Method for increasing plant drought stress tolerance

Abstract

The present invention relates to a method for enhancing drought tolerance of plants, and more specifically, to a method for enhancing drought tolerance of plants by including the step of increasing the expression of RGL1 of the plants, thereby enabling the plants to have high resistance to drought stress.

Description

Technology Field

[0001] The present invention relates to a method for improving the drought resistance of plants. Background Technology

[0002] Proline (Pro) is a multifunctional amino acid found in plants that accumulates in response to drought, high salinity, heavy metal exposure, pathogen infection, low temperatures, and oxidative stress. Additionally, proline acts as a molecular chaperone that stabilizes protein structures and can function as an antioxidant that regulates intracellular redox states and controls free radical levels. Furthermore, recent studies have reported that proline accumulation is regulated by ubiquitination during abiotic stress.

[0003] Protein ubiquitination is an important post-translational modification process found in eukaryotic cells that regulates various cellular and developmental processes. The ubiquitination pathway proceeds through a three-enzyme reaction chain consisting of the activation of the Ub molecule by E1 ubiquitin (Ub) activator, the acceptance of the activated Ub by E2 Ub-binding enzyme, and the facilitation of the transfer of Ub from the E2-Ub intermediate to the target protein by E3 Ub ligase. Most ubiquitination enzymes are E3 ligases, a significant portion of which are of the novel gene ring type. Several Arabidopsis ring E3 ligases are known to be involved in various cellular processes. These processes include plant hormone responses, seed germination, seedling growth, adaptive responses to nitrogen limitation, and sugar responses. In particular, ring-type E3 ligases play a crucial role in responding to environmental stimuli. These responses include photomorphogenesis, defense signaling, senescence, and mechanisms of tolerance to low temperature, drought, salinity, and osmotic stress. Despite recent progress in understanding the role of ubiquitination in plant responses to abiotic stress, the ubiquitination pathway in abiotic stress responses is still not well understood.

[0004] one side AtRZF1 The gene encodes a RING-type E3 Ub ligase subunit and plays an important role in the drought response. AtRZF1 Plants that overexpress it are more sensitive to drought, and atrzf1 Mutations are less sensitive than wild-type (WT) plants, which is AtRZF1 This indicates that the drought response is negatively regulated. atrzf1 Proline accumulation in plants is WT and AtRZF1 It is higher than in overexpressing plants, suggesting that AtRZF1 affects proline content and induces drought sensitivity.

[0005] However, considering that RING-type E3 Ub ligases influence various aspects of drought signaling, the role of AtRZF1 has not yet been fully elucidated. Therefore, it is necessary to identify the components that interact with AtRZF1 in the drought signaling pathway. Prior art literature

[0007] Korean Registered Patent No. 10-2632726 The problem to be solved

[0008] The present invention aims to provide a method for enhancing the drought resistance of plants.

[0009] The present invention aims to provide a plant with enhanced drought resistance. means of solving the problem

[0010] 1. A method for enhancing drought tolerance of a plant, comprising a step of increasing the expression of RGL1 (Rhamnogalacturonan lyase 1) in the plant.

[0011] 2. A method for enhancing drought tolerance of a plant according to 1, comprising the step of transforming the plant with a recombinant vector into which a gene encoding RGL1 is inserted.

[0012] 3. A method for enhancing drought resistance of a plant according to 1 above, comprising the step of treating the plant with an expression promoter of RGL1.

[0013] 4. A method for improving drought resistance of a plant, wherein RGL1 is composed of an amino acid sequence of any one of SEQ ID NOs 1 to 9 in the above 1.

[0014] 5. A method for enhancing drought resistance of a plant, wherein the gene encoding RGL1 in 1 above consists of the nucleotide sequence of SEQ ID NO. 10.

[0015] 6. A method for enhancing drought resistance of a plant, wherein the RGL1 expression promoter in 3 above is any one selected from the group consisting of mannitol, NaCl, ABA, and H2O2.

[0016] 7. A method for enhancing drought resistance of a plant, wherein the plant is selected from the group consisting of Arabidopsis thaliana, rice, corn, barley, cabbage, rapeseed, soybean, tomato, and chili pepper.

[0017] 8. A method for enhancing drought resistance of a plant, wherein the plant is a plant cell, plant seed, or plant body, in accordance with 1 above.

[0018] 9. A method for enhancing drought tolerance of a plant, wherein the recombinant vector in 2 above is a recombinant vector in which the RGL1 gene is operably linked to a promoter.

[0019] 10. A plant with enhanced drought resistance obtained according to the method of any one of claims 1 to 9 above. Effects of the invention

[0021] The drought resistance of plants can be enhanced or increased using the present invention.

[0022] By using the method of the present invention, the expression of RGL1 in plants can be increased, and the plants can be made to have increased drought resistance. Brief explanation of the drawing

[0024] Fig. 1 is atrzf1 and pca31 This is the result of a comparative analysis of drought-induced sensitivity in mutants. Figure 2 is under osmotic stress conditions. atrzf1and pca31 This is the result of analyzing the phenotype of. Fig. 3 is pca31 This is the result of identifying the location where T-DNA is inserted and analyzing the domain of RGL1 accordingly. Figure 4 shows the results of confirming the rhamnogalacturonan degrading enzyme (RGL) activity of RGL1. Figure 5 shows the results of comparing the composition of pectin and monosaccharides between wild-type Arabidopsis and mutants. Fig. 6 is atrzf1 class pca31 This is the result of RNA-seq analysis of the mutant transcript. Figure 7 shows a complementary system ( rgl1 RNAi / atrzf1 This is the result of analyzing the phenotype of ). Figure 8 shows the results of analyzing the expression of RGL1 in Arabidopsis thaliana. Figure 9 shows the results of analyzing the phenotype of the RGL1 transgenic line under osmotic stress conditions. Figure 10 is a schematic diagram of the vascular bundle analysis of the RGL1 transgenic line and the osmotic stress response model including the AtRZF1 and RGL1 pathways. Fig. 11 is RGL1 This is the result of analyzing the phenotype of transgenic plants under normal growth conditions. Specific details for implementing the invention

[0025] The present invention provides a method for improving the drought resistance of plants.

[0026] The present invention relates to a method for enhancing drought tolerance of plants, and more specifically, to a method for enhancing drought tolerance of plants by including the step of increasing the expression of RGL1 of the plants, thereby enabling the plants to have high resistance to drought stress.

[0027] The present invention provides a method for enhancing drought resistance of a plant, comprising the step of increasing the expression of RGL1 (Rhamnogalacturonan lyase 1) of the plant.

[0028] RGL1 is a plant cell wall enzyme and an activated rhamnogalacturonan degrading enzyme. Because it can modify RG-I by breaking the glycosidic bond between rhamnose (Rha) and galacturonic acid (GalA) within the rhamnogalacturonan (RG)-I backbone, RGL1 can influence the composition of the cell wall. Furthermore, RGL1 accumulates proline, which confers resistance to stress, and under dry conditions atrzf1 It can promote root growth and vascular bundle development by activating the drought tolerance phenotype mediated by the mutant genotype of *Arabidopsis thaliana ring zinc finger 1*. Therefore RGL1 The drought resistance of plants can be enhanced through the overexpression of [it].

[0029] The gene encoding RGL1 is a protein belonging to the RGL gene family.

[0030] Arabidopsis thaliana without introns Arabidopsis thaliana The RGL1 coding gene of ) has a translation region of 2034 bp and can encode the RGL1 protein composed of 677 amino acids.

[0031] The above RGL1 may consist of any one of the amino acid sequences of SEQ ID NOs 1 to 9.

[0032] RGL1 of Arabidopsis contains the amino acid sequence of SEQ ID NO. 1.

[0033] rice( Oryza sativa RGL1 of ) contains the amino acid sequence of SEQ ID NO. 2.

[0034] corner( Zea mays RGL1 of ) contains the amino acid sequence of SEQ ID NO. 3.

[0035] barley( Hordeum vulgare RGL1 of ) contains the amino acid sequence of SEQ ID NO. 4.

[0036] napa cabbage( Brassica oleracea RGL1 of ) contains the amino acid sequence of SEQ ID NO. 5.

[0037] rapeseed Brassica napus RGL1 of ) contains the amino acid sequence of SEQ ID NO. 6.

[0038] bean( Glycine max RGL1 of ) contains the amino acid sequence of SEQ ID NO. 7.

[0039] tomato( Solanum lycopersicum RGL1 of ) contains the amino acid sequence of SEQ ID NO. 8.

[0040] pepper( Capsicum annuum RGL1 of ) contains the amino acid sequence of SEQ ID NO. 9.

[0041] The amino acid sequences of SEQ ID NOs 2 to 9 all have high homology with the amino acid sequence of SEQ ID NO. 1, and the amino acid sequences of SEQ ID NOs 1 to 9 all share similar motifs, so the amino acid sequences can be included in the amino acid sequences forming RGL1 of the method for improving drought resistance of plants according to the present invention.

[0042] The gene encoding the above RGL1 may consist of the nucleotide sequence of SEQ ID NO. 10. The gene encoding the above RGL1 protein may consist of the nucleotide sequence of SEQ ID NO. 10 derived from Arabidopsis thaliana, and the wild-type Arabidopsis thaliana genome At2g22620 It may have originated from the gene region.

[0043] The step of increasing the expression of RGL1 in the above-mentioned plant is not limited to a step of increasing the expression amount of RGL1. For example, it may be a step of transforming the above-mentioned plant with a recombinant vector into which a gene encoding for RGL1 is inserted, and the above-mentioned recombinant vector may be a recombinant vector in which the RGL1 gene is operably linked to a promoter.

[0044] The plant body, transformed seed, and plant body of the present invention, which are redifferentiated from the transformed plant cells, are RGL1 It can overexpress, and the above RGL1By introducing a recombinant vector containing a gene, the present in plants RGL1 It can increase the expression of.

[0045] of the above plant RGL1 The step that increases the expression of RGL1 It may further include the step of treating the plant with an expression promoter. RGL1 By treating plants with an expression promoter, in the plants RGL1 It can increase the expression amount of, and the above RGL1 The expression promoter of may be, for example, mannitol, NaCl, ABA, or H2O2. RGL1 Through treatment with an expression promoter RGL1 This may be overexpressed, further increasing dryness resistance.

[0046] “Drought tolerance” in this specification refers to the property of a plant to withstand drought, meaning resistance to drought stress.

[0047] The term “recombinant vector” in this specification refers to a genetic construct comprising an essential regulatory element operably linked to express a gene insert, and includes all conventional vectors including plasmid vectors, cosmid vectors, bacteriophage vectors, and virus vectors.

[0048] The "recombination vector" of the present invention is the above RGL1The vector is functionally linked to an expression regulatory sequence to enable gene expression. For example, in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, the vector may include signal sequences or leader sequences for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose. Additionally, the vector may include selectivity markers, and the vector may self-replicate or be incorporated into host DNA. The vector of the present invention may be manufactured using gene recombination technology well known in the art, and site-specific DNA cleavage and ligation may be performed using enzymes, etc., generally known in the art.

[0049] The above recombinant vector may include an antibiotic resistance marker for the selection of the host into which the vector is introduced, and this may be inherent in the vector or introduced from the outside.

[0050] The recombinant vector of the present invention may use a conventional terminator, examples of which include nophalin synthase (NOS), rice α-amylase RAmy1 A terminator, the octopine gene terminator of Agrobacterium tumefaciens, the phaseoline terminator, and the rrnB1 / B2 terminator of Escherichia coli, but are not limited thereto.

[0051] The recombinant vector of the present invention may preferably include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can typically be selected by chemical methods, and any gene capable of distinguishing transformed cells from non-transformed cells is included. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, hygromycin, chloramphenicol, G418, and bleomycin, and the aadA gene.

[0052] In this specification, "promoter" refers to a region of DNA upstream of a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. "Plant promoter" is a promoter capable of initiating transcription in a plant cell.

[0053] The term "transformation" in this specification refers to a phenomenon in which DNA, which is genetic material, enters a living cell of a different lineage and alters the genetic traits; it is also referred to as transformation, type change, or type change.

[0054] "Transforming" a plant body with the recombinant vector of the present invention may be carried out by transformation techniques known to those skilled in the art. Specifically, a transformation method using Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome-mediated method, in planta transformation, vacuum infiltration method, floral meristem dipping method, or Agrobacterium spraying method ( Agrobacterium A spraying method) can be used, and more specifically, a transformation method using Agrobacterium or an Agrobacterium spraying method ( Agrobacterium A spraying method may be used, but is not limited thereto.

[0055] In the present invention, the plant may be a plant selected from the group consisting of Arabidopsis thaliana, rice, corn, barley, napa cabbage, rapeseed, soybeans, tomatoes, and chili peppers.

[0056] The plants of this specification include plant cells, plant seeds, or plant bodies, and may include not only mature plant bodies but also plant tissues capable of developing into mature plants.

[0057] The present invention provides a plant with enhanced drought resistance obtained according to the method described above.

[0058] The above plant may be a plant selected from the group consisting of Arabidopsis thaliana, rice, corn, barley, cabbage, rapeseed, soybean, tomato, and chili pepper, and the above plant may have enhanced drought tolerance due to increased expression of RGL1.

[0059] The present invention will be described in detail below with reference to examples.

[0061] Examples

[0062] Experimental method

[0063] 1. Plant materials, growth conditions, and stress induction

[0064] Arabidopsis thaliana Columbia (Col-0) and atrzf1 Mutants (Min et al., 2021) were used as background lines for WT and repressor screening, respectively. atrzf1 To screen for inhibitors, seeds were placed in a growth chamber at 22°C at 120 μmol m⁻¹. -2 sec -1 Seeds were sown directly into soil (peat-based soil: vermiculite, 2:1, v / v) under conditions of light intensity, 60% relative humidity, and 16 hours of daylight. For the osmotic stress experiment, 10-day-old A. thaliana Seedlings were immersed in a solution containing 400 mM mannitol and sampled at 0, 6, 12, and 24 hours. For ABA or salt stress experiments, 10-day-old A. thaliana Seedlings were immersed in a solution containing 100 μM ABA or 150 mM NaCl, and samples were taken at 0, 3, 6, and 12 hours. For the oxidative stress experiment, 10-day-old A. thaliana Seedlings were immersed in a solution containing 6 mM H2O2 and sampled at 0, 1, 3, and 6 hours. In each case, the collected seedlings were immediately flash-frozen with liquid nitrogen and stored at -80°C.

[0065] 2. Proline content measurement

[0066] Proline was extracted by grinding 100 mg of plant leaves with 1 mL of 3% sulfosalicylic acid. Then, 200 μL of the extract was mixed with 100 μL of a ninhydrin reagent mixture (80% glacial acetic acid, 6.8% phosphoric acid, 70.17 mM ninhydrin) at 100°C for 60 minutes. The reaction was stopped using ice. Next, 200 μL of toluene was added to the reaction mixture and mixed. The absorbance of the toluene layer was measured at 520 nm using a UV / VIS spectrophotometer (JASCO, Tokyo, Japan). Proline concentration was estimated from a standard curve and calculated on a FW or DW basis using the following formula: [(ng Pro / mL × mL extraction buffer) / 115.5 ng nmol] / g sample = nmol Pro / g FW or DW substance. The leaf FW was measured immediately after cutting, and DW was determined after drying at 60°C for 12 hours.

[0068] 3. Determination of T-DNA insertion sites using Thermal Asymmetric Interlace (TAIL)-PCR

[0069] pca31 gDNA was isolated from mutants, and TAIL-PCR was performed using AD and T-DNA LB end primers. The purified fragments obtained after tertiary TAIL-PCR were cloned into the pGEM T-easy vector (Promega, Madison, WI, USA), and DNA sequencing was performed. The obtained DNA sequences were searched using the NCBI BLAST program (http: / / www.ncbi.nlm.nih.gov). After designing primers, specific fragments were amplified by combining them with T-DNA LB primers, and sequencing was subsequently performed to identify the T-DNA insertion site.

[0071] 4. RGL1 activity analysis

[0072] To express and purify histidine (His)-tagged RGL1, the full length of RGL1 cDNA was amplified by PCR using a primer set. The PCR product was first cloned into the pDONR / ZEO vector and verified by sequencing. The resulting plasmid was cloned into the protein expression vector pET300 using a Gateway system according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). His-fused full-length RGL1 was expressed in E. coli, purified using Ni-NTA agarose (Qiagen, Hilden, Germany), and used for enzymatic analysis. The specific activity of RGL1 was determined as follows: 500 ng of His-RGL1 was added to potato rhamnogalacturonan-I (RG-I; concentrations of 1 μg / mL at 0.05; Megazyme, Wicklow, Ireland) used as a substrate in a solution containing 1 mM CaCl2 in 50 mM Tris-HCl (pH 8.5). The reaction was carried out at 37°C for 1 hour. The enzymatic reaction was stopped by adding three times the volume of 50 mM HCl. RGL activity was analyzed by spectrophotometry by measuring the production of Δ4,5-unsaturated galacturonic acid (GalA) at 235 nm. Enzymatic activity was estimated in units per microgram (μg) of protein. One unit of RGL activity was defined as the amount of enzyme (μmol / min) forming 1 μmol of 4,5-unsaturated GalA product per minute.

[0074] 5. Measurement of pectin and monosaccharide content

[0075] Three-week-old seedlings were immediately flash-frozen with liquid nitrogen and ground into a fine powder. The pectin content of each sample was measured using a total pectin content analysis kit (Cat No. BC1405, Solarbio, Beijing, China). Pectin content was determined based on a standard calibration curve, and the results were expressed in micrograms (μg) per mg of sample material (alcohol-insoluble residue, AIR). The experiment was repeated three times.

[0076] Monosaccharide content was measured according to the method of Caffall et al. (2009). Leaf samples from 3-week-old plants were ground into a fine powder using liquid nitrogen. The samples were serially extracted by washing twice each with solutions of 80% (v / v) ethanol, chloroform:methanol (1:1, v / v), 100% ethanol, and 100% acetone. After centrifugation at 6000 ×g for 10 minutes, the pellets were washed twice with sterile water and acetone, dried using a Speed-Vac rotary evaporator, and weighed. Approximately 200–300 μg of sample material (AIR) was supplemented with 20 μg of alloyositol as an internal standard. The dried samples were hydrolyzed in 1 M methanol-HCl (Supelco, St. Louis, MO, USA) at 80°C for 18 hours, and after cooling, dried twice more with anhydrous methanol. The dried sample was mixed with 200 μL of Tri-Sil TP reagent (Thermo Fisher Scientific, Waltham, MA, USA) and heated at 80°C for 20 minutes. After cooling, the sample was redissolved in 3 mL of hexane and filtered through glass wool. The derived sample was injected into a GC-MS system (GC-7890A / MS-5975C, Agilent, Folsom, CA, USA) and separated on a DB-5MS capillary column (0.25 mm id × 30 m length, 0.25 μm film thickness; Agilent) using helium as the carrier gas at a flow rate of 1 mL / min. The GC temperature gradient was set as follows: 80°C for 2 minutes, 80–140°C at 20°C / min, and 140–200°C.

[0078] 6. RNA-seq analysis

[0079] Total RNA is 2-week-old WT, atrzf1 , and pca31Extracts were obtained from seedlings using the Plant RNeasy Extraction Kit (Qiagen). Three biological copies were prepared for each sample for RNA-seq analysis. RNA quality was evaluated using an Agilent 2100 Bioanalyzer and an RNA nanochip (Agilent Technologies, Amstelveen, Netherlands). The library was constructed using the QuantSeq 3' mRNA-Seq Library Preparation Kit (Lexogen, Austria) according to the manufacturer's instructions. High-throughput sequencing was performed using single-end 75 sequencing with a NextSeq 500 (Illumina, CA, USA). QuantSeq 3' mRNA-Seq read data were aligned using Bowtie 2. Bowtie 2 indices were generated from representative transcriptome sequences to align with the transcriptome. Differentially expressed genes were determined using BEDTools based on counts obtained from unique and multiple alignments. Read count data were processed using the interquartile normalization method at Bioconductor.

[0081] 7. Histological analysis

[0082] For optical microscopy analysis, 3-week-old WT, RGL1 overexpression, and rgl1 Flower stalks of RNAi plants were embedded in LR White resin (London Resin Co., London, UK) and thinly sliced ​​to a thickness of 1 μm. To observe vascular bundles, the sliced ​​sections were stained with 0.1% toluidine blue and observed under an optical microscope (Zeiss 7, Axiolab, Germany).

[0084] 8. Statistical analysis

[0085] Statistical analysis was performed using GraphPad Prism 10.0.1 (GraphPad Software, Inc., CA, USA) and SPSS 23.0 software (IBM Corp., Armonk, NY, USA), and included one-way analysis of variance (ANOVA) and Tukey's multiple range test. Different characters shown in the graph indicate statistically significant differences (P < 0.05).

[0087] Experimental results

[0088] 1. through T-DNA tagging mutagenesis atrzf1 Inhibitor screening

[0089] To investigate the role of AtRZF1 in proline metabolism and water deficit response, approximately 26,000 T-DNA tagged transgenic lines were used with the vector pSKI015, which has four repetitions of the 35S enhancer at the right boundary of the T-DNA. atrzf1 It was generated in the mutant background. Proline is under drought conditions. atrzf1 Since it accumulates excessively, by evaluating proline content in the leaves of 5-week-old T1 transgenic plants exposed to drought stress atrzf1 The inhibitor candidates were separated. Various atrzf1 Among repressor mutants pca31 We selected AtRZF1 to investigate abiotic stress signaling mediated by AtRZF1 (Fig. 1). As shown in Figs. 1a and 1b pca31 The sensitivity to drought in mutants was analyzed, and as shown in Figures 1c and 1d, pca31 The survival rate and proline accumulation of were analyzed.

[0091] 2. pca31 Confirmation of the mutant's drought sensitivity

[0092] pca31 The response of WT to drought stress and atrzf1To compare with, plant survival analysis was performed through drought treatment. 3-week-old WT, atrzf1 , and pca31 The seedlings were kept without water for 10 days, and then watered again for 3 days. After watering, their phenotypes were observed. Under sufficiently watered soil conditions, WT, atrzf1 , and pca31 No phenotypic differences were observed between the seedlings (Fig. 1a). However, when water was withheld for 10 days and then watered again for 3 days, atrzf1 The seedlings are WT and pca31 It showed better growth than the seedlings (Figure 1b). WT on the 3rd day after watering, atrzf1 , and pca31 The survival rate of the seedlings was measured. atrzf1 While the survival rate of was 88.7%, WT and pca31 The survival rates were only 10.7% and 12.7%, respectively (Figure 1c). These results pca31 The second mutation caused by T-DNA insertion in atrzf1 It indicates that it is responsible for suppressing the insensitivity to drought stress. Also pca31 Proline levels under drought conditions atrzf1 It was found to be significantly lower and similar to WT (when calculated based on FW or DW) (Fig. 1d). This result pca31 The second mutation of atrzf1 It suggests that it inhibits drought-induced proline accumulation in mutants.

[0094] 3. pca31 Confirmation of the mutant's osmotic stress response

[0095] WT, atrzf1 , and pca31To investigate potential differences in the response to osmotic stress between them, seeds of each genotype were planted and grown in Murashige and Skoog (MS) medium containing 0 or 400 mM mannitol. During the early growth stages, namely seed germination, cotyledon greening, and primary root growth, in MS medium without mannitol, WT, atrzf1 , and pca31 No phenotypic differences were observed between the plants (Fig. 2). After mannitol treatment for 2–4 days, WT and atrzf1 It showed a similar germination rate, and this pca31 It was higher. This pca31 WT and during this osmotic stress atrzf1 It indicates that germination is delayed compared to. Next, mannitol-induced sensitivity was evaluated by assessing the cotyledon greening rate for 10 days after germination. Under osmotic conditions atrzf1 The cotyledon greening rate of WT and pca31 It was higher, and WT and pca31 [It] showed similar recording rates (Figs. 2a and 2b). WT grown in MS medium containing 400 mM mannitol and pca31 While the cotyledon greening rates of seedlings were only 35.6% and 33.8%, respectively, atrzf1 78.2% of the seedlings exhibited green cotyledons (Fig. 2b). In addition, the inhibition of root growth in the seedlings under dehydration conditions was investigated. WT and pca31 In the case of seedlings, root growth and length were similar in response to 400 mM mannitol, but under osmotic stress conditions atrzf1 The seedlings are WT and pca31 They showed longer root length and better root growth than the seedlings (Figs. 2c and 2d). Collectively, these results pca31 In the response of mutants to osmotic stress atrzf1 It indicates that it acts as an inhibitor of.

[0097] 4. pca31 Identification of T-DNA insertion sites in the genome

[0098] To identify the T-DNA insertion site, pca31 Thermal Asymmetric Interlace (TAIL)-PCR was performed using left-bound (LB) primers and random denaturation (AD) primers on genomic DNA (gDNA) extracted from seedlings (Table S1). The gDNA sequences of the products obtained from the TAIL-PCR analysis were retrieved using the BLAST program (blast.ncbi.nlm.nih.gov / Blast.cgi). As a result, pca31 genome At2g22620 and At2g22630 A single T-DNA insertion was confirmed in the intergene region between genes (Fig. 3a). At2g22620 class At2g22630 The genes encode the RGL gene family and AGAMOUS-LIKE 17, respectively. The T-DNA locations are At2g22620 It was found to be nearby (Fig. 3a). To further confirm the T-DNA insertion site, primers were designed and combined with T-DNA-specific LB3 primers to WT, atrzf1 and pca31 PCR was performed using gDNA (Figs. 3a and 3b). When gDNA-PCR was performed using F1 and R1 primers, the PCR bands were WT and atrzf1 It appeared in, but pca31 It did not appear in. However, when using R1 and LB3 primers pca31 PCR products were detected only in (Fig. 3b). In addition, reverse transcription PCR (RT-PCR) analysis results showed that in 7-day-old WT seedlings AtRZF1 Although a transcript exists, atrzf1 class pca31 It was shown that it is not present in seedlings (Fig. 3c). PCR-based genotyping analysis showed that T-DNA insertion pca31 It was confirmed that it is associated with the phenotype.

[0099] Since the four copies of the enhancement element in the activation tag vector can affect the expression levels of adjacent genes around the T-DNA insertion site, through quantitative PCR (qPCR) experiments At2g22620 and At2g22630 The transcription level of the gene was investigated. pca31 at At2g22630 The gene expression level is WT or atrzf1 There was no change compared to, but pca31 at At2g22620 The gene expression level is WT or atrzf1 It decreased significantly compared to (Fig. 3d). At2g22620 The level of gene transcription is higher than WT atrzf1 Appeared higher in, At2g22620 These results suggest that the expression level of can be regulated through a ubiquitination pathway involving AtRZF1 (Fig. 3d). These results indicate that under dehydrated conditions pca31 related to phenotype At2g22620 It indicates a decrease in the expression level of genes.

[0100] At2g22620 Since the gene belongs to the RGL gene family, it was named RGL1 (Fig. 3e). Unlike RGL, which is known as a plant cell wall enzyme that cleaves polymers, the biological function of RGL1 has not yet been reported. Intron-free RGL1 has a translation region of 2034 bp, encodes a protein composed of 677 amino acids, and has a calculated molecular weight of 78.5 kDa. Amino acid sequence analysis identified three distinct domains within RGL1: the RGL domain, the carbohydrate-binding-like domain (CBDL), and the galactose-binding-like domain (GBDL). These domains A. thaliana It is highly conserved among RGL proteins (Fig. 3e). A. thaliana There are 7 RGLs, and RGL1 and the other 6 A. thalianaThe amino acid sequence identity among RGL members is 54%–63%. The phylogenetic tree constructed using the clustering algorithm A. thaliana It shows that RGLs are classified into 5 subgroups.

[0102] 5. Check RGL activity of RGL1

[0103] Enzymatic analysis was performed to investigate whether At2g22620 (RGL1) possesses RGL activity. As reported, RGL modifies RG-I by cleaving the glycosidic bond between rhamnose (Rha) and galacturonic acid (GalA) within the RG-I backbone via a β-elimination reaction. This process generates an Rha residue at the reducing end of the product and introduces a double bond between C4 and C5 of the non-reducing GalA residue, which can be detected by a spectrophotometer at 235 nm. For enzymatic analysis, Escherichia coli Externally expressed RGL1 purified from [source] was mixed with potato RG-I using it as a substrate. As shown in Figure 4, the reaction produced a typical Michaelis-Menten curve. The Km and Vmax values ​​for potato RG-I were determined using a Lineweaver-Burk plot, and the Km value was found to be 0.014 μg / mL and the Vmax value 33.9 U / μg protein. These results indicate that RGL1 is indeed an activated rhamnogalacturonan degrading enzyme.

[0105] 6. atrzf1 and pca31 Analysis of RG-I composition in the cell wall of mutants

[0106] RGL1 acts as an enzyme that modifies pectin RG-I, affecting the composition of pectin polysaccharides, and the expression level of RGL1 atrzf1 class pca31 Considering the change between (Fig. 3d), atrzf1 and pca31 We analyzed whether there was a change in pectin content in the mutants. pca31 silver atrzf1 Compared to, pectin levels decreased by 28%, and it contained less pectin than WT (Fig. 5a). In addition, through gas chromatography-mass spectrometry (GC-MS), 3-week-old WT, atrzf1 , and pca31 The monosaccharide composition was measured in the seedlings. All measured monosaccharides, namely arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), galactose (Gal), galacturonic acid (GalA), and glucose (Glc), are atrzf1 WT and in mutants pca31 It was found to be higher than the saplings (Figs. 5b to 5h). However, pca31 The levels at were similar to those in WT seedlings. The above results suggest that AtRZF1 and RGL1 have opposing physiological functions in regulating major components of pectin RG-I monosaccharides such as arabinose, rhamnose, galactose, and galacturonic acid. Furthermore, the ratios of other neutral monosaccharides (fucose, xylose, glucose) also atrzf1 class pca31 The results showed clear contrasts between the mutants. Collectively, these monosaccharide analyses suggest that the expression level of RGL1 is important in regulating the amount of pectin monosaccharides.

[0108] 7. RNA sequencing analysis results

[0109] pca31 At this level of osmotic stress response and pectin RG-I composition atrzf1 Because they act as repressors, it was hypothesized that AtRZF1 and RGL1 would inversely regulate various target genes. To verify this hypothesis, through RNA sequencing (RNA-seq) experiments, WT, atrzf1 , and pca31 Transcriptome changes were analyzed. WT cultivated for 2 weeks, atrzf1 , and pca31Biologically distinct RNA samples were extracted from seedlings in triplicate, and each RNA sample was sequenced. Then, the read data were mapped to the Arabidopsis TAIR10 genome version (Arabidopsis.org). The expression levels of each gene were compared between samples ( atrzf1 / WT, pca31 / WT, and pca31 / atrzf1 As a result, 2,116 genes meeting the validity criteria were identified (cutoff for each comparison: fold change ≥ |1.5| and P < 0.05) (Fig. 6a). atrzf1 Most of the differentially regulated genes in pca31 It showed a lower fold change compared to WT (Fig. 6a). atrzf1 When comparing and WT, AtRZF1 It was found that gene mutations caused more than a 1.5-fold change in 517 genes (increased expression in 292 genes, decreased expression in 225 genes) (Figs. 6a and 6b). In particular, atrzf1 Among the 292 genes with increased expression, 75 genes are pca31 at atrzf1 Expression was decreased compared to the mutant (Figs. 6b and 6c). Conversely, atrzf1 Among the 225 genes with reduced expression, 75 genes are pca31 at atrzf1 Expression increased compared to the mutant (Figs. 6b and 6c). Based on gene ontology (GO) analysis, atrzf1 class pca31 The putative molecular functions of 150 genes that were oppositely expressed were investigated. Many of these genes were classified as genes responding to environmental stress, cellular components, RNA metabolism, and signal transduction (Fig. 6c). These results atrzf1 class pca31The 150 genes that are inversely expressed between them suggest that they play an important role in the plant's response to abiotic stress. Furthermore, these results suggest that AtRZF1 and RGL1 each have unique biological functions in addition to their common role in abiotic stress signaling.

[0110] pca31 Less sensitive to WT in this osmotic stress response atrzf1 Because it was isolated as a suppressor mutant, atrzf1 class pca31 We focused on abiotic stress-related genes that exhibit opposite expression patterns between them. Based on GO analysis (Fig. 6c), under normal growth conditions, WT, atrzf1 , and pca31 The transcription levels of all 17 stress-related genes were investigated. As a result of qPCR analysis, atrzf1 class pca31 It was confirmed that there were significant changes in the expression levels of many genes tested between them. Among the tested genes, 9 were atrzf1 class pca31 In between, opposite expression patterns were observed compared to WT. The above nine stress-related genes under osmotic stress conditions atrzf1 class pca31 We further investigated whether they are regulated inversely. Similar to the expression patterns under normal growth conditions, the inverse expression patterns of these genes also occur under osmotic stress. atrzf1 class pca31 It was still observed between, pca31 In this, the expression of 6 genes increased significantly, and 3 genes are atrzf1 Expression decreased compared to. Collectively, these results RGL1 Reduced expression of in Arabidopsis transcripts AtRZF1 It suggests that the effects of mutations are generally suppressed.

[0112] 8. Regarding osmotic stress pca31 Comparative analysis of reactions and complementary systems

[0113] pca31 at RGL1 To determine whether the decreased expression is responsible for the sensitive phenotype induced by osmotic stress, atrzf1 at RGL1 The silencing of genes pca31 An experiment was conducted to verify whether it could exhibit a phenotype similar to . To this end, rgl1 RNAi (ri) constructs atrzf1 Transgenic lines were generated by introducing mutants into a background. The 15 homozygous lines obtained ( rgl1 RNAi / atrzf1 ) middle, pca31 Similar to RGL1 Two independent lineages with reduced expression levels ( rgl1 RNAi / atrzf1 9-3; Com9 and rgl1 RNAi / atrzf1 16-1; Com16) was selected for further investigation into the phenotype under dehydration stress.

[0114] Under normal growth conditions, WT, atrzf1 , pca31 and no clear differences in taproot growth and cotyledon greening were observed between the two selected complementary plants (Fig. 7). Next, the inhibition of seedling root growth was investigated by treatment with 400 mM mannitol. As shown in Figs. 7a, b and the previous Figs. 2c, d, atrzf1 The increase in root length pca31 In response to osmotic stress, it returned to WT levels. Similarly, the root length of complementary lines (Com9 and Com16) decreased, and under mannitol treatment, WT or pca31 No significant difference was observed (Figs. 7a and 7b). In addition, the cotyledon greening rate of complementary lines (Com9 and Com16) was WT or when exposed to osmotic stress for 10 days. pca31 It was similar to, and this atrzf1This indicates that the increased recording rate returned to the WT level (Figs. 7c and 7d). Collectively, these results pca31 A decrease in RGL1 expression levels during the osmotic stress response atrzf1 It proves that it suppresses the insensitivity of.

[0116] 9. A. thaliana at RGL1 Analysis of expression and localization within cell organelles

[0117] To investigate the in vivo function of RGL1, expression patterns in WT plants were evaluated. First, using qPCR with RNA extracted from seedlings aged 1 to 5 weeks RGL1 The level of the warrior was investigated. RGL1 The expression level peaked at the 2-week-old seedling stage, decreased significantly at the 3-week-old seedling stage, and remained at a low level until 5 weeks. In addition, as a result of evaluating transcription levels in various organs, RGL1 It was found that this is primarily expressed in the roots and flowers compared to the stems and leaves. These results RGL1 This indicates that expression is regulated according to the plant's growth stage and organ.

[0118] RGL1 To further investigate whether it is expressed in response to this abiotic stress, qPCR was performed after treatment with 400 mM mannitol, 150 mM NaCl, 100 μM abscisic acid (ABA), and 6 mM hydrogen peroxide (H2O2) RGL1 The expression level of was evaluated. RGL1 The expression levels of were induced by ABA and abiotic stress treatments, peaking at 6 hours under osmotic stress and ABA, and at 3 hours under salt and oxidative stress conditions (Figs. 8a to 8d). As a control for ABA and abiotic stress treatments RAB18 , RD29A , and AtGSTU5 The genes were analyzed (Figs. 8a to 8d). The results were RGL1 The transcript body A. thaliana It indicates that it accumulated in response to ABA, osmotic stress, salt stress, and oxidative stress. RGL1 To gain a deeper understanding of the organ specificity of expression and abiotic stress responses, 1410-bp RGL1 Transgenic plants expressing beta-glucuronidase (GUS) under the control of a promoter were generated. Then, GUS expression patterns in the transgenic plants were observed before and after treatment with mannitol, ABA, and H2O2. Under water (H2O) treatment conditions, GUS expression was weakly detected in shoot apical meristems and roots. However, GUS staining in the transgenic plants was strongly induced in the roots after treatment with mannitol, ABA, and H2O2. During abiotic stress RGL1 To quantitatively analyze the strength of the promoter, 2 weeks old RGL1 The GUS expression levels of promoter-GUS transgenic seedlings were measured by fluorescence analysis, and GUS activity increased in response to abiotic stress. This was observed under ABA treatment and abiotic stress conditions. RGL1 This is consistent with the result that transcriptome levels were induced.

[0119] To investigate the intracellular localization of RGL1, a transfection expressing RGL1-green fluorescent protein (GFP) under the control of the 35S promoter A. thalianaPlants were generated. RGL1 is a plant cell wall enzyme and is expected to be located in the cell wall. To visualize the cell wall, propidium iodide (PI), a red fluorescent dye commonly used as a marker, was used. Transgenic plants expressing RGL1-GFP were stained with PI and observed using a confocal microscope. In the root cells of transgenic plants expressing RGL1-GFP, GFP signals were observed mainly in the cell wall, and some were also weakly observed in the cytoplasm (Fig. 8e). Additionally, when the RGL1-GFP transgenic line was treated with 0.6 M mannitol for 1 hour, GFP fluorescence was observed in the outer regions of root cells with separated plasma membranes (Fig. 8f). These results confirm that RGL1 is located in both the cell wall and the cytoplasm.

[0121] 10. RGL1 Overexpression and rgl1 RNAi lineage dehydration stress response

[0122] To further confirm the association between RGL1 and dehydration, by the 35S promoter RGL1 We generated additional transgenic plants that overexpressed or suppressed the expression of. Two RGL1 Overexpression plants (OX6-2 and OX13-6) and two rgl1 RNAi lineage ri6-6 class ri36-5 Select ) and use qPCR RGL1 Expression levels were evaluated. Compared to WT, the OX6-2 and OX13-6 lines RGL1 The expression level was significantly high, on the other hand ri6-6 class ri36-5 lineage RGL1 The expression level was significantly lower than that of WT (Fig. 11). Under normal growth conditions, WT, rgl1 RNAi, and RGL1 The seeds of the overexpressed plants showed a similar germination rate (Fig. 11). RGL1 To investigate the association between expression levels and dehydration response, WT, rgl1RNAi, and RGL1 Seeds of overexpression plants were grown in MS medium containing 400 mM mannitol. In the presence of 400 mM mannitol, the cotyledon greening rate of WT plants was found to be approximately 40% or less 10 days after germination. However, this greening rate ri6-6 and ri36-5 It decreased in the lineage (10.4%–20.7%), and increased in the OX6-2 and OX13-6 lines (74.7%–79.4%) (Figs. 9a and 9b). These results rgl1 RNAi strains are more sensitive to dehydration stress than WT, and RGL1 The overexpression line showed less sensitivity than WT, indicating that RGL1 acts as a positive regulator of the osmotic stress response.

[0123] Furthermore, the inhibition of root elongation under osmotic stress conditions was investigated. Under normal conditions, WT and RGL1 There was no clear difference in taproot length among overexpressed plants. However, rgl1 The taproot length of the RNAi lines was shorter than WT (Figs. 9c and 9d). This suggests that RGL1 plays an important role in root elongation. When treated with 400 mM mannitol, rgl1 The root length of the RNAi lines (ri6-6 and ri36-5) was similar to WT, but RGL1 Root growth of overexpressing plants is WT and rgl1 It was significantly larger than RNAi plants (Figs. 9c and 9d). These results are based on WT, atrzf1 , and pca31 It is reminiscent of the mannitol-induced root inhibition pattern observed in, which RGL1 It depends on the expression level (Figs. 2c, 2d, and 3d). In conclusion, this suggests that RGL1 levels play an important role in root growth and elongation under osmotic stress.

[0125] 11. RGL1 The effect of reduced expression on vascular tissue development

[0126] According to a recent report, Pectate Lyase-Like (PLL) protein, an enzyme that breaks down pectin, A. thaliana It affects root growth, available sugar levels, and vascular bundle development. RGL1 Considering that the transcription level affects pectin content and taproot elongation, in plant flower stalk stems RGL1 The effect of expression levels on vascular bundle development was investigated. To this end, 3-week-old WT, using a microscope and cross-sections of flower stalks, RGL1 Overexpression, and rgl1 The composition of vascular tissue in RNAi plants was investigated. The overall vascular tissue morphology and stem diameter were WT, RGL1 Overexpression, and rgl1 The number of RNAi transgenic seedlings was similar between them (Fig. 10a). However, the number of vascular bundles was similar between WT and RGL1 Although they were similar among the overexpressed seedlings, rgl1 In RNAi lineages, WT and RGL1 The number of vascular bundles was lower than that of overexpressing plants (Figs. 10a and 10b). From these results, RGL1 It was confirmed that vascular tissue development is inhibited at low expression levels.

Claims

Claim 1 A method for enhancing drought tolerance of plants, comprising a step of increasing the expression of RGL1 (Rhamnogalacturonan lyase 1) in plants. Claim 2 A method for enhancing drought resistance of a plant according to claim 1, comprising the step of transforming the plant with a recombinant vector into which a gene encoding RGL1 is inserted. Claim 3 A method for enhancing drought resistance of a plant according to claim 1, comprising the step of treating the plant with an expression promoter of RGL1. Claim 4 A method for improving drought resistance of a plant according to claim 1, wherein RGL1 is composed of an amino acid sequence of any one of SEQ ID NOs 1 to 9. Claim 5 A method for enhancing drought resistance of a plant according to claim 1, wherein the gene encoding RGL1 consists of the nucleotide sequence of SEQ ID NO.

10. Claim 6 A method for enhancing drought resistance of a plant according to claim 3, wherein the expression promoter of RGL1 is any one selected from the group consisting of mannitol, NaCl, ABA, and H2O2. Claim 7 A method for enhancing drought resistance of a plant according to claim 1, wherein the plant is any one of the group consisting of Arabidopsis thaliana, rice, corn, barley, napa cabbage, rapeseed, soybean, tomato, and chili pepper. Claim 8 A method for enhancing drought resistance of a plant according to claim 1, wherein the plant is a plant cell, a plant seed, or a plant body. Claim 9 A method for enhancing drought resistance of a plant according to claim 2, wherein the recombinant vector is a recombinant vector in which the RGL1 gene is operably linked to a promoter. Claim 10 A plant with enhanced drought resistance obtained according to the method of any one of claims 1 to 9.

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