Generation of heat-tolerant pollen tubes

Abstract

Rising global temperatures negatively impact crop yields by inhibiting seed and fruit production, primarily due to the vulnerability of pollen tube (PT) delivery of sperm cells to ovules under heat stress (HS). Two in vivo assays were developed to investigate how various HS conditions affect pollen-pistil interactions in Arabidopsis, revealing that HS impairs multiple stages of PT growth: pollen germination, growth in the transmitting tract, emergence from the transmitting tract, and ovule targeting, and seed formation. Increased temperatures progressively halted PT growth earlier in its journey toward the ovules. This disruption of pollen-pistil interactions may contribute to reduced crop yields under HS. Additionally described are expression cassettes designed to generate heat-tolerant plants by overexpressing LLG, offering a potential solution to mitigate the adverse effects of heat stress on crop production.

Description

GENERATION OF HEAT-TOLERANT POLLEN TUBESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 664,653 filed June 26, 2024, the specification of which is incorporated herein in its entirety by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 1939255 awarded by the National Science Foundation. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (ARIZ_24.29_PCT_Sequence_Listing.xml; Size: 3,457 bytes; and Date of Creation: June 25, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0004] The present invention features expression cassettes and plants engineered to confer enhanced stress tolerance, such as increased heat resistance.BACKGROUND OF THE INVENTION

[0005] Increasing global temperatures negatively affect crop yield by inhibiting the production of seeds and / or fruits. Creating and growing crop plants that can overcome heat stress to set seed and fruit at higher temperatures is advantageous to growers. Seed and fruit development in nearly all crops depends on fertilization via pollen tubes. Pollen tubes are a critical component of plant reproduction as they deliver sperm cells to ovules for fertilization and subsequent seed and fruit development. However, pollen tube growth is vulnerable to heat stress, resulting in poor growth, fertilization, and reduced seed and fruit development. The invention described here showed that overexpressing a gene from tomatoes in Arabidopsis thaliana (Arabidopsis) (a research model plant) pollen tubes can alter molecular mechanisms, increase pollen tube integrity, and promote growth at high heat stress, with temperatures ranging from 35°C to 37°C (the ideal temperature for growth of this plant is 21 °C). The present invention may also be applied to tomatoes and other crops to enhance pollen tube growth and fertility under heat stress, thereby improving seed and fruit yield at elevated temperatures.BRIEF SUMMARY OF THE INVENTION

[0006] The present invention aims to provide systems, compositions, and methods for generating heat-tolerant pollen, which ultimately makes more seeds, as specified in the independent claims. Embodiments of the invention are given in the dependent claims.Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0007] The assay(s) described in the present invention offer the opportunity to test pollen tubes (PTs) with mutated cell wall components, signaling molecules, or essential proteins for maintaining PT(s) under heat stress (HS). Moreover, complementation of PT mutants or the beneficial effects of ectopic expression or overexpression of pollen-expressed genes in overcoming HS can be visualized and quantified using this assay. It can also be used to conduct mutant screens that identify pollen tubes capable of overcoming HS, potentially resulting in increased fertilization and seed development under such conditions.

[0008] In some embodiments, the present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a plant-specific promoter (e.g., a promoter that is also very active and can express the genes abundantly). In some embodiments, the expression cassette may allow for the overexpression of LLG. In other embodiments, the present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong pollen-specific promoter. In further embodiments, the present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong root hair cell-specific promoter or shoot-specific promoter.

[0009] In some embodiments, the present invention features an expression cassette, wherein the strong-plant specific promoter is selected from a group consisting of a stem-specific promoter, leaf-specific promoter, seed-specific promoter, root-specific promoter, shoot-specific promoter, flower-specific promoter, fruit-specific promoter, root hair cell-specific promoter, or pollen-specific promoter. In some embodiments, the strong plant-specific promoter is selected from a promoter specific to another part of the plant.

[0010] The aforementioned expression cassette may further comprise a reporter (e.g., a fluorescent reporter, a luminescent reporter, or an enzymatic reporter). Thus, in some embodiments, the present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG), a reporter, and a selectable marker, all three operatively linked to a strong plant-specific promoter (e.g., a strong pollen-specific promoter or a strong root hair cell-specific promoter, or shoot-specific promoter.

[0011] In some embodiments, the selectable marker is also present on the plasmid or expression cassette carrying the LLG and the reporter, and is expressed from its own promoter. In other embodiments, the reporter is present on the plasmid or expression cassette carrying theLLG and the selectable marker, and is expressed from its own promoter.

[0012] In some embodiments, the selectable marker is on a separate expression cassette. In embodiments where the selectable marker is on a separate expression cassette, that selectable marker is operatively connected to a promoter on that same expression cassette. In some embodiments, the reporter is on a separate expression cassette. In embodiments where the reporter is on a separate expression cassette, that reporter is operatively connected to a promoter on that same expression cassette.

[0013] In some embodiments, the present invention features an expression cassette comprising a gene comprising a nucleotide sequence encoding a LLG, wherein the nucleotide sequence has 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 99, or 100%) sequence identity to the nucleotide sequence set forth in SEQ ID NO. 1 .

[0014] In some embodiments, the present invention features an expression cassette comprising a gene comprising a nucleotide sequence encoding a LLG, wherein the nucleotide sequence encodes a polypeptide having 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 99, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO. 2.

[0015] In some embodiments, the LLG is LLG2 / 3 from Solanum lycopersicum. In some embodiments, the strong pollen-specific promoter comprises a promoter of a LAT52 gene from Solanum lycopersicum. In some embodiments, the selectable marker comprises a hygromycin resistance gene, a spectinomycin resistance gene, or a combination thereof. In some embodiments, the selectable marker comprises another antibiotic.

[0016] In some embodiments, the present invention may also feature a stress-tolerant transgenic plant (e.g., Solanum lycopersicum or Arabidopsis). The transgenic plant may express an expression cassette as described herein. For example, the expression cassette may comprise a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong plant-specific promoter. In other embodiments, the transgenic plant may overexpress an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong plant-specific promoter.

[0017] In some embodiments, the present invention comprises a transgenic plant comprising an expression cassette. In some embodiments, the expression cassette (e.g., the first expression cassette) comprises a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a plant-specific promoter and a selectable marker operatively linked to a plant-specific promoter. In other embodiments, the expression cassette (e.g., the first expression cassette) comprises a gene for Lorelei-like genes (LLG) and a selectable, both operatively linked to aplant-specific promoter and a reporter operatively linked to a plant-specific promoter. In other embodiments, the present invention comprises a transgenic plant comprising a first expression cassette and a second expression cassette. In some embodiments, the first expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a plant-specific promoter and the second expression cassette comprises a selectable marker operatively linked to a plant-specific promoter. In other embodiments, the first expression cassette comprises a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a plant-specific promoter, and the second expression cassette comprises a reporter operatively linked to a plant-specific promoter.

[0018] In some embodiments, the present invention features a stress-tolerant transgenic plant (e.g., Solanum lycopersicum or Arabidopsis) that expresses an expression cassette comprising a gene for Lorelei-like genes (LLG), a reporter, and a selectable marker, all three operatively linked to a strong plant-specific promoter (e.g., a strong pollen-specific promoter or a strong root hair cell-specific promoter or shoot-specific promoter). In some embodiments, the selectable marker is also present in the plasmid carrying the LLG and the reporter, and is expressed from its own promoter.

[0019] The transgenic plant may express an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong plant-specific promoter. In some embodiments, the strong-plant specific promoter is selected from a group consisting of a stem-specific promoter, leaf-specific promoter, seed-specific promoter, root-specific promoter, shoot-specific promoter, flower-specific promoter, fruit-specific promoter, root hair cell-specific promoter, or pollen-specific promoter.

[0020] In some embodiments, the transgenic plant is heat-tolerant compared to a wild-type plant. In some embodiments, the transgenic plant reduces cell bursting compared to a wild-type plant. In some embodiments, the transgenic plant reduces premature pollen tube bursting compared to a wild-type plant. In some embodiments, the transgenic plant increases seed production compared to a wild-type plant. In some embodiments, the transgenic plant is salt-tolerant compared to a wild-type plant. In some embodiments, the transgenic plant is more tolerant to biotic stress compared to a wild-type plant, wherein biotic stress comprises disease-causing fungi, bacteria, or other microorganisms. In some embodiments, the transgenic plant is more resistant to at least one of disease-causing fungi, bacteria, or other microorganisms.

[0021] In further embodiments, the present invention features a method of producing a stress-tolerant transgenic plant. In some embodiments, the method comprises introducing anexpression cassette as described herein into a plant cell and regenerating the plant cell to produce a stress-tolerant plant. In some embodiments, the expression cassette comprises a strong plant-specific promoter (e.g., a strong pollen-specific promoter or a strong root hair-specific promoter), a Lorelei-like gene (LLG) operatively linked to the strong plant-specific promoter, and a selectable marker linked to the LLG. In other embodiments, the expression cassette comprises a strong pollen-specific Lorelei-like gene (LLG) operatively linked to the strong pollen-specific promoter, and a selectable marker linked to the LLG. In other embodiments, the expression cassette comprises a strong root hair-specific promoter, a Lorelei-like gene (LLG) and a reporter that are operatively linked to the root hair-specific promoter, and a selectable marker linked to the plasmid carrying the expression cassette, and regenerating the plant cell to produce a stress-tolerant plant. In some embodiments, the transgenic plant is salt-tolerant compared to a wild-type plant.

[0022] In some embodiments, the present invention features a method of producing a transgenic plant, comprising transgenically increasing the expression of a gene comprising a Lorelei-like gene (LLG), wherein LLG expression is increased compared to a wild-type plant. In some embodiments, the transgenic plant is stress-tolerant compared to a wild-type plant. In some embodiments, the transgenic plant is heat-tolerant compared to a wild-type plant. In some embodiments, the transgenic plant is salt-tolerant compared to a wild-type plant.

[0023] One unique and inventive technical feature of the present invention is the overexpression of LLG, e.g., in pollen. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for stress-tolerant (e.g., heat-tolerant) plants. None of the presently known prior references or works have the present invention's unique, inventive technical feature.

[0024] Moreover, the prior references teach away from the present invention. For example, previous research focused on understanding the effects of heat stress on plant reproduction, such as altered flowering times and defects in male and female gametes. Pollen development has been identified as particularly vulnerable to heat stress, and pollen-expressed genes have been identified as targets for alteration under high temperatures. Priming of pollen to heat stress, defined as exposure to mild heat stress followed by a return to normal conditions before exposure to chronic heat stress, has been implicated as a method for heat tolerance in pollen tubes. This invention differs from previous work in that it overexpresses an essential protein in pollen tube cell wall integrity and exposes the pollen to acute heat stress for a short duration (12 hours) upon pollination.

[0025] Furthermore, the inventive technical features of the present invention contributed to asurprising result. For example, while the transgenic pollen overcame the negative effects of transient heat stress, there was no guarantee that this advantage would translate to improved seed set and better performance compared to non-transgenic pollen, as the transiently heat-stressed plants (exposed to heat stress for only 12 hours) were returned to optimal temperatures until they set seed. Instead, the Inventors found that the advantages accrued from the better performance of transgenic pollen continued to be beneficial up until seed formation and development. This demonstrated that the better performance of transgenic pollen, even for a short duration, is consequential for seed yield. Additionally, not only were pollen tubes homozygous for the transgene able to travel farther down the pistil, but a higher percentage were able to successfully enter the ovules under heat stress compared to non-transgenic pollen (WT, 6%; Transgenic lines, ~20% of ovules received a pollen tube). Additionally, there was a slight increase in the number of seeds created with pollen heterozygous for the transgene compared to wild-type pollen alone under heat stress (WT, 22.5 seeds / silique; transgenic lines, 25.8 seeds / silique).

[0026] Any feature or combination of features described herein are included within the scope of the present invention, provided that the features included in any such combination are not mutually inconsistent, as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are evident in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0027] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings, in which:

[0028] FIG. 1A and 1 B show a non-limiting example of an LLG2 / 3 construct that may be transformed into any plant but was analyzed first for its effects in wild-type Arabidopsis. pLAT52. strong pollen-specific promoter from tomato; functional in Arabidopsis pollen; SP: Signal peptide from tomato LLG2 / 3; cYFP citrine YFP that is stable and functional in acidic apoplast where LLG2 / 3 localizes; SoLy LLG2 / 3: Tomato has only one LLG that is expressed in pollen and pollen tubes; Hygro R: resistance to hygromycin for identifying transformation events with the transgene in plants; Spec R: resistance to spectinomycin for selecting plasmid DNA transformation into E. coli and Agrobacterium tumifaciens. Additionally, FIG. 1A shows the ectopic expression of the Soly LLG2 / 3 construct in wild-type Arabidopsis pollen, with the expression of cYFP reporter in pollen. The endogenous Arabidopsis LLG2 and LLG3 remain functional in these transgenic pollen tubes, as the construct was introduced into wild-type plants. Thus, homozygous and heterozygous individuals were identified in the three independentlytransformed lines

[0029] FIG. 2 shows the expression of Soly LLG2 / 3 in Arabidopsis pollen (T2 plants).

[0030] FIG. 3 shows a PCR assay, which shows that Line #14 is homozygous for both Arabidopsis Ilg2 and Ilg3 mutations when carrying the pLAT52:YFP-SoLy LLG2 / 3 transgene. Only when functional can the transgene rescue the phenotype and produce this double homozygous mutant individual.

[0031] FIG. 4 shows an illustrative example of the experimental design demonstrating the application of heat stress for 12 hours during pollen tube growth. Stage 12B buds were emasculated on female parents and allowed to mature for 24 hours before pollination with A. thaliana pollen. Immediately after pollination, plants were exposed to either control (21 °C) or heat stress (35°C, 36°C, or 37°C) conditions for 12 hours. After the 12-hour treatment, pistils were either harvested and fixed for aniline blue staining or the plants were returned to control conditions to assess seed set and yield.

[0032] FIG. 5 shows that 36°C is detrimental to wild-type (WT) Arabidopsis pollen tube growth. Note: PT front: the furthest growth by a bulk of pollen tubes; disregarding individual tubes that escape from the front.

[0033] FIG. 6 shows that the overexpression of tomato LLG2 / 3 is not detrimental to Arabidopsis pollen tube growth at normal, non-heat stress conditions (21 °C).

[0034] FIG. 7 shows that the overexpression of tomato LLG2 / 3 can overcome Arabidopsis pollen tube growth inhibition under heat stress. Arabidopsis pollen tubes carrying the tomato LLG2 / 3 construct grow significantly better than wild-type ones under heat stress.

[0035] FIG. 8 shows that overexpression of tomato LLG2 / 3 in wild-type Arabidopsis pollen makes it sire more seeds under heat stress than the non-transgenic wild-type pollen. Upon heat stress, in each heterozygous independent transformant, more seeds were resistant to hygromycin, indicating that transgenic pollen performed better than the non-transgenic pollen. Pollen tube growth of transgenic pollen confers an advantage in producing more seeds under heat stress.

[0036] FIG. 9 shows a genetic transmission analysis of pLAT52:SolyLLG2 / 3. Data analysis was performed using a binomial test, *p<0.05. These results confirm enhanced transmission of transgenic pollen expressing SolyLLG2 / 3 from pLAT52, a pollen-specific promoter, under heat stress.

[0037] FIG. 10 shows transmission of pLAT52:SolyLLG2 / 3- ND. Data analysis was performed using a binomial test, *p<0.05. Overexpression of non-functional tomato LLG2 / 3 is used to test if the transgene has any effect on thermotolerance of pollen tubes. Tomato LLG2 / 3 was mutated at two critical amino acids (ND), highly conserved among all land plant LLGs. Use of a non-functional SolyLLG2 / 3 mutant (with conserved residues mutated) shows that thenon-functional SolyLLG2 / 3 mutant does not confer thermotolerance, confirming that functional protein is required for increased thermotolerance, demonstrating the existence of a structure-function relationship, and identifying critical amino acid residues that cannot be mutated while maintaining increased thermotolerance.

[0038] FIG. 11A-11C shows that overexpression of exogenous Arabidopsis LLG3 genes confers thermotolerance. FIG. 11 A shows that overexpression of Arabidopsis LLG3 (AtLLG3) using the same pollen-specific LAT52 promoter also confers thermotolerance— in the Arabidopsis wild-type background that already has endogenous LLG2 and LLG3 expression. Data analysis was performed using the Kruskal-Wallis Test, ns> 0.05 *p<0.05, **p<0.01. This data suggests that increasing total LLG expression levels, regardless of the species origin of the LLG gene, can confer thermotolerance in pollen tubes. Furthermore, these results suggest that functional redundancy or dose sensitivity plays a key role in pollen tube thermotolerance, suggesting the mechanism may rely on LLG dosage, rather than, or in addition to, LLG identity. Therefore, methods of overexpressing any LLG gene, including endogenous or heterologous LLGs, including as under the control of pollen-specific promoters in wild-type genetic backgrounds, may increase thermotolerance in pollen tubes. FIG. 11 B and FIG. 11C show experimental data obtained regarding the ratio of the length of the pollen tube to the length of the pistil, as depicted in FIG. 11A.

[0039] FIG. 12A-12C shows that three LLG copies (e.g., endogenous + transgene) are more effective in conferring thermotolerance than one or two. Lines overexpressing tomato pollen-specific LLG2 / LLG3 and AtLLG3 in an Arabidopsis wild-type background outperformed non-transgenic wild-type pollen, while lines expressing only one or two LLGs did not show statistically significant improvement. This suggests a threshold or additive model of LLG-mediated thermotolerance, suggesting that dosage-based genetic engineering strategies may be effective, because the degree of thermotolerance correlates with the number of functional LLG copies expressed in pollen tubes, supporting an additive model of gene function.

[0040] FIG. 13A-13C show that higher temperatures during in vivo pollination cause progressive early termination of pollen tube growth. FIG. 13A shows micrographs of aniline blue-stained pistils under control (21 °C) and heat stress conditions (35°C, 36°C, 37°C) with the pollen tube front in each pistil (the point where the bulk of the pollen tube tips were detected) indicated by an arrow. FIG. 13B shows that pollen tube growth was quantified by measuring the length of the pollen tube front relative to the pistil length, starting from the style. FIG. 13C shows that heat stress significantly reduces pollen tube growth; each data point represents an individual pistil, with total pistil number indicated (n). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction, ns> 0.05, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001.

[0041] FIG. 14A-14B show that pollen tube reception is impaired after 12 hours of transient heat stress. FIG. 14A shows that pollen tube reception in the aniline blue assay is defined as the termination of pollen tube growth upon entry into the ovule through the micropyle, reaching the synergid cells for burst and fertilization (indicated by the white arrow). FIG. 14B shows pollen tube (PT) reception is measured as the percentage of ovules that successfully received a pollen tube within a pistil, with each pistil represented as a data point, and n = the total number of pistils measured. Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction, ns> 0.05, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 .

[0042] FIG. 15 shows that the seed set is significantly reduced after exposure to 12 hours of transient heat stress. Pistils were hand-pollinated before exposure to control (21 °C) or heat stress (35°C, 36°C, 37°C) conditions for 12 hours. After 12 hours, plants were returned to control conditions to allow seed set. Approximately 2 weeks after pollination, siliques were collected, and the number of seeds were counted per silique (n). One-Way ANOVA ns> 0.05, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 .DETAILED DESCRIPTION OF THE INVENTION

[0043] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein is related to compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0044] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional techniques well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., ColdSpring Harbor Press, 2001 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, the disclosures of which are incorporated in their entirety by reference herein.

[0045] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0046] The term “vector or construct” may refer to any nucleic acid that acts as a carrier for other (e.g., foreign) nucleic acid sequences that are not native to the vector. When introduced into an appropriate host cell, a vector may replicate itself (and, thereby, the foreign nucleic acid sequence) or express at least a portion of the foreign nucleic acid sequence. In one context, a vector is a linear or circular nucleic acid into which a nucleic acid sequence of interest is introduced (for example, cloned) for replication (e.g., production) and / or manipulation using standard recombinant nucleic acid techniques (e.g., restriction digestion). A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Typical vectors include, for example, plasmids, cosmids, phage, phagemids, artificial chromosomes (e.g., BAC, PAC, HAC, YAC), and hybrids that incorporate features of more than one of these types of vectors. Typically, a vector includes one or more unique restriction sites (and, in some cases, a multi-cloning site) to facilitate the insertion of a target nucleic acid sequence.

[0047] As used herein, the term “strong promoter” refers to a promoter that yields more RNA and thus more protein than a “weak promoter.” Furthermore, a strong promoter initiates transcription of RNA (and thus subsequent production of protein) more frequently and / or efficiently than a weak promoter, while a weak promoter conversely only initiates transcription of RNA (and thus subsequent production of protein) comparatively less frequently and / or less efficiently as compared to a strong promoter. Further still, a strong promoter binds RNA polymerase and transcription factors with higher affinity than a weak promoter, while conversely, a weak promoter binds RNA polymerase and transcription factors with a lower affinity than a strong promoter. A strong promoter exhibits a sequence close to the consensus sequence for iothe organism to which it belongs, while a weak promoter has greater deviation from the consensus sequence than a strong promoter does. Cumulatively, this results in a strong promoter having a greater transcription rate and, thus, a greater level of gene expression than a weak promoter. A strong promoter may be differentiated from a weak promoter through various methods. For example, a strong promoter linked to a fluorescent protein, e.g., cYFP, can be easily seen under a fluorescent microscope, whereas a weak promoter linked in the same way cannot be so easily visualized. Further, a strong promoter in this situation would produce a higher level of fluorescence than a weak promoter. Similarly, a quantitative PCR assay can be used to compare the amount of mRNA expression, wherein strong promoters would produce more mRNA than weak promoters. Similarly, Western Blotting could be used to compare the amount of protein expression, wherein strong promoters would produce more protein than weak promoters. In some embodiments, a strong versus weak promoter may be differentiated via use of use of Relative Promoter Units (RPUs), which can be calculated by dividing the activity of the promoter in question (the dividend) by an activity level of a standard reference promoter (the divisor). In some embodiments, a strong promoter may be defined as a promoter with an RPU value of >1. In some embodiments, a moderate promoter may be defined as a promoter with an RPU value of ~0.3-1.0. In some embodiments, a weak promoter may be defined as a promoter with an RPU value of <0.3.

[0048] The term “transgenic plant” may refer to any plant whose DNA has been modified using genetic engineering techniques, well known in the art, to introduce a new trait to the plant that does not occur naturally in the species.

[0049] Referring now to FIGs. 1A-15, the present invention features systems, compositions, and methods that enable the generation of heat-tolerant pollen, ultimately resulting in increased seed production.

[0050] The present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong plant-specific promoter. In some embodiments, the plant-specific promoter is a strong pollen-specific promoter. In other embodiments, the strong plant-specific promoter is a strong root hair cell-specific promoter or shoot-specific promoter. In some embodiments, the plant-specific promoter is a stem-specific promoter (e.g., a strong stem-specific promoter). In some embodiments, the plant-specific promoter is a leaf-specific promoter (e.g., a strong leaf-specific promoter). In some embodiments, the plant-specific promoter is a seed-specific promoter (e.g., a strong seed-specific promoter). In some embodiments, the plant-specific promoter is a root-specific promoter (e.g., a strong root-specific promoter). In some embodiments, the plant-specific promoter is a flower-specific promoter (e.g., a strong flower-specific promoter). In some iiembodiments, the plant-specific promoter is a shoot-specific promoter (e.g., a strong shoot-specific promoter). In some embodiments, the plant-specific promoter is a fruit-specific promoter (a strong fruit-specific promoter). The present invention is not limited to the aforementioned promoters and may utilize any plant-specific promoter known in the art, such as a plant-specific promoter capable of driving expression in a particular portion of a plant or throughout the whole plant.

[0051] In some embodiments, the present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong pollen-specific promoter. In some embodiments, the present invention features an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong root hair-specific promoter.

[0052] The present invention may feature an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, with both operatively linked to a strong plant-specific promoter. In some embodiments, the expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, with both operatively linked to a strong pollen-specific promoter. In some embodiments, the expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, with both operatively linked to a strong root hair-specific promoter. In some embodiments, the expression cassette is an overexpression cassette. In some embodiments, a selectable marker is also present in the expression cassette (e.g, plasmid) carrying the LLG and the reporter, and is expressed from its own promoter.

[0053] In some embodiments, the selectable marker is on a separate expression cassette. In embodiments where the selectable marker is on a separate expression cassette, that selectable marker is operatively connected to a promoter on that same expression cassette. In some embodiments, the reporter is on a separate expression cassette. In embodiments where the reporter is on a separate expression cassette, that reporter is operatively connected to a promoter on that same expression cassette.

[0054] In some embodiments, the present invention comprises a transgenic plant comprising an expression cassette. In some embodiments, the present invention comprises a transgenic plant comprising a first expression cassette and a second expression cassette. In some embodiments, the first expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a plant-specific promoter and a selectable marker operatively linked to a plant-specific promoter. In some embodiments, the first expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a plant-specific promoter. In some embodiments, the second expression cassette comprises aselectable marker operatively linked to a promoter on that same expression cassette. In some embodiments, the second expression cassette comprises a reporter operatively linked to a promoter on that same expression cassette.

[0055] In some embodiments, the strong-plant specific promoter is selected from a group consisting of a stem-specific promoter, leaf-specific promoter, seed-specific promoter, root-specific promoter, shoot-specific promoter, flower-specific promoter, fruit-specific promoter, root hair cell-specific promoter, and pollen-specific promoter. In some embodiments, the strong plant-specific promoter is selected from a promoter specific to another part of the plant.

[0056] In some embodiments, the pollen-specific promoter comprises the promoter of the LAT52 gene from Solanum lycopersicum. In some embodiments, the pollen-specific promoter comprises the promoter of the Zm13 gene from Zea mays. In some embodiments, the pollen-specific promoter comprises the promoter of the OsGEX2 I OsG6B gene from Oryza sativa. In some embodiments, the pollen-specific promoter comprises the promoter of the AtEXL4 (or AtEXPA4) gene from Arabidopsis thaliana. In some embodiments, the pollen-specific promoter comprises the promoter of the AtPP2-A1 (i.e., Pollen-specific protein 2) gene from Arabidopsis. In some embodiments, the pollen-specific promoter comprises the promoter of the AGP6 / AGP11 (i.e., Arabinogalactan proteins) gene from Arabidopsis. In some embodiments, the pollen-specific promoter comprises the promoter of the NTP303 gene from Nicotiana tabacum. In some embodiments, the pollen-specific promoter comprises another pollen-specific promoter. In some embodiments, the pollen-specific promoter comprises the promoter of the PsPK1 gene from Petunia. In other embodiments, the promoter may be preferentially, but not exclusively, expressed in pollen, for example, ACT11 (Actin 11), ACA9, DUO1 , and / or G10, and the like. In some embodiments, the promoter comprises another promoter that is preferentially, but not exclusively, expressed in pollen.

[0057] The expression cassette may further comprise a reporter. In some embodiments, the report is a yellow fluorescent protein (YFP), a green fluorescent protein (GFP), or a red fluorescent protein (RFP). In some embodiments, the reporter is a luminescent reporter or a bioluminescent reporter, including but not limited to Luciferase family reporters (e.g., Firefly luciferase (Luc, Flue), Renilla luciferase (Rluc), NanoLuc, Gaussia luciferase (Glue), or the like. In some embodiments, the reporter is an enzymatic reporter, e.g., a colorimetric or chemiluminescent reporter, including but not limited to p-galactosidase (LacZ), Alkaline phosphatase (AP), p-glucuronidase (GUS), and the like. In some embodiments, the reporter is a fluorescent dye and tag system, including but not limited to HaloTag, SNAP-tag, CLIP-tag, FIAsH / ReAsH, and the like. In some embodiments, the reporter is a FRET-Based Reporters (Fluorescence Resonance Energy Transfer), including but not limited to CFP / YFP,mTurquoise2 / mVenus pairs, Cameleon calcium sensor, and the like. In some embodiments, the reporter is a transcriptional reporter using a split system, including but not limited to Split luciferase or split GFP and the like. In some embodiments, the reporter is a Fluorescent Timer and / or photoconvertible protein reporter, including but not limited to fluorescent timers (e.g., DsRed-E5), photoconvertible proteins (e.g., Kaede, Dendra2), and the like. In some embodiments, the reporter is a CRISPR-based reporter, including but not limited to CRISPRainbow, CRISPR-FISH, and the like.

[0058] In some embodiments, the LLG is LLG2 / 3 from Solanum lycopersicum (e.g., tomato). In some embodiments, the LLG is LRE or LLG1 from Arabidopsis. In other embodiments, the LLG is LLG2 from Arabidopsis. In further embodiments, the LLG is LLG3 from Arabidopsis. The present invention is not restricted to the aforementioned LLG and may include any LLG derived from a crop proposed for modification. Thus, in some embodiments, LLG may be derived from any crop undergoing modification at the relevant time. Without wishing to limit the present invention to any theory or mechanism, it is believed that all LLGs are interchangeably functional within their respective domains, and LLGs from other plant species, including but not limited to other species within the Brassicaceae family, can perform the corresponding functions in Arabidopsis.

[0059] In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 70% identical to SEQ ID NO: 1 . In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 75% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 80% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 85% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 90% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 95% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 98% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is about 99% identical to SEQ ID NO: 1. In some embodiments, the gene for LLG comprises a nucleotide sequence that is identical to SEQ ID NO: 1 .

[0060] In some embodiments, the selectable marker comprises a hygromycin resistance gene, a spectinomycin resistance gene, a Basta resistance gene, a kanamycin resistance gene, or a combination thereof. Other selectable markers may be used in accordance with the present invention.

[0061] The present invention may also feature a stress-tolerant transgenic plant (e.g., Solanumlycopersicum or Arabidopsis). In some embodiments, the transgenic plant expresses an expression cassette comprising a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a strong pollen-specific promoter. In accordance with the present invention, any flowering plant (including, but not limited to, rice, maize, cotton, soybean, and the like) can be utilized; the present invention is not limited to Solanum lycopersicum or Arabidopsis. In some embodiments, LLG is overexpressed.

[0062] In some embodiments, the present invention features a transgenic plant comprising an expression cassette as described herein. In some embodiments, the present invention features a stress-tolerant transgenic plant expressing an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong pollen-specific promoter. In some embodiments, the stress-tolerant transgenic plant expresses an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong root hair-specific promoter. In some embodiments, a selectable marker is also present in the plasmid carrying the LLG and the reporter, and is expressed from its own promoter.

[0063] In some embodiments, the present invention comprises a transgenic plant comprising an expression cassette. In some embodiments, the present invention comprises a transgenic plant comprising a first expression cassette and a second expression cassette. In some embodiments, the first expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a plant-specific promoter and the second expression cassette comprises a selectable marker operatively linked to a plant-specific promoter. In other embodiments, the first expression cassette comprises a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a plant-specific promoter, and the second expression cassette comprises a reporter operatively linked to a plant-specific promoter.

[0064] The transgenic plant may be heat-tolerant. In some embodiments, the transgenic plant is Solanum lycopersicum. In other embodiments, the transgenic plant is Arabidopsis. In accordance with the present invention, any flowering plant (including, but not limited to, rice, maize, cotton, soybean, and the like) can be utilized; the present invention is not limited to Solanum lycopersicum or Arabidopsis. In some embodiments, LLG is overexpressed.

[0065] In some embodiments, the transgenic plant is heat-tolerant compared to a wild-type plant. In other embodiments, the heat-tolerant transgenic plant reduces pollen tube bursting compared to a wild-type plant. In further embodiments, the heat-tolerant transgenic plant increases seed production compared to a wild-type plant.

[0066] In some embodiments, the present invention features a salt-tolerant transgenic plantcomprising an expression cassette as described herein. In some embodiments, the transgenic plant is salt-tolerant compared to a wild-type plant. In some embodiments, salt stress tolerance may be achieved via transgenic modification of the plant using a root hair cell-specific promoter (e.g., a strong root hair cell-specific). In some embodiments, protein products produced by the LLG gene may modulate the FERONIA receptor, thereby increasing salt tolerance.

[0067] In other embodiments, the present invention features a transgenic plant that is tolerant to biotic stress. In some embodiments, biotic stress tolerance may be achieved via transgenic modification of the plant using a strong root hair cell-specific promoter or a shoot-specific promoter. In some embodiments, biotic stress tolerance may be achieved via modulation of the FERONIA receptor, including but not limited to modulation of the FERONIA receptor using LLG as a ligand. In some embodiments, protein products produced by the LLG gene may modulate the FERONIA receptor, thereby increasing biotic stress tolerance. In some embodiments, the transgenic plant is tolerant and / or resistant to disease-causing fungi, bacteria, and / or other microorganisms.

[0068] In some embodiments, the expression cassettes described herein may be used to determine the number of copies of LLG within transgenic plants. For example, the cYFP expressed in the cassette in mature pollen grains of transgenic plants can be observed with microscopy and is notably brighter compared to the autofluorescence of non-transgenic, wild-type Arabidopsis pollen. Therefore, the number of pollen grains expressing YFP can be counted and compared to the number of non-fluorescent pollen grains in the same sample, if present. The difference in levels of fluorescence can also be quantified using imaging algorithms to distinguish stronger from weaker expression. This assessment can indicate a single insertion in a locus (50% YFP positive) or two insertions in a locus (83% or above), or three insertions in a locus (90% or above).

[0069] The present invention may also feature a method of producing a stress-tolerant transgenic plant. In some embodiments, the method comprises introducing an expression cassette as described herein into a plant cell and regenerating the plant cell to produce a stress-tolerant plant.

[0070] In some embodiments, the method of producing a stress-tolerant transgenic plant comprises introducing an expression cassette into a plant cell, the expression cassette comprising a strong plant-specific promoter, a Lorelei-like gene (LLG) operatively linked to the strong plant-specific promoter, and a reporter linked to the LLG, and regenerating the plant cell to produce a stress-tolerant plant. In some embodiments, the strong plant-specific promoter is a pollen-specific promoter. In some embodiments, the strong plant-specific promoter is a strong root hair-specific promoter. The selectable marker is also present in the plasmid carrying theLLG and the reporter, and is expressed from its own promoter.

[0071] In some embodiments, the selectable marker is also present on the plasmid or expression cassette carrying the LLG and the reporter, and is expressed from its own promoter. For example, in some embodiments, the expression cassette comprises a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a plant-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

[0072] In certain embodiments, the selectable marker is on a separate expression cassette (e.g., a second expression cassette). In embodiments where the selectable marker is on a separate expression cassette, the selectable marker is operatively connected to a promoter on that same expression cassette (e.g., the selectable marker is operatively connected to a promoter on the second expression cassette).

[0073] In other embodiments, the reporter is also present on the plasmid or expression cassette carrying the LLG and the selectable marker, and is expressed from its own promoter. For example, in some embodiments, the expression cassette comprises a gene for Lorelei-like genes (LLG) and a selectable marker, both operatively linked to a plant-specific promoter and a reporter operatively linked to a plant-specific promoter.

[0074] In some embodiments, the reporter is on a separate expression cassette (e.g., a second expression cassette). In embodiments where the reporter is on a separate expression cassette, the reporter is operatively connected to a promoter on that same expression cassette (e.g., the reporter is operatively connected to a promoter on the second expression cassette).

[0075] In some embodiments, the present invention features a method of quantifying pollen tube growth in a pistil of a plant, comprising emasculating a bud, pollinating the pistil with a transgenic pollen that is homozygous for a transgene of the plant, incubating the plant in a heat stress chamber for about 12 hours, fixing and staining the pistil with aniline blue, measuring a ratio of a length of the pollen tube to a length of the pistil of the plant, wherein ratios closer to one indicate greater pollen tube growth than ratios that are closer to zero, and wherein pollen tubes with ratios closer to one indicate enhanced performance of the pollen tube under the heat stress than pollen tubes with ratios that are closer to zero.

[0076] In some embodiments, the present invention features a method of quantifying seed formation in a plant, comprising emasculating a bud, pollinating a pistil of the plant with a transgenic pollen that is hemizygous for a transgene of the plant to create a pollinated plant, incubating the pollinated plant in either a control chamber kept at about an optimal growth temperature for the pollinated plant for about 12 hours or a heat stress chamber at about a temperature greater than the optimal growth temperature for the pollinated plant for about 12 hours, returning the pollinated plant to a control condition for about 3 weeks or until thepollinated plant sets a seed, harvesting the seed, growing the seed on an antibiotic selection media containing an antibiotic to select for transgenic seeds containing a resistance gene that confers resistance to the antibiotic, counting the number of resistant seeds and susceptible seeds, and performing genetic transmission analysis on the seeds formed after the introduction of a heat stressor by the plant kept in the heat stress chamber, wherein the control condition comprises a temperature that is about an optimal growth temperature for the pollinated plant, and wherein resistant seeds are deemed transgenic and susceptible seeds are deemed non-transgenic.

[0077] EXAMPLE 1

[0078] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0079] Rising global temperatures have a detrimental effect on crop yield by inhibiting the production of seeds and fruits. Essential to this yield is the delivery of sperm cells to ovules by pollen tubes (PT), a process highly vulnerable to heat stress (HS). To determine how various HS conditions impact pollen-pistil interactions, the Inventors have developed an in vivo assay that effectively demonstrates the effects of HS on Arabidopsis PT growth. This assay shows how different stages of PT growth were affected; pollen germination, growth in the transmitting tract, emergence from the transmitting tract, ovule targeting, and seed formation were all susceptible to HS. Furthermore, pollen tube growth ceases progressively earlier in its journey toward the ovules as temperature increases. Thus, the failure of pollen-pistil interactions could be one of the reasons for crop yield reduction under HS.

[0080] Arabidopsis has two Lorelei-like genes (LLG) expressed only in pollen and pollen tubes - LLG2 and LLG3. LLG2 and LLG3 are essential for maintaining pollen tube integrity; loss of both genes results in premature pollen tube bursting. Nearly half of the pollen tubes produced by Ilg2 / llg2, Ilg3 / + heterozygous plants burst and never sire a seed. Because double homozygous mutant seeds were never detected in the progeny, it was inferred that double mutant pollen tubes burst, and consequently, they never sired a seed. Thus, double homozygous mutant individuals are never found in the population.

[0081] Contrastingly, tomato has only one LLG expressed in pollen and pollen tubes, named Lorelei-Like Gene 2 / 3 (LLG2 / 3). Tomato pollen tubes exposed to heat stress also experience premature pollen tube bursting. The question arises whether overexpressing tomato LLG2 / 3 can overcome the pollen tube bursting in tomato. To avoid ectopic effects in other tissues, a DNA construct was generated to overexpress the LLG2 / 3 protein, specifically in pollen and pollen tubes (See FIG. 1A and 1B). The LAT52 promoter was chosen as it is well characterized for itsexpression in pollen and pollen tubes. It can abundantly express reporter genes in many plants, including Arabidopsis and tomato, in these tissues.

[0082] Complementation experiments were conducted to demonstrate the functionality of the DNA construct. The construct was introduced into Arabidopsis plants homozygous for the Ilg2 mutation and heterozygous for the Ilg3 mutation (Jlg2 / llg2 Ilg3 / +). If the tomato LLG2 / 3 gene can effectively replace the function of Arabidopsis LLG3, it should rescue the phenotype of the Arabidopsis Ilg2 / llg2 Ilg3 / + mutants. Successful complementation would prevent the premature bursting of double mutant pollen tubes (Jlg2, Ilg3) , allowing them to produce viable seeds and enabling the detection of double homozygous individuals in subsequent generations. Among 15 T1 transformants generated by introducing the construct into Ilg2 / llg2 Ilg3 / + mutants, one T1 individual was identified that exhibited the Ilg2 / llg2 Ilg3 / llg3 genotype, confirming complementation and demonstrating the functionality of the construct (FIG. 3). Similar results were also obtained when progeny of T2 plant Ilg2 / llg2, Ilg3 / + carrying the transgene was analyzed and many Ilg2 / llg2, Ilg3 / llg3 double mutant homozygous individuals were detected.

[0083] Overexpression of tomato LLG2 / 3 in wild-type Arabidopsis pollen tubes confers heat tolerance to pollen tubes when exposed to 36°C for 12 hours. Wild-type Arabidopsis pollen is negatively impacted by heat stress (36°C). Tomato LLG2 / 3 can be expressed in Arabidopsis pollen tubes. Tomato LLG2 / 3 is functional in Arabidopsis pollen tubes, at least under heat stress (36°C).

[0084] EXAMPLE 2

[0085] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0086] Development of a Quantitative In Vivo Assay to Assess Pollen Tube Thermotolerance Under Transient Heat Stress in Arabidopsis thaliana’. Arabidopsis thaliana (A. thaliana) seeds were germinated on MS plates supplemented with 2% sucrose and stratified for 2 days at 4°C. Following stratification, plates were transferred to a growth chamber at 21 °C under continuous light. After 10-14 days, seedlings were transplanted to soil and grown in a chamber with 16 hours of light at 21°C and 8 hours of dark at 18°C. Plants aged 4-6 weeks post-transplantation were used as female or male parents. For pollinations, stage 12b buds on female plants were emasculated 24 hours prior. On the day of pollination, 1-day old flowers were selected as pollen donors, and pistils were pollinated profusely. Within 5-10 minutes of pollination, plants were transferred to either a heat stress chamber (35°C, 36°C, or 37°C), or a control chamber (21°C) and maintained under these conditions for 12 hours during the day. After 12 hours, pollinated pistils were either harvested and fixed for aniline blue staining to assess pollen tube growth orreturned to control conditions to assess seed set and yield. To measure seed set, plants were kept at control conditions for additional two weeks before siliques were collected and seeds counted. ~80% of A. thaliana ovules receive a pollen tube within 12 hours of pollination. Therefore, the 12-hour time point was selected to assess pollen tube progression, ensuring sufficient growth time through the transmitting tract and entry into the ovule.

[0087] Higher Temperatures During In Vivo Pollination Cause Progressive Early Termination of Pollen Tube Growth'. To assess pollen tube growth of heat-stressed pistils, the aniline blue assay was used to stain the callose in the pollen tubes and visualize their growth, using an Axiovert 100 microscope. Images were captured with Metamorph 7, and the pollen tube front length was measured using Fiji in millimeters relative to the length of the pistil, starting from the style to the end of the ovary (FIG. 13A-13B). To ensure that results reflected true changes in pollen tube growth rather than differences in pistil size, pollen tube length was normalized to pistil length (FIG. 13B). This approach accounted for variation in pistil size across samples, developmental stages, or stress conditions, including potential heat stress-induced dehydration.

[0088] Pollen tubes reached the bottom of the pistil within 12 hours in pistils grown under control conditions and had a pollen tube front measurement relative to the pistil length close to 1 (FIG. 13A and 13C). When exposed to 35°C for 12 hours, pollen tube growth significantly decreased, and on average only managed to grow halfway down the pistil (FIG. 13A and 13C). A further significant decrease in pollen tube growth was seen when pistils were exposed to 36°C, and an even greater decrease was seen at 37°C (FIG. 13A and 13C), where most pollen tubes stopped just after growing through the style region of the pistil, and none of the pistils tested had tubes that grew longer than a quarter down the pistil. Taken together, these results show that increasing the temperature to 35°C and above had severe effects on pollen tube growth in the pistil.

[0089] Although pollen tube growth was reduced under heat stress, this does not necessarily imply that pollen tube reception was also impaired. Some pollen tube growth was still observed within the pistil, raising the possibility that ovules adjacent to these regions could remain receptive and successfully receive pollen tubes. To address this, pollen tube reception was assessed following heat stress. As expected, in the pistils that were pollinated under control conditions, nearly 80% of ovules received a pollen tube (FIG. 14B). However, this percentage decreased significantly to about 25% at 35°C, and to 17% at 36°C (FIG. 14B). The most severe decrease was observed at 37°C, with the successful pollen tube reception decreasing to 0% (FIG. 14B). The combined reduction in pollen tube growth (FIG. 13A-C) and pollen tube reception (FIG. 14A-14B) under heat stress suggests that seed set may be compromised, even after the plants are returned to normal conditions.

[0090] Seed Production is Progressively Reduced or Completely Abolished with Increasing Temperature in A. thaliana Self-crosses: This experiment sought to understand the consequences of transient heat stress: although the stress was applied only during the pollen tube growth phase and heat-stressed plants were returned to normal conditions afterward, it was questioned whether pollen tube growth and fertilization would recover sufficiently to complete seed set. To test this, heat-stressed pollinated pistils were moved back to control conditions and allowed to set seed. After approximately 2 weeks, siliques were collected, and the number of seeds were counted. The number of seeds produced at heat stress temperatures was significantly reduced to about 20 seeds per silique at 35°C, 5 seeds per silique at 36°C, and 0-8 seeds per silique at 37°C (FIG. 15). Heat stress at 37°C had such a severe impact on pollen tube growth that only a single silique produced any seeds (n=1). These results indicate that 12 hours of transient heat stress during the critical pollen tube growth phase could have a detrimental impact on seed set in A. thaliana. Furthermore, these results show that pollen tube growth did not merely pause during transient heat stress exposure and resume growth when returned to normal conditions; instead, pollen tubes in heat-stressed pistils likely stalled or burst and were unable to fertilize the heat-stressed ovules.

[0091] Heat Stress Alters Pistil Length Independently of Pollination Status: In addition to the pollen tube growth inhibition observed in FIG. 13A-13C, it was also noticed that pollinated pistils exposed to heat stress appeared shorter than those under control conditions. To examine this further, pistil lengths were measured in the same pollinated samples shown in FIG. 13A-13. It was found that pistils pollinated at 35°C and 36°C were slightly but significantly shorter than those at 21°C, while pistils at 37°C also showed a decrease in length, though this difference was not statistically significant.

[0092] Two possible explanations for this reduction in pistil length in heat stressed samples were considered. One possibility is that heat stress directly affects pistils through mechanisms such as tissue dehydration, reduced cell expansion, or developmental delays. Alternatively, fertilization itself may normally promote pistil elongation, as developing ovules can trigger silique growth. Since FIG. 14A-14B and FIG. 15 show that heat-stressed pistils have impaired pollen tube reception and reduced seed set, it is possible that the lack of fertilization under heat stress also contributes to reduced pistil elongation.

[0093] To explore whether pistil size changes under heat stress independently of pollination, pistils were emasculated and left unpollinated, then exposed to either control or heat stress conditions for 12 hours — the same duration used in FIG. 13. It was observed that unpollinated pistils subjected to 35°C exhibited a slight but significant reduction in length, while those exposed to 36°C and 37°C showed a decrease in length that was not statistically significantcompared to the 21 °C controls. These results suggest that heat stress alone can contribute to reductions in pistil length and increase variability across samples. These results further support the approach of normalizing pollen tube length to pistil length, as it provides a more accurate and reliable assessment of pollen tube growth under heat stress conditions.

[0094] As used herein, the term “about” refers to plus or minus 10% of the referenced number.

[0095] Although there has been shown as the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made to those within the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only, and the dimensions of the figures do not limit the claims. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” include embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such, the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

WHAT IS CLAIMED IS:1 . An expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong plant-specific promoter.

2. The expression cassette of claim 1 , the strong-plant specific promoter is selected from a group consisting of a stem-specific promoter, leaf-specific promoter, seed-specific promoter, root-specific promoter, shoot-specific promoter, flower-specific promoter, fruit-specific promoter, root hair cell-specific promoter, and pollen-specific promoter.

3. The expression cassette claim 1 , wherein the strong plant-specific promoter is a strong pollen-specific promoter or a strong root hair cell-specific promoter.

4. An expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong pollen-specific promoter.

5. An expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong root hair cell-specific promoter.

6. The expression cassette of any one of claims 1-5 further comprising a selectable marker, wherein the selectable marker is operatively linked to a plant-specific promoter.

7. The expression cassette of any one of claims 1-5, wherein a selectable marker is provided on a separate expression cassette.

8. The expression cassette of claim 6 or claim 7, wherein the selectable marker comprises a hygromycin resistance gene, a spectinomycin resistance gene, or a combination thereof.

9. An expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong plant-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

10. The expression cassette of claim 9, wherein the strong plant-specific promoter is a strong pollen-specific promoter.

11. The expression cassette claim 9, wherein the strong plant-specific promoter is a strong root hair cell-specific promoter.

12. An expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong pollen-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

13. An expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong root hair-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

14. The expression cassette of any one of claims 1-13, wherein the expression cassette is an overexpression cassette.

15. The expression cassette of any one of claims 1-14, wherein the gene for LLG comprisesa nucleotide sequence that is about 70% identical to SEQ ID NO: 1.

16. The expression cassette of any one of claims 1-15, wherein the gene for LLG comprises a nucleotide sequence that is about 75% identical to SEQ ID NO: 1.

17. The expression cassette of any one of claims 1-16, wherein the gene for LLG comprises a nucleotide sequence that is about 80% identical to SEQ ID NO: 1.

18. The expression cassette of any one of claims 1-17, wherein the gene for LLG comprises a nucleotide sequence that is about 85% identical to SEQ ID NO: 1.

19. The expression cassette of any one of claims 1-18, wherein the gene for LLG comprises a nucleotide sequence that is about 90% identical to SEQ ID NO: 1.

20. The expression cassette of any one of claims 1-19, wherein the gene for LLG comprises a nucleotide sequence that is about 95% identical to SEQ ID NO: 1.21 . The expression cassette of any one of claims 1-20, wherein the gene for LLG comprises a nucleotide sequence that is about 98% identical to SEQ ID NO: 1.

22. The expression cassette of any one of claims 1-21 , wherein the gene for LLG comprises a nucleotide sequence that is about 99% identical to SEQ ID NO: 1.

23. The expression cassette of any one of claims 1-22, wherein the gene for LLG comprises a nucleotide sequence that is identical to SEQ ID NO: 1.

24. The expression cassette of any one of claims 1-23, wherein the LLG is LLG2 / 3 from Solanum lycopersicum.

25. The expression cassette of any one of claims 1-24, wherein the strong pollen-specific promoter comprises a promoter of a LAT52 gene from Solanum lycopersicum.

26. The expression cassette of any one of claims 1-25, wherein the reporter comprises a fluorescent reporter, a luminescent reporter, or an enzymatic reporter.

27. A transgenic plant comprising an expression cassette of any of claims 1 -26.

28. The transgenic plant of claim 27, wherein the plant is Solanum lycopersicum or Arabidopsis.

29. A stress-tolerant transgenic plant, the transgenic plant expressing an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong plant-specific promoter.

30. The transgenic plant of claim 29, wherein the strong-plant specific promoter is selected from a group consisting of a stem-specific promoter, leaf-specific promoter, seed-specific promoter, root-specific promoter, shoot-specific promoter, flower-specific promoter, fruit-specific promoter, root hair cell-specific promoter, and pollen-specific promoter.

31. The transgenic plant of claim 29 or claim 30, wherein the strong plant-specific promoter is a strong pollen-specific promoter or a strong root hair cell-specific promoter.

32. A stress-tolerant transgenic plant, the transgenic plant expressing an expressioncassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong pollen-specific promoter.

33. A stress-tolerant transgenic plant, the transgenic plant expressing an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong root hair-specific promoter.

34. The transgenic plant of any one of claims 29-33, wherein the expression cassette further comprises a selectable marker, wherein the selectable marker is operatively linked to a plant-specific promoter.

35. The transgenic plant of any one of claims 29-33 further comprising a second expression cassette comprising a selectable marker operatively linked to a plant-specific promoter.

36. The transgenic plant of claim 34 or claim 35, wherein the selectable marker comprises a hygromycin resistance gene, a spectinomycin resistance gene, or a combination thereof.

37. A stress-tolerant transgenic plant, the transgenic plant expressing an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong plant-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

38. The transgenic plant of claim 37, wherein the strong plant-specific promoter is a strong pollen-specific promoter.

39. The transgenic plant of claim 37, wherein the strong plant-specific promoter is a strong root hair cell-specific promoter.

40. A stress-tolerant transgenic plant, the transgenic plant expressing an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong pollen-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

41. A stress-tolerant transgenic plant, the transgenic plant expressing an expression cassette comprising a gene for Lorelei-like genes (LLG) and a reporter, both operatively linked to a strong root hair-specific promoter and a selectable marker operatively linked to a plant-specific promoter.

42. The transgenic plant of any one of claims 29-41 , wherein the expression cassette is an overexpression cassette.

43. The transgenic plant of any one of claims 29-42, wherein the gene for LLG comprises a nucleotide sequence that is about 70% identical to SEQ ID NO: 1.

44. The transgenic plant of any one of claims 29-43, wherein the gene for LLG comprises a nucleotide sequence that is about 75% identical to SEQ ID NO: 1.

45. The transgenic plant of any one of claims 29-44, wherein the gene for LLG comprises a nucleotide sequence that is about 80% identical to SEQ ID NO: 1.

46. The transgenic plant of any one of claims 29-45, wherein the gene for LLG comprises a nucleotide sequence that is about 85% identical to SEQ ID NO: 1.

47. The transgenic plant of any one of claims 29-46, wherein the gene for LLG comprises a nucleotide sequence that is about 90% identical to SEQ ID NO: 1.

48. The transgenic plant of any one of claims 29-47, wherein the gene for LLG comprises a nucleotide sequence that is about 95% identical to SEQ ID NO: 1.

49. The transgenic plant of any one of claims 29-48, wherein the gene for LLG comprises a nucleotide sequence that is about 98% identical to SEQ ID NO: 1.

50. The transgenic plant of any one of claims 29-49, wherein the gene for LLG comprises a nucleotide sequence that is about 99% identical to SEQ ID NO: 1.51 . The transgenic plant of any one of claims 29-50, wherein the gene for LLG comprises a nucleotide sequence that is identical to SEQ ID NO: 1.

52. The transgenic plant of any one of claims 29-51 , wherein the LLG is LLG2 / 3 from Solanum lycopersicum.

53. The transgenic plant of any one of claims 29-52, wherein the strong pollen-specific promoter comprises a promoter of a LAT52 gene from Solanum lycopersicum.

54. The transgenic plant of any one of claims 29-53, wherein the reporter comprises a fluorescent reporter, a luminescent reporter, or an enzymatic reporter.

55. The transgenic plant of any one of claims 29-54, wherein the plant is Solanum lycopersicum or Arabidopsis.

56. The transgenic plant of any one of claims 29-55, wherein the transgenic plant is heat-tolerant compared to a wild-type plant.

57. The transgenic plant of any one of claims 29-56, wherein the transgenic plant reduces cell bursting compared to a wild-type plant.

58. The transgenic plant of any one of claims 29-57, wherein the transgenic plant reduces premature pollen tube bursting compared to a wild-type plant.

59. The transgenic plant of any one of claims 29-58, wherein the transgenic plant increases seed production compared to a wild-type plant.

60. The transgenic plant of any one of claims 29-55, wherein the transgenic plant is salt-tolerant compared to a wild-type plant.

61. The transgenic plant of any one of claims 29-55, wherein the transgenic plant is more tolerant to biotic stress compared to a wild-type plant, wherein biotic stress comprises disease-causing fungi, bacteria, or other microorganisms.

62. The transgenic plant of any one of claims 29-55, wherein the transgenic plant is more resistant to at least one of disease-causing fungi, bacteria, or other microorganisms.

63. A method of producing a stress-tolerant transgenic plant, the method comprising: a) introducing an expression cassette according to any one of claims 1-26 into a plant cell; and b) regenerating the plant cell to produce a stress-tolerant plant.

64. A method of producing a stress-tolerant transgenic plant, the method comprising: a) introducing an expression cassette into a plant cell, the expression cassette comprising: i) a strong plant-specific promoter; ii) a Lorelei-like gene (LLG) operatively linked to the strong plant-specific promoter; and iii) a reporter linked to the LLG; and b) regenerating the plant cell to produce a stress-tolerant plant.

65. The method of claim 64, wherein the strong plant-specific promoter is a pollen-specific promoter.

66. The method of claim 64, wherein the strong plant-specific promoter is a strong root hair-specific promoter.

67. A method of producing a stress-tolerant transgenic plant, the method comprising: a) introducing an expression cassette into a plant cell, the expression cassette comprising: i) a strong pollen-specific promoter; ii) a Lorelei-like gene (LLG) operatively linked to the pollen-specific promoter; and iii) a reporter linked to the LLG; and b) regenerating the plant cell to produce a stress-tolerant plant.

68. A method of producing a stress-tolerant transgenic plant, the method comprising: a) introducing an expression cassette into a plant cell, the expression cassette comprising: i) a strong root hair-specific promoter; ii) a Lorelei-like gene (LLG) operatively linked to the root hair-specific promoter; and iii) a reporter linked to the LLG; and b) regenerating the plant cell to produce a stress-tolerant plant.

69. The method of any one of claims 64-68, wherein the expression cassette further comprises a selectable marker, wherein the selectable marker is operatively linked to a plant-specific promoter.

70. The method of any one of claims 64-68, further comprising introducing a second expression cassette comprising a selectable marker operatively connected to a plant-specific promoter.71 . A method of producing a stress-tolerant transgenic plant, the method comprising: a) introducing a first and a second expression cassette into a plant cell, wherein the first expression cassette comprises i) a strong plant-specific promoter; ii) a Lorelei-like gene (LLG) operatively linked to the strong plant-specific promoter; and iii) a reporter linked to the LLG and the second expression cassette comprises a selectable marker is operatively linked to a plant-specific promoter; and b) regenerating the plant cell to produce a stress-tolerant plant.

72. The method of any one of claims 63-71 , wherein the transgenic plant is heat-tolerant compared to a wild-type plant.

73. The method of any one of claims 63-71 , wherein the transgenic plant is salt-tolerant compared to a wild-type plant.

74. A method of producing a transgenic plant, comprising transgenically increasing the expression of a gene comprising a Lorelei-like gene (LLG), wherein LLG expression is increased compared to a wild-type plant, and wherein the transgenic plant is stress-tolerant compared to a wild-type plant.

75. A method of quantifying pollen tube growth in a pistil of a plant, comprising emasculating a bud, pollinating the pistil with a transgenic pollen that is homozygous for a transgene of the plant, incubating the plant in a heat stress chamber for about 12 hours, fixing and staining the pistil with aniline blue, measuring a ratio of a length of the pollen tube to a length of the pistil of the plant, wherein ratios closer to one indicate greater pollen tube growth than ratios that are closer to zero, and wherein pollen tubes with ratios closer to one indicate enhanced performance of the pollen tube under the heat stress than pollen tubes with ratios that are closer to zero.

76. A method of quantifying seed formation in a plant, comprising emasculating a bud, pollinating a pistil of the plant with a transgenic pollen that is hemizygous for a transgene of the plant to create a pollinated plant, incubating the pollinated plant in either a control chamber kept at about an optimal growth temperature for the pollinated plant for about 12 hours or a heat stress chamber at about a temperature greater than the optimal growth temperature for the pollinated plant for about 12 hours, returning the pollinated plant to a control condition for about 3 weeks or until the pollinated plant sets a seed, harvesting the seed, growing the seed on an antibiotic selection media containing an antibiotic to select for transgenic seeds containing a resistance gene that confersresistance to the antibiotic, counting the number of resistant seeds and susceptible seeds, and performing genetic transmission analysis on the seeds formed after the introduction of a heat stressor by the plant kept in the heat stress chamber, wherein the control condition comprises a temperature that is about an optimal growth temperature for the pollinated plant, and wherein resistant seeds are deemed transgenic and susceptible seeds are deemed non-transgenic.

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