Investigation of the role of strigolactones in barley in response to drought

Strigolactones(SLS)被描述为一类促进根际共生相互作用并调节植物结构和器官发育的植物激素。在大麦中，最近发现脱落酸(ABA)抑制SL生物合成的转录。另一方面，我们的初步数据表明，SL信号突变体对干旱敏感，对ABA处理不敏感。有关水稻、番茄干旱胁迫或L渗透胁迫过程中SLS与ABA相互作用的研究结果表明，SLS在应对非生物胁迫反应中也可能发挥重要作用。拟议项目的总体目标是进一步剖析ABA-SL的相互作用，以揭示它们在作物物种大麦对干旱的反应中所起的作用。在第一次尝试中，现有的大麦突变体hvd14.d在SL信号方面存在缺陷，其亲本将在环境条件和干旱胁迫下种植在土壤中。除了生理和形态分析外，还将进行HvD14的免疫定位研究，以增加我们对其转录位置和/或在植物发育期间和对干旱的反应中可能通过韧皮部或木质部运输蛋白质的了解。与显示HvD14基因序列自然变异的大麦种质的互补性比较研究将被分析，以获得关于增强分蘖和耐旱性的分子机制的知识。为了揭示SLS和ABA之间的直接或间接相互作用，我们将利用CRISPR RNA/Cas9技术和分蘖群体的定向突变，进一步鉴定具有SL生物合成(D27、D17、D10、MAX1)和信号转导(D3、D53、Fc1)基因的突变株。与双子叶植物相比，单子叶植物含有多个拷贝的MAX1基因，大麦的三个独立等位基因的功能将通过特定的基因敲除来证明。此外，还将分析在干旱胁迫下野生型和SL突变体植物中差异表达的基因和转录组的深度测序，并测量SLS、ABA、生长素、油菜素类固醇、细胞分裂素、赤霉素、茉莉酸和水杨酸的浓度，以揭示植物激素之间可能的串扰作用。此外，选定的突变系将用选定的激素处理，如ABA、油菜素类固醇和细胞分裂素，以确认预测的相互作用。最后，在所有工作包中获得的结果将被汇总，以确认SLS与其他植物激素在应对干旱胁迫、影响气孔关闭以及分蘖或侧根形成等发育过程中的串扰假设。特别是，对植物激素含量的分析以及D14和其他被确定为SL对干旱胁迫反应的重要因素的蛋白质的定位研究可能会进一步加强这一假说。

Strigolactones (SLs) are described as a class of plant hormones that promotes symbiotic interactions in the rhizosphere and that regulates plant architecture and organ development. In barley it was recently shown that abscisic acid (ABA) represses transcripts of SL biosynthesis. On the other hand our preliminary data indicate that SL signalling mutant is hypersensitive to drought and insensitive to ABA treatment. Additional reports on interactions of SLs with ABA during drought stress in rice and tomato or osmotic stress in L japonicus suggest that SLs may play also an important role in coping with abiotic stress responses. The overall goal of the proposed project is to further dissect the ABA-SL interaction to reveal their role in the crop species barley in response to drought. In a first attempt the available barley mutant hvd14.d, defective in SL signaling, and its parental line will be grown on soil under ambient conditions and drought stress. Additional to physiological and morphological analyses immunolocalisation studies of HvD14 will be performed to increase our knowledge in regard of place of its transcription and/or possible transport of the protein via phloem or xylem during plant development and in response to drought. Complementary comparative studies with barley germplasm that display a natural variation in the sequence of the HvD14 gene will be analysed to gain knowledge about molecular mechanisms for enhanced tillering and drought tolerance. To reveal the direct or indirect interactions between SLs and ABA we will use targeted mutagenesis with CRISPR RNA/Cas9 technology and a TILLING population, to identify further mutants with modified barley genes of SL biosynthesis (D27, D17, D10, MAX1) and signaling (D3, D53, FC1). As in contrast to dicots, monocots contain several copies of MAX1 gene, the functionality of the three individual alleles of barley will be proven by specific knockout. Furthermore, differentially expressed genes and deep sequencing of transcriptome, completed with measurement of concentrations of SLs, ABA, auxins, brassinosteroids, cytokinins, gibberellins, jasmonic acid and salicylic acid will be analysed in drought-stressed wild-type and SL mutant plants to reveal the role of a possible crosstalk among the phytohormones. Moreover, selected mutant lines will be treated with selected hormones, such as ABA, brassinosteroids and cytokinins to confirm predicted interactions. Finally, results obtained in all work packages will be assembled to confirm an assumed crosstalk of SLs with other phytohormones in response to drought stress, with impact on stomata closure, and developmental processes as tillering or lateral root formation. In particular, analysis of phytohormone content and localization studies of D14 and other proteins that were identified within the studies as important factors in SL response to drought stress may further strengthen this hypothesis.