Functional Characterization Of Stress-Responsive NAC Transcription Factors And Drought Resistance Improvement By Multiple-Gene Assembling In Rice

Rice is an important agricultural commodity with the second-highest worldwide production among cereals.It is also an ideal model plant for scientific study.The growth and production of rice require suitable water supply and environmental temperature.Rice exposed to drought and extreme high temperature can suffer severe yield penalty or death.In response to such abiotic stresses,rice can systematically act at three levels:stress tolerance,stress escape,and stress avoidance.The stress response of rice depends on complex gene networks,in which transcription factors such as NAC and bZIP are hub regulators.There are two main purposes on this research:one is to further elucidate the mechanism behind rice abiotic stress response,the other one is to search for genetic resources for rice stress resistance improvement.To accomplish these goals,we identified and systematically studied SN28,a new member of the stress-responsive NAC family.Meanwhile,we performed in-depth studies on OsPP15 and OsPP18,two PP2C coding genes directly targeted by the previously reported key drought-responsive NAC transcription factor SNAC1.In addition,we co-overexpressed function-known stressresponsive NAC and bZIP transcription factor genes simultaneously in rice and assessed the drought resistance of the multiple-gene assembly transgenic rice in the controlled field.The main results of this research are as follows:1.We identified SN28 as a member close to the stress-responsive NAC subfamily,and its expression was strongly induced by drought and heat stresses.Mutation of SN28 lead to impaired drought and heat resistance in both seedling and booting stages,while overexpressing SN28 conferred increased drought and heat resistance.Besides,overexpressing SN28 did not cause side effect on rice production under normal conditions,but mutation of SN28 had negative effect on yield.Dual luciferase assay performed in rice protoplast indicated that SN28 functioned as a transcription activator.And the results of immunofluorescence and western blot assays indicated that SN28 was translocated to nucleus when treated with drought stress.Transcriptome profiling by RNA-Seq showed that a large number of genes related to very-long-chain fatty acid biosynthesis and transport were up-regulated in the-SN28-overexpressing seedlings,while genes involved in protein folding were greatly induced in the-SN28-overexpressing panicles,in response to drought and heat stress treatment,respectively.By ChIP-Seq analysis,we identified 107 and 1005 SN28-binding genes in the drought and heat stress treatment,respectively.Integrating the RNA-Seq and ChIP-Seq data revealed 5 and 8 SN28-targeting genes in response to drought and heat,respectively.Putative and function-known stress-responsive genes such as OsPIP2;7,OsCYP20-2,and GRAS2 were among those genes.Based on these results,we propose that SN28 functions as a novel positive regulator of drought and heat resistance,emphasizing its potential in the application in rice stress resistance improvement.2.The SNAC1-targeting and drought-induced gene OsPP 18 encodes the only known plant PP2C that positively regulates stress response in an ABA-independent manner.Since previous study failed to identify its substrate with traditional methods,we tried to use phosphoproteomic analysis and succeeded in identifying 128 putative OsPP18 substrates.Combining protein interaction predication and experiments including Y2H,BiFC,and Co-IP,we confirmed the direct interaction between OsPP18 and MAPK6.Western blot analysis on the transgenic materials revealed that OsPP 18 may dephosphorylate MAPK6 via the core motif pTEpY.Then,we further identified an interaction between MAPK6 and OsPIP2;7 by using the similar methods,and constructed the constitutively phosphorylated and dephosphorylated forms of OsPIP2;7 according to the result of the phosphoproteomic analysis.Subcellular localization analysis of the two forms of OsPIP2;7 indicated that the phosphorylation on the C-terminal was essential for the cellular membrane localization of OsPIP2;7.Subsequent yeast H2O2 uptake experiment suggested that the membrane-located OsPIP2;7 can conduct the transport of exogenous H2O2 into yeast cells.Taking these results,we conclude that OsPP 18 can regulate the accumulation of H2O2 through dephosphorylation of MAPK6 and thus inhibit the phosphorylation and membrane localization of OsPIP2;7,which,as a result,abolished the H2O2 uptake via OsPIP2;7.3.Based on the result of ChIP-Seq analysis on SNAC1,we identified another SNAC1-targeting gene OsPP 15 encoding a phosphatase in PP2C A subfamily.Investigation on the transgenic materials indicated that OsPP 15 was a negative regulator of drought response and ABA signal transduction as well as a positive regulator of development and production in normal condition.GA content measurement result showed that the contents of bioactive GAs were significantly raised in OsPP 15 overexpressing seedlings.RNA-Seq analysis revealed the transcriptional up-regulation of key GA biosynthesis genes GA20ox4 and OsCPS3.Investigation on the stomatal closure and the expression level of peroxidase genes in drought condition suggested the negative role of OsPP15 on the regulation of H2O2 induced stomatal closure in response to drought.The results in this part revealed OsPP15 as a key player in the manipulation of the balance between GA and ABA signaling as well as the balance between development and stress resistance.Taken the results on the two SNAC1 targeting genes together,this research elucidated a novel SNAC1 regulatory mechanism on rice development and abiotic stress resistance,which acts through the differential regulation of the two PP2C genes.4.In the last part of this research,we used MISSA vector system to construct a double-gene assembly transgenic rice(XL22)co-overexpressing function-known stressresponsive genes OsbZIP46CAl and SAPK6 as well as a quadruple-gene assembly transgenic rice(XL42)co-overexpressing two NAC transcription factor genes SN28 and SNAC1 on the basis of XL22.Drought testing showed that XL22 had enhanced drought resistance and mostly unaffected agronomic traits in normal condition compared to the single-gene overexpressors.Although XL42 showed even more improved drought resistance than XL22,it was compromised by significant yield loss under normal growth condition.Thus,XL22 was considered as a more promising candidate for application in rice production.The subsequent drought resistance evaluation on XL22 indicated that XL22 maintained higher seed setting rate and yield after recovery from drought stress treatment at booting stage.In addition,XL22 seedlings showed enhanced resistance to extreme temperatures when subjected to heat or cold treatment.RNA-Seq analysis revealed that a set of potential drought-tolerance related genes were up-regulated in the XL22 under drought stress conditions.Based on the results in this part,we conclude that multiple-gene assembly with proper genes may be a promising strategy for drought resistance improvement in rice.And the combination of OsbZIP46CAl and SAPK6 is such an example for pyramiding genes for drought resistance improvement.Taking all the results together,we found SN28 as a novel regulator for improving drought and heat resistance in rice.Meanwhile,we elucidated the novel mechanism on SNAC1 in regulating stress resistance via the two target PP2C genes.And in addition,we provided evidence for a potential application of multiple-gene assembly strategy in drought resistance breeding.These findings expanded our understandings on plant stress response mechanisms and offered novel stress-responsive gene for crop improvement.