Functional Analysis Of Arabidopsis VAC14 In Regulating Stomatal Movement

Stomata are small pores formed by two guard cells that permit the regulation of gaseous and water exchanges between plant interior and its surrounding environment.Expansion and contraction of guard cells lead to the opening and closure of stomata,respectively.Stomatal movement responds to a variety of environmental factors,such as humidity,CO₂,light,and the phytohormone ABA.Illumination promotes the opening of stomata to absorb more CO₂ for increased photosynthesis whereas ABA induces the closure of stomata to reduce water loss in drought.The opening and closure of stomata involve multiple intracellular activities,including the dynamic assembly of actin microfilament or microtubules,the flux of Ca²⁺,K⁺,and other ions,the fusion or fission of vacuoles,and so on,among which the dynamic remodeling of vacuoles plays a vital role.Phosphatidylinositol-3,5-diphosphate[PI(3,5)P₂]is a key factor in the dynamic remodeling of vacuoles.In yeast and metazoans,the level of PI(3,5)P₂ is controlled by a PI(3,5)P₂ metabolic complex that includes a FAB1/PIKfyve kinase,a SAC phosphatase,and a scaffold protein VAC14.Although mutations of the FAB1/PIKfyve kinase in Arabidopsis has been reported to cause hyposensitivity to ABA-induced stomatal closure,it was not clear whether the effect of PI(3,5)P₂ on stomatal movement is cell autonomous and what the physiological consequences of its reduction are.In this study,we used a guard cell-specific promoter to drive artificial microRNAs(amiR)for Arabidopsis VAC14 and by interfering PI(3,5)P₂,so that the role of VAC14 in the autonomous regulation of stomatal movement could be explored.The results we have obtained are as follows:(1)Downregulating VAC14 results in enlarged guard cellsDownregulating VAC14 did not affect stomatal density.However,guard cells of amiR-VAC14 plants were significantly larger than those of wild type,indicating that downregulation VAC14 in guard cells promoted cell expansion.(2)Downregulating VAC14 in guard cells reduces stomatal closureamiR-VAC14 plants were hyposensitive to ABA,dark,or pathogen-induced stomatal closure compared with wild type,indicating that VAC14 plays a crucial role in regulating stomatal closure induced by multiple environmental cues.By contrast,downregulating VAC14did not affect the inhibition of ABA on light-induced stomatal opening.(3)Downregulating VAC14 compromises vacuolar fission during stomatal closureBy using Oregon Green staining and GFP-INT1 labeling,we demonstrated that downregulating VAC14 compromised vacuolar fission during ABA-induced stomatal closure,suggesting that VAC14 mediates vacuolar dynamics.(4)VAC14 positively regulates stomatal movement through the production of PI(3,5)P₂ in guard cellsThe application of a PI(3,5)P₂-inhibitor YM201636 significantly reduced ABA-induced stomatal closure,mimicking the defects in amiR-VAC14 plants,whereas the application of PI(3,5)P₂ rescued the hyposensitive response of amiR-VAC14 stomata to ABA.These results suggested that the effect of amiR-VAC14 on stomatal closure is due to a reduction of PI(3,5)P₂.(5)Downregulating VAC14 in guard cells reduces drought toleranceBy using a detached leaf assay,we demonstrated that downregulating VAC14 caused higher transpiration water loss;by using a leaf thermal profile assay,we demonstrated that downregulating VAC14 caused a reduced leaf temperature;by using a drought assay,we demonstrated that amiR-VAC14 plants were less tolerance to drought.These results indicated that VAC14 positively regulates plant drought tolerance.(6)Overexpressing VAC14 does not affect stomatal closureBy analyzing stomatal movement of VAC14-overexpressing plants,we determined that overexpressing VAC14 alone was not sufficient to enhance ABA-induced stomatal closure or drought tolerance,suggesting that VAC14 acts in a complex to fulfil its function in PI(3,5)P₂production.