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Article

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

by
Zhi Zou
1,*,
Xiaowen Fu
1,
Xiaoping Yi
1,
Chunqiang Li
1,
Jiaquan Huang
1,2,* and
Yongguo Zhao
1,3,*
1
National Key Laboratory for Tropical Crop Breeding/Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions, Institute of Tropical Biosciences and Biotechnology/Sanya Research Institute of Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
2
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication) and College of Tropical Agriculture and Forestry, Hainan University, Sanya 572025, China
3
College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(5), 762; https://doi.org/10.3390/plants14050762
Submission received: 27 December 2024 / Revised: 22 February 2025 / Accepted: 26 February 2025 / Published: 1 March 2025
(This article belongs to the Special Issue Tempo and Mode of Diversification in Plant Evolution)
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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.

1. Introduction

Early methionine-labelled (EM)/D-19 proteins constitute a small family (LEA_5) of late embryogenesis abundant (LEA) proteins, which is defined by the presence of the conserved LEA_5 domain (PF00477) in the Pfam database [1]. This family was first characterized in wheat (Triticum aestivum) embryos [2], and then widely found in all three domains of life, i.e., eukaryotes, bacteria, and archaea [3,4,5]. LEA_5 family proteins are typical for the presence of approximately 20% Gly residues and a preponderance of charged and hydroxylated amino acids (AA), existing largely as random coils in solution and having a potential role in preventing freezing, desiccation, or osmotic stress damage [6,7,8]. In angiosperms, the family is usually present in two members, e.g., rice (Oryza sativa), maize (Zea mays), arabidopsis (Arabidopsis thaliana), papaya (Carica papaya), castor bean (Ricinus communis), and cassava (Manihot esculenta), which preferentially accumulate in embryonic tissues [9,10,11,12,13,14,15]. Their protein accumulation in seeds usually begins late in embryogenesis and the mRNA/protein abundances could be manipulated by abscisic acid (ABA) and osmotic stress [16,17,18]. In arabidopsis, both members (i.e., AtLEA20/EM6 and AtLEA35/EM1) are required for normal seed development, and the atem6-1 mutant was shown to be deficient in maturation drying [19,20]. Whereas AtLEA35 predominantly accumulates in the vascular tissues and the meristem of the embryo, AtLEA20 is expressed throughout the embryo as well as in non-seed organs [10,21,22]. Among two members present in rice, the transcripts of OsLEA21 (EM/EMP1) at the seedling stage could be upregulated by ABA, cold, drought, and osmotic stresses, but downregulated by GA, salt, and flooded standing stresses [12]. Moreover, ABA modulation and seed-specific expression of OsLEA21 was proven to be regulated by OsVP1, an ortholog of Viviparous-1 (VPl) in maize or ABA INSENSITIVE 3 (ABI3) in arabidopsis within the B3 transcription factor family, who works through binding the RY/Sph box or ABA-responsive element (ABRE) present in the promoter region [23,24,25].
Tigernut (Cyperus esculentus L. var. sativus Baeck.) is an oil-bearing tuber plant that belongs to the Cyperaceae family within the monocot clade [26,27,28]. Though it originated in the Mediterranean coast, tigernut is emerging as a novel oil crop widely cultivated in tropical, subtropical, and temperate zones for its potential health benefits, high biomass, and wide adaptability [29,30,31,32]. Unlike other tubers (e.g., potato (Solanum tuberosum)) and tuberous roots (e.g., cassava) that are highly sensitive to desiccation, the water content of mature tigernut tubers can drop to less than 6% without affecting the sprouting capability, which is comparative to orthodox seeds [33,34,35]. Correspondingly, comparative proteomic analysis revealed a seed-like proteome of mature tubers, including significant accumulations of oleosins, caleosins, as well as LEA proteins [34,35,36,37,38]. Among various LEA proteins, only seed maturation protein (SMP) and LEA_1 families have been systematically characterized in tigernut, which were shown to exhibit seed desiccation-like accumulation during tuber maturation [35,38]. Given the essential roles of LEA_5 proteins in desiccation tolerance, in this study, we would like to report a genome-wide identification, evolutionary, and expression analyses of the LEA_5 gene family in tigernut, which revealed the monogenic origin and lineage-specific family evolution in Poales, including the Cyperaceae family. Moreover, our results imply that the desiccation tolerance of tigernut tubers is more likely to be contributed by tuber-specific activation of LEA_5 genes by certain transcription factors different from those of orthodox seeds. These findings will provide valuable information for further functional analyses.

2. Results

2.1. Characterization of Four LEA_5 Family Genes in Tigernut

As shown in Table 1, a total of four genes that encode LEA_5 domain-containing proteins were identified from four scaffolds (Scfs) of the tigernut genome, and all of them were detected in the full-length transcriptome as described before [39], supporting their expression and putative functions. Their CDS (coding sequence) length varies from 252 to 465 bp (base pair), which was predicted to encode 83–154 AA with the MW (molecular weight) of 8.88–16.92 kDa (kilodalton) (Table 1). Though the overall sequence similarity of deduced proteins varies from 36.20% to 59.82% (Table S1), all of them harbor at least one LEA_5 domain, i.e., one for CeLEA5-1 and two for others (Table 1). The theoretical pI (isoelectric point) values were shown to range from 5.22 to 6.02, and the GRAVY (grand average of hydropathicity) values are between −1.313 to −1.582 (Table 1), implying the acidic and hydrophilic features. The hydrophilic feature was also supported by the ProtScale analysis, which revealed similar hydropathicity scales for all four CeLEA5 proteins (Figure 1A). Comparing the AA composition showed that, except for Cys and Trp, other 18 AA were found in at least one of four proteins, which are rich in Gly, Glu, and Arg. Additionally, Phe is only present in CeLEA5-3, whereas Asn and His/Tyr are absent from CeLEA5-1 and -4, respectively (Figure 1B). Compared with CeLEA5-1 and -4, sequence alignment revealed that the longer protein length of CeLEA5-2 and -3 was caused by fragment insertion (Figure 1C).
It is worth noting that the family amounts in tigernut are twice of two present in two well-studied model plants arabidopsis and rice (Table S2), implying species or lineage-specific expansion. To disclose their evolutionary relationships, an unrooted phylogenetic tree was constructed using full-length protein sequences of eight LEA_5 genes in these three species. As shown in Figure 1D, these proteins were clustered into two main groups. Notably, AtLEA20 and -35, which share 53.59% sequence similarity, were clustered in species in Group I. By contrast, OsLEA21 was clustered with CeLEA5-2, exhibiting 72.32% sequence similarity, which is slightly smaller than 77.89% observed between OsLEA21 and CeLEA5-1. Group II includes CeLEA5-4 and OsLEA20, which exhibit 61.70% sequence similarity (Table S1).
To uncover possible structural variation, gene structures and conserved motifs were further compared. In contrast to the invariable exon-intron structure with a single intron in phase 1 (Figure 1E), our MEME analysis revealed distinct motif composition among Ce/Os/AtLEA5 proteins. Among the 10 motifs identified as shown in Figure 1F, Motifs 1 and 4 are highly conserved and shared by all sequences. Whereas Motif 4 is present in a single copy, Motif 1 appears in three copies in both CeLEA5-3 and AtLEA35, implying tandem duplication. Motifs 2, 3, and 5 are also widely distributed, which are only absent from CeLEA5-1/OsLEA20, CeLEA5-4/OsLEA20, and CeLEA5-3/CeLEA5-4/OsLEA20, respectively. Like Motif 1, Motif 3 appears in two copies in both CeLEA5-3 and AtLEA35. By contrast, other motifs are sequence-specific. Whereas Motif 9 is confined to OsLEA20 (in two copies), Motifs 6, 7, 8, and 10 are only present in CeLEA5-2/OsLEA20, CeLEA5-4/OsLEA21, CeLEA5-1/-4, and CeLEA5-1/OsLEA20, respectively (Figure 1F). Notably, except for Motifs 6, 7, 8, and 9, other motifs belong to the LEA_5 domain (Figure 1F), implying possible functional divergence.

2.2. Characterization of LEA_5 Genes from Representative Plant Species and Insights into Lineage-Specific Family Evolution

Since the origin and evolution of CeLEA5 genes were not well resolved by above phylogenetic analysis, a similar approach was also used to identify homologs from 33 representative plant species, which include the basal angiosperm Amborella trichopoda, four core eudicots, 25 core monocots, and three early diverged monocots that did not experience the τ WGD, i.e., Acorus gramineus, eelgrass (Zostera marina), and duckweed (Spirodela polyrhiza) [40,41]. As shown in Table S2, a single member was not only identified in A. trichopoda, a rare example without any recent whole-genome duplication (WGD), but also in eelgrass, duckweed, apostasia (Dendrobium catenatum), and Dioscorea alata, though they were proven to have undergone at least one recent WGD after monocot radiation [40,41,42]. By contrast, 2–6 members were found in other species (Table S2), implying the monogenic origin of the LEA_5 family followed by lineage-specific expansion.
To infer lineage-specific evolution, orthologous genes among different species were clustered using Orthofinder [43]. As shown in Figure 2 and Table S3, a total of five orthogroups were identified. Whereas LEA5a is shared by both monocots and eudicots, LEA5b/LEA5c/LEA5d and LEA5e are specific to monocots and eudicots, respectively. LEA_5 family genes in A. trichopoda, A. gramineus, eelgrass, duckweed, garden asparagus (Asparagus officinalis), and oil palm (Elaeis guineensis) belong to LEA5a, which also includes CeLEA5-1 and OsLEA21 (Table S3), though OsLEA21 was grouped with CeLEA5-2 in the phylogenetic tree as shown in Figure 1D. LEA5b and LEA5d appear to be Poales-specific, whereas LEA5c seems to be confined to Cyperaceae and Juncaceae (Table S3).
To gain insights into the origin of LEA_5 genes, species-specific duplication events were further examined. As shown in Figure 3A, no syntenic relationship was observed between CeLEA5 genes. Instead, CeLEA5-2/-3 and -4 were characterized as dispersed repeats of CeLEA5-1 and -3, respectively, which is similar to that observed in Rhynchospora breviuscula, another Cyperaceae plant. Further interspecific synteny analyses revealed that all CeLEA5 genes have syntelogs in at least one out of 32 species tested in this study, which includes papaya, castor bean, cassava, A. gramineus, and duckweed. Significantly, 1:1 syntenic relationships were observed between tigernut and R. breviuscula, providing direct evidence of early divergence into four groups before Cyperaceae radiation (Figure 3B). Moreover, CeLEA5-1 harbors syntelogs not only in Juncus effuses (a Juncaceae plant within Poales), Sparganium stoloniferum (a Typhaceae plant within Poales), and Joinvillea ascendens (a Joinvilleaceae plant within Poales) (Figure 3C), but also in oil palm, garden asparagus (Figure 3D), A. gramineus, and castor bean (Figure 3E), whereas CeLEA5-2 and -3 have syntelogs in S. stoloniferum/duckweed and J. effuses, respectively (Figure 3C,D). The location of SsLEA5-1, -2, and -3 within syntenic blocks provides direct evidence of LEA5b from LEA5a via WGD, most likely the σ event shared by all Poales plants [44], followed by species-specific expansion via WGD in S. stoloniferum [45]. Notably, though no syntelog was identified for DaLEA5-1 in tigernut, D. alata exhibits 1:1, 1:2, with 1:3 syntenic relationships with duckweed, J. effuses/J. ascendens, and S. stoloniferum, respectively, providing direct evidence of LEA5c from LEA5a via WGD (Figure S1A). Interestingly, in contrast to no syntelog that was identified for all four CeLEA5 genes in A. trichopoda and arabidopsis, A. trichopoda exhibits 1:1 and 1:2 syntenic relationships with arabidopsis/castor bean and A. gramineus (Figure 3E). Moreover, though AtLEA20 and -35 were characterized as dispersed repeats, both of them were shown to be located within syntenic blocks with CpLEA5-1, CpLEA5-2, RcLEA5-1, RcLEA5-2, MeLEA5-1, and MeLEA5-2 (Figure S1B), implying their WGD-derivation followed by species-specific chromosome rearrangement. Interestingly, in contrast to no syntelog that was identified for CeLEA5 genes in all tested Poaceae species, both JaLEA5-1 and -3 were shown to have syntelogs in these species, e.g., Pharus latifolius, rice, and sorghum (Sorghum bicolor). Additionally, tandem duplication was also shown to play a key role in gene expansion of the LEA_5 family in Poales, e.g., J. effuses, J. ascendens, Brachypodium distachyon, barley (Hordeum vulgare), foxtail millet (Setaria italica), and sorghum (Table S2). Notably, despite the occurrence of one additional WGD after the split with sorghum [46], maize has two LEA5a members that were characterized as proximal repeats (Table S2).

2.3. CeLEA5 Genes Have Evolved to Be Preferentially Expressed in Tigernut Tubers

To provide a global view of the expression evolution of LEA_5 genes, the tissue-specific expression profiles were first mined from the Plant Public RNA-seq Database, which includes 28,164, 11,726, and 19,664 libraries for arabidopsis, rice, and maize, respectively. As shown in Figure S2, all of them exhibit a seed/embryo-preferential expression pattern.
Global expression profiles of CeLEA5 genes were examined in seven main tissues/developmental stages, i.e., shoot apex, young leaf, mature leaf, leaf sheath, root, rhizome, and tuber. Interestingly, all four CeLEA5 genes were shown to be predominantly expressed in tubers (Figure 4A), which represent the maturation stage of 120 days after sowing (DAS). Among them, CeLEA5-1 transcripts were most abundant, followed by CeLEA5-4 and -2, and least for CeLEA5-3. Both CeLEA5-1 and -4 were barely expressed in other tissues, whereas CeLEA5-2 and -3 were also expressed in shoot apexes and/or rhizomes, though their transcript levels were considerably lower than those in tubers (Figure 4A). The results support expression and possible functional divergence of CeLEA5 genes and between paralogs.

2.4. CeLEA5 Genes Were Expressed More than Their Orthologs in C. rotundus

The absence of a CeLEA5-1 (the dominant member) ortholog in the tuber transcriptome assembly of purple nutsedge (C. rotundus) (Table S2), a desiccation-sensitive tuber plant close to tigernut, implies a possible less important role of LEA_5 genes in the tuber development of this species. To test this hypothesis, expression profiles of LEA_5 genes in three representative stages of tuber development were compared, i.e., 20, 50, and 90 DAS, which represent tuber initiation, swelling, and maturation, respectively. As shown in Figure 4B, except for CrLEA5-3, the transcripts of the other two CrLEA5 genes were rarely detected. Moreover, CrLEA5-3 transcripts were considerably lower than those of all four CeLEA5 genes (Figure 4B), implying species-specific activation of LEA_5 genes in tigernut tubers, especially at the maturation stage.

2.5. Transcripts of Most CeLEA5 Genes Were Gradually Upregulated During Tuber Development

To learn more about the expression profiles of CeLEA5 genes during tuber development, five typical stages were examined using qRT-PCR, i.e., 1, 10, 20, 25, and 35 days after tuber initiation (DAI), which represent initiation, early swelling, middle swelling, late swelling, and maturation, respectively [28]. The moisture content of the first three stages was characterized as approximately 85%, and the two latter stages were about 75% and 48%, respectively [35]. As shown in Figure 4C, a gradual increase in transcripts during tuber development was observed for CeLEA5-1, -2, and -3, though no significant difference was observed between the two early stages, i.e., 10 vs. 1 DAI. By contrast, CeLEA5-4 transcripts were gradually downregulated at three swelling stages, but also peaked at the maturation stage as other members (Figure 4C), implying their putative roles in the acquisition of desiccation tolerance.

3. Discussion

Desiccation tolerance, an ancient adaptation trait appearing very early in the evolution of terrestrial life, is usually present in spores, pollen, and seeds of vascular plants [47]. The underlying mechanism is usually associated with significant accumulation of LEA proteins, a class of small and extremely hydrophilic proteins that were first discovered in cotton (Gossypium hirsutum) seeds [22]. Among them, LEA_5 domain-containing proteins constitute a small family (usually in two copies) that are essential for stress responses as well as desiccation tolerance and dormancy in orthodox seeds [19,20,22,48]. However, their roles in non-seed storage organs are largely unknown.

3.1. The Tigernut Genome Encodes a High Number of Four LEA_5 Genes, and the Family Expansion Was Contributed by Dispersed Duplication

Tigernut is a unique plant producing desiccation-tolerant tubers, which differs from its relative purple nutsedge, another Cyperaceae plant bearing desiccation-sensitive tubers [34,38]. In this study, the first genome-wide characterization of the LEA_5 gene family was conducted in tigernut, and a high number of four members with one to two conserved LEA_5 domains were obtained. Interestingly, all of them were detected in transcriptomes from both Illumina RNA-seq and PacBio Single-Molecule Real-Time (SMRT) sequencing [39]. By contrast, only three members corresponding to CeLEA5-2, -3, and -4 were identified from the transcriptome de novo assembled from Illumina RNA-seq of purple nutsedge tubers [36]. Notably, similar to arabidopsis and rice, four CeLEA5 genes were also shown to be dispersed repeats.

3.2. Comparative Genomics Analysis Reveals the Monogenic Origin and Lineage-Specific Evolution of the LEA_5 Family in Poales

Tigernut resides in the Cyperaceae family within the Poales order, which also includes the well-known Poaceae family [27,49]. It was proven that after the monocot-eudicot split, all core eudicots shared one so-called γ WGT (whole-genome triplication), whereas all monocots with the exception of Acorales and Alismatales plants (also called core monocots) experienced the τ WGD [44,50]. Furthermore, Poales plants underwent one order-specific σ WGD, and all Poaceae plants shared the ρ WGD [44].
To learn more about the origin and evolution of CeLEA5 genes, a total of 82 homologs were further identified from 33 representative plant species, which belong to 17 plant families, i.e., Amborellaceae (1), Brassicaceae (1), Caricaceae (1), Euphorbiaceae (2), Acoraceae (1), Zosteraceae (1), Araceae (1), Asparagaceae (1), Orchidaceae (1), Dioscoreaceae (1), Arecaceae (1), Bromeliaceae (2), Typhaceae (2), Cyperaceae (7), Juncaceae (2), Joinvilleaceae (1), and Poaceae (7). The data cover the majority of genome-available plant families within the lineage of core monocots, where Acoraceae/Zosteraceae/Araceae (early diverged monocots), Brassicaceae/Caricaceae/Euphorbiaceae (core eudicots), and Amborellaceae (the basal angiosperm) were used as out-groups for core monocots, monocots, and core angiosperms, respectively. A single member found in A. trichopoda, eelgrass (an early diverged monocot in Alismatales), and duckweed (another early diverged monocot in Alismatales) implies the monogenic origin of this gene family in angiosperms. Correspondingly, LEA_5 genes in these species could be assigned into the same orthogroup, i.e., LEA5a. Moreover, though A. gramineus (an early diverged monocot in Acorales) has two members, both of which belong to LEA5a and were characterized as recent WGD repeats, which is similar to oil palm whose family expansion was contributed by the Arecaceae-specific p WGD [51]. By contrast, four CeLEA5 genes were assigned into four out of five orthogroups identified in this study, i.e., LEA5a, LEA5b, LEA5c, and LEA5d. Unlike LEA5a, which is shared by both monocots and eudicots, LEA5e is specific to eudicots, whereas the other three groups appear to be monocot-specific. More exactly, both LEA5b and LEA5d are more likely to be Poales-specific, while LEA5c is limited to Cyperaceae and Juncaceae. Though the origin of LEA5d was not well resolved, both LEA5b and LEA5c are more likely to arise from WGD, because DaLEA5-1, SsLEA5-1, SsLEA5-2, SsLEA5-3, JaLEA5-1, JaLEA5-2, JeLEA5-1, and JeLEA5-4 are still located within syntenic blocks. Whereas LEA5b is more likely to be generated by the σ WGD, LEA5c may be derived from the WGD as described in C. littledalei [52]. That means that both CeLEA5-2 and -3 are more likely to arise from CeLEA5-1 via WGD followed by chromosome rearrangement. Since no syntenic relationship was observed between LEA_5 genes present in all species examined in both Cyperaceae and Juncaceae, the rearrangement is more likely to occur sometime before the split between these two families. On the contrary, the rearrangement between AtLEA20 and -35 may be species-specific, since their orthologs in papaya, castor bean, and cassava are still located within syntenic blocks.
Besides WGD, local duplication such as tandem and proximal duplications also played a role in the family expansion, especially in Poaceae plants, varying from two to five copies. Additionally, gene contraction was also frequently observed and a good example is the Juncaceae family. Among two species examined in this family, J. effusus has four members that belong to LEA5a (1), LEA5b (2), and LEA5c (1), whereas Luzula sylvatica harbors only two members that belong to LEA5a (1) and LEA5d (1). However, the biological significance needs to be further studied.

3.3. CeLEA5 Genes Underwent Apparent Expression and Functional Divergence

Seed-preferential expression of LEA_5 genes has been documented in a high number of plant species, including wheat, maize, rice, barley, arabidopsis, castor bean, and papaya [9,10,11,13,14,22]. Correspondingly, our large-scale transcriptional profiling conducted in arabidopsis, rice, and maize indeed supports a seed/embryo-predominant expression pattern of LEA_5 genes. By contrast, four CeLEA5 genes were shown to have evolved to be predominantly expressed in the tigernut tubers, implying their neofunctionalization in vegetative tissues. Interestingly, their orthologs in purple nutsedge showed low expression in the tuber transcriptomes of three representative developmental stages, i.e., 20, 50, and 90 DAS. Since purple nutsedge tubers are highly sensitive to desiccation [34], species rather than tuber-specific activation of LEA_5 genes in tigernut could be speculated. The distinct patterns may explain the difference in tuber desiccation tolerance between these two close species. Moreover, during tuber development, except for CeLEA5-4, gradual upregulation of other CeLEA5 genes since 10 DAI was observed, which is negatively correlating with the dynamics of water content [27,35]. Sharp transcript increase for all four CeLEA5 genes during tuber maturation is highly similar to that observed in orthodox seeds, which is accompanied by the acquisition of desiccation tolerance and dormancy [20,47]. It has been suggested that LEA_5 proteins could replace water and thus protect the embryo during the phase of desiccation [53]. A similar role of CeLEA5 proteins in tigernut tubers could be speculated. Further gene knockout and overexpression of CeLEA5 genes in purple nutsedge and potato tubers may provide more evidence. Additionally, despite the central role of ABI3/VP1 in regulating LEA_5 genes in seeds [23,24,25,54], a distinct regulatory mechanism may be present for CeLEA5 genes, since no evidence is available for the expression of an ABI3/VP1 homolog (CESC_01781) in tigernut tubers as well as other tissues with available transcriptome data. Moreover, we could not isolate its mRNA by using the PCR technology. Thereby, further identifying transcription factors that mediate tuber-specific activation of CeLEA5 genes is of particular interest.

4. Conclusions

This study presents the first genome-scale analysis of the LEA_5 gene family in tigernut, a Cyperaceae plant producing desiccation-tolerant tubers. Identification and comparison of 86 members from 34 plant species representing 17 plant families support the monogenic origin in angiosperms and lineage-specific family evolution in Poales. Though four CeLEA5 genes were characterized as dispersed repeats, they belong to four out of five orthogroups, which were derived from WGD (LEA5b, LEA5c, and LEA5e) and dispersed duplication (LEA5d). Whereas LEA5e is specific to eudicots, LEA5b and LEA5d appear to be Poales-specific, and LEA5c is limited to Cyperaceae and Juncaceae. In contrast to seed/embryo-preferential expression of LEA_5 genes in other species, CeLEA5 genes have evolved to be predominantly expressed in tubers, exhibiting seed desiccation-like accumulation during tuber maturation. These findings highlight the lineage-specific evolution of the LEA_5 family and putative roles of CeLEA5 genes in acquiring desiccation tolerance of tigernut tubers, which facilitates further functional analysis and genetic improvement in tigernut and other species.

5. Materials and Methods

5.1. Identification of LEA_5 Family Genes from Datasets

Genome sequences of representative plant species were downloaded from Genome Warehouse (https://ngdc.cncb.ac.cn/gwh/, accessed on 20 November 2024), TAIR11 (https://www.arabidopsis.org/, accessed on 20 November 2024), Phytozome v13 (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 20 November 2024), and NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 20 November 2024): A. trichopoda (v2.1), A. thaliana (Araport11), C. papaya (Sunset), R. communis (WT05), M. esculenta (v8), A. gramineus (v1), Z. marina (v3.1), S. polyrhiza (v2), D. alata (v1), D. catenatum (v1), A. officinalis (v1.1), E. guineensis (v3), A. comosus (v3), Puya raimondii (v1), S. stoloniferum (v1), Typha latifolia (v1), O. sativa (v7.0), H. vulgare (Morex V3), B. distachyon (v3.2), S. italica (v2.2), S. bicolor (v5.1s), Z. mays (RefGen_V4), C. esculentus (v1), C. littledalei (v1), C. breviculmis (v1), C. scoparia (v1), S. tabernaemontani (v1), and B. planiculmis (v1). Transcriptome data of tigernut were accessed from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 20 November 2024), whereas for purple nutsedge, the de novo assembled transcriptome described before [36] was adopted. To identify LEA_5 family genes, HMMER (v3.3.2, http://hmmer.janelia.org/, accessed on 20 November 2024) searches were performed using the Pfam profile PF03760 (v35.0, http://pfam.xfam.org/, accessed on 20 November 2024). Gene models of candidates were manually revised with mRNAs when available, whereas gene structures were displayed using GSDS 2.0 [55]. The presence of the conserved LEA_5 domain in deduced proteins was confirmed using Pfam Search (http://pfam.xfam.org/, accessed on 20 November 2024), and biochemical parameters were calculated using ProtParam (http://web.expasy.org/protparam/, accessed on 20 November 2024). Expression data of arabidopsis, rice, and maize were accessed from the Plant Public RNA-seq Database (http://plantrnadb.com/, accessed on 20 November 2024).

5.2. Phylogenetic and Conserved Motif Analyses

Multiple sequence alignments were carried out using Muscle v5.1 [56], and phylogenetic tree construction was performed using RAxML (http://www.phylo.org/portal2/home.action#, accessed on 20 November 2024) with the maximum likelihood method and bootstrap of 1000 replicates. Conserved motifs were identified using MEME (v5.4.1, http://meme-suite.org/tools/meme, accessed on 20 November 2024) with the parameters as follows: any number of repetitions; the maximum number of motifs, 10; and, the optimum width of each motif, between 5 and 50 residues.

5.3. Synteny Analysis and Definition of Orthogroups

Orthologous genes were clustered using Orthofinder (v2.3.8) [43]. Synteny analysis was conducted as previously described [57], and gene duplication modes were identified using the DupGen_finder pipeline [58].

5.4. Plant Materials

The growing conditions of tigernut plants (Reyan3) were as previously described [27]. Tubers were collected at 1, 10, 20, 25, and 35 DAI, which represent tuber initiation, three stages of swelling (early, middle, and late), and maturation as described before [28]. Once collected, samples with three biological replicates were frozen with liquid nitrogen and stored at −80 °C for further use.

5.5. Gene Expression Analysis Based on RNA-Seq

Global expression profiles of Ce/CrLEA5 genes were analyzed using the Illumina RNA-seq datasets PRJNA703731 and PRJNA671562, which are 150 bp paired-end reads with three biological replicates. Except for mature tubers that were collected at 120 DAS, other tissues (i.e., shoot apex, young leaf, mature leaf, sheath of mature leaf, root, and rhizome) were collected at 85 DAS. Different stages of tuber development in tigernut and purple nutsedge were collected at 20, 50, and 90 DAS, respectively. Quality control of raw RNA-seq reads and subsequent read mapping were conducted as previously described [48], and the relative gene expression level was presented as FPKM [59].

5.6. Gene Expression Analysis Based on qRT-PCR

Total RNA extraction, integrity and concentration detection, and synthesis of the first-strand cDNA were carried out as described before [27,35]. Primers used for qRT-PCR analysis are shown in Table S3, where CeTIP41 and CeUCE2 [27] are two reference genes. PCR reaction, relative gene abundance calculation, and statistical analysis were conducted as previously described [60], where differences among means were tested following Duncan’s one-way multiple-range post hoc ANOVA in Data Processing System software v20.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14050762/s1, Figure S1: Synteny analyses within and between between D. alata/J. effuses/S. stoloniferum/J. ascendens (A) and A. thaliana/C. papaya/R. communis/M. esculenta (B). Shown are LEA_5 gene-encoding chromosomes/scaffolds and only syntenic blocks containing LEA_5 genes are marked, where red and purple lines for intra- and inter-species, respectively. The scale is in Mb. (At: A. thaliana; Cp: C. papaya; Da: D. alata; Ja: J. ascendens; Je: J. effuses; LEA: late embryogenesis-abundant; Mb: megabase; Me: M. esculenta; Rc: R. communis; Ss: S. stoloniferum). Figure S2: Global expression profiles of AtLEA5, OsLEA5, and ZmLEA5 genes. (At: A. thaliana; LEA: late embryogenesis-abundant; Os: O. sativa; Zm: Z. mays). Table S1: Percent similarity within and between CeLEA5s, OsLEA5s, and AtLEA5s. (At: A. thaliana; Ce: C. esculentus; LEA: late embryogenesis-abundant; Os: O. sativa). Table S2: LEA_5 family genes identified in representative plant species. (AA: amino acid; Ac: A. comosus; Ag: A. gramineus; Ao: Asparagus officinalis; At: A. thaliana; Atr: A. trichopoda; Bd: B. distachyon; Bp: B. planiculmis; Cb: C. breviculmis; Ce: C. esculentus; Chr: chromosome; Cl: C. littledalei; Cr: C. rotundus; Cs: C. scoparia; Eg: Elaeis guineensis; Hv: H. vulgare; Ja: J. ascendens; Je: J. effusus; LEA: Late embryogenesis abundant; Ls: L. sylvatica; Pl: P. latifolius; Pr: Puya raimondii; Rb: R. breviuscula; Scf: scaffold; Sb: S. bicolor; Si: S. italica; Sp: Spirodela polyrhiza; Ss: S. stoloniferum; St: S. tabernaemontani; Tl: T. latifolia; WGD: whole-genome duplication; Zm: Zea mays). Table S3: Species-specific distribution of LEA_5 genes in five orthogroups identified in this study. (LEA: late embryogenesis-abundant). Table S4: Primers used in this study. (Ce: C. esculentus; LEA: late embryogenesis-abundant).

Author Contributions

Z.Z. conceived the idea and designed the project outline. Z.Z., X.F., X.Y., C.L., J.H., and Y.Z. performed the experiments and analyzed the data. Z.Z., J.H., and Y.Z. prepared and refined the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Hainan Province Science and Technology Special Fund (ZDYF2024XDNY171 and ZDYF2024XDNY156), the National Natural Science Foundation of China (32460342 and 31971688), and the Project of National Key Laboratory for Tropical Crop Breeding (NKLTCB202325). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability Statement

Transcriptome data used in this study is under the NCBI accession numbers PRJNA703731 and PRJNA671562.

Acknowledgments

The authors appreciate those contributors who make the related genome and transcriptome data accessible in public databases.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, D412–D419. [Google Scholar] [CrossRef] [PubMed]
  2. Cuming, A.C.; Lane, B.G. Protein synthesis in imbibing wheat embryos. Eur. J. Biochem. 1979, 99, 217–224. [Google Scholar] [CrossRef] [PubMed]
  3. Baker, J.; Van Dennsteele, C.; Dure, L., III. Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant Mol. Biol. 1988, 11, 277–291. [Google Scholar] [CrossRef] [PubMed]
  4. Campos, F.; Cuevas-Velazquez, C.; Fares, M.A.; Reyes, J.L.; Covarrubias, A.A. Group 1 LEA proteins, an ancestral plant protein group, are also present in other eukaryotes, and in the archeae and bacteria domains. Mol. Genet. Genomics 2013, 288, 503–517. [Google Scholar] [CrossRef]
  5. Artur, M.A.S.; Zhao, T.; Ligterink, W.; Schranz, E.; Hilhorst, H.W. Dissecting the genomic diversification of late embryogenesis abundant (LEA) protein gene families in plants. Genome Biol. Evol. 2019, 11, 459–471. [Google Scholar] [CrossRef]
  6. McCubbin, W.D.; Kay, C.M.; Lane, B.G. Hydrodynamic and optical properties of the wheat-germ Em protein. Can. J. Biochem. Cell Biol. 1985, 63, 803–811. [Google Scholar] [CrossRef]
  7. Russouw, P.S.; Farrant, J.; Brandt, W.; Lindsey, G.G. The most prevalent protein in a heat-treated extract of pea (Pisum sativum) embryos is an LEA group I protein; its conformation is not affected by exposure to high temperature. Seed Sci. Res. 1997, 7, 117–123. [Google Scholar] [CrossRef]
  8. Soulages, J.L.; Kim, K.; Walters, C.; Cushman, J.C. Temperature-induced extended helix/random coil transitions in a group 1 late embryogenesis-abundant protein from soybean. Plant Physiol. 2002, 128, 822–832. [Google Scholar] [CrossRef]
  9. Williams, M.E.; Tsang, A. A maize gene expressed during embryogenesis is abscisic acid-inducible and highly conserved. Plant Mol. Biol. 1991, 16, 919–923. [Google Scholar] [CrossRef]
  10. Gaubier, P.; Raynal, M.; Hull, G.; Huestis, G.M.; Grellet, F.; Arenas, C.; Pages, C.; Delseny, M. Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation. Mol. Gen. Genet. 1993, 238, 409–418. [Google Scholar] [CrossRef]
  11. Stacy, R.A.; Espelund, M.; Saebøe-Larssen, S.; Hollung, K.; Helliesen, E.; Jakobsen, K.S. Evolution of the Group 1 late embryogenesis abundant (Lea) genes: Analysis of the Lea B19 gene family in barley. Plant Mol. Biol. 1995, 28, 1039–1054. [Google Scholar] [CrossRef] [PubMed]
  12. Wang, X.S.; Zhu, H.B.; Jin, G.L.; Liu, H.L.; Wu, W.R.; Zhu, J. Genome-scale identification and analysis of LEA genes in rice (Oryza sativa L.). Plant. Sci. 2007, 172, 414–420. [Google Scholar] [CrossRef]
  13. Zou, Z.; Huang, Q.X.; An, F. Genome-wide identification, classification and phylogenetic analysis of LEA gene family in castor bean (Ricinus communis L.). Chin. J. Oil Crop. Sci. 2013, 35, 637–643. [Google Scholar]
  14. Zou, Z.; Guo, J.Y.; Zheng, Y.J.; Xiao, Y.H.; Guo, A.P. Genomic analysis of LEA genes in Carica papaya and insight into lineage-specific family evolution in Brassicales. Life 2022, 12, 1453. [Google Scholar] [CrossRef]
  15. Wu, C.; Hu, W.; Yan, Y.; Tie, W.; Ding, Z.; Guo, J.; He, G. The late embryogenesis abundant protein family in cassava (Manihot esculenta Crantz): Genome-wide characterization and expression during abiotic stress. Molecules 2018, 23, 1196. [Google Scholar] [CrossRef]
  16. Morris, P.C.; Kumar, A.; Bowles, D.J.; Cuming, A.C. Osmotic stress and abscisic acid induce expression of the wheat Em genes. Eur. J. Biochem. 1990, 190, 625–630. [Google Scholar] [CrossRef]
  17. Espelund, M.; Saebøe-Larssen, S.; Hughes, D.W.; Galau, G.A.; Larsen, F.; Jakobsen, K.S. Late embryogenesis-abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress. Plant J. 1992, 2, 241–252. [Google Scholar] [CrossRef]
  18. Miyoshi, K.; Kagaya, Y.; Ogawa, Y.; Nagato, Y.; Hattori, T. Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice. Plant Cell Physiol. 2002, 43, 307–313. [Google Scholar] [CrossRef]
  19. Manfre, A.J.; Lanni, L.M.; Marcotte, W.R. The Arabidopsis group 1 LATE EMBRYOGENESIS ABUNDANT protein ATEM6 is required for normal seed development. Plant Physiol. 2006, 140, 140–149. [Google Scholar] [CrossRef]
  20. Manfre, A.J.; LaHatte, G.A.; Climer, C.R.; Marcotte, W.R. Seed dehydration and the establishment of desiccation tolerance during seed maturation is altered in the Arabidopsis thaliana mutant atem6-1. Plant Cell Physiol. 2009, 50, 243–253. [Google Scholar] [CrossRef]
  21. Vicient, C.M.; Hull, G.; Guilleminot, J.; Devic, M.; Delseny, M. Differential expression of the Arabidopsis genes coding for Em-like proteins. J. Exp. Bot. 2000, 51, 1211–1220. [Google Scholar] [CrossRef] [PubMed]
  22. Hundertmark, M.; Hincha, D.K. LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genom. 2008, 9, 118. [Google Scholar] [CrossRef] [PubMed]
  23. McCarty, D.R.; Hattori, T.; Carson, C.B.; Vasil, V.; Lazar, M.; Vasil, I.K. The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell 1991, 66, 895–905. [Google Scholar] [CrossRef]
  24. Giraudat, J.; Hauge, B.M.; Valon, C.; Smalle, J.; Parcy, F.; Goodman, H.M. Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 1992, 4, 1251–1261. [Google Scholar] [CrossRef]
  25. Hattori, T.; Terada, T.; Hamasuna, S. Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: Analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1. Plant J. 1995, 7, 913–925. [Google Scholar] [CrossRef]
  26. Zou, Z.; Xiao, Y.; Zhang, L.; Zhao, Y. Analysis of Lhc family genes reveals development regulation and diurnal fluctuation expression patterns in Cyperus esculentus, a Cyperaceae plant. Planta 2023, 257, 59. [Google Scholar] [CrossRef]
  27. Zou, Z.; Zheng, Y.J.; Xiao, Y.H.; Liu, H.Y.; Huang, J.Q.; Zhao, Y.G. Molecular insights into PIP aquaporins in tigernut (Cyperus esculentus L.), a Cyperaceae tuber plant. Tropical Plants 2024, 3, e027. [Google Scholar] [CrossRef]
  28. Zou, Z.; Fu, X.; Li, C.; Yi, X.; Huang, J.; Zhao, Y. Insights into the stearoyl-acyl carrier protein desaturase (SAD) family in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant. Plants 2025, 14, 584. [Google Scholar] [CrossRef]
  29. Makareviciene, V.; Gumbytea, M.; Yunik, A. Opportunities for the use of chufa sedge in biodiesel production. Ind. Crop. Prod. 2013, 50, 633–637. [Google Scholar] [CrossRef]
  30. Codina-Torrella, I.; Guamis, B.; Trujillo, A.J. Characterization and comparison of tiger nuts (Cyperus esculentus L.) from different geographical origin. Ind. Crop. Prod. 2015, 65, 406–414. [Google Scholar] [CrossRef]
  31. Yang, X.; Niu, L.; Zhang, Y.; Ren, W.; Yang, C.; Yang, J.; Xing, G.; Zhong, X.; Zhang, J.; Slaski, J.; et al. Morpho-agronomic and biochemical characterization of accessions of tiger nut (Cyperus esculentus) grown in the north temperate zone of China. Plants 2022, 11, 923. [Google Scholar] [CrossRef] [PubMed]
  32. Yu, Y.; Lu, X.; Zhang, T.; Zhao, C.; Guan, S.; Pu, Y.; Gao, F. Tiger nut (Cyperus esculentus L): Nutrition, processing, function and applications. Foods 2022, 11, 601. [Google Scholar] [CrossRef]
  33. Turesson, H.; Marttila, S.; Gustavsson, K.E.; Hofvander, P.; Olsson, M.E.; Bülow, L.; Stymne, S.; Carlsson, A.S. Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues. Am. J. Bot. 2010, 97, 1884–1893. [Google Scholar] [CrossRef]
  34. Niemeyer, P.W.; Irisarri, I.; Scholz, P.; Schmitt, K.; Valerius, O.; Braus, G.H.; Herrfurth, C.; Feussner, I.; Sharma, S.; Carlsson, A.S.; et al. A seed-like proteome in oil-rich tubers. Plant J. 2022, 112, 518–534. [Google Scholar] [CrossRef]
  35. Zou, Z.; Zhao, Y.; Zhang, L.; Xiao, Y.; Guo, A. Analysis of Cyperus esculentus SMP family genes reveals lineage-specific evolution and seed desiccation-like transcript accumulation during tuber maturation. Ind. Crop. Prod. 2022, 187, 115382. [Google Scholar] [CrossRef]
  36. Zou, Z.; Zheng, Y.J.; Zhang, Z.T.; Xiao, Y.H.; Xie, Z.N.; Chang, L.L.; Zhang, L.; Zhao, Y.G. Molecular characterization oleosin genes in Cyperus esculentus, a Cyperaceae plant producing oil in underground tubers. Plant Cell Rep. 2023, 42, 1791–1808. [Google Scholar] [CrossRef]
  37. Zou, Z.; Fu, X.W.; Huang, J.Q.; Zhao, Y.G. Molecular characterization of CeOLE6, a diverged SH oleosin gene, preferentially expressed in Cyperus esculentus tubers. Planta 2024, 260, 122. [Google Scholar] [CrossRef]
  38. Zhao, Y.; Fu, X.; Zou, Z. Insights into genes encoding LEA_1 domain-containing proteins in Cyperus esculentus, a desiccation-tolerant tuber plant. Plants 2024, 13, 2933. [Google Scholar] [CrossRef]
  39. Zou, Z.; Zhao, Y.G.; Zhang, L.; Kong, H.; Guo, Y.L.; Guo, A.P. Single-molecule real-time (SMRT)-based full-length transcriptome analysis of tigernut (Cyperus esculentus L.). Chin. J. Oil Crop Sci. 2021, 43, 229–235. [Google Scholar] [CrossRef]
  40. Wang, W.; Haberer, G.; Gundlach, H.; Gläßer, C.; Nussbaumer, T.; Luo, M.C.; Lomsadze, A.; Borodovsky, M.; Kerstetter, R.A.; Shanklin, J.; et al. The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat. Commun. 2014, 5, 3311. [Google Scholar] [CrossRef]
  41. Olsen, J.L.; Rouzé, P.; Verhelst, B.; Lin, Y.C.; Bayer, T.; Collen, J.; Dattolo, E.; De Paoli, E.; Dittami, S.; Maumus, F.; et al. The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea. Nature 2016, 530, 331–335. [Google Scholar] [CrossRef] [PubMed]
  42. Bredeson, J.V.; Lyons, J.B.; Oniyinde, I.O.; Okereke, N.R.; Kolade, O.; Nnabue, I.; Nwadili, C.O.; Hřibová, E.; Parker, M.; Nwogha, J.; et al. Chromosome evolution and the genetic basis of agronomically important traits in greater yam. Nat. Commun. 2022, 13, 2001. [Google Scholar] [CrossRef] [PubMed]
  43. Emms, D.M.; Kelly, S. OrthoFinder: Phylogenetic orthology inference for comparative genomics. Genome Biol. 2019, 20, 238. [Google Scholar] [CrossRef] [PubMed]
  44. Jiao, Y.; Li, J.; Tang, H.; Paterson, A.H. Integrated syntenic and phylogenomic analyses reveal an ancient genome duplication in monocots. Plant Cell 2014, 26, 2792–2802. [Google Scholar] [CrossRef]
  45. Zou, Y.; Wei, Z.; Xiao, K.; Wu, Z.; Xu, X. Genomic analysis of the emergent aquatic plant Sparganium stoloniferum provides insights into its clonality, local adaptation and demographic history. Mol. Ecol. Resour. 2023, 23, 1868–1879. [Google Scholar] [CrossRef]
  46. Schnable, P.S.; Ware, D.; Fulton, R.S.; Stein, J.C.; Wei, F.; Pasternak, S.; Liang, C.; Zhang, J.; Fulton, L.; Graves, T.A.; et al. The B73 maize genome: Complexity, diversity, and dynamics. Science 2009, 326, 1112–1115. [Google Scholar] [CrossRef]
  47. Oliver, M.J.; Farrant, J.M.; Hilhorst, H.W.M.; Mundree, S.; Williams, B.; Bewley, J.D. Desiccation tolerance: Avoiding cellular damage during drying and rehydration. Annu. Rev. Plant Biol. 2020, 71, 435–460. [Google Scholar] [CrossRef]
  48. Verdier, J.; Lalanne, D.; Pelletier, S.; Torres-Jerez, I.; Righetti, K.; Bandyopadhyay, K.; Leprince, O.; Chatelain, E.; Vu, B.L.; Gouzy, J.; et al. A regulatory network-based approach dissects late maturation processes related to the acquisition of desiccation tolerance and longevity of Medicago truncatula seeds. Plant Physiol. 2013, 163, 757–774. [Google Scholar] [CrossRef]
  49. Zou, Z.; Zheng, Y.J.; Chang, L.L.; Zou, L.P.; Zhang, L.; Min, Y.; Zhao, Y.G. TIP aquaporins in Cyperus esculentus: Genome-wide identification, expression profiles, subcellular localizations, and interaction patterns. BMC Plant Biol. 2024, 24, 298. [Google Scholar] [CrossRef]
  50. Jiao, Y.; Leebens-Mack, J.; Ayyampalayam, S.; Bowers, J.E.; McKain, M.R.; McNeal, J.; Rolf, M.; Ruzicka, D.R.; Wafula, E.; Wickett, N.J.; et al. A genome triplication associated with early diversification of the core eudicots. Genome Biol. 2012, 13, R3. [Google Scholar] [CrossRef]
  51. Singh, R.; Ong-Abdullah, M.; Low, E.T.L.; Manaf, M.A.A.; Rosli, R.; Nookiah, R.; Ooi, L.C.-L.; Ooi, S.E.; Chan, K.L.; Azizi, N.; et al. Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds. Nature 2013, 500, 335–339. [Google Scholar] [CrossRef] [PubMed]
  52. Can, M.; Wei, W.; Zi, H.; Bai, M.; Liu, Y.; Gao, D.; Tu, D.; Bao, Y.; Wang, L.; Chen, S.; et al. Genome sequence of Kobresia littledalei, the first chromosome-level genome in the family Cyperaceae. Sci. Data 2020, 7, 175. [Google Scholar] [CrossRef] [PubMed]
  53. Walters, C.; Ried, J.L.; Walker-Simmons, M.K. Heat soluble proteins extracted from wheat embryos have tightly bound sugars and unusual hydration properties. Seed Sci. Res. 1997, 7, 125–134. [Google Scholar] [CrossRef]
  54. Delahaie, J.; Hundertmark, M.; Bove, J.; Leprince, O.; Rogniaux, H.; Buitink, J. LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance. J. Exp. Bot. 2013, 64, 4559–4573. [Google Scholar] [CrossRef]
  55. Hu, B.; Jin, J.; Guo, A.Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: An upgraded gene feature visualization server. Bioinformatics. 2015, 31, 1296–1297. [Google Scholar] [CrossRef]
  56. Edgar, R.C. MUSCLE v5 Enables Improved Estimates of Phylogenetic Tree Confidence by Ensemble Bootstrapping; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, USA, 2021. [Google Scholar] [CrossRef]
  57. Zou, Z.; Yang, J.H. Genomic analysis of Dof transcription factors in Hevea brasiliensis, a rubber-producing tree. Ind. Crops Prod. 2019, 134, 271–283. [Google Scholar] [CrossRef]
  58. Qiao, X.; Li, Q.; Yin, H.; Qi, K.; Li, L.; Wang, R.; Zhang, S.; Paterson, A.H. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019, 20, 38. [Google Scholar] [CrossRef]
  59. Mortazavi, A.; Williams, B.A.; McCue, K.; Schaeffer, L.; Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat. Methods 2008, 5, 621–628. [Google Scholar] [CrossRef]
  60. Zou, Z.; Gong, J.; An, F.; Xie, G.S.; Wang, J.K.; Mo, Y.Y.; Yang, L.F. Genome-wide identification of rubber tree (Hevea brasiliensis Muell. Arg.) aquaporin genes and their response to ethephon stimulation in the laticifer, a rubber-producing tissue. BMC Genom. 2015, 16, 1001. [Google Scholar] [CrossRef]
Plants 14 00762 g001
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 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).
Plants 14 00762 g001
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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 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).
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Plants 14 00762 g003
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 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).
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Plants 14 00762 g004
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).
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).
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Table 1. LEA_5 family genes identified in C. esculentus. (AA: amino acid; Ce: C. esculentus; GRAVY: grand average of hydropathicity; kDa: kilodalton; LEA: Late embryogenesis abundant; MW: molecular weight; pI: isoelectric point; Scf: scaffold).
Table 1. LEA_5 family genes identified in C. esculentus. (AA: amino acid; Ce: C. esculentus; GRAVY: grand average of hydropathicity; kDa: kilodalton; LEA: Late embryogenesis abundant; MW: molecular weight; pI: isoelectric point; Scf: scaffold).
Gene NameLocus IDPositionAAMW (kDa)pIGRAVYLEA_5 LocationDuplicateMode
CeLEA5-1CESC_21264Scf50:743435..744190(−)848.88 5.43 −1.398 2..67-
CeLEA5-2CESC_23041Scf10:593714..594313(−)11212.34 5.61 −1.388 16..58,
57..105		CeLEA5-1Dispersed
CeLEA5-3CESC_08006Scf17:1581661..1582217(+)15416.92 6.02 −1.582 2..84,
82..148		CeLEA5-1Dispersed
CeLEA5-4CESC_16081Scf34:1210271..1210837(−)838.96 5.22 −1.313 9..49,
47..70		CeLEA5-3Dispersed
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MDPI and ACS Style

Zou, Z.; Fu, X.; Yi, X.; Li, C.; Huang, J.; Zhao, Y. Integrative Analysis Provides Insights into Genes Encoding LEA_5 Domain-Containing Proteins in Tigernut (Cyperus esculentus L.). Plants 2025, 14, 762. https://doi.org/10.3390/plants14050762

AMA Style

Zou Z, Fu X, Yi X, Li C, Huang J, Zhao Y. Integrative Analysis Provides Insights into Genes Encoding LEA_5 Domain-Containing Proteins in Tigernut (Cyperus esculentus L.). Plants. 2025; 14(5):762. https://doi.org/10.3390/plants14050762

Chicago/Turabian Style

Zou, Zhi, Xiaowen Fu, Xiaoping Yi, Chunqiang Li, Jiaquan Huang, and Yongguo Zhao. 2025. "Integrative Analysis Provides Insights into Genes Encoding LEA_5 Domain-Containing Proteins in Tigernut (Cyperus esculentus L.)" Plants 14, no. 5: 762. https://doi.org/10.3390/plants14050762

APA Style

Zou, Z., Fu, X., Yi, X., Li, C., Huang, J., & Zhao, Y. (2025). Integrative Analysis Provides Insights into Genes Encoding LEA_5 Domain-Containing Proteins in Tigernut (Cyperus esculentus L.). Plants, 14(5), 762. https://doi.org/10.3390/plants14050762

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