DcABF3, an ABF transcription factor from carrot, alters stomatal density and reduces 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.
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