Genome-scale identification, classification, and tissue specific expression analysis of late embryogenesis abundant (LEA) genes under abiotic stress conditions in Sorghum bicolor L.

Abstract

Late embryogenesis abundant (LEA) proteins, the space fillers or molecular shields, are the hydrophilic protective proteins which play an important role during plant development and abiotic stress. The systematic survey and characterization revealed a total of 68 LEA genes, belonging to 8 families in Sorghum bicolor. The LEA-2, a typical hydrophobic family is the most abundant family. All of them are evenly distributed on all 10 chromosomes and chromosomes 1, 2, and 3 appear to be the hot spots. Majority of the S. bicolor LEA (SbLEA) genes are intron less or have fewer introns. A total of 22 paralogous events were observed and majority of them appear to be segmental duplications. Segmental duplication played an important role in SbLEA-2 family expansion. A total of 12 orthologs were observed with Arabidopsis and 13 with Oryza sativa. Majority of them are basic in nature, and targeted by chloroplast subcellular localization. Fifteen miRNAs targeted to 25 SbLEAs appear to participate in development, as well as in abiotic stress tolerance. Promoter analysis revealed the presence of abiotic stress-responsive DRE, MYB, MYC, and GT1, biotic stress-responsive W-Box, hormone-responsive ABA, ERE, and TGA, and development-responsive SKn cis-elements. This reveals that LEA proteins play a vital role during stress tolerance and developmental processes. Using microarray data, 65 SbLEA genes were analyzed in different tissues (roots, pith, rind, internode, shoot, and leaf) which show clear tissue specific expression. qRT-PCR analysis of 23 SbLEA genes revealed their abundant expression in various tissues like roots, stems and leaves. Higher expression was noticed in stems compared to roots and leaves. Majority of the SbLEA family members were up-regulated at least in one tissue under different stress conditions. The SbLEA3-2 is the regulator, which showed abundant expression under diverse stress conditions. Present study provides new insights into the formation of LEAs in S. bicolor and to understand their role in developmental processes under stress conditions, which may be a valuable source for future research.

About the Authors:

M. Nagaraju

Roles Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing

Affiliation: Department of Genetics, Osmania University, Hyderabad, India

S. Anil Kumar

Roles Formal analysis, Investigation, Methodology, Visualization

Affiliation: Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research, Vadlamudi, Guntur, Andhra Pradesh, India

Palakolanu Sudhakar Reddy

Roles Formal analysis, Software, Visualization, Writing – original draft

Affiliation: International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Hyderabad, India

ORCID logo http://orcid.org/0000-0002-5341-187X

Anuj Kumar

Roles Data curation, Formal analysis, Software

Affiliation: Advance Center for Computational & Applied Biotechnology, Uttarakhand Council for Biotechnology (UCB), Silk Park, Prem Nagar, Dehradun, India

ORCID logo http://orcid.org/0000-0002-5023-7618

D. Manohar Rao

Roles Conceptualization, Formal analysis, Resources, Supervision, Writing – original draft

Affiliation: Department of Genetics, Osmania University, Hyderabad, India

P. B. Kavi Kishor

Roles Conceptualization, Formal analysis, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation: Department of Genetics, Osmania University, Hyderabad, India

ORCID logo http://orcid.org/0000-0002-9587-7547

Introduction

Environmental stresses such as drought, salinity, high and low temperatures, metals, radiation, and diseases cause extensive damage to crop plants by bringing about the changes in gene regulation and metabolism leading to reduced productivity [1]. To combat the stress conditions, plants develop defense-responsive pathways with the help of regulatory and functional genes [2]. Among them, the functional group of genes, mostly the Ca2+-dependent signaling molecules activate the late embryogenesis abundant (LEA)-type genes, which are protective proteins that help in damage repair of plants under diverse abiotic stress conditions [3]. LEA proteins are characterized by repeated motifs and disordered structure [4], first discovered in cotton seeds [5] during embryo development. Under desiccation, expressions of LEAs were high in embryos during seed maturation [6–7]. LEAs have been reported to be responsive to various developmental processes and also to abiotic stresses like drought, low temperature, salt, and ABA [8–9]. LEAs act as membrane protectors and stabilizers, ion chelators, hydration buffers and antioxidants [9].

Based on their amino acid sequence, homology, and conserved motifs, LEA proteins are classified into eight groups LEA-1, LEA-2, LEA-3, LEA-4, LEA-5, LEA-6, dehydrins, and seed maturation proteins (SMP) [10–11]. The groups 1–5 represent the major groups, and are present in most of the plants [12]. The LEA-1 proteins contain a 20-amino-acid motif (GGETRKEQLGEEGYREMGRK) with a high content of Gly, Glu, and Gln residues [13]. The group 2 dehydrins consist of a motif called K-segment (EKKGIMDKIKEKLPG), which gives chaperone activity to protect proteins during abiotic stress [6]. The LEA-3 proteins have 11-amino acid sequences (TAQAAKEKAGE) repeated 13-times and most of their functions were studied in transgenics [12]. The LEA-4 group does not show any conserved motif or repeats but have a conserved structure at the N-terminus which forms α-helical structure [7]. The LEA-5 has lesser amino acid homology, but participates in seed maturation and dehydration [6]. The LEA-6 proteins are characterized by their small size and two highly conserved motifs, motif 1 with LEDYK but replaced by proline and threonine at positions 6 and 7 in motif 2. Members of dehydrins are intrinsically unstructured, expressed during the late embryogenesis stage and stable under heat stress [14]. LEA proteins are ubiquitous and localized in cytoplasm, nucleus, chloroplast, mitochondria, and endoplasmic reticulum [15]. LEAs do not have a specific localization and their particular functions depend on their intra-cellular locations. For example, the mitochondrial localized pea LEA3 proteins protect rhodanese and fumarase from inactivation under dehydration [16]. On the other hand, the nuclear localized LEA2, LEA4, and LEA7 proteins display DNA binding [17]. The histidine-containing motifs in LEA2 and LEA4 proteins are responsible for binding divalent cations and ion sequestration [18]. LEA proteins are rich in glycine, glutamate, lysine and threonine but lack cysteine and tryptophan residues. Due to the presence of highly charged amino acids like alanine, serine/threonine, they are highly hydrophilic in nature. The secondary structures in LEA proteins are detected by the presence of repeated motifs [9]. Though highly disordered, they acquire structural folding into α-helical conformations under desiccation [19]. Based on the sequence similarity and conserved motif sites, 51 LEA genes belonging to 9 different groups were identified so far in Arabidopsis [6–7], 108 in Brassica napus [20], 53 in Populus [21], 36 in Brachypodium distachyon [22], 34 in Oryza sativa [23], 30 in Prunus mume [24], 29 in Solanum tuberosum [25], and 27 in tomato [26].

Over expression of LEAs confer abiotic stress tolerance in different plants like Arabidopsis, tobacco, rice, wheat, and lettuce [27]. The NtLEA7-3 shows resistance to drought, salt, and cold in Arabidopsis thaliana [28]. In yeast, tomato LEA25 enhances the salt and chilling stress tolerance [29]. The HVA1 promotes drought and salt stress tolerance in wheat and rice [30–31]. Heterologous expression of BnLEA4-1 in E. coli shows tolerance to heat and salt stress [32]. The citrus dehydrin acts as radical scavenger and reduces the metal toxicity [18]. Likewise, two soybean LEA4 proteins bind to Fe and are associated strongly in reducing oxidative damage induced by abiotic stress [33]. Further, it was shown that loss of LEA4 proteins result in drought susceptibility in Arabidopsis [34]. The Arabidopsis LEA2 protein alters the pathogenesis-related protein expression and confers defense response [35]. Similarly, the group 3 LEA proteins in maize confer tolerance to bacterial infection. While their heterologous expression in tobacco exhibit tolerance to Pseudomonas syringae [36], wheat TaLEA2 and TaLEA3 in yeast enhance the salt and freezing stress tolerance [37]. Lin et al [38] found that VrDhn1 stabilizes the DNA under seed desiccation. Thus, it appears overexpression of diverse LEA proteins offer tailored protection against abiotic stress in a wide range of plants [15].

Sorghum bicolor is the fifth most important cereal crop, used as food, feed, fuel, fibre, and fertilizer. It is moderately tolerant to drought, salinity, water logging conditions as well as high temperature [39–42]. The knowledge about the number of LEA proteins and their families, structure characterization, tissue specific expression, and chromosomal location is meagre in S. bicolor. Hence, in the present investigation, comprehensive genome-scale identification of LEAs, their structural characterization, chromosomal location, and promoter analysis alongside the tissue specific gene expressions were carried out under varied abiotic stress conditions.

Material and methods

Identification, chromosomal localization, and gene structure analysis of LEA in S. bicolor

In the present study, 34 Oryza [23] and 51 Arabidopsis [7] LEA gene sequences were retrieved from NCBI database and searched (using TBLASTN) against Sorghum bicolor genome in Gramene database (http://www.gramene.org/) to find out their homologs. Genscan (http://genes.mit.edu/GENSCAN.html) program was used to retrieve the coding and protein sequences. Based on homology, Sorghum LEA sequences were analyzed by SMART program (http://smart.embl-heidelberg.de/) [43] for the presence of conserved domains. MOTIF search (http://www.genome.jp/tools/motif/) tools were used to check the reliability of conserved domains. Chromosomal locations of LEAs were determined with the information obtained from Gramene database and the physical map was drawn based on their positions. Gene characterization was studied using Gene Structure Display Server (http://gsds.cbi.pku.edu.cn) [44].

In silico characterization of SbLEA proteins

The molecular weight (MW), isoelectric point (pI), and GRAVY (grand average of hydropathicity), instability and aliphatic indices were calculated using ProtParam of Expasy tools [45] (http://web.expasy.org/protparam). The NetPhos3.1 software was used to determine the phosphorylation sites within the LEA family [46]. The protein subcellular localization of LEA family members was identified by using WoLF PSORT programs (http://wolfpsort.org/) [47]. The putative trans-membrane helices were identified by using TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/) [48]. The conserved motif structures of LEA family genes were retrieved by using Multiple Em for Motif Elicitation (MEME) software (http://meme-suite.org/) with default parameters: number of motifs (1–10), motif width of 5–50, and the number of motif sites (5–10) [49]. The putative miRNAs in targeting the SbLEA genes were identified using psRNATarget server [50] with default parameters.

Promoter analysis of SbLEA family, phylogenetic analysis, and estimation of synonymous and non-synonymous substitution rates

The 1000 bp genomic sequence upstream of start codon of SbLEA genes were examined using PLACE [51] software to check for the presence of cis-elements responsible for development, biotic, and abiotic stresses. The NJ phylogenetic trees for LEA protein family of S. bicolor, O. sativa, and A. thaliana were generated using MEGA 6.2 software [52] with default parameters like Poisson correction, pairwise deletion, and bootstrap value (1,000 replicates). Paralogues and orthologues were identified using phylogeny and InParanoid 8 (the orthology analysis software) [53] with default parameters like 0.01 cut off E value, 50 or higher cut off score values. Synonymous and non-synonymous sites and substitution rates of paralogous and orthologous gene pairs were calculated using PAL2NAL software (http://www.bork.embl.de/pal2nal/) [54].

In-silico expression profiling of SbLEAs

Expression analysis for the identified SbLEA genes was performed using Affymetrix whole-transcriptome Sorghum array data accessible from the SorghumFDB [55]. The Genevestigator platform [56] was used to perform the microarray analysis for SbLEAs genes under several environmental stresses (drought, salt, heat, and cold) with different samples embedded in the platform. The expression profiles of SbLEA genes identified from Sorghum array was used for cluster analysis. A heat map of expression profiling was developed by using hierarchical clustering tool embedded in Genevestigator platform [57].

Plant material and stress conditions

The seeds of S. bicolor BTx623 variety were sown in pots containing 4.5 kg of black clay soil under glass house conditions at 28/20 oC day/night temperatures. After 40 days, the plants were subjected to drought and salt stresses by treating with 1 liter each of 150 mM mannitol and NaCl individually for 4 h. The cold stress was applied by keeping the plants at 4°C for 4 h and heat stress by exposing the plants to 40°C for 4 h in a growth chamber. The respective controls were maintained under identical conditions. Roots, stems, and leaves were collected and snap frozen immediately in liquid nitrogen and stored at -80°C until further use.

RNA extraction and qRT-PCR analysis for transcriptional profiling of SbLEA genes

The MACHEREY-NAGEL kit was used to isolate the total RNA from roots, stems, and leaves by following the manufacturer’s instructions. The first strand cDNA was synthesized from total RNA (3 μg concentration) using first strand synthesis kit (Thermo Scientific). Gene specific primers were designed by using NCBI PRIMER Blast (www.ncbi.nlm.nih.gov/tools/primer-blast/) [58] and Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) [59] with the default parameters: 57–60°C annealing temperature, 18–22 bp primer length, 50–55% GC contents, and 80–140 bp amplicon length (S1 Table). The SYBR Green Master Mix (2X) (Takara) was used according to the manufacturer's recommendations. Two biological duplicates with three technical replicates were taken for qRT-PCR analysis in Mx3000p (Agilent Technologies) with the following thermal cycles: 1 cycle at 95 oC for 10 min, followed by 40 cycles alternatively at 95 oC for 15 sec and 60 oC for 1 min. The amplicon dissociation curves were recorded with fluorescence lamp after 40th cycle by heating from 58 to 95 oC within 20 min. Transcript levels of SbAcp and SbEP-F genes were used as internal controls [60]. Relative gene expressions were calculated by employing Rest software [61] and average values are represented. Statistical significance of the expression values was determined by using t-test.

Results

Identification, chromosomal localization and gene structure analysis of SbLEA genes

A total of 68 LEA genes were identified in the genome of S. bicolor based on rice and Arabidopsis LEA homologs. Their reliability was checked for the presence of conserved domain using SMART and MOTIF tools. The genes are grouped into 8 sub-families like LEA 1–6, dehydrins, and SMP based on their conserved domains and Pfam nomenclature. Among all the families, SbLEA2 was found as the largest family with 40 genes (SbLEA2-1 to SbLEA2-40), followed by SbLEA3 with 7 genes (SbLEA3-1 to SbLEA3-7), and SbDHNs with 6 genes (SbDHN1- SbDHN6). Both SbLEA1 and SbLEA4 families contain 5 genes each, while SMP has only 3 members. The smallest families are SbLEA5 and SbLEA6 with one member each (Table 1). SbLEA genes were distributed on all the chromosomes. Out of 68 genes, 13 genes are localized on chromosome 1; 11 on 2, 10 on 3, 6 on 4, 3 on 5, 7 on 6, 4 on 7, 3 on 8, 8 on 9, and 3 on 10 (Table 1 and Fig 1). All the members of SMPs have only 1 intron and 2 exons. A total of 22 genes out of 40 in the group SbLEA2 lack introns. SbLEA2-9 showed a maximum of 8 exons. Out of 68 SbLEA genes, only one exon was observed in 31 genes, 2 exons in 19 genes, 3 in 6, 4 in 5, and 5 in 5, 6 in 1, and 8 in 1. A total of 22 genes out of 40 in the group SbLEA2 lack introns (Table 1 and Fig 2).

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Fig 1. Chromosomal distribution of LEA genes in Sorghum.

Duplications are illustrated by different colors (Segmental) and regional duplications are linked with line.

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Fig 2. Distribution of exons, introns, upstream and downstream regions in SbLEAs.

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Table 1. Identified LEA genes in Sorghum bicolor exhibiting family, number of amino acids, chromosomal location, iso-electricpoint (pI/molecular weight (MW), DNA binding domains (DBD), no. of exons, number of transmembrane helices, localization, GRAVY, instability index, and aliphatic index.

https://doi.org/10.1371/journal.pone.0209980.t001

In silico characterization of SbLEA proteins

The SbLEA family genes encode polypeptides ranging from 79 to 624 amino acids in length. While SbLEA3-3 and SbLEA7 contain 79 amino acids (aa), 624 aa are present in SbLEA2-23. Accordingly, the predicted molecular weights range between 8.14 to 67.77 kDa. Among all, the LEA-2 family members show the highest molecular weights (Table 1). Physicochemical analysis reveal that the theoretical pI values range between 4.77 (SbSMP-2) - 11.48 (SbLEA2-27). A total of 50 out of 68 proteins (73.52%) are of basic in nature, while remaining 18 (26.47%) of them are acidic (Table 1). Likewise, SMP group was found to be the most acidic, and similarly in LEA-2, 6 (15%) were identified as acidic. The instability index ranges between 8.54 (SbLEA1-3) and 69.39 (SbLEA2-27) depending upon the group. Nearly 42.64% of SbLEA proteins have a low instability index (> 40), but the LEA-2 group appears unstable (62.5%). The GRAVY of SbLEA proteins vary between -1.282 (SbLEA2-24) to 0.378 (SbLEA2-15). While most of the LEA proteins are hydrophilic, 24 out of 40 (60%) LEA-2 family proteins appear hydrophobic. But, LEA-1, LEA-4, and SbDHNs are completely hydrophilic in nature. The aliphatic index of SbLEA proteins ranges from 29.05 (SbDHN-6) to 104.41 (SbLEA2-15), and SbLEA-2 exhibits the highest from the rest. Contrarily, SbDHNs show the least aliphatic index. It is found that LEA proteins localise mostly to chloroplast (44.11%), followed by cytoplasm (16.17%), nucleus (14.70%), mitochondria (7.35%), plastid (4.41%), and the rest in extra cellular matrix, cytoplasm-nucleus, chloroplast-nucleus, chloroplast-mitochondria, and chloroplast-cytoplasm as revealed by Wolfpsort tool. Majority of SbLEA-2 group members appear to target to chloroplast (~60%). Proteins in the group SbLEA-1 are ~70 aa residues long, with conserved DNA binding domain, whereas in the case of LEA-2 and DHNs family, proteins are 100 residues long. In LEA-3, they are 90 residues long, but in LEA-4 group, they are the smallest with ~30–40 residues. The putative transmembrane helices were identified by using TMHMM server. Only the SbLEA-2 family proteins contain transmembrane helices, some exceptions being SbLEA4-2, SbLEA5-1, and SbDHN-3 (Table 1).

Majority of the SbLEA proteins phosphorylate at serine and threonine sites and very few of them at tyrosine residue. In case of SMP group members, phosphorylation occurs at threonine. Protein kinase C (PKC) and unsp are the most dominant types present in higher amounts in all the SbLEA proteins. Next to PKC, cdc2, PKA, DNAPK, P38MAPK, and PKG are the most common kinases associated with phosphorylation. The highest number of cdc2 was found in LEA-2 family (S2 Table).

Conserved motif analysis

Sixty eight SbLEAs did not share high similarity, and each family was submitted to MEME separately and in combination for domain or motif structure analysis. Ten conserved motifs were identified for each family except SbLEA-6, which contains only 7 (Fig 3 and S1 Fig). The paralogs and closely related genes exhibit similar motif compositions. The composition of the motifs is similar in each family but varies among different families. Motif 3 in LEA-1, motif 5 in LEA-2, and motif 5 and 6 in DHNs appeared as the biggest motifs. Fifty four SbLEA proteins exhibit common motifs and motif 1 is the most common and conserved structural motif present in majority of the proteins. Motifs 9 and 10 are the key features of DHN sequences. For recognition of SbDHN proteins, K-segment in motifs 1 and 3, S-segment in motif 2, and Y-segment in motif 4 were used (Fig 3 and S1 Fig). Conserved motifs were not observed in LEA-1, 4, 5, 6, and SMP families. Next to motif 1, motif 3 is the most conserved and located at C terminus. While in LEA-3 group, motif 5 is the most conserved, in LEA-2 family, motif 7 is the structural motif conserved at N terminus (S2 and S3 Figs).

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Fig 3. Conserved motif patterns of different SbLEA families.

The scale represents the lengths of the proteins and motifs.

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In silico prediction of miRNAs targeting LEAs

Our analyses identified that 25 different SbLEA genes namely SbLEA2-1, 3, 5, 8, 14, 15, 18, 24, 25, 27, 31, 32, 33, 35, 36, 37, 38, and 40, SbLEA3-2, 3, 5, 6, and 7, SbSMP-3 and SbDHN-5 are the targets for 15 different miRNAs. It appears that miRNAs target 18 genes in SbLEA-2 group, and 5 in SbLEA-3. While six miRNAs target LEA3-5, 3 of them target SbLEA2-35 group. Sbi-miR6225, sbi-miR437x, sbi-miR5568, and sbi-miR6220 appear as the most common miRNAs that target SbLEA genes and participate in cleavage and translation (S3 Table).

Promoter analysis of SbLEA genes

Promoter analysis revealed that SbLEA genes have potential cis-regulatory elements, which are further divided into abiotic stress-responsive (DRE, DPBF, MYC, MYB, HSE, LTRE, GT1GM, Cu responsive, Sp1, G-box, and I-box), hormone specific (ABRE, TCA, ERE, etc.), biotic stress-responsive (WBox), development specific (pollen, endosperm specific) and guard cell specific elements (CGCG). The Myb and Myc are the most conserved elements present in all the genes. The salt-responsive elements were observed in SbLEA-2 family, whereas DRE, and DPBF in all other families and very few of them in SbLEA-2. At least one heat shock element (HSE) was identified in all the SbLEA families with an exception of dehydrins. ABA-responsive elements (ABRE) and TCA are the most dominant elements present in the highest numbers in all the families. Among all, SMP group exhibits the highest number of ABRE elements (S4 Table).

Phylogenetic analysis of LEA family proteins

Phylogenetic analysis was carried out for 68 SbLEA proteins to analyse the evolutionary relationships within and between the groups (Fig 4). Different families of SbLEAs exhibit high similarity and cluster into 2 major clades (Fig 4). A total of 23 SbLEA genes belonging to LEA-1, LEA-3, LEA-4, SMP, and DHNs form a cluster in clade 1, while the other 45 members of SbLEA-2 family appear in clade 2. Out of 6 SbDHNs, 4 form a cluster into clade 1 (SbDHN-1, 2, 4, and 6), and remaining 2 into clade 2 (SbDHN-3 and 5). The SbLEA1-1, 2, and 4 are grouped into clade 1, whereas SbLEA1-3 and 5 into clade 2. Among the 22, 4 regional paralogs were noticed within SbLEAs. On the other hand, SbLEA2-11/13 on chromosome 2, SbLEA3-2/4 on chromosome 3, SbSMP-1/2 on chromosome 1, and SbDHN-1/2 on chromosome 3, and 18 appear as segmental duplications (Figs 1 and 4). SbLEA-2, the most dominant group present in Sorghum, shows 13 paralogs. SbLEA-3, 4, and DHNs show two paralogous events each, while SbLEA-1 and SMP exhibit one event (Fig 4 and Table 2).

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Fig 4. Phylogenetic analysis of 68 SbLEAs.

LEA gene families were classified based on their homology and are distinguished by different colors.

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Table 2. Non-synonymous to synonymous substitution ratios of LEA paralogs.

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To know the evolutionary relationship and find ortholog pairs, another phylogenetic tree was constructed with Arabidopsis and Oryza (Fig 5). In this, LEA proteins are grouped into 2 clades, while LEA-2 family of Sorghum, Oryza and Arabidopsis fall into clade 2, others into clade 1. Thirty eight out of 68 from Sorghum, 9 out of 39 from rice, and 7 out of 51 from Arabidopsis fall into clade 2, but SbLEA-2 family appears as the most dominant group. A total of 11 paralogs each are observed in Sorghum and Arabidopsis, but only 7 in Oryza. The SbLEA shows 12 orthologs with Arabidopsis and 13 with Oryza. The Oryza and Arabidopsis share only six orthologs among them (Fig 5 and S5 Table). From the InParanoid, the orthology analysis of SbLEAs exhibits ortholog relationship with Setaria, Oryza, Hordeum and Brachypodium (S6 Table).

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Fig 5. N-J phylogenetic tree showing the relationship between LEA proteins in Oryza, Arabidopsis and Sorghum.

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Estimation of non-synonymous and synonymous substitution rates of LEA

The non-synonymous (dN) versus synonymous (dS) substitutions (dN/dS) were estimated for SbLEA genes which show duplication events within Sorghum as paralogs (Table 2). The paralogous events, exhibit divergence substitution rates. While the number of synonymous sites (S) ranges between 50.2 (SbLEA1-2/4) and 297 (SbLEA2-6/23), it ranges between 176.5 (SbLEA3-2/4) and 1017 (SbLEA2-6/23) in non-synonymous sites (N). In contrast, synonymous substitution rate (dS) ranges between 0.0441 (SbLEA4-3/5) and 81.9485 (SbSMP-1/2), and non-synonymous (dN) between 1.0265 (SbSMP-1/2) and 17.1860 (SbLEA2-27/35) (Table 2). Most of the paralogs dN/dS were found to be below <1 (Table 2). The paralogous synonymous and non-synonymous substitution calculations were extended to orthologous LEA gene pairs between Arabidopsis, Oryza and S. bicolor. Out of 25 orthologs, Sorghum shows 12 events with Arabidopsis of which 4 duplications share same chromosomes (Sb01g046000/At1g72100 on chromosome 1; Sb02g028010/At2g41260 on chromosome 2; Sb03g012950/At3g50980 on chromosome 3; and Sb04g023155/At4g15910 on chromosome 4). No such events were observed in Oryza. Only 5 orthologs of Arabidopsis show dN/dS substitution ratios with 99.00. Of the 13 ortholog pairs of Sorghum and Oryza exhibit dN/dS ratios, 5 events show 99, while the remaining vary from 0.0273 to 21.2957 (S5 Table). The orthology analysis of SbLEAs with Oryza, Setaria, Brachypodium and Hordeum shows that majority of them exhibit Darwinian selection, and the dN/dS ratio is greater than 1 (S6 Table).

Microarray-based gene expression profiling in different tissues and different developmental stages under abiotic stress conditions

Of the 68 sorghum SbLEAs, microarray data for 65 SbLEA genes were available on the Genevestigator platform, these were further utilized for expression analysis. Expression of these 65 SbLEA genes in six tissues (roots, pith, rind, internode, shoot, and leaf) was analyzed under normal and abiotic stress conditions using microarray data (Fig 6A). The expression level was higher in root, pith and in the leaf tissues.

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Fig 6.

Digital expression analysis of SbLEA genes a) in different tissues; b) in various developmental stages; c) under diverse abiotic stress conditions. Colour scale represents % expression, down and upregulation.

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The expression profiles of SbLEAs genes were analyzed at five different development stages, including stem elongation, booting, flowering, dough, and seedling. SbLEA genes were found expressed in all developmental stages (either up-regulated or down-regulated) (Fig 6B). However, the expression of SbLEA genes in the booting and flowering stages demonstrated a slightly different pattern, particularly SbLEA-2 members displayed the dominant expression profile compared to other developmental stages. High expression of SbLEA genes during booting and flowering stages might have been caused by booting-related cellular deteriorations, leading to substantial metabolic or physiological changes that significantly affect the overall regulation under abiotic stresses.

Hierarchical clustering based on the above expression profiles of individual SbLEA genes under various abiotic stress conditions allowed grouping of the 65 SbLEA genes into two major clusters. One of these clusters contained the only SbLEA2-22 gene which shows very high up-regulation under different stress conditions. The remaining SbLEA genes were distributed among other sub-clusters of the second major cluster (Fig 6C). The heat map of different SbLEA genes following abiotic stresses showed significantly altered expression (either up-regulation or down-regulation) up to 2.5-folds (Fig 2). Members of the SbLEA-2 (SbLEA2-22, SbLEA2-24, SbLEA2-32, SbLEA2-33, and SbLEA2-37) were up-regulated under stress conditions. Similarly, SbLEA3-1 and SbLEA3-2 members were up-regulated under salt, cold, and drought stresses.

Quantitative expression analysis of SbLEAs

To investigate the differential gene expressions in vegetative tissues of Sorghum, a systematic analysis of quantitative real-time (qRT)—PCR was carried out for a group of 23 SbLEA genes. qRT-PCR expression analysis of 23 SbLEA genes in different tissues under drought, salt, heat, and cold stresses reveals their comprehensive roles in stress tolerance mechanism, as well as in growth and development. The differential expression patterns in roots, stems, and leaves are shown in the Figs 7 and 8A. Most of the LEA genes exhibit the highest expression levels in stem tissues (SbLEA1-5, 2–9, 2–13, 2–18, 2–37, 3–7, and 4–1) (Fig 8A). Compared to leaf and stems, root tissues show lower expression values under the stress conditions. The SbLEA3-2 show the highest expression levels in leaf tissues under salt (82-folds), cold (434-folds), and drought stresses (52-folds), and in stem under salt stress (445-folds). On the other hand, SbLEA2-23 show several-folds increase in leaf tissues under drought (191.78-folds), and cold (340.93-folds), whereas in roots under heat stress (369.64-folds). Surprisingly, members of SbLEA-2 (the major family) display high expression under all stresses in leaves compared to other tissues, the SbLEA2-37 exhibit 48.95-folds in drought-exposed and 99.27-folds in cold-treated leaves. Expression of LEA1-5 in roots is better under drought stress (11.28-folds), and in stems under cold stress (15.06-folds). The LEA-4 family members exhibit the highest expression in stems under stress compared to other tissues; the LEA4-3 exhibits the highest expression in stems under cold stress (39.4-folds). Interestingly under heat stress, expression of majority of the SbLEAs was high in roots, the SbLEA1-2 exhibits 14.22-folds, SbLEA2-9 28.24-folds, SbLEA2-23 369.64-folds, SbLEA2-37 111.43-folds, and SbLEA3-2 133.43-folds. Expression of SMP-2 is high in root tissues under drought (16.99-folds) and cold (10.85-folds) stresses, but the leaf tissues display high activity (11.65-folds) under salt stress (Figs 7 and 8B and S7 Table).

[Figure omitted. See PDF.]

Fig 7. The relative expression values of SbLEA genes in roots, stems and leaf tissues under drought, salt, heat and cold stress.

Error bars indicate ± SD. * indicate significant differences calculated by t-test (*P ≤ 0.05).

https://doi.org/10.1371/journal.pone.0209980.g007

[Figure omitted. See PDF.]

Fig 8.

qRT-PCR expression patterns of SbLEAs a) tissue specific expression of SbLEAs in roots, stems, and leaf tissues; b) transcriptional expression analysis of SbLEA genes in roots, stems, and leaf tissues under drought, salt, heat and cold stresses. (DR: Drought Root, DS: Drought Stem, DL: Drought Leaf, SR: Salt Root, SS: Salt Stem, SL: Salt Leaf, HR: Heat Root, HS: Heat Stem, HL: Heat Leaf, CR: Cold Root, CS: Cold Stem, CL: Cold Leaf). The expression represents log2 values.

https://doi.org/10.1371/journal.pone.0209980.g008

Discussion

Genome-wide analysis of Sorghum bicolor for LEA genes reveals 68 SbLEAs that belong to 8 families. Similar studies in other plant species showed different number of LEAs; 23 in Phyllostachys [62], 27 in tomato [26], 29 in potato [25], 30 in Prunus [24], 32 in maize [63], 34 in rice [23], 36 in soybean [64], 51 in Arabidopsis [7], 53 in poplar [21], 61 in Cucumis melo (melon) and 73 in Citrullus lanatus (water melon) [65], 72 in sweet orange [66], 79 in cucumber [67], 108 in Brassica [20], 136 in Gossypium arboreum, 142 in G. raimondii, and 242 in G. hirsutum [68]. It is puzzling to note that the number of LEA genes is very large, abundant and diversely distributed across different taxa. The abundance perhaps indicates their conservative role under abiotic stress conditions as well as during growth and development. It is interesting to observe that aquatic plants have less number of LEAs because they do not suffer from drought stress. Thus, the present and previous research findings are consistent with the results of Kamisugi and Cuming [69] regarding the wider distribution and function of LEA proteins in terrestrial plants. Generally, the LEA families with close taxonomic relationships exhibit the same number and distribution of genes. However, the number of the LEA genes varies in Sorghum, maize, and rice. This occurrence may be due to the evolutionary variations of the whole genomes and wide changes in the environment. Comparison of SbLEAs with rice and maize show divergence signals which are associated with selected traits and are functionally stress-responsive. This indicates that stress adaptation in maize is possible by evolution of protein coding sequences [70]. The divergence of LEA families in Zea and Oryza occurred due to evolutionary changes, the large number of LEA genes and evolution of LEA-2 family members may be meant for adaptation of Sorghum to stress conditions. The LEA family proteins are further classified into 8 subfamilies among all the crops based on their conserved domain and phylogenetic tree analysis. But, Arabidopsis holds an extra subgroup named as AtM [7]. In Sorghum, the most dominating LEA-2 family has the highest number of genes (58.8%), but dicots such as Arabidopsis (codes for 35%), Populus (49%) and Brassica (23%) are rich in LEA-4 members [7, 20, 21]. Similarly, DHNs and SMP groups also show variations among monocots and dicots. Arabidopsis consists of 10 DHNs, and 6 SMPs [7], Oryza 8 DHNs, 5 SMPs [23], Brassica 23 DHNs, 16 SMPs [20], and Sorghum 6 DHNs [71], and 3 SMP genes. The expansion of gene family depends on segmental, tandem duplications, and transposition events [72]. In the present study, 22 paralogs were observed including 4 regional duplications, and 13 paralogous pairs (SbLEA-2 family) with segmental duplication events. This indicates that segmental and tandem duplications are responsible for SbLEA gene family expansion [20, 26]. Lan et al. [21] pointed out that stress-responsive genes generally contain very less number of introns. In the present study, 45.58% of LEA genes lack introns (especially 55% of genes in the major SbLEA-2 group) and 27.94% hold one intron. Similar results were recorded in Brassica [20]. This supports the earlier view that introns delay the gene expression and extend the transcript length, which results in an additional burden on the process of transcription [73].

Filiz et al. [22] and Altunoglu et al. [62] pointed out that LEA4, LEA5, and LEA6 group proteins are acidic while most of the LEA proteins are basic in nature. Present study shows that 73.52% are basic in nature, but 85% of proteins from SbLEA-2 are basic thus corroborating the earlier findings. In contrast, SMPs are found to be acidic in nature which is in agreement with the findings of Liang et al. [20] in Brassica. The grand average of hydropathy values of SbLEA proteins are highly hydrophilic, except SbLEA-2 family. Previous studies report only one or two proteins with hydrophobicity [7, 20], while 85% of SbLEA-2 group proteins are hydrophobic, similar to cotton LEA2 members [68]. Hydrophilic nature and high net charge are the characteristic features of LEAs [74], which makes them disordered, and act like molecular chaperones under stress in plants [75].

Instability index shows majority of the SbLEA proteins are stable like that of SiLEAs as noticed by Cao and Li [26]. LEA proteins are not transmembrane proteins [76] and are located in mitochondria, chloroplasts, nucleus, and cytoplasm. Contrarily, SbLEA-2 family members exhibit transmembrane helices, which are hydrophobic in nature. Detection of transmembrane helices in proteins indicate their expression in subcellular compartments. SbLEA-2 shows high aliphatic index inferring the relative volume occupied by aliphatic side chains like alanine, valine, isoleucine and leucine, which enhance the thermostability of proteins [77]. Majority of the SbLEA-2 family members are localized in chloroplasts, like in cotton [68]. The wide distribution within subcellular compartments leads to interaction with cellular membranes under stress and establish protective mechanism for stress tolerance [15].

Generally, the diversity of structure and conserved motifs cause the evolution of multigene families [78]. It is the amino acid composition that causes disordered structure in LEAs [79]. Our analysis revealed that SbLEA proteins show group-specific conserved motifs. Identical results were reported earlier for LEA proteins in Arabidopsis [7], Prunus [24], poplar [21], Solanum [26], maize [63], Brassica [20], and cotton [68]. Specific conserved motifs and their number indicate that they are evolved from the gene expansion within their specific families, and the motif compositions vary from one family to the other. While glycine-rich regions are noticed in AtLEA-2, other LEA members are rich in lysine [7]. But, conserved motifs in SbLEA-2 family are rich with cystine and lysine in contrast to hydrophilins that lack tryptophan and cysteine [80]. The intrinsically disordered proteins which are small in size play several important roles in cells that help in structural flexibility, binding of DNA, RNA, proteins, macro molecules, and membrane proteins to protect and maintain the cellular stability under stress [75, 81, 34]. Phosphorylation helps LEA and dehydrin proteins in binding to calcium, iron and other divalent cations [82, 83]. Phosphorylation of YnSKn type DHNs by PKCs, and SKn DHNs by CK2s, maintains the activity of DHNs conferring tolerance to stress. Eriksson and Harryson [84] and Nagaraju et al. [71] pointed out that such phosphorylation enhances the membrane binding activity of DHNs.

Micro RNAs (miRNAs) are the large group of small, noncoding regulatory elements, which play pivotal roles in gene regulation by disturbing the transcripts of genes and mediate the plants adaptation under abiotic stress [85–87]. For example, expression of rice miR319a in creeping bent grass confers tolerance against salt and drought stresses [88]. Also, salt stress alters the expression of miR396c and miR394 [89]. Sb-miR437, found in majority of SbLEA genes has also been identified earlier in Oryza, maize, and sugarcane but absent in Arabidopsis and Populus. This suggests that miR437 is monocot specific [90]. Sorghum miRNAs may target transcription factors like SPB, zinc finger, WRKY, WD-40, NAC, MYB, HSFs, GRAS, ARFs, and bHLH families [91], which play important roles in growth, development, metabolism, biotic and abiotic stresses [92–94].

Present study identifies several abiotic stress-responsive elements, hormone specific, development specific, and biotic stress-responsive elements, as also noticed in other crop plants [26, 68]. The cis-elements responsive to phytohormones increase the plants potentiality to survive under environmental changes. It is known that ABRE play an important role in ABA signalling and abiotic stress tolerance. Similarly, DRE/CRT/LTRE (drought responsive/C-repeat/low temperature-responsive) elements enhance the drought, cold and salt-responsive gene expression, by controlling transcription factors like CBF/DREB1 [95, 96]. Multiple CGCG cis-elements present in all the SbLEAs bind to calmodulin/Ca2+ and are responsible for eliciting multiple signaling pathways [97]. SbLEAs also contain biotic stress-responsive cis-elements; WBOXNTERF3, WBOXATNPR1, and CGTCA that respond to wounds, pathogens and salicylic acid [98, 99]. GT1GMSCAM4 cis-elements, rich in GAAAAA, were detected, and play a crucial role in salt and pathogen-induced gene expression and tolerance [100]. The MYB cis-acting promoter elements identified in the present study play a key role in the abscisic acid-dependent signaling pathway in response to drought, salt, and cold as pointed out by Li et al. [101]. Identification of wide range of cis-elements in the Sorghum paralogous gene promoter regions perhaps indicate the variation in expression between paralogous duplicated genes, neo-functionalization or sub-functionalization, which is an important evolutionary mechanism [102]. The presence of these cis-elements in SbLEA genes represent that they play important roles in different stresses.

Based on the phylogenetic analysis, SbLEA genes were classified into 8 groups, similar to other plants [7, 20, 68]. While SbLEA2 is the largest group, SbLEA5 and 6 represent fewer genes, consistent with Arabidopsis [7]. Interestingly, LEA6 group is absent in rice [23]. The present study revealed 25 ortholog gene relationships with Arabidopsis and Oryza. Generally, Sorghum exhibits relationship with Oryza, being the common monocot ancestor, but the present study reveals that S. bicolor LEA proteins are phylogenetically close to Arabidopsis also. The phylogenetic tree depicts common evolutionary origin of LEA-1, 3, 4, 5, 6, and SMP [6], which is consistent with potato and cotton [25, 68]. Genome-wide analysis in few plants reveals the differences among LEAs in monocots and dicots. In dicots, LEA4 and DHNs are the most abundant [7, 20, 26], but analysis of Sorghum reveals LEA2 is a big, atypical, hydrophobic group. A recent study in rice and poplar reports higher number [66]. The phylogenetic analysis reveals that whole genome duplication contributes to expansion of SbLEA family. Indeed, rice (monocot ancestor) genome contains 34 LEA genes [23], and the whole genome duplication event is expected to generate 68 genes as seen in Sorghum. Similar results were observed in Arabidopsis, Brassica, and cotton also. Out of a total of 22 paralogous duplication events, 1 segmental and 4 tandem duplications are observed in Sorghum. As pointed out by Salih et al. [103], the abundance of LEA proteins mainly occur through segmental duplication events during evolution, similar to Arabidopsis, Brassica, and cotton. It is known that the synonymous (dS) and nonsynonymous (dN) values reveal the selective pressure on SbLEA duplicated genes. While greater than 1 dN/dS value indicates positive selection, less than 1 functional constraint, and equal to 1 neutral selection [104]. The dN/dS ratio analysis of SbLEA 22 paralogous pairs reveal that only 11 events had ratios of which one shows more than 1, and remaining very low values, similar to Brassica [20], melon [65], and cotton [68]. This infers that during evolution, the purifying selection influences the SbLEA genes and specifically LEA2 shows conserved structures and functions under selective pressure [105].

Gene expression analysis provides new insights into their function [106, 107]. Microarray data from the databases show high expression of SbLEA genes in different tissues. This indicates that abiotic stresses and/or high metabolic activity generally lead to up-regulation of SbLEA genes in different tissues in a tissue-specific manner. These results agree with the results of quantitative real-time expression analysis carried out for a set of SbLEA genes in the present study. SbLEA gene expressions in different tissues exhibit variations, which reveal their role during growth and development. Both SlLEA9 and SlLEA23 show high expression levels in tomato flower buds, suggesting their roles in reproductive development [26]. The At5g27980 regulates pollen germination and tube growth due to its abundant expression in the mature pollen [108, 109]. Expression of ZmLEA3 group in root, stem, and leaf tissues also suggests their role in growth and development [63]. Present study shows abundant expression of LEA2 group genes in vegetative tissues, akin to cotton LEAs [68]. Majority of the SbLEAs are expressed in leaf tissues, consistent with the observations of Liang et al. [20] in Brassica. Native expression of paralogous genes in different tissues implies distinct divergence and evolution of duplicated genes for different functions during plant growth and development. SbLEA genes expression was further assessed under drought, salt, heat, and cold in different tissues, which gives new insights into their critical roles under abiotic stress conditions. These results show significant changes in expression levels under diverse stresses implying their association with stress tolerance. They act as molecular chaperones, protect, stabilize, prevent aggregation and denaturation of proteins under stress conditions [110]. Among different tissues, roots are first affected under many abiotic stresses [111], followed by leaves. Leaves wilt or become chlorotic and lead to disruption of photosynthesis and yield losses [112]. The paralogs also show expression variations similar to previous studies by Du et al. [24]. Expression of ZmLEA3 at the transcriptional level was reported under biotic and abiotic stresses and its over-expression in tobacco exhibit tolerance against osmotic and oxidative stresses by participating in protein protection mechanism and by binding to metal ions [36]. Similarly, SbLEA3-2 upregulates in leaf tissues under all stresses, acting as regulatory gene that participates in stress tolerance mechanism. SbLEA1-5, SMP-1, SMP-2, LEA3-2, LEA4-3, and many members of the SbLEA-2 group upregulate in stem under heat, drought, and salt stresses. Over expression of SiLEA14 enhances abiotic stress tolerance in foxtail millet [113]. While overexpression of tomato LEA25 enhances salt and chilling stress tolerance in yeast [29], NtLEA7-3 displays tolerance against cold, drought, and salt stresses in Arabidopsis [28]. The Brassica BnLEA4-1 expressed in E. coli exhibits tolerance to temperature and salt stresses [32]. The SbLEA-2 family members, a typical hydrophobic proteins, upregulate under different stresses, and the results are consistent with that of cotton which show high expression under drought stress [68]. The Medicago MtPM25, a hydrophobic protein participates in disaggregation of proteins under stress, but unable to protect membranes [114]. Thus, the abundant presence of LEA-2 genes under stress conditions indicates that they act as key factors in plant adaptation mechanism under diverse environmental stresses.

Conclusion

A systematic genome-wide analysis resulted in the identification of a total of 68 LEA genes in Sorghum, which are classified into 8 groups and distributed on all the chromosomes. For the first time in monocots, a typical hydrophobic group SbLEA2 is identified with large number of genes like that of dicots. Present study helps in understanding the evolution and functions of an important major family SbLEA2 by functional analysis. It appears that segmental and whole genome duplication plays an important role in their expansion. The gene organization and motif compositions of the LEAs are highly conserved which indicate their conserved functional roles. Alongside the abiotic stress-responsive elements, hormone specific, developmental, biotic and other cis-elements were identified, indicating their complex regulatory mechanism. Further, the diversified and tissue specific expression profiles provide a further insight into the possible functional divergence in SbLEA gene family. The transcriptional profiling under abiotic stress indicates they might play an essential role in stress tolerance. Taken together, present study lays the foundation for further investigations of the specific functions of these Sorghum LEA genes, especially LEA2 family, in other monocots with reference to abiotic stress tolerance.

Supporting information

[Figure omitted. See PDF.]

S1 Fig. MEME identified motif sequences of LEA proteins in Sorghum.

https://doi.org/10.1371/journal.pone.0209980.s001

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S2 Fig. Motif distribution of LEA proteins in Sorghum.

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S3 Fig. Web logos of SbLEA proteins conserved motifs.

https://doi.org/10.1371/journal.pone.0209980.s003

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S1 Table. SbLEA gene primers used in the gene expression analysis.

https://doi.org/10.1371/journal.pone.0209980.s004

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S2 Table. Types of protein kinases in the phosphorylation of SbLEAs.

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S3 Table. miRNAs targets SbLEA genes.

https://doi.org/10.1371/journal.pone.0209980.s006

(DOCX)

S4 Table. Conserved cis-acting elements in LEA promoters of Sorghum.

https://doi.org/10.1371/journal.pone.0209980.s007

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S5 Table. Non-synonymous to synonymous substitution ratios of LEA orthologs.

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S6 Table. dN/dS ratios of SbLEA orthologs between Sorghum, Setaria, Oryza, Brachypodium and Hordeum.

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(DOCX)

S7 Table. Native and relative expression analysis of SbLEAs.

https://doi.org/10.1371/journal.pone.0209980.s010

(DOCX)

Acknowledgments

MN is thankful to the UGC, New Delhi, for providing fellowship. PBK is thankful to the CSIR, New Delhi, for providing CSIR-Emeritus Scientist Fellowship.

Citation: Nagaraju M, Kumar SA, Reddy PS, Kumar A, Rao DM, Kavi Kishor PB (2019) Genome-scale identification, classification, and tissue specific expression analysis of late embryogenesis abundant (LEA) genes under abiotic stress conditions in Sorghum bicolor L. PLoS ONE 14(1): e0209980. https://doi.org/10.1371/journal.pone.0209980

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