【作者】 王慧；

【导师】 杨中宝；

【作者基本信息】 山东大学 ， 生物与医药（专业学位）， 2023， 硕士

【摘要】 铝毒害是酸性土壤中植物生长的主要限制因素之一,其主要抑制根系的生长。根尖是铝毒害的主要作用位点。在长期演绎过程中,植物逐渐形成外部排斥和内部耐受机制来抵御铝毒害,其中外部排斥机制在大多数植物中占据主导地位。铝胁迫下根系有机酸的分泌被证明为植物耐铝性的重要排斥机制之一。在拟南芥中,苹果酸转运蛋白ALMT1和柠檬酸转运蛋白MATE介导的根系苹果酸和柠檬酸的分泌在铝胁迫耐性中发挥着重要的作用,其中前者占据主导地位。而C2H2型锌指转录因子STOP1被证明在调控铝胁迫耐性中发挥关键性作用,其参与了对ALMT1、MATE和ALS3等基因表达的调控。氮元素作为植物有机体的重要组成部分,已被证明能够通过整合多种环境信号进而参与对植物适应外界环境的调控过程中。我们前期研究证明了铝胁迫和硝酸盐信号之间具有一定的互作关系,其中NRT1.1在该过程中发挥着重要作用。但对于NRT1.1介导的硝酸盐信号究竟如何调控铝胁迫信号,以及具体的生理和分子调控机制是什么,目前不是非常清楚。基于这些科学问题和前期研究基础,本研究进一步对NRT1.1介导的硝酸盐信号调控铝胁迫响应的生理和分子机制进行了研究。目前已获得以下主要结果:(1)NRT1.1响应铝胁迫信号并参与铝胁迫下植物根伸长抑制的调控为探究硝酸盐信号能否响应铝胁迫,我们通过对铝胁迫下野生型和nrt1.1突变体的根生长表型进行分析,发现NRT1.1参与了铝胁迫下植物根生长抑制的调控,且该调控过程依赖于硝酸盐的存在。通过对不同硝酸盐供应下铝胁迫对硝酸盐转运相关基因的表达进行分析,发现NRT1.1、NRT2.1、NRT2.4以及NRT2.5都能在不同程度上响应铝胁迫信号。(2)NRT1.1参与铝胁迫下STOP1和ALMT1表达以及根系有机酸分泌的调控,但在对铝胁迫下根生长抑制调控中存在非STOP1-ALMT1依赖性路径在不同浓度硝酸盐供应和铝胁迫下,nrt1.1突变体根系苹果酸和柠檬酸的分泌量显著低于野生型。同时结合苏木精染色对根尖铝积累量进行定性分析,发现nrt1.1突变体根尖的铝积累量显著高于野生型。这些结果表明铝胁迫下NRT1.1能够通过调控根系有机酸的分泌进而影响根尖铝的积累。随后的研究发现,铝胁迫下nrt1.1-1突变体根中ALMT1和STOP1在转录水平上的表达均较野生型相比显著下调。进一步研究发现,NRT1.1也参与了铝胁迫下STOP1蛋白在根尖的积累。然而,进一步结合遗传学手段我们发现,NRT1.1与STOP1和ALMT1共同参与调控铝胁迫下根生长的抑制。(3)NIN样蛋白6和7(NLP6/7)位于NRT1.1上游参与铝胁迫下STOP1和ALMT1表达、根系有机酸分泌以及根生长抑制的调控通过对铝胁迫下根系中NLP6和NLP7基因的表达,以及铝胁迫下NLP6和NLP7对根生长抑制的调控研究,我们证明了NLP6和NLP7能够在转录水平响应铝胁迫信号,进而参与对铝胁迫下根生长抑制的调控,且该过程依赖于硝酸盐信号的存在。进一步对铝胁迫下NLP6和NLP7在根系有机酸分泌、根尖铝积累、STOP1在根中的转录和蛋白水平变化、ALMT1的转录表达变化以及与STOP1和ALMT1介导的铝胁迫抑制根生长中的调控作用进行研究,发现与NRT1.1类似,NLP6和NLP7同样也参与了以上这些过程的调控。而进一步的遗传学分析结果表明,NLP6和NLP7位于NRT1.1上游介入以上过程的调控。(4)CBLs相互作用蛋白激酶23(CIPK23)位于NRT1.1上游参与铝胁迫下STOP1和ALMT1表达、根系有机酸分泌以及根生长抑制的调控鉴于CIPK23在NRT1.1磷酸化过程中的关键作用,为了验证其是否也参与了铝胁迫下NLP6/NLP7-NRT1.1介导的根系有机酸分泌、根尖铝积累、STOP1在根中的转录和蛋白水平变化、ALMT1的转录表达变化以及与STOP1和ALMT1介导的铝胁迫抑制根生长等过程中的调控,我们也进行了同NLP6和NLP7一节中的研究,结果证实了 CIPK23确实参与了对上述过程的调控。且遗传学关系分析表明,CIPK23也位于NRT1.1上游参与调控上述过程。(5)硝酸盐还原酶编码基因NIA1和NIA2响应铝胁迫信号参与铝胁迫下根生长抑制的调控根据上述研究结果,我们可以发现NRT1.1、NLP6/NLP7和CIPK23均参与了铝胁迫下根系有机酸分泌的调控,尽管它们均参与了对STOP1和ALMT1转录或蛋白水平的调控,但对铝胁迫下根生长抑制的调控却存在不依赖于STOP1-ALMT1的路径。鉴于碳氮代谢在有机酸合成中的重要作用,我们进一步对该代谢调控路径中参与硝酸盐信号传递的下游关键基因硝酸盐还原酶编码基因NIA1和NIA2的表达进行了检测。结果发现,铝胁迫均显著诱导了这两个基因的表达。且表型分析显示,NIA1和NIA2均参与了铝胁迫下根生长抑制的调控。下一步我们将对这两个基因是否参与了 NRT1.1介导的硝酸盐信号调控铝胁迫响应的过程中进行深入探讨。综上,我们的研究证明了 NRT1.1介导的硝酸盐信号在铝胁迫抑制根生长中的调控作用,揭示了 NLP/NLP7、CIPK23与NRT1.1在该调控路径中的关系。下一步我们将进一步重点围绕NRT1.1如何与STOP1和ALMT1协同调控铝胁迫下根生长抑制的分子机制进行深入研究。更多还原

【Abstract】 Aluminum toxicity is one of the main limiting factors for plant growth in acidic soils,which mainly inhibits the growth of the root system.The root tip is the main site of action of aluminum poisoning.In the long-term deduction process,plants gradually form external rejection and internal tolerance mechanisms to resist aluminum poisoning,in which external rejection mechanisms dominate most plants.The secretion of root organic acids under aluminum stress proved to be one of the important rejection mechanisms of plant aluminum tolerance.In Arabidopsis,the malate transporter ALMT1 and the citric acid transporter MATE-mediated secretion of root malic acid and citric acid play an important role in aluminum stress tolerance,with the former dominating.The C2H2 zinc finger transcription factor STOP1 has been shown to play a key role in regulating aluminum stress tolerance,and it is involved in the regulation of ALMT1,MATE and ALS3 gene expression.As an important part of plant organisms,nitrogen has been shown to participate in the regulation of plant adaptation to the external environment by integrating multiple environmental signals.Our previous studies demonstrated that there is a certain interaction between aluminum stress and nitrate signaling,in which NRT1.1 plays an important role in this process.However,it is not very clear how NRT1.1mediated nitrate signaling regulates aluminum stress signaling,and what the specific physiological and molecular regulatory mechanisms are.Based on these scientific questions and previous research,the physiological and molecular mechanisms of NRT 1.1-mediated nitrate signaling in regulating the response to aluminum stress were further studied.The following key results have been obtained:(1)NRT1.1 responds to aluminum stress signals and participates in the regulation of plant root elongation inhibition under aluminum stressIn order to explore whether nitrate signaling can respond to aluminum stress,we analyzed the root growth phenotypes of wild-type and nrt1.1 mutants under alu-minum stress,and found that NRT1.1 was involved in the regulation of plant root growth inhibition under aluminum stress,and this regulatory process depended on the presence of nitrate.By analyzing the expression of nitrate transport-related genes under aluminum stress under different nitrate supply,it was found that NRT1.1,NRT2.1.NRT2.4 and NRT2.5 could all respond to aluminum stress signals to varying degrees.(2)NRT1.1 was involved in the regulation of STOP1 and ALMT1 expression and root organic acid secretion under aluminum stress,but there was a non-STOP1-ALMT1-dependent path in the regulation of root growth inhibition under aluminum stressUnder different concentrations of nitrate supply and aluminum stress,the secretion and secretion of malic acid and citric acid in the root of nrt1.1 mutants were significantly lower than that of wild type.At the same time,the qualitative analysis of the aluminum accumulation at the root tip combined with hematoxylin staining showed that the aluminum accumulation at the root tips of nrt1.1 mutants were significantly higher than that of the wild type.These results show that NRT1.1 can affect the accumulation of apical aluminum by regulating the secretion of root organic acids under aluminum stress.Subsequent studies found that the expression of ALMT1 and STOP1 at the transcription level in the roots of nrt1.1-1 mutant under aluminum stress was significantly downregulated compared with the wild type.Further studies found that NRT1.1 was also involved in the accumulation of STOP1 protein at the root tip under aluminum stress.However,further combined with genetic methods,we found that NRT1.1,STOP1 and ALMT1 were involved in regulating the inhibition of root growth under aluminum stress.(3)NIN-like proteins 6 and 7(NLP6/7)were located upstream of NRT1.1 and participated in the regulation of STOP1 and ALMT1 expression,root organic acid secretion and root growth inhibition under aluminum stressBy studying the expression of NLP6 and NLP7 genes in roots under aluminum stress and the regulation of root growth inhibition by NLP6 and NLP7 under aluminum stress,we demonstrate that NLP6 and NLP7 can respond to aluminum stress signals at the transcriptional level,and then participate in the regulation of root growth inhibition under aluminum stress,and this process depends on the presence of nitrate signals.Furthermore,NLP6 and NLP7 under aluminum stress on root organic acid secretion,root apical aluminum accumulation,STOP1 transcription and protein level changes in roots,ALMT1 transcriptional expression changes,and STOP1 and ALMT1-mediated aluminum stress inhibition of root growth were studied,and it was found that NLP6 and NLP7 were also involved in the regulation of these processes similar to NRT1.1.Further genetic analysis showed that NLP6 and NLP7 were located upstream of NRT1.1 and intervened in the regulation of the above processes.(4)CBLs-interacting protein kinase 23(CIPK23)is located upstream of NRT1.1 and participates in the regulation of STOP1 and ALMT1 expression,root organic acid secretion and root growth inhibition under aluminum stressGiven the key role of CIPK23 in the phosphorylation of NRT1.1,in order to verify whether it is also involved in the NLP6/NLP7-NRT1.1-mediated root organic acid secretion,root apical aluminum accumulation,STOP1 transcription and protein level changes in roots,ALMT1 transcriptional expression changes,and STOP1 and ALMT1-mediated aluminum stress inhibition of root growth under aluminum stress,we also conducted the same NLP6 and NLP7 studies.The results confirmed that CIPK23 was indeed involved in regulating the above process.The genetic relationship analysis showed that CIPK23 was also located upstream of NRT1.1 to participate in the regulation of the above process.(5)Nitrate reductase encoding genes NIA1 and NIA2 respond to aluminum stress signals to participate in the regulation of root growth inhibition under aluminum stressBased on the above results,we can find that NRT1.1,NLP6/NLP7 and CIPK23 are involved in the regulation of root organic acid secretion under aluminum stress.although they are all involved in the regulation of STOP1 and ALMT1 transcription or protein levels,but there is a pathway independent of STOP1-ALMT1 for root growth inhibition under aluminum stress.In view of the important role of carbon and nitrogen metabolism in organic acid synthesis,we further examined the expression of the genes NIA1 and NIA2 encoded by nitrate reductase,downstream key genes involved in nitrate signaling in this metabolic regulatory pathway.It was found that aluminum stress significantly induced the expression of these two genes.Phenotypic analysis showed that both NIA1 and NIA2 were involved in the regulation of root growth inhibition under aluminum stress.In the next step,we will explore whether these two genes are involved in NRT1.1-mediated nitrate signaling in regulating the response to aluminum stress.In summary,our study proves the regulatory role of NRT1.1-mediated nitrate signaling in the inhibition of root growth under aluminum stress,and reveals the relationship between NLP/NLP7,CIPK23 and NRT1.1 in this regulatory pathway.In the next step,we will further focus on how NRT1.1 works with STOP1 and ALMT1 to regulate the molecular mechanism of root growth inhibition under aluminum stress.更多还原