Elsevier

Plant Physiology and Biochemistry

Volume 130, September 2018, Pages 54-60
Plant Physiology and Biochemistry

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

https://doi.org/10.1016/j.plaphy.2018.06.034Get rights and content

Highlights

  • Transgenic Arabidopsis expressing the genetic construction CspMYB15:CsMYB61 shown an increase in water use efficiency.

  • The transgenic lines exhibited a smaller opening of the stomata pore, lower stomatal conductance and respiration rate.

  • The transgenic plants shown an enhance sensitivity to exogenous ABA and high drought stress tolerance.

Abstract

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 CsMYB15, 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.

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)

  • T. Hsieh et al.

    Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress

    Plant Physiol.

    (2002)
  • Cited by (16)

    • Comparative physiological and root transcriptome analysis of two annual ryegrass cultivars under drought stress

      2022, Journal of Plant Physiology
      Citation 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
    View all citing articles on Scopus
    View full text