Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress. c c c c c Figure 8. Wax ester contents and compositions on Arabidopsis stems of wild-type and wsd mutant plants after exposure to drought. Waxes were isolated from stems and purified by solid-phase extraction. Wax esters were quantified by Q-TOF mass spectrometry with 17:0ol-18:0 as internal standard. (a) wsd1-1 and wsd1-2. (b) wsd6-1 and wsd6-2. (c) wsd7-1 and wsd7-2. Means AE SD, n = 5. The letters a, b indicate significant differences to wild-type under control or stress conditions, respectively. The letter c depicts significant differences between control and stress conditions of the same line (Student's t-test, P < 0.05).The WSD6 and WSD7 cDNAs from pJET1.2/blunt were ligated into the SpeI and BamHI sites of pMDC83 (Curtis and Grossniklaus,