FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis.
FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis.
复制标题
基于FRET的记者直接可视化脱甲酸浓度变化和拟南芥的分布。
DOI：
10.7554/elife.01739
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发表时间：
2014-04-15
期刊：
影响因子：
7.7
通讯作者：
Schroeder JI
中科院分区：
文献类型：
--
作者：
Waadt R;Hitomi K;Nishimura N;Hitomi C;Adams SR;Getzoff ED;Schroeder JI
脱落酸（阿坝）是一种植物激素，调节植物的生长发育和介导非生物胁迫反应。直接细胞监测阿坝浓度的动态变化，响应于环境的线索是必不可少的了解阿坝的行动。我们开发了ABAleons：ABA特异性光遗传学报告基因，其瞬时转化阿坝受体与PP 2C型磷酸酶的植物激素触发的相互作用，以响应阿坝发送荧光共振能量转移（FRET）信号。我们报告了阿坝亲和力在100-600 nM范围内的ABAleons的设计，工程和使用，以空间和时间分辨率绘制植物组织中阿坝浓度的变化。ABAleon高表达可部分抑制拟南芥阿坝反应。ABAleons报告阿坝浓度的差异，在不同的细胞类型，阿坝浓度增加，响应低湿度和NaCl的保卫细胞和NaCl和渗透胁迫的根和阿坝运输从下胚轴的地上部和根。http://dx.doi.org/10.7554/eLife.01739.001植物能够对环境中的有害变化做出反应，例如，当水变得稀缺或土壤变得太咸时，以最小化这些变化造成的压力和损害的方式。激素是在这种情况下触发植物反应的化学物质。脱落酸是一种调节植物对干旱和盐胁迫的反应并控制植物在这些条件下生长的激素。在过去，可以测量给定组织中这种激素的平均水平，但不能测量活植物中单个细胞的水平。此外，很难直接跟踪脱落酸如何在植物细胞、组织或器官之间移动。现在，Waadt等人（以及独立的Jones等人）已经开发出可以真实的测量活植物中单个细胞内脱落酸水平的工具。这些植物经过基因工程改造，产生了具有两种特性的传感器蛋白：它们可以以可逆的方式与脱落酸结合，并且它们含有两种在不同波长下发出荧光的“标签”。将特定波长的光照射到植物上，只被其中一个标签吸收，实际上会导致传感器蛋白上的两个标签都发出荧光。然而，当传感器与脱落酸结合时，它们在一个波长处发出更多的荧光，而当它们不与脱落酸结合时，它们在另一个波长处发出更多的荧光。因此，测量由传感器蛋白发出的光中这两个波长的比率可以用作植物细胞中脱落酸浓度的测量。Waadt等人开发了称为“ABAleons”的传感器蛋白，并使用其中一种来分析脱落酸在模式植物拟南芥（Arabidopsis thaliana）的不同组织中的吸收、分布和移动。脱落酸水平的变化可以在单个植物细胞的水平上检测到，并且随着时间的推移，从几秒到几小时。ABAleons还发现，当湿度水平较低或盐含量较高时，保卫细胞中脱落酸的浓度会增加。低水含量或高盐含量也会缓慢增加植物根部脱落酸的浓度。此外，Waadt等人发现脱落酸从茎的基部向上移动到芽中，并向下移动到根中。Waadt等人还报道，ABAleons使植物对脱落酸的反应较低，可能是因为与ABAleons的结合减少了可用于执行其作为激素的作用的脱落酸的量。下一个挑战是设计ABAleons，使这种不必要的副作用最小化，然后继续使用ABAleons研究环境条件和参与植物激素反应的蛋白质。DOI：http://dx.doi.org/10.7554/eLife.01739.002网站
Abscisic acid (ABA) is a plant hormone that regulates plant growth and development and mediates abiotic stress responses. Direct cellular monitoring of dynamic ABA concentration changes in response to environmental cues is essential for understanding ABA action. We have developed ABAleons: ABA-specific optogenetic reporters that instantaneously convert the phytohormone-triggered interaction of ABA receptors with PP2C-type phosphatases to send a fluorescence resonance energy transfer (FRET) signal in response to ABA. We report the design, engineering and use of ABAleons with ABA affinities in the range of 100–600 nM to map ABA concentration changes in plant tissues with spatial and temporal resolution. High ABAleon expression can partially repress Arabidopsis ABA responses. ABAleons report ABA concentration differences in distinct cell types, ABA concentration increases in response to low humidity and NaCl in guard cells and to NaCl and osmotic stress in roots and ABA transport from the hypocotyl to the shoot and root. DOI: http://dx.doi.org/10.7554/eLife.01739.001 Plants are able to respond to detrimental changes in their environment—when, for example, water becomes scarce or the soil becomes too salty—in ways that minimize stress and damage caused by these changes. Hormones are chemicals that trigger the plant’s response under these circumstances. Abscisic acid is the hormone that regulates how plants respond to drought and salt stress and that controls the plant growth in these conditions. In the past, it was possible to measure the average level of this hormone in a given tissue, but not the level in individual cells in a living plant. Moreover, it was difficult to follow directly how abscisic acid moved between the plant cells, tissues or organs. Now, Waadt et al. (and independently Jones et al.) have developed tools that can measure the levels of abscisic acid within individual cells in living plants and in real time. The plants were genetically engineered to produce sensor proteins with two properties: they can bind to abscisic acid in a reversible manner, and they contain two ‘tags’ that fluoresce at different wavelengths. Shining light onto the plant at a specific wavelength that is only absorbed by one of the tags actually causes both of the tags on the sensor proteins to fluoresce. However, the sensors fluoresce more at one wavelength when they are bound to abscisic acid, and more at the other wavelength when they are not bound to abscisic acid. Hence, measuring the ratio of these two wavelengths in the light that is given off by the sensor proteins can be used as a measure of the concentration of abscisic acid in a plant cell. Waadt et al. developed sensor proteins called ‘ABAleons’, and used one of these to analyze the uptake, distribution and movement of abscisic acid in different tissues in the model plant Arabidopsis thaliana. Changes in the level of abscisic acid could be detected at the level of an individual plant cell, and over time scales of fractions of seconds to hours. ABAleons also revealed that the concentration of abscisic acid in guard cells—specialized cells that help stop the loss of water vapor from a leaf—increases when humidity levels are low, or when salt levels are high. Low water levels, or high salt levels, also slowly increased the concentration of abscisic acid in the roots of the plant. Furthermore, Waadt et al. saw that abscisic acid moved long distances from the base of the stem up into the shoot, and down to the root. Waadt et al. also report that the ABAleons made plants less responsive to abscisic acid, possibly because binding to the ABAleons reduced the amount of abscisic acid that was available to perform its role as a hormone. The next challenge is to engineer ABAleons that minimize this unwanted side effect, and then go on to use ABAleons to study environmental conditions and proteins involved in plant hormone responses. DOI: http://dx.doi.org/10.7554/eLife.01739.002