OsMDH12: A Peroxisomal Malate Dehydrogenase Regulating Tiller Number and Salt Tolerance in Rice

Salinity is an important environmental factor influencing crop growth and yield. Malate dehydrogenase (MDH) catalyses the reversible conversion of oxaloacetate (OAA) to malate. While many MDHs have been identified in various plants, the biochemical function of MDH in rice remains uncharacterised, and its role in growth and salt stress response is largely unexplored. In this study, the biochemical function of OsMDH12 was determined, revealing its involvement in regulating tiller number and salt tolerance in rice. OsMDH12 localises in the peroxisome and is expressed across various organs. In vitro analysis confirmed that OsMDH12 converts OAA to malate. Seedlings of OsMDH12-overexpressing (OE) plants had shorter shoot lengths and lower fresh weights than wild-type (WT) plants, while osmdh12 mutants displayed the opposite. At maturity, OsMDH12-OE plants had fewer tillers than WT, whereas osmdh12 mutants had more, suggesting OsMDH12’s role in tiller number regulation. Moreover, OsMDH12-OE plants were sensitive to salt stress, but osmdh12 mutants showed enhanced salt tolerance. The Na+/K+ content ratio increased in OsMDH12-OE plants and decreased in osmdh12 mutants, suggesting that OsMDH12 might negatively affect salt tolerance through influencing the Na+/K+ balance. These findings hint at OsMDH12’s potential as a genetic tool to enhance rice growth and salt tolerance.


Introduction
Rice (Oryza sativa) is one of the most important food crops worldwide, with over half the global population relying on it as a staple.Tillering is a key agronomic trait that influences grain yield and is affected by both genetic and physiological factors.Therefore, identifying key genes for tiller development is crucial for breeding high-yield crops.
Salinity is an important global environmental factor that limits plant growth and crop productivity.To respond to salt stress, plants have evolved several mechanisms to combine endogenous developmental cues with exogenous salinity stress signals to balance growth and stress responses optimally.Ion toxicity and osmotic stress are major factors in the inhibition of plant growth caused by salt stress [32].To mitigate the damage caused by osmotic stress under salt stress, plants promote the accumulation of protective metabolites in the cytoplasm [33].These metabolites can function as compatible osmolytes that do not interfere with plant metabolism, including abscisic acid, flavonoids, proline, sugar, glycine betaine, polyamines, and α-amino nitrogen [34,35].Under salt stress, these osmolytes play a crucial role in osmoregulation by decreasing cellular osmotic potential and stabilizing proteins and cellular structures [35,36].The metabolomic profile of various plants (Arabidopsis, rice, and lotus) shows that the balance between amino acids and organic acids is a conserved response under salt stress [34,37].Malate is one of the organic acids that affects plant growth, fruit acidity, and nutrient quality and plays an important role in response to salt stress [38][39][40][41].Malate is primarily produced in the cytosol and transported to the vacuole for storage as malic acid [42], an intermediate metabolite of the citric and glyoxylate cycles [43].Many studies have shown that salt stress leads to the accumulation of malic acid in various plants, including Arabidopsis [43], Pear [44], apple [45], grape [46,47], rice [48], and tomato [49].When plants are exposed to salinity stress, malic acid accumulates and is transported into mitochondria, where it feeds into the tricarboxylic acid cycle to promote mitochondrial ATP production and maintain respiratory flux [38,44,50].Malate dehydrogenase (MDH) is a class of oxidoreductase enzymes that utilise NAD or NADP(H) as cofactors to catalyse the reversible reaction between malic acid and OAA [51].This reaction is vital for cellular metabolic processes such as the tricarboxylic acid cycle and the malate-aspartate shuttle in both plants and mammals [52].Various MDHs have been identified in numerous plant species, including Arabidopsis thaliana [43], apple [53], Chinese Fir (Cunninghamia lanceolata) [54], cotton [55], maize [56], melon [57], poplar (Populus trichocarpa) [58], tobacco [59], tomato [60], soybean [61], Stylosanthes (Stylosanthes guianensis) [62], and rice [48].Some have been found to be essential for several anabolic and catabolic processes, converting OAA to malic acid.For instance, ZmMDH4 [63], GhmMDH1 [64], and MdcyMDH1 [40] are known to catalyse the conversion of OAA to malate in maize, cotton, and apples.While the biochemical function of MDH is established, its biological function in plants is not yet fully understood.
In rice, twelve MDH (OsMDH1-OsMDH12) members are present.OsMDH1 is a plastid-localised protein that negatively regulates salt tolerance through affecting vitamin B6 content [65].OsMDH10/FLO16 encodes a NAD-dependent cytosolic malate dehydrogenase.Knockout of OsMDH10/FLO16 notably reduces the starch content in grains, whereas its overexpression significantly increases grain weight, suggesting that OsMDH10 is vital for starch biosynthesis and seed development [66].Recent studies indicate that natural variations in the promoter region of OsMDH8 may be linked to salt tolerance during the rice seedling stage [48].Most MDH family genes are significantly induced by salt stress [48], implying a potential role in rice's salt stress response.However, the role In this study, we functionally characterised OsMDH12, which is localised in the peroxisome.We found that OsMDH12 serves as a negative regulator of both tiller number and salt tolerance in rice.

Phylogenetic Tree, Expression Profiles, and Subcellular Localization of OsMDH12
The full-length 1071 bp cDNA sequence of OsMDH12 (Os12g0632700) encodes a malate dehydrogenase that is predicted to be 357 amino acids long, according to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu(accessed on 1 May 2021)).
A phylogenetic tree was constructed using the amino acid sequences of OsMDH12 and malate dehydrogenases from other plants, such as Arabidopsis, maize, and tomato.OsMDH12 displays high amino acid sequence similarity to AtpMDH1, AtpMDH2, Zm-MDH12, ZmMDH13, SlMDH2, SlMDH3, and OsMDH3 (Figure 1A).

Phylogenetic Tree, Expression Profiles, and Subcellular Localization of OsMDH12
The full-length 1071 bp cDNA sequence of OsMDH12 (Os12g0632700) encodes a malate dehydrogenase that is predicted to be 357 amino acids long, according to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu(accessed on 1 May 2021).
A phylogenetic tree was constructed using the amino acid sequences of OsMDH12 and malate dehydrogenases from other plants, such as Arabidopsis, maize, and tomato.OsMDH12 displays high amino acid sequence similarity to AtpMDH1, AtpMDH2, ZmMDH12, ZmMDH13, SlMDH2, SlMDH3, and OsMDH3 (Figure 1A).
To assess the function of OsMDH12, we first analysed its expression pattern in various organs.Quantitative real-time PCR (qRT-PCR) revealed that OsMDH12 is expressed in all tissues examined, including roots, stems, leaves, and spikes (Figure 1B).Subsequently, we explored the expression of OsMDH12 under abiotic stress conditions.The transcription level of OsMDH12 in rice leaves and roots was detected after salt and cold treatment.The results indicated that the transcript level of OsMDH12 was strongly induced by both salt and cold stress (Figure 1C,D).
To ascertain the subcellular localization of OsMDH12, the coding sequence of the full-length OsMDH12 was fused to Green Fluorescent Protein (GFP) under the control of the CaMV35S promoter.Colocalization with the peroxisome protein AtpMDH1 [67] in the leaf epidermal cells of Nicotiana benthamiana confirmed a peroxisomal location for Os-MDH12 (Figure 1E).To assess the function of OsMDH12, we first analysed its expression pattern in various organs.Quantitative real-time PCR (qRT-PCR) revealed that OsMDH12 is expressed in all tissues examined, including roots, stems, leaves, and spikes (Figure 1B).Subsequently, we explored the expression of OsMDH12 under abiotic stress conditions.The transcription Plants 2023, 12, 3558 4 of 13 level of OsMDH12 in rice leaves and roots was detected after salt and cold treatment.The results indicated that the transcript level of OsMDH12 was strongly induced by both salt and cold stress (Figure 1C,D).
To ascertain the subcellular localization of OsMDH12, the coding sequence of the full-length OsMDH12 was fused to Green Fluorescent Protein (GFP) under the control of the CaMV35S promoter.Colocalization with the peroxisome protein AtpMDH1 [67] in the leaf epidermal cells of Nicotiana benthamiana confirmed a peroxisomal location for OsMDH12 (Figure 1E).

Enzyme Analysis of OsMDH12
Malate dehydrogenase (MDH) has been shown to catalyse the reversible reaction between malic acid and OAA, using NAD or NADP(H) as cofactors (Figure 2A).To ascertain the biochemical function of OsMDH12, we conducted enzymatic assays in vitro using recombinant OsMDH12 proteins.Our results demonstrated that OsMDH12 displayed high activity when OAA was used as the donor (Figure 2B).Conversely, no activity was detected when malate served as the donor (Figure 2B), suggesting that OsMDH12 catalyses the conversion of OAA to malate.

Enzyme Analysis of OsMDH12
Malate dehydrogenase (MDH) has been shown to catalyse the reversible reaction between malic acid and OAA, using NAD or NADP(H) as cofactors (Figure 2A).To ascertain the biochemical function of OsMDH12, we conducted enzymatic assays in vitro using recombinant OsMDH12 proteins.Our results demonstrated that OsMDH12 displayed high activity when OAA was used as the donor (Figure 2B).Conversely, no activity was detected when malate served as the donor (Figure 2B), suggesting that OsMDH12 catalyses the conversion of OAA to malate.

Molecular Characterization of MDH12-OE and osmdh12 Knockout Mutant Plants
To investigate the function of OsMDH12 in rice, we generated OsMDH12-overexpressing (OsMDH12-OE1 and OsMDH12-OE2) (Figure 3A) and osmdh12 CRISPR (os-mdh12-1 and osmdh12-2) lines (Figure 3B)in the japonica cultivar Zhonghua11 (ZH11) background.osmdh12-1 carried a one-base insertion (T) in the target site, and osmdh12-1 carried a two-base deletion (TC) in the target site, which all truncated the OsMDH12 open reading frame (Figure 3A).At the seedling stage, growth in the OsMDH12-OE plants was inhibited, showing significantly decreased shoot lengths and fresh weights (Figure 3C-E).In contrast, the osmdh12 mutants displayed a significant increase in shoot lengths and fresh weights(Figure 3C-E).Additionally, malate content was reduced in the osmdh12 mutant lines compared to the WT (Figure 3F).These results suggested that MDH12 negatively regulates growth but positively regulates malic acid content in rice.

Molecular Characterization of MDH12-OE and osmdh12 Knockout Mutant Plants
To investigate the function of OsMDH12 in rice, we generated OsMDH12-overexpressing (OsMDH12-OE1 and OsMDH12-OE2) (Figure 3A) and osmdh12 CRISPR (osmdh12-1 and osmdh12-2) lines (Figure 3B)in the japonica cultivar Zhonghua11 (ZH11) background.osmdh12-1 carried a one-base insertion (T) in the target site, and osmdh12-1 carried a twobase deletion (TC) in the target site, which all truncated the OsMDH12 open reading frame (Figure 3A).At the seedling stage, growth in the OsMDH12-OE plants was inhibited, showing significantly decreased shoot lengths and fresh weights (Figure 3C-E).In contrast, the osmdh12 mutants displayed a significant increase in shoot lengths and fresh weights(Figure 3C-E).Additionally, malate content was reduced in the osmdh12 mutant lines compared to the WT (Figure 3F).These results suggested that MDH12 negatively regulates growth but positively regulates malic acid content in rice.

MDH12 Negatively Regulates Tiller Numbers in Rice
To further explore the physiological functions of OsMDH12, we observed the phenotypes of OsMDH12-OE and osmdh12 mutant plants at the mature stage.When grown in a paddy field, no significant difference in plant height was observed between the osmdh12 mutant and the WT (Figure 4A,B).However, OsMDH12-OE plants displayed fewer tillers compared to WT plants (Figure 4C).Conversely, osmdh12 plants had a greater number of tillers, indicating that OsMDH12 negatively regulates tiller numbers in rice (Figure 4C).We then searched a publicly available genetic co-expression database (CREP, http://crep.ncpgr.cn/(accessed on 1 June 2022)) to identify genes co-expressed with OsMDH12 that are involved in regulating tiller number in rice.OsMDH12 exhibited a high correlation coefficient with three genes (PAY1, TN1, and OsTOM2) implicated in tiller number [31,68,69] (Figure 4D, Table S2).To investigate whether the expression levels of PAY1, TN1, and OsTOM2 were affected in osmdh12 mutant plants, we performed qRT-PCR analysis.The results revealed that the expression of PAY1 and TN1 in the shoots was not significantly different between OsMDH12-OE and osmdh12 mutant plants compared to WT (Figure 4E,F).However, the expression of OsTOM2 was repressed in the shoots of OsMDH12-OE plants but was upregulated in the roots of the osmdh12 mutant (Figure 4G).These findings suggest that OsMDH12 regulates tiller number through affecting the expression of OsTOM2 in rice.

MDH12 Negatively Regulates Tiller Numbers in Rice
To further explore the physiological functions of OsMDH12, we observed the phenotypes of OsMDH12-OE and osmdh12 mutant plants at the mature stage.When grown in a paddy field, no significant difference in plant height was observed between the osmdh12 mutant and the WT (Figure 4A,B).However, OsMDH12-OE plants displayed fewer tillers compared to WT plants (Figure 4C).Conversely, osmdh12 plants had a greater number of tillers, indicating that OsMDH12 negatively regulates tiller numbers in rice (Figure 4C).We then searched a publicly available genetic co-expression database (CREP, http://crep.ncpgr.cn/(accessed on 1 June 2022)) to identify genes co-expressed with OsMDH12 that are involved in regulating tiller number in rice.OsMDH12 exhibited a high correlation coefficient with three genes (PAY1, TN1, and OsTOM2) implicated in tiller number [31,68,69] (Figure 4D, Table S2).To investigate whether the expression levels of PAY1, TN1, and OsTOM2 were affected in osmdh12 mutant plants, we performed qRT-PCR analysis.The results revealed that the expression of PAY1 and TN1 in the shoots was not significantly different between OsMDH12-OE and osmdh12 mutant plants compared to WT (Figure 4E,F).However, the expression of OsTOM2 was repressed in the shoots of OsMDH12-OE plants but was upregulated in the roots of the osmdh12 mutant (Figure 4G).These findings suggest that OsMDH12 regulates tiller number through affecting the expression of OsTOM2 in rice.

OsMDH12 Negatively Regulates Salt Tolerance in Rice
To explore the role of OsMDH12 in salt tolerance, we induced salinity stress in OsMDH12-OE and osmdh12 mutant plants.Under normal growth conditions, no noticeable phenotypic differences were observed between OsMDH12 transgenic and WT plants (Figure 5A).However, when subjected to 150 mM NaCl treatment (Figure 5B,C), OsMDH12-OE plants exhibited salt-sensitive phenotypes, characterised by lower survival rates (Figure 5D) and increased ROS accumulation (Figure 5E).Conversely, osmdh12 mutants displayed enhanced salt stress tolerance.We then analysed the Na + /K + content ratio in both OsMDH12 transgenic and WT plants.The results revealed that the Na + /K + content ratio significantly increased in OsMDH12-OE plants but decreased in osmdh12 mutant plants (Figure 5F).These findings suggest that OsMDH12 negatively regulates salt tolerance through affecting the Na + /K + ratio in rice.

OsMDH12 Negatively Regulates Salt Tolerance in Rice
To explore the role of OsMDH12 in salt tolerance, we induced salinity stress in OsMDH12-OE and osmdh12 mutant plants.Under normal growth conditions, no noticeable phenotypic differences were observed between OsMDH12 transgenic and WT plants (Figure 5A).However, when subjected to 150 mM NaCl treatment (Figure 5B,C), OsMDH12-OE plants exhibited salt-sensitive phenotypes, characterised by lower survival rates (Figure 5D) and increased ROS accumulation (Figure 5E).Conversely, osmdh12 mutants displayed enhanced salt stress tolerance.We then analysed the Na + /K + content ratio in both OsMDH12 transgenic and WT plants.The results revealed that the Na + /K + content ratio significantly increased in OsMDH12-OE plants but decreased in osmdh12 mutant plants (Figure 5F).These findings suggest that OsMDH12 negatively regulates salt tolerance through affecting the Na + /K + ratio in rice.

Discussion
In this study, we characterised the function of OsMDH12, a member of the rice MDH gene family, and identified its negative regulatory role in controlling tiller number and salt tolerance.Malate dehydrogenase catalyses the reversible conversion of malate and oxaloacetate (OAA) using NADH or NADPH as coenzymes.NADP-dependent MDH isozymes are primarily localised in chloroplasts, while NAD-dependent MDH isozymes are found in other organelles, including the cytoplasm, mitochondria, glycosomes, peroxisomes, and chloroplasts [43].Our data demonstrate that OsMDH12 is an NADPdependent MDH isoform which is localised to the peroxisome and catalyses the conversion of OAA to malate in rice (Figure 1 and Figure 2).
Members of the MDH family are crucial for root growth [57], seed germination [66] and maturation [70], and embryo development [43].In Arabidopsis, the suppression of NAD + -dependent plastidial MDH (pdnad-mdh) leads to embryonic lethality, while reduced expression of pdnad-mdh results in dwarfism, diminished chlorophyll levels, lowered photosynthetic rates, reduced daytime carbohydrate levels, and chloroplast disorganization [51,68].In rice, OsMDH10 has been implicated in starch biosynthesis and seed development [40].OsMDH12 shares high homology with Arabidopsis AtpMDH1 and AtpMDH2, suggesting functional similarities (Figure 1).In Arabidopsis, the mmdh1/mmdh2 double mutants exhibit significantly reduced growth and photosynthesis rates compared to the WT [69,70].Similar phenotypes were observed in osmdh12 mutants during the seedling stage, including decreased plant height and fresh weight (Figure 3).These findings suggest that the functions of MDH genes are conserved in both rice and

Discussion
In this study, we characterised the function of OsMDH12, a member of the rice MDH gene family, and identified its negative regulatory role in controlling tiller number and salt tolerance.Malate dehydrogenase catalyses the reversible conversion of malate and oxaloacetate (OAA) using NADH or NADPH as coenzymes.NADP-dependent MDH isozymes are primarily localised in chloroplasts, while NAD-dependent MDH isozymes are found in other organelles, including the cytoplasm, mitochondria, glycosomes, peroxisomes, and chloroplasts [43].Our data demonstrate that OsMDH12 is an NADP-dependent MDH isoform which is localised to the peroxisome and catalyses the conversion of OAA to malate in rice (Figures 1 and 2).
Members of the MDH family are crucial for root growth [57], seed germination [66] and maturation [70], and embryo development [43].In Arabidopsis, the suppression of NAD +dependent plastidial MDH (pdnad-mdh) leads to embryonic lethality, while reduced expression of pdnad-mdh results in dwarfism, diminished chlorophyll levels, lowered photosynthetic rates, reduced daytime carbohydrate levels, and chloroplast disorganization [51,68].In rice, OsMDH10 has been implicated in starch biosynthesis and seed development [40].OsMDH12 shares high homology with Arabidopsis AtpMDH1 and AtpMDH2, suggesting functional similarities (Figure 1).In Arabidopsis, the mmdh1/mmdh2 double mutants exhibit significantly reduced growth and photosynthesis rates compared to the WT [69,70].Similar phenotypes were observed in osmdh12 mutants during the seedling stage, including decreased plant height and fresh weight (Figure 3).These findings suggest that the functions of MDH genes are conserved in both rice and Arabidopsis.However, unlike previous studies on MDH genes in plants [43,57,66,71], we found that OsMDH12 plays a pivotal role in regulating rice tiller number.
Tiller number, a crucial yield component, is a target for genetic improvement in crops [72].Elements, both macronutrients and micronutrients, significantly influence plant growth and the degree of tillering in rice [68,73].For example, iron (Fe) limitation hampers rice growth, reduces height, and restricts tiller numbers.OsTOM2, part of the major facilitator superfamily (MFS), is a 2'-deoxymugineic acid (DMA) transporter that mediates DMA secretion from roots to the rhizosphere and is involved in Fe mobilization via DMA secretion into the vascular bundles for phloem/xylem metal loading [74].Inhibition of OsTOM2 expression markedly reduces tiller number, dry weight, and yield in rice, implying that OsTOM2 is essential for optimal growth [68].We observed similar phenotypes in OsMDH12-overexpressing plants, which displayed fewer tillers and reduced dry weights (Figure 4).Moreover, the expression of OsTOM2 was significantly decreased in OsMDH12overexpressing plants, while it increased in OsMDH12 mutant plants, suggesting that OsMDH12 may regulate tiller number via OsTOM2 expression.The role of OsMDH12 in metal mobilization warrants further investigation.
Several studies have demonstrated that MDH genes play significant roles in plant responses to various abiotic stresses [40,54,57], particularly in salt stress resistance [48,65].For instance, the overexpression of plastid maize NADP-malate dehydrogenase (ZmNADP-MDH) in Arabidopsis enhances its salt stress tolerance [70].In apples, overexpressing cytosolic NAD-malate dehydrogenase (MdcyMDH) results in increased salt tolerance and decreased ROS levels [63].Recent research has shown that OsMDH1, a plastid NADdependent dehydrogenase, negatively regulates salt stress in rice [64].Further analysis indicated that OsMDH1 modulates the response to salt stress through affecting vitamin B6 levels [64].Despite the differing subcellular localizations of OsMDH1 and OsMDH12, the phenotypes of transgenic plants overexpressing either gene were similar (Figure 5).Lines overexpressing OsMDH1 or OsMDH12 are sensitive to salt stress, while lines with a knockout of OsMDH1 or OsMDH12 show tolerance to salt stress.In contrast to OsMDH1, our findings suggest that OsMDH12-mediated salt tolerance is closely linked to an imbalance in the K + /Na + ratio.Whether OsMDH12 is involved in the regulation of vitamin B6 synthesis remains an open question for further study.
In conclusion, we have uncovered the biological function of OsMDH12, a member of the malate dehydrogenase family.Our results show that OsMDH12 encodes a peroxisomal malate dehydrogenase that converts OAA to malate.Moreover, we demonstrate that OsMDH12 plays an essential role in regulating both plant growth and the response to salt stress.

Plant Materials and Stress Treatments
All plant materials used in this study were either ZH11 or derived from this cultivar.The osmdh12 mutants were generated using the CRISPR-Cas9 system, while OsMDH12-OE plants were produced under the control of the maize ubiquitin promoter in ZH11.The full-length coding sequence of OsMDH12 was amplified from Nipponbare (NIP) cultivar and cloned into the pJC034 vector.For physiological analyses, seedlings were cultivated in paddy fields in Lingshui, Hainan Province, China.For salt treatments, seeds were Plants 2023, 12, 3558 9 of 13 germinated for 3 days at 37 • C and then incubated in culture solution at 28 • C for 7 days.Following this, the seedlings were transferred to a 150 mmol/L NaCl solution for an additional 7 days.Seeds were then allowed to recover in a nutrient solution for 7 days [75].Shoot and root growth seedings were estimated as surviving seedings.Thirty plants of ZH11 and OsMDH12 transgenic lines for each experiment were used for subsequent statistical calculations.For cold treatment, the seedlings were exposed to 6 • C [76].Leaves were sampled at 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h intervals.

Gene Transcription Analysis
qRT-PCR was conducted to assess gene expression following salt or cold treatments.Leaves from various treatment groups were harvested at different time points post-treatment.All samples were processed using the RNA isolator Total RNA Extraction Reagent (Vazyme, Nanjing, China), and first-strand cDNA was synthesised using EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix (TRANSGEN, Beijing, China), as per the manufacturer's instructions.Primers for gene specificity were designed using Primer 5.0 and are listed in Supplemental Table S1.The internal reference used was the Ubiquitin5 gene.qRT-PCR was carried out on an ABI 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA), utilizing a SYBR Premix Ex Taq Kit in accordance with the manufacturer's guidelines.All reactions were conducted in a minimum of three biological replicates.

OsMDH Activity Assays
The full-length coding sequence of OsMDH12 was cloned into the pDEST15 vector using the Gateway recombination reaction (Invitrogen, Waltham, MA, USA).Expression constructs were introduced into BL21 (DE3) cells, and the expression of the fusion proteins was induced with 0.1 mM IPTG when the bacterial culture reached an OD600 of 0.5.After incubation at 16 • C for 14 h, cells were harvested via centrifugation and resuspended in 50 mL of Lysis Buffer containing Tris-HCl (50 mM), NaCl (50 mM), 5% glycerol, and PMSF (0.1 mM).Cells were disrupted using a pressure crusher, and the soluble protein fractions were subsequently centrifuged [77].Purification was carried out and monitored using a GST resin filter column for GST-tagged proteins.OsMDH12 activity was assessed through the spectrophotometric measurement of NADH consumption at 340 nm.The reaction was conducted in a 250 mM HEPES (pH 8.0) buffer containing MgCl 2 (2 mM), NAD + /NADH (0.25 mM), and either OAA or malate (2.5 mM).Purified OsMDH12-GST or GST (5 µg) was added to a 200 µL reaction volume.All reactions were conducted in a minimum of three replicates.

Measurement of Malate
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) system (QTRAP 6500, AB SCIEX, Toronto, ON, Canada) was used to determine the content of malate as previously described [73].All samples were rushed using a high-throughput tissue grinder (MM400, Retsch, Haan, Germany) after freeze-drying.The dry powder was extracted with 70% aqueous methanol containing lidocaine as the internal standard (0.1 mg/L) overnight at 4 • C. Following centrifugation at 1000× g for 5 min, the lipids were absorbed and filtered using a 0.22 µm pore size nylon syringe filter (SCAA-104, ANPEL, Shanghai, China) before the LC-MS analysis.For LC-MS analysis, a C18 column (Shim-pack GISS, SHIMADZU, Kyoto, Japan, 2.1 mm × 100 mm, 1.9 µm) was equipped.A 2 µL sample of OsMDH genes in plant development and in responding to salt stress remains largely unexplored.

Figure 1 .
Figure 1.Expression pattern of OsMDH12.(A) Phylogenetic relationship of MDH proteins in rice, Arabidopsis, and tomato.Bootstrap values from 1000 trials are indicated.(B) Expression levels of OsMDH12 in different rice organs.Samples were taken from Zhonghua11 grown in a paddy field.(C,D) Time-course expression of OsMDH12 under salt (C) and cold (D) treatment.Standard deviations are based on three biological replicates.(E) Subcellular localization of OsMDH12 protein in leaves of N. benthamiana.35S:GFP-OsMDH12 and 35S:RFP:AtpMDH1 were co-transformed into the leaves of Nicotiana benthamiana.AtpMDH1 serves as a peroxisome marker.The scale is set at 50 µm.

Plants 2023 , 14 Figure 1 .
Figure 1.Expression pattern of OsMDH12.(A) Phylogenetic relationship of MDH proteins in rice, Arabidopsis, and tomato.Bootstrap values from 1000 trials are indicated.(B) Expression levels of OsMDH12 in different rice organs.Samples were taken from Zhonghua11 grown in a paddy field.(C,D) Time-course expression of OsMDH12 under salt (C) and cold (D) treatment.Standard deviations are based on three biological replicates.(E) Subcellular localization of OsMDH12 protein in leaves of N. benthamiana.35S:GFP-OsMDH12 and 35S:RFP:AtpMDH1 were co-transformed into the leaves of Nicotiana benthamiana.AtpMDH1 serves as a peroxisome marker.The scale is set at 50 µm.

Figure 2 .
Figure 2. OsMDH12 catalyses OAA to malate.(A) Schematic diagram of the enzyme-catalysed reaction by Bend OsMDH12.(B) Assays of OsMDH12 activity.The assay was conducted in the presence of GST-OsMDH12, GST, NADH, and OAA.Data are expressed as the decrease in OD at 340 nm over time.

Figure 2 .
Figure 2. OsMDH12 catalyses OAA to malate.(A) Schematic diagram of the enzyme-catalysed reaction by Bend OsMDH12.(B) Assays of OsMDH12 activity.The assay was conducted in the presence of GST-OsMDH12, GST, NADH, and OAA.Data are expressed as the decrease in OD at 340 nm over time.

Figure 3 .
Figure 3. Appearance and malate contents of OsMDH12 transgenic lines at the seedling stage.(A) The osmdh12 knockout mutants were generated using CRISPR gene editing technology.(B) Expression level of OsMDH12 in two independent overexpressing lines.(C) Phenotype comparison of ZH11, OsMDH12-OE lines, and osmdh12 mutants (two different CRISPR lines) at the seedling stage.The scale is set at 2 cm.(D,E) Plant height (D) and fresh weight (E) in ZH11, OsMDH12-OE, and osmdh12 mutants.(F) Malate content in ZH11, OsMDH12-OE, and osmdh12 mutants.Values are presented as mean ± SD (n = 10); ** p < 0.01 (t-test).

Figure 3 .
Figure 3. Appearance and malate contents of OsMDH12 transgenic lines at the seedling stage.(A) The osmdh12 knockout mutants were generated using CRISPR gene editing technology.(B) Expression level of OsMDH12 in two independent overexpressing lines.(C) Phenotype comparison of ZH11, OsMDH12-OE lines, and osmdh12 mutants (two different CRISPR lines) at the seedling stage.The scale is set at 2 cm.(D,E) Plant height (D) and fresh weight (E) in ZH11, OsMDH12-OE, and osmdh12 mutants.(F) Malate content in ZH11, OsMDH12-OE, and osmdh12 mutants.Values are presented as mean ± SD (n = 10); ** p < 0.01 (t-test).

Figure 5 .
Figure 5. Performance of OsMDH12 transgenic plants under salt stress.(A-C) Two-week-old rice seedlings from ZH11, OsMDH12-OE, and osmdh12 plants (A) were treated with 150 mM NaCl for seven days (B) and then allowed to recover for three days (C).The scale is set at 5 cm.(D) Survival rate under salt stress.(E) Phenotype revealed by NBT staining after 24 h of salt stress.(F) Na + /K + ratio in ZH11, OsMDH12-OE, and osmdh12 mutants.Values represent the mean ± SD (n = 3); * p < 0.05, ** p < 0.01 (t-test).

Figure 5 .
Figure 5. Performance of OsMDH12 transgenic plants under salt stress.(A-C) Two-week-old rice seedlings from ZH11, OsMDH12-OE, and osmdh12 plants (A) were treated with 150 mM NaCl for seven days (B) and then allowed to recover for three days (C).The scale is set at 5 cm.(D) Survival rate under salt stress.(E) Phenotype revealed by NBT staining after 24 h of salt stress.(F) Na + /K + ratio in ZH11, OsMDH12-OE, and osmdh12 mutants.Values represent the mean ± SD (n = 3); * p < 0.05, ** p < 0.01 (t-test).