DataSheet_8_Barley ABI5 (Abscisic Acid INSENSITIVE 5) Is Involved in Abscisic Acid-Dependent Drought Response.docx

ABA INSENSITIVE 5 (ABI5) is a basic leucine zipper (bZIP) transcription factor which acts in the abscisic acid (ABA) network and is activated in response to abiotic stresses. However, the precise role of barley (Hordeum vulgare) ABI5 in ABA signaling and its function under stress remains elusive. Here, we show that HvABI5 is involved in ABA-dependent regulation of barley response to drought stress. We identified barley TILLING mutants carrying different alleles in the HvABI5 gene and we studied in detail the physiological and molecular response to drought and ABA for one of them. The hvabi5.d mutant, carrying G1751A transition, was insensitive to ABA during seed germination, yet it showed the ability to store more water than its parent cv. “Sebastian” (WT) in response to drought stress. The drought-tolerant phenotype of hvabi5.d was associated with better membrane protection, higher flavonoid content, and faster stomatal closure in the mutant under stress compared to the WT. The microarray transcriptome analysis revealed up-regulation of genes associated with cell protection mechanisms in the mutant. Furthermore, HvABI5 target genes: HVA1 and HVA22 showed higher activity after drought, which may imply better adaptation of hvabi5.d to stress. On the other hand, chlorophyll content in hvabi5.d was lower than in WT, which was associated with decreased photosynthesis efficiency observed in the mutant after drought treatment. To verify that HvABI5 acts in the ABA-dependent manner we analyzed expression of selected genes related to ABA pathway in hvabi5.d and its WT parent after drought and ABA treatments. The expression of key genes involved in ABA metabolism and signaling differed in the mutant and the WT under stress. Drought-induced increase of expression of HvNCED1, HvBG8, HvSnRK2.1, and HvPP2C4 genes was 2–20 times higher in hvabi5.d compared to “Sebastian”. We also observed a faster stomatal closure in hvabi5.d and much higher induction of HvNCED1 and HvSnRK2.1 genes after ABA treatment. Together, these findings demonstrate that HvABI5 plays a role in regulation of drought response in barley and suggest that HvABI5 might be engaged in the fine tuning of ABA signaling by a feedback regulation between biosynthetic and signaling events. In addition, they point to different mechanisms of HvABI5 action in regulating drought response and seed germination in barley.

ABA不敏感5（ABA INSENSITIVE 5，ABI5）是一类参与脱落酸（abscisic acid，ABA）信号通路的碱性亮氨酸拉链（basic leucine zipper，bZIP）转录因子，可在非生物胁迫下被激活。然而，大麦（Hordeum vulgare）ABI5在ABA信号传导中的精确作用及其在胁迫条件下的功能仍有待阐明。本研究证实HvABI5参与大麦干旱胁迫响应的ABA依赖型调控。我们筛选获得了HvABI5基因上携带不同等位变异的大麦TILLING突变体，并针对其中一株突变体的干旱与ABA响应开展了详尽的生理与分子机制分析。携带G1751A碱基转换的hvabi5.d突变体在种子萌发阶段对ABA不敏感，但在干旱胁迫下的储水能力高于其野生型亲本‘Sebastian’（WT）。与野生型相比，该突变体的耐旱表型与其在胁迫下更优异的膜保护能力、更高的类黄酮含量以及更快的气孔闭合速率密切相关。芯片转录组分析显示，突变体中与细胞保护机制相关的基因表达上调。此外，HvABI5的靶基因HVA1与HVA22在干旱处理后表达水平更高，这或许暗示hvabi5.d突变体对胁迫的适应性更强。另一方面，hvabi5.d的叶绿素含量低于野生型，这与其干旱处理后观测到的光合效率降低现象一致。为验证HvABI5的ABA依赖型作用模式，我们分析了干旱与ABA处理后hvabi5.d与其野生型亲本中若干ABA通路相关基因的表达情况。ABA代谢与信号传导关键基因的表达在突变体与野生型中的响应存在显著差异。与‘Sebastian’相比，干旱诱导的HvNCED1、HvBG8、HvSnRK2.1及HvPP2C4基因的表达量在hvabi5.d中上调了2~20倍。我们还观测到，ABA处理后hvabi5.d的气孔闭合更快，且HvNCED1与HvSnRK2.1基因的诱导表达幅度显著更高。综上，上述研究结果表明HvABI5参与调控大麦的干旱胁迫响应，并提示HvABI5可能通过合成与信号通路间的反馈调控机制，精细调节ABA信号传导。此外，本研究还揭示了HvABI5在大麦干旱胁迫响应与种子萌发调控中存在不同的作用机制。