DataSheet_2_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）是一类碱性亮氨酸拉链（basic leucine zipper, bZIP）转录因子，参与脱落酸（abscisic acid, ABA）信号通路，并在逆境胁迫响应中被激活。然而，栽培大麦（Hordeum vulgare）中ABI5（HvABI5）在ABA信号通路中的确切功能，及其在胁迫条件下的作用机制仍不明确。本研究证实，HvABI5参与ABA依赖型的大麦干旱胁迫响应调控。我们筛选获得了HvABI5基因携带不同等位变异的大麦TILLING（Targeting Induced Local Lesions In Genomes）突变体，并对其中一株突变体的干旱与ABA响应的生理及分子机制进行了详细解析。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，该突变体的储水能力优于其野生型亲本"Sebastian"（WT）。hvabi5.d的耐旱表型与其在胁迫下相较于野生型更优异的膜保护能力、更高的类黄酮含量以及更快的气孔关闭速率相关。芯片转录组分析显示，该突变体中与细胞保护机制相关的基因表达显著上调。此外，HvABI5的靶基因HVA1与HVA22在干旱处理后表达量更高，这暗示hvabi5.d对胁迫环境的适应性更强。但另一方面，hvabi5.d的叶绿素含量低于野生型，这与其经干旱处理后观测到的光合效率降低相吻合。为验证HvABI5以ABA依赖型方式发挥功能，我们分别在干旱与ABA处理条件下，检测了hvabi5.d及其野生型亲本中与ABA通路相关的部分基因的表达水平。胁迫条件下，突变体与野生型中参与ABA代谢与信号通路的关键基因表达模式存在显著差异。相较于野生型"Sebastian"，hvabi5.d中干旱诱导的HvNCED1、HvBG8、HvSnRK2.1及HvPP2C4基因的表达上调幅度可达2~20倍。此外，我们还观测到，在ABA处理后，hvabi5.d的气孔关闭速率更快，且HvNCED1与HvSnRK2.1基因的诱导表达量显著升高。综上，本研究结果证实HvABI5参与调控大麦的干旱胁迫响应，并暗示HvABI5可能通过合成与信号通路间的反馈调节，对ABA信号通路进行精细调控。此外，本研究还揭示了HvABI5在调控大麦干旱胁迫响应与种子萌发过程中，通过不同的分子机制发挥功能。