Intact leaf gas exchange provides a robust method for measuring the kinetics of stomatal conductance responses to abscisic acid and other small molecules in Arabidopsis and grasses
Intact leaf gas exchange provides a robust method for measuring the kinetics of stomatal conductance responses to abscisic acid and other small molecules in Arabidopsis and grasses
复制标题
完整的叶子气体交换提供了一种可靠的方法来测量拟南芥和草中脱落酸和其他小分子的气孔导度反应动力学
DOI：
10.1186/s13007-019-0423-y
复制
发表时间：
2019-04-17
期刊：
影响因子：
5.1
通讯作者：
Schroeder, Julian I.
中科院分区：
文献类型：
--
作者：
Ceciliato, Paulo H. O.;Zhang, Jingbo;Schroeder, Julian I.
背景保卫细胞感知外部和内部刺激，调节植物的气孔导度。通过使用气体交换分析仪，可以测量气孔导度对光强、[CO2]浓度和相对湿度变化的时间分辨响应。这在测量气孔对植物激素脱落酸(ABA)或细菌鞭毛蛋白22(Flg22)等可溶小分子的响应时更加困难，特别是在研究具有响应表型的突变体时。结果提出了一种使用气体交换分析仪以时间分辨的方式评估小分子对气孔导度的动态影响的方法。通过在装有水的微离心管中向完整叶片的蒸腾流中添加ABA来研究ABA诱导的气孔关闭。在野生型拟南芥叶片中，强烈的ABA反应以时间和剂量依赖的方式被分解，而在ABA不敏感的突变体开放气孔1-3(Ost1-3)的叶片中没有观察到同样的反应。此外，当质膜内源蛋白(PIP)水孔蛋白四倍体突变体PIP1；1 PIP1；2 PIP2；1 PIP2；2的叶片被检测时，观察到了对ABA的强大的野生型反应。在完整野生型叶片的蒸腾流中加入细菌肽flg22后，观察到了flg22诱导的气孔关闭效应。结论由于拟南芥气孔大小不一，且离体表皮条中气孔的动态响应有限，因此评价小分子对气孔生理的影响具有挑战性，在某些情况下会导致结果不一致。此外，当使用表皮剥离来评估气孔开度响应时，来自叶肉的潜在信号丢失。在这里，我们提出了一种侵入性较小的技术，允许以时间分辨的方式测量对小分子的气孔导度响应，该小分子对拟南芥和远缘短柄蕨叶片都是优化的。
BackgroundGuard cells perceive external and internal stimuli and regulate stomatal conductance in plants. With the use of gas exchange analyzers, time-resolved stomatal conductance responses to light intensity, [CO2] concentration and relative humidity changes can be measured. This is more difficult to achieve when measuring stomatal responses to small soluble molecules such as the plant hormone abscisic acid (ABA) or the bacterial peptide flagellin 22 (flg22), in particular when investigating mutants with response phenotypes.ResultsA method to evaluate thedynamic effects of small molecules on stomatal conductance in a time-resolved fashion using gas exchange analyzers is presented here. ABA-induced stomatal closure was investigated by adding ABA to the transpiration stream of intact leaves placed in a microcentrifuge tube containing water. Strong ABA responses were resolved in time- and in a dose-dependent manner in wild-type Arabidopsis leaves, whereas the same response was not observed in leaves of the ABA-insensitive mutant open stomata 1-3 (ost1-3). Moreover, when leaves of the Plasma membrane Intrinsic Protein (PIP) aquaporin quadruple mutant pip1;1 pip1;2 pip2;1 pip2;2 were tested, robust wild-type-like responses to ABA were observed. When the bacterial peptide flg22 was added to the transpiration stream of intact wild-type leaves, a strong flg22-induced stomatal closure effect was observed. Finally, the proposed technique was further developed and optimized for evaluation of stomatal conductance responses to small molecules in leaves of grasses using the reference plant Brachypodium distachyon.ConclusionsDue to the variable size of stomata in Arabidopsis and the limited dynamic response of stomata in isolated epidermal strips, evaluation of the effect of small molecules on stomatal physiology has been challenging and has led in some cases to inconsistent results. Moreover, potentialsignals from the mesophyll are missing when using epidermal peels to evaluate stomatal aperture responses. Here we propose a less invasive technique which allows for time-resolved measurements of stomatal conductance responses to small molecules optimized for both Arabidopsis and Brachypodium distachyon leaves.