Abstract
The basic helix-loop-helix (bHLH) transcription factors (TFs) comprise one of the largest gene families in plants, and participate in various physiological processes, but the physiological role and regulatory function of the majority of bHLHs remain poorly understood. Here, a total of 56 putative CsbHLH genes were identified in sweet orange (Citrus sinensis) based on a genome-wide analysis. The CsbHLH genes, except four members, were distributed throughout nine chromosomes and divided into 19 subgroups. Most of the CsbHLH genes were responsive to cold stress, with the greatest up-regulation being observed in CsbHLH18. CsbHLH18 is localized in the nuclei and has transcriptional activation activity. Overexpression of CsbHLH18 conferred enhanced cold tolerance in transgenic tobacco. The transgenic plants accumulated significantly less reactive oxygen species (ROS), concurrent with increased activities and transcript levels of antioxidant enzymes. In contrast, knockdown of bHLH18 by RNAi in trifoliate orange promoted cold susceptibility, accompanied by down-regulation of antioxidant genes and accumulation of more ROS. Protein-DNA interaction assays demonstrate that CsbHLH18 directly and specifically binds to and activates the promoter of CsPOD. Taken together, these findings indicate that CsbHLH18 plays a positive role in cold tolerance through, at least partly, modulation of ROS homeostasis by directly regulating the antioxidant gene.
Highlights
Plants are routinely exposed to a range of abiotic factors, such as extreme temperatures, drought, and high salinity, which have adverse effects on plant growth and may substantially reduce crop productivity
They were named according to their orders in the nine chromosomes (Fig. 1A; Supplementary Table S2).The percentage of CsbHLH genes per chromosome varied from 0.15% on chromosome 6 to 0.37% on chromosomes 9, with an average of 0.24% (Table 1), indicating an uneven distribution of the basic helix–loop–helix (bHLH) genes.The greatest number of CsbHLH genes was on chromosome 5 (10 genes), followed by seven on chromosomes 1 and 2, six on chromosomes 3, 7, and 9, four on chromosome 4, and three on chromosomes 6 and 8
Multiple sequence alignment using the bHLH domains was carried out to understand the degree of conservation and relative frequency of corresponding amino acids at each position (Fig. 1B).We observed that 22 amino acid residues were highly conserved with at least a 50% consensus ratio; in particular, the residues Arg-13, Arg-14, Leu-24, Pro-29, and Leu-56 had a consensus ratio >90%
Summary
Plants are routinely exposed to a range of abiotic factors, such as extreme temperatures, drought, and high salinity, which have adverse effects on plant growth and may substantially reduce crop productivity. Transcription factors (TFs) are important regulatory proteins that function to control the expression of target genes through binding to specific cis-acting elements within the promoters (Golldack et al, 2011).identification and characterization of stress-responsive TFs is crucial to elucidate the molecular network associated with stress response and has been widely adopted as a strategy for unraveling valuable candidate genes for improving stress tolerance using genetic engineering (Liu et al, 2014; Zhu, 2016). The HLH region is composed of two amphipathic helices connected by a divergent loop, and functions in facilitating formation of homo- and/or heterodimeric complexes between proteins (Nair and Burley, 2000; Buck and Atchley, 2003)
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