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Figure 1.Induction of ROS production in Arabidopsis guard cells’ chloroplasts by ABA and MJ. (A) Leaf disks were prepared from wild-type plants and treated with ABA and MJ. After incubation of leaf discs in solution with the phytohormones, cells were loaded with H2DCFDA as described in methods. Shown are representative images of single section confocal microscopy (1 μm thick) of guard cells from the different treatments. Scale bar = 10 µm. (B) Quantification of the H2DCFDA-dependent fluorescence of individual chloroplasts shown in (A). Quantification was done using ImagePRO-Plus program from projected images of 2–3 confocal Z stack sections of individual chloroplasts, as described in methods. Shown is a representative one of three replicate experiments. n = 30 chloroplasts from 10 guard cells sampled from three different young rosette leaves (± SE). R.U. relative unites.
Zooming into sub-organellar localization of reactive oxygen species in guard cell chloroplasts during abscisic acid and methyl jasmonate treatments
Regulation of stomata movements is crucial for plants ability to cope with their changing environment. Guard cells’ (GC) water potential directs water flux inside/outside this cell, which eventually is causing the stoma to open or close, respectively. Some of the osmolytes which accumulates in the GC cytoplasm and are known to play a role in stomata opening are sugars, arising from chloroplast starch degradation. During stomata closure, the accumulated osmolytes are removed from the GC cytoplasm. Surprisingly little is known about prevention of starch degradation and forming additional sugars which may interfere with osmotic changes that are necessary for correct closure of stomata.
One of the early events leading to stomata closure is production of reactive oxygen species (ROS) in various subcellular sites and organelles of the stoma. Here we report that ROS production during abscisic acid (ABA) and methyl jasmonate (MJ) stimuli in Arabidopsis GC chloroplasts were more than tripled. Moreover, ROS were detected on the suborganelle level in compartments that are typically occupied by starch grains. This observation leads us to suspect that ROS function in that particular location is necessary for stomata closure. We therefore hypothesize that these ROS are involved in redox control that lead to the inactivation of starch degradation that takes place in these compartments, thus contributing to the stoma closure in an additional way.
Drought sensitivity and water loss in wild-type and AtVAMP711 transgenic plants. (A) Increased drought sensitivity of antisense AtVAMP711transgenic lines. Irrigation was stopped in 3-month-old plants, grown under a short-day light regime. The picture was taken after 10 d without water. The upper row shows a representative picture from a triplicate experiment (line 2091, middle pot; 2092, right pot). The lower row, shows plants that remained to be irrigated. The phenotype was also observed in other antisense line (2096, data not shown). (B) Water loss measurements in detached leaves of wild-type and transgenic plants. Rosette leaves of AtVAMP711 antisense (lines 2091 and 2092) or overexpressing (line 7+, 12+), were detached from irrigated, 2-month-old plants and placed in weighing dishes. Loss of fresh weight was monitored at the indicated times, as described in the Materials and methods. Shown is a representative triplicate experiment (n=4 leaves from four individual plants ±SE).
Reduced expression of the v-SNAREs AtVAMP71/AtVAMP7C gene family in Arabidopsis reduces drought tolerance by suppression of abscisic acid-dependent stomatal closure
by Leshem Y., Golani Y., Kaye Y., Levine A. (2010)
Induction of stomatal closure by ABA and sorbitol. (A) Induction of stomatal closure by ABA in wild-type and antisense VAMP711 lines. Leaf discs from young rosette leaves were treated with ABA (20 μM) for 3 h. Stomatal aperture was measured by the microscope optical micrometer as described in the Materials and methods. Shown are representative triplicate experiments. The different small letters denote statistical significance after F test (P <0.05), N=60 ±SE. (B) Stomatal closure by osmotic conditions. Young rosette leaves were detached and loaded with Neutral Red as a vacuolar stain. Leaf discs were incubated in 1 M sorbitol for 1 h. ABA was applied as above in (A). Stomatal aperture was observed and measured using light microscopy as in (A). Different small letters within columns denote statistical significance after F test (P<0.05), N=60 ±SE. (C–F) Representative images from (B), above, showing control and sorbitol-treated stomata of wild-type (C, D, respectively) and antisense AtVAMP711 line 2091 plants (E, F).
Abstract
Stomatal closure during water stress is a major plant mechanism for reducing the loss of water through leaves. The opening and closure of stomata are mediated by endomembrane trafficking.
The role of the vacuolar trafficking pathway, that involves v-SNAREs of the AtVAMP71 family (formerly called AtVAMP7C) in stomatal movements, was analysed. Expression of AtVAMP711–14 genes was manipulated in Arabidopsis plants with sense or antisense constructs by transformation of the AtVAMP711 gene. Antisense plants exhibited decreased stomatal closure during drought or after treatment with abscisic acid (ABA), resulting in the rapid loss of leaf water and tissue collapse.
Induction and localization of intracellular ROS in guard cells after treatment with ABA. (A) Total ROS accumulation in guard cells. Leaf discs of wild-type or antisense plants were treated with ABA as in Fig. 2. ROS were detected by 2′,7′-dichlorodihydrofluorescein diacetate as described in the Materials and methods. Confocal Z-sections of each guard cell (compiled 8-10 sections, each 1 μm thick) were projected to quantify the mean intensity of H2DCFDA in a whole guard cell. Z section projections and fluorescence measurements were done using the ImagePro Plus program. Different letters within column denote statistical significance after F test (P <0.05, NS, not significant), N=15 ±SE. (B) Intracellular localization of ROS induced by ABA in wild-type and antisense AtVAMP711 guard cells. Simultaneous staining of ROS by H2DCFDA (green filter) and intracellular membranes by MitoFluor Red 589 (red filter) in guard cells of wild-type and antisense plants (line 2091). Leaf discs were treated with ABA for 2 h as in (A). Loading of dyes and fluorescence detection are described in the Materials and methods. Shown are representative confocal images of a single section in guard cells of control (top panel), or ABA-treated plants (lower panel). For a complete Z stack of each image see Supplementary Fig. S2 at JXB online. Similar results were obtained in the other VAMP711 antisense lines (data not shown). (C, D) Nuclear localization of ABA-induced ROS in wild-type guard cells and epidermal cells. Simultaneous staining of nuclei (DAPI, blue), ROS (H2DCFDA, green) (D) and intracellular membranes [MitoFluo Red 589 (C)] in guard cells (C) and epidermal pavement cells (D). Leaf discs of wild-type and antisense plants (line 2091) were treated with ABA for 2 h as in (A). Shown are representative confocal images (C, D). Control images are presented in the top panel of (B). Similar results were obtained in the antisense lines (data not shown).
No improvement was seen in plants overexpressing the AtVAMP711 gene, suggesting that wild-type levels of AtVAMP711expression are sufficient. ABA treatment induced the production of reactive oxygen species (ROS) in guard cells of both wild-type and antisense plants, indicating that correct hormone sensing is maintained. ROS were detected in nuclei, chloroplasts, and vacuoles. ABA treatment caused a significant increase in ROS-containing small vacuoles and also in plastids and nuclei of neighbouring epidermal and mesophyll cells.
Taken together, our results show that VAMP71 proteins play an important role in the localization of ROS, and in the regulation of stomatal closure by ABA treatment. The paper also describes a novel aspect of ROS signalling in plants: that of ROS production in small vacuoles that are dispersed in the cytoplasm.
PLANT STOMATA ENCYCLOPEDIA 
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