A DUF966 gene family member OsDSR3 positively regulates alkali stress tolerance in rice.

Rice growth and production are severely constrained by alkali stress. However, the mechanism underlying the rice tolerance to alkali stress is unclear. OsDSR3, a novel gene from the domains of unknown function 966 (DUF966) family, was identified and characterized for its function in the response of rice to alkali stress. The result of this study clearly showed that alkali stress significantly induced OsDSR3 expression level. Moreover, the expression of OsDSR3 was up-regulated by drought, salt, cold, H2O2 and abscisic acid (ABA), and down-regulated by gibberellic acid (GA3), and 2,4-Dichlorophenoxyacetic acid (2,4-D) treatments. Subcellular localization exhibited that OsDSR3 was detected in the nucleus and membrane. OsDSR3-overexpressing (OsDSR3-OE) plants showed higher tolerance to alkali stress than the wild-type (WT). In contrast, OsDSR3 knockout (OsDSR3-KO) mutants were more vulnerable to alkali stress. The differentially expressed genes (DEGs) among OsDSR3-OE and WT seedlings were mainly enriched in porphyrin and chlorophyll, starch and sucrose, and carotenoid metabolic pathways. Among these DEGs, 26 were identified as potential alkali stress-responsive genes, including several up-regulated genes like OsHAK5, OsGRX23 and OsNIR2. Consistent with the expression profiles of metabolic pathways-related genes, most of the metabolite contents and metabolite synthases activities were improved in OsDSR3-OE lines and decreased in OsDSR3-KO lines compared to WT. This may explain the higher tolerance of OE lines and lower tolerance of KO lines to alkali stress. These findings suggested that OsDSR3 positively regulates rice tolerance to alkali stress, which will help to elucidate the molecular mechanism underlying rice alkali tolerance.

Domain of unknown function (DUF) proteins in plants: function and perspective.

Domains of unknown function (DUFs), which are deposited in the protein family database (Pfam), are protein domains with conserved amino acid sequences and uncharacterized functions. Proteins with the same DUF were classified as DUF families. Although DUF families are generally not essential for the survival of plants, they play roles in plant development and adaptation. Characterizing the functions of DUFs is important for deciphering biological puzzles. DUFs were generally studied through forward and reverse genetics. Some novelty approaches, especially the determination of crystal structures and interaction partners of the DUFs, should attract more attention. This review described the identification of DUF genes by genome-wide and transcriptome-wide analyses, summarized the function of DUF-containing proteins, and addressed the prospects for future studies in DUFs in plants.

The suppressed expression of a stress responsive gene 'OsDSR2' enhances rice tolerance in drought and salt stress.

Rice is a crucial staple food crop in many countries, yet, abiotic factors like salt and drought impact its growth. The Domain of Unknown Function 966 (DUF966) gene family may be crucial in how rice plants respond to abiotic stress. Our earlier research showed that overexpression of OsDSR2 (DUF966-stress repressive gene 2 in Oryza sativa) decreased resistance to salt and drought stress. To further understand how OsDSR2 negatively affects rice tolerance to salt and drought stress, transgenic rice plants with decreased OsDSR2 expression levels were created employing the RNAi technique. We investigated alterations in rice phenotype, physiology, and differentially expressed genes (DEGs) using a combination of physio-biochemical measurement and RNA-seq analysis. The results of the study demonstrated that rice seedling lines with OsDSR2 knockdown exhibited improved salt and drought stress tolerance. Statistical analysis revealed that the transgenic plants' survival rate (56-68%) was higher than the control plants (30%), in addition to a roughly 3 fold, 3.5 fold, 20% and 10.5% reduction in cell membrane permeability, malondialdehyde (MDA), superoxide anion radical (O2-) and hydrogen peroxide (H2O2) contents, respectively. However, the proline content and antioxidant enzymes (superoxide dismutase (SOD) and peroxidase (POD)) activities were considerably increased by about 5.5 fold, 3.5 fold, and 4.5 fold, respectively, at physiological levels. There were 115 up-regulated and 173 down-regulated DEGs in the leaves of the transgenic lines on the transcriptional regulation under the combined salt-drought stress. Among these, both up-regulation DEGs (e.g., OsHAK5, OsIAA25) and the down-regulation DEGs (e.g., OsbZIP23, OsERF48, OsAP2-39, etc.) may be related to the enhanced tolerance of the transgenic lines under combined salt-drought stress. This possibly depended on the involvement of abscisic acid (ABA) and indoleacetic acid (IAA) signaling pathways. These findings further confirmed that OsDSR2 negatively affected rice's ability to withstand salt and drought, suggesting that it could be a helpful gene for CRISPR-Cas9 technology-based genetic modification of rice's ability to withstand abiotic stress.

Melatonin Enhances Drought Tolerance in Rice Seedlings by Modulating Antioxidant Systems, Osmoregulation, and Corresponding Gene Expression.

Rice is the third largest food crop in the world, especially in Asia. Its production in various regions is affected to different degrees by drought stress. Melatonin (MT), a novel growth regulator, plays an essential role in enhancing stress resistance in crops. Nevertheless, the underlying mechanism by which melatonin helps mitigate drought damage in rice remains unclear. Therefore, in the present study, rice seedlings pretreated with melatonin (200 µM) were stressed with drought (water potential of -0.5 MPa). These rice seedlings were subsequently examined for their phenotypes and physiological and molecular properties, including metabolite contents, enzyme activities, and the corresponding gene expression levels. The findings demonstrated that drought stress induced an increase in malondialdehyde (MDA) levels, lipoxygenase (LOX) activity, and reactive oxygen species (ROS, e.g., O2- and H2O2) in rice seedlings. However, the melatonin application significantly reduced LOX activity and the MDA and ROS contents (O2- production rate and H2O2 content), with a decrease of 29.35%, 47.23%, and (45.54% and 49.33%), respectively. It activated the expression of ALM1, OsPOX1, OsCATC, and OsAPX2, which increased the activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), respectively. Meanwhile, the melatonin pretreatment enhanced the proline, fructose, and sucrose content by inducing OsP5CS, OsSUS7, and OsSPS1 gene expression levels. Moreover, the melatonin pretreatment considerably up-regulated the expression levels of the melatonin synthesis genes TDC2 and ASMT1 under drought stress by 7-fold and 5-fold, approximately. These improvements were reflected by an increase in the relative water content (RWC) and the root-shoot ratio in the drought-stressed rice seedlings that received a melatonin application. Consequently, melatonin considerably reduced the adverse effects of drought stress on rice seedlings and improved rice's ability to tolerate drought by primarily boosting endogenous antioxidant enzymes and osmoregulation abilities.

Exogenous Melatonin Alleviates Alkaline Stress by Removing Reactive Oxygen Species and Promoting Antioxidant Defence in Rice Seedlings.

Saline-alkali stress seriously restricts rice growth, development, and production in northern China. The damage of alkaline stress on rice is much greater than that of salt due to ion toxicity, osmotic stress, and especially high pH. As a signal molecule, melatonin (N-acetyl-5-methoxytryptamine, MT) mediates many physiological processes in rice and participates in protecting rice from abiotic stress. The potential mechanism of exogenous melatonin-mediated alkaline stress tolerance is still largely unknown. In this study, the effects of melatonin on the morphological change, physiological property, and corresponding genes expression in rice seedlings were analyzed under alkaline stress (20 mmol L-1, pH 9.55). The results showed that the expression levels of MT synthesis genes (TDC2, T5H, SNAT, ASMT1, and ASMT2) were induced by both exogenous MT and alkaline stress treatment. The cell membrane was protected by MT, and the MT furtherly play role in scavenging reactive oxygen species (ROS), reducing lipoxygenase (LOX) activity, and malondialdehyde (MDA) content. The scavenging of ROS by melatonin is attributed to the coupling of the improvement of redox homeostasis and the enhancement of antioxidant enzyme activity and antioxidant content by upregulating the transcriptional levels of antioxidase genes. In the meantime, MT pretreatment promoted the accumulation of free proline, sucrose, and fructose by regulating the OsP5CS, OsSUS7, and OsSPS1 gene expression level and increased chlorophyll content upregulating the expression of chlorophyll synthesis-related genes. Ultimately, the alleviating effect of exogenous melatonin on alkaline stress was reflected in increasing the leaf relative water content (RWC) and root-shoot ratio and reducing the leaf tip wilt index (TWI) through a series of physiological and biochemical changes. Melatonin pretreatment changed the expression level of MT synthesis genes which might contribute to MT synthesis in rice, consequently, activated the ROS scavenging system and alleviating the damage of alkaline stress on rice seedlings. Our study comprehensively understands the alleviating effect of exogenous melatonin on rice under alkaline stress.