Abscisic acid dynamics in roots detected with genetically encoded FRET sensors.
Abscisic acid dynamics in roots detected with genetically encoded FRET sensors.
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DOI：
10.7554/elife.01741
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发表时间：
2014-04-15
期刊：
影响因子：
7.7
通讯作者：
Frommer WB
中科院分区：
文献类型：
--
作者：
Jones AM;Danielson JA;Manojkumar SN;Lanquar V;Grossmann G;Frommer WB
胞质激素水平必须严格控制在内流、外排、合成、降解和区隔水平。为了确定单细胞水平上的ABA动态，使用结构导向设计和高通量筛选平台设计了覆盖范围为0.2-800µM的FRET传感器（ABACUS）。当ABACUS1在酵母中表达时，检测由AIT1/NRT1.2转运体介导的高浓度ABA摄取。表达ABACUS1-2µ（Kd ~ 2µM）和ABACUS1-80µ（Kd ~ 80µM）的拟南芥根以浓度依赖的方式对ABA灌注做出反应。观察到的ABA在根中的积累特性似乎与已知的ABA转运体（AIT1, ABCG40）的活性不相容。ABACUS揭示了外部ABA对体内平衡的影响，即ABA触发的ABA降解、修饰或区室化的诱导。ABACUS可用于研究突变体对ABA的反应，定量监测ABA的易位和调控，识别缺失成分。传感器筛选平台有望实现ABA传感器的快速微调和植物和动物激素传感器的工程，以促进我们对激素信号的理解。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反应的激素，也控制着植物的生长发育。过去，人们可以测量给定组织中这种激素的平均水平，但无法测量活植物中单个细胞的水平，也无法测量细胞内特定区域的水平。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，Jones等人（以及独立的Waadt等人）开发了一种工具，可以实时测量活植物中单个细胞特定区域内脱落酸的水平。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，并且它们含有两个发出不同波长荧光的“报告者”。将特定波长的光照射到植物上，只被其中一个报告蛋白吸收，就会导致传感器蛋白上的两个报告蛋白都发出荧光。然而，当传感器与脱落酸结合时，两种报告蛋白发出不同的荧光。具体来说，一个报告细胞发出更多的荧光，而另一个报告细胞发出更少的荧光。因此，测量传感器蛋白发出的光中这两种波长的比值可以用来测量植物细胞中脱落酸的浓度。Jones等人使用高通量平台设计了五种传感器蛋白，用于检测大范围浓度的脱落酸。在活的植物中使用这些“ABACUS”传感器可以跟踪根细胞对脱落酸的吸收，并揭示细胞内的激素浓度保持在低于外部提供的水平。由于已知的脱落酸转运蛋白能够将细胞内的激素浓度提高到高于细胞外的水平，因此脱落酸可能通过尚未发现的转运蛋白转运到植物根部。Jones等人也表明，根细胞能迅速清除脱落酸，向根中添加额外的脱落酸可在几分钟内提高清除速度。植物也被改造成将传感器蛋白特异性地靶向细胞核。将来，将这些传感器定位到细胞壁上，应该可以跟踪这种激素在细胞间的运动。进一步的目标包括使用ABACUS来追踪处于逆境中的植物中的脱落酸，并使用高通量平台开发新的传感器来追踪活生物体（包括动物）中的其他激素。DOI: http://dx.doi.org/10.7554/eLife.01741.002
Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2–800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI: http://dx.doi.org/10.7554/eLife.01741.001 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 also controls growth and development. 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, nor in specific compartments within a cell. Moreover, it was difficult to follow directly how abscisic acid moved between the plant cells, tissues or organs. Now, Jones et al. (and independently Waadt et al.) have developed tools that can measure the levels of abscisic acid within defined compartments of 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 ‘reporters’ that fluoresce at different wavelengths. Shining light onto the plant at a specific wavelength that is only absorbed by one of the reporters causes both of the reporters on the sensor proteins to fluoresce. However, the two reporters fluoresce differently when the sensor binds to abscisic acid. Specifically, one reporter fluoresces more and the other less. 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. Jones et al. used a high-throughput platform to engineer five sensor proteins that detect abscisic acid over a wide range of concentrations. Using these ‘ABACUS’ sensors in living plants could track the uptake of abscisic acid into root cells, and revealed that the concentration of the hormone inside the cell stayed below the levels provided on the outside. Since known abscisic acid-transporters are capable of raising the hormone concentration inside a cell above that provided on the outside, abscisic acid transport into plant roots may occur via as-yet-undiscovered transporter proteins. Jones et al. also show that root cells rapidly eliminate abscisic acid, and that adding extra abscisic acid to the roots increases the rate of elimination within minutes. Plants were also engineered to target the sensor proteins specifically to the cell nucleus. In the future, targeting these sensors to the cell wall should allow tracking of the cell-to-cell movement of this hormone. Further aims include using ABACUS to track abscisic acid in plants undergoing stress, and to use the high-throughput platform to develop new sensors to track other hormones in living organisms (including animals). DOI: http://dx.doi.org/10.7554/eLife.01741.002