Assessing stomatal response to live bacterial cells using whole leaf imaging.
Assessing stomatal response to live bacterial cells using whole leaf imaging.
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DOI：
10.3791/2185
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发表时间：
2010-10-02
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影响因子：
--
通讯作者：
Melotto, Maeli
中科院分区：
文献类型：
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作者：
Chitrakar, Reejana;Melotto, Maeli
气孔是植物表皮的自然开口，负责植物内部和环境之间的气体交换。它们由一对保卫细胞形成，能够响应许多外部因素（包括光强度、二氧化碳浓度和相对湿度 (RH)）而关闭气孔。气孔也是病原体进入叶片的主要途径，是疾病发展的关键步骤。最近的研究表明，关闭孔可有效减少拟南芥植物细菌性疾病的发生。植物先天免疫的一个组成部分。此前，我们使用表皮来评估气孔对活细菌的反应（Melotto et al. 2006）；然而，维持植物表皮和细菌细胞有利的环境条件一直具有挑战性。使用 MES 缓冲液（10 mM KCl、25 mM MES-KOH、pH 6.15）可以保持叶表皮的活力和健康，用于保卫细胞的电生理实验。然而，该缓冲液不适合获得细菌悬浮液。另一方面，细菌细胞可以在水中保持活力，这不适合长时间维持表皮。当表皮浮在水面上时，暴露在空气中的表皮细胞会在 4 小时内干燥，从而限制了进行实验的时间。评估特定刺激对保卫细胞的影响的理想方法应该尽可能减少对气孔生理学和植物自然环境的干扰。因此，我们开发了一种新方法来评估气孔对活细菌的反应，其中叶片受伤和操作大大减少，旨在提供易于重复且可靠的气孔测定。该方案基于用碘化丙啶 (PI) 对完整叶子进行染色、用细菌悬浮液孵育叶子染色以及在激光扫描共聚焦显微镜下观察叶子。最后，该方法允许使用密切模仿植物被病原体攻击的自然条件的条件在较长时间内观察相同的活叶样本。
Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.