Elsevier

Plant Science

Volume 302, January 2021, 110699
Plant Science

DcABF3, an ABF transcription factor from carrot, alters stomatal density and reduces ABA sensitivity in transgenic Arabidopsis

https://doi.org/10.1016/j.plantsci.2020.110699Get rights and content

Highlights

  • DcABF3, an ABA-responsive element-binding transcription factor, was cloned and functionally characterized.

  • DcABF3 alters drought tolerance by increasing stomatal density in Arabidopsis.

  • DcABF3 reduces ABA sensitivity in transgenic Arabidopsis.

Abstract

Abscisic acid-responsive element (ABRE)-binding factors (ABFs) are important transcription factors involved in various physiological processes in plants. Stomata are micro channels for water and gas exchange of plants. Previous researches have demonstrated that ABFs can modulate the stomatal development in some plants. However, little is known about stomata-related functions of ABFs in carrots. In our study, DcABF3, a gene encoding for ABF transcription factor, was isolated from carrot. The open reading frame of DcABF3 was 1329 bp, encoding 442 amino acids. Expression profiles of DcABF3 indicated that DcABF3 can respond to drought, salt or ABA treatment in carrots. Overexpressing DcABF3 in Arabidopsis led to the increase of stomatal density which caused severe water loss. Expression assay indicated that overexpression of DcABF3 caused high expression of stomatal development-related transcription factor genes, SPCH, FAMA, MUTE and SCRMs. Increased antioxidant enzyme activities and higher expression levels of stress-related genes were also found in transgenic lines after water deficit treatment. Changes in expression of ABA synthesis-related genes and AtABIs indicated the potential role of DcABF3 in ABA signaling pathway. Under the treatment of exogenous ABA, DcABF3-overexpression Arabidopsis seedlings exhibited increased root length and germination rate. Our findings demonstrated that heterologous overexpression of DcABF3 positively affected stomatal development and also reduced ABA sensitivity in transgenic Arabidopsis.

Introduction

Stomata are small pores gradually evolved by land plants for adapting to photosynthetic gas exchange [1]. Stomata are formed by guard cells and pores on plant epidermis, making them the main gas exchange channels [2]. During plant development, the balance of photosynthesis and water loss is maintained by regulating stomatal density and stomatal aperture [3]. It is generally known that stomatal changes are directly related to the water use efficiency (WUE) and drought resistance of plants [4,5]. Especially the stomatal density, plants adjust stomatal density to cope with long-term water deficits [[6], [7], [8]]. High WUE is conducive to improving the biomass production and water stress tolerance [9]. The growth and development of plants are constantly challenged by several environmental conditions, of which drought is one of the major obstacles to plant survival, distribution and productivity [10,11]. Therefore, stomata have become one of the ideal models to explore the physiological mechanism of responding to drought stress in plants. In recent years, a relatively clear understanding of stomatal development has been found in plants, especially in Arabidopsis [12,13]. Several basic helix-loop-helix (bHLH) transcription factors are confirmed to participate in the regulation of stomatal cell fate transition and morphological changes, such as SPCH, MUTE, FAMA and SCRM [[14], [15], [16], [17]]. Regulation functions on stomata of other proteins, such as SDD1, EPF, and CAS, which could interact with bHLH transcription factors, have also been identified gradually [7,18].

In addition to the regulation of endogenous signals, stomatal development is also affected by environmental signals and endogenous hormones. Several hormones have been found to be involved in the regulation of stomatal development through the interaction between hormone metabolism and stomatal development pathway [19,20]. As a major chemical signal regulating the stomatal closure, abscisic acid (ABA) plays a central role in regulating the development of stomata [3]. A protein kinase, MAPK in ABA signaling pathway can regulate stomatal closure by inducing H2O2 production in guard cells [21]. By reducing turgor pressure, cell division rate and cell wall extensibility, ABA appears to slow leaf expansion rates and further decrease the stomatal index [22]. The regulation of stomatal density by ABA is also related to its inhibition to bHLH transcription factors [23]. Some researchers have demonstrated the changes of stomatal ABA sensitivity during the development of Arabidopsis. With the increase of leaf age, the content of ABA decreased, while the sensitivity of ABA rose progressively [24].

ABA-responsive elements (ABRE)-binding transcription factors (ABFs) are a group of transcription factors which belong to the A subfamily of basic leucine zipper motif (bZIP) transcription factor family [25]. Previous researches have confirmed that ABFs can regulate a wild range of target genes and appear to function in ABA signal transduction [[26], [27], [28]]. More and more ABF genes and their potential functions were identified from various plants [[29], [30], [31], [32]]. In Arabidopsis, a total of 13 members of bZIP family were identified as ABF transcription factors, some of them were confirmed to be involved in responses to different stresses [25]. TRAB1 of rice can activate ABA response genes to control seed maturation and dormancy [30]. Another ABF in rice, OsABI5, were up-regulated by ABA and salt but down-regulated by drought and low temperature [33]. Due to the great quantity of ABFs in different plant species, current understanding of the role of ABFs is substantially to be supplemented.

Carrot (Daucus carota L.) is a worldwide cultivated root vegetable, which originated from the southwest of Asia [34]. With the release of carrot genome data [35], members of bZIP transcription factor family were identified and classified. Ten of the bZIP transcription factor family members were classified into subfamily A, ABF transcription factors [36,37]. Till now, few of them was functionally characterized in carrot. An ABF transcription factor, DcABF3 (also known as DcAREB3) [38] was identified in our study. Its potential role was further functionally characterized in transgenic Arabidopsis. Our research can provide a reference for the functional identification of ABF transcription factors in carrot.

Section snippets

Plant materials and treatments

Seedlings of carrot ‘Kurodagosun’ were grown in the artificial room at Nanjing Agricultural University (Nanjing, China). The growth conditions were kept at 25 °C for 14 h light and 18 °C for 10 h dark photoperiod with a 60∼70 % relative humidity and 240 μmol·m-2·s-1 light intensity. Forty-day-old carrot seedlings were used for drought (200 g·L-1 PEG), salt (0.2 M NaCl) or ABA (0.1 M ABA) treatment. Seedlings treated with the same amount of distilled water were set as control. Leaves of the

Cloning and bioinformatics analysis of DcABF3

By RT-PCR, a gene DcABF3 encoding ABF transcription factor was cloned. The length of DcABF3 was 1329 bp, encoding 442 amino acids. The molecular weight of DcABF3 protein was 49.08 kDa and its pI was 8.97. A high conserved bZIP domain was found at the C-terminus of DcABF3 protein, which contained a 25 amino acid basic region and a parallel leucine zipper. The results of sequence alignment between DcABF3 and Arabidopsis ABFs showed that three highly conserved regions (C1, C2 and C3) were located

Discussion

Plants have complicated regulatory networks to resist changes of the external environmental conditions. In previous studies, researchers have made great advances to deeply understand the ABA-mediated network [47,48]. As major transcription factors involved in ABA signaling pathway, ABFs play irreplaceable roles [49]. Previous studies have demonstrated that ABFs could function in responding to adversity stresses, but different ABF transcription factors possess their own unique functions [26,28,50

Conclusion

A gene encoding an ABRE-binding factor, DcABF3, was cloned from carrot. Sequence analysis showed that DcABF3 contained a bZIP domain and had the closest phylogenetic relationship with AtABF1 and AtABF4. DcABF3 was identified to localize in nucleus by subcellular localization. Expression analysis indicated that DcABF3 could respond to drought, ABA and salt treatment in carrot. Overexpressing DcABF3 in Arabidopsis increased the stomatal density and affected the water deficit tolerance. ABA

Funding

The research was supported by National Natural Science Foundation of China (31872098), Open Fund of the State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University (ZW201905), and Priority Academic Program Development of Jiangsu Higher Education Institutions Project (PAPD).

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

Xiong AS, Wang YH and Que F initiated and designed the research; Wang YH, Que F, Zhang RR and Ahmed K performed the experiments; Wang YH, Que F, Li T, Tian YS and Xu ZS analyzed the data; Xiong AS contributed reagents/materials/analysis tools; Wang YH wrote the paper; Xiong AS revised the paper. All authors read and approved the final manuscript.

References (59)

  • P.J. Rudall et al.

    Several developmental and morphogenetic factors govern the evolution of stomatal patterning in land plants

    New Phytol.

    (2013)
  • L.J. Pillitteri et al.

    The bHLH protein, MUTE, controls differentiation of stomata and the hydathode pore in Arabidopsis

    Plant Cell Physiol.

    (2008)
  • C.C. Chater et al.

    Putting the brakes on: abscisic acid as a central environmental regulator of stomatal development

    New Phytol

    (2014)
  • X.J. Song et al.

    Bar the windows: an optimized strategy to survive drought and salt adversities

    Gene Dev.

    (2009)
  • C.Y. Yoo et al.

    The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating stomatal density via transrepression of SDD1

    Plant Cell

    (2010)
  • C. Xie et al.

    Overexpression of MtCAS31 enhances drought tolerance in transgenic Arabidopsis by reducing stomatal density

    New Phytol.

    (2012)
  • R.K. Upadhyay et al.

    SlERF36, an EAR-motif-containing ERF gene from tomato, alters stomatal density and modulates photosynthesis and growth

    J Exp Bot.

    (2013)
  • P.J. Franks et al.

    Increasing water-use efficiency directly through genetic manipulation of stomatal density

    New Phytol.

    (2015)
  • J. Krasensky et al.

    Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks

    J Exp Bot

    (2012)
  • L.J. Pillitteri et al.

    Stomatal development in Arabidopsis

    Arabidopsis Book

    (2013)
  • E. Lee et al.

    Deep functional redundancy between FAMA and FOUR LIPS in stomatal development

    Plant J.

    (2014)
  • C.A. MacAlister et al.

    Sequence and function of basic helix-loop-helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants

    Evol Dev.

    (2011)
  • C.A. MacAlister et al.

    Transcription factor control of asymmetric cell divisions that establish the stomatal lineage

    Nature

    (2007)
  • K. Ohashi-Ito et al.

    Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development

    Plant Cell

    (2006)
  • M.M. Kanaoka et al.

    SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation

    Plant Cell

    (2008)
  • S. Morales-Navarro et al.

    Overexpression of a SDD1-like gene from wild tomato decreases stomatal density and enhances dehydration avoidance in Arabidopsis and cultivated tomato

    Front Plant Sci.

    (2018)
  • T.W. Kim et al.

    Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway

    Nature

    (2012)
  • L. Villalobos et al.

    A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin

    Nat Chem Biol.

    (2012)
  • G.E. Gudesblat et al.

    Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide

    New Phytol.

    (2007)
  • Cited by (25)

    • Characterization of three tandem-duplicated calcium binding protein (CaBP) genes and promoters reveals their roles in the phytohormone and wounding responses in citrus

      2023, International Journal of Biological Macromolecules
      Citation Excerpt :

      One of the changes is the level of cytosolic free calcium that acts as a vital intracellular messenger inducing the downstream changes. Mounting evidences have shown that a fast and transient elevation in cytoplasmic calcium concentration often occurs in plant cells in their early responses to a large number of hormonal stimuli and various stresses [4–7]. Increases in calcium ion concentration are normally sensed by a class of calcium binding proteins (CaBPs) [8–10].

    • Involvement of three ABRE-binding factors in the gametophytic self-incompatibility reaction in pear

      2022, Scientia Horticulturae
      Citation Excerpt :

      High-throughput sequencing is an efficient approach for isolating DEGs responsive to the GSI reaction, but little is known about the molecular pathway by which self S-RNase influences the expression of these DEGs. The ABF transcription factor, which belongs to the subfamily of bZIP-type transcription factors (Jakoby et al., 2002), has been widely studied in various physiological processes including stomatal density (Wang et al., 2021) and abiotic stress tolerance (Huang et al., 2010; Liu et al., 2019; Tang et al., 2012). The overexpression of SlAREB1 enhances the tolerance to a series of abiotic stresses and promotes the expression of biotic and abiotic stress-related genes in tomato (Orellana et al., 2010; Yáñez et al., 2009).

    • Genome-wide analysis of MdABF Subfamily and functional identification of MdABF1 in drought tolerance in apple

      2022, Environmental and Experimental Botany
      Citation Excerpt :

      An increasing number of studies have reported that a large number of transcription factors can regulate stomata by interacting with bHLH transcription factors. For instance, DcABF3 could increase stomatal density through bHLH signaling (Wang et al., 2021). Similarly, Poncirus trifoliate experiments showed that PtrABF interacted with downstream factors to reduce stomatal density (Zhang et al., 2015).

    View all citing articles on Scopus
    View full text