Genome-wide identification of bHLH transcription factors and functional analysis in salt gland development of the recretohalophyte sea lavender (Limonium bicolor)

Abstract

Transcription factors with basic helix-loop-helix (bHLH) structures regulate plant growth, epidermal structure development, metabolic processes, and responses to stress extensively. Sea lavender (Limonium bicolor) is a recretohalophyte with unique salt glands in the epidermis that make it highly resistant to salt stress, contributing to the improvement of saline lands. However, the features of the bHLH transcription factor family in L. bicolor are largely unknown. Here, we systematically analyzed the characteristics, localization, and phylogenetic relationships of 187 identified bHLH family genes throughout the L. bicolor genome, as well as their cis-regulatory promoter elements, expression patterns, and key roles in salt gland development or salt tolerance by genetic analysis. Nine verified L. bicolor bHLH genes are expressed and the encoded proteins function in the nucleus, among which the proteins encoded by Lb2G14060 and Lb1G07934 also localize to salt glands. Analysis of CRISPR-Cas9-generated knockout mutants and overexpression lines indicated that the protein encoded by Lb1G07934 is involved in the formation of salt glands, salt secretion, and salt resistance, indicating that bHLH genes strongly influence epidermal structure development and stress responses. The current study lays the foundation for further investigation of the effects and functional mechanisms of bHLH genes in L. bicolor and paves the way for selecting salt-tolerance genes that will enhance salt resistance in crops and for the improvement of saline soils.

Figure 1

Chromosomal locations of 178 LbbHLH family genes in L. bicolor. The chromosome number is displayed above each chromosome. Chromosome length is expressed in Mb.

Figure 2

Intraspecific and interspecific collinearity analysis of bHLH genes. a Interchromosomal relationships of LbbHLH genes shown in a schematic. Gray lines indicate collinear blocks and red lines indicate bHLH family members in collinear blocks. b Interspecific analysis of bHLH genes between Arabidopsis, B. vulgaris, and F. tataricum and L. bicolor. Red lines indicate collinear blocks of bHLH genes within the Arabidopsis, B. vulgaris, F. tataricum, and L. bicolor genomes.

Figure 3

Localization of bHLH gene expression. a Positioning of 35S::LbbHLH-GFP in Arabidopsis protoplasts. The GFP-LbbHLH recombinant proteins were expressed only in the nucleus. Scale bar = 10 μm. b Negative control: the probe could not detect any transcripts and did not hybridize with any nucleic acid sequence. Localization of bHLH genes: LbbHLH transcripts were detected by an antisense probe (labeled with digoxin) that produces a blue–purple color. Scale bar = 50 μm.

Figure 4

Growth status and indicators of CR lines of L. bicolor. a Growth status of transgenic plants growing in 200 mM NaCl medium at 0, 10, and 20 days. Scale bar = 5 mm. b Leaf lethality rates of transgenic lines. Data are means ± standard deviation of nine replicates. c Staining of leaves of regenerated lines. Scale bar = 1 mm. d Statistics of the degree of staining. Data are means ± standard deviation of nine replicates. SPSS was used to determine the statistical significance of the data in the t-test. ^***P < 0.001. e Sequence alignment of Lb1G07934 from the CRISPR mutants CR-2 and CR-4, showing DNA alignments. Red lines indicate the CRISPR target sites; yellow boxes indicate the PAM sequence. f Appearance comparison of three lines before and after treatment with 200 mM NaCl. Scale bar = 5 cm. g Na⁺, K⁺, MDA, and proline contents in three lines before and after 200 mM NaCl treatment. Data are means ± standard deviation of three replicates. SPSS was used to determine the statistical significance of the data. Different letters indicate significant differences (p = 0.05; Duncan’s multiple range test).

Figure 5

Appearance of salt glands and measurement of salt secretion in different Lb1G07934 lines. a Phenotypes of salt glands in Mock-OE, Mock-CR, OE, and CR lines. b Density of salt glands in Mock-OE, Mock-CR, OE, and CR lines. Scale bar = 100 μm. Data are means ± standard deviation of 10 replicates. c Expression levels of Lb1G07934 in Lb1G07934-CR and Lb1G07934-OE lines. The Mock-CR and Mock-OE lines were used as a control. d Secretion status and DAB, Evans Blue, and NBT staining of leaf discs after 24 h of treatment. e Quantitative analysis of DAB, Evans Blue, and NBT staining. Data are means ± standard deviation of three replicates. SPSS was used to determine the statistical significance of the data (b and e) in the t-test. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. f Statistics of the volume of secretions, Na⁺ contents in secretions, and the rate of Na⁺ secretion by a single salt gland. SPSS was used to determine the statistical significance of the data (c and f) in Duncan’s multiple range test (P = 0.05).