Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage.
ABSTRACT: Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.
Project description:Inositol is a cyclic polyol that is involved in various physiological processes, including signal transduction and stress adaptation in plants. l-myo-inositol monophosphatase (IMPase) is one of the metal-dependent phosphatase family members and catalyzes the last reaction step of biosynthesis of inositol. Although increased IMPase activity induced by abiotic stress has been reported in chickpea plants, the role and regulation of the IMP gene in rice (Oryza sativa L.) remains poorly understood. In the present work, we obtained a full-length cDNA sequence coding IMPase in the cold tolerant rice landraces in Gaogonggui, which is named as OsIMP. Multiple alignment results have displayed that this sequence has characteristic signature motifs and conserved enzyme active sites of the phosphatase super family. Phylogenetic analysis showed that IMPase is most closely related to that of the wild rice Oryza brachyantha, while transcript analysis revealed that the expression of the OsIMP is significantly induced by cold stress and exogenous abscisic acid (ABA) treatment. Meanwhile, we cloned the 5' flanking promoter sequence of the OsIMP gene and identified several important cis-acting elements, such as LTR (low-temperature responsiveness), TCA-element (salicylic acid responsiveness), ABRE-element (abscisic acid responsiveness), GARE-motif (gibberellin responsive), MBS (MYB Binding Site) and other cis-acting elements related to defense and stress responsiveness. To further investigate the potential function of the OsIMP gene, we generated transgenic tobacco plants overexpressing the OsIMP gene and the cold tolerance test indicated that these transgenic tobacco plants exhibit improved cold tolerance. Furthermore, transgenic tobacco plants have a lower level of hydrogen peroxide (H?O?) and malondialdehyde (MDA), and a higher content of total chlorophyll as well as increased antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), when compared to wild type (WT) tobacco plants under normal and cold stress conditions.
Project description:Low temperature is an environmental stress factor that is always been applied in research on improving crop growth, productivity, and quality of crops. Polyunsaturated fatty acids (PUFAs) play an important role in cold tolerance, so its genetic manipulation of the PUFA contents in crops has led to the modification of cold sensitivity. In this study, we over-expressed an ?-3 fatty acid desaturase from Glycine max (GmFAD3A) drove by a maize ubiquitin promoter in rice. Compared to the wild type (ZH11), ectopic expression of GmFAD3A increased the contents of lipids and total PUFAs. Seed germination rates in GmFAD3A transgenic rice were enhanced under low temperature (15 °C). Moreover, cold tolerance and survival ratio were significantly improved in GmFAD3A transgenic seedlings. Malondialdehyde (MDA) content in GmFAD3A transgenic rice was lower than that in WT under cold stress, while proline content obviously increased. Meanwhile, the activities of superoxide dismutase (SOD), hydroperoxidase (CAT), and peroxidase (POD) increased substantially in GmFAD3A transgenic rice after 4 h of cold treatment. Taken together, our results suggest that GmFAD3A can enhances cold tolerance and the seed germination rate at a low temperature in rice through the accumulation of proline content, the synergistic increase of the antioxidant enzymes activity, which finally ameliorated the oxidative damage.
Project description:Jasmonates (JAs) and abscisic acid (ABA) are phytohormones known play important roles in plant response and adaptation to various abiotic stresses including salinity, drought, wounding, and cold. JAZ (JASMONATE ZIM-domain) proteins have been reported to play negative roles in JA signaling. However, direct evidence is still lacking that JAZ proteins regulate drought resistance. In this study, OsJAZ1 was investigated for its role in drought resistance in rice. Expression of OsJAZ1 was strongly responsive to JA treatment, and it was slightly responsive to ABA, salicylic acid, and abiotic stresses including drought, salinity, and cold. The OsJAZ1-overexpression rice plants were more sensitive to drought stress treatment than the wild-type (WT) rice Zhonghua 11 (ZH11) at both the seedling and reproductive stages, while the jaz1 T-DNA insertion mutant plants showed increased drought tolerance compared to the WT plants. The OsJAZ1-overexpression plants were hyposensitive to MeJA and ABA, whereas the jaz1 mutant plants were hypersensitive to MeJA and ABA. In addition, there were significant differences in shoot and root length between the OsJAZ1 transgenic and WT plants under the MeJA and ABA treatments. A subcellular localization assay indicated that OsJAZ1 was localized in both the nucleus and cytoplasm. Transcriptome profiling analysis by RNA-seq revealed that the expression levels of many genes in the ABA and JA signaling pathways exhibited significant differences between the OsJAZ1-overexpression plants and WT ZH11 under drought stress treatment. Quantitative real-time PCR confirmed the expression profiles of some of the differentially expressed genes, including OsNCED4, OsLEA3, RAB21, OsbHLH006, OsbHLH148, OsDREB1A, OsDREB1B, SNAC1, and OsCCD1. These results together suggest that OsJAZ1 plays a role in regulating the drought resistance of rice partially via the ABA and JA pathways.
Project description:Plants, as sessile organisms, survive environmental changes by prioritizing their responses to the most life-threatening stress by allocating limited resources. Previous studies showed that pathogen resistance was suppressed under abiotic stresses. Here, we show the mechanism underlying this phenomenon. Phosphorylation of WRKY45, the central transcription factor in salicylic-acid (SA)-signalling-dependent pathogen defence in rice, via the OsMKK10-2-OsMPK6 cascade, was required to fully activate WRKY45. The activation of WRKY45 by benzothiadiazole (BTH) was reduced under low temperature and high salinity, probably through abscisic acid (ABA) signalling. An ABA treatment dephosphorylated/inactivated OsMPK6 via protein tyrosine phosphatases, OsPTP1/2, leading to the impaired activation of WRKY45 and a reduction in Magnaporthe oryzae resistance, even after BTH treatment. BTH induced a strong M. oryzae resistance in OsPTP1/2 knockdown rice, even under cold and high salinity, indicating that OsPTP1/2 is the node of SA-ABA signalling crosstalk and its down-regulation makes rice disease resistant, even under abiotic stresses. These results points to one of the directions to further improve crops by managing the tradeoffs between different stress responses of plants.
Project description:Salicylic acid (SA) levels increase in Arabidopsis upon exposure to low temperature for more than a week. This cold-induced SA biosynthesis was found to proceed through the isochorismate synthase (ICS) pathway. The sid2-1 mutant is deficient in ICS1 and does not make SA at low temperature. We used microarray analysis to examine the genes differentially regulated by low temperature that were dependent on SA We used the sid2-1 mutant and a WT control at 22?C and after 3 weeks at 4?C. All samples were in triplicate.
Project description:In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH-overexpressing apple plants possessed improved cold and salt tolerance compared to wild-type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation-reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2 O2 content, but a low O2·- production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt- and cold-tolerant trees.
Project description:Abscisic acid (ABA) plays versatile functions in regulating plant development and tolerance to various biotic and abiotic stresses. Towards elucidating the functions of one of the ABA receptors (ABARs) in rice, OsPYL10 was cloned from drought tolerant rice cv. Nagina 22 and was overexpressed under stress inducible RD29A promoter in a mega rice variety MTU1010 by using Agrobacterium mediated genetic transformation. Four single copy transgenic lines selected based on Southern blot analysis were used for physiological and molecular analysis. PYL10 receptor appears to regulate its ligand ABA accumulation as PYL10 overexpressing transgenics accumulated 2-3.3-fold higher levels of ABA than that of WT in flag leaf at anthesis under non-stress conditions. The enhanced accumulation of ABA was associated with enhanced expression of genes for ABA biosynthesis viz., ZEP1, NCED1, NCED2, NCED3, and NCED4 in transgenics than in WT plants. At seedling stage, PYL10 transgenics showed significantly higher survival rate under cold stress as compared with WT plants. qRT-PCR analysis showed that expression levels of cold responsive genes viz., DREB1F, MYB3R2, TPP1, COR410, DEHYDRIN, and LEA3 were significantly higher in PYL10 overexpressing transgenic lines as compared to WT plants under cold stress. PYL10 transgenic and WT plants grown in the same pot were subjected to -80 kPa drought stress and recovery treatments at vegetative and reproductive stages. At vegetative stage drought stress, three overexpressing lines showed significantly higher grain yield (40-58%) and at reproductive stage drought stress one of these overexpression lines showed two-fold higher grain yield than that of WT plants. Excised leaf water loss analysis showed that PYL10 transgenic lost about 20% less water than WT plants. At reproductive stage, OsPYL10 transgenic maintained higher RWC, membrane stability index, chlorophyll content, and accumulated lower amount of MDA and H2O2 as compared with WT plants. qRT-PCR analysis showed that expression levels of RAB16, Dehydrin, LEA3, and ABA45 were higher in PYL10 transgenics as compared with WT plants under drought stress. Thus, overall results showed that OsPYL10 overexpression has potential to improve both drought and cold stress tolerance of indica rice.
Project description:Salicylic acid (SA) can induce plants to actively enhance abiotic stress resistance. Spraying SA to prevent cold stress in flue-cured tobacco fields can provide theoretical support and practical guidance for the actual protection from cold stress in fields at high altitude in Yunnan. The experiment was performed in Jianchuan County Yunnan Province, China. Honghuadajinyuan, a flue-cured tobacco variety with cold resistance, was used as the research object. SA was tested at two concentrations (0.05 [SA-1] and 0.1 [SA-1] mol L-1) relative to an untreated control (Control) to compare the quality of fresh tobacco leaves, curing characteristics, enzyme activity of antioxidants, and quality of the first-cured tobacco leaves. The tissue structure thickness, SPAD, and plastid pigment content of fresh tobacco leaves were least in the control; there was no significant difference between SA-1 and SA-2. The change of moisture content during curing was SA-1 > SA-2 > Control, and the water loss rate was Control > SA-2 > SA-1, and both varied greatly at 38-48°C. In each curing stage, the carbon and nitrogen metabolites and polyphenols changed most rapidly at 38°C, and the sugar metabolites changed as follows: Control > SA-1 > SA-2. The activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase in fresh tobacco leaves were SA-1 > SA-2 > Control. Malondialdehyde content and the inactivation rate of antioxidant enzymes during curing was Control > SA-2 > SA-1. The economic character and sensory smoking quality of flue-cured tobacco leaves were SA-1 > SA-2 > Control. In high-altitude tobacco planting areas prone to cold stress in the field, early warning weather forecast and field spraying 0.05 mol L-1 SA are beneficial to protect and improve the quality of fresh tobacco leaves, curing characteristics, antioxidant system enzyme activities, and the quality of flue-cured tobacco leaves.
Project description:Leaf senescence is one of the most common factors that affects the growth and yield of rice. Although numerous genes affecting leaf senescence have been identified, few involved in cuticular wax synthesis have been described for rice premature leaf senescence. Here, we cloned and characterized Premature Leaf Senescence 4 (PLS4) in rice (Oryza sativa), which encodes a putative 3-oxoacyl-reductase in the fatty acid biosynthetic pathway. Subcellular localization of OsPLS4 was observed in the chloroplast. A single nucleotide substitution in OsPLS4 reduced leaf cuticular wax, and the expression levels of most wax biosynthesis-associated genes were downregulated. TEM showed chloroplast development were defective in the pls4 mutant. Further investigation revealed that the chlorophyll (Chl) content was reduced in the pls4 mutant compared with the WT and that the photosynthesis rate was lower, which caused ROS dramatic accumulation at the heading stage. These results confirmed premature leaf senescence in pls4 plants. Cold treatment indicated that the mutant was more sensitive than the WT was to cold stress. Together, all the above results indicate that the OsPLS4 mutation affects cuticular wax biosynthesis and chloroplast development in rice, causing reduced cuticular wax and premature leaf senescence.
Project description:BACKGROUND:Abscisic acid (ABA) acts as a signaling hormone in plants against abiotic stress, but its function in energy homeostasis under heat stress is unclear. RESULTS:Two rice genotypes, Nipponbare (wild-type, WT) with flat leaves and its mutant high temperature susceptibility (hts) plant with semi-rolled leaves, were subjected to heat stress. We found significantly higher tissue temperature, respiration rate, and ABA and H2O2 contents in leaves as well as a lower transpiration rate and stomatal conductance in hts than WT plants. Additionally, increased expression of HSP71.1 and HSP24.1 as well as greater increases in carbohydrate content, ATP, NAD (H), and dry matter weight, were detected in WT than hts plants under heat stress. More importantly, exogenous ABA significantly decreased heat tolerance of hts plants, but clearly enhanced heat resistance of WT plants. The increases in carbohydrates, ATP, NAD (H), and heat shock proteins in WT plants were enhanced by ABA under heat stress, whereas these increases were reduced in hts plants. CONCLUSION:It was concluded that ABA is a negative regulator of heat tolerance in hts plants with semi-rolled leaves by modulating energy homeostasis.