Modified crop plant having increased expression of one or more one transcription factors specific for the plant's ESR or betl

Abstract

The present invention relates to a modified crop plant or part thereof which exhibits increased or ectopic expression of one or more one transcription factors, which one or more transcription factors are specifically expressed in the plant's ESR and / or in the plant's BETL, or modulate gene expression of one or more ESR and / or BETL-specific genes.

Description

[0001] Modified crop plant having increased expression of one or more one transcription factors specific for the plant’s ESR or BETL

[0002] REFERENCE TO SEQUENCE LISTING

[0003] Pursuant to the EFS-web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an xml 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference, for the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

[0004] FIELD OF THE INVENTION

[0005] The present application relates to transcription factors that are specifically expressed in a plant’s ESR and / or in the plant’s BETL. In particular, it relates to the MADS-box transcription factors which are specifically expressed in plant ESR and / or BETL regions of the endosperm. The loss of function of the MADS-box gene results in a decrease in seed length and width, indicating the role of MADS-box genes in the development of plant endosperm and embryo.

[0006] BACKGROUND The endosperm is a tissue in plants that serves as a repository for agronomic reserve substances such as starch and proteins. Its condition is decisive for nutritional content and quality of the plant’s respective organs or parts.

[0007] During early development, the endosperm of e.g. maize as well as of other crop plants forms four distinct domains with specialized functions: (i) the starchy endosperm, which is a storage site for nutrients; (ii) the aleurone layer, which activates reserve metabolites during germination; (iii) the basal endosperm transfer layer (BETL), facilitating efficient nutrient transfer from maternal source organs; and (iv) the embryo surrounding region (ESR), with roles in nutrient transfer to the embryo, defense, and signaling.

[0008] Among these, the BETL shows substantial potential for influencing grain quality, as transporting metabolites from maternal tissues into the endosperm for storage is critical for grain yield. Optimizing its development or function is interesting for controlling grain-filling and enhancing overall grain quality and yield (Sosso et al., 2015).

[0009] Similarly, the ESR holds interest. Consider e.g., maize, in which, in contrast to the endosperm, which mostly contains starch and proteins, the embryo contains around 85% of the grain’s oil. In view of oil’s higher caloric density compared to starch, enlarging the embryo could potentially enhance a crop plant’s energy yield. As the ESR likely aids in nutrient transfer to the embryo, optimizing its development or functionality could either regulate embryo size or grain oil content (Li et al., 2022).

[0010] It is hence one object of the present invention to provide approaches to exploit the potential that modulating the ESR or BETL, or modulating the expression of ESR- or BETL-specific genes provide.

[0011] It is one other object of the present invention to provide means for grain improvement in terms of nutrient content or size enlargement in crop plants.

[0012] It is one other object of the present invention to provide means for seed quality improvement in crop plants. These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred under particular circumstances. Likewise, the specification discloses further embodiments of the invention which may be preferred under particular circumstances.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] Note that in this specification, the terms

[0015] • MADS66 and PHE1L3

[0016] • MADS66 and PHERESl-like3

[0017] • ZmMADS66 and ZmPHElL3

[0018] • MADS66 and zmphelI3

[0019] • MADS66 and ZmPHEL3 are used synonymously and interchangeably.

[0020] Figure 1. Overview of endosperm compartments on the example of maize. AL, aleurone layer; BETL, basal endosperm transfer layer; EAS, endosperm adjacent to scutellum; EM, embryo; ESR, embryo-surrounding region; SE, starchy endosperm.

[0021] Figure 2: Expression levels of maize PHE1 like genes; ZmPHElLl, ZmPHElL2, ZmMADS66, and ZmPHElL4 (Zm00001d013792) in maize seeds at different days after pollination (DAP) as indicated.

[0022] Figure 3: Maize PHE1L1 promoter translational fusion reporter construct pZmPHElLl ::GFP map. BdUbilO defines the promoter and intron from Brachypodium Ubiquitin 10 gene. Nos-t defines the nos terminator. 3’35S defines the 35S terminator.

[0023] Figure 4: Maize PHE1L2 promoter translational fusion report construct pZmPHElL2::GFP map. BdUbilO defines the promoter and intron from Brachypodium Ubiquitin 10 gene. Nos-t defines the nos terminator. 3’35S defines the 35S terminator.

[0024] Figure 5: Maize MADS66 promoter translational fusion report construct pZmMADS66::GFP map. BdUbilO defines the promoter and intron from Brachypodium Ubiquitin 10 gene. Nos-t defines the nos terminator. 3’35S defines the 35S terminator. Note that the map uses the term “pZmPHElL3::GFP” instead of the term “pZmMADS66::GFP”. However, both terms mean the same.

[0025] Figure 6. Expression patterns of ZmPHElLl in endosperm of the immature kernels harvested 7 days after pollination (DAP). The ZmPHElLl expression and the protein localization were revealed by the fluorescence signals from the ZmPHELl-GFP translational fusion reporter. The pictures show whole seeds and zoom ins of the indicated regions of the endosperm in 7 DAP kernels.

[0026] Figure 7. Expression patterns of ZmPHElL2 in endosperm of the immature kernels harvested 7 days after pollination (DAP). The ZmPHElL2 expression and the protein localization were revealed by the fluorescence signals from the ZmPHEL2-GFP translational fusion reporter. The pictures show whole seeds and zoom ins of the indicated regions of the endosperm in 8 DAP kernels.

[0027] Figure 8. Expression patterns of ZmMADS66 in endosperm of the immature kernels harvested 7 days after pollination (DAP). The ZmMADS66 expression and the protein localization were revealed by the fluorescence signals from the ZmPHEL3-GFP translational fusion reporter. The pictures show whole seeds and zoom ins of the indicated regions of the endosperm in 4 DAP kernels.

[0028] Figure 9. Genome editing nuclease LbCpfl expression construct GEMT505 map. tDT defines the flourescence tdTomato gene. LpCpfl-RR defines the maize codon-optimized CDS of the Lachnospiraceae bacterium CRISPR / Cpfl (LbCpfl) gene.

[0029] Figure 10. Multiplex genome editing crRNA construct pZmPHElLl CrRNA map. CrRNA-1 to CrRNA-5 defines the 5 crRNA guide RNAs, which target to maize endogenous PHEL1 gene (ZmPHElLl) at the MADS-box domain. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0030] Figure 11. Multiplex genome editing crRNA construct pZmPHElL2_CrRNA map. CrRNA-1 to CrRNA-5 defines the 5 crRNA guide RNAs, which target to maize endogenous PHEL2 gene (ZmPHElL2) at the MADS-box domain. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator.

[0031] Figure 12. Multiplex genome editing crRNA construct pZmMADS66_CrRNA map. CrRNA- 1 and CrRNA-2 defines the 2 crRNA guide RNAs, which target to maize endogenous MADS66gene (Zm MADS66) at the MADS-box domain. ZmUbil defines the promoter and intron from maize Ubiquitin 1 gene. Tnos defines the nos terminator. Note that the map uses the term “ZmPHElL3_CrRNA” instead of the term “ZmMADS66_ CrRNA”. However, both terms mean the same.

[0032] Figure 13. KWS RBP8 expression construct pABM-BdEFl_RBP8 map. KWS RBP8 is driven by the strong constitutive EFl promoter from Brachypodium (pBdEFl).

[0033] Figure 14. Corn Al 88 immature tassels bombardment. A: a fresh isolate immature tassel ready for bombardment; B: a fluorescent image of the immature tassels 20 hours after bombardment.

[0034] Figure 15: Multiple Sequence Alignment of ZmPHElLl gene from the two edited mutants and the WT A188.

[0035] Figure 16: Multiple Sequence Alignment of ZmPHElL2 gene from the two edited mutants and the WT A188.

[0036] Figure 17: Multiple Sequence Alignment of ZmMADS66 gene from the two edited mutants and the WT A188.

[0037] Figure 18. Kernel size measurements in homozygous mutants zmphelll _#1 and zmphelll _#2. Length (A) and width (B) of immature maize kernels were measured at 12 days after pollination (DAP). Differences in kernel dimensions reflect the potential role of ZmPHElLl in regulating seed size.

[0038] Figure 19. Kernel size measurements in homozygous mutants zmphell2 i \ and zmphell2Ji2. Length (A) and width (B) of immature maize kernels were measured at 12 days after pollination (DAP) for zmphell2 i \ mutant, while the length (C) and width (D) of immature maize kernels were measured at 7 days after pollination (DAP) for zmphell2Ji2. Differences in kernel dimensions reflect the potential role of ZmPHElL2 in regulating seed size.

[0039] Figure 20. Kernel size measurements in homozygous mutants zmMADS66_#l and zmMADS66_#2. Length (A) and width (B) of immature maize kernels were measured at 4 days after pollination (DAP). Differences in kernel dimensions reflect the potential role of ZmMADS66 in regulating seed size.

[0040] Figure 21 : Selected targets of PHE1L1 and PHE1L2..

[0041] Figure 22: Selected targets of PHE1L1 and PHE1L2.

[0042] DETAILED DESCRIPTION OF EMBODIMENTS

[0043] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

[0044] It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done. Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

[0045] According to one aspect of the invention, a modified crop plant or part thereof is provided which exhibits altered or ectopic expression of one or more one transcription factors, which one or more transcription factors a) are specifically expressed in the plant’s ESR and / or in the plant’s BETL, or b) modulate gene expression of one or more ESR and / or BETL-specific genes.

[0046] In one embodiment said modified crop plant or part thereof is engineered or technically modified to exhibit increased or ectopic expression of said one or more one transcription factors. Such engineering or modification may encompass, in embodiments, the use of genomic engineering or editing technologies, including the use of transposases, restriction endonucleases or site directed nucleases (SDN).

[0047] As used herein, the term “altered expression” means that, compared to a non-modified crop plant of the same species or variety, or part thereof, the expression of one or more ESR- and / or BETL-specific transcription factors is changed or altered. In a specific embodiment, the term “altered expression” means “increased expression”. In said embodiment, the expression of one or more ESR- and / or BETL-specific transcription factors is increased compared to a nonmodified crop plant of the same species or variety, or part thereof.

[0048] As used herein, the term ’’ectopic expression” refers to the expression of one or more ESR- and / or BETL-specific transcription factors at times and locations when / where they are not typically or naturally expressed.

[0049] In one embodiment, said modified crop plant or part thereof has been edited, transiently or non- transiently, to exhibit such altered expression. a) According to another aspect of the invention, a modified crop plant or part thereof is provided which comprises one or more modifications in at least one transcription factor gene, which transcription factor gene is specifically expressed in the plant’s ESR and / or in the plant’s BETL, or b) modulates gene expression of one or more ESR and / or BETL-specific genes, which modification results in altered expression of one or more ESR and / or BETL-specific genes.

[0050] In one embodiment, said modified crop plant or part thereof has been edited, transiently or non- transiently, to comprises one or more modifications.

[0051] Such effect can be achieved either by altered interaction between the transcription factor and the promoter or the enhancer of a ESR and / or BETL-specific gene. Such effect can also be achieved by modifying the transcription factor to facilitate the recruitment of activating or repressing chromatin complexes, by e.g. fusing the transcription factor to activating or repressing domains (e.g. VP 16 domain , EAR domain), respectively.

[0052] According to another aspect of the invention, a modified crop plant or part thereof is provided which comprises one or more modifications in at least one promoter or at least one enhancer of a ESR and / or BETL-specific gene, which modification results in altered activation by one or more transcription factors, which transcription factor is specifically expressed in the plant’s ESR and / or in the plant’s BETL, or modulates gene expression of one or more ESR and / or BETL-specific genes

[0053] In one embodiment, said modified crop plant or part thereof has been edited, transiently or non- transiently, to comprises one or more modifications.

[0054] Transcription factors are proteins involved in the process of converting, or transcribing, DNA into RNA. Transcription factors include a wide number of proteins that initiate and regulate the transcription of genes. One distinct feature of transcription factors is that they have DNA- binding domains that give them the ability to bind to specific sequences of DNA called enhancer or promoter sequences. Some transcription factors bind to a DNA promoter sequence near the transcription start site and help form the transcription initiation complex. Other transcription factors bind to regulatory sequences, such as enhancer sequences, and can either stimulate or repress transcription of the related gene. These regulatory sequences can be thousands of base pairs upstream or downstream from the gene being transcribed. Regulation of transcription is the most common form of gene control. The action of transcription factors allows for unique expression of each gene in different cell types and during development. According to embodiments, the expression of one or more of the plant’s own respective transcription factors has been altered, or increased, e.g., by introducing copies of heterologous or homologous genes encoding for ESR- and / or BETL-specific transcription factors.

[0055] According to further embodiments, one or more promoters of genes encoding for ESR- and / or BETL-specific transcription factors have been edited or modified so as to drive expression of the said genes.

[0056] As used herein, the term “transcription factors specifically expressed in the plant’s ESR and / or in the plant’s BETL” relates to transcription factors which are specifically expressed in ESR and / or BET (which does yet not exclude that such transcription factors can also be expressed in other tissues of the plant).

[0057] As used herein, the term “modifying or modulating the expression of a gene” can mean increasing, reducing or stabilizing, the transcription (RNA production) and / or translation (protein expression of the respective gene.

[0058] As used herein, the term “ESR and / or BETL-specific gene” relates to genes which are specifically expressed in ESR and / or BET (which does yet not exclude that such genes can also be expressed in other tissues of the plant).

[0059] The terms “transcription factors specifically expressed in the plant’s ESR and / or in the plant’s BETL“; “ESR- and / or BETL-specific transcription factors” and “Transcription factors specific to ESR and / or BETL” are used synonymously herein.

[0060] As used herein, the term ESR (“embryo surrounding region” relates to a defined region within the endosperm which is in close proximity of the embryo, and which is cytologically different from the remaining endosperm. Gene expression restricted to the ESR reinforces the notion of a specialized endosperm domain in many plants. The ESR is a dynamic structure that is set apart prior to cellularisation and starts to disappear with the onset of reserve accumulation in the developing seed. During later developmental stages it is frequently succeeded by a liquid filled space around the embryo. As used herein, the term BETL (“basal endosperm transfer layer)” relates to an interface specialized in nutrient transfer from maternal phloem terminals to the endosperm. The hexose transporter SWEET4c is preferentially expressed in the BETL, and loss of function of Sweet4c results in the production of a shrivelled endosperm.

[0061] In general, as can be seen in Figure 1, the endosperm, which in Maize occupies 70% of the kernel volume at the end of early development, has been described as differentiating only four main cell types. The basal endosperm transfer layer (BETL) and the aleurone layer (AL) are two peripheral cell types in contact with maternal tissues. The embryo-surrounding region (ESR) is formed of small densely cytoplasmic cells encircling the young embryo. The starchy endosperm (SE) corresponds to the central region of the endosperm, which subsequently accumulates huge amounts of storage compounds before undergoing progressive programmed cell death.

[0062] As used herein, the term “crop plant” relates to plants grown and tended or cared for in a field so as to obtain a harvest.

[0063] According to embodiments, such crop plant is selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oriza sativa, Oryza austr aliensis, Oryza alta, Triticum aestivum, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis ar enosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Astragalus sinicus, Lotus japonicas, Torenia fournieri, Spinacia oleracea, Vicia faba, Phaseolus vulgaris, Allium cepa, Allium fistulosum, Allium sativum, and Allium tuberosum. As used herein, the term “part of a crop plant” relates to at least one of seedlings, shoots, seeds, cells, callus or germplasm.

[0064] The inventors consider that increased expression of such transcription factor may result in at least one of a) grain improvement in terms of nutrient content or size enlargement, and / or b) seed quality improvement.

[0065] According to another aspect of the invention, the use of one or more transcription factors which a) are specifically expressed in the plant’s ESR and / or in the plant’s BETL, or b) modulate gene expression of one or more ESR and / or BETL-specific genes. is provided for at least one of

[0066] • modulating the expression of at least one ESR and / or BETL-specific gene

[0067] • grain improvement in terms of nutrient content or size enlargement, and / or

[0068] • seed quality improvement.

[0069] Increased expression of such transcription factors specific to ESR and / or BETL offers an approach for selectively targeting genes with pivotal functions in endosperm development and function. The resulting modified endosperm cellular composition can significantly influence grain traits of interest in plant breeding. Targeting gene expression in the endosperm is hence a viable approach which so far has not been extensively explored.

[0070] While certain ESR and BETL specific genes were described (outlined in Table 1), they encode proteins other than Transcription factors, and moreover, their identification relied on techniques like as in situ hybridization of GUS assays, which reveal transcript localization or promoter activity, but not protein localization.

[0071] Table 1. Known BETL and ESR specific genes.

[0072] According to embodiments of the invention, the modified crop plant or part thereof a) comprises an artificially introduced recombinant, heterologous or homologous Nucleic Acid Molecule encoding for at least one such transcription factor b) has been transiently transfected with an artificially introduced recombinant, heterologous or homologous Nucleic Acid Molecule encoding for at least one such transcription factor c) is modified or treated so as to exhibit altered, or increased, expression of at least one such intrinsic transcription factor

[0073] As used herein, the term “recombinant Nucleic Acid Molecule” relates to a man-made, nonnatural Nucleic Acid Molecule which has been generated and introduced into the plant or part thereof for the purpose of the expression of a specific protein.

[0074] As used herein, the term “heterologous Nucleic Acid Molecule” relates to a Nucleic Acid Molecule that encodes a protein that is e not normally produced in vivo by the plant or part thereof in which it is expressed or that mediates or encodes mediators that alter expression of endogenous Nucleic Acid Molecule by affecting transcription, translation, or other regulatable biochemical processes or is not present in the exact orientation or position as the corresponding non-heterologous Nucleic Acid Molecule.

[0075] As used herein, the term “artificially introduced homologous Nucleic Acid Molecule” relates to a Nucleic Acid Molecule that is identical to naturally occurring Nucleic Acid Molecule in the plant or part thereof, yet has been artificially introduced so as to increase the copy number of the respective Nucleic Acid Molecule

[0076] As used herein, the term “intrinsic transcription factor” relates to the plant or plant part’s naturally occurring transcription factor.

[0077] According to embodiments of the invention, the modified crop plant belongs to one taxon selected from the group consisting of

[0078] • Poaceae

[0079] • Amaranthaceae

[0080] • Brassicaceae, and / or

[0081] • Asteraceae

[0082] According to embodiments of the invention, the modified crop plant is maize (Zea mays), sugar beet (Beta vulgaris), rapeseed (Brassica napus) or sunflower (Helianthus annus).

[0083] According to embodiments of the invention, the transcription factor is at least one selected from the group consisting of

[0084] • PHERESl-likel (PHE1L1) or homologues, orthologues, paralogues or aliases thereof

[0085] • PHERESl-like2 (PHE1L2) or homologues, orthologues, paralogues or aliases thereof, and / or

[0086] • PHERESl-like3 (MADS66) or homologues, orthologues, paralogues or aliases thereof

[0087] The two first transcription factors are denoted as PHERESl-likel (PHE1L1) and PHERES1- like2 (PHE1L2) because of their sequence resemblance to the Arabidopsis thaliana TF PHERES1 (PHE1). Identification and characterization of these two transcription factors specific to the ESR and BETL enables the precise targeting of key regulatory elements involved in grain or plant development, introducing an approach to plant breeding or plant engineering which allows for more focused genetic manipulation, minimizing off-target effects, and has the potential to improve grain quality and yield.

[0088] The following table shows the respective transcription factors or homologues, orthologues, paralogues or aliases thereof. A homologous gene (or homolog) is a gene inherited in two species from a common ancestor. While homologous genes can be similar in sequence, similar sequences are not necessarily homologous. Orthologous genes are homologous genes where a gene diverges after a speciation event, but the gene and its main function are conserved. If a gene is duplicated in a species, the resulting duplicated genes are paralogs of each other, even though over time they might become different in sequence composition and function. The data base “gramene.org” provides information regarding homologues, orthologues and paralogues of a given plant gene.

[0089] Table 2. Transcription factors and examples of homologues, orthologues, paralogues or aliases thereof

[0090] All the above genes are members of the MADS box gene family. MADS box genes are an example of a family of highly conserved TFs that have diverse roles in plant development. Although these TFs play a pivotal role in determining floral organ identity, they are also important regulators of vegetative development. P0TM1 (potato MADS box, GenBank accession number U23757) is a member of the SQUA-like family of plant MADS box genes isolated from an early-stage tuber cDNA library and is expressed in apical and axillary meristems.

[0091] The following table shows a non-limiting selection of BETL and ESR specific genes that are targets of PHE1L1 and PHE1L2, as identified by Cut and Tag (see example 4).

[0092] AGL-like genes play a role in flowering. ARF-like genes are Auxin Response Factors mediating the auxin response. YUCCA-like and TAR-like genes play a role in auxin biosynthesis.

[0093] According to embodiments of the invention, the modified crop plant or part thereof is at least one selected from a seedling, shoot, seed, cell, callus and germplasm.

[0094] Examples

[0095] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0096] Example 1. PHE1 like genes expression analysis by real time qPCR PHE1L1 and PHE1L2 expression levels were analyzed by qPCR in maize seeds at different days after pollination (DAP). The RNAs were isolated from the endosperm of immature seeds at different days after pollination (DAP) as indicated. The RNA isolation, reverse transcription, and qPCR were carried out following a standard protocol. The results showed that these genes are expressed immediately after the initiation of the cellularization (around 4 DAP), when the ESR and BETL start to differentiate (Wu et al., 2022; He et al., 2024). Results are shown in Figure 2.

[0097] Example 2: Producing maize PHE like gene promoter report lines in maize A188 via Agrobacterium-mediated stable transformation.

[0098] Expression patterns of PHE1L1, PHE1L2 and MADS66 were studied using GFP-tagged reporter lines. These lines were generated by incorporating the coding sequences (CDS) of the genes along with 2.5 kb of sequence upstream of the CDS. As these genes lack introns, the sequences were directly amplified from maize genomic Nucleic Acid Molecule. Subsequently, these sequences were inserted into a destination vector containing GFP at the C-terminus and the NOS terminator. These vectors were used to transform Al 88 plants.

[0099] Construct generation: the Nucleic Acid Molecule fragment of 2-2.5 kb of the 5’ upstream promoter region and the full CDS sequence we as synthesized and in frame fused to GFP and cloned into a binary vector by GenScript (Piscataway, NJ 08854, USA). The constructs are shown in Figures 3 to 5.

[0100] The plasmid was transformed into Agrobacterium LBA4404 via electroporation transformation. Maize transformation was performed using maize Al 88 immature embryos, size 0.8-1.5 mm and following a standard Agrobacterium-mediated transformation protocol (Ishida et al 1996). Briefly, 9-12 days after pollination, immature embryos were isolated septically and inoculated with the Agrobacterium LB A4404 containing a construct of interest. After 7 days of co-culture the embryos and resulted regenerated cells and tissues were under herbicide selection for transformation events. Nucleic Acid Molecule of the putative transgenic plants was extracted and analyzed through qPCR for the presence of the gene expression cassette of interest. At least three independent transgenic lines with single copy of the transgene cassette from each of the constructs were moved into soils and grown in greenhouse for T1 seed production. Example 3: Expression patterns were studied using GFP-tagged reporter lines.

[0101] The transgenic T1 plants were grown in soil in greenhouse. The immature seeds were harvested at different days after pollination (DAP) as indicated. GFP localization was observed using confocal microscopy. The analyses showed that PHE1L1 is expressed uniquely in the ESR (Figure 6), while PHE1L2 is expressed in both the ESR and BETL (Figure 7). This expression pattern is consistent with previously reported mRNA localization data for both genes based on in situ hybridization (He et al., 2024). Maize MADS66 is expressed throughout the immature endosperm (Figure 8).

[0102] Example 4: Identification of the downstream targets of maize PHE1 like genes

[0103] Targets of the maize PHE1L1 and PHE1L2 transcription factors were identified by CUT&Tag Sequencing. CUT&Tag is a method used to analyse protein interactions with Nucleic Acid Molecule. CUT&Tag Sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel Nucleic Acid Molecule sequencing to identify the binding sites of Nucleic Acid Molecule-associated proteins

[0104] For the CUT&Tag experiment, nuclei were extracted from frozen kernels at 8 DAP using a nuclei extraction buffer. Following filtration and washing steps, nuclei were incubated overnight at 4°C with primary antibodies against targeting GFP and histone H3. The following day, nuclei were incubated with secondary antibody in digitonin-containing buffer, followed by binding of protein A-Tn5 transposase. Tagmentation was performed at 37°C, and DNA was released using SDS and subsequently quenched with Triton X-100. Library concentration and quality were assessed with Qubit and TapeStation, respectively. Sequencing was performed on an Illumina NextSeq 1000 / 2000 using a P2 flow cell and P2 Reagent Cartridge. Raw sequencing reads were assessed for quality using FastQC, and adapter trimming was performed with Trim Galore. High-quality reads were aligned to the maize reference genome (Zm-A188- REFERENCE-KSU-1.0) using Bowtie2. PCR duplicates and low-quality alignments were removed prior to peak calling with MACS2, using parameters optimized for transcription factor binding site detection. Genomic feature overlaps and peak annotations were carried out using BEDTools. Downstream analyses included peak visualization and integration with gene annotation datasets for functional interpretation. CUT&Tag profiling revealed that transcription factor binding sites were predominantly enriched in promoter regions, suggesting a regulatory role near transcription start sites. Motif analysis showed an enrichment of MADS-box binding motifs within the peaks, consistent with the activity of MADS-domain transcription factors. Several target genes included other MADS-box transcription factors as well as genes involved in auxin biosynthesis, both of which are known regulators of seed growth. These results point to a regulatory network in which MADS transcription factors may promote seed growth by activating other key developmental regulators. In these experiments, whole seeds at 8 DAP were used as the transcription factors are located uniquely in the endosperm. Among the targets genes were identified with roles in endosperm proliferation (see Table 3 and Table 4).

[0105] Table 4: Selected targets of ZmPHElLl and ZmPHElL2 (Part II). Zm00056aa033204 Zm00001d037126

[0106] Example 5: Multiplex transient genome editing SDN-1 via particle bombardment and maize immature tassel meristem regeneration system

[0107] 1. Maize immature tassel preparation

[0108] Immature tassels from the selected lines at the developmental stages of late V6 to late V7 are used. It takes 25-32 days (most likely 28 days) to reach these stages after seed planting. The developmental stages of immature tassels are determined using a Zeiss stereo microscope. The immature tassel is manually isolated under aseptic conditions.

[0109] 2. Multiplex genome editing SDN-1 via transient co-bombardment

[0110] The fresh isolated immature tassels were placed onto an osmotic medium plate (e.g N6 0SM medium) for 4 hours. Particle bombardment is conducted using a Bio-Rad PDS- 1000 / He particle gun. The bombardment conditions were: 28-30 mm / Hg vacuum, 1100 psi helium pressure. Per bombardment, 100-150 ng each of CRISPR constructs (Cas nuclease, multiplex crRNAs; Figure 9-12), and at least one regeneration booster construct (e.g., RBP8; Figure 13) are co-coated onto 100 pg of 0.6 pm gold particles using calciumspermidine method. crRNA constructs are designed to target PHE1L1 (Figure 10), maize PHE1L2 (Figure 11), and maize MADS66 (Figure 12).

[0111] Four bombardments per sample plate are performed. The bombarded immature tassels were kept on the osmotic medium plate for another 16-20 hours after the bombardment. The fluorescence report gene, tDTomato was used for monitoring biolistic transformation and the gene expression (Figure 14). . Plant regenerati on 16-20 hours after the bombardment the Al 88 immature tassel was subj ect to regeneration, which comprising the steps of:

[0112] - Embryogenic callus induction: cut the bombarded immature tassel into a segment of 2-5 mm in length, with a sharp blade, and place onto a callus induction medium (e.g., N6_5Ag) in petro dish plate (25 x 100 mm) with the bombarded surface up. Seal the plate with surgical tape and culture at 27°C, dark, for about 1 week.

[0113] - Embryogenic callus development: transfer the segments from callus induction onto a fresh MRM3 medium, and incubate the plate at 27 °C, dark for another 2 weeks for embryogenic callus development.

[0114] - Shoot development: separate embryogenic calluses into small pieces of 2-5 mm in diameter and transfer the calluses onto a Shooting medium plate (25x 100mm). Seal the plate with surgical tape and culture at 25°C, weak light, for ~3-5 days (20-100 pmol m2s ') and then full light (>100 pmol m2s ') for another ~7 days.

[0115] - Root and plantlet development: transfer the developing shoots onto a rooting medium in a phytotray and culture the shoots in the full light chamber at 25 °C. The regenerated To plants are ready for sampling and moving to soil in 5-7 days.

[0116] 4. Molecular screening for SDN-1 events in the regenerated To plants

[0117] A 10-15 mm leaf tip from each of the leaves of a To event are collected for Nucleic Acid Molecule extraction. Genome editing SDN-1 in the regenerated TO plants are screened by TaqMan real-time PCR (qPCR), marker capillary electrophoresis analysis, and TaqMan Digital Droplet PCR. Site-specific modification is further conformed by next generation sequencing (NGS) or Sanger sequencing.

[0118] 5. Grow the edited TO plants and T1 seed production

[0119] After the molecular screening and confirmation, the selected TO plants are transferred to soil, and grown in a growth chamber or greenhouse under the suitable growth conditions. TO plants are phenotypically analyzed and grown for T1 seed production by self-cross

[0120] 6. Genotyping the edited T1 plants

[0121] One month after growth in green house a leaf disk was sampled from each of the three youngest leaves. The three leaf disks from the same plant were combined for Nucleic Acid Molecule isolation. Biallelic edited T1 plants were selected by targeted amplification and Sanger sequencing. Homozygous editing in the three PHE1 like genes was demonstrated in Multiple Sequence Alignment of the edited lines was demonstrated in Figure 15 to 17. A summary of the maize PHE1 like genes and the edited mutants in the MADS-box function domain is demonstrated in Table 5.

[0122] Table. 5: List of the maize PHE1 like genes and their homozygous edited mutants in MADS- box domain

[0123] Gene Name Locus (B73 GRAMENE v4.0) Edited mutants Deletion

[0124] Example 6: Phenotyping of PHE1L1, PHE1L2 and MADS66 maize mutants

[0125] Maize kernel sizes from the T2 homozygous mutants for PHE1L1, PHE1L2, and MADS66 were assessed through imaging of immature kernels using a Leica S9i stereomicroscope. Length and width measurements were carried out using Fiji software. All mutants exhibited reduced kernel size in comparison to the wild type, with MADS66 mutants demonstrating the most significant reduction, followed by PHE1L1, and then PHE1L2. Variations in kernel dimensions suggest that maize PHEl-like genes are involved in controlling seed size.

[0126] References

[0127] • Ling Meng. Immature inflorescence meristem editing US2023081632 (Al)

[0128] • Sosso D, Luo D, Li QB, Sasse J, Yang J, Gendrot G, Suzuki M, Koch KE, McCarty DR, Chourey PS, Rogowsky PM, Ross-Ibarra J, Yang B, Frommer WB. (2015). Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport. Nat Genet 47: 1489-1493.

[0129] • Li X, Wang M, Zhang R, Fang H, Fu Z, Yang X, Li J. (2022). Genetic architecture of embryo size and related traits in maize. The Crop Jounal 10:204-215.

[0130] • Wu H, Becraft PW, Dannenhoffer JM. (2022). Maize Endosperm Development: Tissues, Cells, Molecular Regulation and Grain Quality Improvement. Front Plant Sci 13:852082. • He J, Wang J, Zhang Z. (2024). Toward unveiling transcriptome dynamics and regulatory modules at the maternal / filial interface of developing maize kernel. Plant J. doi:org / 10.1111 / tpj.16733.

[0131] • Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens Nat Biotechnol 14:745-750)

[0132] • Opsahl-Ferstad HG, Le Deunff E, Dumas C, Rogowsky PM. (1997). ZmEsr, a novel endosperm-specific gene expressed in a restricted region around the maize embryo. Plant J 12:235-246.

[0133] • Bonello JF, Opsahl-Ferstad HG, Perez P, Dumas C, Rogowsky PM. (2000). Esr genes show different levels of expression in the same region of maize endosperm. Gene 246:219-227.

[0134] • Bate NJ, Niu X, Wang Y, Reimann KS, Helentjaris TG. (2004). An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development. Plant Physiol 134:246-254.

[0135] • Magnard JL, Le Deunff E, Domenech J, Rogowsky PM, Testillano PS, Rougier M, Risueno MC, Vergne P, Dumas C. (2000). Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize. Plant Molecular Biology 44:559-574.

[0136] • Hueros G, Varotto S, Salamini F, Thompson RD. (1995). Molecular Characterization of BET 1, a Gene Expressed in the Endosperm Transfer Cells of Maize. Plant Cell 7:747-757.

[0137] • Hueros G, Gomez E, Cheikh N, Edwards J, Weldon M, Salamini F, Thompson RD. (1999a). Identification of a promoter sequence form the BETL1 gene cluster able to confer transfer-cell-specific expression in transgenic maize. Plant Physiol 121 : 1143 - 1152.

[0138] • Batista RA, Moreno-Romero J, Qiu Y, van Boven J, Santos-Gonzalez J, Figueiredo DD, Kohler C. The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons. Elife. 2019 Dec 2;8:e50541.

[0139] SEQUENCES The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.

[0140] In some cases, signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https : / / services. healthtech. dtu.dk / services / SignalP-6.0. The same applies to His tags or C-Myc tags, if existing.

[0141] Table 6: Sequences

Claims

What is claimed1. A modified crop plant or part thereof which exhibits altered or ectopic expression of one or more one transcription factors, which one or more transcription factors a) are specifically expressed in the plant’s ESR and / or in the plant’s BETL, or b) modulate gene expression of one or more ESR and / or BETL-specific genes.

2. A modified crop plant or part thereof which comprises one or more modifications in at least one transcription factor gene, which transcription factor gene is a) specifically expressed in the plant’s ESR and / or in the plant’s BETL, or b) modulates gene expression of one or more ESR and / or BETL-specific genes, which modification results in altered expression of one or more ESR and / or BETL- specific genes.

3. A modified crop plant or part thereof which comprises one or more modifications in at least one promoter or at least one enhancer of a ESR and / or BETL-specific gene, which modification results in altered activation by one or more transcription factors, which transcription factor is a) specifically expressed in the plant’s ESR and / or in the plant’s BETL, or b) modulates gene expression of one or more ESR and / or BETL-specific genes4. Use of one or more transcription factors which a) are specifically expressed in the plant’s ESR and / or in the plant’s BETL, or endosperm, or b) modulate gene expression of one or more ESR and / or BETL-specific genes, for at least one of• modulating the expression of at least one ESR and / or BETL-specific gene• grain improvement in terms of nutrient content or size enlargement, and / or• seed quality improvement.

5. The modified crop plant or part thereof or the use according to any one of the aforementioned claims, wherein the plant or part thereofa) comprises an artificially introduced recombinant, heterologous or homologous Nucleic Acid Molecule encoding for at least one such transcription factor b) has been transiently transfected with an artificially introduced recombinant, heterologous or homologous Nucleic Acid Molecule encoding for at least one such transcription factor c) is modified or treated so as to exhibit altered expression of at least one such intrinsic transcription factor6. The modified crop plant or part thereof or the use according to any one of the aforementioned claims, wherein the modified crop plant belongs to one taxon selected from the group consisting of• Poaceae• Amaranthaceae• Brassicaceae, and / or• Asteraceae7. The modified crop plant or part thereof or the use according to any one of the aforementioned claims, wherein the modified crop plant is maize (Zea mays), sugar beet (Beta vulgaris), rapeseed (Brassica napus) or sunflower (Helianthus annus).

8. The modified crop plant or part thereof or the use according to any one of the aforementioned claims, wherein the transcription factor is at least one selected from the group consisting of• PHERESl-likel (PHE1L1) or homologues, orthologues, paralogues or aliases thereof• PHERESl-like2 (PHE1L2) or homologues, orthologues, paralogues or aliases thereof, and / or• PHERESl-like3 (PHERES1L3) or homologues, orthologues, paralogues or aliases thereof9. The modified crop plant or part thereof or the use according to any one of the aforementioned claims, wherein the plant or part thereof is at least one selected from a seedling, shoot, seed, cell, callus and germplasm.

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