Research articleStomata regulation by tissue-specific expression of the Citrus sinensis MYB61 transcription factor improves water-use efficiency in Arabidopsis
Introduction
Regulation of the opening and closing of stomata is a key physiological function for the proper development and survival of plants (Taiz and Zeiger, 2002). Water use efficiency (WUE), which relates biomass generation to water use (Tardieu et al., 2018), is a common metric of plant performance under drought stress. Advances in biotechnological tools have generated plants that consume less water and perform superiorly under water deficit conditions. The use of such engineered plants promises agricultural benefits through reduced water demand and offers the potential for establishment of crops in less exploited arid zones (Cominelli et al., 2010; Cominelli and Tonelli, 2010). Citruses are one of the most important fruit crops in tropical and subtropical regions, but citrus producing areas are frequently subject to water deficiency, affecting both vegetative and reproductive processes (Vu and Yelenosky, 1988). Considering that water scarcity will likely increase as a product of global climate change, drought tolerance is a desirable characteristic for citrus sustainability and/or expansion. Among plant strategies for drought stress tolerance, higher regulation of stomata could be a good physiological strategy (Blatt et al., 2017).
Transcription factors that participate in the control of stomatal movements have been identified and characterized. The MYB transcription factors represent one of the largest families in plants and share the MYB DNA binding domain. In Arabidopsis, four transcription factors of the R2R3-MYB family that are involved in the modulation of stomatal movements have been characterized: AtMYB15, AtMYB44, AtMYB61, and AtMYB60 (Cominelli et al., 2005; Liang et al., 2005; Jung et al., 2008; Ding et al., 2009; Galbiati et al., 2011). The first three play a role in stomatal closure, while MYB60 participates in stomatal opening. Previous studies in our laboratory contributed to characterization of the MYB60 gene in Vitis vinifera where its participation in stomatal opening was demonstrated (Galbiati et al., 2011). In contrast, AtMYB61 is expressed specifically in the guard cells where it acts as a transcriptional regulator of stomatal closure, having a role in the regulation of stoma pore size through an ABA-independent pathway and in the light-to-dark transition (Liang et al., 2005). Thermography analysis revealed that 35S:MYB61 plants were approximately 0.5 °C warmer than WT plants, whereas myb61 mutant plants were approximately 0.5 °C cooler than WT plants. This suggests that MYB61 alone is necessary and sufficient for the partial closure of stomata (Liang et al., 2005).
Transcription factors that participate in the regulation of stomatal opening and closing in the citrus genus have not yet been described. Identifying citrus MYB61 homologs is relevant to developing genetic tools for WUE improvement in these water intensive fruit crops. In this work, the coding sequence of MYB61 and 1.2 kb of the putative promoter of the MYB15 – a light- and ABA-inducible gene – from Citrus sinensis was identified and cloned. A cisgenic construction using both sequences was developed and its role in WUE improvement and drought tolerance was evaluated in Arabidopsis plants.
Section snippets
Plant material
Arabidopsis thaliana (Col-0) were grown under long day conditions (16 h light/8 h dark cycle and 70–80 μmol m−2 s−1) on Petri dishes containing 0.5X MS salts, 1% w/v sucrose and 0.7% agar. Fourteen day-old Arabidopsis plants were transferred to pots with sterile substrate (peat:vermiculite 2:1) and irrigated regularly with tap water supplemented with B5 mineral nutrients.
Cloning of genomic sequences of pCsMYB15 and CsMYB61
The genomic sequences of pCsMYB15 and CsMYB61 were identified using the Citrus sinensis Annotation Project (Huazhong
Tissue-specific expression of putative CsMYB15 promoter
Genetic regulation of stomatal movement depends mainly on an efficient gene expression control system. Previous reports have shown that the promoter of MYB15 is active not only in vegetative and reproductive organs but also in the guard cells of stomata in Arabidopsis during the day (Ding et al., 2009). With the aim to drive specific expression of a transcriptional regulator of stomatal closure, a putative promoter (1.2 kb upstream of the annotated ATG start codon) of MYB15 from Citrus sinensis
Discussion
Harnessing transcription factors, which regulate the expression of the stress-responsive genes, represents a powerful strategy to modulate drought resistance and prevent plant productivity loss. In the present study, the expression of CsMYB61 under transcriptional control of pCsMYB15 in Arabidopsis significantly increased WUE under water limited conditions. The higher pCsMYB15:CsMYB61 expression lines exhibited significantly lower opening of their stomata compared to the WT plant and this
Authors and contributors
JLRR conceived the study and designed the experiments with PAJ. JLRR, CIB, DO, MRD and JPM performed the experiments and analyzed the data. JLRR, FA, PG and PAJ drafted the paper. All authors contributed to the revision of the manuscript and approved the final version.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Acknowledgment
The authors thank Valeria M. Borjas Mendez for technical assistance. Jesús L. Romero-Romero was supported by PhD fellowship CONACYT (SC392465), COTEBAL: 1865 and CONICYT Doctoral Scholarship for foreigners (63130094). This work was supported by FONDECYT 1150220 to PAJ, CAPES FB-002-2014 to FA and CONICYT PAI 82140040 to JPM. We thank Alyssa Grube for assistance in language support.
References (23)
- et al.
A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance
Curr. Biol.
(2005) - et al.
Transgenic crops coping with water scarcity
N. Biotech.
(2010) - et al.
Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana
Journal of Genetics and Genomics
(2009) - et al.
AtMYB61, an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana
Curr. Biol.
(2005) - et al.
TRAUCO, a Trithorax-group gene homologue, is required for early embryogenesis in Arabidopsis thaliana
J. Exp. Bot.
(2010) - et al.
Small pores with a big impact
Plant Physiol.
(2017) - et al.
Functional characterization of Citrus macrophylla BOR1 as a boron transporter
Physiol. Plantarum
(2013) - et al.
Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
Plant J.
(1998) - et al.
Transcription factors controlling stomatal movements and drought tolerance
Transcription
(2010) - et al.
The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress
BMC Plant Biol.
(2011)
Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiol.
Cited by (16)
Comparative physiological and root transcriptome analysis of two annual ryegrass cultivars under drought stress
2022, Journal of Plant PhysiologyCitation Excerpt :In our study, we found MYB61, MYBS2 and MYBS3 were down-regulated in DS only. Overexpression of MYB61 reduced stomatal conductance and improved water use efficiency, thus enhancing drought tolerance in Arabidopsis thaliana (Romero-Romero et al., 2018). It has been reported that overexpression of MYBS2 enhanced plant growth, stress tolerance and grain weight of rice, and overexpression MYBS3 conferred rice with cold tolerance (Chen et al., 2019; Su et al., 2010).
MicroRNA: A Dynamic Player from Signalling to Abiotic Tolerance in Plants
2023, International Journal of Molecular Sciences