KR20240167569A - OsBGAL9 gene and use thereof for promoting rice growth and increasing stress tolerance - Google Patents

Abstract

The present invention relates to the OsBGAL9 gene for promoting rice growth and increasing stress tolerance, and to uses thereof. Transgenic plants overexpressing the OsBGAL9 gene derived from rice of the present invention have strong tolerance to high temperature and low temperature stress, and have the effect of resisting blast disease and white leaf blight. In addition, it has the effect of promoting plant stem growth. Therefore, the OsBGAL9 gene is expected to be very useful for the development of transgenic plants with increased stress tolerance and promoted plant growth, and since plants with strong stress tolerance and promoted plant stem growth can be obtained by using the gene, it is expected to greatly contribute to increasing the yield of economic crops, etc.

Description

{OsBGAL9 gene and use thereof for promoting rice growth and increasing stress tolerance}

The present invention relates to the OsBGAL9 gene and its use for promoting rice growth and increasing stress tolerance.

Rice ( Oryza sativa ) is one of the most important crops in the world for the purpose of seed production, but more than 60 types of diseases occur in rice, and depending on the year, when the damage is severe, it can reduce production by 50-90%. In addition, abnormal climate phenomena are gradually becoming more severe as the average temperature of the earth rises due to the increase in greenhouse gases caused by industrialization and deforestation. As a result, crop production is decreasing, and this is likely to lead to a food problem. Such damage has emerged as the biggest obstacle to securing a stable food supply for mankind, and in Korea, for example, in 1978, rice head blast disease occurred in the Tongil variety, causing a serious problem in terms of food security, and as a result, a large amount of rice was imported from abroad, which became a social problem for a while.

To solve these problems, plants with increased stress tolerance gene function were produced, but the problem occurred that stress tolerance was increased but plant growth was inhibited. Similarly, the problem occurred that stress tolerance was weakened when plant growth was promoted.

Accordingly, the inventors of the present invention discovered OsBGAL9 for the development of transgenic plants with increased stress tolerance and promoted plant growth, and determined that it has high agricultural utility as a gene that promotes rice growth while simultaneously regulating complex resistance to the environment and pathogens, and thus completed the present invention.

Republic of Korea Patent Publication No. 10-1985668 (May 29, 2019)

The purpose of the present invention is to provide a recombinant vector for increasing stress tolerance or promoting growth of a plant, which comprises a gene encoding BGAL9 (beta-galactosidase 9).

In addition, another object of the present invention is to provide a transgenic plant into which a gene encoding BGAL9 (beta-galactosidase 9) is introduced or overexpressed.

In addition, another object of the present invention is to provide seeds of transgenic plants.

In addition, another object of the present invention is to provide a method for increasing stress tolerance or promoting growth of a plant, which comprises a step of introducing or overexpressing a gene encoding BGAL9 (beta-galactosidase 9) in the plant.

In addition, another object of the present invention is to provide a method for producing a transgenic plant with increased stress tolerance or accelerated growth, which comprises a step of introducing a recombinant vector containing a gene encoding BGAL9 (beta-galactosidase 9) into a plant and transforming the plant.

To achieve the above purpose, the present invention provides a recombinant vector for increasing stress tolerance or promoting growth of a plant, which comprises a gene encoding BGAL9 (beta-galactosidase 9).

Next, the present invention provides a transgenic plant into which a gene encoding BGAL9 (beta-galactosidase 9) is introduced or overexpressed.

Furthermore, the present invention provides seeds of transgenic plants.

In addition, the present invention provides a method for increasing stress tolerance or promoting growth of a plant, comprising a step of introducing or overexpressing a gene encoding BGAL9 (beta-galactosidase 9) in the plant.

Finally, the present invention provides a method for producing a transgenic plant with increased stress tolerance or accelerated growth, comprising the step of introducing a recombinant vector containing a gene encoding BGAL9 (beta-galactosidase 9) into the plant and transforming the plant.

The transgenic plant overexpressing the rice-derived OsBGAL9 gene of the present invention has strong tolerance to high temperature and low temperature stress, and has the effect of resisting blast disease and white leaf blight. In addition, it has the effect of promoting plant stem growth. Therefore, the OsBGAL9 gene is expected to be very useful for the development of transgenic plants with increased stress tolerance and promoted plant growth, and since plants with strong stress tolerance and promoted plant stem growth can be obtained by using this, it is expected to greatly contribute to increasing the yield of economic crops, etc.

Figure 1 is a diagram showing the expression of OsSPL7 and OsBGAL9 in WT (Wild Type), OsSPL7-OX, and Osspl7 plants according to one embodiment of the present invention. A and B used rice ACTIN as an internal control. C and D used rice UBQ5 as an internal control.
(A), (C) Relative expression levels of OsSPL7.
(B), (D) Relative expression levels of OsBGAL9.
FIG. 2 is a diagram showing a phylogenetic tree and sequence analysis of OsBGAL9 according to one embodiment of the present invention.
(A) Diagram of the OsBGAL9 gene (black boxes: exons, lines: introns, red triangles: HSE binding sites).
(B) Phylogenetic relationship of OsBGAL9 and other family GH35 proteins.
(C) Protein sequence of OsBGAL9.
FIG. 3 is a diagram showing the identification of the OsSPL7 binding site in the OsBGAL9 promoter according to one embodiment of the present invention.
(A) Diagram of the two HSE binding sites in the OsBGAL9 promoter.
(B) Schematic diagram of the constructs used for transcriptional activity analysis in rice protoplasts.
(C) Relative LUC/GUS activity in transformed protoplasts.
(D) Y1H assay showing that OsSPL7 binds to the OsBGAL9 promoter.
(E) EMSA analysis to detect OsSPL7 binding to P-type HSE in the OsBGAL9 promoter.
FIG. 4 is a diagram showing the production of OsBGAL9 loss-of-function and overexpression lines in one embodiment of the present invention.
(A) Diagram of the CRISPR/Cas9 target and T-DNA insertion sites in OsBGAL9.
(B) Diagram of the OsBGAL9 overexpression construct.
(C) Sequence analysis of the target site in Osbgal9 mutants generated by CRISPR/Cas9.
(D) Relative OsBGAL9 expression levels in HY (Wild Type) and T-DNA insertion loss-of-function plants Osbgal9-t.
(E) Relative OsBGAL9 expression levels in overexpression and CRISPR/Cas9 loss-of-function plants determined by RT-qPCR.
FIG. 5 is a diagram showing the phenotypes of OsBGAL9 overexpressing and Osbgal9 loss-of-function plants according to one embodiment of the present invention.
(A) Growth phenotypes of paddy-grown OsBGAL9-OX and Osbgal9 plants at mature stage.
(B) Plant heights of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 at the mature stage.
(C) Internode morphology of DJ (Wild Type), OsBGAL9-OX, and Osbgal9.
(D) The length between each node and the relative contribution of each node to the total stem length.
(E) Representative cross-section of the second segment.
(F) Cell length of the second segment observed in cross-section.
FIG. 6 is a diagram showing the phenotype of an Osbgal9-t T-DNA mutant plant compared to a wild-type control HY in one embodiment of the present invention.
(A) Growth phenotypes of Osbgal9-t and HY plants grown in paddy fields at the mature stage.
(B) Plant height of Osbgal9-t and HY plants.
(C) Internode morphology of HY (Wild Type) and Osbgal9-t.
(D) Relative contribution of each node to the total stem length of HY (Wild Type) and Osbgal9-t plants.
(E) Representative cross-section of the second segment.
(F) Cell length of the second segment observed in cross-section.
FIG. 7 is a diagram showing histochemical GUS staining of an OsBGAL9pro:GUS plant according to one embodiment of the present invention.
(A) Tissue stained at some stage.
(B) Tissue stained at the flowering stage.
(C) Relative GUS and OsBGAL9 transcript levels in different tissues.
(D) Stained tissue after exposure to low temperature (4 °C) or high temperature (42 °C) for 24 hours.
(E) Relative transcript levels of GUS, OsBGAL9, and OsSPL7 in plants exposed to low and high temperatures.
(F) Tissue stained 48 hours after infection with Magnaporthe oryzae .
(G) Relative transcript levels of GUS, OsBGAL9, and OsSPL7 after M. oryzae infection.
FIG. 8 is a diagram showing the expression of OsBGAL9 in OsSPL7 overexpressing and loss-of-function plants under stress conditions in one embodiment of the present invention.
(A) Relative expression levels of OsBGAL9 in plants of the indicated genotypes under stress conditions.
(B) Relative expression levels of OsBGAL9, OsBGAL2, and OsBGAL4 in DJ (Wild Type) plants under stress conditions.
Figure 9 is a diagram showing the disease resistance of OsBGAL9-OX and Osbgal9 plants according to one embodiment of the present invention. The red asterisk indicates the basal tip of a water-soaked lesion.
(A) Representative disease phenotypes of OsBGAL9-OX and Osbgal9 leaves after inoculation with M. oryzae PO6-6 .
(B) Lesion lengths on OsBGAL9-OX and Osbgal9 leaves after inoculation with M. oryzae PO6-6 .
(C) Representative disease phenotypes of OsBGAL9-OX and Osbgal9 leaves after inoculation with Xanthomonas oryzae pv. oryzae ( Xoo ) PXO99 .
(D) Lesion lengths on OsBGAL9-OX and Osbgal9 leaves after inoculation with Xoo PXO99 .
Figure 10 is a diagram showing the disease resistance of Osbgal9-t plants in one embodiment of the present invention. The red asterisk indicates the basal tip of a water-soaked lesion.
(A) Representative disease phenotypes of Osbgal9-t leaves after inoculation with M. oryzae PO6-6 .
(B) Lesion length on Osbgal9-t leaves after inoculation with M. oryzae PO6-6 .
(C) Representative disease phenotypes of Osbgal9-t leaves after inoculation with Xoo PXO99 .
(D) Lesion length on Osbgal9-t leaves after inoculation with Xoo PXO99 .
FIG. 11 is a diagram showing the cold resistance and heat resistance of OsBGAL9-OX and Osbgal9 plants in one embodiment of the present invention.
(A) Phenotypes of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 plants after cold exposure.
(B) Phenotypes of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 plants after high temperature exposure.
(C) Survival rates of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 plants after low temperature exposure.
(D) Survival rates of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 plants after exposure to high temperature.
FIG. 12 is a diagram showing the heat resistance and cold resistance of Osbgal9-t and HY (Wild Type) in one embodiment of the present invention.
(A) Phenotypes of HY (Wild Type) and Osbgal9-t seedlings after 1 week of recovery from cold stress treatment for 72 h at 4 ℃.
(B) Phenotypes of HY (Wild Type) and Osbgal9-t seedlings after 1 week of recovery from high temperature stress treatment at 42°C for 24 h.
(C) Survival rate of Osbgal9-t seedlings after recovery from cold stress.
(D) Survival rate of Osbgal9-t seedlings after recovery from high temperature stress.
Figure 13 is a diagram showing the intracellular localization of OsBGAL9 according to one embodiment of the present invention. Blue arrows indicate cells that have undergone plasmolysis, and white arrows indicate GFP signals in the cell wall.
FIG. 14 is a diagram showing Bgal activity in recombinant OsBGAL9 and OsBGAL9-OX and Osbgal9 plants according to one embodiment of the present invention.
(A) SDS-PAGE and Coomassie Brilliant Blue staining of purified recombinant OsBGAL9 produced in HEK293 cells.
(B) Bgal activity assay of recombinant OsBGAL9 using X-Gal or ONPG as substrate.
(C) In-gel activity analysis of recombinant OsBGAL9.
(D) Analysis of Bgal activity toward ONPG in OsBGAL9-OX and OsBGAL9 loss-of-function plants.
FIG. 15 is a diagram showing Bgal activity for ONPG in Osbgal9-t mutant and HY (Wild Type) according to one embodiment of the present invention.
(A) Bgal activity assay for ONPG in the second node.
(B) Bgal activity assay for ONPG in leaves.
FIG. 16 is a diagram showing the relative expression levels of BGAL in leaves of OsBGAL9 overexpressing and Osbgal9 loss-of-function plants in one embodiment of the present invention.
FIG. 17 is a diagram showing MALDI-TOF mass spectra of xyloglucan oligomers after incubation with heat-denatured recombinant OsBGAL9 (control; upper panel) and activated OsBGAL9 (lower panel) in one embodiment of the present invention.
FIG. 18 is a diagram showing immunodetection of cell wall components in the cross-section of the second segment of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 in one embodiment of the present invention.
FIG. 19 is a diagram showing the immunodetection of cell wall components in the second internode cross-section of HY (Wild Type) and Osbgal9-t in one embodiment of the present invention.
FIG. 20 is a diagram showing Yariv staining of AGP in the second internode of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 mutant plants in one embodiment of the present invention.
(A) Staining with β-D-glucosyl Yariv reagent (β-GlcY).
(B) Staining with β-D-galactosyl Yariv reagent (β-GalY).
FIG. 21 is a diagram showing Yariv staining of AGP in the second node of HY (Wild Type) and Osbgal9-t mutant plants in one embodiment of the present invention.
(A) Staining with β-D-glucosyl Yariv reagent (β-GlcY).
(B) Staining with β-D-galactosyl Yariv reagent (β-GalY).
FIG. 22 is a diagram showing a comparison of the cell wall composition between the second nodes of DJ (Wild Type), OsBGAL9-OX, and Osbgal9 mutant plants in one embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to the attached drawings as implementation examples of the present invention. However, the following implementation examples are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to “include” a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless specifically stated otherwise.

Throughout this specification, '%' used to indicate the concentration of a particular substance, unless otherwise stated, is solid/solid (w/w) %, solid/liquid (w/v) %, and liquid/liquid (v/v) %.

In one aspect, the present invention provides a recombinant vector for increasing stress tolerance or promoting growth of a plant, comprising a gene encoding BGAL9 (beta-galactosidase 9).

The term “BGAL9 (beta-galactosidase 9)” used in the present invention refers to an enzyme that promotes the breakdown of lactose into galactose, and breaks down the sugar into glucose and galactose.

The term “OsBGAL9 (Oryza sativa beta-galactosidase 9)” used in the present invention plays an important role in the growth and development of rice and in certain biological situations. The activity of this gene is regulated by certain stress conditions, thereby helping rice adapt to and survive changes in the external environment.

In one embodiment of the present invention, the OsBGAL9 may be, but is not limited to, a protein localized to the cell wall.

In one embodiment of the present invention, the BGAL9 (beta-galactosidase 9) may be derived from rice ( Oryza sativa ), but is not limited thereto.

In the present invention, the term “rice” has the scientific name Oryza sativa L. and is an annual herbaceous plant. The rice in the present invention may be any one selected from the group consisting of the Japonica type, the Indica type, and the Javanica type.

In one embodiment of the present invention, the BGAL9 (beta-galactosidase 9) may be represented by the base sequence of sequence number 1, but is not limited thereto.

In one embodiment of the present invention, the stress may be, but is not limited to, biological and abiotic stress.

In one embodiment of the present invention, the biological stress may be at least one disease selected from the group consisting of blast, white leaf blight, leg blight, grain blight, seed spot or sheath blight, more preferably, infection with blast or white leaf blight, but is not limited thereto.

In one embodiment of the present invention, the abiotic stress may be, but is not limited to, high temperature or low temperature stress.

In one embodiment of the present invention, the high temperature stress may be, but is not limited to, a temperature of 30° C. or higher, preferably 35° C. or higher, more preferably 42° C. or higher.

In one embodiment of the present invention, the low temperature stress may be, but is not limited to, a temperature of 15° C. or lower, preferably 10° C. or lower, more preferably 4° C. or lower.

In one aspect, the present invention provides a transgenic plant into which a gene encoding BGAL9 (beta-galactosidase 9) is introduced or overexpressed.

In one embodiment of the present invention, the plant may be at least one selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onions, more preferably rice, but is not limited thereto.

In one aspect, the present invention provides seeds of transgenic plants.

In one aspect, the present invention provides a method for increasing stress tolerance or promoting growth of a plant, comprising the step of introducing or overexpressing a gene encoding BGAL9 (beta-galactosidase 9) in the plant.

In one aspect, the present invention provides a method for producing a transgenic plant with increased stress tolerance or accelerated growth, comprising the step of introducing a recombinant vector containing a gene encoding BGAL9 (beta-galactosidase 9) into the plant and transforming the plant.

In one embodiment of the present invention, the recombinant OsBGAL9 and OsBGAL9-OX and Osbgal9 plants may have Bgal activity, but are not limited thereto.

In one embodiment of the present invention, the OsBGAL9 may hydrolyze galactose residues of AGPs, but is not limited thereto.

In one embodiment of the present invention, the modification of the AGPs may be, but is not limited to, to grow a plant.

As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other backward inventions or other embodiments included within the scope of the spirit of the present invention by adding, changing, deleting, etc. other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are exemplary and not restrictive in all respects. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modifications derived from the meaning and scope of the claims and the equivalent concept should be interpreted as being included in the scope of the present invention.

<Example 1> Production of OsBGAL9 mutant

Osbgal9-t (PFG_1C-06424.L), a T-DNA insertion loss-of-function mutant of OsBGAL9 represented by the nucleotide sequence of SEQ ID NO: 1, was verified by isolating a homozygous strain using Hwayoung (HY, wild type), a japonica rice seed type, by genomic PCR using T-DNA and gene-specific primers (SEQ ID NOs: 2 and 3) (see Table 1).

Loss-of-function mutant lines of OsBGAL9, Osbgal9-1 and Osbgal9-2, were generated by CRISPR/Cas9 gene editing, using the rice cultivar Dongjin (DJ, wild type). The CRISPR direct program was used to screen possible target sequences to find effective Protospacer Adjacent Motifs (PAMs) and avoid off-target effects. A single guide RNA (sgRNA, SEQ ID NOs: 4 and 5) was cloned into the entry vector pOs-sgRNA and then cloned into the destination vector pH-Ubi-cas9-7 using the Gateway system. The resulting constructs were transformed into DJ by Agrobacterium tumefaciens -mediated transformation. The mutant genotypes were determined by Sanger sequencing of the genomic regions flanking the target region after PCR amplification with specific primers (SEQ ID NOs: 6 and 7).

In addition, OsBGAL9-OX, an OsBGAL9 overexpression line, was generated by containing the OsBGAL9 coding sequence using the maize Ubiquitin1 (UBI) promoter in the pGA3438 recombinant vector. The cDNA of OsBGAL9 is represented by the nucleotide sequence of SEQ ID NO: 25. The OsBGAL9 overexpression recombinant vector was transformed into DJ (Wild Type) by Agrobacterium tumefaciens -mediated transformation. Rice plants were grown in a greenhouse under a light/dark cycle of 14 h at 30 °C during the day and 10 h at 20 °C during the night or under natural environmental conditions in summer.

Primer name Primer Sequence Sequence number Bgal_T-DNA F TGCCATTGTGCAAGTGTTACCC Sequence number 2 R GCCGCATGTGAAATCTGGATCC Sequence number 3 Bgal9_sgRNA F GGCAAAAATTGAAAGAGCTAGTTA Sequence number 4 R AAACTAACTAGCTCTTTCAATTTT Sequence number 5 Bgal9 PCR primer F GGAGTATGGGGACGTGCATAAT Sequence number 6 R TTCTTACCTGGCCCATCAGTTC Sequence number 7

<Experimental Example 1> OsBGAL9 expression and function analysis

In a previous study, it was confirmed that the expression of OsBGAL9 was increased in plants overexpressing OsSPL7 (OsSPL7-OX), and in the present invention, the expression of OsBGAL9 was confirmed in plants overexpressing OsSPL7 (OsSPL7-OX) and plants with OsSPL7 function loss (Osspl7).

To confirm the expression of OsBGAL9, RNA isolation and RT-qPCR analysis were performed. Total RNA was extracted from the collected samples using the Qiagen RNeasy mini kit according to the manufacturer's instructions. 2 μg of total RNA was synthesized into primary cDNA using ReverTra Ace ^® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). qPCR was performed on a Rotor-Gene 6000 real-time amplification system (Corbett Research, Sydney, Australia) using specific primers (SEQ ID NOs: 23 and 24) and Prime Q-Master Mix 2X (GENTBIO). UBIQUITIN5 (OsUBQ5; LOC_Os01g22490) (SEQ ID NOs: 8 and 9) and ACTIN (OsACT; LOC_Os03g61970) (SEQ ID NOs: 10 and 11) genes were used as referenced in Table 2 below. The results confirming the expression of OsBGAL9 are shown in Figure 1.

Primer name Primer Sequence Sequence number OsBGAL9 F GGGGATTTCCACCTTGGTTACT Sequence number 23 R CTTCTGGCAACTTCAACAAGGT Sequence number 24 OsUBQ5 F GCACAAGCACAAGAAGGTGA Sequence number 8 R GCCTGCTGGTTGTAGACGTA Sequence number 9 OsACT F GTCTTGTGAGCTTTGCATGGT Sequence number 10 R ACAAACGAGCTTGGATCTACC Sequence number 11

As shown in Fig. 1, it was confirmed that the expression of OsBGAL9 was increased in OsSPL7-OX. Figs. 1A and B used rice ACTIN as an internal control, and Figs. 1C and D used rice UBQ5 as an internal control.

Through this, we confirmed that the expression of OsBGAL9 is regulated by the transcription factor OsSPL7.

A BLAST search of the OsBGAL9 promoter in NCBI and PlantPAN 3.0 online databases revealed the presence of two P-type HSE binding sites (TTCNNGAANNTTC) located 900 bp and 701 bp upstream from the ATG, respectively, as shown in Fig. 2A.

To confirm the function of OsBGAL9, Arabidopsis thaliana , mustard ( Brassica campestri ), sweet potato ( Ipomoea batatas ), Protein sequences of rice ( Oryza sativa ) and radish were analyzed, and a phylogenetic tree was reconstructed based on the analysis. The phylogenetic tree showed that OsBGAL9 belongs to group A together with AtBGAL17 of Arabidopsis thaliana, BcBGAL17 of radish, and IbBGAL17 of sweet potato, while other BGALs belong to group B, and this is shown in Fig. 2B.

Through this, we showed that OsBGAL9 belongs to a cluster that is not related to other plant-specific BGALs, and confirmed that it may have a function similar to BGALs in animals.

The protein sequence of OsBGAL9 was analyzed to determine whether N-glycosylation sites exist. As shown in Fig. 2C, five putative N-glycosylation sites (marked in red) were identified in the protein sequence analysis using NetNGlyc-1.0, suggesting that N-glycosylation may be required for OsBGAL9 activity. N-glycosylation is a process in which sugar molecules are attached to proteins, and sugar molecules can be inserted into proteins to affect the structure and function of the protein.

Through this, we confirmed that N-glycosylation may be required for the activity of OsBGAL9, which means that it may play an important role in regulating the function of the protein.

<Experimental Example 2> Confirmation of interaction between OsSPL7 and OsBGAL9

2-1. Confirmation of OsBGAL9 regulation by transcription factor OsSPL7

To determine whether OsBGAL9 is directly regulated by the transcription factor OsSPL7, transcriptional activity analysis was performed using rice protoplasts isolated from Oc suspension cells.

The OsBGAL9 promoter (SEQ ID NOs: 12 and 13) was cloned upstream of the firefly luciferase (LUC) reporter gene, and the OsSPL7 coding sequence was cloned as an effector construct downstream of the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 3B).

As shown in Figure 3C, overexpression of OsSPL7 from 35S:OsSPL7 induced significant LUC activity compared to the OsBGAL9pro:LUC reporter construct.

Through this, we confirmed that OsBGAL9 is directly regulated by the transcription factor OsSPL7.

2-2. Confirmation of interaction between OsSPL7 and OsBGAL9 promoters

To confirm the direct interaction between OsSPL7 and OsBGAL9 promoters (SEQ ID NOs: 12 and 13), a yeast one-hybrid (Y1H) system was performed using the HIS reporter gene.

The Y1H system is an experimental technique to investigate the interaction between a DNA-binding protein and a DNA sequence of a gene, and is used to determine whether a specific protein binds to the promoter region of a gene. The full-length coding sequence of OsSPL7 was amplified using PCR primers containing NdeI (SEQ ID NO: 14) and BamHI (SEQ ID NO: 15) restriction enzyme sites (Table 3) and cloned in-frame with the sequence encoding the yeast GAL4 activation domain in pGADT7 (Clontech, USA), generating pGADT7-OsSPL7. The 1,079-bp promoter region of OsBGAL9 was amplified using PCR primers (SEQ ID NOs: 6 and 7) and cloned into the pHIS2 reporter vector (Clontech, USA) to generate the reporter construct pHIS2-OsBGAL9pro:HIS3. This was introduced into the yeast strain AH109 and transformed, and the transformants were selected on SD (Synthetic Defined) medium lacking Trp and Leu (SD-Trp/-Leu) at 30°C. To confirm the binding ability of positive transformants, they were spotted on SD-Trp/-Leu/-His medium containing 5 mM 3-AT (Amino Triazole) to test the binding ability. Yeast cells containing a combination of empty pGADT7 and pHIS2-OsBGAL9pro were used as a negative control. The results of the Y1H experiment are shown in Fig. 3D.

As shown in Fig. 3D, the growth of transformants showing that OsSPL7 binds to the OsBGAL9 promoter fragment was confirmed in the presence of 3-AT (SD/-Leu/-Trp/-His/+3AT) in the absence of Leu, Trp, and His in SD medium.

Through this, we confirmed that OsSPL7 directly interacts with the OsBGAL9 promoter.

Primer name Primer Sequence Sequence number SPL7-NdeI F CCCATATGATGGAGAGTTCCAACCTGGG Sequence number 14 SPL7-BamHI R CGGGATCCTCAGGTATGCAGAGTCTGCT Sequence number 15 SPL7-EcorI F GGAATTCCACATGGAGAGTTCCAACCTGGG Sequence number 16

2-3. Confirmation of OsBGAL9 promoter HSE binding of OsSPL7

To confirm whether recombinant OsSPL7 binds to the HSE (SEQ ID NOs: 17 and 18) of the OsBGAL9 promoter, an Electrophoretic Mobility Shift Assay (EMSA) was performed.

EMSA is an experimental technique to investigate the interaction between proteins and DNA, and is used to confirm the DNA-binding ability of proteins and visually analyze the formation of protein-DNA complexes. The coding sequence of OsSPL7 was amplified using PCR primers containing EcoRI (SEQ ID NO: 16) and BamHI (SEQ ID NO: 15) restriction enzyme sites (Table 3) and cloned in-frame and downstream of the maltose-binding protein (MBP) sequence in the pMAL-c2X vector and transformed into E. coli strain BL21. The recombinant MBP-OsSPL7 was purified using amylose magnetic beads according to the manufacturer's protocol (New England BioLabs, Korea). The 3' biotin-labeled probe of HSE cis-elements (SEQ ID NO: 19) was synthesized by Bionics (Daejeon, Korea). DNA gel shift analysis was performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The mobility of the labeled probe in the presence of recombinant OsSPL7 fused to MBP-OsSPL7 is shown in Figure 3E.

As shown in Figure 3E, the intensity of the shifted band decreased as the unlabeled probe was added. In contrast, OsSPL7 did not bind to the probe with mutations in the HSE fragment, as evidenced by the absence of a shifted band.

Through this, we confirmed that OsSPL7 directly binds to the P-type HSE element of the OsBGAL9 promoter and activates the transcription of OsBGAL9.

<Experimental Example 3> Analysis of phenotypic characteristics of OsBGAL9 loss-of-function and overexpression plants

3-1. Measurement of OsBGAL9 expression levels in plants with loss of OsBGAL9 function and overexpression of OsBGAL9

To understand the function of OsBGAL9, we obtained Osbgal9-t, a T-DNA mutant of OsBGAL9, and obtained two OsBGAL9 loss-of-function mutant lines (Osbgal9-1, Osbgal9-2) through CRISPR/Cas9 gene editing. Two OsBGAL9 overexpression lines (OsBGAL9-OX1, OsBGAL9-OX2) were produced for phenotypic testing (Fig. 4A and B). Sequencing of the target fragments in the OsBGAL9 loss-of-function mutant lines (Osbgal9-1 and Osbgal9-2) detected a single nucleotide insertion in each mutant. Osbgal9-1 had a cytosine (C) insertion, and Osbgal9-2 had a thymine (T) insertion (Fig. 4C). In addition, the expression levels of OsBGAL9 in Osbgal9 mutant and OsBGAL-OX plants were decreased in Osbgal9 mutant plants and increased in OsBGAL-OX plants (Fig. 4D and E).

3-2. Analysis of OsBGAL9 phenotypic characteristics of OsBGAL9 loss-of-function and overexpression plants

To analyze the phenotypic characteristics of OsBGAL9 loss-of-function and overexpression plants, histological analysis and internode cell length were measured.

At the flowering stage, 5-mm-thick tissues from the second nodes of rice plants grown in a rice field were fixed in FAA (50:5:10:35 by volume of 37% formaldehyde/acetic acid/95% ethanol/ion-exchange resin-treated water). The tissues fixed overnight were dehydrated with sequentially increasing concentrations of ethanol. The tissues were embedded in Paraplast Plus (Sigma, St. Louis, MO, USA) and sectioned to 8–10 µm thickness using a microtome. The microsectioned tissues were stained with 0.1% (w/v) toluidine blue and observed using an Olympus BX61 microscope (Olympus, Tokyo, Japan). Cell size was quantified in the longitudinal sections of the second nodes using ImageJ software.

In a rice field, 10-week-old Osbgal9-1 and Osbgal9-2 plants exhibited a dwarf phenotype compared to the wild-type control, Dongjin (DJ) cultivar (Fig. 5A and B). This was similar to what was observed in the Osbgal9-t plants, a T-DNA insertion loss-of-function mutant (Fig. 6A and B). In contrast, OsBGAL-OX plants exhibited increased plant height (Fig. 5A and B). As the plant height decreased, the internode length was significantly reduced in the Osbgal9 mutant (Osbgal9-1, Osbgal9-2) plants compared to the OsBGAL9-OX and DJ plants (Fig. 5C and D). Likewise, the internode length was reduced in the Osbgal9-t plants compared to the wild-type control, HY (Fig. 6C and D). Cross-sections of the second nodes of DJ, OsBGAL9-OX, and Osbgal9 mutant plants were prepared to measure the cell size. OsBGAL9-OX showed a 9–14% increase in the average cell length at the second node, while Osbgal9-1 and Osbgal9-2 showed a 30–37% decrease in the average cell length at the second node (Fig. 5E and F). Similarly, Osbgal9-t plants showed a 45.9% decrease in the average cell length at the second node compared to HY (Fig. 6E and F).

Through this, we confirmed that OsBGAL9 plays an important role in cell elongation of internode tissue and causes changes in plant height in OsBGAL9 overexpressing and loss-of-function plants.

<Experimental Example 4> Analysis of OsBGAL9 expression pattern

4-1. Analysis of temporal and spatial expression patterns of OsBGAL9

To investigate the temporal and spatial expression pattern of OsBGAL9, histochemical staining of transgenic rice plants expressing ß-GLUCURONIDASE (GUS) under the control of the OsBGAL9 promoter (SEQ ID NOs: 12 and 13) was performed. GUS PCR primers are shown in SEQ ID NOs: 21 and 22.

GUS analysis is one of the marker analysis methods to investigate the location and level of gene expression, and measures the activity of GUS enzyme that indicates transcriptional activity by linking the GUS gene to the promoter region of the desired gene. The OsBGAL9pro:GUS construct was obtained by cloning the 1.9-kb promoter region (SEQ ID NO: 20) upstream of the start codon of OsBGAL9 upstream of GUS into the modified pCAMBIA1300-Gateway-GUSPlus vector. The resulting plasmid was introduced into rice using Agrobacterium -mediated transformation. T3 homozygous lines were selected based on the hygromycin resistance of all progeny. Tissues from 8-week-old plants were harvested, incubated in 90% (v/v) acetone on ice for 30 min, and placed under vacuum at room temperature for 10 min, and analyzed by GUS staining assay.

GUS staining analysis results showed that strong GUS signals were detected in internode tissues in flowering-stage rice, but not in 2-week-old rice (Fig. 7A and B). Reverse transcription-quantitative PCR (RT-qPCR) analysis of GUS and OsBGAL9 was performed, and the results showed that OsBGAL9 is preferentially expressed during stem elongation, which causes the differences in plant height observed in OsBGAL9 overexpression and loss-of-function mutants (Fig. 7C).

4-2. Analysis of biological and abiotic stress responses of OsBGAL9

Since OsSPL7 functions in biotic and abiotic stress responses, the potential co-expression of OsSPL7 and OsBGAL9 was investigated. Two-week-old rice plants harboring OsBGAL9pro:GUS reporter were exposed to biotic and abiotic stresses such as low temperature (4 °C), high temperature (42 °C), and infection with the rice blast fungus Magnaporthe oryzae , and then the expression of OsSPL7 and OsBGAL9 was measured.

To expose rice to abiotic stresses, 20 homozygous OsBGAL9-overexpressing and Osbgal9-loss-function plants germinated on half-strength MS (Murashige and Skoog) medium were transferred to field conditions for abiotic stress treatments. For heat tolerance evaluation, 3-week-old rice plants were exposed to 42 °C for 24 h in a growth room with high relative humidity (> 90%) and maintained under field conditions for recovery for 1 week. To evaluate cold tolerance, 3-week-old rice plants were exposed to 4 °C for 72 h and maintained under field conditions for recovery. Survival rate was calculated based on the number of surviving plants after 1 week. Three biological replicates were used per genotype.

When rice plants were exposed to abiotic stress, GUS activity increased in the rice plants exposed to abiotic stress, while no GUS activity was observed in the control rice plants (Fig. 7D). In addition, the expression of OsBGAL9 and OsSPL7 increased in the rice plants exposed to abiotic stress compared to the control group (Fig. 7E).

To expose rice to biotic stress, the rice blast fungus ( Magnaporthe oryzae ) PO6-6 strain was cultured on agar medium (V8 Juice medium, 80 mL/L) (Campbell's Soup Company, Camden, NJ), and then used to inoculate rice with blast disease. After culturing for 2 weeks under continuous fluorescent light, the spores were collected and suspended in water until the spore concentration reached 5 × 10 ⁶ mL ^-1 . The spore suspension was spot inoculated onto injured rice leaves at 5 weeks of age. Ten days post-inoculation (DPI), leaf samples were collected and photographed, and the length of the disease lesions (brown area) was measured using ImageJ software.

When rice was exposed to biotic stress, GUS activity increased in the rice exposed to biotic stress (Fig. 7F), and the expression of OsBGAL9 and OsSPL7 increased compared to the control group (Fig. 7G).

In the absence of OsSPL7, the induction of OsBGAL9 expression in response to biotic and abiotic stresses was significantly reduced, while overexpression of OsSPL7 promoted the expression of OsBGAL9 (Fig. 8A). In contrast, other BGALs, such as OsBGAL2 and OsBGAL4, showed little or no change in expression in response to stress treatment (Fig. 8B).

These results suggest that OsBGAL9 exhibits a dual expression mode, being expressed primarily within the stem under normal growth conditions and throughout the whole plant under extreme temperatures and pathogen attack.

<Experimental Example 5> Analysis of Biological and Abiotic Stress Responses in OsBGAL9 Loss-of-Function and Over-expression Plants

To analyze the biotic and abiotic stress responses of OsBGAL9 loss-of-function and overexpression plants, OsBGAL9 loss-of-function and overexpression rice plants were infected with the rice blast fungus Magnaporthe oryzae or the bacterial leaf blight fungus Xanthomonas oryzae pv. oryzae ( Xoo ) and exposed to low temperature (4 °C) or high temperature (42 °C).

To expose rice to biological stress, it was infected with the rice blast fungus ( Magnaporthe oryzae ) or the white leaf blight fungus ( Xanthomonas oryzae pv. oryzae ( Xoo )). The rice blast fungus infection was performed in the same manner as in Experimental Example 4-2. The Xanthomonas oryzae pv. oryzae ( Xoo ) PXO99 strain was cultured on peptin sucrose agar medium (10 g/L sucrose, 10 g/L peptone, 1 g/L L-glutamate, 15 g/L agar, pH 6.8) and suspended in sterilized water until the OD ₆₀₀ reached 0.6. To inoculate 5-week-old rice plants, the leaf cutting method was used and the success of Xoo infection was assessed based on the length of water-soaked lesions, which was observed and measured after 12 days (DPI).

When OsBGAL9 loss-of-function and overexpression-of-function rice plants were exposed to biotic stress, OsBGAL9-OX plants exhibited enhanced resistance to rice blast fungus ( M. oryzae ) at 10 days post-inoculation (DPI), whereas Osbgal plants were more susceptible to rice blast fungus than DJ, the control (Fig. 9A and B). Similarly, OsBGAL9-OX plants exhibited shorter water-soaked lesions at 10 days post-inoculation (DPI) with the bacterium Xoo, whereas Osbgal9 plants exhibited longer lesions (Fig. 9C and D). In addition, when Osbgal9-t plants were infected with M. oryzae or Xoo , they were more susceptible to rice blast fungus than HY, the control (Fig. 10A to D).

Through this, we confirmed that OsBGAL9 plays an important role in improving resistance to rice blast fungus ( M. oryzae ) and white leaf blight fungus ( Xoo ).

Exposure to abiotic stresses, low temperature (4 ℃) or high temperature (42 ℃), was performed in the same manner as in Experimental Example 4-2, and when OsBGAL9 function-loss and overexpression rice plants were exposed to abiotic stresses, the survival rate of OsBGAL9-OX plants was significantly higher than that of DJ for one week after low temperature exposure. In contrast, only a small number of rice plants survived in Osbgal9 plants (Fig. 11 A and C). After high temperature exposure for one week, Osbgal9-1 and Osbgal9-2 plants showed severe wilting and chlorophyll deficiency (Fig. 11 B and D). Similarly, Osbgal9-t plants showed greater sensitivity to low and high temperature stress than HY when exposed to low (Fig. 12 A and C) or high temperature (Fig. 12 B and D).

Through this, we confirmed that increased OsBGAL9 expression enhanced resistance to low and high temperature stress.

<Experimental Example 6> Analysis of the intracellular localization of OsBGAL9

To analyze the subcellular localization of OsBGAL9, the GFP signal associated with the OsBGAL9-GFP (Green Fluorescent Protein) fusion was traced in transgenic plants harboring the 35S:OsBGAL9-GFP transgene to investigate the subcellular localization of OsBGAL9.

To analyze the subcellular localization of OsBGAL9, the full-length coding sequence of OsBGAL9 lacking the stop codon was amplified using PCR primers (SEQ ID NOs: 6 and 7) and cloned into the pENTR™/D-TOPO™ vector (Invitrogen, Gaithersburg, MD, USA). The OsBGAL9 cDNA was confirmed by sequencing and recombined into the binary destination vector pH7FWG2 with a GFP tag located at the C terminus using Gateway LR Clonase (Invitrogen, Gaithersburg, MD, USA). The OsBGAL9-GFP construct was transformed into rice callus by Agrobacterium-mediated transformation. The subcellular localization of OsBGAL9 was observed using a confocal microscope (LSM510 META, Carl Zeiss, Jena, Germany). GFP fluorescence was excited at 488 nm and detected with a band-pass filter at 490–540 nm (GFP panel).

Subcellular localization analysis of OsBGAL9 detected GFP fluorescence at the border of root epidermal cells to determine plasma membrane or cell wall localization. To clarify the localization of OsBGAL9, rice plants were cultured in 30% (w/v) sucrose solution for 15 min to induce plasma lysis before confocal scanning. GFP signal remained in the cell wall, whereas the plasma membrane shrank and detached from the cell wall (Fig. 13). This suggests that OsBGAL9 is a cell wall-localized protein.

<Experimental Example 7> Confirmation of Bgal activity in recombinant OsBGAL9, OsBGAL9 loss-of-function and overexpression plants

7-1. Generation of recombinant OsBGAL9

To confirm that OsBGAL9 is BGAL, recombinant OsBGAL9 was produced and in vitro activity assays were performed.

Although production of recombinant OsBGAL9 in Escherichia coli and Pichia pastoris failed, recombinant OsBGAL9 (amino acids 27-673) without a signal peptide was successfully produced in a secreted form in HEK293 cells. The OsBGAL9 coding sequence was cloned in-frame between the murine Igκ chain leader sequence and the C-terminal myc epitope/6 tandem histidine tags for protein secretion. It was inserted into the pSecTag2 mammalian expression vector (Invitrogen, MA, USA). The OsBGAL9 expression construct was transcribed into HEK293 cells using the FreeStyle 293 expression system (Invitrogen) according to the manufacturer's instructions. After culturing for 5 days, the secreted recombinant OsBGAL9 was purified by passing the culture medium through a nickel resin column. The purified protein was confirmed by SDS-PAGE and Coomassie Brilliant Blue staining (Fig. 14A).

7-2. Analysis of Bgal activity of recombinant OsBGAL9

To analyze the Bgal activity of the generated recombinant OsBGAL9, the activity assay was performed with two typical synthetic Bgal substrates, X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) and ONPG (ortho-Nitrophenyl-ß-galactoside). X-Gal forms a blue product when Bgal enzyme decomposes it, and ONPG forms a yellow product when Bgal enzyme decomposes it. Therefore, when X-Gal and ONPG are used as Bgal substrates, the activity of Bgal can be visually confirmed.

For the activity assay in solution, the reaction mixture (100 μl) contained 10 μg recombinant OsBGAL9, 100 mM sodium phosphate buffer (pH 7.0), and 0.5 mM X-Gal or 1 mM ONPG as substrate. Control reactions used purified recombinant OsXOAT12 produced in HEK293 cells instead of OsBGAL9. After incubating the reaction mixture for 20 min at 37 °C, the color change of the reaction mixture was evaluated.

The Bgal activity of OsBGAL9 was analyzed using synthetic Bgal substrates, X-Gal and ONPG. Recombinant OsBGAL9 formed characteristic blue and yellow products, respectively, confirming that it could degrade both substrates. In contrast, the control, recombinant OsXOAT12, did not degrade either substrate, confirming that it did not show any color change (Fig. 14B).

To analyze the Bgal activity of the generated recombinant OsBGAL9, an in-gel activity assay was performed. The in-gel activity assay is one of the techniques for measuring the enzyme activity of a protein, and means confirming a specific substrate or enzyme activity within a gel after separating the protein through electrophoresis.

In-gel activity assay was performed using clear-native PAGE followed by incubation with X-Gal substrate. Purified recombinant OsBGAL9 (15 μg) was run on a native PAGE 4–16% Bis-Tris gel (Invitrogen) using gel running buffer (50 mM Bis-Tris, 50 mM tricine, pH 7.0) at 4 °C together with recombinant OsXOAT12 as a control. The gel was briefly rinsed in 100 mM sodium phosphate buffer (pH 7.0) and incubated for 1 h in the same buffer containing 0.5 mM X-Gal to detect Bgal activity. After recording the in-gel activity image, the same gel in the same buffer was stained for protein with Coomassie Brilliant Blue. Three individual transfections were performed, and the purified recombinant OsBGAL9 from these transfections were used as biological replicates for the activity assay, and representative images are shown.

The Bgal activity of OsBGAL9 was analyzed by performing an in-gel activity assay, and it was confirmed that the OsBGAL9 protein band exhibited Bgal activity toward the X-Gal substrate (Fig. 14C).

7-3. Analysis of Bgal activity in OsBGAL9 loss-of-function and overexpression plants

To analyze Bgal activity in OsBGAL9 loss-of-function and overexpression plants, proteins were extracted from rice and analyzed for Bgal activity.

Approximately 2 g of tissue collected from the second node or leaf was ground in liquid nitrogen. The ground powder was added to a solution of 50 mM sodium acetate buffer (pH 4.5), 1 M sodium chloride, and 1X protease inhibitor (Sigma, Mannheim, Germany) and kept on ice for 1 h. After centrifugation at 4 °C, 3,220 RCF for 20 min, the supernatant was transferred to an Amicon Ultra centrifugal filter device (Millipore) and centrifuged at 4 °C, 3,220 RCF for 10 min to remove the flow-through. After removal, the column was washed by adding 10 mL of 20 mM sodium acetate buffer (pH 5.0) and centrifuged at 4 °C, 3,220 RCF for 20 min. Finally, 10 mL of 20 mM sodium acetate buffer (pH 5.0) containing protease inhibitor solution and 2 mL of the liquid remaining in the Amicon Ultra filter device were collected by centrifugation at 4 °C, 3,220 RCF for 5 min. The total protein amount was quantified by the Bradford assay (Thermo fisher Scientific, Korea) according to Bradford (1976). Bgal activity was measured using ONPG as a substrate. The reaction mixture contained 400 ㎕ of 20 mM sodium acetate buffer (pH 5.0), 500 ㎕ of 2 mM substrate, and 100 ㎕ of protein extract (100 ㎍/mL). The reaction mixture was incubated at 37 °C for 60 min. The reaction was stopped by adding 500 ㎕ of 0.5 M sodium carbonate, and the absorbance was measured at 420 nm.

Analysis of Bgal activity in OsBGAL9 loss-of-function and overexpression plants using a synthetic Bgal substrate, ONPG, revealed that OsBGAL9-OX plants showed 50% higher Bgal activity toward ONPG in the internodes compared with DJ plants, whereas Osbgal9 plants showed about 70% reduction compared with DJ (Fig. 14D). However, Bgal activity in leaves was not correlated with OsBGAL9 expression, as both OsBGAL9-OX and Osbgal9 plants showed higher activity than DJ (Fig. 14D). Similarly, Osbgal9-t plants showed reduced Bgal activity in the internodes (Fig. 15A) and increased Bgal activity in the leaves (Fig. 15B) compared with the control, HY.

The expression levels of all Bgals were measured in the leaves of OsBGAL9-OX and Osbgal9 plants, and the results showed that the expression of OsBGAL1, OsBGAL3, OsBGAL6, and OsBGAL7 was increased in the leaves of OsBGAL9-OX and Osbgal9 plants compared to DJ, and higher activities toward ONPG were observed in the leaves of OsBGAL9-OX and Osbgal9 plants (Fig. 16). This confirmed that the reason why the activity of Bgal in the leaves was higher than that of DJ was due to the expression of other BGAL genes.

<Experimental Example 8> Confirmation of BGAL9 activity according to cell wall composition of OsBGAL9 loss-of-function and overexpression plants

8-1. OsBGAL9 activity assay on xyloglucan oligomers

Xyloglucan oligomers are polysaccharide components found in cell walls, where “X” represents a xylose-substituted G and L represents a galactose-xylose-substituted G.

Since AtBGAL10, one of the BGALs from Arabidopsis thaliana , can hydrolyze the galactose residue located at the second substituted glucose (G) at the non-reducing end of the xyloglucan oligomer XLLG, to determine whether OsBGAL9, which has a similar hydrolytic activity, was cultured with a mixture of xyloglucan oligomers containing XXXG, XLXG/XXLG, and XLLG, which were produced by hydrolyzing xyloglucan extracted from the Arabidopsis mur2 ( murus 2 ) mutant with endo-β-1,4-glucanase (E-CELTR, Megazyme, Wicklow, Ireland).

To analyze OsBGAL9 activity toward xyloglucan oligomers, the reaction mixture (40 μL) contained 100 mM sodium phosphate buffer (pH 7.0), 10 μg of recombinant OsBGAL9, and 10 μg of xyloglucan oligomers. After incubation at 37 °C for 24 h, the mass of xyloglucan oligomers in the reaction mixture was analyzed by Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry using a Bruker Autoflex ToF mass spectrometer (Billerica, MA, USA) in reflectance mode. The spectra were averaged from at least 500 laser shots. Xyloglucan oligomers were prepared by hydrolyzing xyloglucan extracted from the Arabidopsis mur2 ( murus 2 ) mutant with endo-β-1,4-glucanase.

Examination of the mass spectrum using a mass spectrometer confirmed that there was no significant difference in the relative signal intensities corresponding to the masses XXLG (m/z 1247) and XLLG (m/z 1409) when comparing the samples cultured with heat-denatured recombinant OsBGAL9 (control) and recombinant OsBGAL9 (Fig. 17).

Through this, we confirmed that the galactose residue of xyloglucan is unlikely to be a target of OsBGAL9.

8-2. Activity analysis of OsBGAL9 on galactose residues

Galactose residues are also present in other cell walls (rhamnogalacturonan I (RG-I) and AGP, which are pectic galactans in the cell wall). Therefore, to further investigate the target of the hydrolytic activity of OsBGAL9 on galactose residues, immunoblot analysis was performed using the second node of OsBGAL9-OX and Osbgal9 plants with several monoclonal antibodies, such as LM5, LM26 (for β-(1 → 4)-linked in pectic RG-I side chain), and JIM16 (for β-(1 → 4)-linked in pectic RG-I side chain).

The second nodes of 7-week-old DJ, OsBGAL9-OX, and Osbgal9 plants were collected, cut into small pieces of 1–3 mm, vacuum infiltrated for 60 min, and then placed in a fixative solution (1.6% (w/v) paraformaldehyde, 0.2% (w/v) glutaraldehyde, and 1% (v/v) tryptone (Triton X-100) in 25 mM sodium phosphate buffer) at 4 °C for 24 h. The samples were washed three times with 25 mM sodium phosphate buffer (pH 7.1) and then washed with water. The samples were dehydrated with sequential concentrations of ethanol, cleared with xylene, and embedded in paraffin. Tissue sections (20 μm) were cut with a paraffin slicer (RM2265, Leica). Finally, dewaxing was performed in the following order: 100% xylene, xylene:ethanol (1:1, v/v), ethanol (100, 95, 85, 70, 50, 30% (v/v)) for 5 to 10 minutes.

Sections were blocked in 1X sodium phosphate buffer (PBS, pH 7.1) containing 3% (w/v) nonfat dry milk for 30 min and incubated with LM5, LM26, or JIM16 (Agrisera, Sweden) for 60 min at room temperature. Sections were washed at least three times with PBS, pH 7.1, and incubated with secondary Fluor 488 anti-mouse antibody (Thermo Fisher Scientific). Sections were washed several times with PBS, then further washed with distilled water, dried, and counterstained using mounting agent (CitiFluor™ AF1, Germany). Fluorescence images were obtained using a confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan) excited at 488 nm.

In Fig. 18, VB (Vascular Bundle) means vascular bundle, and Epi means epidermis. The levels of LM5 and LM26 in the vascular bundle and epidermis of DJ, OsBGAL9-OX, and Osbgal9 plants were similar. However, the marker for JM16 increased in Osbgal9 and decreased in OsBGAL9-OX compared to DJ (Fig. 18). In addition, the levels of LM5 and LM26 were similar in Osbgal9-t plants, but the marker for JM16 increased compared to the control, HY (Fig. 19).

8-3. OsBGAL9 activity analysis on arabinogalactone proteins (AGPs)

Arabinogalactan proteins (AGPs) are a family of hydroxyproline (Hyp)-rich glycoproteins found in the plant extracellular matrix as plasma membrane-anchored or extracellularly secreted proteins, linked to carbohydrate chains. AGPs play an important role in cell growth and development.

To investigate the activity against AGPs present in the cell wall of OsBGAL9, the cells were stained and analyzed with β-D-glucosyl Yariv reagent (β-GlcY) and β-D-glucosyl Yariv reagent (β-GalY), chemicals widely used to distribute and quantify AGPs.

Transverse sections of the second node of DJ, OsBGAL9-OX and Osbgal9 plants at the flowering stage were used. Hand-cut sections were incubated in βGlcY (2 mg/mL β-D-glucosyl in 0.15 M NaCl) (Biosupplies Australia, Cat#100-2) and βGalY (2 mg/mL β-D-galactosyl in 0.15 M NaCl) (Biosupplies Australia, Cat#100-8a) reagents for 60 min at room temperature and washed three times with distilled water. Afterwards, photographs were taken using an Olympus BX61 microscope (Olympus, Tokyo, Japan).

Analysis of the activity of OsBGAL9 on AGPs revealed that AGPs were significantly increased in the stems of Osbgal9 plants and decreased in OsBGAL9-OX plants (Fig. 20). Similarly, AGPs were found to be increased in Osbgal9-t plants compared to the control group, HY (Fig. 21).

The alcohol insoluble residue (AIR) fractions of cell walls from DJ, OsBGAL9-OX, and Osbgal9 plants were analyzed by gas-liquid chromatography.

Frozen samples obtained from the second node tissues of DJ, OsBGAL9-OX and Osbgal9 plants were ground under liquid nitrogen and suspended three times in 70% (v/v) ethanol using a homogenizer (Polytron homogenizer). AIRs were collected by centrifugation at 10,000 g for 5 min and washed sequentially with absolute ethanol and 100% acetone. The final residue was dried in a vacuum oven at 60 °C. To quantify cell wall neutral sugars (alditol acetates), 5 mg of AIRs were reacted with 70% sulfuric acid solution for 30 min at room temperature, then inositol was added as an internal standard and diluted with water to a final concentration of 6% (v/v) sulfuric acid. After heating at 105 °C for 120 min, they were treated with 25% (w/v) ammonium solution. After reduction with sodium borohydride in DMSO, the solution was heated at 40 °C for 90 min and treated sequentially with glacial acetic acid, acetic anhydride, 1-methylimidazole, dichloromethane, and water. The organic layer containing alditol acetates of hydrolyzed cell wall sugars was washed three times with water, and the sugars were analyzed using a gas-liquid chromatograph (model 6890; Hewlett-Packard) equipped with a 30 m × 0.25 mm (i.d.) silica capillary column DB 225 (Alltech Assoc., Deerfield, IL, USA).

Analysis of sugars using gas-liquid chromatography revealed no significant differences in sugar contents (glucose, galactose, arabinose, and xylose) in the cell wall alcohol-insoluble residue (AIR) fractions of DJ, OsBGAL9-OX, and Osbgal9 plants (Fig. 22).

Through this, we confirmed that OsBGAL9 functions in hydrolyzing galactose residues of AGPs.

These results confirm that OsBGAL9 alters the composition of AGPs but does not affect pectin or xyloglucan. Loss of OsBGAL9 function led to a reduced stem growth phenotype associated with the accumulation of AGP glycans, whereas Osbgal9 mutants did not show significant changes in the composition of the second internode cell wall sugars. This suggests that OsBGAL9 does not play an important role in cell wall biosynthesis, whereas the modification of AGPs is important for cell elongation. These results imply that arabinogalactan may be involved in the rigidification of the cell wall by oxidative cross-linking, which influenced the altered tolerance of OsBGAL9-OX and Osbgal9 to temperature stress.

The present invention has been described so far with reference to preferred embodiments and experimental examples. Those skilled in the art will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the above description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

Attach electronic file of sequence list

Claims (11)

A recombinant vector for increasing stress tolerance or promoting growth of a plant, comprising a gene encoding BGAL9 (beta-galactosidase 9).
In the first paragraph,
The above BGAL9 (beta-galactosidase 9) is a recombinant vector for increasing stress tolerance or promoting growth of plants, characterized in that it is derived from rice ( Oryza sativa ).
In the second paragraph,
The above BGAL9 (beta-galactosidase 9) is a recombinant vector for increasing stress tolerance or promoting growth of plants, represented by the base sequence of sequence number 1.
In the first paragraph,
A recombinant vector for increasing stress tolerance or promoting growth of a plant, characterized in that the above stresses are biological and abiotic stresses.
In paragraph 4,
A recombinant vector for increasing stress tolerance or promoting growth of a plant, characterized in that the above biological stress is infection by at least one disease selected from the group consisting of blast, white leaf blight, leg blight, grain blight, seed spot and sheath blight.
In paragraph 4,
A recombinant vector for increasing stress tolerance or promoting growth of a plant, characterized in that the abiotic stress is high temperature or low temperature stress.
A transgenic plant in which a gene encoding BGAL9 (beta-galactosidase 9) is introduced or overexpressed.
In Article 7,
A transgenic plant, characterized in that the plant is at least one species selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onions.
Seeds of the transformed plant of Article 7.
A method for increasing stress tolerance or promoting growth of a plant, comprising the step of introducing or overexpressing a gene encoding BGAL9 (beta-galactosidase 9) in a plant.
A method for producing a transformed plant with increased stress tolerance or accelerated growth, comprising the step of introducing a recombinant vector containing a gene encoding BGAL9 (beta-galactosidase 9) into the plant and transforming the plant.