Patent Application
20240124886
The present invention relates to OsBCAT2 (Oryza sativa BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE 2) gene controlling drought stress of plant and uses thereof.
This work was supported by the Starting growth Technological R&D Program (TIPS Program (No. S3307687)) funded by the Ministry of SMEs and Startups (MSS, Korea) in 2022.
Unpredictable weather events caused by climate change occur worldwide. Abiotic stress is a natural incident with disastrous consequences for nearby living things. Moreover, extreme weather disasters lead to crop yield decreases. The drought stress significantly influenced international crop production. According to the reports, in rice (Oryza sativa. L.) plants, drought stress inflicts irreversible damage during the whole period of growth and decreases the grain yield drastically. Specifically, drought stress in the vegetative stage of rice (Yangliangyou 6, Hanyou 113) caused a yield reduction of 23-24%.
Plants have various strategies to combat abiotic stress. At the molecular level, plants regulate the expression of genes involving in stress responses through the stress-specific signaling pathway. Physiological responses lead to morphological and endogenous biochemical adjustments. For instance, plant amino acids play an essential role in minimizing the damage by abiotic stress. So far, proline is best characterized amino acid that accumulates in plant cells to confront a wide range of abiotic stress. It has been documented that proline accumulates in response to drought, salt, heat, low temperature, and nutrient deficient stress in various plants. γ-Aminobutyric acid (GABA) alleviates drought and heat stress through regulating antioxidant genes in sunflower. Besides, glycine, taurine, arginine, and serine were reported as stress-induced amino acids which act as osmolytes. Interestingly, among plant amino acids, Branched-chain amino acids (BCAA), leucine (Leu), isoleucine (Ile), and valine (Val) have been reported to accumulate under stress conditions in plants. Fold change value of BCAA quantity under drought conditions was higher than that of proline, a well-known drought inducible amino acid.
BCAA has a critical role in protein biosynthesis and plant development. Moreover, they are also involved in the formation of diverse secondary metabolites, such as cyanogenic glycosides, glucosinolate, and acyl-sugars. Branched-chain amino acids synthesis is carried out with two distinct substrates. The preliminary pathway of valine and leucine starts with pyruvate created by glycolysis. On the other hand, the synthesis of isoleucine starts with 2-ketobutyrate after the threonine deamination by threonine deaminase (TD, EC 4.2.1.16). The first engaged enzyme for Val, Leu production, is acetohydroxyacid synthase (AHAS also known as acetolactate synthase ALS, EC 2.2.1.6), which catalyzes both two pyruvate molecules for the constitution of acetolactate for Val and α-ketobutyrate with one pyruvate to form acetohydroxybutyrate as a precursor of isoleucine. Ketol-acid reductoisomerase (KARI, EC 1.1.1.86), and dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9), consecutively catalyze a fundamental two-step reaction for BCAA synthesis. The branched-chain amino acid aminotransferase (BCATs, EC 2.6.1.42) is in charge of the final step of BCAA biosynthesis in plastids.
BCAA catabolic reactions mainly occur in mitochondria. BCAT is an enzyme involved in the first step of degradation and the last step in synthesizing BCAA. BCAT, which belongs to Class IV of the pyridoxal 5′phosphate-dependent aminotransferases, converts Val, Ile, or Leu to α-keto acids KIV, KIC, or KMV. Branched-chain keto acid dehydrogenase (BCKDH also known as BCKDH complex, EC 1.2.4.4) catalyzes the branched-chain α-keto acids to isobutyryl-CoA, isovaleryl-CoA, or methylbutyryl-CoA (Taylor et al. 2004). Then, Isovaleryl-CoA dehydrogenase (IVD, EC 1.3.99.10), identified in several plant species, catalyzes acyl-CoA to enoyl-CoA. Although this enzyme has not been fully elucidated, the substrate specificity appears to be different among these proteins of other species.
Among these catabolic enzymes, Interestingly, plant species have various BCAT isoform families. In Arabidopsis thaliana and Solanum lycopersicum, seven and six distinct isoforms of BCAT have been validated, respectively. It has been reported that AtBCAT2 (At1g10070), SlBCAT1 (SGN-U569828), and SlBCAT2 (SGN-U569830) are localized to mitochondria, where it conducts a vital function in BCAA catabolism. Nevertheless, the pathways related to gene and catabolic protein networks have not been fully elucidated in plants.
Meanwhile, in Korean Patent Publication No. 2021-0063574, “Drought resistant transgenic plant inhibiting AtPR5K2 protein expression” is disclosed, and in Korean Patent Publication No. 2022-0083464, “Novel branched-chain amino acid aminotransferase variant and method of producing L-isoleucine using the same” is disclosed. However, OsBCAT2 gene controlling plant drought tolerance of the present invention and uses thereof have not been described.
The present invention is devised according to the necessities that are explained in the above. Specifically, based on previous research examining the role played by branched-chain amino acids (BCAA) on plant tolerance under abiotic stress conditions, inventors of the present invention studied the function of OsBCAT2 (BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE 2) gene, which is derived from rice.
By using CRISPR-Cas9 system, the inventors of the present invention prepared loss of function rice OsBCAT2 mutants (i.e., osbcat2). In addition, as a result of examining the drought tolerance exhibited by the osbcat2, the inventors found that osbcat2 mutants show enhanced tolerance to drought and enhanced grain yield over those of wild type under various drought stress conditions. The present invention is completed accordingly.
To solve the problems that are described in the above, the present invention provides a method of controlling tolerance to drought stress in a plant comprising regulating expression of a gene encoding OsBCAT2 (BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE 2) protein derived from rice in a plant cell.
The present invention further provides a method of producing a transgenic plant having controlled tolerance to drought stress comprising: transforming a plant cell with a recombinant vector containing a gene encoding OsBCAT2 protein derived from rice; and regenerating a transgenic plant from the transformed plant cell.
The present invention further provides a transgenic plant having controlled tolerance to drought stress produced by the aforementioned method of producing a transgenic plant, and a transgenic seed thereof.
The present invention further provides a method of producing a genome-edited rice plant having enhanced tolerance to drought stress comprising: (a) carrying out genome editing by introducing guide RNA specific to target nucleotide sequence in a gene encoding OsBCAT2 protein derived from rice, and endonuclease protein to a rice plant cell; and (b) regenerating a plant from the genome-edited rice plant cell.
The present invention further provides a genome-edited rice plant having enhanced tolerance to drought stress produced by the aforementioned method of producing a genome-edited rice plant, and a genome-edited seed thereof.
The present invention still further provides a composition for controlling tolerance to drought stress in a plant comprising, as an effective component, a gene encoding OsBCAT2 protein derived from rice.
Because having crops with tolerance to environmental stress is considered to be very important in the field of agriculture, it is expected that OsBCAT2 (branched-chain amino acid aminotransferase 2) gene derived from rice, which is described in the present invention, can be advantageously used for enhancing the plant tolerance to drought stress.
To achieve the object of the present invention, the present invention provides a method of controlling tolerance to drought stress in a plant comprising regulating expression of a gene encoding OsBCAT2 (branched-chain amino acid aminotransferase 2) protein derived from rice in a plant cell.
With regard to the method of controlling tolerance to drought stress according to one embodiment of the present invention, the OsBCAT2 protein derived from rice may preferably consist of the amino acid sequence of SEQ ID NO: 2, but it is not limited thereto.
Also included in the scope of the OsBCAT2 protein derived from rice according to the present invention are the protein having the amino acid sequence represented by SEQ ID NO: 2, and functional equivalents thereof. As described herein, the term “functional equivalents” means a protein which has, as a result of addition, substitution, or deletion of an amino acid, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 2, and it indicates a protein which exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2. The expression “substantially the same physiological activity” indicates an activity of controlling plant tolerance to drought stress.
Also included in the present invention is a gene which encodes OsBCAT2 protein derived from rice, and the gene has the scope to encompass all genomic DNA, cDNA, and synthetic DNA which encode OsBCAT2 protein derived from rice. Preferably, the gene encoding OsBCAT2 protein derived from rice described in the present invention may comprise the OsBCAT2 nucleotide sequence derived from rice that is represented by SEQ ID NO: 1. Furthermore, homologs of the aforementioned sequence are also within the scope of the present invention. Specifically, the aforementioned gene may include a nucleotide sequence which has 70% or higher, more preferably 80% or higher, even more preferably 90% or higher, and most preferably 95% or higher sequence homology with the nucleotide sequence of SEQ ID NO: 1. The “sequence homology %” of the polynucleotide is identified by comparing two sequences that are optimally aligned. In this regard, a part of the polynucleotide in comparative region may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any addition or deletion) in the optimized alignment of the two sequences.
The method of controlling plant tolerance to drought stress according to the present invention may involve the inhibition of expression of a gene encoding OsBCAT2 protein derived from rice in plant cell so as to have higher plant tolerance to drought stress compared to a non-transgenic plant, but it is not limited thereto.
According to one embodiment of the present invention, the inhibition of expression of a gene encoding OsBCAT2 protein derived from rice can be an inhibition (suppression) of the gene encoding OsBCAT2 protein derived from rice based on insertion of T-DNA to the gene encoding OsBCAT2 protein derived from rice, induction of mutation using endogenous transposon or irradiation of X-ray or γ-ray, or use of RNAi or antisense RNA, or CRISPR/Cas9 gene editing system, but it is not limited thereto. Any method typically employed in the pertinent art to have inhibited gene expression can be possibly used.
With regard to the recombinant vector of the present invention, the promoter is a promoter which is suitable for transformation. Preferably, it may be any one of CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter. Preferably, it may be CaMV 35S promoter, but it is not limited thereto.
The recombinant expression vector may preferably contain one or more selective marker. The selective marker is a nucleotide sequence having a property of allowing vector selection by a common chemical method. Any gene that can be used for identifying transformed cells from non-transformed cells can be a selective marker. The marker gene can be a dominant drug-resistance gene, but it is not limited thereto.
In case of transforming an eukaryotic cell with the vector of the present invention, yeast (Saccharomyce cerevisiae), an insect cell, human cell (e.g., CHO (Chinese hamster ovary) cell line, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell line), a plant cell, or the like can be used as a host cell. Preferably, the host cell is a plant cell.
The present invention further provides a method of producing a transgenic plant having controlled tolerance to drought stress comprising: transforming a plant cell with a recombinant vector containing a gene encoding OsBCAT2 protein derived from rice; and regenerating a transgenic plant from the transformed plant cell.
With regard to the method of producing a transgenic plant according to the present invention, the OsBCAT2 protein derived from rice may preferably include a protein consisting of the amino acid sequence of SEQ ID NO: 2, and functional equivalents thereof. In this regard, the detailed information is the same as those described above.
Further, with regard to the method of producing a transgenic plant described in the present invention, the method for transforming a plant cell is the same as described in the above. As for the method for regenerating a transgenic plant from a transformed plant cell, a method well known in the pertinent art can be used. The transformed plant cell needs to be regenerated into a mature plant. Techniques for regeneration into a mature plant by culture of callus or protoplast are well known in the pertinent art for various species.
With regard to the method of producing a transgenic plant according to one embodiment of the present invention, by inhibiting the expression of the gene, which encodes OsBCAT2 protein derived from rice, in plant cell, higher plant tolerance to drought stress compared to a non-transgenic plant, i.e., wild type, can be obtained, but it is not limited thereto.
Inhibiting the expression of the gene which encodes OsBCAT2 protein derived from rice can be achieved by transforming a plant cell with a recombinant vector containing sense or antisense DNA, or microRNA for OsBCAT2 gene derived from rice to inhibit the expression of the gene which encodes OsBCAT2 protein derived from rice. However, the inhibition is not limited thereto, and any gene expression inhibition technique well known in the pertinent art can be employed.
The present invention further provides a transgenic plant having controlled tolerance to drought stress, which is produced by the aforementioned method of producing a transgenic plant, and a transgenic seed thereof.
The transgenic plant according to the present invention is characterized in that, as expression of the gene encoding OsBCAT2 protein derived from rice is inhibited, the plant exhibits enhanced tolerance to drought stress. The plant can be a monocot plant such as rice, barley, wheat, rye, corn, sugar cane, oat, or onion, or a dicot plant such as Arabidopsis thaliana, potato, eggplant, tobacco, pepper, tomato, burdock, crown daisy, lettuce, balloon flower, spinach, chard, yam, carrot, water parsley, Chinese cabbage, cabbage, Raphanus sativus for. raphnistroides MAK, watermelon, oriental melon, cucumber, zucchini, gourd, strawberry, soybean, mung bean, kidney bean, or sweet pea. Preferably, it can be a monocot plant, and more preferably rice plant. However, it is not limited thereto.
The present invention further provides a method of producing a genome-edited rice plant having enhanced tolerance to drought stress comprising: (a) carrying out genome editing by introducing guide RNA specific to target nucleotide sequence in a gene encoding OsBCAT2 protein derived from rice, and enconuclease protein to a rice plant cell; and (b) regenerating a plant from the genome-edited rice plant cell.
As described herein, the term “genome/gene editing” means a technique for introducing a target-oriented mutation to a genome nucleotide sequence of a plant or an animal cell including human cell. Specifically, it indicates a technique for knock-out or knock-in of a specific gene by deletion, insertion, or substitution of one more nucleic acid molecules by DNA cutting, or a technique for introducing a mutation even to a non-coding DNA sequence which does not produce any protein. According to the purpose of the present invention, the genome editing may be an introduction of a mutation to a plant by using an endonuclease, for example, Cas9 (CRISPR associated protein 9) protein, and a guide RNA. Furthermore, the term “gene editing” may be interchangeably used with “gene engineering”.
Furthermore, the term “target gene” described herein means part of DNA which is present in the genome of a plant and to be edited according to the present invention. Type of the target gene is not limited, and it may include a coding region and also a non-coding region. Depending on the purpose, a person who is skilled in the pertinent art may select the target gene according to the mutation of a plant that is desired to be produced by genome editing.
Furthermore, the term “guide RNA” described herein means a ribonucleic acid which includes RNA specific to a target DNA in nucleotide sequence encoding the target gene and, according to complementary binding between the whole or partial sequence of the guide RNA and the nucleotide sequence of target DNA, the guide RNA plays the role of guiding an endonuclease to the target DNA nucleotide sequence. The guide RNA represents two types of RNA, i.e., a dual RNA having crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as a constitutional element; or a single chain guide RNA (sgRNA) comprising the first site which includes a sequence that is fully or partially complementary to the nucleotide sequence in the target gene and the second site which includes a sequence interacting with RNA-guide nuclease, but those in the form in which the RNA-guide nuclease is active for the target nucleotide sequence are also within the scope of the invention without any limitation. Considering the type of an endonuclease to be used together, microorganisms from which the endonuclease is derived, or the like, the guide RNA may be suitably selected according to the technique that is well known in the pertinent art.
Furthermore, the guide RNA may be those transcribed from a plasmid template, those obtained by in vitro transcription (e.g., oligonucleotide double strand), or those obtained by synthesis, or the like, but it is not limited thereto.
Furthermore, with regard to the method of producing a genome-edited rice plant according to the present invention, the endonuclease protein may be one or more selected from a group consisting of Cas9, Cpf1 (CRISPR from Prevotella and Francisella 1), TALEN (Transcription activator-like effector nuclease), ZFN (Zinc Finger Nuclease), and a functional analog thereof. It may be preferably Cas9 protein, but it is not limited thereto.
Furthermore, the Cas9 protein may be one or more selected from a group consisting of Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from S. thermophilus, Cas9 protein derived from S. aureus, Cas9 protein derived from Neisseria meningitides, Cas9 protein derived from Pasteurella multocida, Cas9 protein derived from Francisella novicida, and the like, but it is not limited thereto. Information of Cas9 protein or gene of Cas9 protein can be obtained from a known database like GenBank of NCBI (National Center for Biotechnology Information).
Cas9 protein is an RNA-guided DNA endonuclease enzyme which induces breakage of a double stranded DNA. In order for Cas9 protein to cause DNA breakage after precise binding to a target nucleotide sequence, a short nucleotide sequence consisting of three nucleotides, which is known as PAM (Protospacer Adjacent Motif), should be present next to the target nucleotide sequence, and Cas9 protein causes the breakage by assuming the position between the 3rd and the 4th base pairs from the PAM sequence (NGG).
In the present invention, the guide RNA and endonuclease protein may function as RNA gene scissors (RNA-Guided Engineered Nuclease, RGEN) by forming a ribonucleic acid-protein (i.e., ribonucleoprotein) complex.
The CRISPR/Cas9 system employed in the present invention is a method of gene editing based on NHEJ (non-homologous end joining) mechanism in which insertion-deletion (InDel) mutation resulting from incomplete repair, which is induced during a process of DNA repair, is caused by introducing breakage of a double strand at specific site of a specific gene to be edited.
With regard to the method of producing a genome-edited rice plant according to the present invention, introducing guide RNA and endonuclease protein to a rice plant cell of the aforementioned step (a) may involve use of: a complex (i.e., ribinucleoprotein) of a guide RNA specific to the target nucleotide sequence in OsBCAT2 gene derived from rice and an endonuclease protein; or a recombinant vector having a DNA encoding a guide RNA specific to the target nucleotide sequence in OsBCAT2 gene derived from rice and a nucleic acid sequence encoding an endonuclease protein, but it is not limited thereto.
Furthermore, with regard to the method of producing a genome-edited rice plant according to one embodiment of the present invention, the target nucleotide sequence in OsBCAT2 gene derived from rice may be the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, but it is not limited thereto.
The present invention further provides a genome-edited rice plant having enhanced tolerance to drought stress, which is produced by the aforementioned method of producing a genome-edited rice plant, and a genome-edited seed thereof.
The genome-edited rice plant according to the present invention is characterized in that, as the gene encoding BCAT2 protein derived from rice is knocked-out, the plant exhibits enhanced tolerance to drought stress.
The present invention still further provides a composition for controlling tolerance to drought stress in plant comprising, as an effective component, a gene encoding OsBCAT2 protein derived from rice.
The composition of the present invention comprises, as an effective component, a gene encoding OsBCAT2 protein derived from rice or a mutant of the gene encoding OsBCAT2 protein derived from rice, and, by transforming a plant cell with a recombinant vector containing a gene encoding OsBCAT2 protein derived from rice or a mutant of the gene encoding OsBCAT2 protein derived from rice, plant tolerance to drought stress can be controlled.
Hereinbelow, the present invention is explained in greater detail in view of the Examples. However, it is evident that the following Examples are given only for exemplification of the present invention and by no means the present invention is limited to the following Examples.
Materials and Method
1. Plant Materials
To generate a loss of function OsBCAT2 (Os03g0231600, LOC_Os03g12890) mutants (osbcat2), rice (Oryza sativa L. ssp. Japonica cv. Dongjin) was edited using the CRISPR-Cas9 system through two guide RNAs. Guide RNA was designed via the web-based tool CRISPR RGEN Tools (http://www.rgenome.net). As described previous report, the method was performed (Chung et al. 2020, Int. J. Mol. Sci. 21(24); 9606). We obtained osbcat2 mutants with a total of 18 genotypes. Among these, we selected osbcat2 mutants (#3, #4, #5, #10, and #15), which have premature stop codons before 89 amino acids were used in this research.
The Japonica rice cultivar Dongjin (Oryza sativa L.) grown under the same conditions as mutants (osbcat2), was used as the non-transgenic control.
2. Abiotic Stress and Phytohormone Treatments
On a Murashige-Skoog (MS) solid medium, rice seeds (Oryza sativa cv. Dongjin) were germinated for four days at 28° C. in the dark. The seedlings were then transported to a growth chamber with a 16 h light/8 h dark cycle, 200 μmol m2 s−1 of light intensity, and 70% relative humidity. One-week-old seedlings were transferred to a Yoshida solution and grown for two more weeks for gene expression analysis. Non-transgenic (NT) (Oryza sativa L. ssp. Japonica cv. Dongjin) seedlings were used to study the phytohormone-dependent response of OsBCAT2 and were transferred to 50 ml tubes containing 100 μM ABA (abscisic acid), JA (jasmonic acid), and SA (salicylic acid) solution (Sigma, USA). Seedlings were harvested every 2 hours until 6 hours after each hormone treatment for RNA extraction.
To confirm the transcription levels of the OsBCAT2 gene under various abiotic stresses, non-transgenic (NT) plants were grown in soil for two weeks under standard greenhouse conditions (16 h light/8 h dark cycles at 28-30° C.). For stress treatments, remove the soil from the roots of the seedling completely, and drought stress was induced by air-drying the seedlings, while salinity stress was imposed by incubating the seedlings in water containing 400 mM NaCl at 28° C. Low-temperature stress was treated by incubating the seedlings in water at 4° C. (1.5° C.).
3. Amino Acid Feeding and Water Deficit Stress Treatment
Non-transgenic plants (Oryza sativa cv. Dongjin) were sown on MS (Murashige and Skoog) media and incubated in a dark growth chamber for four days at 28° C. Seedlings were then transferred to a growth chamber with a light/dark cycle of 16 h light/8 h dark at 30° C. and grown to the two-week-old seedling. The seedlings were acclimatized in water for one day before being transferred to the Yoshida solution. They were then grown to three-week-old plants in Yoshida solution.
Three-week-old plants grown in Yoshida liquid media were fed with 10 mM randomly selected FAAs, including L-valine, leucine, and isoleucine (Sigma, USA) by dipping their roots into a corresponding FAA solution for 24 hrs. Plants pretreated with BCAA were harvested for amino acid analysis. Plants were then transferred to a 50 ml tube containing 30 mL of 21% PEG 8000 (Sigma) solution. PEG-induced visual symptoms such as leaf rolling and wilting were monitored by imaging plants at the indicated time points using an a5000 (Sony) camera. Air-drying-induced drought phenotypes of FAA pretreated NT plants were recorded through relative water content (RWC) and symptoms analysis. Calculation formula of RWC (%)=(FW−DW)/(TW−DW)×100. FW: fresh weight, DW: dry weight, TW: turgid weight.
4. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Analysis
To investigate the spatiotemporal gene transcript patterns of OsBCAT2, total RNA samples were extracted from the roots, stem, leaf, and flowers from different developmental stages of rice plants using a Hybrid-R RNA purification kit (GeneAll, Seoul, Korea) according to the manufacturer's instructions. To measure the expression levels of OsBCAT2, total RNA samples were isolated from the shoots and roots using a Hybrid-R RNA purification kit (GeneAll, Seoul, Korea). The cDNAs of total and/or immunoprecipitated RNAs were synthesized using the RevertAid™ First Strand cDNA Synthesis Kit with an oligo (dT) primer (Thermo Scientific, Waltham, MA, USA). 20 ng of cDNA was utilized as a template for qRT-PCR analysis. qRT-PCR was carried out using 2× qRT-PCR Pre-mix with 20× EvaGreen (SolGent, Seoul, Korea) and ROX dye (Promega, Madison, WI, USA). The amplification reactions were carried out at 95° C. for 10 minutes, followed by 40 cycles at 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds in a 20 μL volume mix containing 1 μL EvaGreen Mix. The rice UBIQUITIN1 (Os06g0681400) were used as an internal standard.
5. Drought Stress Treatment and Stress Measurement at the Vegetative Stage
For 4 days, osbcaO2 and NT (Oryza sativa L. ssp. Japonica cv. Dongjin) seedlings were germinated on the Murashige-Skoog (MS) medium in a dark growth chamber at 28° C. Before the transplant, germinated plants were acclimated to light conditions for one day. The 5-day-old seedlings were transplanted into soil pots (4×4×6 cm, three plants per pot) within a container (59×38.5×15 cm) and grown for five weeks in greenhouse conditions at the automatically controlled glass-greenhouse condition (light/dark cycle of 16 h light/8 h dark at 30° C.±3° C.). Drought treatment for observation or measurement was uniformly imposed by withholding water and re-watering at the vegetative phase of rice. Soil moisture was monitored during the test and measured by a soil moisture sensor (SM159, Delta-T Devices Cambridge, United Kingdom).
To measure the drought stress of osbcat2 and NT, we evaluated the efficiency of photosystems I and II through JIP test (Strasser, R. J., Tsimilli-Michael, M., Srivastava, A. (2004). Analysis of the Chlorophyll a Fluorescence Transient. In: Papageorgiou, G. C., Govindjee (eds) Chlorophyll a Fluorescence. Advances in Photosynthesis and Respiration, vol 19. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-3218-9_12). These data were recorded using a Handy-PEA fluorimeter (Plant Efficiency Analyzer Hansatech Instruments, UK) in dark conditions to impose optimal dark adaptation (at least 20 min). Ten leaves were collected and calculated using the Handy PEA software (version 1.31) and analyzed according to the calculation formula of the JIP test.
6. Analysis of Agronomic Traits of Rice in a Rice Paddy Field
To evaluate the agronomic characteristics and traits of osbcat2 and NT rice plants, five independent T1 homozygous mutants and NT (Oryza sativa L. ssp. Japonica cv. Dongjin) plants were cultivated in the paddy field at the Kyungpook National University, Gunwi (128:34E/36:15N), Korea. We divided the paddy field into three sections to minimize the influence of microclimate, and each plot was planted with ten plants per line. The experimental environment was created for agronomic evaluation of osbcat2 and NT plants under dehydration conditions. Plants were grown in an artificially created reservoir with a rain-off shelter. Water deficit conditions is imposed to plants before and after the heading phase by controlling water levels. When phenotypes caused by drought stress were observed in NT, re-irrigation was performed for recovery. The yield components of 10 plants per line from three different plots for the drought field conditions were measured.
7. DAB and NBT Staining
For DAB staining, 50 mg 3,3′-Diaminobenzidine (Sigma) was dissolved in 45 ml H2O2 then the pH was adjusted to 3.8 with 0.1 N HCl. To this solution, 25 μL of Tween20 (0.05% v/v) and 2.5 ml of Na2HPO4 (200 mM) were added and filled with H2O2 to make a total volume of 50 ml. Leaf sections of accurately 2.5 cm in length were cut and soaked in a 1% DAB solution. After 30 min vacuum infiltrating, the immersed leaves were incubated in the dark for 20 h at room temperature. And then, the leaves were bleached by bathing and boiling ethanol until the brown spots appeared clearly. The area of brown spots represents the DAB reaction degree to H2O2.
In order to detect superoxide accumulation using a 0.2% solution of NBT in a 10 mM potassium phosphate buffer (pH 7.7), roughly 2.5 cm-long leaf pieces were cut. After 15 minutes of vacuum infiltration, the submerged leaves were incubated at room temperature for overnight. After incubation, the leaves were fixed and clarified in alcoholic lacto-phenol (2:1:1, % ethanol:lactic acid:phenol) at 65° C. for 30 minutes, rinsing with 50% ethanol, and then rinsing with water. In leaves, a blue precipitate form when NBT reacts with superoxide.
For 12 genes including BCATs of Arabidopsis thaliana (AtBCATs) and BCATs of tomato (SlBCATs) that are taken as a subject, multiple amino acid sequence alignment was carried out. As a result of the phylogenetic analysis, very high homology was found among BCAT genes of Arabidopsis thaliana, tomato, and rice, including AtBCAT2 and SlBCAT2 that are known to be present in mitochondria. Amino acid sequence homology of BCAA aminotransferase domain among them was more than 80% and it was recognized that difference of the amino acids sequence was found only in the signal peptide sequence region at the N terminus which determined the subcellular localization of the protein.
In order to find the drought-induced branched-chain amino acid aminotransferase genes, we used RNA-sequencing (RNA-seq) in a previous study (Chung et al. 2016 BMC Genomics. 17:563), 5 drought-responsive (2 up and 3 down-regulated, respectively, with a log 2 ratio≥2.0 and ≤−2.0) BCAA transferase-related gene were identified. Among these, OsBCAT2 was up-regulated by drought stress.
To confirm the transcription levels in OsBCAT2 in various abiotic conditions, 2-week-old rice seedlings were exposed to drought, high salt, abscisic acid (ABA), and low temperature. These analyses show that OsBCAT2 transcript level was strongly induced within 6 hr of exposure to drought stress in leaves than in other abiotic stress ((a) of
Amino acids are involved in most metabolic pathways and known to regulate various developmental processes in plants. We performed the transcription analysis of OsBCAT2 by qRT-PCR. The expression level was checked in various tissues at different developmental stages, starting from vegetative to developmental stage. These data showed that OsBCAT2 transcripts were up-regulated from all the rice tissues. OsBCAT2 showed higher expression during the developmental stage than at the vegetative stage, specifically in leaves in dark conditions (
It is known that major enzymes involved in biosynthesis and degradation of BCAA are present in chloroplast and mitochondria, respectively. Accordingly, by expressing OsBCAT2-GFP fusion protein in rice protoplast, the inventors of the present invention investigated the subcellular localization of OsBCAT2 in order to speculate whether OsBCAT2 is involved in the synthesis or degradation of BCAA. As a result of confocal laser scanning microscopy analysis, the fluorescent signal of OsBCAT2-GFP protein was observed only from mitochondria. In addition, as a result of subcellular localization analysis, it was found that the expression of OsBCAT2 protein is similar to the expression of mitochondria marker (
A previous study about OsDIAT overexpressing transgenic plants showed that increasing BCAA content in leaf tissue confers enhanced tolerance to drought stress (U.S. U.S. Pat. No. 11,046,970B2). To investigate the biological functions of osbcat2 mutants under drought stress, we designed two types of single guide RNA (sgRNA) for CRISPR-Cas9 systems technology to achieve knockout mutants in OsBCAT2 gene and obtained homozygous five individual mutants (#3, #4, #5, #10, #15) ((a) of
We conducted a drought tolerance test on osbcat2 mutants to examine their response to drought stress and biological roles. In the greenhouse, 4-week-old plants were subjected to drought stress by withholding water for several days ((a) of
It was reported that Arabidopsis plants increased BCAA in response to drought and osmotic stress. We evaluated whether BCAA levels in NT and osbcat2 mutant are different under drought conditions. For High-performance liquid chromatography (HPLC) analysis, we exposed them (6-week-old plants) to drought stress conditions by air drying (6 hr). We measured the branched-chain amino acid content under normal and drought conditions. Compared to normal conditions samples, BCAA levels significantly increased under drought stress conditions. Specifically, osbcat2 mutants accumulated much higher BCAA than NT plants ((a) of
In order to further confirm whether the OsBCAT2 gene is involved in BCAA catabolism, we analyzed BCAA content in dried rice seed. The result showed that BCAA content exponentially increased in osbcat2 mutants than that in NT plants. Overall, these results indicate that drought stress leads to BCAA accumulation in rice plants and the expression of OsBCAT2, involving in BCAA catabolism, is induced by drought stress.
To determine the effect of amino acid pre-treatment in osmotic stress tolerance, various amino acids (i.e., proline, alanine, aspartic acid, methionine, isoleucine, leucine, valine, and BCAA compounds) were applied to NT rice for 24 hours before osmotic stress treatment. The pre-treated plants were examined by HPLC analysis. The result showed that the internal amino acid level has been significantly elevated by external application ((a) of
Furthermore, to determine the effect of amino acid accumulation in rice under severe osmotic stress, an air-drying test was carried out. First, a pre-treatment with amino acids was carried out for 24 hours in the same manner as the treatment method described in the above. After the air-drying treatment for 10 hours, it appears that significant damages are caused by drought stress in all plants. However, the plant treated with BCAA or proline showed less damage compared to the plant groups which have been pre-treated with other amino acids ((a) of
In order to further investigate the effect of supplying exogenous BCAA, level of moisture loss caused by air drying was monitored every hour. The results showed that the moisture loss was reduced by pre-treatment of any amino acid compared to that of plant with mock treatment ((b) of
The above study revealed that loss of function osbcat2 mutant confers drought tolerance. To evaluate the yield performance of osbcat2 mutants under drought stress conditions, osbcat2 and NT plants were grown in the field conditions with rain-off shelters at Gunwi (128:34E/36:15N), Korea, for 100 days. Then, the sequential drought stress at the flowering and milk stage were applied by halting irrigation and drying 2-weeks. Under normal conditions, five independent osbcat2 were slightly lower than NT in the number of spikelets (NS) and Filling rate (FR). On the other hand, under drought conditions, osbcat2 significantly improved number of total seed (NTS) and total grain weight (TGW) ((a) of
Proline is one of the best characterized amino acids involving in drought tolerance in plants, and the mechanism of abiotic stress responses by proline has been studied well. However, the tolerance mechanism against abiotic stress by BCAA has been poorly understood so far.
It has been well documented that biological active form of JA is JA-Ile, conjugated form with isoleucine in plants, and it is mediated by JASMONATE RESISTANT1 (JAR1). To identify the function of the OsBCAT2 in JA signaling, we checked the transcription level of rice JAR1 under drought stress. JAR1 expression level was significantly higher in osbcat2 than in NT plants ((a) of
Expression of JA responsive antioxidant genes, such as APX, SOD, and POD, was highly up-regulated in osbcat2 ((d) of
The contents of the electronic sequence listing (sequencelisting.xml; Size: 45,949 bytes; and Date of Creation: Oct. 14, 2022) is herein incorporated by reference in its entirety.
