Biol. 29, 995-1004), said one or more NEENA molecule is preferablyBASF Agricultural Solutions US LLC 231727WO01 inserted between nt 685 and 686 , nt 771 and 772, nt 821 and 822, nt 871 and 872 or nt 921 and 922, most preferably between nt 871 and 872 of the pNelF-4A10 promoter (SEQ ID NO: 131).

[0171] When said one or more NEENA molecule is functionally linked to the promoter sequence of the soybean CYP1 gene (Glyma.llG098700), said one or more NEENA molecule is preferably inserted between nt 1509 and 1510, nt 1609 and 1610, nt 1666 and 1667, nt 1725 and 1726, nt 1779 and 1780, nt 1819 and 1820, nt 1852 and 1853 or nt 1879 and 1880, more preferably between nt 1779 and 1780, nt 1819 and 1820 or nt 1879 and 1880, most preferably between nucleotide 1879 and 1880 of the pCYPl promoter (SEQ ID NO: 142).

[0172] In an embodiment of the invention, more than one NEENA is functionally linked to the promoter of a nucleic acid molecule the expression of which is under the control of that promoter. The individual NEENA molecules can be present in any orientation relative to each other. Accordingly, they may be present in one or more of the configurations selected from the group consisting of: head-to-head, head- to-tail, tail-to-head, tail-to-tail, and combinations thereof. The NEENA molecules referred to in the methods described herein may be contiguous with reference to each other, may be separated by a part of the promoter to which they are functionally linked, by a part of the 5' UTR or by a part of the first intron or may be separated from one another by a spacer sequence, which may comprise about 1 to 50 nucleotides. Preferably, the spacer sequence comprises about 1 to 40 nucleotides, more preferably about 1 to 30 nucleotides, even more preferably about 1 to 20 nucleotides, most preferably about 1 to 10 nucleotides. The spacer sequence may comprise one or more nucleotides which are naturally flanking the selected NEENA molecule or functional fragment thereof.

[0173] Another embodiment of the invention comprises a recombinant expression construct comprising one or more NEENA molecule functionally linked to a promoter and one or more expressed nucleic acid molecules, wherein said NEENA molecule is heterologous to said promoter and wherein said NEENA molecule comprises a nucleic acid as defined above in (i) to (iv): (i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or (ii) a nucleic acid having at least 90 % sequence identity to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or (iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement or reverse complement, or (iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of (i) to (iii).

[0174] "Expression construct" and "expression cassette" are used herein interchangeably and refer to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate part of a plant or plant cell, comprising a promoter functional in said part of a plant or plant cell into which it will be introduced, operably linked to the nucleotide sequence of interest which is - optionally - operablyBASF Agricultural Solutions US LLC 231727WO01 linked to termination signals. If translation is required, it also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region may code for a protein of interest but may also code for a functional RNA of interest, for example RNAa, siRNA, snoRNA, snRNA, microRNA, ta- siRNA or any other noncoding regulatory RNA, in the sense or antisense direction. The expression construct comprising the nucleotide sequence of interest may be chimeric, meaning that one or more of its components is heterologous with respect to one or more of its other components.

[0175] The expression construct may be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e. the particular DNA sequence of the expression construct does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor thereof by a transformation event. An example of a heterologous expression construct comprising a nucleic acid molecule and one or more regulatory nucleic acid molecule (such as a promoter or a transcription termination signal) linked thereto, is a construct originating by experimental manipulations in which the nucleic acid molecule, the regulatory nucleic acid molecule or both, are not located in their natural (native) genetic environment, or have been modified by experimental manipulations. Experimental manipulations include for example a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the sequence of the nucleic acid molecule is preferably retained at least in part. The genetic environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, more preferably at least 1,000 bp, most preferably at least 5,000 bp, in length. A naturally occurring expression construct - for example the naturally occurring combination of a promoter with the corresponding gene - becomes a transgenic expression construct when it is modified by non-natural, synthetic or artificial methods such as, for example, mutagenization. Such methods have been described for example in US5565350 and W00015815.

[0176] The expression of the nucleotide sequence in the expression construct may be under the control of a constitutive promoter or of an inducible promoter, which may initiate transcription only when the host cell is exposed to some particular external stimulus. In the case of a plant or plant cell, the promoter can also be specific to a particular tissue or organ or stage of development.

[0177] The term "recombinant" with respect to nucleic acid molecules refers to nucleic acid molecules produced by recombinant DNA techniques. Recombinant nucleic acid molecules may also comprise molecules which as such do not exist in nature but are modified, changed, mutated or otherwise manipulated by man. Preferably, a "recombinant nucleic acid molecule" is a non-naturally occurring nucleic acid molecule that differs in sequence from a naturally occurring nucleic acid molecule by at leastBASF Agricultural Solutions US LLC 231727WO01 one nucleic acid. A "recombinant nucleic acid molecule" may also comprise a "recombinant construct" which comprises a sequence of, preferably operably linked, nucleic acid molecules which do not naturally occur in that order. Preferred methods to produce said recombinant nucleic acid molecule may comprise cloning techniques, directed or non-directed mutagenesis, synthesis or recombination techniques.

[0178] Optionally, the one or more NEENA molecule in the recombinant expression construct may also be heterologous to the expressed nucleic acid molecule which is under the control of the promoter to which the NEENA molecule is functionally linked. The expression construct may comprise one or more, preferably two or more, such as for example 3 or more or 4 or more combinations of promoters functionally linked to one or more NEENA molecule and a nucleic acid molecule to be expressed heterologous to the respective NEENA molecule. The expression construct may also further comprise additional promoters which are not functionally linked to one or more NEENA molecule and which control the expression of nucleic acid molecules homologous or heterologous to the respective promoter.

[0179] Another embodiment of the invention provides a recombinant expression vector comprising one or more recombinant expression constructs as defined above. A multitude of expression vectors that may be used in the present invention are known to a skilled person. Methods for introducing a vector comprising such an expression construct, comprising for example a promoter functionally linked to one or more NEENA molecule and optionally other elements such as a terminator, into the genome of a plant and for recovering transgenic plants from a transformed cell are also well known in the art. Depending on the method used for the transformation of a plant or plant part the entire vector might be integrated into the genome of said plant or plant part or certain components of the vector might be integrated into the genome, such as, for example a T-DNA.

[0180] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a genomic integrating vector or "integrating vector", which can transport a nucleic acid molecule, such as an expression construct or expression cassette, which becomes integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors". In the present specification, "plasmid" and "vector" are used interchangeably unless otherwise clear from the context. Expression vectors designed to produce RNAs as described herein in vitro or in vivo may contain sequences recognized by any RNA polymerase, including mitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III. These vectors can be used to transcribe the desired RNA molecule in the cell according to this invention. A plant transformation vector is to be understood as a vector suitable in the process of plant transformation.BASF Agricultural Solutions US LLC 231727WO01

[0181] The term "gene" as used herein refers to a region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term "structural gene" as used herein refers to a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

[0182] The term "genome" or "genomic DNA" refers to the heritable genetic information of a host organism. Said genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the plastids (e.g. chloroplasts) and other cellular organelles (e.g. mitochondria). Preferably, the term genome (or genomic DNA) refers to the chromosomal DNA of the nucleus.

[0183] A transgenic cell or transgenic plant or transgenic plant part comprising one or more heterologous NEENA molecule, wherein the NEENA molecule comprises a nucleic acid as defined above in (i) to (iv), is also enclosed in this invention. A NEENA molecule is understood to be heterologous to the plant if it is synthetic or if it is derived from another organism or from the same organism but its natural genomic location is modified compared to a control plant (for example a wild-type plant). It is to be understood, that a modified genomic location means the NEENA is located on another chromosome or on the same chromosome but dislocated for 10 kb or more, for example 10 kb, preferably 5 kb or more, for example 5 kb, more preferably 1000 bp or more, for example 1000 bp, even more preferably 500 bp or more, for example 500 bp, even more preferably lOObp or more, for example 100 bp, most preferably 10 bp or more from its natural genomic location in a wild-type plant.

[0184] The term "transgene" as used herein, refers to any nucleic acid sequence, which is introduced into the genome of a cell by experimental manipulations. A transgene may be an "endogenous DNA sequence," or a "heterologous DNA sequence" (i.e., "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell into which it is introduced as long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence. The term "transgenic" when referring to an organism means transformed, preferably stably transformed, with a recombinant DNA molecule that preferably comprises a suitable promoter operably linked to a DNA sequence of interest.

[0185] Accordingly in another aspect, a method for producing a plant, plant part or plant cell with enhanced expression of one or more nucleic acid molecules, as compared to a respective control plant, plant part or plant cell is provided herein, comprising the steps of:BASF Agricultural Solutions US LLC 231727WO01(a) Making a recombinant expression construct comprising one or more NEENA molecule as described above functionally linked to a promoter and one or more expressed nucleic acid molecules, or making a recombinant expression vector comprising one or more of said recombinant expression constructs;(b) introducing said expression construct into the plant, plant part or plant cell; and(c) optionally, regenerating a plant or plant part comprising said one or more NEENA molecule functionally linked to the promoter from said plant, plant part or plant cell.

[0186] Methods for introducing a recombinant expression construct or vector comprising such an expression construct into the genome of a plant, for recovering transgenic plants from a transformed cell and for regenerating a plant or plant part are also well known in the art.

[0187] A transgenic cell, transgenic plant or transgenic plant part comprising a recombinant expression vector as defined above or a recombinant expression construct as defined above is a further embodiment of the invention. The transgenic cell, transgenic plant or transgenic plant part may be selected from the group consisting of bacteria, fungi, yeasts or plant, insect or mammalian cells or plants. Preferably, the transgenic cells are bacteria, fungi, yeasts or plant cells. Preferred bacteria are Enterobacteria such as E. coli and bacteria of the genus Agrobacteria, for example Agrobacterium tumefaciens and Agrobacterium rhizogenes.

[0188] The present invention may be used in any plant species, including, but not limited to, monocotyledonous or dicotyledonous plants and plant cells. Examples of species of interest include corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, turnip, mustard, tobacco, barley, oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers. Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia , and chrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.). Especially preferred dicotyledonous crop plants are soy, canola, cotton or potato. Especially preferred monocotyledonous crop plants are corn, wheat and rice.

[0189] In an embodiment, the plant, plant part or plant cell is a canola or soybean plant, plant part or plant cell. In a further embodiment, the one or more NEENA molecule according to the invention is selected from the group consisting of: (i) a nucleic acid having the sequence of any one of SEQ ID NOs: 1 or 3 or their complement, (ii) a nucleic acid having at least 90% sequence identity to any one of SEQ IDBASF Agricultural Solutions US LLC 231727WO01NOs: 1 or 3 or their complement, (iii) a nucleic acid hybridizing under stringent conditions to SEQ ID NOs: 1 or 3 or their complement, and (iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii).

[0190] In another embodiment, the invention provides a transgenic cell culture, transgenic seed, transgenic plant parts or propagation material derived from a transgenic cell or transgenic plant or transgenic plant part as defined above comprising one or more heterologous NEENA molecule comprising a nucleic acid as defined above in (i) to (iv). In yet another embodiment, the invention provides a transgenic cell culture, transgenic seed, transgenic plant part or propagation material derived from a transgenic cell or transgenic plant or transgenic plant part as defined above comprising a recombinant expression construct or a recombinant expression vector as described above. Transgenic plant parts or propagation material, as used herein, comprise all tissues and organs of a plant comprising the respective NEENA molecule, recombinant expression construct or recombinant vector including, but not limited to, leaf, stem and fruit as well as material that is useful for propagation and / or regeneration of plants such as cuttings, scions, layers, branches and shoots.

[0191] In another further aspect, the invention provides the use of a transgenic cell culture, transgenic seed, transgenic plant part or propagation material derived from a transgenic cell or transgenic plant or plant part as defined above for the production of foodstuffs, animal feeds, seeds, pharmaceuticals or fine chemicals.

[0192] Figure 1: Cloning strategy of the MPRA expression library (A). An oligo library consisting of query sequences (q), each provided with 7 unique 11-nt barcode sequences (b), a cut site (c) and adaptor sequences (a), was PCR amplified and subsequently cloned in-between the CaMV 35S enhancer (EN35S-I, e) and the 3' pin2 of pBasl (p). Finally, a DNA fragment containing the minimal 35S promoter (m), the petunia cab 22L leader sequence (I) and the gus coding sequence (g) was cloned between the query sequence (q) and the barcode (b). The composition of the vectors to test individual candidate enhancers is shown in panel B.

[0193] Figure 2: Impact of candidate NEENA molecules on the activity of a CaMV 35S promoter enhancer- minimal promoter combination in transiently transformed canola mesophyll protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA as follows: a: SOY-II, b: SOY-16, c: SOY-10, d: SOY-3, e: SOY-5, f: SOY-8, g: SOY-9, h: SOY-15, i: SOY-6, j: SOY-14, k: SOY-2, I: SOY-12, m: SOY-7, n: SOY-1, o: SOYA, p: SOY-13, q: MISC-5.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid.BASF Agricultural Solutions US LLC 231727WO01Activity of the promoter with a control sequence (q: MISC-5.1) was set at 1.

[0194] Figure 3: Impact of the EN-35S2-1.1 sequence on the activity of the Pbdc7 promoter in transiently transformed soybean protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the Pbdc7 promoter (SEQ ID NO: 28) between which the EN-35S2- 1.1 fragment was inserted as follows: a: nt 402-403, b: nt 628-629, c: nt 827-828, d: nt 1052-1053, e: nt 1188-1189, f: nt 1300-1301, g: nt 1413-1414, h: nt 1508-1509, i: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (i: none) was set at 1.

[0195] Figure 4: Impact of candidate NEENA molecules on the activity of the Pbdc7 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1508 and 1509 of the Pbdc7 sequence (SEQ ID NO: 28) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY- 5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2-1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a cointroduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0196] Figure 5: Impact of candidate NEENA molecules on the activity of the Pbdc7 promoter in transiently transformed soybean protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1508 and 1509 of the Pbdc7 sequence (SEQ ID NO: 28) as follows: a: SOY-3, b: SOY-5, c: SOY-9, d: SOY-6, e: SOY-2, f: SOY-1, g: EN-35S2-1.1 and h: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (h: none) was set at 1.

[0197] Figure 6: Impact of candidate NEENA molecules and introns on the activity of the Pbdc7 promoter in transiently transformed canola (A) or soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the introns whereas the series legend shows the candidate NEENA molecules that were inserted between nucleotide position 1508 and 1509 of the Pbdc7 sequence (SEQ ID NO: 28) as follows: a: SOY-3, b: SOY-5, c: SOY-6, d: SOY-1, e: no NEENA, f: NEENAc5, g: NEENAcl8, h: NEENAssl3, i: intronl_h3At and j: no intron. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without any inserted sequences (e / j: no NEENA / no intron) was set at 1.

[0198] Figure 7: Comparison of the activity of the PubilOAt promoter and a Pbdc7 promoter functionally linked to candidate NEENA SOY-1 and intron NEENAc5 in transiently transformed canola protoplasts. TheBASF Agricultural Solutions US LLC 231727WO01 vertical axis shows the relative promoter activity. The horizontal axis shows the promoter variant as follows: a: PubilOAt and b: Pbdc7+SOY-l+NEENAc5. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the PubilOAt promoter was set at 100. Panel A and B show the results from 2 independent experiments.

[0199] Figure 8: Impact of the EN-35S2-1.1 sequence on the activity of the Pbdcl6 promoter in transiently transformed soybean protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the Pbdcl6 promoter (SEQ ID NO: 61) between which the EN-35S2-1.1 fragment was inserted as follows: a: 66-67, b: 335-336, c: 493-494, d: 600-601, e: 695- 696, f: 729-730, g: 767-768, h: 833-834, i: 857-858, j: 887-888, k: 925-926 and I: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a cointroduced pGVEc plasmid. Activity of the promoter without an inserted sequence (I: none) was set at 1.

[0200] Figure 9: Impact of candidate NEENA molecules on the activity of the Pbdcl6 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecule that was inserted between nucleotide position 925 and 926 of the Pbdcl6 promoter (SEQ ID NO: 61) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2-1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a cointroduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0201] Figure 10: Impact of candidate NEENA molecules on the activity of the Pbdcl6 promoter in transiently transformed soybean protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecule that was inserted between nucleotide position 925 and 926 of the Pbdcl6 promoter (SEQ ID NO: 61) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2-1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a cointroduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0202] Figure 11: Impact of the EN-35S2-1.1 sequence on the activity of the GmUbi9 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the GmUbi9 promoter (SEQ ID NO: 73) between which the EN-35S2-1.1 fragment was inserted as follows: a: 1585-1586, b: 1632-1633, c: 1689-1690, d: 1740- 1741, e: 1799-1800, f: 1830-1831, g: 1862-1863, h: 1902-1903, i: none - promoter missing the first 400 nt, and j: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (j: none) was set at 1.BASF Agricultural Solutions US LLC 231727WO01

[0203] Figure 12: Impact of the EN-35S2-1.1 sequence on the activity of the GmUbi2 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the GmUbi2 promoter (SEQ ID NO: 85) between which the EN-35S2-1.1 fragment was inserted as follows: a: 918-919, b: 967-968, c: 1078-1079, d: 1138-1139, e: 1188-1189, f: 1228-1229, g: 1256-1257, h: 1289-1290, and i: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (i: none) was set at 1.

[0204] Figure 13: Impact of the EN-35S2-1.1 sequence on the activity of the GmUbi7 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the GmUbi7 promoter (SEQ ID NO: 96) between which the EN-35S2-1.1 fragment was inserted as follows: a: 897-898, b: 996-997, c: 1071-1072, d: 1140-1141, e: 1190-1191, f: 1221-1222, g: 1253-1254, h: 1282-1283, and i: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (i: none) was set at 1.

[0205] Figure 14: Impact of the EN-35S2-1.1 sequence on the activity of the pCH3 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the pCH3 promoter (SEQ ID NO: 107) between which the EN-35S2-1.1 fragment was inserted as follows: a: 1470-1471, b: 1560-1561, c: 1607-1608, d: 1660-1661, e: 1709-1710, f: 1740-1741, g: 1775-1776, h: 1805-1806, i: none, and j: none - promoter with T1590C mutation. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (i: none) was set at 1.

[0206] Figure 15: Impact of the EN-35S2-1.1 sequence on the activity of the pCH5 promoter in transiently transformed canola protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the pCH5 promoter (SEQ ID NO: 119) between which the EN-35S2-1.1 fragment was inserted as follows: a: 1515-1516, b: 1572-1573, c: 1632-1633, d: 1695-1696, e: 1755-1756, f: 1796-1797, g: 1833-1834, h: 1866-1867, i: none, and j: none - promoter missing the first 200 nt. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (i: none) was set at 1.

[0207] Figure 16: Impact of the EN-35S2-1.1 sequence on the activity of the pNelF-4A10 promoter in transiently transformed soybean protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the pNelF-4A10 promoter (SEQ ID NO: 131) betweenBASF Agricultural Solutions US LLC 231727WO01 which the EN-35S2-1.1 fragment was inserted as follows: a: 571-572, b: 625-626, c: 685-686, d: 721-722, e: 771-772, f: 821-822, g: 871-872, h: 921-922, and i: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (i: none) was set at 1.

[0208] Figure 17: Impact of the EN-35S2-1.1 sequence on the activity of the pCYPl promoter in transiently transformed soybean protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the nucleotide positions on the pCYPl promoter (SEQ ID NO: 142) between which the EN-35S2-1.1 fragment was inserted as follows: a: 1509-1510, b: 1609-1610, c: 1666-1667, d: 1725- 1726, e: 1779-1780, f: 1819-1820, g: 1852-1853, h: 1879-1880, and i: none. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0209] Figure 18: Impact of candidate NEEN A sequences on the activity of the soybean GmUbi9 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1462 and 1463 of the sequence of the GmUbi9 promoter lacking the first 400 nt (SEQ ID NO: 74) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2-1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0210] Figure 19: Impact of candidate NEENA sequences on the activity of the soybean GmUbi2 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1228 and 1229 of the GmUbi2 sequence (SEQ ID NO: 85) as follows: a: SOY-11, b: SOY- 10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2- 1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0211] Figure 20: Impact of candidate NEENA sequences on the activity of the soybean GmUbi7 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1282 and 1283 of the GmUbi7 sequence (SEQ ID NO: 96) as follows: a: SOY-11, b: SOY- 10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2- 1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferaseBASF Agricultural Solutions US LLC 231727WO01 activities of a co-introduced pGVE3 plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0212] Figure 21: Impact of candidate NEENA sequences on the activity of the Arabidopsis pCH3 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1775 and 1776 of the pCH3 sequence having a T-to-C mutation (SEQ ID NO: 108) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2-1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0213] Figure 22: Impact of candidate NEENA sequences on the activity of the Arabidopsis pCH5 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1633 and 1634 of the sequence of the pCH5 promoter lacking the first 200 nt (SEQ ID NO: 120) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2-1.1. The horizontal axis shows the name of the NEENA sequence. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0214] Figure 23: Impact of candidate NEENA sequences on activity of the tobacco NelF-4A10 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 871 and 872 of the NelF-4A10 sequence (SEQ ID NO: 131) as follows: a: SOY-11, b: SOY-10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN- 35S2-1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVE3 plasmid. Activity of the promoter without an inserted sequence (k: none) was set at 1.

[0215] Figure 24: Impact of candidate NEENA sequences on the activity of the soybean CYP1 promoter in transiently transformed canola (A) and soybean (B) protoplasts. The vertical axis shows the relative promoter activity. The horizontal axis shows the candidate NEENA molecules that were inserted between nucleotide position 1879 and 1880 of the CYP1 sequence (SEQ ID NO: 142) as follows: a: SOY-11, b: SOY- 10, c: SOY-3, d: SOY-5, e: SOY-9, f: SOY-15, g: SOY-6, h: SOY-2, i: SOY-7, j: SOY-1, k: none and I: EN-35S2- 1.1. GUS activities were corrected for variation in protoplast transfection efficiency using the luciferase activities of a co-introduced pGVEc plasmid. Activity of the promoter without an inserted sequence (k:BASF Agricultural Solutions US LLC 231727WO01 none) was set at 1.

[0216] Figure 25: Nucleotide sequence of candidate NEENA molecules disclosed herein with SEQ ID NOs: 1-9 (A) and SEQ ID NOs: 10-15 (B).

[0217] Figure 26: mRNA expression profile for the native soybean gene from which the pCYPl promoter is derived. The y-axis indicates the transcript abundance in Transcripts Per Kilobase Million (TPM). The x- axis indicates the specific tissue as follows: a: stem, b: leaf, c: root, d: flower, e: pod and seed and f: seed.

[0218] Figure 27: Impact of candidate NEENA sequence SOY-3 and the NEENAc5 intron on the activity of the soybean Pbdc7 promoter in stably transformed soybean plants. Activity of the soybean Pbdc7 promoter with the NEENAc5 intron and the SOY-3 enhancer (c: pBas81, 13 lines) compared to that of the PubilOAt promoter (a: pBas80, 2 lines) in stably transformed soybean plants, b: non-transgenic soybean plants (soybean cultivar Thorne). The vertical axis shows the average GUS activity levels in leaves of TO plants (Fl / min.pg sol protein, Fl: Fluorescence Intensity).

[0219] Figure 28: Impact of candidate NEENA sequence SOY-3 and the NEENAc5 intron on the activity of the soybean Pbdc7 promoter in stably transformed soybean plants in the leaves (A) , roots (B) or stems (C). Activity of the soybean Pbdc7 promoter with the NEENAc5 intron and the SOY-3 enhancer (a: pBas81) compared to that of the PubilOAt promoter (b: pBas80) in stably transformed soybean plants, c: non- transgenic soybean plants (soybean cultivar Thorne). The vertical axis shows the average GUS activity levels (Fl / min.pg sol protein) in leaves (A), roots (B) and stems (C) of homozygous T0S1 plants from singlecopy lines (5 plants per line, Fl = Fluorescence Intensity).

[0220] Figure 29: Impact of candidate NEENA sequences on the activity of various promoters in stably transformed soybean plants. Activity of NEENA-containing promoters (see Table 11) measured in between 10 and 14 lines per vector, compared to the activity of the PubilOAt promoter (5 lines) in stably transformed soybean plants, a: pBas82; b: pBas83; c: pBas87; d: pBas86; e: pBas85; f: pBas84; g: pBas80, h: non-transgenic soybean plants (soybean cultivar Thorne). The vertical axis shows the average GUS activity levels (Fl / pg soluble protein) in leaves of TO plants (Fl = Fluorescence Intensity).Reference to sequence listing

[0221] The sequence listing of the present application is submitted electronically as an XML file named "231727WO01_SEQLISTING_St26.xml", which is 429 kilobytes (size as measured in Microsoft Windows®), contains 157 sequences SEQ ID NO: 1 through SEQ ID NO: 157. This sequence listing submitted electronically is an integral part of the specification and is incorporated herein by reference in its entirety.BASF Agricultural Solutions US LLC 231727WO01

[0222] In the description and examples, reference is made to the following sequences:SEQ ID NO: 1: Nucleotide sequence of NEENA SOY-1SEQ ID NO: 2: Nucleotide sequence of NEENA SOY-2SEQ ID NO: 3: Nucleotide sequence of NEENA SOY-3SEQ ID NO: 4: Nucleotide sequence of NEENA SOYASEQ ID NO: 5: Nucleotide sequence of NEENA SOY-5SEQ ID NO: 6: Nucleotide sequence of NEENA SOY-6SEQ ID NO: 7: Nucleotide sequence of NEENA SOY-7SEQ ID NO: 8: Nucleotide sequence of NEENA SOY-8SEQ ID NO: 9: Nucleotide sequence of NEENA SOY-9SEQ ID NO: 10: Nucleotide sequence of NEENA SOY-10SEQ ID NO: 11: Nucleotide sequence of NEENA SOY-11SEQ ID NO: 12: Nucleotide sequence of NEENA SOY-12SEQ ID NO: 13: Nucleotide sequence of NEENA SOY-13SEQ ID NO: 14: Nucleotide sequence of NEENA SOY-14SEQ ID NO: 15: Nucleotide sequence of NEENA SOY-15SEQ ID NO: 16: Nucleotide sequence of NEENA SOY-16SEQ ID NO: 17: Nucleotide sequence of lambdal-1.9 control sequence from lambda phageSEQ ID NO: 18: Nucleotide sequence of MISC-5.1 control sequence from lambda phageSEQ ID NO: 19: Nucleotide sequence of EN35S-1, enhancer fragment (-421 to -93 nt) of theCaMV 35S RNA promoter (Kay et al. 1987, Science 236, 1299-1302)SEQ ID NO: 20: Nucleotide sequence of 3'pin2, 3' untranslated region of the potato proteinase inhibitor II gene (Keil et al. 1986, Nucleic Acids Res 14, 5641- 5650)SEQ ID NO: 21: Nucleotide sequence of 5'cab22L, 5' untranslated region of the chlorophyll a / b binding protein gene of Petunia x hybrida (Harpster MH. Et al. 1988, Mol Gen Genet 212, 182-190)SEQ ID NO: 22: Nucleotide sequence of gus, coding sequence of the beta-glucuronidase gene of Escherichia coli, including the second intron of the potato ST-LS1 gene (Vancanneyt et al. 1990, Mol Gen Genet 220, 245-250)SEQ ID NO: 23: Nucleotide sequence of P-35S-1.9 or P35Smin, minimal CaMV 35S RNA promoter (nt -48 to +1) (Odell JT. Et al. 1985, Nature 313, 810-812)SEQ ID NO: 24: Nucleotide sequence of pBasl cloning vector for MPRA librarySEQ ID NO: 25: Nucleotide sequence of luc, coding sequence of the luciferase gene from firefly (Photinus pyralis)BASF Agricultural Solutions US LLC 231727WO01SEQ ID NO: 26: Nucleotide sequence of pGVEc control vectorSEQ ID NO: 27: Nucleotide sequence of P35S2, promoter region of the 35S transcript gene of Cauliflower mosaic virus (Odell JT. Et al. 1985, Nature 313, 810-812)SEQ ID NO: 28: Nucleotide sequence of Pbdc7, promoter region of a plasma intrinsic protein 1 gene of Glycine max (WO2014150449)SEQ ID NO: 29: Nucleotide sequence of EN-35S2-1.1, enhancer fragment (-208 to -65 nt) of the CaMV 35S RNA promoterSEQ ID NO: 30 Nucleotide sequence of Pbdc7>SOY-3> NEENAc5SEQ ID NO: 31 Nucleotide sequence of Pbdc7>SOY-3>NEENAcl8SEQ ID NO: 32 Nucleotide sequence of Pbdc7>SOY-3> NEENAssl3SEQ ID NO: 33 Nucleotide sequence of Pbdc7>SOY-3>intronl_h3AtSEQ ID NO: 34 Nucleotide sequence of Pbdc7>SOY-5> NEENAc5SEQ ID NO: 35 Nucleotide sequence of Pbdc7>SOY-5 > NEENAC18SEQ ID NO: 36 Nucleotide sequence of Pbdc7>SOY-5 >NEENAssl3SEQ ID NO: 37 Nucleotide sequence of Pbdc7>SOY-5 >intronl_h3AtSEQ ID NO: 38 Nucleotide sequence of Pbdc7>SOY-6 > NEENAc5SEQ ID NO: 39 Nucleotide sequence of Pbdc7>SOY-6 >NEENAcl8SEQ ID NO: 40 Nucleotide sequence of Pbdc7>SOY-6 >NEENAssl3SEQ ID NO: 41 Nucleotide sequence of Pbdc7>SOY-6 >intronl_h3AtSEQ ID NO: 42 Nucleotide sequence of Pbdc7>SOY-l > NEENAc5SEQ ID NO: 43 Nucleotide sequence of Pbdc7>SOY-l >NEENAcl8SEQ ID NO: 44 Nucleotide sequence of Pbdc7>SOY-l > NEENAssl3SEQ ID NO: 45 Nucleotide sequence of Pbdc7>SOY-l >intronl_h3AtSEQ ID NO: 46 Nucleotide sequence of Pbdc7>SOY-llSEQ ID NO: 47 Nucleotide sequence of Pbdc7>SQY-10SEQ ID NO: 48 Nucleotide sequence of Pbdc7>SOY-3SEQ ID NO: 49 Nucleotide sequence of Pbdc7>SOY-5SEQ ID NO: 50 Nucleotide sequence of Pbdc7>SOY-9SEQ ID NO: 51 Nucleotide sequence of Pbdc7>SOY-15SEQ ID NO: 52 Nucleotide sequence of Pbdc7>SOY-6SEQ ID NO: 53 Nucleotide sequence of Pbdc7>SOY-2SEQ ID NO: 54 Nucleotide sequence of Pbdc7>SOY-7SEQ ID NO: 55 Nucleotide sequence of Pbdc7>SOY-lSEQ ID NO: 56 Nucleotide sequence of Pbdc7> NEENAc5SEQ ID NO: 57 Nucleotide sequence of Pbdc7>NEENAcl8BASF Agricultural Solutions US LLC 231727WO01SEQ ID NO: 58: Nucleotide sequence of Pbdc7> NEENAsslBSEQ ID NO: 59: Nucleotide sequence of Pbdc7>intronl_h3AtSEQ ID NO: 60: Nucleotide sequence of PubilOAt, promoter region of the ubiquitin 10 gene of Arabidopsis thalianaSEQ ID NO: 61: Nucleotide sequence of Pbdcl6, promoter region of a peptidyl-prolyl cistrans isomerase gene of Glycine max (Pbdcl6-1.2, WO2021183803)SEQ ID NO: 62: Nucleotide sequence of Pbdcl6>SOY-llSEQ ID NO: 63: Nucleotide sequence of Pbdcl6>SOY-10SEQ ID NO: 64: Nucleotide sequence of Pbdcl6>SOY-3SEQ ID NO: 65: Nucleotide sequence of Pbdcl6>SOY-5SEQ ID NO: 66: Nucleotide sequence of Pbdcl6>SOY-9SEQ ID NO: 67: Nucleotide sequence of Pbdcl6>SOY-15SEQ ID NO: 68: Nucleotide sequence of Pbdcl6>SOY-6SEQ ID NO: 69: Nucleotide sequence of Pbdcl6>SOY-2SEQ ID NO: 70: Nucleotide sequence of Pbdcl6>SOY-7SEQ ID NO: 71: Nucleotide sequence of Pbdcl6>SOY-lSEQ ID NO: 72: Nucleotide sequence of pBas2 gus expression vector containing the MISC-5.1 control sequence in-between the EN35S-1 enhancer and the minimal 35S promoterSEQ ID NO: 73: Nucleotide sequence of the promoter region of the GmUbi9 ubiquitin gene of Glycine max (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237)SEQ ID NO: 74: Nucleotide sequence of PGmUbi9 (lacking the first 400 nt of SEQ ID NO: 73)SEQ ID NO: 75: Nucleotide sequence of PGmUbi9>SOY-llSEQ ID NO: 76: Nucleotide sequence of PGmUbi9>SOY-10SEQ ID NO: 77: Nucleotide sequence of PGmUbi9>SOY-3SEQ ID NO: 78: Nucleotide sequence of PGmUbi9>SOY-5SEQ ID NO: 79: Nucleotide sequence of PGmUbi9>SOY-9SEQ ID NO: 80: Nucleotide sequence of PGmUbi9>SOY-15SEQ ID NO: 81: Nucleotide sequence of PGmUbi9>SOY-6SEQ ID NO: 82: Nucleotide sequence of PGmUbi9>SOY-2SEQ ID NO: 83: Nucleotide sequence of PGmUbi9>SOY-7SEQ ID NO: 84: Nucleotide sequence of PGmUbi9>SOY-lSEQ ID NO: 85: Nucleotide sequence of the promoter region of the GmUbi2 ubiquitin gene of Glycine max (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237)SEQ ID NO: 86: Nucleotide sequence of PGmUbi2>SOY-llBASF Agricultural Solutions US LLC 231727WO01SEQ ID NO: 87: Nucleotide sequence of PGmUbi2>SOY-10SEQ ID NO: 88: Nucleotide sequence of PGmUbi2>SOY-3SEQ ID NO: 89: Nucleotide sequence of PGmUbi2>SOY-5SEQ ID NO: 90: Nucleotide sequence of PGmUbi2>SOY-9SEQ ID NO: 91: Nucleotide sequence of PGmUbi2>SOY-15SEQ ID NO: 92: Nucleotide sequence of PGmUbi2>SOY-6SEQ ID NO: 93: Nucleotide sequence of PGmUbi2>SOY-2SEQ ID NO: 94: Nucleotide sequence of PGmUbi2>SOY-7SEQ ID NO: 95: Nucleotide sequence of PGmUbi2>SOY-lSEQ ID NO: 96: Nucleotide sequence of the promoter region of the GmUbi7 ubiquitin gene of Glycine max (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237)SEQ ID NO: 97: Nucleotide sequence of PGmUbi7>SOY-llSEQ ID NO: 98: Nucleotide sequence of PGmUbi7>SOY-10SEQ ID NO: 99: Nucleotide sequence of PGmUbi7>SOY-3SEQ ID NO: 100: Nucleotide sequence of PGmUbi7>SOY-5SEQ ID NO: 101: Nucleotide sequence of PGmUbi7>SOY-9SEQ ID NO: 102: Nucleotide sequence of PGmUbi7>SOY-15SEQ ID NO: 103: Nucleotide sequence of PGmUbi7>SOY-6SEQ ID NO: 104: Nucleotide sequence of PGmUbi7>SOY-2SEQ ID NO: 105: Nucleotide sequence of PGmUbi7>SOY-7SEQ ID NO: 106: Nucleotide sequence of PGmUbi7>SOY-lSEQ ID NO: 107: Nucleotide sequence of the promoter region of the serine hydroxymethyltransferase 4 gene of Arabidopsis thaliana (pCH3, Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545)SEQ ID NO: 108: Nucleotide sequence of pCH3 with a T1590C mutationSEQ ID NO: 109: Nucleotide sequence of pCH3>SOY-llSEQ ID NO: 110: Nucleotide sequence of pCH3>SOY-10SEQ ID NO: 111: Nucleotide sequence of pCH3>SOY-3SEQ ID NO: 112: Nucleotide sequence of pCH3>SOY-5SEQ ID NO: 113: Nucleotide sequence of pCH3>SOY-9SEQ ID NO: 114: Nucleotide sequence of pCH3>SOY-15SEQ ID NO: 115: Nucleotide sequence of pCH3>SOY-6SEQ ID NO: 116: Nucleotide sequence of pCH3>SOY-2SEQ ID NO: 117: Nucleotide sequence of pCH3>SOY-7SEQ ID NO: 118: Nucleotide sequence of pCH3>SOY-lBASF Agricultural Solutions US LLC 231727WO01SEQ ID NO: 119: Nucleotide sequence of the promoter region of the Arabinogalactan protein 15 gene of Ara bidopsis thaliana (pCH5, Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545)SEQ ID NO: 120: Nucleotide sequence of pCH5 lacking the first 200 ntSEQ ID NO: 121: Nucleotide sequence of pCH5>SOY-llSEQ ID NO: 122: Nucleotide sequence of pCH5>SQY-10SEQ ID NO: 123: Nucleotide sequence of pCH5>SOY-3SEQ ID NO: 124: Nucleotide sequence of pCH5>SOY-5SEQ ID NO: 125: Nucleotide sequence of pCH5>SOY-9SEQ ID NO: 126: Nucleotide sequence of pCH5>SOY-15SEQ ID NO: 127: Nucleotide sequence of pCH5>SOY-6SEQ ID NO: 128: Nucleotide sequence of pCH5>SOY-2SEQ ID NO: 129: Nucleotide sequence of pCH5>SOY-7SEQ ID NO: 130: Nucleotide sequence of pCH5>SOY-lSEQ ID NO: 131: Nucleotide sequence of the promoter region of the NelF-4A10 gene of Nicotiana tabacum (Mandel et al. 1995, Plant Mol. Biol. 29, 995-1004)SEQ ID NO: 132: Nucleotide sequence of pNelF-4A10>SQY-llSEQ ID NO: 133: Nucleotide sequence of pNelF-4A10>SQY-10SEQ ID NO: 134: Nucleotide sequence of pNelF-4A10>SQY-3SEQ ID NO: 135: Nucleotide sequence of pNelF-4A10>SQY-5SEQ ID NO: 136: Nucleotide sequence of pNelF-4A10>SQY-9SEQ ID NO: 137: Nucleotide sequence of pNelF-4A10>SQY-15SEQ ID NO: 138: Nucleotide sequence of pNelF-4A10>SQY-6SEQ ID NO: 139: Nucleotide sequence of pNelF-4A10>SQY-2SEQ ID NO: 140: Nucleotide sequence of pNelF-4A10>SQY-7SEQ ID NO: 141: Nucleotide sequence of pNelF-4A10>SQY-lSEQ ID NO: 142: Nucleotide sequence of the promoter region of the CYP1 gene of Glycine maxSEQ ID NO: 143: Nucleotide sequence of pCYPl>SOY-llSEQ ID NO: 144: Nucleotide sequence of pCYPl>SQY-10SEQ ID NO: 145: Nucleotide sequence of pCYPl>SOY-3SEQ ID NO: 146: Nucleotide sequence of pCYPl>SOY-5SEQ ID NO: 147: Nucleotide sequence of pCYPl>SOY-9SEQ ID NO: 148: Nucleotide sequence of pCYPl>SOY-15SEQ ID NO: 149: Nucleotide sequence of pCYPl>SOY-6BASF Agricultural Solutions US LLC 231727WO01SEQ ID NO: 150: Nucleotide sequence of pCYPl>SOY-2SEQ ID NO: 151: Nucleotide sequence of pCYPl>SOY-7SEQ ID NO: 152: Nucleotide sequence of pCYPl>SOY-lSEQ ID NO: 153: Nucleotide sequence of NEENAc5 intron (nucleotides 31-584 of SEQ ID NO:3 of WO 2011 / 023537)SEQ ID NO: 154: Nucleotide sequence of NEENAssl3 intron (nucleotides 35-490 of SEQ ID NO: 12 of WO 2011 / 023800)SEQ ID NO: 155: Nucleotide sequence of NEENAcl8 intron (nucleotides 38-737 of SEQ ID NO:4 of WO 2011 / 023537)SEQ ID NO: 156: Nucleotide sequence of pBas80 T-DNA vector containing the gus gene under the control of PubilOAt and the 2m-epsps selectable marker geneSEQ ID NO: 157: Nucleotide sequence of pCYPl>SOY-3>NEENAcl8Chemicals and common methods

[0223] Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, Ligation of nucleic acids, transformation, selection and cultivation of bacterial cells were performed as described in Sambrook et al., 1989, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Standard materials and methods for PCR can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany. Sequence analyses of recombinant DNA were performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the Sanger technology (Sanger et al., 1977). Unless described otherwise, chemicals and reagents were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, Wl, USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, CA, USA). Restriction endonucleases were from New England Biolabs (Ipswich, MA, USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides were synthesized by Eurofins MWG Operon (Ebersberg, Germany). The term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as modified or substituted oligonucleotides, including two or more nucleo-monomers covalently coupledBASF Agricultural Solutions US LLC 231727WO01 to each other by linkages (e.g. phosphodiesters) or substitute linkages.1: Identification of new candidate NEENA molecules from the

[0224] An MPRA library was constructed to test soybean genomic sequences for enhancer activity.

[0225] Using gene coordinates from version 4 of the Williams 82 soybean genome sequence (https: / / phytozome-next.jgi.doe.gov / info / Gmax_Wm82_a4_vl), intergenic regions were selected which were at least 244-nucleotide (nt) long (after removal of stretches of Ns). This yielded 2235 sequences totaling 3.35 Mbp. These sequences and 2 control sequences from lambda phage (lambdal-1.9 SEQ ID NO: 17 and MISC-5.1 SEQ ID NO: 18) were digested in silico with Kpnl, Xbal and Sfil, and split into 244-nt long fragments with a 20-nt overlap, except for the 2 most 3' fragments for which the overlap is such that the 3'end of the last 244-nt fragment coincides with the 3'end of the original sequence. The resulting 15,272 244-nt long query sequences, except for 3 sequences that would result in an Sfil site or an additional Kpnl / Xbal site when inserted in the MPRA oligo, were cloned into an MPRA library (Melnikov et al. 2014, J. Vis. Exp.(90), e51719).

[0226] Each query sequence was linked to 7 different unique 11-nt long barcodes. The barcodes do not contain AATAAT or restriction sites for Xbal or Kpnl, differ by at least 2 nt, have no base repeats longer than 2 nt, and do not start with TC. 300-nt long oligos were synthesized containing each of the 106,904 query sequence-barcode combinations and the sequences required for amplification and cloning of the library into the expression vectors (Figure 1). In a first cloning step, the amplified oligo library was cloned in-between the CaMV 35S enhancer (EN35S-1; SEQ ID NO: 19) and the 3' pin2 (SEQ ID NO: 20) of pBasl (SEQ ID NO: 24) using the Sfil restriction recognition sites. Next, a Kpn-Nhel fragment containing the minimal 35S promoter (P-35S-1.9 SEQ ID NO: 23), the petunia cab22L leader sequence (SEQ ID NO: 21) and the gus coding sequence (SEQ ID NO: 22) from pBasl, was cloned in the Kpnl and Xbal sites between the query sequence and the barcode. The resulting plasmid library contained the query sequences inbetween the 35S enhancer and the minimal 35S promoter, and the linked barcodes in the 3'UTR of the GUS gene. Sequencing of 48 clones from the plasmid library showed that 41 of the clones contained the expected query sequences whereas 3 clones contained shorter-than-expected inserts and 4 clones did not contain an insert.

[0227] The resulting plasmid library was transfected into soybean mesophyll protoplasts. Protoplasts were isolated from the unifoliate leaves of 6-day-old seedlings. Healthy leaves were cut into fine strips with a sharp razor blade. The strips were infiltrated with cell wall-dissolving enzyme solution (1.5% cellulase RIO and 0.75% macerozyme RIO in 10 mM KCI and 0.6 M mannitol, pH 7.5) and incubated overnight in the dark with gentle shaking (40 rpm) at 24°C. After enzymatic digestion, the releasedBASF Agricultural Solutions US LLC 231727WO01 protoplasts were collected by filtering the mixture through 40-pm nylon meshes and resuspended in W5 solution. The resuspended protoplasts were kept on ice and allowed to settle by gravity, after which the cell pellet was resuspended in MMG. For transfection, 200 pl of protoplasts (l x 106cells) were mixed with 40 pl (80 pg) plasmid library DNA and 220 pl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated for 13-15 min in the dark. After removing the PEG solution, the protoplasts were resuspended in 2 ml of W5 solution and incubated at 24°C. 6 transfected protoplast samples were incubated for 6h and collected for RNA isolation. Total RNA was isolated and PCR products were prepared from the barcode containing regions of the RNA and of the plasmid library DNA and sequenced on the MiSeq as described in WO202148316. 31 x 106and 42 x 106reads were obtained for the plasmid DNA and the RNA, respectively. From these data, the frequency of each barcode within the RNA of the transfected protoplasts, as well as within the transfected plasmid DNA library, was deduced. The ratio of the barcode abundance in the RNA versus the abundance in the plasmid library DNA is a measure for the expression enhancing activity of the test sequence that is linked to the specific barcode. As each test sequence is linked to 7 different barcodes, each test sequence has 7 RNA / DNA ratios. The median RNA / DNA value was used as a measure for the enhancer activity of the tested sequence. A paired t-test was used to test the significance (p<0.05) of the expression increase of specific sequences.

[0228] The 4 control sequences (nucleotides 1-244 and nucleotides 225-468 of SEQ ID NOs: 17 and 18) that are derived from lambda phage DNA, showed median RNA / DNA ratios of 1.15, 1.08, 0.51 and 0.02. A total of 16 soybean sequences (see Table 1) which had an RNA / DNA ratio higher than 7 and a p-value below 0.05 were selected for testing their potential promoter enhancing activity.Table 1: Abundance of the selected query sequences in the RNA expressed in soybean mesophyll protoplasts compared to their abundance in the plasmid library.BASF Agricultural Solutions US LLC 231727WO01Example 2: Selected NEENAs enhance promoter activity in canola leaf protoplasts

[0229] All of the 244-nt long sequences whose SEQ ID NO are listed in Table 1 were cloned downstream of the 35S enhancer and upstream of the minimal 35S promoter and the gus coding sequence by replacing the MISC-5.1 control sequence in plasmid pBas2 (SEQ ID NO: 72) as shown in Figure 1. The resulting plasmids were introduced in canola mesophyll protoplasts. Protoplast were isolated from the leaves of 4- to 7-week-old aseptically grown canola plants as described in Example 1 for soybean. Protoplasts were transfected in microtiter plates using a semi-automated robotic platform. In brief, 30 pl of cells (5 x 104cells) were mixed with 6 pl total plasmid DNA before adding 32 pl of PEG 1500 solution. The plates were then centrifuged for 1 min at 1000 rpm and incubated at room temperature for 15 min. After removing the PEG solution, transfected protoplasts were resuspended in 140 pl of W5 solution and incubated in the dark at 24°C. Protein was extracted and GUS activities determined following an overnight incubation of the protoplasts. To correct for differences in introduction efficiency, GUS activities of transfected protoplasts were divided by the luciferase activities from a co-introduced control vector (pGVEc, SEQ ID NO: 26) having the firefly luciferase gene (pGVEc nt 834-2486, luc, SEQ ID NO: 25) under control of the CaMV 35S promoter (pGVEc nt 237-764, P35S2, SEQ ID NO: 27). The resulting data show that several of the candidate NEENAs effectively increased expression from the 35S enhancer-promoter combination up to 11-fold (Figure 2).Example 3: Identification of suitable NEENA insertion sites in the soybean Pbdc7 promoter in soybean leaf protoplasts

[0230] The promoter of the bdc7 soybean gene (Glyma.02G255000, Pbdc7, SEQ ID NO: 28) is a constitutive promoter that is suitable for transgene expression (WQ2014150449). However, the expression level is relatively low and it would be beneficial to have promoter variants with higher activity. One way to engineer such promoter variants with increased activity is to insert a NEENA in the promoter. However, one important question is what could be a suitable position within the promoter to insert suchBASF Agricultural Solutions US LLC 231727WO01NEENA. To answer that question, we inserted a sequence with known expression enhancing activity derived from the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) at different positions in the Pbdc7 promoter. The resulting promoters were cloned upstream of the gus coding sequence by replacing nt 409- 1409 of pBas2 (SEQ ID NO: 72). Testing of the resulting plasmids in soybean protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the Pbdc7 promoter, with the highest increase being more than 200-fold when the enhancer is inserted between nt 1508 and 1509 of the Pbdc7 promoter sequence (Figure 3).Example 4: Selected NEENAs enhance expression derived from the Pbdc7 promoter

[0231] The results from Example 3 show that the position between nt 1508 and 1509 of the Pbdc7 promoter is very well suited to insert expression enhancing fragments. Therefore, 10 different soybean candidate NEENA sequences were inserted at this position in the promoter (Table 2) and the activity of the resulting promoters was tested in protoplasts.Table 2: List of GUS expression vectors used to test the impact of various NEENA molecules and introns on Pbdc7 promoter activity.BASF Agricultural Solutions US LLC 231727WO01

[0232] Results in canola leaf protoplasts showed the candidate NEENA molecules increased the activity of the Pbdc7 promoter, with six NEENA molecules (SOY-3, SOY-5, SOY-9, SOY-6, SOY-2, and SOY-1) increasing promoter activity at least 20-fold (Figure 4).

[0233] In soybean cell suspension protoplasts, these six NEENA sequences (SOY-3, SOY-5, SOY-9, SOY-6, SOY-2, and SOY-1) increased Pbdc7 promoter activity between 6- and 34-fold (Figure 5). This shows that the activity of the soybean Pbdc7 promoter can be increased substantially by functionally linking a heterologous NEENA sequence to the promoter according to an embodiment of the invention.

[0234] In plants, promoter activity can be increased by the presence of an intron in the 5'UTR. As the Pbdc7 promoter does not contain an intron, four different introns (NEENAc5 (SEQ ID NO: 153; WO 2011 / 023537 Al), NEENAcl8 (SEQ ID NO: 155; WO 2011 / 023537 Al), NEENAssl3 (SEQ ID NO: 154; WO 2011 / 023800 Al) or intronl_h3At (Chaubet et al. 1992, J. Mol. Biol. 225, 569-574)) were inserted just downstream of the Pbdc7 promoter and of promoter variants where Pbdc7 is functionally linked to one of four different NEENA molecules (SOY-3, SOY-5, SOY-6 or SOY-1) to test whether the activity of the Pbdc7 promoter variants can be further increased by inserting an intron in the 5'UTR. A list of expression vectors and the various promoter variants tested is provided in Table 2. Figure 6A shows that the insertion of any of these four introns further increased the activity of the promoter variants, wherein Pbdc7 is functionally linked to a NEENA, between 2 and 4 times in canola protoplasts. In soybean protoplasts, activity of the promoter variants, wherein Pbdc7 is functionally linked to a NEENA was further increased up to 8-fold (Figure 6B).BASF Agricultural Solutions US LLC 231727WO01

[0235] In canola protoplasts, the activity of the strongest promoter variant (Pbdc7 functionally linked to NEENA SOY-1 and intron NEENAc5) was between 20% and 70% of the activity of the very strong PubilO promoter from Arabidopsis (SEQ ID NO: 60, see Figure 7). These results show that a very weak promoter, such as for example the soybean Pbdc7 promoter, can be transformed into a strong promoter by functionally linking a NEENA molecule and an intron sequence to the promoter.Example 5: Identification of suitable NEENA insertion sites in the soybean Pbdcl6 promoter in protoplasts from soybean cell suspension cultures

[0236] The promoter of the bdcl6 soybean gene (Glyma.l3G258500, SEQ ID NO: 61) is a constitutive promoter that is suitable for transgene expression (WQ2021183803). However, the expression level is relatively low and it would be beneficial to have promoter variants with higher activity. To determine what is a suitable position within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the Pbdcl6 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2 (SEQ ID NO: 72). Testing of the resulting plasmids in soybean protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the Pbdcl6 promoter, with the highest increase being more than 90-fold when the enhancer is inserted between nt 925 and 926 of the Pbdcl6 promoter sequence (Figure 8).Example 6: Selected NEENAs enhance expression derived from the Pbdcl6 promoter

[0237] The results from Example 5 show that the position between nt 925 and 926 of the Pbdcl6 promoter is very well suited to insert expression enhancing fragments. Therefore, 10 different soybean candidate NEENA sequences were inserted at this position in the promoter (Table 3) and the activity of the resulting promoters was tested in protoplasts.Table 3: List of GUS expression vectors used to test the impact of various NEENA molecules on Pbdcl6 promoter activity.BASF Agricultural Solutions US LLC 231727WO01

[0238] Results in canola leaf protoplasts confirmed that candidate NEENA sequences increased the activity of the Pbdcl6 promoter, with four NEENA molecules (SOY-3, SOY-5, SOY-9, and SOY-1) increasing promoter activity more than 30-fold (Figure 9).

[0239] In soybean protoplasts, these four NEENA molecules (SOY-3, SOY-5, SOY-9 and SOY-1) increased the activity of the Pbdcl6 promoter between 10- and 85-fold (Figure 10).

[0240] This shows that the activity of the soybean Pbdcl6 promoter can be increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention.Example 7: Identification of suitable NEENA insertion sites in the soybean GmUbi9 promoter

[0241] The promoter of the GmUbi9 soybean gene (Glyma.l3G138600, SEQ ID NO: 73) is a constitutive promoter that is suitable for transgene expression (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237). To determine preferred positions within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the GmUbi9 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting plasmids in canola protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the GmUbi9 promoter, with the highest increase being 10-fold when the enhancer is inserted between nt 1862 and 1863 of the GmUbi9 promoter sequence (Figure 11). A promoter variant lacking the first 400 nt that overlap with the coding sequence of the upstream soybean gene (SEQ ID NO: 74) showed about half of the activity of the 2-kb promoter fragment (Figure 11: i).Example 8: Selected NEENAs enhance expression derived from the GmUbi9 promoter

[0242] The results from Example 7 show that the position between nt 1862 and 1863 of the GmUbi9 promoter is very well suited to insert expression enhancing fragments. Therefore, different soybean candidate NEENA sequences were inserted at this position in the GmUbi9 promoter which lacks the firstBASF Agricultural Solutions US LLC 231727WO01400 nt. The activity of the resulting promoter variants (see Table 4) was tested in protoplasts.Table 4: List of GUS expression vectors used to test the impact of various NEENA molecules on GmUbi9 promoter activity.

[0243] In canola protoplasts, SOY-1 increased the activity of the GmUbi9 promoter about 8-fold (Figure 18A) to a level that is similar to that of PubilOAt. In soybean protoplasts, the strongest expression increase (about 24-fold) was obtained by insertion of SOY-3 (Figure 18B), resulting in an activity that is about 3- fold below that of PubilOAt. These results show that the activity of the soybean GmUbi9 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention.Example 9: Identification of suitable NEENA insertion sites in the soybean GmUbi2 promoter

[0244] The promoter of the GmUbi2 soybean gene (Glyma.l3G117900, SEQ ID NO: 85) is a constitutive promoter that is suitable for transgene expression (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237). To determine suitable positions within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the GmUbi2 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting plasmids in canola protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the GmUbi2 promoter, with the highest increase being more than 30-fold when the enhancer is inserted between nt 1228 and 1229 of the GmUbi2 promoter sequence (Figure 12).BASF Agricultural Solutions US LLC 231727WO01Example 10: Selected NEENAs enhance expression derived from the GmUbi2 promoter

[0245] The results from Example 9 show that the position between nt 1228 and 1229 of the GmUbi2 promoter is very well suited to insert expression enhancing fragments. Therefore, soybean candidate NEENA sequences were inserted at this position in the promoter (Table 5) and the activity of the resulting promoters was tested in protoplasts.Table 5: List of GUS expression vectors used to test the impact of various NEENA molecules on GmUbi2 promoter activity.

[0246] In canola protoplasts, SOY-1 and SOY-3 increased the activity of the GmUbi2 promoter about 10- fold (Figure 19A) to a level that is about 70% of that of PubilOAt. In soybean protoplasts, the strongest expression increase (about 6-fold) was obtained by insertion of SOY-3 (Figure 19B), resulting in an activity that is about 80% of that of PubilOAt. These results showed that the activity of the soybean GmUbi2 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention. Example 11: Identification of suitable NEENA insertion sites in the soybean GmUbi7 promoter

[0247] The promoter of the GmUbi7 soybean gene (Glyma.l7G042100, SEQ ID NO: 96) is a constitutive promoter that is suitable for transgene expression (Hernandez-Garcia et al. 2010, BMC Plant Biology 10, 237). To determine preferred positions within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positionsBASF Agricultural Solutions US LLC 231727WO01 in the GmUbi7 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting plasmids in canola protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the GmUbi7 promoter in all positions tested, with the highest increase being more than 10-fold when the enhancer is inserted between nt 1282 and 1283 of the GmUbi7 promoter sequence (Figure 13).Example 12: Selected NEENAs enhance expression derived from the GmUbi7 promoter

[0248] The results from Example 11 show that the position between nt 1282 and 1283 of the GmUbi7 promoter is very well suited to insert expression enhancing fragments. Acordingly, soybean candidate NEENA sequences were inserted at this position in the promoter (Table 6) and the activity of the resulting promoters was tested in protoplasts.Table 6: List of GUS expression vectors used to test the impact of various NEENA molecules on GmUbi7 promoter activity.

[0249] In canola protoplasts, SOY-1 increased the activity of the GmUbi7 promoter about 2.5-fold (Figure 20A) to a level that is about 40% of that of PubilOAt. In soybean protoplasts, an expression increase of about 2-fold was obtained by insertion of SOY-3 or SOY-1 (Figure 20B), resulting in an activity that is similar to that of PubilOAt. These results show that the activity of the soybean GmUbi7 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention.BASF Agricultural Solutions US LLC 231727WO01Example 13: Identification of suitable NEENA insertion sites in the Arabidopsis pCH3 promoter

[0250] The pCH3 promoter of the Arabidopsis serine hydroxymethyltransferase 4 gene (AT4G13930, SEQ ID NO: 107) is a constitutive promoter that is suitable for transgene expression (Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545). To determine preferred positions within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the pCH3 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting expression vectors in canola protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the pCH3 promoter in all positions tested, with the highest increase being almost 6-fold when the enhancer is inserted between nt 1775 and 1776 of the pCH3 promoter sequence (Figure 14). A promoter variant having a T-to-C mutation at nt 1590 (SEQ ID NO: 108) had similar activity as the original promoter (Figure 14: j).Example 14: Selected NEENAs enhance expression derived from the pCH3 promoter

[0251] The results from Example 13 show that the position between nt 1775 and 1776 of the pCH3 promoter is very well suited to insert expression enhancing fragments. Accordingly, soybean candidate NEENA sequences were inserted at this position in the pCH3 promoter having a T-to-C mutation at nt 1590 (Table 7) and the activity of the resulting promoter variants was tested in protoplasts.Table 7: List of GUS expression vectors used to test the impact of various NEENA molecules on pCH3 promoter activity.BASF Agricultural Solutions US LLC 231727WO01

[0252] In canola protoplasts, SOY-1 increased the activity of the pCH3 promoter about 2.5-fold (Figure 21A) to a level that is about 3-fold below that of PubilOAt. In soybean protoplasts, the strongest expression increase (about 12-fold) was obtained by insertion of SOY-5 (Figure 21B), resulting in an activity that is about 3-fold below that of PubilOAt. These results show that the activity of the Arabidopsis pCH3 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention.Example 15: Identification of suitable NEENA insertion sites in the Arabidopsis pCH5 promoter

[0253] The pCH5 promoter of the Arabidopsis Arabinogalactan protein 15 gene (AT5G11740, SEQ ID NO: 119) is a constitutive promoter that is suitable for transgene expression (Zhou et al. 2023, ACS Synth. Biol. 12, 1533-1545). To determine preferred positions within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the pCH5 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting expression vectors in canola protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the pCH5 promoter in all positions tested, with the highest increase being almost 7-fold when the enhancer is inserted between nt 1833 and 1834 of the pCH5 promoter sequence (Figure 15). A promoter variant lacking the first 200 nt that overlap with the coding sequence of the upstream Arabidopsis gene (SEQ ID NO: 120) had similar activity as the 2-kb promoter fragment (Figure 15).Example 16: Selected NEENAs enhance expression derived from the pCH5 promoter

[0254] The results from Example 15 show that the position between nt 1833 and 1834 of the pCH5 promoter is very well suited to insert expression enhancing fragments. Accordingly, soybean candidate NEENA sequences were inserted at this position in the pCH5 promoter lacking the first 200 nt (Table 8) and the activity of the resulting promoter variants was tested in protoplasts.Table 8: List of GUS expression vectors used to test the impact of various NEENA molecules on pCH5 promoter activity.BASF Agricultural Solutions US LLC 231727WO01

[0255] In canola protoplasts, SOY-1 increased the activity of the pCH5 promoter about 1.5-fold (Figure 22A) to a level that is about 3-fold below that of PubilOAt. In soybean protoplasts, the strongest expression increase (about 10-fold) was obtained by insertion of SOY-3 (Figure 22B), resulting in an activity that is about 88% of that of PubilOAt. These results show that the activity of the Arabidopsis pCH5 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention.Example 17: Identification of suitable NEENA insertion sites in the promoter of the tobacco NelF-4A10 gene

[0256] The promoter of the tobacco NelF-4A10 gene (SEQ ID NO: 131) is a constitutive promoter that is suitable for transgene expression (Mandel et al. 1995, Plant Mol. Biol. 29, 995-1004). To determine preferred positions within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the NelF-4A10 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting plasmids in canola protoplasts showed that insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the NelF-4A10 promoter, with the highest increase being 3.5-fold when the enhancer is inserted between nt 871 and 872 of the NelF-4A10 promoter sequence (Figure 16).Example 18: Selected NEENAs enhance expression derived from the NelF-4A10 promoter

[0257] The results from Example 17 show that the position between nt 871 and 872 of the NelF-4A10 promoter is very well suited to insert expression enhancing fragments. Therefore, soybean candidate NEENA sequences were inserted at this position in the promoter (Table 9) and the activity of the resulting promoters was tested in protoplasts.BASF Agricultural Solutions US LLC 231727WO01Table 9: List of GUS expression vectors used to test the impact of various NEENA molecules on NelF-4A10 promoter activity.

[0258] In canola protoplasts, SOY-1 increased the activity of the tobacco NelF-4A10 promoter about 6- fold (Figure 23A) to a level that is about 40% of that of PubilOAt. In soybean protoplasts, the strongest expression increase (about 10-fold) was obtained by insertion of SOY-1 and SOY-3 (Figure 23B), resulting in an activity that is about 3-fold below that of PubilOAt. These results show that the activity of the tobacco NelF-4A10 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention. Example 19: Identification of suitable NEENA insertion sites in the promoter of the soybean CYP1 gene

[0259] RNA-seq was performed at GENEWIZ (South Plainfield, NJ) using RNAs isolated from various soybean tissues (cultivar Williams82). As shown in Figure 26, the mRNA expression profile of the CYP1 gene (Glyma.llG098700) shows that the gene is expressed in nearly all plant tissues including stem, leaf, root, flower, pod and seed. The pCYPl promoter is therefore suitable for transgene expression in plants. The promoter sequence of the soybean CYP1 gene (Glyma.llG098700, SEQ ID NO: 142) was isolated.

[0260] To determine what is a suitable position within the promoter to insert candidate NEENA molecules, the enhancer of the CaMV 35S RNA promoter (EN-35S2-1.1, SEQ ID NO: 29) was inserted at different positions in the CYP1 promoter and the different promoter variants were cloned upstream of the gus coding sequence by replacing nt 409-1409 of pBas2. Testing of the resulting plasmids in canolaBASF Agricultural Solutions US LLC 231727WO01 protoplasts showed that the CYP1 promoter is active in canola and that the insertion of the EN-35S2-1.1 enhancer results in a substantial increase in the activity of the CYP1 promoter, with the highest increase being more than 20-fold when the enhancer is inserted between nt 1879 and 1880 of the CYP1 promoter sequence (Figure 17). Example 20: Selected NEENAs enhance expression derived from the CYP1 promoter

[0261] The results from Example 19 show that the position between nt 1879 and 1880 of the CYP1 promoter is very well suited to insert expression enhancing fragments. Therefore, the 10 soybean candidate NEENA sequences were inserted at this position in the promoter (Table 10) and the activity of the resulting promoters is tested in protoplasts. Table 10: List of GUS expression vectors used to test the impact of various NEENA molecules on CYP1 promoter activity.

[0262] In canola protoplasts, several of the soy enhancers increased the activity of the soybean CYP1 promoter about 2-fold (Figure 24A) to a level that is about 10% of that of PubilOAt. In soybean protoplasts, the strongest expression increase (about 140-fold) was obtained by insertion of SOY-3 (Figure 24B), resulting in an activity that is about 20% of that of PubilOAt. These results show that the activity of the soybean CYP1 promoter was increased substantially by functionally linking a heterologous NEENA sequence to the promoter, according to an embodiment of the invention.BASF Agricultural Solutions US LLC 231727WO01Example 21: Selected NEENAs enhance Pbdc7 promoter activity in transgenic soybean plants

[0263] The plant transformation vector pBas80 (SEQ ID NO: 156) contains the GUS gene under the control of the PubilOAt promoter and the 2m-EPSPS gene under the control of the Arabidopsis thaliana Ph4a748_ABC histone promoter (Chaboute et al., 1987) and H3 histone intron (Chaubet et al. 1992, J. Mol. Biol. 225, 569-574) to allow for selection on glyphosate. To determine the activity of the Pbdc7 promoter with the NEENAc5 intron and the SOY-3 enhancer in transgenic plants, the PubilOAt promoter of pBas80 was replaced by this promoter variant to create plant transformation vector pBas81 (see Table 11). Both recombinant expression vectors were used for stable transformation of mature seeds of soybean cultivar Thorne. Seeds were surface sterilized in a desiccator for about 16 hrs using chlorine gas as described by Di et al. 1996 (Plant Cell Rep. 15: 746-750). Agrobacterium transformation using half seed explants was essentially as described by Paz and Wang 2006 (US7473822B1) and Luth et al. 2015 (Agrobacterium Protocols. Methods in Molecular Biology, 275-284). The disarmed Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993, Transgenic Research 2, 208-218) harbouring the T-DNA vector was used for co-cultivation of the half seed explants. After 5 to 6 days of co-cultivation, glyphosate resistant shoots were selected on a medium containing 0.075mM Glyphosate. Transgenic shoots were analysed for copy number of the 2m-epsps gene and plants containing a single-copy insert were analysed for GUS activity levels in the leaves (Jefferson et al. 1987, EMBO J 6, 3901-3907). The result of the GUS activity in TO plants is shown in Figure 27, with the activity of the Pbdc7 promoter with NEENAc5 intron and SOY-3 enhancer being similar to that of the PubilOAt promoter. To determine whether this Pbdc7 promoter variant is active in different tissues, GUS activity was determined in leaves, stems and roots of homozygous T0S1 plants. Table 12 and Figure 28 show that the activity of the Pdc7 promoter with NEENAc5 intron and SOY-3 enhancer is between 28 and 34% of that of PubilOAt. These data demonstrate that this promoter has good activity in transgenic soybean plants.Table 11: List of GUS T-DNA vectors used to determine the activity of various promoter-NEENA combinations.BASF Agricultural Solutions US LLC 231727WO01Table 12: GUS activity levels of homozygous T0S1 soybean plants containing single-copy inserts of a promoter>GUS T-DNA. Fl = Fluorescence Intensity.Example 22: Selected NEENAs enhance activity of various promoters in transgenic soybean plants

[0264] To determine the activity of NEENA-containing promoters in transgenic plants, the PubilOAt promoter of pBas80 was replaced by various NEENA-containing promoters to create plant transformation vectors pBas82 to pBas87 (see Table 11). The resulting vectors were used for stable transformation of mature seeds of soybean cultivar Thorne as described in Example 21. Transgenic shoots were analysed for copy number of the 2m-epsps gene and plants containing a single-copy insert were analysed for GUS activity levels in the leaves. The result of the GUS activity in TO plants is shown in Figure 29, with the activity of the NEENA-containing promoters being between 22 (pGmUbi9-SOY-3) and 52 (pGmUbi2-SOY- 3) % of that of the PubilOAt promoter. These data demonstrate that these promoters are active in transgenic soybean plants.

Claims

1. BASF Agricultural Solutions US LLC 231727WO01Claims1. An isolated nucleic acid expression enhancing nucleic acid (NEENA) molecule selected from the group consisting of: i) a nucleic acid having a sequence of any one of SEQ ID NOs: 1-15 or their complement, ii) a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 1-15 or their complement, iii) a nucleic acid hybridizing under stringent conditions to any one of SEQ ID NOs: 1-15 or their complement, and iv) a functional fragment of 30 or more consecutive bases of a nucleic acid of i) to iii), wherein said NEENA molecule has expression enhancing activity.

2. A recombinant nucleic acid sequence for regulating expression of a polynucleotide of interest, comprising a nucleic acid having promoter activity functionally linked to one or more NEENA molecules as defined in claim 1, wherein said NEENA molecule is heterologous to the nucleic acid having promoter activity.

3. A method for enhancing expression derived from a plant promoter, comprising functionally linking to the promoter one or more heterologous NEENA molecules as defined in claim 1.

4. A method for producing a plant, plant part or plant cell with enhanced expression of one or more nucleic acid molecules, as compared to a respective control plant, plant part or plant cell, comprising the steps of: a) introducing into the plant, plant part or plant cell one or more heterologous NEENA molecule as defined in claim 1, and b) functionally linking said one or more NEENA molecule to a promoter and to a nucleic acid molecule being under the control of said promoter, wherein said NEENA molecule is heterologous to said promoter.

5. The method according to claims 3 or 4, comprising the steps of a) introducing said one or more NEENA molecule into a plant, plant part or plant cell, and b) integrating said one or more NEENA molecule into the genome of said plant, plant part or plant cell whereby said one or more NEENA molecule is functionally linked to an endogenous promoter heterologous to said one or more NEENA molecule and, optionally, c) regenerating a plant or plant part comprising said one or more NEENA molecule functionally linked to the promoter from said plant, plant part or plant cell.

6. The method according to claim 5, wherein said one or more NEENA molecule is integrated into the genome of a plant, plant part or plant cell by applying genome editing technologies.74BASF Agricultural Solutions US LLC 231727WO017. The method according to claim 6 wherein the genome editing technology comprises the introduction of single or double strand breaks near to the position where a NEENA molecule is to be integrated into the genome using targeted nucleases and the introduction of a DNA repair template comprising said NEENA molecule and at its 3' and 5' end sequences essentially identical or complementary to the sequences upstream and downstream of the single or double strand break facilitating recombination at the position of the single or double strand break.

8. The method according to claim 6, wherein the genome editing technology comprises introduction of point mutations in the genome of the plant, plant part or plant cell thereby introducing the sequence of said NEENA molecule in said genome.

9. The method according to any one of claims 3 to 6 comprising the steps of a) providing to a plant, a plant part or a plant cell an expression construct comprising one or more NEENA molecules as defined in claim 1, functionally linked to said promoter heterologous to said one or more NEENA molecule, and b) integrating said expression construct into the genome of said plant, plant part or plant cell and, optionally, c) regenerating a plant or plant part comprising said one or more expression constructs from said transformed plant, plant part or plant cell.

10. The method according to any one of claims 3 to 9, wherein said one or more NEENA molecule is functionally linked to said promoter upstream or downstream of the transcriptional start site of a nucleic acid molecule the expression of which is under control of said promoter.

11. The method according to any one of claims 3 to 10, wherein said one or more NEENA molecule is functionally linked to said promoter within the 5'UTR or within an intron of the nucleic acid molecule the expression of which is under control of said promoter.

12. The method according to any one of claims 3 to 11, wherein said promoter is a tissue specific, developmental specific or inducible promoter of a nucleic acid molecule the expression of which is under control of said promoter.

13. A recombinant expression construct comprising one or more NEENA molecule functionally linked to a promoter and one or more expressed nucleic acid molecules, wherein said one or more NEENA molecule is heterologous to said promoter and comprises a nucleic acid as defined in claim 1.

14. A recombinant expression vector comprising one or more recombinant expression constructs according to the previous claim.75BASF Agricultural Solutions US LLC 231727WO0115. A transgenic cell or transgenic plant or transgenic plant part comprising a) a recombinant expression construct according to claim 13, b) a recombinant expression vector according to claim 14, or c) one or more heterologous NEENA molecule wherein said one or more NEENA molecule comprises a nucleic acid as defined in claim 1.

16. The transgenic cell according to the previous claim , selected or derived from the group consisting of bacteria, fungi, yeasts or plants.

17. A transgenic cell culture, transgenic seed or propagation material derived from the transgenic cell or transgenic plant or transgenic plant part according to claims 15 or 16 comprising a recombinant expression construct according to claim 13, a recombinant expression vector according to claim 14 or one or more heterologous NEENA molecule wherein said NEENA molecule comprises a nucleic acid as defined in claim 1.

18. Use of a NEENA molecule according to claim 1 or a recombinant construct according to claim 13, or a recombinant vector according to claim 14 for enhancing expression in plants or in plant parts.

19. Use of a transgenic cell culture, transgenic seed, transgenic plant, transgenic plant part or propagation material derived from a transgenic cell or plant according to claim 17 for the production of foodstuffs, animal feeds, seeds, pharmaceuticals or fine chemical.76

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