Integrative Analysis Provides Insights into Genes Encoding LEA_5 Domain-Containing Proteins in Tigernut (Cyperus esculentus L.)

Abstract

LEA_5 domain-containing proteins constitute a small family of late embryogenesis-abundant proteins that are essential for seed desiccation tolerance and dormancy. However, their roles in non-seed storage organs such as underground tubers are largely unknown. This study presents the first genome-scale analysis of the LEA_5 family in tigernut (Cyperus esculentus L.), a Cyperaceae plant producing desiccation-tolerant tubers. Four LEA_5 genes identified from the tigernut genome are twice of two present in model plants Arabidopsis thaliana and Oryza sativa. A comparison of 86 members from 34 representative plant species revealed the monogenic origin and lineage-specific family evolution in Poales, which includes the Cyperaceae family. CeLEA5 genes belong to four out of five orthogroups identified in this study, i.e., LEA5a, LEA5b, LEA5c, and LEA5d. Whereas LEA5e is specific to eudicots, LEA5b and LEA5d appear to be Poales-specific and LEA5c is confined to families Cyperaceae and Juncaceae. Though no syntenic relationship was observed between CeLEA5 genes, comparative genomics analyses indicated that LEA5b and LEA5c are more likely to arise from LEA5a via whole-genome duplication. Additionally, local duplication, especially tandem duplication, also played a role in the family expansion in Juncus effuses, Joinvillea ascendens, and most Poaceae plants examined in this study. Structural variation (e.g., fragment insertion) and expression divergence of LEA_5 genes were also observed. Whereas LEA_5 genes in A. thaliana, O. sativa, and Zea mays were shown to be preferentially expressed in seeds/embryos, CeLEA5 genes have evolved to be predominantly expressed in tubers, exhibiting seed desiccation-like accumulation during tuber maturation. Moreover, CeLEA5 orthologs in C. rotundus showed weak expression in various stages of tuber development, which may explain the difference in tuber desiccation tolerance between these two close species. These findings highlight the lineage-specific evolution of the LEA_5 family, which facilitates further functional analysis and genetic improvement in tigernut and other species.

Keywords: Cyperaceae; desiccation tolerance; late embryogenesis-abundant protein; phylogenomics; underground tuber; vegetative tissue.

Figure 1

Structural and phylogenetic analyses of LEA_5 family genes in C. esculentus. (A) Kyte–Doolittle hydrophobicity plots of CeLEA5 proteins using ProtScale (v1). (B) Amino acid composition of CeLEA5 proteins. (C) Multiple sequence alignment of CeLEA5 proteins using MUSCLE (v5.1). Identical and similar amino acids are highlighted in black or dark grey, respectively, whereas conserved LEA_5 domains are boxed in red. (D) An unrooted phylogenetic tree resulting from full-length Ce/Os/AtLEA5 proteins with RAxML (maximum likelihood method and bootstrap of 1000 replicates), where the distance scale denotes the number of amino acid substitutions per site. The name of each clade (i.e., I and II) is indicated next to the corresponding group. (E) The exon-intron structures. “1” represents the intron phase that is located between the first and second bases of a codon. (F) The distribution of conserved motifs among Ce/Os/AtLEA5 proteins, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif. (At: A. thaliana; Ce: C. esculentus; LEA: Late embryogenesis abundant; Os: Oryza sativa).

Figure 2

Species-specific distribution of five orthogroups in 34 representative plant species. The species tree is referred to NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/taxonomy, accessed on 20 November 2024) and well-established recent WGDs are marked: γ represents the whole-genome triplication event shared by all core eudicots; β and α represent two WGDs that are specific to Brassicaceae; β″ and α″ represent two Araceae-specific WGDs; τ represents the WGD shared by all core monocots; p represents the Arecaceae-specific WGD; σ represents the Poales-specific WGD; and ρ represents the Poaceae-specific WGD. Names of tested plant families are indicated next to the corresponding branches. (LEA: Late embryogenesis abundant; WGD: whole-genome duplication).

Figure 3

Synteny analyses within and between C. esculentus and representative plant species. (A) Chromosomal localization and duplication events of the LEA_5 family genes in C. esculentus and R. breviuscula. (B) Synteny analyses within and between C. esculentus, C. littledalei, C. scoparia, and R. breviuscula. (C) Synteny analyses within and between C. esculentus, J. effusus, S. stoloniferum, A. comosus, and J. ascendens. (D) Synteny analyses within and between C. esculentus, E. guineensis, A. officinalis, and D. alata. (E) Synteny analyses within and between C. esculentus, A. gramineus, A. trichopoda, A. thaliana, and R. communis. (F) Synteny analyses within and between J. ascendens, P. latifolius, O. sativa, and S. bicolor. Shown are LEA_5 gene-encoding chromosomes/scaffolds and only syntenic blocks containing LEA_5 genes are marked, where red and purple lines indicate intra- and inter-species, respectively. The scale is in Mb. (Ac: A. comosus; Ag: A. gramineus; Ao: A. officinalis; At: A. thaliana; Atr: A. trichopoda; Bd: B. distachyon; Ce: C. esculentus; Cl: C. littledalei; Cs: C. scoparia; Da: D. alata; Eg: E. guineensis; Ja: J. ascendens; Je: J. effuses; Mb: megabase; Os: O. sativa; Pl: P. latifolius; Rb: R. breviuscula; Rc: R. communis; Sb: S. bicolor; Ss: S. stoloniferum).

Figure 4

Expression profiles of Ce/CrLEA5 genes. (A) Tissue-specific expression profiles of five CeLEA5 genes. (B) Expression profiles of Ce/CrLEA5 genes at three representative stages of tuber development. (C) Expression profiles of CeLEA5-1, -2, -3, and -4 at different stages of tuber development. The heatmap was generated using the R package (v2) implemented with a row-based standardization. Color scale represents FPKM normalized log₂ transformed counts, where blue indicates low expression and red indicates high expression. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan’s one-way multiple-range post hoc ANOVA (p < 0.01). (Ce: C. esculentus; Cr: C. rotundus; DAI: days after tuber initiation; DAS: days after sowing; FPKM: Fragments per kilobase of exon per million fragments mapped).