Function and stimulation of the transient increase of the leaf apoplastic pH during exposure to chlorine-stress

质外体是位于质膜(PM)外的一个胞外隔室。外界胁迫因素会影响叶质外体液膜的pH值。以蚕豆(Vicia Faba L.)的质外体pH(PHapo)为例。根暴露在压力剂量的氯化钠下几分钟后，叶子就会增加。这种增加是暂时的，持续1-3个小时。这主要归因于氯化物(Cl-−)，而不是盐胁迫的钠或渗透组分。尽管越来越多的迹象表明，PHAPO的上升可能是对盐胁迫的有益反应的一部分，但在之前的研究中，它几乎没有受到关注。一方面，它可能是没有功能含义的次要效应，即由跨PM的Cl−运动引起的另一方面，对蚕豆的研究结果表明，氯−诱导的叶片PHapo的瞬时升高有助于富含脱落酸的保卫细胞。ABA是一种控制气孔开度的植物激素。关于这种富集性，尚不清楚叶片pHapo的瞬时增加是否代表了对ClABA-胁迫的反应，即仅凭这一点就足以诱导GC中−的积累。为了澄清这一点，在设想的项目中计划人为地增加叶片的PHapo，使之以一种短暂的方式增加。这是通过pH缓冲液的渗透来实现的。通过这种方法，可以在植物不受Cl-−胁迫的背景下，研究瞬时pH升高对GC中脱落酸浓度的影响。这项研究将提供信息，即单独存在的瞬时氯−诱导的PHapo上升是否足以导致脱落酸在GC中的积累。如果不是这样的话，GC-ABA的增加很可能是Cl-−胁迫引起的PHAPO变化和由Cl-−-胁迫引起的其他胁迫反应相互作用的结果。PLANTA H+成像中的比率计量法用于非侵入性地定量PHAPO。保卫细胞将从表现出PHapo瞬时升高的叶片中纯化出来。对这些GC样品进行了不同的分析：首先，通过质谱仪(MS)分析ABA的浓度。此外，基于MS的鸟枪蛋白质组学被应用于研究PHAPO对催化GC自主合成ABA或介导ABA流入GC的蛋白质丰度的影响。此外，该项目寻求扩大关于阴离子刺激剂的知识，这些阴离子刺激剂不同于Cl-−，当在胁迫剂量下使用时，可以诱导叶质外体增加pH。这可能是硫酸盐，它是许多盐渍土壤中的主要阴离子。最后，我们研究了根质外体pH对Cl-−胁迫剂量的响应，因为我们对Cl-−胁迫过程中根PHapo的调节还缺乏了解。

The apoplast is an extracellular compartment that lies outside the plasma membrane (PM). External stress factors can influence the pH of the fluid film of the leaf apoplast. For instance, the apoplastic pH (pHapo) of field bean (Vicia faba L.) leaves increases minutes after exposing the roots to a stressful dose of NaCl. This increase is transient and lasts over a period of 1-3 hours. It is ascribable to the chloride (Cl−) but not to the sodium or the osmotic component of the NaCl-stress. Although indications increase that this rise of the pHapo could be part of a beneficial response to salt stress, it has received little attention in the previous research. On the one hand, it could be a secondary effect without functional implication, arising i.e. from Cl− movements across the PM. On the other hand, results derived from studies on Vicia faba show that the Cl−-induced transient increase of the leaf pHapo is instrumental for enriching guard cells (GCs) with abscisic acid (ABA). ABA is a phytohormone that controls stomatal aperture. Regarding this enrichment, it is unclear whether the transient increase of the leaf pHapo represents a response to Cl−-stress that alone is sufficient to induce an accumulation of ABA in the GCs. To clarify this, it is planned in the envisaged project to artificially increase the leaf pHapo to increase in a transient way. This is done via the infiltration of a pH buffer. By this means, the effect of the transient pHapo increase on the ABA concentration in the GCs can be investigated in a background in which plants are not Cl−-stressed. This investigation will provide information as to whether the sole presence of the transient Cl−-induced pHapo rise was sufficient to cause the accumulation of ABA in the GCs. If this is not the case, it is likely that the GC ABA enrichment is rather the outcome of an interaction between the Cl− stress-induced pHapo change and other stress responses that arise from Cl−-stress. Ratiometric in planta H+-imaging is used for the non-invasive quantification of the pHapo. Guard cells will be purified from leaves that have shown the transient increase of the pHapo. These GC samples are subjected to different analysis: First, ABA concentration is analyzed via mass spectrometry (MS). Moreover, MS-based shotgun proteomics is applied to investigate pHapo-based effects on abundances of proteins that catalyse the GC-autonomous ABA synthesis or mediate ABA influx into GCs. Furthermore, the project seeks to expand knowledge concerning anionic stimulants other than Cl− that can induce the leaf apoplast to increase pH, when applied at a stressful dose. This could be sulfate, which is a dominant anion in many saline soils. Lastly, the response of the root apoplastic pH to a stressful dose of Cl− is investigated, as our understanding about the regulation of the root pHapo during exposure to Cl−-stress is rudimentary.