| Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds
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Author:
Date:
2018-07-05
[Abstract] Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation ...
[摘要] 植物液泡是植物细胞中最大的隔室，占细胞体积的80％以上。各种蛋白质，糖，色素和其他代谢物存储在这些细胞器中（Paris et al。，1996; Olbrich et al。，2007）。花产生多种特殊代谢物，其中一些是该器官特有的，如授粉综合征的成分， ie ，气味挥发物和黄酮类化合物（Hoballah et al。， 2007; Cna'ani et al。，2015）。为了研究存储在花液泡中的化合物，必须将该隔室与细胞的其余部分分开。为了能够分离液泡，首先通过将刺穿的花冠与纤维素酶和macrozyme酶一起孵育来产生原生质体。在过滤和几个离心步骤后，通过显微镜观察显示原生质体与碎片和受损/破裂的原生质体分离。裂解浓缩的原生质体，并通过Ficoll梯度离心提取液泡。 Vacuoles用于隔离代谢物的定量GC-MS分析。这种方法使我们能够将空泡识别为糖基化挥发性苯丙酸类的亚细胞聚集位点，并假设共轭气味化合物在通向顶空的途径中被隔离（Cna'ani et al。，2017） 。
【背景】植物空泡占植物细胞中细胞体积的80％。这些细胞器对植物生长和发育至关重要，在整个植物的生命中具有不同的功能。 ...
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| Detection of Alternative Oxidase Expression in Arabidopsis thaliana Protoplasts Treated with Aluminium
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Author:
Date:
2015-07-05
[Abstract] Aluminium (Al), a non-essential metal widespread in the environment that is known to be toxic to humans as well as to plants, can cause damage not only to the roots but also to the aerial parts of plants. Its toxicity has been recognized as one of the major factors that limit crop production on acid soil. Alternative oxidase, the respiratory terminal oxidase in plants, which contributes to maintain the electron flux and reduce mitochondrial ROS levels, is often dramatically induced to make plants to adapt better to stress conditions like Al stress. In this protocol, the expression of alternative oxidase induced by Al treatment was detected in Arabidopsis protoplasts using an adaptation of previous methods (Yamamoto et al., 2002; Li et al., 2011; Liu et al. ...
[摘要] 铝（Al），一种已知对人类以及对植物有毒的环境中普遍存在的非必需金属，不仅对根而且对植物的地上部分都造成损害。 其毒性已被认为是限制作物在酸性土壤上生产的主要因素之一。 替代氧化酶，植物中的呼吸末端氧化酶，有助于维持电子通量和降低线粒体ROS水平，经常被显着诱导，使植物更好地适应应力条件，如铝胁迫。 在该方案中，使用先前方法的改编，在拟南芥原生质体中检测由A1处理诱导的替代氧化酶的表达（Yamamoto等人，2002; et al。，2011; Liu et al。，2014），它们有助于研究Al处理中替代氧化酶的机制。
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| Measurement of Endogenous H2O2 and NO and Cell Viability by Confocal Laser Scanning Microscopy
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Author:
Date:
2013-10-05
[Abstract] Recently, there is compelling evidence that hydrogen peroxide (H2O2) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses including stomatal movement. Thus, the choice of sensitive methods for detection of endogenous H2O2 and NO in guard cells are very important for understanding the role of H2O2 and NO in guard cell signaling. In addition, besides stomatal closure caused by interfering guard cell signaling, it can also be caused by widespread, nonspecific damage to guard cells. To determine whether stomatal movement is caused by damage to guard cells, sensitive methods for detection of guard cell viability are often required.
The oxidatively sensitive fluorophore ...
[摘要] 最近，有令人信服的证据表明，过氧化氢（H 2 O 2 O 2）和一氧化氮（NO）作为植物中的信号分子起作用，介导一系列反应，包括气孔运动。因此，选择用于检测保卫细胞中内源性H 2 O 2 O 2和NO的灵敏方法对于理解H 2 sub的作用是非常重要的> O 2和保守细胞信号中的NO。此外，除了由干扰保卫细胞信号传导引起的气孔闭合之外，它还可以由保护细胞的广泛的非特异性损伤引起。为了确定气孔运动是否由保卫细胞的损伤引起，通常需要用于检测保卫细胞存活力的灵敏方法。
氧化敏感荧光团2'，7'-二氯荧光素（H 2 DCF）通常用于测量细胞内H 2 O 2 O 2的变化，/sub>级别。 H 2 DCF的非极性二乙酸酯（H 2 DCFDA）进入细胞，并水解成极性更强的非荧光化合物H 2，/sub> DCF，因此，它被捕获。随后通过过氧化物酶催化的H 2 O 2 O 2对H 2 DCF的氧化产生高度荧光的DCF。类似地，细胞可渗透的NO-敏感性荧光探针4,5-二氨基荧光素二乙酸酯（DAF-2DA）广泛用于在动物和植物细胞中直接检测NO存在。非极性DAF-2DA进入细胞，并被胞质酯酶水解为极性更强的非荧光化合物DAF-2，其在NO存在下转化为高度荧光的三唑衍生物DAF-2T。荧光指示剂染料荧光素二乙酸酯（FAD）和碘化丙啶（PI）广泛用于检测细胞活力。 ...
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