Regulation of xanthophyll conversion and the function of zeaxanthin in land plants

叶黄素玉米黄质(ZX)作为光系统II(PSII)中的热能(非光化学猝灭，NPQ)和类囊体膜中的抗氧化剂，在陆地植物中起着重要的光保护作用。在叶黄素循环的脱环氧化反应中，紫黄质(VX)在强光(HL)下由紫黄质(VX)在类囊体膜上生成ZX，在基质定位的ZX环氧化反应(ZEP)催化下，ZX在弱光或黑暗的环氧化反应中被还原为VX。ZEP活性决定了在HL胁迫后叶绿体中保持高ZX含量的时间，从而决定了依赖ZX的NPQ的持续时间。我们发现，ZEP活性随着HL胁迫的增加和PSII的光抑制而逐渐下调，并且ZEP蛋白在PSII光抑制时伴随着D1蛋白的降解。HL诱导的ZEP活性下调确保了ZX的高水平被保持以响应延长的HL周期，以允许NPQ有效地重新激活(或保留)，从而在波动光条件下的瞬时弱光周期之后(或期间)进行光保护。因此，ZX被认为是与先前的光氧化胁迫相关的叶绿体“记忆”中的一个关键成分。因此，对ZEP活性的功能和(光)调节的详细了解对于理解植物中光保护NPQ机制适应HL或波动光是至关重要的。在前期的工作中，我们提供了证据表明，HL诱导的ZEP失活可能是由过氧化氢引起的，过氧化氢是在HL胁迫下叶绿体中经常形成的一种活性氧物种(ROS)。对ZEP体内和体外活性的分析进一步表明，ZEP的活性可能需要类囊体膜上特定的蛋白质相互作用，以及未知的辅助因子。为了在分子水平上详细地描述ZEP的性质和调控，我们将解决计划中的三个关键问题：(I)ZEP在类囊体膜上的准确定位和相互作用伙伴，(Ii)HL诱导ZEP失活的分子基础，以及(Iii)ZEP的详细生化性质，重点是关键辅助因子。这种方法应该能够在分子水平上理解ZEP在光保护中的功能。

The xanthophyll zeaxanthin (Zx) serves central photoprotective functions in land plants as regulator of the dissipation of excess light energy as heat (non-photochemical quenching, NPQ) in photosystem II (PSII) and as antioxidant in the thylakoid membrane. Zx is formed in the thylakoid membrane from violaxanthin (Vx) under high light (HL) in the de-epoxidation reactions (catalyzed by the lumen-localized Vx de-epoxidase) of the xanthophyll cycle and is reconverted back to Vx under low light or in darkness in the epoxidation reactions, catalyzed by the stroma-localized Zx epoxidase (ZEP). ZEP activity determines the period of the maintenance of a high Zx amount, and thus of Zx-dependent NPQ, in the chloroplast after HL stress. We have shown that ZEP activity is gradually down-regulated in response to increasing HL stress and photoinhibition of PSII, and that ZEP protein is degraded concomitant with the D1 protein upon PSII photoinhibition. The HL-induced down-regulation of ZEP activity ensures that high levels of Zx are retained in response to prolonged HL periods to allow for efficient reactivation (or retention) of NPQ and thus photoprotection after (or during) transient low light periods under fluctuating light conditions. Therefore, Zx is supposed to be a key component in the “memory” of the chloroplast with respect to preceding photo-oxidative stress. Detailed knowledge of the functioning and (light) regulation of ZEP activity is thus central for the understanding of the acclimation of photoprotective NPQ mechanisms in plants to HL or to fluctuating light. In preliminary work, we provided evidence that the HL-induced inactivation of ZEP may be caused by hydrogen peroxide, a reactive oxygen species (ROS) that is frequently formed in chloroplasts under HL stress. Analyses of the in vivo and in vitro activity of ZEP further showed that ZEP activity likely requires specific protein interactions in the thylakoid membrane and yet unknown cofactors. To characterize the properties and regulation of ZEP in detail at the molecular level, we will address three key issues in the planned project: (i) the exact localization and interaction partners of ZEP in the thylakoid membrane, (ii) the molecular basis of HL-induced ZEP inactivation, and (iii) the detailed biochemical properties of ZEP with focus on essential cofactors. This approach should allow the understanding of the functioning of ZEP in photoprotection at the molecular level.