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Chinese Science Bulletin, Volume 64, Issue 1: 95-106(2019) https://doi.org/10.1360/N972018-00745

The microtubule-associated protein WDL3 mediates ABA-induced stomatal closure in Arabidopsis

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  • ReceivedJul 25, 2018
  • AcceptedOct 8, 2018
  • PublishedNov 22, 2018

Abstract

Stomatal movements control CO2 uptake for plant photosynthesis and water loss by transpiration, then determine plant productivity and water utilization efficiency. The microtubule dynamics is widely recognized to be essential for guard cell function. However, the molecular mechanisms underlying this process remain largely unknown. WDL3 belongs to the microtubule-associated protein WAVE-DAMPENED 2(WVD2)/WVD2-LIKE (WDL) family, which binds to and stabilizes microtubules against low-temperature and dilution-induced depolymerization. In the presence of WDL3, tubulins assemble into large microtubule bundles in vitro, otherwise only single filament patterns form. It has been intensively investigated that WDL3 participates in COP1(CONSTITUTIVE PHOTOMORPHOGENIC 1)-mediated hypocotyl cell elongation in darkness, but whether and how it modulates the microtubule behaviors during guard cell signaling transduction is still an open question. In this study, we dissected the interplay among WDL3, microtubule dynamics and Ca2+ in ABA-induced stomatal closure. We found that transpirational water loss from detached leaves occurred slowly in the WDL3 RNA interference transgenic line. Stomatal bioassay revealed that guard cell sensitivity to ABA was promoted in the WDL3 RNAi seedlings, and this phenotype could be partially blocked by paclitaxel, a microtubule stabilizing agent. On the other hand, the microtubule-disrupting drug oryzalin, enhanced ABA-triggered stomatal closure even further, and the WDL3 RNAi guard cells were more sensitive to oryzalin treatment. Based on the pharmacological results above, we next tested the effect of WDL3 on cortical microtubules in guard cells directly by Confocal microscopy. The differences in the configurations of microtubule filaments between WDL3 RNAi and wild type (WT) were analyzed after ABA treatment. Microscopic images showed that ABA-induced microtubule disassembly took faster in WDL3 RNAi guard cells than in WT, and the extent of filament bundling decreased significantly in WDL3 RNAi seedlings, as evaluated by the Image J software. This was consistent with the rapid ABA-induced stomatal closing in WDL3 RNAi material. Moreover, we examined the potential role of Ca2+ in this signal pathway. The cytosolic Ca2+ chelator—BAPTA generally alleviated ABA effects in both WT and WDL3 RNAi materials. ABA-induced stomatal closure and microtubule remodeling were much more delayed in WDL3 RNAi seedlings, indicating that Ca2+ acted upstream in the WDL3 mediated-ABA signaling pathway for stomatal movement. In addition, when exogenous ABA was applied, the Ca2+ influx monitored by the non-invasive micro-test technique (NMT) in WDL3 RNAi guard cells was more pronounced than in WT.

Taken together, our results suggest that WDL3, probably coordinated with Ca2+, is involved in the precise regulation of microtubule architecture and dynamics, to accurately execute ABA-stimulated signaling transduction during stomatal movement. Thus it contributes to the proper control of leaf transpiration.


Funded by

国家自然科学基金(31470011)

国家自然科学基金(31871351)


Acknowledgment

感谢中国农业大学袁明、毛同林教授赠予WDL3 RNAi, Tubulin5A-YFP种子以及毛同林教授课题组对本试验的指导, 感谢北京师范大学李杰婕教授对Image J软件使用的指导.


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  • Figure 1

    The WDL3 RNAi leaves show delayed transpirational water loss. (a) The wilty phenotypes of WDL3 RNAi and wild-type (Col); (b) water loss from detached rosette leaves of wild-type (Col) and WDL3 RNAi plants. Data are means of three independent experiments ±SE (with 15 leaves in each experiment). Statistical analysis was performed by paired t test (*P<0.05)

  • Figure 2

    Effects of microtubule specific drugs on WDL3-involved stomatal closure in response to ABA. Data are means of three independent experiments ±SD (with 20−40 stomata in each experiment). Statistical analysis was performed by paired t test (**P<0.01)

  • Figure 3

    The depolymerization of microtubule filaments is enhanced in WDL3 RNAi guard cells in response to ABA. (a) Three typical patterns of microtubules organization (Types 1 to 3, from up to down) were observed in guard cells. Bar=10 μm; (b) percentage of guard cells displaying the indicated type of microtubules at each condition. Data are from three independent experiments at least (at least 100 guard cells for each sample)

  • Figure 4

    (Color online) The microtubule density and bundling are greatly altered in WDL3 RNAi guard cells. (a) Continuous fluorescent intensity of the YFP signal along the line in guard cells with Type1 microtubule orientation was measured in wild type and WDL3 RNAi. Filament numbers of Type 1 were determined by the number of fluorescent peaks with an intensity higher than 50; (b) skeletonized microtubule arrays and the measurement of their occupancy and skewness in guard cells. Data are presented as the mean ±SE. At least 30 guard cells were analyzed for each sample. The P-values (*P<0. 05, **P<0. 01) were relative to that control in the same type. Bar=10 μm

  • Figure 5

    Effect of BAPTA on WDL3-involved ABA signaling during stomatal closure. Data are means of three independent experiments ±SD (with 20−40 stomata in each experiment). Statistical analysis was performed by paired t test (**P<0.01)

  • Figure 6

    Effects of Ca2+ chelator (BAPTA) on microtubule reorganization in ABA-induced stomatal closure. (a) Arrangement of cortical microtubules in guard cells from wild type and WDL3 RNAi treated with ABA plus BAPTA; (b) percentage of guard cells displaying the indicated type of microtubules at each condition. Data are from three independent experiments at least (at least 100 guard cells for each sample)

  • Figure 7

    WDL3 RNAi guard cells show significantly higher ABA-induced Ca2+ influx than wild type. (a) The integrated Ca2+ flux pattern. (b) The mean Ca2+ fluxes of Col and WDL3 RNAi guard cells before and after exposure to ABA. Each point represents the mean for 6–8 individual guard cells and bars represent the standard error of the mean. Statistical analysis was performed by paired t test (**P<0.01)

  • Figure 8

    Function model of WDL3 mediating ABA-induced stomatal closure

  • Table 1   Three typical patterns of microtubules organization in guard cells

    排布类型

    特点

    辐射状

    (Type1)

    微管骨架均匀而有序地由保卫细胞腹壁向背壁方向呈辐射状排列, 微管束之间大多相互平行, 多出现于开放气孔的保卫细胞中

    网状

    (Type2)

    规则整齐的微管束开始出现不同程度交叉、倾斜, 相互交错成网状排布, 为微管动态转换中的过渡状态

    解聚状

    (Type3)

    细胞内微管趋于解聚、碎片化, 弥散在细胞中, 清晰可辨的微管束很少, 多见于关闭气孔的保卫细胞

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