Stomata regulation by tissue-specific expression of the Citrus sinensis MYB61 transcription factor improves water-use efficiency in Arabidopsis

Water-use efficiency (WUE) is a quantitative measurement of biomass produced per volume of water transpired by a plant. WUE is an important physiological trait for drought response to mitigate the water deficiency. In this work, a cisgenic construction from Citrus sinensis was developed and its function in the improvement of WUE was evaluated in Arabidopsis. Sequences of the CsMYB61 coding region, a transcription factor implicated in the closure of stomata, together with a putative stomata-specific promoter from CsMYB.1.5, were identified and cloned. The protein encoded in the CsMYB61 locus harbors domains and motifs characteristic of MYB61 proteins. In addition, a 1.2 kb promoter region of the gene CsMYB15 (pCsMYB15) containing regulatory elements for expression in guard cells and in response to Abscisic Acid (ABA) and light was isolated. In Arabidopsis, pCsMYB15 directs the expression of the reporter gene GUS in stomata in the presence of light. In addition, transgenic lines expressing the CsMYB61 coding region under transcriptional control of pCsMYB15 have a normal phenotype under in vitro and greenhouse conditions. These transgenic lines exhibited a smaller opening of the stomata pore, lower stomatal conductance and respiration rate, enhanced sensitivity to exogenous ABA, and high drought stress tolerance. Our results indicate that stomata-specific expression of CsMYB61 enhances water use efficiency under drought conditions in Arabidospis.