两种富含甘氨酸的RNA结合蛋白参与选择性剪接实现对水稻耐热性的昼夜节律调控

Rice(Oryza sativa L.)production is threatened by global warming associated with extreme high temperatures,and rice heat sensitivity is differed when stress occurs between daytime and nighttime.However,the underlying molecular mechanism are largely unknown.We show here that two glycine-rich RNA binding proteins,Os GRP3 and Os GRP162,are required for thermotolerance in rice,especially at nighttime.The rhythmic expression of Os GRP3/Os GRP162 peaks at midnight,and at these coincident times,is increased by heat stress.This is largely dependent on the evening complex component Os ELF3-2.We next found that the double mutant of Os GRP3/Os GRP162 is strikingly more sensitive to heat stress in terms of survival rate and seed setting rate when comparing to the wild-type plants.Interestingly,the defect in thermotolerance is more evident when heat stress occurred in nighttime than that in daytime.Upon heat stress,the double mutant of Os GRP3/Os GRP162 displays globally reduced expression of heat-stress responsive genes,and increases of m RNA alternative splicing dominated by exon-skipping.This study thus reveals the important role of Os GRP3/Os GRP162 in thermotolerance in rice,and unravels the mechanism on how Os GRP3/Os GRP162 regulate thermotolerance in a diurnal manner.

Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application

The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction（PCR） analyses demonstrated that foliar treatment of bentazon（500 mg/L） greatly induced expression of CYP81A6 in both wild-type（Jiazhe B） and its knockout mutant（Jiazhe m B）： a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to 20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl（500 mg/L） treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-（3H）-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuronmethyl itself. Use of a promoter-GUS reporter construct（CYP81A6Pro：：GUS） demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides.

Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa1 in rice

The OsLpa1 gene（LOC_Os02g57400） was identified to be involved in phytic acid（PA） metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa1 in rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter：：GUS transgenic plants（GUS： β-glucuronidase）; GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts（OsLpa1.1, OsLpa1.2, OsLpa1.3） are produced due to alternative splicing; quantitative reversetranscriptase polymerase chain reaction（RT-PCR） analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa1.2 in all tissues. OsLpa1.2 is predominant in germinating seeds（about 5 times that of OsLpa1.1）, but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa1.2 in both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF（day after flowering） seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein（GFP） in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.

bZIP17 regulates heat stress tolerance at reproductive stage in Arabidopsis

High temperature elicits a well-conserved response called the unfol ded protein response(UPR)to bring protein homeostasis in the endoplasmic reticulum(ER).Two key UPR regulators bZIP28 and bZIP60 have been shown to be essential for maintaining fertility under heat stress conditions in Arabidopsis,however,the function of transcriptional activator bZIP17,a paralog of bZIP28,in heat stress response at reproductive stage is not reported.Here we found that bzip17 mutant plants were sensitive to heat stress in terms of silique length and fertility comparing to that of wildtype(WT)Arabidopsis plants,and transcri ptomic analysis showed that 1380 genes were specifically up-regulated and 493 genes were specifically down-regulated by heat stress in the flowers of WT plants comparing to that in bzip17 mutant plants.These bZIP17-dependent up-regulated genes were enriched in responses to a biotic stresses such as water deprivation and salt stress.Further chromatin immuno-preci pitation coupled with high-throughput sequencing(ChIP-Seq)uncovered 1645 genes that were direct targets of bZIP17 in MYC-bZIP17 expressing seedlings subjected to heat stress.Among these 1645 genes,ERSE-II cis-element was enriched in the binding peaks of their promoters,and the up-regulation of 113 genes by heat stress in flowers was dependent on bZIP17.Our results revealed direct targets of bZIP17 in flowers during heat stress responses and demonstrated the important role of bZIP17 in maintai ning fertility upon heat stress in plants.

Disruption of three polyamine uptake transporter genes in rice by CRISPR/Cas9 gene editing confers tolerance to herbicide paraquat

Weeds are a major biotic constraint that can cause dramatic crop production losses.Herbicide technology has been widely used by farmers as the most cost-effective weed control measure,and development of new strategy to improve herbicide tolerance in plants is urgently needed.The CRISPR/Cas9-based genome editing tool has been used in diverse applications related to agricultural technology for crop improvement.Here we identified three polyamine uptake transporter(PUT)genes in rice that are homologous to the Arabidopsis AtRMV1.We successfully demonstrate that CRISPR/Cas9-targeted mutagenesis of OsPUT1/2/3 greatly improves paraquat resistance in rice without obvious yield penalty.Therefore,manipulation of these loci could be valuable for producing transgene-free rice with improved herbicide resistance in future.

Roles of plant hormones in thermomorphogenesis

Global warming has great impacts on plant growth and development,as well as ecological distribution.Plants constantly perceive environmental temperatures and adjust their growth and development programs accordingly to cope with the environment under non-lethal warm temperature conditions.Plant hormones are endogenous bioactive chemicals that play central roles in plant growth,developmental,and responses to biotic and abiotic stresses.In this review,we summarize the important roles of plant hormones,including auxin,brassinosteroids(BRs),Gibberellins(GAs),ethylene(ET),and jasmonates(JAs),in regulating plant growth under warm temperature conditions.This provides a picture on how plants sense and transduce the warm temperature signals to regulate downstream gene expression for controlling plant growth under warm temperature conditions via hormone biosynthesis and signaling pathways.