OsTrxL1 Interacts with OsCYP89A2 to Cooperatively Regulate Drought Stress Responses in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2026-0193

Abstract

Abstract:

Objective This study aims to elucidate the biological function of the rice thioredoxin-like protein gene OsTrxL1 in response to drought stress and its potential molecular regulatory mechanisms, providing a theoretical basis for a deeper understanding of rice abiotic stress responses. Methods CRISPR/Cas9 gene editing and transgenic techniques were used to obtain knockout and overexpression lines. The biological function of OsTrxL1 was analyzed by measuring physiological indicators under drought stress in rice. In combination with subcellular localization analysis, RT-qPCR, yeast two-hybrid, and split luciferase assays, the molecular regulatory mechanism of OsTrxL1 in drought stress response was elucidated. Results OsTrxL1 protein was localized in the endoplasmic reticulum and nucleus. RT-qPCR analysis showed that this gene was expressed in different rice tissues including roots, stems, and leaves, and was induced by drought, cold, and salt stress. Overexpression of OsTrxL1 significantly enhanced rice tolerance to abiotic stress, reflected by increased post-stress survival rate, reduced membrane lipid peroxidation, and enhanced osmotic regulation ability, whereas OsTrxL1 knockout mutants exhibited reduced stress resistance. OsTrxL1 physically interacts with the cytochrome P450 family member OsCYP89A2. Conclusion OsTrxL1 may maintain redox homeostasis within the endoplasmic reticulum lumen and stabilize the protein structure of OsCYP89A2 under stress conditions, thereby positively regulating rice response and adaptation to abiotic stress, providing an important candidate gene resource for crop stress-resistance molecular breeding.

Key words: OsTrxL1, OsCYP89A2, drought stress, endoplasmic reticulum, rice

ZHONG Jiao, REN Qiong, FAN Zhao-rui, DUAN Yu, QIN Tong, KANG Zhen-hui. OsTrxL1 Interacts with OsCYP89A2 to Cooperatively Regulate Drought Stress Responses in Rice[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0193.

Figures/Tables 7

Table 1 Candidate interacting proteins of OsTrxL1 identified by the yeast two-hybrid system

NO.	Protein names	Identities	Notes
1	40S ribosomal protein S14	151/151（100%）	Sequence ID： XP_015627702.1
2	Cytochrome P450 89A2	249/249（100%）	GeneID：4349105
3	DNA-binding protein MNB1B	157/157（100%）	Sequence ID： XP_015643271.1
4	FLO/LFY-like protein， partial	247/248（99%）	Sequence ID： AFA43522.1
5	Hypothetical protein DAI22_04g114500	92/95（97%）	Sequence ID： KAF2933812.1
6	Hypothetical protein EJB05_02252， partial	26/34（76%）	Sequence ID： TVU50860.1
7	Pleckstrin homology （PH） domain-containing protein-like	82/82（100%）	Sequence ID： BAD82044.1
8	Putative chloroplastic RNA-binding protein， with alternative splicing isoforms	271/288（94%）	Sequence ID： AAS01974.1
9	Ribosomal L1 domain-containing protein 1	266/268（99%）	GeneID：4339582
10	Tubulin alpha-1 chain， putative， expressed	129/129（100%）	Sequence ID： ABF98641.1
11	U4/U6 small nuclear ribonucleoprotein Prp31 homolog isoform X1	265/265（100%）	GeneID：4336619

Fig. 1 Phylogenetic tree, conserved motifs, and gene structuresA: Phylogenetic tree of the rice Trx gene family; B: expression of conserved motifs in rice Trx proteins; C: exon-intron structures of the rice Trx gene family

Fig. 2 Subcellular localization and expression pattern of OsTrxL1A: Subcellular localization of OsTrxL1 in tobacco epidermal cells, bar=20 μm. B: Colocalization of OsTrxL1 in tobacco epidermal cells, green fluorescence indicates GFP signal, red fluorescence indicates chloroplast autofluorescence and nuclear autofluorescence (NLS), bright field (Bright), bar=20 μm. C: Expression levels of OsTrxL1 in different rice tissues (vs. Root). D: Gene expression levels of OsTrxL1 under different stress treatments (20% PEG6000, 150 mmol/L NaCl). E: Gene expression levels of OsTrxL1 at different time points under drought stress (vs. Control). F: OsTrxL1pro::GUS staining. Data are presented as mean± standard deviation. Data were analyzed using t-test, (n=3; *P<0.05; **P<0.01; ***P<0.001; ****P<0.000 1), the same below

Fig. 3 Seedling growth and water loss phenotypes of OsTrxL1 transgenic linesA: Sequence comparison diagram of the CRISPR/Cas9-edited T1 generation. The underlined sequences represent the sgRNA and PAM sequences, and red text indicates nucleotide sequence changes. B: Expression level of OsTrxL1 in the OE (overexpression) line at the T2 generation and WT plants. C: Seedling development of wild-type (WT), overexpression (OE), and knockout (KO) lines under control conditions, 200 mmol/L mannitol, 150 mmol/L NaCl (bar=5 cm), 100 μmol/L ABA, and 4 ℃ (bar=5 mm). D: Phenotypes of each line after drought stress treatment for 5 and 10 days. E: Seedling height. F: Adventitious root number under 200 mmol/L mannitol and 150 mmol/L NaCl. G: Leaf water loss rate. H: Survival rate of plants under 25% PEG6000 treatment

Fig. 4 Agronomic traits related to OsTrxL1 transgenic linesA-B: Mature plant height, Bar=30 cm. C, D: Spike length, Bar=2 cm. E, G: Grain length. F, H: Grain width, Bar=2 cm. I: Thousand-grain weight. J: Tillering number. K: Flag leaf length. L: Flag leaf width

Fig. 5 Physiological and biochemical indicators of OsTrxL1 transgenic lines under 20% PEG6000 simulated drought treatment for 3 hoursA: Malondialdehyde. B: Superoxide dismutase. C: Peroxidase. D: NBT staining. E: DAB staining, PEG: 20%PEG6000 treatment for 3 h, Bar=5 mm

Fig. 6 Yeast two-hybrid screening and luciferase protein complementation assay for OsTrxL1 and OsCYP89A2A: Self-activation and functional validation of the bait. B: Yeast two-hybrid confirmation. C: Luciferase protein complementation assay.

References 39

[1]	He ZM, Yan Y, Guo XJ, et al. Trp31 residue of trx-1 is essential for maintaining antioxidant activity and cellular redox defense against oxidative stress [J]. Antioxidants, 2025, 14(3): 257.
[2]	Sugiura K, Yokochi Y, Fu NE, et al. The thioredoxin (Trx) redox state sensor protein can visualize Trx activities in the light/dark response in chloroplasts [J]. J Biol Chem, 2019, 294(32): 12091-12098.
[3]	Xu LG, Zhou Y, Cheng J, et al. Identification of thioredoxin genes and analysis of their expression under abiotic stresses in Medicago truncatula [J]. Acta Physiol Plant, 2022, 44(11): 120.
[4]	Nikkanen L, Rintamäki E. Chloroplast thioredoxin systems dynamically regulate photosynthesis in plants [J]. Biochem J, 2019, 476(7): 1159-1172.
[5]	Lee ES, Park JH, Wi SD, et al. Demyristoylation of the cytoplasmic redox protein trx-h2 is critical for inducing a rapid cold stress response in plants [J]. Antioxidants, 2021, 10(8): 1287.
[6]	You J, Chan ZL. ROS regulation during abiotic stress responses in crop plants [J]. Front Plant Sci, 2015, 6: 1092.
[7]	Hägglund P, Finnie C, Yano H, et al. Seed thioredoxin h [J]. BBA Proteins Proteom, 2016, 1864(8): 974-982.
[8]	Sainz MM, Filippi CV, Eastman G, et al. Analysis of thioredoxins and glutaredoxins in soybean: evidence of translational regulation under water restriction [J]. Antioxidants, 2022, 11(8): 1622.
[9]	Yokochi Y, Fukushi Y, Wakabayashi KI, et al. Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana [J]. Proc Natl Acad Sci U S A, 2021, 118(51): e2114952118.
[10]	Sekiguchi T, Yoshida K, Wakabayashi KI, et al. Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins [J]. J Biol Chem, 2022, 298(11): 102541.
[11]	Fukushi Y, Yokochi Y, Hisabori T, et al. Overexpression of thioredoxin-like protein ACHT2 leads to negative feedback control of photosynthesis in Arabidopsis thaliana [J]. J Plant Res, 2024, 137(3): 445-453.
[12]	Yokochi Y, Yoshida K, Hahn F, et al. Redox regulation of NADP-malate dehydrogenase is vital for land plants under fluctuating light environment [J]. Proc Natl Acad Sci U S A, 2021, 118(6): e2016903118.
[13]	Zhang HH, Liu XQ, Zhang HB, et al. Thioredoxin-like protein CDSP32 alleviates Cd-induced photosynthetic inhibition in tobacco leaves by regulating cyclic electron flow and excess energy dissipation [J]. Plant Physiol Biochem, 2021, 167: 831-839.
[14]	Sachdev S, Ansari SA, Ansari MI, et al. Abiotic stress and reactive oxygen species: generation, signaling, and defense mechanisms [J]. Antioxidants, 2021, 10(2): 277.
[15]	Gechev T, Petrov V. Reactive oxygen species and abiotic stress in plants [J]. Int J Mol Sci, 2020, 21(20): 7433.
[16]	Han SC, Wang YL, Li YX, et al. The OsNAC41-RoLe1-OsAGAP module promotes root development and drought resistance in upland rice [J]. Mol Plant, 2024, 17(10): 1573-1593.
[17]	Wei SB, Li X, Lu ZF, et al. A transcriptional regulator that boosts grain yields and shortens the growth duration of rice [J]. Science, 2022, 377(6604): eabi8455.
[18]	Uga Y, Sugimoto K, Ogawa S, et al. Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions [J]. Nat Genet, 2013, 45(9): 1097-1102.
[19]	Perreau F, Frey A, Effroy-Cuzzi D, et al. ABSCISIC ACID-DEFICIENT4 has an essential function in both cis-violaxanthin and cis-neoxanthin synthesis [J]. Plant Physiol, 2020, 184(3): 1303-1316.
[20]	Williams D, De Luca V. Plant cytochrome P450s directing monoterpene indole alkaloid (MIA) and benzylisoquinoline alkaloid (BIA) biosynthesis [J]. Phytochem Rev, 2023, 22(2): 309-338.
[21]	Pandian BA, Sathishraj R, Djanaguiraman M, et al. Role of cytochrome P450 enzymes in plant stress response [J]. Antioxidants, 2020, 9(5): 454.
[22]	Tamiru M, Undan JR, Takagi H, et al. A cytochrome P450, OsDSS1, is involved in growth and drought stress responses in rice (Oryza sativa L.) [J]. Plant Mol Biol, 2015, 88(1-2): 85-99.
[23]	Wang M, Yuan JR, Qin LM, et al. TaCYP81D5, one member in a wheat cytochrome P450 gene cluster, confers salinity tolerance via reactive oxygen species scavenging [J]. Plant Biotechnol J, 2020, 18(3): 791-804.
[24]	Liu B, Song ZY, Qi XQ. Plant cytochrome P450 enzymes for bioactive metabolites biosynthesis, growth regulation, and stress adaptation [J]. Plant Physiol, 2025, 199(1): kiaf297.
[25]	Bang SW, Lee DK, Jung H, et al. Overexpression of OsTF1L, a rice HD-Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance [J]. Plant Biotechnol J, 2019, 17(1): 118-131.
[26]	Riaz M, Ali Q, Yan L. Calcium-L-aspartate nanoparticles mitigate Boron toxicity in rice seedlings by modulating physiological, antioxidant, and cell wall mechanisms [J]. Sci Rep, 2025, 15: 42439.
[27]	Zhang SX, Yu Y, Song TQ, et al. Genome-wide identification of foxtail millet’s TRX family and a functional analysis of SiNRX1 in response to drought and salt stresses in transgenic Arabidopsis [J]. Front Plant Sci, 2022, 13: 946037.
[28]	Ji MG, Park HJ, Cha JY, et al. Expression of Arabidopsis thaliana Thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance [J]. Plant Physiol Biochem, 2020, 147: 313-321.
[29]	Chen JL, Zhang J, Fang C, et al. OsPDIL1-5: dual role in promoting growth and development while modulating drought stress tolerance in rice (Oryza sativa L.) [J]. Front Plant Sci, 2024, 15: 1479726.
[30]	Jung SE, Kim TH, Shim JS, et al. Rice NAC17 transcription factor enhances drought tolerance by modulating lignin accumulation [J]. Plant Sci, 2022, 323: 111404.
[31]	Sun Y, Gu XY, Qu CF, et al. OsPUB75-OsHDA716 mediates deactivation and degradation of OsbZIP46 to negatively regulate drought tolerance in rice [J]. Plant Physiol, 2024, 197(1): kiae545.
[32]	Liu J, Shi K, Wang SP, et al. MsCYP71 is a positive regulator for drought resistance in alfalfa [J]. Plant Physiol Biochem, 2023, 203: 107999.
[33]	Johnson SM, Lim FL, Finkler A, et al. Transcriptomic analysis of Sorghum bicolor responding to combined heat and drought stress [J]. BMC Genom, 2014, 15(1): 456.
[34]	Rao MJ, Xu YT, Tang XM, et al. CsCYT75B1, a Citrus CYTOCHROME P450 gene, is involved in accumulation of antioxidant flavonoids and induces drought tolerance in transgenic Arabidopsis [J]. Antioxidants, 2020, 9(2): 161.
[35]	Luo YH, Zhong JJ, Xiao H. Mechanism and engineering of endoplasmic reticulum-localized membrane protein folding in Saccharomyces cerevisiae [J]. Metab Eng, 2025, 90: 43-56.
[36]	Brignac-Huber LM, Park JW, Reed JR, et al. Cytochrome P450 organization and function are modulated by endoplasmic reticulum phospholipid heterogeneity [J]. Drug Metab Dispos, 2016, 44(12): 1859-1866.
[37]	Chong W, Shastri M, Eri R. Endoplasmic reticulum stress and oxidative stress: a vicious nexus implicated in bowel disease pathophysiology [J]. Int J Mol Sci, 2017, 18(4): 771.
[38]	Chen JS, Yap MC, Bassot A, et al. The ER thioredoxin-related transmembrane protein TMX2 controls redox-mediated tethering of ER-mitochondria contacts [J]. Cell Rep, 2025, 44(11): 116486.
[39]	Ong G, Logue SE. Unfolding the interactions between endoplasmic reticulum stress and oxidative stress [J]. Antioxidants, 2023, 12(5): 981.