Composition for regulating epidermal cell transdifferentiation and fruit growth in abscission zone of plant and, comprising MYB74

Abstract

The present invention relates to a technology for promoting plant growth through regulation of gene expression. According to the present invention, food production can be increased by regulating the activity of the MYB74 transcription factor, which transdifferentiates residual cells in an abscission zone into epidermal cells after organ abscission in a plant, thereby forming a protective layer at the abscission site while allowing fruit to continue growing.

Description

Composition for regulating epidermal cell transformation differentiation and fruit growth in plant abscission regions containing MYB74

[0001] The present invention relates to the regulation of plant growth through the regulation of gene expression, and specifically, to a method for promoting the growth of a plant's flower stalk and fruit using a recombinant vector comprising a MYB74 transcription factor or a nucleic acid encoding the same that converts non-epidermal cells in the abscission region into epidermal cells, a composition comprising the recombinant vector for this purpose, and a transgenic plant.

[0002] The present invention was carried out under the support of the Samsung Electronics Foundation for Future Technology, under project number SSTF-BA2101-10 and project number 2021060007, with the research management agency for the above project being the Samsung Electronics Foundation for Future Technology, the research project title being “Study on the Causes and Mechanisms of Novel Formation of Epidermal Cells in Plant Abscess Regions,” the lead institution being the Daegu Gyeongbuk Institute of Science and Technology, and the research period being 2021-06-01 ~ 2026-05-31.

[0003] The present invention was carried out under the support of the Ministry of Science and ICT under project number 1711177335 and project number 2022010288, with the research management agency for the above project being Daegu Gyeongbuk Institute of Science and Technology, the research project title being “Cell Fate Regulation and Re-adjustment”, the lead institution being Daegu Gyeongbuk Institute of Science and Technology, and the research period being 2022-01-01 ~ 2022-12-31.

[0004] The present invention was carried out under the support of the Ministry of Science and ICT under project number 1711184799 and project number 2023030057, the research management agency for the above project is the National Research Foundation of Korea, the research project name is “Research on Deciphering Cellular Precision of Plant Development”, the lead institution is Daegu Gyeongbuk Institute of Science and Technology, and the research period is 2023-03-01 ~ 2024-02-29.

[0005] The present invention was carried out under the support of the Ministry of Science and ICT under project number 2710063648 and project number 2024030029, the research management agency for the above project is the National Research Foundation of Korea, the research project name is “Research on Deciphering Cellular Precision of Plant Development”, the lead institution is Daegu Gyeongbuk Institute of Science and Technology, and the research period is 2024-03-01 ~ 2025-02-28.

[0006] Finally, the present invention was carried out under the support of the Ministry of Science and ICT under project number 1711154181 and project number 2022030117, the research management agency for the said project was the National Research Foundation of Korea, the research project name was “Research on Deciphering Cellular Precision of Plant Development”, the lead institution was Daegu Gyeongbuk Institute of Science and Technology, and the research period was 2022-03-01 ~ 2023-02-28.

[0007] The plant epidermis is a protective barrier that shields internal tissues from the external environment. Plant epidermal cells not only prevent dehydration by blocking indiscriminate water loss to the atmosphere but also act as a barrier protecting the plant body from the invasion of various pathogens and harmful toxic molecules. The key to this barrier function is the waxy cuticle layer formed on the outer surface of epidermal cells, which enables plants to survive and thrive in dry terrestrial environments.

[0008] Furthermore, epidermal cells are not merely passive defense mechanisms; they also serve as channels for transmitting external physical and chemical stimuli to internal tissues and are involved in regulating the differentiation of internal tissues and the overall growth of the plant. For example, epidermal cells located in the apical meristem of plants form organ shapes by regulating cell division patterns in response to mechanical stimuli. Therefore, if defects occur in the differentiation of these epidermal cells or the development of the cuticle layer, the plant becomes vulnerable to external stimuli and experiences problems such as organ deformation or growth inhibition.

[0009] It is generally known that the differentiation of plant epidermal cells is determined only during the embryonic stage. When abscission occurs—the shedding of leaves, flowers, fruits, and seeds—non-epidermal cells within the abscission zone, where the organ was attached but the epidermis is absent, become exposed to the outside. These newly exposed surfaces are called remnants, and they form a protective layer to shield the plant from sudden environmental changes.

[0010] The most common method plants use to protect exposed internal tissues is to block pathogen invasion and dehydration by forming scar tissue through the accumulation of suberin at the wound site and the induction of lignin in cell walls. However, cell walls hardened by lignin lose flexibility, restricting tissue expansion and growth in the affected area; this growth restriction directly leads to reduced productivity, particularly in areas requiring continuous growth for fruit setting, such as flower stalks. Therefore, in these areas, instead of accumulating lignin, the remaining exposed cells undergo differentiation into new epidermal cells to prevent a decline in productivity.

[0011] The inventors revealed that the transcription factor MYB74 plays a role in regulating this transdifferentiation pathway and confirmed that MYB74 downregulates the expression of photosynthesis-related genes in residual cells while simultaneously upregulating the expression of genes essential for cuticle formation. Transdifferentiation of residual cells by MYB74 is a method to maintain growth capacity while protecting plants, and it is expected that regulating the expression and function of MYB74 will increase crop fruit size and improve productivity, thereby contributing to increased food production and food security.

[0012] The present invention relates to a technology for converting and differentiating residual cells into epidermal cells in the abscission zone (AZ) after organ abscission in plants through the MYB74 transcription factor. The vector capable of controlling the expression of the MYB74 transcription factor of the present invention and the composition containing the same can induce the formation of a cuticle layer in the abscission area and significantly increase the growth of the flower stalk and fruit.

[0013]

[0014] Accordingly, the objective of the present invention is to provide a composition for regulating plant growth comprising a MYB74 expression promoter containing the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

[0015] Another object of the present invention is to provide a method for regulating plant growth comprising an increasing step of increasing the expression or activity of MYB74 containing the nucleotide sequence of SEQ ID NO. 1 within a plant body.

[0016] Another aspect of the present invention is to provide a transformed plant using a recombinant vector comprising a nucleic acid encoding a MYB74 transcription factor comprising the nucleotide sequence of SEQ ID NO. 1.

[0017]

[0018] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0019] One aspect of the present invention is a composition for regulating plant growth comprising a MYB74 expression promoter containing the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

[0020] In one embodiment of the present invention, the expression promoter may comprise one or more selected from the group consisting of a polynucleotide containing MYB74, a polynucleotide complementary to MYB74, and a vector expressing MYB74.

[0021] In one embodiment of the present invention, the vector may include the nucleotide sequence of SEQ ID NO. 4.

[0022] In one embodiment of the present invention, the vector may be activated by β-estradiol.

[0023] In one embodiment of the present invention, the composition may induce the conversion differentiation of non-epidermal cells into epidermal cells in the absorption zone (AZ).

[0024] In one embodiment of the present invention, the plant may be one or more selected from the group consisting of fruit, flower, leaf, stem, root, and seed.

[0025] In one embodiment of the present invention, the composition may promote the formation of a cuticle on the exposed surface after exfoliation.

[0026] In one embodiment of the present invention, the composition may inhibit the expression of photosynthesis-related genes.

[0027] In one embodiment of the present invention, the photosynthesis-related gene may be one or more selected from the group consisting of Rubisco activase (RCA), Chlorophyll A / B binding protein 1 (CAB1), and Rubisco small subunit 1A (RBCS1A).

[0028] In one embodiment of the present invention, the plant may be one or more selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat, sorghum, Arabidopsis thaliana, napa cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, carrot, ginseng, tobacco, cotton, sesame, sesame, sugarcane, sugar beet, perilla, peanut, rice bran, rapeseed, apple tree, pear tree, jujube tree, peach, grape, citrus fruit, persimmon, plum, apricot, lemon, banana, rose, carnation, chrysanthemum, lily, sunflower, cosmos and tulip, etc.

[0029] In one embodiment of the present invention, the composition may increase the diameter of the flower stalk or the length of the fruit.

[0030] Another aspect of the present invention is a method for regulating plant growth comprising an increasing step of increasing the expression or activity of MYB74 containing the nucleotide sequence of SEQ ID NO. 1 within a plant body.

[0031] In one embodiment of the present invention, the increase step may be by a vector containing the nucleotide sequence of SEQ ID NO. 4.

[0032] The present invention relates to a technology for promoting plant growth through the regulation of gene expression. According to the present invention, by regulating the activity of the MYB74 transcription factor, which converts and differentiates residual cells in the abscission region into epidermal cells after organ abscission of a plant, a protective layer is formed over the abscission region, and at the same time, epidermal cells having a cuticle undergo cell expansion and growth, thereby enabling the growth of the flower stalk and allowing the fruit to grow continuously, thereby increasing food production.

[0033]

[0034] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0035] Figure 1 is a diagram confirming the expression of Exordium-like 1 (EXL1) according to the developmental stage of the flower.

[0036] Figure 2 is a diagram showing the results of classifying the residual cells of the flower stalk into three developmental states.

[0037] Figure 3 is a graph showing the results of the Gene Ontology (GO) richness analysis according to the three differentiation states of the flower.

[0038] Figure 4 is a diagram showing the cell types of longitudinal and transverse sections of the absorption zone (AZ).

[0039] Figure 5 is a diagram showing the changes in expression of Rubisco activase (RCA), Chlorophyll A / B binding protein 1 (CAB1), and Rubisco small subunit 1A (RBCS1A) in the severance region before and after severance.

[0040] Figure 6 is a diagram showing the surface of residual cells at the stage where a new cuticle layer is formed, observed using a scanning electron microscope.

[0041] Figure 7 is a diagram confirming that cuticle development genes (BDG1 and GPAT4) are expressed in the outer remnants of a transgenic plant.

[0042] Figure 8 is a diagram showing the results of comparing the genes expressed in the remaining cells with the MYB74 target gene.

[0043] Figure 9 is a diagram showing that 329 of the genes presumed to be targets of MYB74 are expressed specifically in cell states.

[0044] Figure 10 is a graph showing the expression level of the MYB74 gene according to the abscission stage of the plant.

[0045] Figure 11 is a graph showing the expression levels of photosynthesis-related genes and stress response genes according to the extraction stage.

[0046] Figure 12 is a diagram showing the presence or absence of cuticles when the expression of MYB74 is induced using β-estradiol and when it is not.

[0047] Figure 13 is a diagram confirming whether a cuticle layer is formed according to the developmental stage of a plant containing prMYB74:MYB74-SRDX and a wild type.

[0048] Figure 14 is a diagram showing whether a cuticle layer is formed according to the developmental stage of a plant containing prMYB74:MYB74-SRDX and the wild type through auramine O staining and scanning electron microscopy.

[0049] Figure 15 is a diagram confirming that the expression of MYB74-SRDX inhibits the expression of GPAT4.

[0050] Figure 16 is a figure comparing the degree of ATML1 expression in wild-type and prMYB74:MYB74-SRDX plants.

[0051] Figure 17 is a graph showing that the diameter of the flower stalk decreases when MYB74 expression is inhibited.

[0052] Figure 18 is a graph comparing the fruit lengths of wild-type and MYB74-expression-inhibited plants.

[0053] A composition for regulating plant growth, comprising a MYB74 expression promoter containing the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

[0054] One aspect of the present invention is a composition for regulating plant growth comprising a MYB74 expression promoter containing the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

[0055] The term “expression promoter” as used in this specification refers to a substance that acts directly or indirectly on MYB74 to improve, induce, stimulate, and increase the expression or activity of MYB74.

[0056] In some embodiments of the present invention, the expression promoter may comprise one or more selected from the group consisting of polynucleotides containing MYB74, polynucleotides complementary to MYB74, and vectors expressing MYB74. Any expression promoter capable of increasing the level of MYB74 in a plant may be used without limitation in addition to those described above.

[0057] In the present invention, the mechanism for increasing the expression or activity of MYB74 is not particularly limited as long as it is known in the art, and may, for example, increase gene expression such as transcription or translation, or convert an inactive form into an active form.

[0058] The term “MYB74” as used in this specification refers to a type of MYB transcription factor that controls the gene transcription process within a plant body. It signifies a regulatory protein that directly binds to specific DNA sequences through a conserved MYB domain to activate or inhibit the expression of downstream genes. MYB74 primarily plays a role in regulating the physiological state of plants by recognizing external environmental stimuli, such as changes in salinity and osmotic pressure, and responding to them.

[0059] In some embodiments of the present invention, MYB74 may include a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the nucleotide sequence of SEQ ID NO. 1.

[0060] The terms “vector” or “recombinant vector” as used herein refer to a carrier comprising a polynucleotide sequence encoding a target polypeptide operably linked to an expression control region to enable the expression of the target polypeptide within a host.

[0061] The expression control region of the vector may include a promoter capable of initiating transcription, an operator for regulating transcription, and a sequence for regulating the termination of transcription and translation.

[0062] After being transformed into a host cell, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0063] In some embodiments of the present invention, one of floral dip, leaf disk, vacuum infiltration, co-infiltration, particle bombardment, and electroportation may be used as a method for transforming MYB74 into a host cell. For example, floral dip may be used.

[0064] The flower immersion method is one of the common methods used for plant transformation, utilizing the infection ability of Agrobacterium to deliver a target gene. When the flower part of a plant is immersed in a solution containing surfactants and sugars to induce infection and the plant is grown normally, the seeds acquire foreign genes, and the harvested seeds are grown in a selective medium to obtain a transformed plant.

[0065] The flower immersion method allows for the immediate acquisition of transformed plants using plant seeds, making the transformation process very easy and simple. Furthermore, it enables the simultaneous transformation of a large number of plants without the need for expensive equipment, thereby allowing for the efficient production of transformed plants.

[0066] In some embodiments of the present invention, the vector may be selected from the group consisting of plasmids in a natural or recombinant state, binary vectors for plants, cosmids, viruses, and bacteriophages.

[0067] In some embodiments of the present invention, the phage vector or cosmid vector may be selected from the group consisting of pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A.

[0068] In some embodiments of the present invention, the plasmid vector may be selected from the group consisting of pDZ-based, pDC-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors. For example, it may be pDZ, pDC, pACYC177, pACYC184, pCL, pCL1920, pSHK130, pDCM2, pECCG117, pUC19, pBR322, pMW118, or pCC1BAC.

[0069] In some embodiments of the present invention, the plant binary vector may be selected from the group consisting of pMDC, pCAMBIA, pPZP, pBI, and pGW systems. For example, it may be pMDC7, pMDC32, pMDC43, or pMDC160.

[0070] In some embodiments of the present invention, the vector for the expression of MYB74 may be pMDC7.

[0071] pMDC7 is a binary vector for Agrobacterium-mediated plant transformation that can rapidly introduce target polynucleotides using a gateway cloning system. The pMDC7 vector is equipped with an XVE inducible expression system that responds to β-estradiol, so it can induce transcription of downstream genes only in the presence of β-estradiol, and thus control the expression of target genes at specific times or tissues.

[0072] In some embodiments of the present invention, the vector may additionally include a marker to confirm whether the target gene has been inserted. The marker is intended for selecting transformed cells and may be a marker that confers a phenotype such as drug resistance or resistance to cytotoxic agents.

[0073] In some embodiments of the present invention, the marker may include a reporter marker capable of confirming whether transformation has occurred and the pattern of gene expression through visual methods or optical analysis. The reporter marker may be used to monitor transformed cells in real time by emitting light of a specific wavelength or inducing a developmental response in cells into which the target gene has been inserted, or to isolate specific cell populations using a flow cytometer or the like.

[0074] In some embodiments of the present invention, the reporter marker may be Green Fluorescent Protein (GFP), mCherry, β-glucuronidase (GUS), or luciferase.

[0075] In some embodiments of the present invention, the vector may include the nucleotide sequence of SEQ ID NO. 4.

[0076] In some embodiments of the present invention, the vector may include the nucleotide sequence of SEQ ID NO. 4 or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the nucleotide sequence of SEQ ID NO. 4.

[0077] In some embodiments of the present invention, the composition may induce the conversion differentiation of non-epidermal cells into epidermal cells in the absorption zone (AZ).

[0078] The term “ablation region” in this specification refers to a layer of cells formed at a point where an organ, such as a leaf, flower, fruit, or seed, is separated from the main body of the plant at a specific time or in response to environmental stimuli. The abslation region consists of small, dense cells that are distinct from surrounding normal cells and forms a band-like layer that precisely controls the shedding of the organ during the plant's development.

[0079] The term “residual cells” as used in this specification refers to a group of cells that remain at the boundary of the abscission region after the abscission process is completed and form the outer surface of the plant body. Until the organ is shed, the residual cells exist as part of the plant’s internal tissue, but they may be exposed to the external environment upon the occurrence of abscission.

[0080] As used herein, the term “transdifferentiation” refers to a developmental process in which a cell that has already completed differentiation into a specific function is directly changed into a cell with different traits without passing through an undifferentiated state. In the event of transdifferentiation, the gene expression network of the existing cell is suppressed while the expression program of the new cell is activated, and the cell's structure, metabolism, and physiological functions are completely altered; this contributes to the survival and growth of the individual by rapidly regenerating damaged tissues or responding to sudden environmental changes.

[0081] In the present invention, the transformation differentiation of non-epidermal cells into epidermal cells refers to the process in which non-epidermal residual cells at the abscission site acquire a new identity as epidermal cells. This transformation differentiation results in the formation of a cuticle layer in place of ligninization, thereby reconstructing a flexible epidermal layer that protects the plant from external stimuli while enabling continuous growth of the flower stalk and fruit.

[0082] In particular, transformative differentiation in the receptacle is a critical process that influences fruit growth and final yield. The receptacle serves as the primary pathway for the transport of water and nutrients from the plant body to the fruit, as well as the underlying tissue that physically supports the fruit. If lignin forms after abscission, the overall growth of the receptacle is inhibited, which can limit the supply of water and nutrients to the fruit. However, by inducing transformative differentiation, a flexible epidermal layer can be formed on the surface of the receptacle, ensuring a continuous supply of water and nutrients. Consequently, this leads to a significant increase in fruit size and weight, thereby boosting food production.

[0083] In some embodiments of the present invention, the plant may be one or more selected from the group consisting of fruit, flower, leaf, stem, root, and seed.

[0084] In some embodiments of the present invention, the composition may inhibit the expression of photosynthesis-related genes.

[0085] In some embodiments of the present invention, the photosynthesis-related gene may be one or more selected from the group consisting of Rubisco activase (RCA), Chlorophyll A / B binding protein 1 (CAB1), and Rubisco small subunit 1A (RBCS1A).

[0086] In some embodiments of the present invention, the composition may increase the expression of cuticle development genes.

[0087] In some embodiments of the present invention, the cuticle development gene may be one or more selected from the group consisting of glycerol-3-phosphate acyltransferase 4 (GPAT4), BDG1, LCR, KCS6, CER1, and CER6.

[0088] In some embodiments of the present invention, the composition may increase the expression of genes related to lipid biosynthesis.

[0089] In some embodiments of the present invention, the lipid biosynthesis-related genes may be one or more selected from the group consisting of KCS6, KCS20, GPAT4, FAR3, LCR, and CER5.

[0090] In some embodiments of the present invention, the plant body may be selected from angiosperms including dicotyledonous plants or monocotyledonous plants.

[0091] In some embodiments of the present invention, the plant body may be one or more selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat, sorghum, Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, carrot, ginseng, tobacco, cotton, sesame, sesame, sugarcane, sugar beet, perilla, peanut, rice bran, rapeseed, apple tree, pear tree, jujube tree, peach, grape, citrus fruit, persimmon, plum, apricot, lemon, banana, rose, carnation, chrysanthemum, lily, sunflower, cosmos and tulip, etc.

[0092] The inventors confirmed that when the MYB74 gene is expressed in the abscission site of Arabidopsis thaliana, the remaining cells differentiate into epidermal cells, and consequently, the length of the fruit increases.

[0093] Arabidopsis thaliana is a widely used plant model for studying the genetic and physiological mechanisms of plants, and the transcription factor MYB74 of the present invention is commonly found in various plants as well as Arabidopsis thaliana. Therefore, the conversion differentiation of residual cells into epidermal cells and the resulting growth-promoting effect observed in the abscission site of Arabidopsis thaliana are likely effects that occur throughout the plant body, not just in Arabidopsis thaliana, and the composition of the present invention can be widely applied to various plants, particularly agricultural crops and fruit trees, in addition to Arabidopsis thaliana.

[0094] In some embodiments of the present invention, the composition may increase the diameter of the flower stalk or the length of the fruit.

[0095] Another aspect of the present invention is a method for regulating plant growth comprising an increasing step of increasing the expression or activity of MYB74 containing the nucleotide sequence of SEQ ID NO. 1 within a plant body.

[0096] In some embodiments of the present invention, the increase step may be by a vector containing the nucleotide sequence of SEQ ID NO. 4.

[0097] Another aspect of the present invention is a plant transformed using a recombinant vector comprising a nucleic acid encoding a MYB74 transcription factor containing the nucleotide sequence of SEQ ID NO. 1.

[0098] The plant growth regulating method and the transgenic plant of the present invention are based on a recombinant vector comprising a nucleic acid encoding the MYB74 transcription factor, similar to the plant growth regulating composition described above; details common to the above are omitted to avoid excessive complexity in this specification.

[0099]

[0100] The present invention will be explained in more detail below through the following examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[0101]

[0102] Example 1: Plant materials and growth conditions

[0103] Arabidopsis thaliana was planted in soil in a growth chamber and grown under the following conditions: 22 ± 2℃, light intensity 110 μmol m⁻² -2 s -1, photoperiod 16 hours. In all experiments, seeds were stratified under dark conditions at 4°C for 2 to 5 days. All mutants used in this invention were obtained from the Arabidopsis Biological Resource Center and include ida-12 (SALK_133209), hae-1 (SALK_105975C), hsl2-1 (SALK_057117), myb74 (SM3.16165), myb2 (SALK_045455) and myb102 (SM3.41645).

[0104]

[0105] Example 2: Plasmid construction and production of transgenic plants

[0106] Plasmids were constructed using the Gateway Cloning (Invitrogen) protocol. The constructed plasmids were introduced into plants by the floral dip method, and transgenic plants were selected from a medium containing antibiotics. Specifically, Arabidopsis flower buds were immersed for 2 to 10 seconds in a solution containing 5% (w / v) sucrose, 0.05% (v / v) Silwet L-77, and Agrobacterium tumefaciens, and then kept in the dark overnight under high humidity.

[0107] To evaluate the effect of MYB74-SRDX on marker gene expression, plasmid constructs prATML1:nls-GFP-GUS and prGPAT4:nls-GFP-GUS were introduced into T2 transgenic plants harboring prMYB74:MYB74-SRDX and prUBQ10:XVE>>MYB74-SRDX, respectively. To confirm the co-expression of marker genes, elite lines of prGPAT4 nls-mCherry and prHAE:nls-mCherry were crossed with the prMYB74:nls-GFP-GUS marker line, and the F1 plants were analyzed.

[0108]

[0109] Example 3: Protoplast Preparation and Fluorescence-Activated Cell Classification

[0110] Protoplasts of remnant remnant cells were isolated from the abscission zone (AZ) of Arabidopsis plants containing the prEXL1:nls-GFP plasmid. Specifically, remnants were collected from approximately 520 plants. The collected remnants were cut twice at high speed for 30 seconds each using a Waring WPB05 blender in distilled spirits, and then collected on Kimwipes (KIMTECH) to remove excess water. The cut material was mixed with 0.5% macerozyme R-10, 1.2% cellulose R-10, 400 mM mannitol, 10 mM CaCl2, 4 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.7), 20 mM KCl, 2.5 mM β-mercaptoethanol, 0.1% bovine serum albumin, 40 U ml -1 RNase inhibitor, 10 μg ml -1 After transferring to an enzyme solution containing actinomycin D, the mixture was reacted for 4 hours under dark conditions at room temperature. A vacuum of 100 mmHg was applied for 30 minutes to ensure proper permeation of the enzyme solution, and subsequently, the separated protoplast was cooled and Ca 2+ It was diluted to an equal volume with a wash and culture solution without urea and filtered through a 40 μm cell strainer. The resulting solution was concentrated by centrifuging at 500 g for 5 minutes.

[0111] The precipitated protoplast was washed in a 15 ml tube and resuspended in 5 ml of culture solution, and GFP-positive protoplast was collected using a FACSAris III cell sorter. A 100 μm nozzle was used, and the system pressure was maintained below 20 psi.

[0112]

[0113] Example 4: scRNA-seq

[0114] scRNA-seq libraries were constructed using 10X Genomics' Chromium Single Cell 3′ Library and Gel Bead Kit v2 (PN-120267), Chromium Single Cell A Chip kit (PN-1000009), and Chromium i7 Multiplex Kit (PN-120262). Library preparation was performed according to the manufacturer's instructions specified in CG00052 Chromium Single Cell 3′ Reagent Kits v2 User Guide Rev B.

[0115] First, count the number of protoplasts, then the concentration is 5 x 10 5 Up to 2 x 10 6 cells ml -1 It was adjusted. Approximately 13,000 protoplasts were mixed with reverse transcription master mix to capture 8,000 cells, which were then loaded onto a Single Cell A Chip (PN-1000009). Subsequently, a Single Cell 3′ Gel Bead suspension and separation oil were injected into designated wells. After loading all reagents, the Single Cell A Chip was mounted on a Chromium Controller to generate a Gel Bead-In-Emulsion (GEM).

[0116] The generated GEM was collected in tubes, and a reverse transcription reaction (GEM-RT) was performed using a thermocycler. During GEM-RT, the read 1 primer sequence (R1) for Illumina library sequencing was introduced into the cDNA. After purifying the GEM-RT reaction, the cDNA was amplified using a thermocycler. The amplified cDNA was qualitatively analyzed using an Agilent Bioanalyzer High Sensitivity chip (Agilent).

[0117] Enzymatic cleavage and size selection were performed to optimize the size of the cDNA amplicons. During the library construction phase, end repair, A-tailing, adapter ligation, and sample index PCR were carried out to ensure the inclusion of P5, P7, read 2 primer sequences (R2), and the sample index. Finally, the libraries were sequenced using the 2 × 100 bp paired-end method on the Illumina HiSeq 4000 platform, aiming for an average of 50,000 read pairs per cell.

[0118]

[0119] Example 5: scRNA-seq Data Preprocessing and Quality Control

[0120] After sequencing, raw reads were mapped to the Arabidopsis reference genome using Cell Ranger version 2.1.1 and the Ensembl GTF file (TAIR10.40). Empty droplets were removed using the DropletUtils version 1.14.2 R package based on a false discovery rate (FDR) < 0.05. Cells with fewer than 1,000 unique molecular identifiers (UMIs) or where more than 10% of UMIs mapped to mitochondrial and chloroplast genes were classified as low quality and excluded from subsequent analysis. Raw UMI counts were normalized by cell-specific size correction factors estimated using the scran version 1.22.1 R package with all options set to default values, and then log2 transformed using a pseudo-count of 1. Highly variable genes were defined as genes satisfying the conditions of an FDR of less than 0.1 and biological variability greater than 0.01 using the scran modelGeneVar function. Subsequently, after removing abscission cells, guard cells, and vascular cells, principal component analysis was performed on the scaled expression matrix of highly variable genes.

[0121]

[0122] Example 6: Signature score

[0123] Gene set signature scores were calculated using the AddModuleScore function of the Seurat version 4.1.1 R package. For cells other than remnant cells, signature scores were calculated using standardized marker genes. State-specific MYB74 target gene scores were estimated based on potential MYB74 target genes associated with photosynthesis (GO:0015979), stress response (GO:0006950), and cuticle synthesis (GO:0010143, GO:0160062, GO:0042335, GO:0010025).

[0124]

[0125] Example 7: GO abundance analysis of scRNA-seq data

[0126] For each transition differentiation state, marker genes were identified using the FindAllMarkers function from the Seurat package with default options. Genes with a corrected P-value of less than 0.05 were considered marker genes specific to that state. The GO abundance levels for each state were calculated using the gProfiler2 version 0.2.1 R package on the obtained marker genes, with all options set to their default values.

[0127]

[0128] Example 8: Trajectory inference

[0129] To infer the state transitions of transform differentiation, trajectory inference was performed using the Palantir version 1.0.1 Python package. Diffusion components were calculated using Palantir's run_diffusion_maps function based on the first 10 principal components. Next, a k-nearest neighbor graph with k = 50 was constructed using the first 10 diffusion components. To visualize these results, a 2D ForceAtlas2 (FA) plot was calculated using the tl.draw_graph function from the scanpy version 1.9.1 Python package with all options set to their default values. Pseudotime was inferred using Palantir's core.run_palantir function with the option num_waypoints = 500. The cell with the highest photosynthetic signature score was selected as the starting cell.

[0130]

[0131] Example 9: RNA velocity

[0132] To support the inferred trajectories, RNA velocities were estimated using the Python package scVelo (version 0.2.3). Count matrices for spliced ​​and non-spliced ​​transcripts were generated using the Python package velocyto (version 0.17.17). After removing genes with low expression levels, all matrices were normalized using the pp.filter_and_normalize function and converted to natural logarithms. The options were set to n_top_genes = 500 and min_shared_counts = 5. Moments for velocity estimation were calculated for each cell using the pp.moments function, with the options set to n_neighbors = 30 and n_pcs = 15.

[0133] RNA velocity was estimated using the tl.velocity function with all options set to their default values. Subsequently, based on the estimated velocity, a velocity graph was calculated using the tl.velocity_graph function, also using default options. Finally, the RNA velocity results were visualized on a 2D ForceAtlas2 (FA) plot.

[0134]

[0135] Example 10: Cell-type label transfer

[0136] Cell type label transfer was performed using the Seurat package to predict the identity of residual cells in the flower receptacle. To facilitate label transfer between different datasets, anchors corresponding to mutual nearest neighbors were identified in the reference and query datasets. Anchors serve as reference points that align the datasets and enable the transfer of cell type labels.

[0137] Anchors between the reference scRNA-seq dataset and remnant reproductive cells were identified via canonical correlation analysis (CCA) using Seurat's FindTransferAnchors function. The parameter reduction was set to "cca," while the remaining parameters were kept at their default values. Finally, cell type labels from the reference dataset were transferred to the remnant reproductive cells using the first five canonical correlation vectors.

[0138]

[0139] Example 11: Induction of expression of MYB74 (iMYB74) or MYB74-SRDX (iMYB74-SRDX) in Arabidopsis

[0140] The coding sequences of MYB74 and MYB74-SRDX were cloned into pMDC7, an estradiol-derived binary vector. Subsequently, the pMDC7-MYB74 and pMDC7-MYB74-SRDX plasmids were introduced into Arabidopsis using an Agrobacterium-mediated flower immersion method. To evaluate the effects of iMYB74 on the transcriptome, flower stalks were collected from 5 to 6-week-old iMYB74 plants. The collected flower stalks were transferred to 1 / 2 MS medium supplemented with 10 μM β-estradiol and incubated at 70 rpm for 10 hours. After incubation, total RNA was measured using the RNeasy Mini Kit (Qiagen), and total RNA concentration was measured using a Nanodrop spectrophotometer (Thermo). RNA quality was evaluated using a Bioanalyzer (Agilent Technologies) equipped with an RNA Pico chip. To investigate the effect of iMYB74 or iMYB74-SRDX on the transformative differentiation of remnant cells in the flower stalk, main inflorescence stems were collected from 5-week-old iMYB74 or iMYB74-SRDX plants and placed in 2 ml tubes containing 1 / 2 MS medium with or without 10 μM β-estradiol. After performing this treatment for 1 to 5 days, the flower stalks were observed under a confocal microscope.

[0141]

[0142] Example 12: RNA-seq Data Processing and Analysis

[0143] Paired-end sequencing reads were generated using the Illumina NovaSeq sequencing platform. Prior to subsequent analysis, adapter sequences were removed and low-quality nucleotides were trimmed using Trimmomatic version 0.38. The purified reads were aligned to the Arabidopsis thaliana genome assembly (TAIR10.1) using HISAT version 2.1.0. This process was performed based on the HISAT and Bowtie2 implementations. Reference genome sequences and gene annotation data were downloaded from the NCBI Genome Assembly and NCBI RefSeq databases, respectively. The aligned data were aligned and indexed using SAMtools version 1.9.

[0144] After alignment, transcripts were assembled and quantified using StringTie version 2.1.3b. Finally, quantification was performed at the gene and transcriptome levels to calculate raw read counts, FPKM values, and TPM values.

[0145]

[0146] Example 13: Differential expression gene analysis

[0147] DESeq2 was used for statistical analysis of differential gene expression. Quality control was performed by selecting only genes with non-zero counts from all samples, and multidimensional scaling plots were generated to observe expression similarity between samples. Relative Log Expression (RLE) normalization was applied to the filtered dataset to correct for variation caused by differences in library size between samples.

[0148] Subsequently, the statistical significance of differential gene expression was evaluated using the nbinom Wald Test in DESeq2, and fold change and P-values ​​were extracted from the Wald test results. All P-values ​​were corrected using the Benjamini-Hochberg algorithm to control for false discovery. Genes with an absolute fold change of 2 or greater and a raw P-value less than 0.05 were defined as significant differentially expressed genes. Hierarchical clustering analysis was performed on the significant genes using rlog-transformed values, with the parameters used as follows: distance metric = Euclidean distance, linkage method = complete.

[0149] Next, gene abundance analysis and functional annotation were performed on significant differentially expressed genes using gProfiler, based on the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway databases. The corrected P-values ​​reported in the gProfiler results were derived based on a one-sided elementary distribution test, and the statistical tests were further corrected using the Benjamini-Hochberg method.

[0150]

[0151] Example 14: Scanning Electron Microscopy and Histological Analysis

[0152] Scanning electron microscopy analysis was performed on the abscission zone (AZ) of the flower organ. Prior to fixation, the sepals and petals were removed from the flower. Subsequently, critical point drying was performed using an EM CPD300 dryer (Leica), after which the tissue sample was attached to a steel stub, coated with osmium, and observed using a Hitachi S-4800 scanning electron microscope.

[0153] Histological analysis of the abscission zone was performed using specimens embedded in Technovit 7100 resin. The embedded specimens were cut into 2 μm thick sections using an RM2255 microtome and a TC65 tungsten carbide disposable blade. The sections were stained with 0.1% toluidine blue and observed under a Zeiss axiovert 40 inverted microscope. Additionally, the radius of the receptacle from the vascular bundle to the center of the stem in the abscission zone was measured on the sections using AxioVision software from the acquired images.

[0154]

[0155] Example 15: Gene expression analysis using promoter-GUS reporter

[0156] Promoter-induced β-glucuronidase (GUS) activity was analyzed. Flowers at various developmental stages were collected and cultured in GUS solution at 37°C for 1 to 24 hours, followed by chemical fixation, dehydration using an ethanol concentration gradient, and embedding in Technovit 3040 solution. The embedded samples were sectioned and imaged, and AxioVision software was used for photography.

[0157]

[0158] Example 16: Tissue Clearing and Confocal Laser Scanning Microscopy Observation

[0159] The expression of marker genes was analyzed using nls-GFP or nls-mCherry reporters driven by proprietary promoters. To evaluate expression, flowers of the marker lines were sectioned longitudinally or transversely, fixed with 4% PFA, cleared with ClearSee solution, and counterstained with a cell wall dye (0.01% calcofluor white). After washing, the cleared samples were mounted on slides with ClearSee solution and observed using a Zeiss LSM710 confocal scanning microscope.

[0160]

[0161] Example 17: Light Sheet Microscope Observation

[0162] Lightsheet images were obtained from flowers of the marker lineage. The flowers were first fixed with 4% PFA, cleared in ClearSee solution for 2 weeks, and then counterstained with 0.1% calcofluor white. Next, the cleared flowers were washed with ClearSee solution, the samples were embedded in a glass capillary filled with 1% low-melting point agarose, and imaged using a Lightsheet Z.1 microscope (Carl Zeiss).

[0163]

[0164] Example 18: Analysis of Permeability of the Detachment Region

[0165] The permeability of the abscission zone was evaluated by the degree of penetration of 0.03% toluidine blue. Flowers of wild-type and transgenic plants were collected and simultaneously incubated in a 0.03% toluidine blue solution for 2 to 3 minutes. The stained flowers were washed with water and photographed using a Zeiss V12 microscope; this process was repeated three times. The stained area was measured using ImageJ.

[0166]

[0167] Example 19: Measurement of receptacle width and fruit (silique) length

[0168] To measure the dimensions of the receptacle and fruit, the fruit was aligned using double-sided adhesive tape so that the median nectary faced upward. Images were taken using a Zeiss V12 microscope equipped with an Axiocam 512 color camera, and the width of the receptacle and the length of the fruit were determined from the acquired photographs.

[0169]

[0170] Example 20: Analysis of double luciferase and transient expression in Nicotiana benthamiana

[0171] The GPAT4 promoter sequence and the MYB74 coding sequence were cloned into the pGreenII 0800-LUC Gateway vector and the pEarlyGate100 (pEG100) vector, respectively. The TSK108-derived multiple cloning site inserted into the pEG100 vector was used as an empty vector control. Cultures of Agrobacterium (GV3101) transformed with each of the pGreenII 0800-GPAT4p-LUC (prGPAT4:LUC), pr35S:MYB74, and pr35S:MCS (TSK108 multiple cloning site) plasmids were resuspended in buffer solution. Subsequently, 5-week-old N. benthamiana leaves were infiltrated with a mixture of TF set and GPAT4p-LUC in a 1:9 ratio and P19.

[0172] Three days after infiltration, the leaves were rapidly frozen in liquid nitrogen, ground into a powder using a TissueLyser, and diluted in 100 μl of lysis buffer. Next, the extract was centrifuged at 8,000 g for 1 minute, and 65 μl of the supernatant was taken and analyzed using a Dual-Glo Luciferase Assay System (Promega). The luminescence signal was detected using an Infinite M200 PRO multimode plate reader (Tecan).

[0173]

[0174] Example 21: RT-qPCR Analysis

[0175] Total RNA was extracted from the receptacles using the RNeasy Micro Kit (QIAGEN). Subsequently, cDNA was synthesized using 1 μg of total RNA and qScript cDNA SuperMix (QUANTABIO). Relative expression levels were measured in triplicate, and qBase+ (Biogazelle) was used for data analysis. Expression levels were normalized using UBQ10 and eIF4A as reference genes.

[0176]

[0177] Example 22: Statistical Analysis

[0178] All quantitative data were analyzed using Origin2021 or GraphPad Prism, and graphs were created using Origin2021, GraphPad, or ggplot2 (v. 3.4.3). Statistical significance was determined using Student's t-test or one-way ANOVA, with the significance level set at P < 0.05; individual P-values ​​are presented in the results. Bar graphs represent the mean ± standard deviation, while box plots display the 25th to 75th percentiles, the full data range, and the median. Open or filled points represent individual samples in both bar graphs and box plots. All experiments were repeated independently at least three times, and representative images are presented unless otherwise specified in the figures. Detailed information regarding the number of samples and biological replicates is included in each figure.

[0179] For the analysis of transgenic plants, two to three representatives were selected after confirming that the expression pattern or phenotypic characteristics of the introduced plasmid were consistent. All analyses were performed on the T2 or T3 generation of transgenic plants.

[0180]

[0181] Experimental Example 1: Three states appearing during the process of transformative differentiation of remnant cells in the flower receptacle

[0182] To investigate changes in remnant cells of the flower stalk during the process of transformational differentiation, single-cell RNA sequencing analysis was performed using remnant cell protoplasts obtained from the abscission region of transgenic plants containing prEXL1:nls-GFP. EXORDIUM-LIKE 1 (EXL1) is expressed in cells in the abscission region before and after abscission (see Fig. 1), and thus, information regarding cellular processes before and after abscission can be confirmed through transgenic plants expressing prEXL1:nls-GFP.

[0183] 2,823 remaining cells that passed quality control standards were profiled, and abscission cells, guard cells, and vascular bundle cells were excluded. The 2,823 cells were classified into three developmental states through unsupervised cluster analysis (see Fig. 2).

[0184] To analyze the characteristics changing across three developmental states, label transfer analysis was performed between the scRNA-seq data of the present invention and the publicly available data of Arabidopsis leaves. As a result, it was found that the remaining cells in State 1 mainly possessed the characteristics of mesophyll cells. The remaining cells in State 2 exhibited characteristics observed in four different cell types, including guard cells, mesophyll cells, vascular cells, and epidermal cells, while the majority of the remaining cells in State 3 exhibited characteristics of epidermal cells.

[0185] To analyze the functional characteristics of genes expressed in each state, a Gene Ontology (GO) abundance analysis was performed on state-specific genes (see Fig. 3). The analysis revealed that genes related to photosynthesis were highly expressed in State 1, whereas they were low in States 2 and 3. The longitudinal and transverse section diagrams in Fig. 4 illustrate cell types within the abscission zone; specifically, looking at the longitudinal section, it was observed that the expression of RUBISCO ACTIVASE (RCA), CHLOROPHYLL A / B BINDING PROTEIN 1 (CAB1), and RUBISCO SMALL SUBUNIT 1A (RBCS1A) was detected in all cells within the abscission zone prior to abscission, but disappeared from the outermost cells of the abscission zone after abscission was complete (see Fig. 5). This indicates that non-epidermal remnant cells have acquired epidermal cell-related characteristics.

[0186]

[0187] Experimental Example 2: Remnant cells of the flower stalk upregulating stress response genes

[0188] To visualize epidermal formation, the inventors examined the surface of residual cells during the accumulation of a new cuticle layer. Observations using a scanning electron microscope confirmed that unfilled spaces existed between cells before being filled with cuticle (see Fig. 6). During the temporary absence of the cuticle layer on the surface of the residual cells, the cells became vulnerable to environmental damage, including plant pathogens; consequently, it was confirmed that the expression of genes related to stress responses became abundant in state 2 (see Fig. 3).

[0189] To verify these changes in vivo, the expression patterns of the stress response genes ABA-INSENSITIVE 5 (ABI5) and ETHYLENE RESPONSE FACTOR 1A (ERF1A) in residual cells were investigated. It was confirmed that AB15 and ERF1A were expressed before and after abscission in transgenic plants containing prABI5:nls-GFP or prERF1A:nls-GFP, and that expression in the outermost cells of the abscission region decreased after a protective cuticle layer was formed on the surface.

[0190]

[0191] Experimental Example 3: Determination of the New Fate of Epidermal Cells

[0192] The inventors confirmed the in vivo expression of cuticle development genes using transgenic plants and found that the cutin biosynthetic gene BODYGUARD 1 (BDG1) and GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE 4 (GPAT4) were expressed in the outermost remnants (see Fig. 7). Not only were the cuticle development-related genes increased, but the epidermal cell marker ATML1 was also increased in the outermost cells (see Fig. 7), which supports the hypothesis that non-epidermal remnants undergo transformative differentiation to become epidermal cells.

[0193]

[0194] Experimental Example 4: MYB74 functioning as a key factor in residual cell differentiation

[0195] The inventors investigated transcription factors exhibiting cell state-specific expression patterns to identify molecular factors that regulate the process of residual cells differentiating into epidermal cells. First, they identified transcription factors that are preferentially expressed during State 2, which includes the transition from non-epidermal cells to epidermal cells. As a result, most transcription factors that appear abundantly in State 2 were associated with stress responses, which is consistent with cell activity in State 2.

[0196] To verify the potential function of MYB74 during the process of transformative differentiation, the inventors examined the expression of MYB74 in residual cells of plants containing prMYB74:nls-GFP-GUS. Analysis revealed that MYB74 began to be expressed before the completion of abscission, reached a peak expression level during the transitional state, and then disappeared from cells in the abscission region. In longitudinal and transverse sections of the transformed flower stalks, MYB74 was present across at least three cell layers during the pre- and post-abscission stages, but was almost absent in the post-abscission stage.

[0197] MYB74 is known to be involved in salt stress and osmotic stress responses, but its role in plant development is largely unknown. Accordingly, to determine whether MYB74 is associated with the transformative differentiation of remnant cells into epidermal cells, the inventors constructed prUBQ10:XVE>>MYB74 (iMYB74) transgenic plants expressing MYB74 using a β-estradiol induction system. RNA-seq analysis was performed after treating the flower stalks during abscission with β-estradiol. The results confirmed that 1,763 genes were upregulated and 3,175 genes were downregulated in remnant cells due to the induction of MYB74 expression. Furthermore, while 58 photosynthesis-related genes were downregulated, 95 genes related to cuticle development and lipid biosynthesis were upregulated, indicating that MYB74 is involved in the transformative differentiation of remnant cells. In addition, as a result of comparison with genes presumed to be targets of the MYB74 gene, 2,176 of the 2,834 presumed target genes were detected in the residual cell transcripts (see Fig. 8), and it was confirmed that 329 of the 2,176 genes (about 15%) were expressed specifically in terms of cell state (see Fig. 9).

[0198] Analysis of the expression levels of MYB74 and MYB74 target genes in individual cells revealed that the expression level of the MYB74 gene was low during the early stages of abscission, reached a peak during the mid-stage, and decreased during the late stage (see Fig. 10). Photosynthesis-related genes showed a bicorrelation with MYB74 expression, while stress response genes showed a strong correlation with MYB74 expression levels during the mid-stage of abscission and a significant decrease in expression during the late stage (see Fig. 11). Additionally, it was confirmed that when MYB74 expression was induced using β-estradiol, cuticle material accumulated in the cell layer beneath the epidermal cells (see Fig. 12).

[0199] In other words, the inventors confirmed that MYB74 converts non-epidermal residual cells with photosynthetic ability into epidermal cells having a cuticle on their surface.

[0200]

[0201] Experimental Example 5: Transcription inhibitor MYB74-SRDX inhibiting fruit growth

[0202] The inventors further analyzed mutations of MYB74 that had lost their function. MYB102 was selected as the subject of analysis because it is the closest related to MYB74 in the phylogenetic tree, and MYB2 was also selected at random. Within the abscission region, the expression patterns of MYB102 and MYB2 were very similar to those of MYB74. However, no distinct phenotypic changes in the abscission region were observed for the myb74 single mutation, the myb74myb102 double mutation, or the myb74myb102myb2 triple mutation. It was predicted that this was because the MYBs expressed in the remaining cells were functionally redundant.

[0203] As an alternative to this, the inventors used an inhibition strategy to induce inhibition of the target gene by expressing a chimeric MYB74 inhibitor under the MYB74 intrinsic promoter. Two transgenic plants containing prMYB74:MYB74-SRDX expressed the chimeric MYB74 inhibitor at high levels.

[0204] As the first step in the analysis of plants containing prMYB74:MYB74-SRDX, the inventors confirmed the formation of a cuticle layer in the abscission region of the fruit after abscission was complete. Compared to the wild type, plants containing prMYB74:MYB74-SRDX were stained by toluidine blue up to a much more mature stage (see Fig. 13), indicating that cuticle formation was inhibited by suppressing the function of MYB74. Scanning electron microscopy observations of the surface of the abscission region also showed a difference in cuticle formation between the wild type and plants containing prMYB74:MYB74-SRDX (see Fig. 14).

[0205] GPAT4 is a gene encoding glycerol-3-phosphate acyltransferase in the cutin synthesis pathway and is specifically expressed in the outermost remnants of cells. The inventors confirmed through dual luciferase reporter analysis that GPAT4 is a target gene of MYB74 and confirmed that GPAT4 is co-expressed with MYB74 in new epidermal cells. Furthermore, it was confirmed that the expression of MYB74-SRDX, driven by the UBQ10 promoter and induced by β-estradiol treatment, reduces the expression of GPAT4 (see Fig. 15).

[0206] In addition, we further verified that residual cells differentiate into epidermal cells induced by MYB74 by analyzing the expression of ATML1, which is essential for epidermal cell differentiation. In the wild type, ATML1 was detected in the abscission region from the early stages of abscission, whereas ATML1 was hardly expressed in the abscission region of plants containing prMYB74:MYB74-SRDX. Furthermore, ATML1 expression was reduced in transgenic plants induced by β-estradiol treatment under the UBQ10 promoter. This demonstrates that inhibition of MYB74 function inhibits differentiation into epidermal cells and cuticle formation.

[0207] After abscission, the receptacle grows radially, which is driven by the growth of vascular bundles and the pith. Since it was confirmed that residual cells in the receptacle of MYB74-SRDX plants did not differentiate into epidermal cells, the effect of inhibiting the function of MYB74 after abscission on receptacle growth was investigated. As a result, MYB74-SRDX inhibited radial growth, reducing the diameter of the receptacle (see Fig. 17) and decreasing the distance from the center of the pith to the vascular bundles in the abscission region. Additionally, the fruit length of plants containing prMYB74:MYB74-SRDX was also shorter (see Fig. 18).

[0208] After the petals, stamens, and sepals fall off, at least 70 to 80% of the receptacle becomes covered with new surface cells, and as a result, a significant number of these new surface cells differentiate into epidermal cells. Considering that epidermal cells contribute to the differentiation of internal tissues and plant growth, these results imply that newly differentiated epidermal cells in the abscission area also play a role in promoting plant growth. In summary, inhibition of MYB74 expression limits fruit growth by inhibiting differentiation into epidermal cells and receptacle growth.

[0209] The present invention relates to the regulation of plant growth through the regulation of gene expression, and specifically, to a method for promoting the growth of a plant's flower stalk and fruit using a recombinant vector comprising a MYB74 transcription factor or a nucleic acid encoding the same that converts non-epidermal cells in the abscission region into epidermal cells, a composition comprising the recombinant vector for this purpose, and a transgenic plant.

Claims

A composition for regulating plant growth, comprising a MYB74 expression promoter containing the nucleotide sequence of SEQ ID NO. 1 as an active ingredient. In paragraph 1, A composition for regulating plant growth, wherein the above-mentioned expression promoter comprises one or more selected from the group consisting of a polynucleotide containing MYB74, a polynucleotide complementary to MYB74, and a vector expressing MYB74. In paragraph 2, A composition for regulating plant growth, wherein the above vector comprises the nucleotide sequence of SEQ ID NO.

4. In paragraph 2, A composition for regulating plant growth, wherein the above vector is activated by β-estradiol. In paragraph 1, The above composition is a composition for regulating plant growth that induces the conversion differentiation of non-epidermal cells into epidermal cells in the absorption zone (AZ). In paragraph 1, A composition for regulating plant growth, wherein the above-mentioned plant is one or more selected from the group consisting of fruit, flower, leaf, stem, root, and seed. In paragraph 1, The above composition is a plant growth regulating composition that promotes the formation of a cuticle on a surface exposed after abscission. In paragraph 1, The above composition is a composition for regulating plant growth that inhibits the expression of photosynthesis-related genes. In paragraph 8, A composition for regulating plant growth, wherein the above photosynthesis-related gene is one or more selected from the group consisting of Rubisco activase (RCA), Chlorophyll A / B binding protein 1 (CAB1), and Rubisco small subunit 1A (RBCS1A). In paragraph 1, A composition for regulating plant growth, wherein the above-mentioned plants are one or more selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, sorghum, Arabidopsis thaliana, Chinese cabbage, radish, chili peppers, strawberries, tomatoes, watermelons, cucumbers, cabbage, Korean melons, pumpkins, green onions, onions, carrots, ginseng, tobacco, cotton, sesame, sesame seeds, sugarcane, sugar beets, perilla seeds, peanuts, rice bran, rapeseed, apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, bananas, roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips. In paragraph 1, The above composition is a composition for regulating plant growth, which increases the diameter of the flower stalk or the length of the fruit. A method for regulating plant growth comprising an increasing step of increasing the expression or activity of MYB74 containing the nucleotide sequence of SEQ ID NO. 1 within a plant. In Paragraph 12, A method for controlling plant growth, wherein the above-mentioned increase step is performed by a vector containing the nucleotide sequence of SEQ ID NO.

4. In Paragraph 12, A method for controlling plant growth, wherein the above-mentioned plant growth includes an increase in the diameter of the flower stalk or the length of the fruit.

Images