The WD40 gene family in recretohalophyte Limonium bicolor: genomic identification and functional analysis in salt gland development and salinity tolerance

Abstract

Introduction: Developing salt-tolerant crops is critical for utilizing saline soils in agriculture. Limonium bicolor, a recretohalophyte with epidermal salt glands, represents a valuable genetic resource for salt tolerance engineering. Although WD40 proteins are known regulators of plant stress responses, their roles in L. bicolor remain unexplored.

Methods: We performed a genome-wide analysis of WD40 genes in L. bicolor, including phylogenetic classification, subcellular localization prediction, cis-element analysis, and expression profiling during salt stress. Functional validation was conducted using virus-induced gene silencing (VIGS).

Results: Among 367 identified WD40 genes (distributed across all chromosomes), Subfamily 6 was the largest. Two key members (Lb1G05968 and Lb3G17197, localized in cytoplasm) showed significant involvement in salt gland development and stress tolerance, as demonstrated by VIGS-induced phenotypic defects.

Discussion: Our findings reveal the WD40 family's expansion in L. bicolor and its functional specialization in salt adaptation. The identified genes (e.g., Lb1G05968, Lb3G17197) provide targets for engineering salt-tolerant crops. This study establishes a foundation for further research on halophyte developmental genetics.

Keywords: Limonium bicolor; WD40 gene family; feature description; salt gland development; salt tolerance; subcellular localization.

Figure 1

Phylogenetic tree of the WD40 family members from Limonium bicolor and Arabidopsis. A multiple amino acid sequence alignment was obtained with MUSCLE (employing gap penalties of -400 for opening and 0 for extension, with UPGMB clustering); The phylogenetic tree was reconstructed based on the maximum-likelihood method in MEGA 7.0. The scale bar indicates number of amino acid substitutions per site. Bootstrap values of significant branches within the six subfamilies (>50%) from 1,000 replicates are shown at key nodes. The red dots represent WD40 family members from Limonium bicolor, while the green dots denote WD40 family members from Arabidopsis thaliana.

Figure 2

Chromosomal distribution of LbWD40 gene family members in Limonium bicolor. Chromosomal localization analysis was performed for 367 LbWD40 gene family members. The chromosome numbers are indicated at the top of each corresponding chromosome. Chromosome lengths in the figure are measured in megabases (Mb).

Figure 3

Chromosomal relationship and homology analysis of the Limonium bicolor WD40 gene family. (a) Chromosomal relationship of WD40 genes in L. bicolor. The gray lines represent colinear blocks in the genomes of L. bicolor, while the red lines emphasize colinear LbWD40 gene pairs. (b) Homology analysis of the WD40 gene family of L. bicolor and three representative plant species: Arabidopsis thaliana, sugarbeet (Beta vulgaris), and Tartary buckwheat (Fagopyrum tataricum).

Figure 4

Exploring the roles of Lb1G05968 and Lb3G17197 based on expression patterns and silencing in Limonium bicolor leaves. SPSS was used to determine the statistical significance of the data. Different letters indicate significant differences (p = 0.05; Duncan’s multiple range test). (a) The Phenotype images of wild type and mutant under salt stress. Scale bars, 2 cm. (b) The total leaf area changes and increase ratio (%) before and after NaCl treatment in the TRV:: 0 control and Lb1G05968 and Lb3G17197 silence lines. (c) Representative photographs of salt glands on the epidermis of leaves from L. bicolor plants infiltrated with the control pTRV::0 vector or the silencing constructs for Lb1G05968 (pTRV::Lb1G05968) or Lb3G17197 (pTRV::Lb3G17197). Two independent plants are shown for each VIGS (Virus-Induced Gene Silencing construct): TRV::Lb1G05968 #1, TRV::Lb1G05968 #3, TRV::Lb3G1719 #1, and TRV::Lb3G17197 #2. Scale bars, 50 μm. (d) Salt gland density in the leaf epidermis of plants infiltrated with pTRV::0, pTRV::Lb1G05968, or pTRV::Lb3G17197. Two independent plants are shown for each VIGS construct: TRV::Lb1G05968 #1, TRV::Lb1G05968 #3 and TRV::Lb3G17197 #1, TRV::Lb3G17197 #3. (e) Relative expression levels of Lb1G05968 and Lb3G17197 in TRV::0 control plants and TRV::Lb1G05968 or TRV::Lb3G17197 plants, as determined by RT-qPCR. (f) Salt secretion indicators for the leaves of TRV::0 and TRV::Lb1G05968 or TRV::Lb3G17197 plants: number of salt glands, concentration of secreted Na+, and volume of secreted fluid. (g) Assessment of salt secretion by the leaf disc method. Representative images of leaf discs show the salt secretion fluid collecting on the disc surface. Leaf disc damage under salt treatment was estimated by DAB and NBT staining in leaf discs from control (TRV::0) and LbWD40-silenced leaves (TRV::Lb1G05968 #1, TRV::Lb1G05968 #3 and TRV::Lb3G17197 #1, TRV::Lb3G17197 #2). (h) Quantitative analysis of staining degree based on average optical density data measured by ImageJ. (i) Localization of Lb1G05968 and Lb3G17197. Each coding sequence was cloned in-frame and upstream of GFP (Green Fluorescent Protein); the resulting construct was infiltrated into Nicotiana benthamiana leaves. Scale bars, 10 μm. Nuclei were stained with DAPI.

Figure 5

The protein interaction map of Lb1G05968 and Lb3G17197 and the yeast interaction, regulatory model with LbHLH (Lb1G04899). (a) Protein–protein interaction network of Lb1G05968 in Arabidopsis. (b) Protein–protein interaction network of Lb3G17197 in Arabidopsis. (c) Yeast two-hybrid assay showing that the proteins encoded by Lb1G05968 and Lb3G17197 interact with LbHLH. AD, GAL4 activation domain from the pGADT7 vector; BD, GAL4 DNA-binding domain from the pGADT7 vector. The pGBKT7-LbHLH construct was introduced in yeast cells to test the autoactivation of LbHLH. The constructs pGADT7-Lb1G05968 and pGADT7-Lb3G17197 were co-introduced into yeast cells together with pGBKT7-LbHLH to test interaction. All transformants were plated on double dropout (DDO) medium (synthetic defined (Ananieva et al.)/−Leu/−Trp). Positive transformants were spotted onto DDO and quadruple dropout (QDO) medium (SD/−Ade/−His/−Leu/−Trp/X-α-gal. (d) Schematic diagram of the interaction between Lb1G05968, Lb3G17197 and LbHLH in regulating salt gland development.