A rice really interesting new gene H2‐type E3 ligase,OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programmes. Here, we characterized the rice (Oryza sativa) really interesting new gene (RING) H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na⁺ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared with wild type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na⁺ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

Overexpression of Rice Sphingosine-1-Phoshpate Lyase Gene OsSPL1 in Transgenic Tobacco Reduces Salt and Oxidative Stress Tolerance

Sphingolipids, including sphingosine-1-phosphate (S1P), have been shown to function as signaling mediators to regulate diverse aspects of plant growth, development, and stress response. In this study, we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response. Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA), and decreased tolerance to salt and oxidative stress, when compared with the wild type. Furthermore, the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress, indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress. Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses.     

OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64)

To overcome the salinity‐induced loss of crop yield, a salinity‐tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T₁ and T₂ sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T₂ transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H₂O₂ production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity‐induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice.     

Molecular characterization of rice arsenic‐induced RING finger E3 ligase 2 (OsAIR2) and its heterogeneous overexpression in Arabidopsis thaliana

Arsenic (As) accumulation adversely affects the growth and productivity of plants and poses a serious threat to human health and food security. In this study, we identified one As‐responsive R eally I nteresting N ew G ene (RING) E3 ubiquitin ligase gene from rice root tissues during As stress. We named it Oryza sativa As‐Induced RING E3 ligase 2 (OsAIR2). Expression of OsAIR2 was induced under various abiotic stress conditions, including heat, salt, drought and As exposure. Results of an in vitro ubiquitination assay showed that OsAIR2 possesses an E3 ligase activity. Within the cell, OsAIR2 was found to be localized to the Golgi apparatus. Using yeast two‐hybrid (Y2H) assay, the 3‐ketoacyl‐CoA thiolase (KAT) protein was identified as an interaction partner. We found that the O. sativa KAT1 (OsKAT1) is localized to the cytosol and peroxisomes. Moreover, in vitro pull‐down assay verified the physical interaction between OsAIR2 and OsKAT1. Interestingly, in vitro ubiquitination assay and in vivo proteasomal degradation assay revealed that OsAIR2 ubiquitinates OsKAT1 and promotes the degradation of OsKAT1 via the 26S proteasome degradation pathway. Heterogeneous overexpression of OsAIR2 in Arabidopsis improved the seed germination and increased the root length under arsenate stress conditions. Therefore, these results suggest that OsAIR2 may be associated with the plant response to As stress and acts as a positive regulator of As stress tolerance.     

Overexpression of the trehalose-6-phosphate synthase gene <Emphasis Type="Italic">OsTPS1</Emphasis> enhances abiotic stress tolerance in rice

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants.     

Rice plastidial NAD‐dependent malate dehydrogenase 1 negatively regulates salt stress response by reducing the vitamin B6 content

Salinity is an important environmental factor that adversely impacts crop growth and productivity. Malate dehydrogenases (MDHs) catalyse the reversible interconversion of malate and oxaloacetate using NAD(H)/NADP(H) as a cofactor and regulate plant development and abiotic stress tolerance. Vitamin B6 functions as an essential cofactor in enzymatic reactions involved in numerous cellular processes. However, the role of plastidial MDH in rice (Oryza sativa) in salt stress response by altering vitamin B6 content remains unknown. In this study, we identified a new loss‐of‐function osmdh1 mutant displaying salt stress‐tolerant phenotype. The OsMDH1 was expressed in different tissues of rice plants including leaf, leaf sheath, panicle, glume, bud, root and stem and was induced in the presence of NaCl. Transient expression of OsMDH1‐GFP in rice protoplasts showed that OsMDH1 localizes to chloroplast. Transgenic rice plants overexpressing OsMDH1 (OsMDH1OX) displayed a salt stress‐sensitive phenotype. Liquid chromatography–mass spectrometry (LC‐MS) metabolic profiling revealed that the amount of pyridoxine was significantly reduced in OsMDH1OX lines compared with the NIP plants. Moreover, the pyridoxine content was higher in the osmdh1 mutant and lower in OsMDH1OX plants than in the NIP plants under the salt stress, indicating that OsMDH1 negatively regulates salt stress‐induced pyridoxine accumulation. Furthermore, genome‐wide RNA‐sequencing (RNA‐seq) analysis indicated that ectopic expression of OsMDH1 altered the expression level of genes encoding key enzymes of the vitamin B6 biosynthesis pathway, possibly reducing the level of pyridoxine. Together, our results establish a novel, negative regulatory role of OsMDH1 in salt stress tolerance by affecting vitamin B6 content of rice tissues.     

The wheat TaGBF1 gene is involved in the blue‐light response and salt tolerance

Light and abiotic stress both strongly modulate plant growth and development. However, the effect of light‐responsive factors on growth and abiotic stress responses in wheat (Triticum aestivum) is unknown. G–box binding factors (GBFs) are blue light‐specific components, but their function in abiotic stress responses has not been studied. Here we identified a wheat GBF1 gene that mediated both the blue light‐ and abiotic stress‐responsive signaling pathways. TaGBF1 was inducible by blue light, salt and exposure to abscisic acid (ABA). TaGBF1 interacted with a G–box light‐responsive element in vitro and promoted a blue‐light response in wheat and Aradidopsis thaliana. Both TaGBF1 over‐expression in wheat and its heterologous expression in A. thaliana heighten sensitivity to salinity and ABA, but its knockdown in wheat conferred resistance to high salinity and ABA. The expression of AtABI5, a key component of the ABA signaling pathway in A. thaliana, and its homolog Wabi5 in wheat was increased by transgenic expression of TaGBF1. The hypersensitivity to salt and ABA caused by TaGBF1 was not observed in the abi5 mutant background, showing that ABI5 is the mediator in TaGBF1‐induced abiotic stress responses. However, the hypersensitivity to salt conferred by TaGBF1 is not dependent on light. This suggests that TaGBF1 is a common component of blue light‐ and abiotic stress‐responsive signaling pathways.     

Leaf cell membrane stability‐based mechanisms of zinc nutrition in mitigating salinity stress in rice

• Excess salt affects about 955 million ha of arable land worldwide, and 49% of agricultural land is Zn‐deficient. Soil salinity and zinc deficiency can intensify plant abiotic stress. The mechanisms by which Zn can mitigate salinity effects on plant functions are not well understood.
• We conducted an experiment to determine how Zn and salinity effects on rice plant retention of Zn, K⁺ and the salt ion Na⁺ affect chlorophyll formation, leaf cell membrane stability and grain yield. We examined the mechanisms of Zn nutrition in mitigating salinity stress by examining plant physiology and nutrition. We used native Zn‐deficient soils (control), four salinity (EC ) and Zn treatments – Zn 10 mg·kg^?1 (Zn₁₀), EC 5 dS ·m^?1 (EC ₅), Zn₁₀+EC ₅ and Zn₁₅+EC ₅, a coarse rice (KS ‐282) and a fine rice (Basmati‐515) in the study.
• Our results showed that Zn alone (Zn₁₀) significantly increased rice tolerance to salinity stress by promoting Zn/K⁺ retention, inhibiting plant Na⁺ uptake and enhancing leaf cell membrane stability and chlorophyll formation in both rice cultivars in native alkaline, Zn‐deficient soils (P  <  0.05). Further, under the salinity treatment (EC ₅), Zn inputs (10–15 mg·kg^?1) could also significantly promote rice plant Zn/K⁺ retention and reduce plant Na⁺ uptake, and thus increased leaf cell membrane stability and grain yield. Coarse rice was more salinity‐tolerant than fine rice, having significantly higher Zn/K⁺ nutrient retention.
• The mechanistic basis of Zn nutrition in mitigating salinity impacts was through promoting plant Zn/K⁺ uptake and inhibiting plant Na⁺ uptake, which could result in increased plant physiological vigour, leaf cell membrane stability and rice productivity.

     

A maize calcineurin B‐like interacting protein kinase ZmCIPK42 confers salt stress tolerance

Calcineurin B‐like (CBL) and CBL‐interacting protein kinase (CIPK) play a crucial role in biotic and abiotic stress responses. However, the roles of different CIPKs in biotic and abiotic stress responses are less well characterized. In this study, we identified a mutation leading to an early protein termination of the maize CIPK gene ZmCIPK42 that undergoes a G to A mutation at the coding region via searching for genes involved in salt stress tolerance and ion homeostasis from maize with querying the EMS mutant library of maize B73. The mutant zmcipk42 plants have less branched tassel and impaired salt stress tolerance at the seedling stage. Quantitative real‐time PCR analysis revealed that ZmCIPK42was expressed in diverse tissues and was induced by NaCl stress. A yeast two‐hybrid screen identified a proteinase inhibitor (ZmMPI) as well as calcineurin B‐like protein 1 and protein 4 (ZmCBL1, ZmCBL4) as interaction partners of ZmCIPK42. These interactions were further confirmed by bimolecular fluorescence complementation in plant cells. Moreover, over‐expressing ZmCIPK42 resulted in enhanced tolerance to high salinity in both maize and Arabidopsis. These findings suggest that ZmCIPK42 is a positive regulator of salt stress tolerance and is a promising candidate gene to improve salt stress tolerance in maize through genetic manipulation.     

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice

Recent genomic and genetic analyses based on Arabidopsis suggest that ubiquitination plays crucial roles in the plant response to abiotic stress and the phytohormone abscisic acid (ABA). However, few such studies have been reported in rice as a monocotyledonous model plant. Taking advantage of strategies in biochemistry, molecular cell biology and genetics, the RING-finger containing E3 ligase OsSDIR1 (Oryza sativa SALT-AND DROUGHT-INDUCED RING FINGER 1) was found to be a candidate drought tolerance gene for engineering of crop plants. The expression of OsSDIR1 was detected in all tissues of rice and up-regulated by drought and NaCl, but not by ABA. In vitro ubiquitination assays demonstrated that OsSDIR1 is a functional E3 ubiquitin ligase and that the RING finger region is required for its activity. OsSDIR1 could complement the drought sensitive phenotype of the sdir1 mutant and overexpressing transgenic Arabidopsis were more sensitive to ABA, indicating that the OsSDIR1 gene is a functional ortholog of SDIR1. Upon drought treatment, the OsSDIR1-transgenic rice showed strong drought tolerance compared to control plants. Analysis of the stomata aperture revealed that there were more closed stomatal pores in transgenic plants than those of control plants. This result was also confirmed by the water loss assay and leaf related water content (RWC) measurements during drought treatment. Thus, we demonstrated that monocot- and dicot- SDIR1s are conserved yet have diverse functions.