Guangdong Provincial Key Laboratory of Biotechnology for Plant Development and Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring, School of Life Sciences, South China Normal University, Guangzhou, China.
Department of Biological Food and Environment, Hefei University, China.
FEBS Open Bio. 2021 May;11(5):1428-1439. doi: 10.1002/2211-5463.13151. Epub 2021 Apr 7.
Abscisic acid (ABA) plays a vital role in many developmental processes and the response to adaptive stress in plants. Under drought stress, plants enhance levels of ABA and activate ABA receptors, but under harsh environmental stress, plants usually cannot efficiently synthesize and release sufficient quantities of ABA. The response of plants to harsh environmental stress may be improved through ABA-independent activation of ABA receptors. The molecular basis of ABA-independent inhibition of group A protein phosphatases type 2C (PP2Cs) by pyrabactin resistance/Pyr1-like (PYR1/PYLs) is not yet clear. Here, we used our previously reported structures of PYL3 to first obtain the monomeric PYL3 mutant and then to introduce bulky hydrophobic residue substitutions to promote the closure of the Gate/L6/CL2 loop, thereby mimicking the conformation of ABA occupancy. Through structure-guided mutagenesis and biochemical characterization, we investigated the mechanism of ABA-independent activation of PYL3. Two types of PYL3 mutants were obtained: (a) PYL3 V108K V107L V192F can bind to ABA and effectively inhibit HAB1 without ABA; (b) PYL3 V108K V107F V192F, PYL3 V108K V107L V192F L111F and PYL3 V108K V107F V192F L111F cannot recognize ABA but can greatly inhibit HAB1 without ABA. Intriguingly, the ability of PYL3 mutants to bind to ABA was severely compromised if any two of three variable residues (V107, V192 and L111) were mutated into a bulky hydrophobic residue. The introduction of PYL3 mutants into transgenic plants will help elucidate the functionality of PYL3 in vivo and may facilitate the future production of transgenic crops with high yield and tolerance of abiotic stresses.
脱落酸（ABA）在植物的许多发育过程和适应应激反应中起着至关重要的作用。在干旱胁迫下，植物会提高 ABA 的水平并激活 ABA 受体，但在恶劣的环境胁迫下，植物通常无法有效地合成和释放足够数量的 ABA。通过 ABA 非依赖性激活 ABA 受体，可以改善植物对恶劣环境胁迫的响应。ABA 非依赖性抑制 A 组蛋白磷酸酶 2C（PP2C）的吡喃并哒嗪受体/吡喃并嘧啶类（PYR1/PYLs）的分子基础尚不清楚。在这里，我们使用之前报道的 PYL3 结构，首先获得单体 PYL3 突变体，然后引入大的疏水性残基取代以促进门/L6/CL2 环的闭合，从而模拟 ABA 占据的构象。通过结构导向的诱变和生化特性分析，我们研究了 PYL3 非 ABA 依赖性激活的机制。获得了两种类型的 PYL3 突变体：（a）PYL3 V108K V107L V192F 可以结合 ABA，并在没有 ABA 的情况下有效抑制 HAB1；（b）PYL3 V108K V107F V192F、PYL3 V108K V107L V192F L111F 和 PYL3 V108K V107F V192F L111F 不能识别 ABA，但在没有 ABA 的情况下可以极大地抑制 HAB1。有趣的是，如果三个可变残基（V107、V192 和 L111）中的任意两个突变为大的疏水性残基，PYL3 突变体结合 ABA 的能力会严重受损。将 PYL3 突变体引入转基因植物将有助于阐明 PYL3 在体内的功能，并可能有助于未来生产具有高产和耐受非生物胁迫的转基因作物。
