Cotton Late Embryogenesis Abundant (LEA2) Genes Promote Root Growth and Confer Drought Stress Tolerance in Transgenic Arabidopsis thaliana.

Late embryogenesis abundant (LEA) proteins play key roles in plant drought tolerance. In this study, 157, 85 and 89 candidate LEA2 proteins were identified in G. hirsutum, G. arboreum and G. raimondii respectively. LEA2 genes were classified into 6 groups, designated as group 1 to 6. Phylogenetic tree analysis revealed orthologous gene pairs within the cotton genome. The cotton specific LEA2 motifs identified were E, R and D in addition to Y, K and S motifs. The genes were distributed on all chromosomes. LEA2s were found to be highly enriched in non-polar, aliphatic amino acid residues, with leucine being the highest, 9.1% in proportion. The miRNA, ghr-miR827a/b/c/d and ghr-miR164 targeted many genes are known to be drought stress responsive. Various stress-responsive regulatory elements, ABA-responsive element (ABRE), Drought-responsive Element (DRE/CRT), MYBS and low-temperature-responsive element (LTRE) were detected. Most genes were highly expressed in leaves and roots, being the primary organs greatly affected by water deficit. The expression levels were much higher in G. tomentosum as opposed to G. hirsutum The tolerant genotype had higher capacity to induce more of LEA2 genes. Over expression of the transformed gene Cot_AD24498 showed that the LEA2 genes are involved in promoting root growth and in turn confers drought stress tolerance. We therefore infer that Cot_AD24498, CotAD_20020, CotAD_21924 and CotAD_59405 could be the candidate genes with profound functions under drought stress in upland cotton among the LEA2 genes. The transformed Arabidopsis plants showed higher tolerance levels to drought stress compared to the wild types. There was significant increase in antioxidants, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) accumulation, increased root length and significant reduction in oxidants, Hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations in the leaves of transformed lines under drought stress condition. This study provides comprehensive analysis of LEA2 proteins in cotton thus forms primary foundation for breeders to utilize these genes in developing drought tolerant genotypes.

Drought stress is one of the major abiotic stress factors with deleterious effects in plant growth and development (Sofia ). With the ever changing environmental condition and erratic precipitation levels, plant production is projected to undergo further decline, that meeting the demands and needs of the growing population will be a challenge in the near future (Tilman ). Plants being sessile, the effects caused by the various abiotic stresses are enormous thus threatening their existence (Rejeb ). Plants have developed various coping strategies for continued survival under these extreme conditions, one of which is through the induction of various transcriptome factors (TFs) with the aim of boosting their tolerance level (Xiong and Ishitani 2006). One of the transcriptome factor (TF) that has a functional role under various abiotic stress conditions is a member of the late embryogenesis abundant (LEA) proteins (Rodriguez-Salazar ). LEA proteins are basically grouped into eight (8) sub families, named as LEA1, LEA2, LEA3, LEA4, LEA5, LEA6, seed maturation proteins (SMPs) and dehydrins (Battaglia and Covarrubias 2013). In several studies conducted on the genome wide identification, the proteins encoding the late embryogenesis abundant (LEA) genes have been found to be the most abundant among all the other LEA protein families (Yang and Xia 2011).

LEA2 proteins are the members of a larger protein family of the late embryogenesis abundant (LEA) (Hundertmark and Hincha 2008). As the name suggests, this group of proteins are found to in large quantities in seeds at the late stages of embryo development (Dure ). Even though, the LEA proteins are synonymous with the seeds, a number of LEA proteins have been detected in the other plant tissues, such as the vegetative tissues (de Nazaré Monteiro Costa ). The distribution of LEA proteins is not restricted to plants only, but have been found in animals (10) (Denekamp ) and in bacteria (11) (Espelund ). The LEA protein families basically have universal structural architecture, high hydrophilicity, low proportion of cysteine (Cys) and tryptophan (Trp) residues and high contents of arginine (Arg), lysine (Lys), glutamate (Glu), alanine (Ala), threonine (Thr) and glycine (Gly). Due to the unique and common features of the LEA proteins, the LEA proteins are mainly referred as hydrophilins with a hydrophilicity index of more than 1 and a glycine (Gly) content of more than 6% (Battaglia ).

The late embryogenesis abundant (LEA) proteins have been positively correlated with several of abiotic stress, and have been found to confer tolerance in plants such as Brassica napus (Dalal ), rice (He ) and Fagus sylvatica (Jiménez ). For instance, overexpression of Arabidopsis LEA gene, AtLEA3 have been found to enhance tolerance to drought and salinity stresses (Zhao ). Overexpression of a rice LEA gene type, OsLEA3-1 was found to confer drought tolerance (Xiao ). Similarly, the LEA gene HVA1 LEA gene from barley, was found to confer dehydration tolerance in transgenic rice (Babu ). In addition, SiLEA14, a novel gene was found to be highly expressed in the roots of foxtail millet under drought condition (Wang ). However, the precise roles of LEA proteins are still not well understood. A number of proposals have been made to explain the possible roles of the LEA proteins in plants during water deficit conditions, such as enzyme protection (Hand ), molecular shield (Furuki ), hydration buffer (Hundertmark ) and membrane interactions (Olvera-Carrillo ). To date, a number of studies have been conducted in trying to determine the distribution and characterization of the LEA proteins in various plants, for instance Arabidopsis (Hundertmark and Hincha 2008), Brassica napus (Dalal ), water melon (Celik Altunoglu ) among other plants. Despite all the significance of the LEA genes, little has been done to investigate their putative role in cotton in relation to drought stress tolerance.

Cotton (Gossypium hirsutum) is an economically important fiber and oil crop cultivated in many tropical and subtropical areas of the world, where they are constantly exposed to a range of abiotic stresses which includes drought, extreme temperature and high salinity (Mahajan ). The completion and publication of the draft genome sequences of upland cotton G. hirsutum (Li ), Gossypium arboreum (Li ) and Gossypium raimondii (Wang ) has become a valuable tool in elucidating the transcriptome factors (TFs) in cotton genomes. There is a paucity of information available about LEA2 sub family in upland cotton. Therefore, in this study we carried out the identification, characterization of the LEA2 genes in three cotton genomes and transformed a novel LEA2 gene, Cot_AD24498 into Arabidopsis thaliana, in which we further investigated the expression levels of the transformed gene in both the transgenic lines and the wild type (WT) under drought stress condition.

Materials and methods

Identification, Sequence Analysis, Phylogenetic Tree Analysis and Subcellular Location Prediction of The LEA2 Proteins In Cotton

G. hirsutum, tetraploid (AD) genome LEA2 protein sequences were downloaded from the Cotton Research Institute website (http://mascotton.njau.edu.cn). The G. arboreum of A genome LEA2 protein sequences were downloaded from the Beijing Genome Institute database (https://www.bgi.com/), and G. raimondii of D genome was obtained from Phytozome (http://www.phytozome.net/). The conserved domain of LEA2 protein (PF03168) was downloaded from Pfam protein families (http://pfam.xfam.org). The hidden Markov model analysis (HMM) profile of LEA2 protein was queried to carry out the HMMER search (http://hmmer.janelia.org/) (Finn ) against G. hirsutum, G. raimondii and G. arboreum protein sequences. The amino acids sequences were analyzed for the presence of the LEA2 protein domains by ScanProsite tool (http://prosite.expasy.org/scanprosite/) and SMART program (http://smart.embl-heidelberg.de/). The three cotton genomes LEA2 proteins together with the LEA2 proteins from Arabidopsis (http://www.arabidopsis.org/) and rice (http://rice.plantbiology.msu.edu/index.shtml) were used to investigate the evolutionary history and patterning in relation to orthology or paralogy among the proteins encoding LEA2 genes. A phylogenetic tree was constructed, the multiple sequence alignments of all the LEA2 proteins were done by Clustal omega, MEGA 7.0 software using default parameters as described by Higgins et al., (Higgins ). The physiochemical characteristics of all the obtained LEA2 proteins were determined through an online ExPASy Server tool (http://www.web.xpasy.org/compute_pi/). In addition, subcellular location prediction for all the upland cotton LEA2 proteins were determined through Wolfpsort (https://www.wolfpsort.hgc.jp/) (Horton ). The subcellular prediction results were further validated through other two online tools TargetP1.1 server (Emanuelsson ) and Protein Prowler Subcellular Localization Predictor version 1.2 (http://www.bioinf.scmb.uq.edu.au/pprowler_webapp_1-2/) (Bodén and Hawkins 2005).

Analysis of promoter regions, chromosomal locations and miRNA target prediction of LEA2 genes

To identify the presence of drought stress-responsive cis-acting regulatory elements in LEA2 promoter regions, 1 kb up and down stream region from the translation start site of the LEA2 genes were analyzed using the PLACE database (http://www.dna.affrc.go.jp/place/signalscan.html) (Higo ). The physical locations in base pair (bp) of each LEA2 genes were determined through BLASTN searching against the local database. Mapchart software (https://www.wur.nl/en/show/Mapchart.htm) (Voorrips 2002), was used to plot the gene loci on G. hirsutum, G. arboreum and G.raimondii chromosomes. Finally we analyzed the miRNA targeting the LEA2 genes by submitting all the coding sequences (CDS) of all the LEA2 genes to the psRNATarget database (http://plantgrn.noble.org/psRNATarget/).

Expression analysis of LEA2 genes and determination of the gene to be transformed

The qRT-PCR analysis was used to determine the expression changes of the LEA2 genes in response to drought stress in the two parental lines used. the upland elite cultivar, G. hirsutum is known to be drought sensitive while the wild tetraploid cotton, G. tomentosum is a drought tolerant (Zheng ). The two cotton genotypes were treated for drought stress for 14 days. The samples for RNA extraction were obtained from the leaves, stem and roots, at 0, 7 and 14 days of stress exposure. All the samples were taken in three biological replicates in both control and treated seedlings. In order to get the best sets of the LEA2 genes for carrying out qRT-PCR validation, we had to rely on the RNA-sequencing data profiled under drought stress condition. The RNA-Sequence data were downloaded from cotton research institute website (http://mascotton.njau.edu.cn/html/Data). RNAs were reversely transcribed to first strand cDNA by use of TransCript-All-in-One-First-Strand cDNA synthesis Super Mix for qPCR (TransGen, Beijing, China). The fluorescent quantitative primers were designed for the selected genes (24 up and 24 down regulated genes) using Primer Premier 5 (Supplemental Table S1). Actin gene served as a reference. The synthesized cDNA was pre-incubated at 95° for 15 sec, followed by 40 cycles of denaturation at 95° for 5 sec and extension at 60° for 34 sec. The fluorescence quantitative assay was used to analyze expression level of the LEA2 genes in root, leaves and stem tissues of cotton plant, and expression changes in G. hirsutum and G. tomentosum under drought stress. The assay was designed with three replicates and the results were analyzed with the double delta Ct method.

Transformation and Screening of Novel gene Cot_AD24498 (LEA2) in the Model Plant Arabidopsis thaliana (Ecotype Colombia-0) Lines

The gene was transformed into model plant, A. thaliana ecotype Colombia-0 (Col-0). The upland cotton, G. hirsutum, accession number CRI-12 (G09091801–2) was used to confirm for the presence of the Cot_AD24498 gene in various tissues. The pWM101-35S:Cot_AD24498 (LEA2) construct in Agrobacterium tumefaciens GV3101 was confirmed by gene specific primer, the forward primer sequence Cot_AD24498 (5′CGGATCCATGTCGGTAAAAGAGTGCGGC3′) and reverse primer sequence pair of Cot_AD24498 (5′GGTCGACTTACACGCTAACACTGCATCT3′), synthesized from Invitrogen, Beijing, China. The Arabidopsis Wild-type (WT) plants were transformed by use of floral dip method (Clough SJ und Bent A 1998). Infiltration media mainly composed of 4.3 g/l, sucrose 50 g/l (5%), 2-(4-morpholino) ethane sulfonic acid (MES) 0.5 g/l, Silwet-77 200 µl/l (0.02%), 6-benzylaminopurine (6-BA) 0.01 mg/l with pH of 5.7. Transformed lines of A. thaliana were selected by germinating seeds on 50% (0.5) MS (PhytoTechnology Laboratories, Lenexa, USA), containing 50 mg/l hygromycin B (Roche Diagnostics GmbH, Mannheim, Germany) for a duration of three (3) days at temperature of 4° to optimize germination. Upon which the seedlings were transferred to Arabidopsis conditioned growth room set at 16 hr light and 8 hr dark. After 7 days in selection medium, and at three true leaves stage, the seedlings were transplanted into small plastic containers filled with vermiculite and humus in equal ratios. The seedlings at generation T0 were grown to set seeds, the seeds obtained were generation T1. The T1 seeds were germinated in selective antibiotic medium; the one-copy lines were identified by determining the segregation ratio of 3:1 of the antibiotics-selectable marker. The 3:1 ratio of the segregated lines (T2) seeds were again germinated in antibiotics-selective medium, only the lines with 100% were selected for the development of T3 generation. The T3 homozygous progeny was bred from a T2 population after real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the selection of three out of the eight successfully transformed overexpressed lines (L2, L3, and L4) was done by using Cot_AD24498 (LEA2) forward primer sequence (5′CGAACATCCATCCCTCCAAC3′) and Cot_AD24498 (LEA2) reverse primer sequence (5′ATCATCAAGAAAACCGACCC3′) with total complementary DNA (cDNA) as template. The phenotypic investigations were carried out in T3 homozygous generation.

qRT-PCR Analysis of the Expression of Drought-Responsive Genes in Transgenic Arabidopsis

We assessed the action of the transformed gene in the transgenic lines and the wild type of the model plant, A. thaliana by carrying out expression analysis of two drought responsive genes. ABRE-binding factor 4 (ABF4) gene; forward sequence 5′AACAACTTAGGAGGTGGTGGTCAT3′ and reverse sequence 5′TGTAGCAGCTGGCGCAGAAGTCAT3′ and responsive to desiccation 29A (RD29A) gene with forward sequence 5′TGAAAGGAGGAGGAGGAATGGTTGG3′ and the reverse sequence 5′ACAAAACACACATAAACATCCAAAGT3′. Total RNA was isolated from four-week-old transgenic Arabidopsis seedlings and wild type (Columbia ecotype) grown under normal conditions (CK) and 15% PEG6000 treatments for 4 days. RNA extraction and real-time RT-PCR (qRT-PCR) analyzed was applied as described in the section” Expression analysis of LEA2 genes and determination of the gene to be transformed”, cotton Actin2 forward sequence 5′ATCCTCCGTCTTGACCTTG3′ and reverse sequence 5′TGTCCGTCAGGCAACTCAT3′ applied as the reference gene.

Quantification of oxidant and antioxidants in transgenic lines and the wild type

When plants are exposed to any form of stress, there are drastic changes which occurs both at molecular and cellular level in order to tolerate the stress factors (Gill ). Reactive oxygen species is an oxidant substance being produced continuously from the respiring cells, and plants have an elaborate mechanism to keep the level within nontoxic limit, but when stresses such as drought sets in, the ROS equilibrium shifts leading to excessive production. In this research work, we undertook to evaluate the various oxidants and antioxidants levels between the transgenic lines (L1, L2 and L3) compared to the wild type when exposed to drought stress condition. Catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), Malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels were quantified according to the method described by Bartosz (Bartosz 2005). The seeds for transgenic and the wild types were grown in0.5 MS for eight (8) days, then transferred to small conical containers filled with a mixture vermiculite and sand in the ratio of 1:1 and grown for 21 days. After 21 days, water was totally withdrawn from drought treated plants for a period of 8 days, while the controlled plants were watered normally. The leaf samples were then harvested for antioxidants and oxidant determination after 8 days of post stress exposure. The samples were obtained in triplicate, in which each represented a biological repeat.

Availability of Data Statement

The author do affirms that all the data supporting the conclusions of this research work are represented fully within the manuscripts and its supplementary files. Supplemental material available at Figshare: https://doi.org/10.25387/g3.6626849.

Results and discussion

LEA2 protein encoding genes in the cotton genome and other plants

In the identification of the LEA2 proteins in the three cotton genomes, we employed the Hidden Markov Model (HMM profile) of the Pfam LEA2 domains PF03168, as keyword to search the three cotton genome sequences databases. Based on the Pfam domain search, we obtained 200 LEA2 genes in G. hirsutum of AD genome, 101 LEA2 genes in G. raimondii of D genome and 110 LEA genes in G. arboreum of A genome. In order to ascertain the various genes obtained for the three cotton genomes, we carried out manual search through SMART (http://smart.embl.de/smart/) and PFAM database (http://pfam.xfam.org) to verify the presence of the LEA2 gene domain. Upon removal of the redundant sequences with no functional domain or those that lacked the LEA2 domains, we eventually obtained 157, 85 and 89 LEA2 proteins in G. hirsutum, G. arboreum and G. raimondii, respectively. The confirmed domains of the LEA2 proteins in the three cotton genomes were further analyzed for their functional domain attributes of the LEA2 proteins, by use of an online tool, conserved domain database (CDD) tool hosted in the NCBI database. The results showed that the LEA2 proteins were members of c112118 super family with E values ranging from 0 to 0.008 (Supplementary Table S2) and all contained transmembrane domain (Supplementary Table S3) The association of the LEA2s with transmembrane domain could possibly explain the reason why the LEA proteins are found in high concentrations in seeds at late stages of seed development, this possibly to aid in maintaining the stability of the cell membrane under dehydration state. Similar results have also been reported in some of the drought and salt enhancing genes such as Salicornia brachiata SNARE-like superfamily protein (SbSLSP), has been reported to be localized in the plasma membrane (Singh ). LEA2 proteins could be playing an integral role in maintaining non-lethal level of reactive oxygen species (ROS homeostasis) in order to minimize oxidative damages to cellular membranous and macromolecules, in addition, LEA2s could also be playing similar roles as the aquaporin’s, the water channel proteins, which are responsible in the regulation of water movement channels such as plasmodesmata and xylem vessels (Buckley 2015). Aquaporin’s (AQPs) have been associated with salt and drought stress tolerance in plants, the aquaporin’s share similar functional domain with LEAs, being basically membrane proteins (Li ).

The number of proteins encoding the LEA2 genes found in G. arboreum, G. raimondii and G. hirsutum were relatively higher than the number recorded in other plants, the entire repertoire of LEA proteins in the 8 LEA families outlined in (Hundertmark and Hincha 2008) have been found to be 34 in rice (Wang ), 30 in Chinese plum (Du ), 27 in tomatoes (Cao and Li 2014), 53 in poplar (Lan ) and 29 in potatoes (Charfeddine ), which is far below the individual numbers of LEA2 in the three cotton genome. The abundance of cotton proteins encoding the LEA2 genes could be possibly due to their unique characteristics of being more hydrophobic than other LEA2 proteins from other species and or they could have evolved much later after other transcriptome factors. The genome size of plants and animal is constant, and high abundance of a particular gene family gives an indication of their integral role in enhancing the survival of the plants. The ever changing environmental conditions, plants are constantly faced with hearse environmental condition and disadvantaged by their sessile nature. The survival of the plants under these extreme environmental conditions therefore is through the increase of more stress tolerance genes or integrating a more complex gene interaction in initiating adaptive response mechanisms aimed at increased tolerance levels (Avramova 2015).

Phylogenetic analyses of LEA2 proteins in G. hirsutum, G. arboreum and G. raimondii

Phylogenetic tree analysis provides valuable knowledge on the lines of evolutionary descent of different genes or proteins from a common ancestor, since its inception, it has remained a powerful tool for structuring classifications, biological diversity and for providing insight into events that occurred during gene evolution (Gregory 2008). In this study a total of 157, 85 and 89 LEA2 proteins were identified from G. hirsutum, G. arboreum and G. raimondii, respectively (Table 1). All the LEA2 proteins were aligned by the neighbor joining (NJ) method in ClustalW. The various LEA2 proteins from upland cotton, G. arboreum, G. raimondii, A. thaliana, T. cacao and G. max were analyzed. The inclusion of A. thaliana, T. cacao and G. max in the analysis of the cotton LEA2s was due to fact that Theobroma cacao share ancestral origins with cotton, A. thaliana and G. max have undergone whole genome duplication similar to cotton plant. The resulting phylogenetic tree showed that the cotton LEA genes tend to cluster together. Based on the clustering pattern, the LEA2 genes were sub-divided into 6 groups, namely group 1 with three sub-groups, group 2, group 3 with two sub-groups, group 4, group 5 and finally group 6 with 5 sub-groups. Groups 1, 2, 4 and 5 were entirely LEA2 proteins from the three cotton genomes.

Table 1

The identified LEA2 genes and their nomenclatural description

In this work	Hundertmark & Hincha (2008)	G. hirsutum	G. arboreum	G. raimondii	V. vinifera	B.napus	G. max	Arabidopsis
LEA2	LEA_2	157	85	89	1	4	5	3

The LEA2s seems to have evolved later among all the LEA genes, in the analysis of the LEA genes in sweet orange, the highest among all the 8 members of the LEA genes were members of the LEA2 (Muniz Pedrosa ), this kind of observation was replicated in a number of plants. More than a half of the phylogenetic tree was mainly covered by the cotton LEA2 proteins, with no presence of LEA2s from other plants used in the analysis of the phylogenetic tree. Theobroma cacao, being evolutionary related to cotton, a few members of the LEA proteins clustered with cotton, while majority of the proteins encoding the LEA2 genes from Theobroma cacao clustered together.

The late embryogenesis abundant (LEA2) proteins from A. thaliana were found to cluster with those of cotton LEA2s in group 3 and 6 (3-2 and 6-1) while Glycine max LEA2 proteins were predominantly found in group 6-1 (Figure 1). No ortholog gene pairs were detected between the proteins encoding the cotton LEA2 genes of cotton to any of the plants used. All the ortholog gene pairs occurred between G. hirsutum and G. arboreum, G. hirsutum and G. raimondii and G. arboreum and G. raimondii. Interestingly, even Theobroma cacao, which is evolutionary related to Gossypium species, had their LEA2 proteins clustered together.

Figure 1

Phylogenetic relationship of LEA2 genes in three cotton species with Arabidopsis, T. cacao and G. max. Neighbor-joining phylogeny of 157 genes for G. hirsutum, 85 genes for G. arboreum, 89 genes for G. raimondii, 9 genes for T. cacao, 5 G. max and 3 Arabidopsis LEA protein sequences, as constructed by MEGA7.0.

The abundance of LEA2s in plants can be explained by either being the last members of the LEA genes to evolve and or due to duplication. Upland cotton is a tetraploid cotton, having emerged through whole genome duplication (WGD) between the two diploid cotton of A and D genomes. The high number of LEA2 genes, have also been observed in Arabidopsis (Hundertmark and Hincha 2008). Therefore, we could infer that LEA2 proteins might have evolved later after species divergence and the presence of ortholog genes in the cotton genome could be due to the whole genome duplication event coupled with chromosome rearrangement. It is generally assumed that ortholog genes have the same biological functions in different species (Tatusov 1997), and duplication makes room for paralogous gene pairs to evolve new functions (Ohno 1970). LEA2 genes could be functionally-oriented ortholog groups consisting of orthologous pair which plays the same biological role in the three different cotton genomes.

Physio-chemical analysis, subcellular localization and amino acid composition of the LEA2 genes in upland cotton

In the analysis of the physio-chemical properties of the LEA2 genes in upland cotton, the proteins encoding the LEA2 genes had varied molecular formulae though with similar elemental composition, carbon (C), hydrogen (H), oxygen (O), nitrogen (N) and sulfur (S) in varying proportions. Molecular weights ranged from 11.5384 to 73.5831 kD, Pl values from 4.63 to 10.35, aliphatic index from 19.78 to 65.4, instability index from 6.91 to 63.52, protein lengths ranged from 100 to 661 bp and the grand average of hydropathy (GRAVY) values ranged from 0.574 to 1.04. The grand average hydropathy (GRAVY) values showed that almost all the LEA2s are hydrophobic proteins, the hydrophobic nature of proteins is integral for their biological functions, allows the proteins to fold spontaneously into complex three-dimensional structures that are significant for biological activity (Gosline ). The hydrophobic nature of the proteins enables the removal of nonpolar amino acids from solvent and their burial in the core of the protein, this attribute is common among the aquaporin’s (AQPs), water channel proteins, are highly hydrophobic and known to have a functional role in water and salt stress tolerance in plants (Sreedharan ). In the sub cellular localization prediction, 10 different sites were detected, in which majority of the LEA2 proteins were found to be localized within the chloroplast with 73 genes. Further analysis by TargetP and Pprowler, more than 70% of the genes were found to be associated with secretory pathway and chloroplast (Table 2 and Supplementary Table S4). The high number of these genes in chloroplast explains their significant role in drought stress, since chloroplast plays a central role in plant response to stress (Gläßer ). The connection between different stress responses and organellar signaling pathways such as reactive oxygen species, emanate from the chloroplast (Kmiecik ). Chloroplasts being semi-autonomous organelles provide complex communication channel that allow for effective coordination of gene expression since most plastid localized proteins are nuclear-encoded, thus ensuring an effective functioning of overall cellular metabolism (Pfannschmidt ). Numerous and vital cellular processes such as aromatic amino acids, fatty acids and carotenoids biosynthesis and sulfate assimilation pathways are harbored within the chloroplast, in addition to photosynthesis, these cellular processes are known to be key factors in plants response to stress. The chloroplast acts as a sensor to abiotic stress thus initiates different cell functions in response to stress factor, enhancing adaptability of the plant to the environmental stress (Mittler 2006). Higher proportions of LEA2 genes were found to be localized within the cytoplasm, nucleus and mitochondrion, with 24, 20 and 16 genes respectively, which further provided a stronger evidence of the importance of these genes in enhancing drought tolerance ability in cotton. The following cell structures contained low numbers of LEA2 genes, endoplasmic reticulum (E.R) with 3, extracellular structures with 5, Golgi body 6, plasma 4 and vacuole with 3 genes each. The result obtained for the subcellular localization of the LEA2 genes is in agreement to previous findings in which the highest proportions of LEA2 genes were found to be localized within the cytoplasm and chloroplast, accounting for 35.7% and 30.9% of the total LEA2 genes in sweet orange, while others were found to target endoplasmic reticulum (E.R) and mitochondrion (Muniz Pedrosa ). Similarly, abiotic stress related gene, plasma membrane protein 3 (PMP3), a member of the small hydrophobic polypeptides with high sequence similarity, and have been functionally characterized to be responsible for salt, drought, cold, and abscisic acid, have been found to be sub localized in the nucleus, cytoplasm, and cell membrane (Fu ).

Table 2

Physiochemical properties of LEA2 gene in upland cotton, G. hirsutum, subcellular location prediction and chromosome position

Gene Id	Molecular Formula	Atoms Numbers	Instability Index	Aliphatic Index	Gravy	Length (Aa)	Pl	Mw (Aa)	Chr No	Sub Cellular Localization
										Wolfpsort	TargetP	Prowler
CotAD_ 00275	C2550H4266N832O1061S220	8929	49.24	24.58	0.824	274	10	29834.66	Dt09_chr23	chlo	S	sp
CotAD_ 00465	C2809H4694N922O1183S186	9794	38.68	27.5	0.704	304	10	33689.28	Dt09_chr23	chlo	C	sp
CotAD_ 00799	C3119H5215N1021O1297S196	10848	42.14	31.89	0.776	337	9	38982.02	scaffold26.1	golg	C	sp
CotAD_ 00808	C2114H3538N688O893S149	7382	38.49	25.51	0.698	226	10	26011.22	scaffold26.1	cyto	_	sp
CotAD_ 01033	C1868H3118N616O781S132	6515	37.57	27.69	0.749	202	9	22587.14	Dt10_ch20	chlo	S	sp
CotAD_ 01298	C1996H3326N664O833S142	6961	35.29	27.79	0.754	218	10	24021.4	Dt10_ch20	cyto	_	other
CotAD_ 01321	C2138H3550N724O880S189	7481	48.5	25.62	0.855	238	10	26020.28	Dt10_ch20	cyto	S	sp
CotAD_ 01385	C2253H3753N751O944S189	7890	53.4	22.96	0.757	247	7	27497.03	Dt09_chr23	cyto	S	sp
CotAD_ 01700	C2382H3972N790O976S223	8343	52.12	25.76	0.914	260	9	28399.83	Dt09_chr23	cyto	_	sp
CotAD_ 02652	C2022H3396N646O835S184	7083	63.52	25.47	0.898	212	10	23764.43	Dt09_chr23	mito	S	sp
CotAD_ 03037	C2465H4132N796O1011S239	8643	54.69	25.19	0.943	262	9	28472.57	Dt05_chr19	cyto	S	sp
CotAD_ 03649	C2938H4904N970O1220S232	10264	42.02	27.07	0.811	320	10	35345.6	At_chr09	cyto	S	sp
CotAD_ 03784	C1076H1792N358O453S53	3732	26.71	31.74	0.644	116	7	13537.66	Dt07_chr16	chlo	_	other
CotAD_ 05724	C1834H3065N601O771S128	6399	46.85	26.71	0.719	197	10	22442.51	At_chr09	chlo	_	sp
CotAD_ 05725	C2229H3732N724O935S169	7789	50.4	25.76	0.755	238	10	27552.78	At_chr09	nucl	_	sp
CotAD_ 06037	C1893H3159N625O802S134	6613	45.22	24.24	0.668	205	10	22125.81	Dt13_ch18	chlo	_	sp
CotAD_ 07087	C1926H3222N628O819S106	6701	43.9	28.43	0.622	206	10	22853.64	At_chr02	plas	_	other
CotAD_ 08181	C1864H3110N616O780S135	6505	43.71	26.87	0.745	202	9	22460.02	Dt09_chr23	cyto	S	sp
CotAD_ 08350	C1894H3182N604O790S142	6612	49.06	27.91	0.802	198	5	22266.98	scaffold190.1	chlo	_	sp
CotAD_ 08837	C2300H3853N745O961S220	8079	55.29	20.73	0.825	245	9	26376.34	scaffold280.1	golg	S	sp
CotAD_ 09578	C2381H3970N790O977S223	8341	50.46	25.38	0.905	260	9	28406.84	At_chr09	chlo	_	sp
CotAD_ 09685	C2306H3847N763O928S220	8064	61.17	29.7	1.024	251	10	27153.8	Dt09_chr23	chlo	_	sp
CotAD_ 09732	C2198H3688N706O923S164	7679	47.07	26.14	0.755	232	9	25906.5	Dt09_chr23	chlo	C	sp
CotAD_ 10376	C2568H4293N841O1038S271	9011	60.05	25.86	1.033	277	10	30152.74	Dt01_chr15	chlo	S	sp
CotAD_ 11658	C2438H4075N799O1007S165	8484	34.86	31.99	0.823	263	10	29835.19	Dt08_chr24	cyto	_	sp
CotAD_ 11875	C1627H2717N535O682S94	5655	33.71	30.96	0.706	175	7	20070.28	scaffold42.1	chlo	S	sp
CotAD_ 11876	C1942H3245N637O798S180	6802	50.01	25.51	0.904	209	10	23563.32	scaffold42.1	chlo	_	other
CotAD_ 11878	C2121H3552N688O886S165	7412	55.97	26.24	0.785	226	10	25841.73	scaffold42.1	chlo	S	sp
CotAD_ 11879	C1215H2031N397O519S61	4223	41.32	28.35	0.574	129	10	15037.05	scaffold42.1	chlo	S	sp
CotAD_ 12375	C1765H2948N580O727S157	6177	61.3	25.95	0.879	190	9	21328.78	At_chr09	chlo	_	other
CotAD_ 13115	C1791H2994N586O760S122	6253	39.07	24.83	0.659	192	9	20770.35	Dt08_chr24	extr	_	sp
CotAD_ 13584	2310H3858N760O957S190	8075	46.59	26.65	0.832	250	10	28048.83	Dt06_chr25	golg	S	sp
CotAD_ 13827	C3342H5592N1090O1370S299	11693	55.07	27.48	0.922	360	8	40945.87	Dt12_ch26	E.R.	_	sp
CotAD_ 14147	C2022H3396N646O838S180	7082	61.99	25.16	0.871	212	10	23855.54	At_chr07	mito	S	sp
CotAD_ 15892	C2861H4789N931O1209S186	9976	40.47	27.23	0.688	307	8	34741.21	Dt12_ch26	chlo	_	sp
CotAD_ 16731	C2370H3954N784O980S202	8290	47.46	26.09	0.845	258	10	28519.44	Dt09_chr23	chlo	S	sp
CotAD_ 17044	C1387H2309N463O581S100	4840	43.02	26.25	0.725	151	5	16422.87	At_chr07	cyto	_	other
CotAD_ 17045	C2199H3654N742O907S185	7687	48.71	26.49	0.838	219	10	23930.18	At_chr07	cyto	_	other
CotAD_ 17062	C2047H3416N676O852S170	7161	50.56	25.07	0.802	244	10	27393.16	At_chr07	chlo	S	sp
CotAD_ 17101	C1958H3277N637O811S177	6860	53.57	24.41	0.86	222	9	25294.09	At_chr06	mito	_	sp
CotAD_ 17102	C2435H4063N805O1008S182	8493	41.98	29.02	0.817	209	10	23661.48	At_chr06	nucl	_	sp
CotAD_ 17103	C2213H3709N715O930S187	7754	61.46	22.72	0.767	265	7	30299.29	At_chr06	mito	_	sp
CotAD_ 17649	C1849H3077N619O759S131	6435	37.22	31.77	0.843	235	9	26726.9	At_chr10	chlo	S	sp
CotAD_ 18210	C1850H3079N619O757S134	6439	35.57	32.09	0.865	203	10	22501.33	scaffold377.1	cyto	_	other
CotAD_ 18233	C1630H2729N529O675S118	5681	40.59	29.98	0.822	203	10	22406.26	scaffold377.1	chlo	_	other
CotAD_ 18546	C2571H4299N841O1038S270	9019	58.81	26.34	1.04	173	10	19695.85	Dt09_chr23	chlo	_	sp
CotAD_ 18729	C1990H3320N658O828S137	6933	43.32	29.42	0.772	277	10	30227.97	scaffold336.1	chlo	S	sp
CotAD_ 19078	C1684H2807N559O714S128	5892	45.25	22.26	0.669	216	10	24007.7	At_chr12	nucl	S	sp
CotAD_ 19107	C2766H4629N901O1165S184	9645	42.16	27.59	0.71	183	9	20031.24	At_chr12	chlo	_	other
CotAD_ 19205	C941H1570N310O394S56	3271	35.65	30.19	0.703	297	7	33395.7	At_chr12	chlo	_	sp
CotAD_ 19213	C1704H2853N553O707S109	5926	45.8	32.12	0.793	100	10	11538.35	At_chr10	chlo	_	sp
CotAD_ 19214	C2114H3541N685O887S125	7352	35.76	30.89	0.719	181	9	20628.72	At_chr10	nucl	C	other
CotAD_ 19375	C2310H3858N760O958S187	8073	46.32	26.78	0.823	225	9	25956.2	Dt11_ch21	golg	S	sp
CotAD_ 20020	C1807H3029N583O761S123	6303	36.84	27.19	0.717	250	10	27947.68	At_chr06	mito	S	sp
CotAD_ 20308	C2201H3658N742O909S184	7694	46.29	26.35	0.83	191	10	21054.44	Dt06_chr25	chlo	_	sp
CotAD_ 21731	C2426H4054N796O986S230	8492	60.23	27.71	0.975	244	10	27381.21	Dt05_chr19	nucl	S	sp
CotAD_ 21924	C1845H3069N619O756S138	6427	38.31	30.96	0.86	262	10	28411.4	Dt11_ch21	nucl	S	sp
CotAD_ 23646	C2458H4115N799O1036S200	8608	53.5	22.84	0.738	204	10	21921.93	Dt07_chr16	nucl	_	other
CotAD_ 24019	C1624H2711N535O680S94	5644	33.71	31.14	0.711	203	10	22391.06	Dt06_chr25	mito	S	sp
CotAD_ 24497	C1941H3243N637O796S181	6798	50.1	25.83	0.916	263	9	29247.79	Dt10_ch20	chlo	S	sp
CotAD_ 24499	C2118H3546N688O883S170	7405	56.27	25.95	0.801	175	8	20026.25	scaffold238.1	chlo	_	sp
CotAD_ 25271	C2240H3751N727O937S188	7843	48.67	24	0.79	209	10	23559.33	scaffold238.1	nucl	S	sp
CotAD_ 26038	C1695H2826N562O718S127	5928	45.53	22.86	0.673	226	9	25852.71	scaffold238.1	chlo	_	sp
CotAD_ 26981	C1423H2384N460O593S106	4966	51.34	27.73	0.79	274	10	29936.66	At_chr09	chlo	C	sp
CotAD_ 27453	C2034H3390N676O861S160	7121	44.82	21.96	0.686	239	10	26994.13	scaffold477.1	mito	_	sp
CotAD_ 27789	C2367H3951N781O998S201	8298	53.3	21.31	0.731	184	9	20135.39	scaffold699.1	E.R.	_	sp
CotAD_ 28249	C2260H3788N730O947S140	7865	33.99	30.49	0.736	150	9	16764.6	At_chr09	nucl	_	sp
CotAD_ 28252	C2177H3646N706O916S180	7625	48.77	22.87	0.752	222	9	24982.77	At_chr07	mito	S	sp
CotAD_ 28872	C1387H2306N466O578S109	4846	48.91	25.43	0.764	257	9	26949.97	Dt03_chr17	nucl	_	sp
CotAD_ 29279	C1875H3141N607O784S137	6544	47.67	27.44	0.769	305	10	34588.47	Dt13_ch18	chlo	_	other
CotAD_ 31344	C2277H3795N757O932S181	7942	42.47	30.2	0.887	101	6	11711.01	scaffold1346.1	chlo	S	sp
CotAD_ 31535	C2944H4916N970O1223S231	10284	41.3	27.17	0.809	240	8	27649.86	At_chr05	vacu	S	sp
CotAD_ 31536	C2047H3416N676O854S171	7164	52.02	24.33	0.789	210	9	23875.63	scaffold1346.1	plas	S	sp
CotAD_ 31537	C1956H3273N637O809S177	6852	54.13	24.72	0.868	254	10	27558.52	scaffold1841.1	nucl	_	sp
CotAD_ 31780	C2649H4422N874O1100S195	9240	40.01	28.67	0.799	310	10	34525.38	Dt08_chr24	chlo	_	sp
CotAD_ 31782	C1944H3258N628O812S139	6781	46.14	28.27	0.774	210	8	23638.39	Dt09_chr23	chlo	S	sp
CotAD_ 31860	C4139H6916N1360O1727S338	14480	44.89	25.26	0.795	206	10	22839.69	scaffold257.1	cyto	_	sp
CotAD_ 31906	C1914H3198N628O804S148	6692	47.49	24.6	0.739	232	10	26256.38	scaffold769.1	cyto	C	sp
CotAD_ 31936	C2627H4393N859O1089S219	9187	47.93	26.37	0.838	152	5	16462.97	Dt01_chr15	mito	S	sp
CotAD_ 32487	C1940H3238N640O815S167	6800	51.19	21.79	0.753	305	10	33718.76	At_chr11	mito	_	sp
CotAD_ 32645	C1845H3066N622O771S148	6452	42.79	24.35	0.753	199	9	22785.41	Dt06_chr25	chlo	S	sp
CotAD_ 32847	C1752H2928N574O730S100	6084	39.49	32.87	0.745	249	10	27707.74	At_chr09	extr	S	sp
CotAD_ 33143	C3449H5767N1129O1433S246	12024	46.47	29.55	0.8	305	10	34544.43	Dt02_chr14	chlo	S	sp
CotAD_ 33144	C1970H3298N640O818S163	6889	54.12	26.18	0.83	240	9	27655.92	Dt05_chr19	chlo	_	sp
CotAD_ 34476	C2374H3959N787O982S206	8308	47.91	25.48	0.844	320	10	35579.84	Dt09_chr23	cyto	_	sp
CotAD_ 34798	C2925H4884N964O1214S245	10232	51.69	25.78	0.826	222	9	25253.03	Dt06_chr25	nucl	S	sp
CotAD_ 35069	C2296H3827N763O944S159	7989	48.49	32.19	0.84	209	10	23628.4	Dt06_chr25	chlo	S	sp
CotAD_ 35091	C2037H3411N661O855S133	7097	42.17	28.83	0.728	288	7	32755.52	Dt06_chr25	extr	_	sp
CotAD_ 35514	C1704H2853N553O708S110	5928	46.78	31.58	0.785	206	6	23420.27	Dt05_chr19	mito	S	sp
CotAD_ 36328	C1970H3298N640O819S162	6889	53.13	26.02	0.821	450	5	49131.5	scaffold821.1	chlo	C	other
CotAD_ 36446	C1628H2725N529O673S119	5674	44.25	30.17	0.833	231	10	24949.39	Dt08_chr24	chlo	_	other
CotAD_ 36583	C2954H4936N970O1224S234	10318	40.36	27.69	0.829	206	9	22761.2	scaffold821.1	chlo	_	sp
CotAD_ 37776	C1843H3062N622O768S149	6444	46.57	24.84	0.769	202	9	22357.93	Dt09_chr23	chlo	S	sp
CotAD_ 37888	C2554H4274N832O1063S219	8942	50.77	24.7	0.823	283	10	31410.18	At_chr08	chlo	S	sp
CotAD_ 38978	C2819H4711N925O1184S205	9844	42.1	26.22	0.734	210	10	22644.27	Dt08_chr24	nucl	S	sp
CotAD_ 39064	C969H1623N313O399S81	3385	56.09	27.65	0.874	210	10	23699.74	Dt01_chr15	chlo	S	sp
CotAD_ 39719	C1971H3300N640O818S160	6889	54.8	26.8	0.83	191	6	20961.07	Dt01_chr15	nucl	S	sp
CotAD_ 40324	C2364H3954N772O959S228	8277	54.49	27.79	0.995	204	10	21780.76	At_chr07	plas	_	sp
CotAD_ 41569	C2875H4808N940O1188S244	10055	50.55	26.76	0.862	208	10	22559.45	At_chr13	chlo	_	sp
CotAD_ 41571	C1947H3252N640O803S171	6813	59.76	26.02	0.87	270	10	30627.54	Dt09_chr23	chlo	_	sp
CotAD_ 41925	C1928H3226N628O816S110	6708	46.13	29.07	0.656	188	9	21941.4	scaffold1231.1	nucl	_	other
CotAD_ 42599	C2794H4661N925O1169S206	9755	43.38	26.65	0.752	373	10	43118.75	scaffold1231.1	cyto	_	other
CotAD_ 44357	C2819H4711N925O1183S209	9847	44.41	26	0.743	210	9	23874.6	scaffold1088.1	cyto	C	sp
CotAD_ 45324	C2259H3786N730O944S141	7860	34.69	31.04	0.754	256	10	28431.93	Dt11_ch21	chlo	S	sp
CotAD_ 46873	C2117H3529N703O871S205	7425	56.14	23.68	0.894	259	10	28603.52	At_chr09	vacu	S	sp
CotAD_ 47322	C1862H3106N616O776S139	6499	43.14	27.2	0.773	220	10	24666.72	At_chr03	chlo	S	sp
CotAD_ 47454	C1973H3304N640O818S176	6911	53.21	24.61	0.854	661	6	73583.12	scaffold1851.1	cysk	S	sp
CotAD_ 47495	C1754H2923N583O719S178	6157	55.01	23.06	0.922	318	10	35234.15	Dt07_chr16	chlo	S	sp
CotAD_ 47749	C1922H3208N634O818S131	6713	42.78	23.89	0.636	251	9	27769.63	Dt07_chr16	chlo	S	sp
CotAD_ 48050	C2571H4320N820O1053S198	8962	50.08	32.15	0.921	217	9	24968.87	Dt10_ch20	mito	_	sp
CotAD_ 48069	C2356H3932N778O994S159	8219	43.83	26.42	0.689	181	10	20577.73	Dt10_ch20	extr	S	sp
CotAD_ 48336	C2036H3400N670O835S177	7118	47.96	27.69	0.9	211	9	23479.93	Dt04_chr22	nucl	S	sp
CotAD_ 48753	C6218H10441N1993O2614S448	21714	47.81	27.02	0.752	210	9	23676.69	At_chr06	mito	_	sp
CotAD_ 48769	C1998H3351N643O829S165	6986	56.41	26.68	0.843	304	10	33675.21	At_chr09	nucl	_	sp
CotAD_ 49818	C2811H4698N922O1186S183	9800	36.96	27.39	0.691	317	5	35274.16	scaffold2616.1	cyto	S	sp
CotAD_ 53045	C2922H4881N961O1224S173	10161	37.17	30.97	0.72	206	8	22650.27	Dt10_ch20	cyto	S	sp
CotAD_ 53263	C1938H3246N628O811S135	6758	44.09	28.27	0.756	251	10	27168.81	At_chr09	chlo	_	other
CotAD_ 53981	C2316H3867N763O933S219	8098	61.7	29.83	1.021	247	7	27715.29	scaffold3326.1	mito	_	sp
CotAD_ 54337	C2251H3749N751O943S189	7883	54.62	22.96	0.757	152	5	16453.02	At_chr07	chlo	_	sp
CotAD_ 55224	C1390H2312N466O579S109	4856	50.61	25.65	0.768	210	10	23769.83	Dt03_chr17	mito	S	sp
CotAD_ 56356	C1954H3266N640O822S101	6783	33.97	32.13	0.677	173	10	19737.98	At_chr09	chlo	_	other
CotAD_ 56696	C1963H3275N649O822S113	6822	33.29	31.22	0.71	213	10	23750.48	Dt03_chr17	nucl	S	sp
CotAD_ 58358	C1600H2547N445O483S11	5086	61.19	65.4		209	10	23626.51	Dt12_ch26	chlo	S	sp
CotAD_ 59405	C1936H3233N637O793S189	6788	54.41	24.72	0.93	320	10	35457.72	Dt05_chr19	chlo	_	sp
CotAD_ 60279	C2316H3879N751O968S220	8134	53.86	20.83	0.82	247	9	26619.63	scaffold2414.1	chlo	S	sp
CotAD_ 60435	C2292H3819N763O938S163	7975	49.59	32.72	0.869	251	10	27952.81	At_chr01	chlo	S	sp
CotAD_ 60617	C1977H3312N640O820S177	6926	54.15	24.45	0.855	210	10	23780.9	Dt01_chr15	mito	S	sp
CotAD_ 61173	C1964H3271N655O821S137	6848	38.49	27.72	0.739	215	10	24043	At_chr04	chlo	_	other
CotAD_ 61391	C1753H2921N583O718S179	6154	54.1	23.06	0.928	191	6	20884.97	Dt01_chr15	chlo	S	sp
CotAD_ 62996	C2926H4886N964O1214S245	10235	51.58	25.88	0.828	318	10	35356.25	At_chr01	nucl	S	sp
CotAD_ 63174	C3526H5909N1141O1460S281	12317	44.13	28.18	0.85	377	10	41228.93	scaffold3177.1	E.R.	S	sp
CotAD_ 64004	C2020H3371N667O833S157	7048	48.11	29.02	0.845	219	10	23825.02	Dt07_chr16	chlo	_	sp
CotAD_ 64120	C2001H3336N664O837S143	6981	33.36	27.19	0.743	218	10	24050.43	At_chr12	chlo	S	other
CotAD_ 64346	C1963H3284N640O817S168	6872	52.92	24.61	0.818	210	9	23572.5	Dt06_chr25	chlo	_	other
CotAD_ 64347	C2142H3567N715O883S231	7538	59.98	19.78	0.901	235	9	26111.93	Dt06_chr25	plas	_	sp
CotAD_ 64657	C2431H4064N796O990S225	8506	59.04	27.96	0.961	262	10	28516.58	At_chr11	vacu	_	sp
CotAD_ 65119	C1908H3186N628O800S147	6669	42.93	25.08	0.747	206	9	22733.19	Dt08_chr24	golg	S	sp
CotAD_ 65370	C1019H1668N278O359S3	3327	61.44	49		326	10	36098.18	scaffold3528.1	chlo	S	sp
CotAD_ 66245	C4148H6934N1360O1732S337	14511	45.38	25.26	0.792	450	5	48836.2	Dt08_chr24	chlo	C	sp
CotAD_ 66538	C1991H3337N643O823S168	6962	59.16	26.99	0.866	211	10	23424.96	At_chr04	chlo	S	sp
CotAD_ 66551	C2086H3485N685O872S114	7242	18.66	32.8	0.72	225	9	25226.24	scaffold3976.1	cyto	_	sp
CotAD_ 66774	C1993H3326N658O830S137	6944	42.93	29.27	0.768	216	10	24090.84	Dt08_chr24	chlo	S	sp
CotAD_ 66775	C2066H3445N685O872S139	7207	32.12	26.21	0.682	225	10	25078.29	Dt08_chr24	chlo	_	other
CotAD_ 67823	C2035H3392N676O841S191	7135	53.53	23.44	0.861	222	10	23928.26	At_chr08	cyto	S	sp
CotAD_ 68063	C2031H3396N664O856S167	7114	50.86	22.36	0.733	218	9	23245.72	At_chr03	cyto	_	sp
CotAD_ 68189	C1936H3242N628O808S135	6749	44.73	28.91	0.772	206	7	22579.21	At_chr10	chlo	S	sp
CotAD_ 69737	C1966H3281N649O821S117	6834	32.83	31.38	0.732	213	10	23867.69	scaffold2095.1	chlo	S	sp
CotAD_ 69738	C1956H3270N640O824S101	6791	32.31	31.82	0.669	210	10	23893.04	scaffold2095.1	chlo	S	sp
CotAD_ 70003	C1807H3029N583O761S120	6300	6.91	27.71	0.713	191	10	20942.44	At_chr12	cyto	_	sp
CotAD_ 70190	C3927H6552N1300O1658S217	13654	30.66	30.05	0.661	430	5	48185.02	scaffold4817.1	cyto	_	other
CotAD_ 70192	C1226H2050N400O509S77	4262	34.45	31.91	0.776	130	5	14420.49	scaffold4817.1	nucl	C	other
CotAD_ 71431	C1743H2916N568O719S152	6098	46.46	26.33	0.874	186	10	20579.98	Dt05_chr19	extr	C	sp
CotAD_ 72458	C1788H2988N586O760S119	6241	39.96	24.83	0.644	192	10	20613.31	scaffold3083.1	cysk	_	sp
CotAD_ 72913	C2901H4845N955O1214S173	10088	38.37	31.06	0.726	315	5	35071.89	scaffold4398.1	cysk	_	other
CotAD_ 73966	C2955H4938N970O1228S230	10321	41.06	27.38	0.809	320	10	35484.73	At_chr12	chlo	S	sp
CotAD_ 74713	C1998H3351N643O829S165	6986	56.41	26.68	0.843	211	9	23479.93	Dt08_chr24	golg	S	sp
CotAD_ 76129	C1937H3235N637O793S190	6792	54.41	24.72	0.935	209	10	23626.51	At_chr12	chlo	_	sp

The cell compartmentalization of stress related genes is fundamental to their functional role (Osman ), the presence of the proteins encoding LEA2 genes in the chloroplast, could be responsible for maintaining osmotic balance and suppression of reactive oxygen species (ROS) production in the guard cells (Wang ), while those present in the membrane, could be responsible for the protection of the membrane integrity (Guo ). In addition, the sub cellular localized proteins encoding LEA2 genes embedded in the channeling or transporter organelles such endoplasmic reticulum, are likely to aid in the process of the ions sequestration (Porcel ). Based on various findings, the LEA protein families are known to have a universal structure, with varying proportions of the various amino acids (Hong-bo ). In order to verify the LEA2 proteins due to their unique hydrophobic property, we found that the LEA2s are rich in non-polar aliphatic amino acid residues, in which the highest proportion was noted in leucine with 9.2%, Valine with 8.2%, isoleucine (6.3%), alanine (5.9%) and the least was proline (5.7%). The high proportions of the non-polar residues, indicated that the LEA2 proteins are mainly embedded within the membrane, non-polar amino acids are found in the center of water soluble proteins while the polar amino acids are found at the surface (Petukhov ). The second in proportions were the polar, non-charged residues such as serine (8.9%), threonine (6.4%), cysteine 1.9%), methionine (2.2%), asparagine (5.0%) and glutamine (3.4%) The high proportions of the polar residues have been found to be predominant among the stress related proteins, such as the heat shock proteins (HSPs) (Wang ), therefore the presence of the polar residue, indicated that the LEA2 proteins could be responsible for coating the cellular macromolecules with a cohesive water layer and in turn protect the membrane and the membrane bounds multiprotein complexes from unfolding and aggregation during drought stress condition.

Genomic organization and motif detection of LEA2 proteins in cotton

Analysis of the exon-intron structure of all the 157 LEA2 genes was done using the gene structure displayer (http://gsds.cbi.pku.edu.cn/), a greater percentage of the LEA2 genes and their exons were highly conserved within the group (Supplementary Figure S1). Most of the LEA2 genes were intronless, with 114 genes, accounting for over 73%, of the LEA2s found to be intronless. The existence of introns in a genome is argued to cause enormous burden on the host (Wahl ). The burden is because the introns requires a spliceosome, which is among the largest molecular complexes in the cell, comprising of 5 small nuclear RNAs and more than 150 proteins (Wahl ). Intron transcription is costly in terms of time and energy (Lane and Martin 2010). Due to various stresses in which the plants are exposed to, the energy demand for survival is relatively high, thus various gene actions within the plant has to function under conserved energy demand threshold (Timperio ). A plant under stress condition requires to survive the effects caused by overload of excessive production of reactive oxygen species (ROS), 3,4-Methylenedioxyamphetamine (MDA) and low levels of Peroxidase (PODs) activities, therefore most of the genes responsible for stress tolerance either lack introns or possess significantly reduced number of introns within their gene structure (Jeffares ). Being the transcription process of the intron laden genes requires a lot of time and energy, which is hypothesized to cause or results into deleterious effect on gene expression (Calderwood ). Conserved motifs in the 157 LEA2 proteins were identified through an online tool MEME (Supplementary Figure S1). The motif lengths identified by MEME (http://meme-suite.org/), were between 14 and 112 amino acids in LEA2 proteins, similar results of conserved motif with lengths between 11 and 164 amino acids were obtained in cotton MYBs protein (He ). The homology in motif lengths with that of MYBs provided significant evidence supporting the possible role of the LEA2s in response to water stress which includes the regulation of stomatal movement, the control of suberin and cuticular waxes synthesis and the regulation of flower development (He ). Most of the LEA2 proteins had distinctive motifs, which are valuable for their identification, the common motifs identified for the cotton LEA proteins were; motif 1 (FFVLFSVFSLILWGASRPQKPKITMKSIKFENFKIQAGSDFSGVPTDMITMNSTVKMTYRNTATFFGVHVTSTPLDLSYSQJTIASG), motif 2 (WLVFRPKKPKFSLQSVTVYAL), motif 3 (NFQVTVTARNPNKRIG IYYD), motif 5 (TVKNPNFGSFKYDNSTVSVNYRGKVVGEA) and motif 14 (RRRSCCCCCCLWTLJ) (Supplementary Figure S2).

The number of the conserved motifs in each LEA2s varied between 1 and 7. The majority of close members in the phylogenetic tree exhibited common motif compositions, which suggested they have a functional similarity within the same subgroup. The alignment results of the LEA2 proteins showed various segments such as Y-segment, K-segment and S-segments (Supplementary Figure S3), which have been previously described in dehydrins (Hanin ). Other unique segments identified were E, R and D segments. The K segment has been found to form an amphipathic α-helix (Monera ). The K-segments assumes α-helical structure identical to class A2 amphipathic α-helices mainly found in apolipoproteins, apolipoproteins facilitate the transportation of water-insoluble lipids in plasma, and α-synucleins (Rorat 2006). The conformation of the protein structure in turn leads to functional change (Dyson and Wright 2005). Drought stress alters the protein ambient microenvironment, leading to protein conformational and functional changes (Mahdieh ). The amphipathic α-helices have the ability to interact with the dehydrated surfaces of various other proteins and biomembranes (Cornell and Taneva 2006). The binding of dehydrins to the dehydrated surface of other proteins enhances formation of amphipathic α-helices which protects other proteins from further loss of water. The presence of this K segment in LEA2 revealed the significant role played by these proteins in plants during drought stress. It has been suggested that the protective role of the LEA proteins is due to their ability to form α-helices which enables them to interact with other proteins and or biomembranes (Koag 2003). Kovacs et al., (Kovacs ), reported the protective activities of two dehydrin proteins isolated from A. thaliana, early response to dehydration 10 (ERD10) and early response to dehydration 14 (ERD14), against thermal inactivation of alcohol dehydrogenase and thermal aggregation of citrate synthase.

Chromosomal location and duplication events of cotton LEA2 genes

A gene’s location on a chromosome plays a significant role in shaping how an organism’s traits vary and evolve (Lazazzera and Hughes 2015). Chromosomes hold thousands of genes, with some situated in the middle of their linear structure and others at either end (Bickmore and Van Steensel 2013). Therefore, for us to understand the gene distribution and mapping positions of the LEA2 genes, the positions of each LEA2 genes were mapped on the A, D and AD cotton chromosome by carrying out homology search against the full-lengths of G. arboreum (A-genome), G. raimondii (D-genome) and G. hirsutum (AD genome) assembly. The LEA2 genes were mapped in all the 26 chromosomes in G. hirsutum, 13 chromosomes in G. arboreum and 12 chromosomes in G.raimondii. In diploid cotton genome, G. arboreum and G. raimondii, the gene distribution pattern was almost identical to the tetraploid cotton gene distribution (Supplementary Figure S4). In chromosome 9 in G. arboreum and its homolog chromosome in G. raimondii, a significant level of gene loss was observed in which only a single gene was contained in chr09 of G. arboreum compared to 10 genes in chr09. But more interestingly, there was total gene loss in chr13 of G. raimondii. The lack of LEA2 genes in chr13 in G. raimondii could only be accounted for due to either gene loss or gene deletion, for most of the LEA genes are found in every chromosome. The occurrences of LEA2 genes on every chromosome indicated that the genes are widely distribution on the entire cotton genome. However, the density of these loci was variable across the 26 chromosomes of upland and 13 chromosomes in A and D diploid cotton. The largest number of genes were located on chromosomes At09 (chr09) and Dt09 (chr23), with 12 and 14 genes respectively, followed by chromosome, Dt08 (chr24) with 10 genes, Dt 06 (chr25) with 9 genes, At07 and At12 with 12 genes each. The lowest loci ranged from 1 to 5 genes, with chromosome At02, At05, At09, Dt02 (chr14) and Dt04 (chr22) had a single gene each (Supplementary Figure S5). A total of 39 genes were not mapped and thus grouped as scaffold. The distribution of the genes on the chromosomes appeared to be uneven.

In general, the central sections of chromosomes were located with less LEA2 genes and relatively high densities of upland cotton LEA2s were observed in the top and bottom sections of most chromosomes. Similar gene loci clustering pattern was also observed in GrMYB genes distribution in which most of the genes were clumped either on the upper or lower regions of the chromosomes (He ). A gene’s location on a chromosome plays a significant role in shaping how an organism’s traits vary and evolve (Sexton and Cavalli 2015). It has been found that evolution is less a function of what a physical trait is, but more of where the genes that affect that trait are located in the genome (Sexton and Cavalli 2015). The distribution of this subset of LEA genes across the whole cotton genome provided a significant role played by these genes within the plant.

The main cause of gene expansion in a genome or organism is either due to segmental or tandem duplication (Cannon ). Two or more genes located on the same chromosome, one following the other, confirms a tandem duplication event, while gene duplication on different chromosomes is designated as segmental duplication event (Yu ). In the present study, cluster formations by the LEA2 genes explained the mechanism behind their expansion in cotton. Most of the duplicated genes were between G. hirsutum and its ancestors, G. arboreum (53) and G. raimondii (11) (Table 3). The tetraploid cotton, G. hirsutum evolved due to whole genome duplication resulting into polyploidy cotton. The Ka/Ks values ranged from 0 to 2.17333, with an average value of 0.4238, which implied that majority of the gene pair had Ka/Ks values of less than 1, which indicated that the LEA2 genes have been influenced extensively by purifying selection during the process of their evolution.

Table 3

Gene duplication, synonymous (Ks), nonsynonymous (Ka) and Ka/Ks values calculated for paralogous LEA2 gene pairs in cotton genome

Gene type	Paralogous gene pairs		Length (aa)	Ka	Ks	Ka/Ks	Negative/purifying selection	P-Value (Fisher)
LEA2	CotAD_59405	CotAD_76129	627	0	0.00654	0	YES	0
LEA2	CotAD_20020	Cotton_A_01845	750	0	0.00568	0	YES	0
LEA2	CotAD_19078	Cotton_A_23172	648	0	0.00672	0	YES	0
LEA2	CotAD_08181	Cotton_A_27543	606	0	0.00697	0	YES	0
LEA2	CotAD_48976	Cotton_A_29779	660	0	0.00642	0	YES	0
LEA2	CotAD_35514	Gorai.010G176400.1	543	0	0.00822	0	YES	0
LEA2	CotAD_31536	Cotton_A_13470	627	0.00211	0.03373	0.06246	YES	0.00360292
LEA2	CotAD_37888	Cotton_A_08663	960	0.04378	0.55839	0.07841	YES	1.73E-37
LEA2	CotAD_03649	CotAD_37888	960	0.04522	0.54142	0.08352	YES	9.32E-36
LEA2	CotAD_03649	Cotton_A_14478	960	0.04592	0.52972	0.08668	YES	3.29E-35
LEA2	CotAD_03649	CotAD_73966	960	0.04597	0.527	0.08723	YES	4.70E-35
LEA2	CotAD_17102	CotAD_31536	627	0.00422	0.03365	0.12547	YES	0.0107355
LEA2	CotAD_44941	Gorai.005G203000.1	720	0.00175	0.01368	0.12779	YES	0.0998325
LEA2	CotAD_08181	CotAD_46550	606	0.00654	0.04975	0.1315	YES	0.00250188
LEA2	CotAD_17101	Cotton_A_13469	666	0.00195	0.01318	0.14805	YES	0.121749
LEA2	CotAD_09578	Cotton_A_02196	780	0.0903	0.59944	0.15064	YES	7.07E-24
LEA2	CotAD_35069	CotAD_62996	954	0.00551	0.03643	0.15116	YES	0.0017334
LEA2	CotAD_59405	Cotton_A_40363	627	0.00636	0.04016	0.15842	YES	0.00848415
LEA2	CotAD_17045	Cotton_A_14354	657	0.00201	0.01262	0.15958	YES	0.13409
LEA2	CotAD_09685	CotAD_53981	753	0.00711	0.04386	0.16211	YES	0.00252472
LEA2	CotAD_01700	Cotton_A_02196	780	0.09992	0.58986	0.16939	YES	8.68E-22
LEA2	CotAD_17062	CotAD_21731	732	0.00719	0.04161	0.17276	YES	0.00506705
LEA2	CotAD_35069	Cotton_A_24356	954	0.00551	0.03178	0.17329	YES	0.00508945
LEA2	CotAD_10376	Cotton_A_05625	831	0.00645	0.03444	0.18723	YES	0.00723285
LEA2	CotAD_21924	Cotton_A_18919	786	0.01028	0.05219	0.19697	YES	0.00026749
LEA2	CotAD_31535	Gorai.006G150200.1	666	0.00391	0.01981	0.19743	YES	0.082505
LEA2	CotAD_25271	Cotton_A_14676	405	0.00647	0.03234	0.20023	YES	0.085476
LEA2	CotAD_09685	Cotton_A_05444	753	0.0089	0.04387	0.20282	YES	0.00516244
LEA2	CotAD_46888	Cotton_A_09596	573	0.00922	0.0453	0.20351	YES	0.0147038
LEA2	CotAD_08181	Gorai.009G305100.1	606	0.00435	0.02103	0.20672	YES	0.090366
LEA2	CotAD_19078	CotAD_66774	648	0.01009	0.04842	0.20844	YES	0.00834864
LEA2	CotAD_32487	Cotton_A_13240	630	0.00425	0.01917	0.22185	YES	0.103356
LEA2	CotAD_23118	CotAD_74061	1215	0.01611	0.06882	0.23405	YES	5.00E-05
LEA2	CotAD_36328	CotAD_64346	630	0.01777	0.07564	0.23489	YES	0.000973496
LEA2	CotAD_32847	CotAD_39064	612	0.01106	0.0461	0.23994	YES	0.0153075
LEA2	CotAD_46873	CotAD_60617	630	0.00835	0.03452	0.24185	YES	0.0372109
LEA2	CotAD_46873	Cotton_A_09615	630	0.00835	0.03452	0.24185	YES	0.0372109
LEA2	CotAD_18546	CotAD_37776	519	0.01016	0.04195	0.24212	YES	0.0375368
LEA2	CotAD_19375	Cotton_A_06435	675	0.01345	0.05541	0.24268	YES	0.00759106
LEA2	CotAD_46888	CotAD_61391	573	0.01387	0.05313	0.26111	YES	0.0175133
LEA2	CotAD_23118	Cotton_A_38117	1215	0.01611	0.06077	0.26514	YES	0.000321992
LEA2	CotAD_19214	Cotton_A_30889	543	0.00237	0.0083	0.28598	YES	0.347253
LEA2	CotAD_31535	Cotton_A_13469	666	0.01377	0.04718	0.2919	YES	0.0234164
LEA2	CotAD_21924	CotAD_64657	786	0.01373	0.04693	0.29247	YES	0.0120925
LEA2	CotAD_31140	Cotton_A_15998	747	0.00174	0.0058	0.30099	YES	0.356655
LEA2	CotAD_30219	Cotton_A_32495	597	0.01105	0.03626	0.30482	YES	0.0618481
LEA2	CotAD_46873	Gorai.001G124400.1	630	0.00208	0.00674	0.30909	YES	0.361889
LEA2	CotAD_46888	Gorai.001G122700.1	573	0.0046	0.0148	0.31039	YES	0.238274
LEA2	CotAD_28252	CotAD_53263	492	0.01356	0.04285	0.31656	YES	0.069282
LEA2	CotAD_14147	Cotton_A_02370	636	0.00416	0.01312	0.3169	YES	0.244174
LEA2	CotAD_23646	Cotton_A_27300	609	0.04249	0.13135	0.32348	YES	0.000630664
LEA2	CotAD_09578	Cotton_A_07036	780	0.00342	0.01037	0.33004	YES	0.256013
LEA2	CotAD_17045	CotAD_64004	657	0.02247	0.06523	0.34445	YES	0.0157104
LEA2	CotAD_37888	CotAD_73966	960	0.01528	0.0442	0.34576	YES	0.0157353
LEA2	CotAD_37888	Cotton_A_14478	960	0.01247	0.03528	0.3534	YES	0.0321315
LEA2	CotAD_23646	Gorai.006G199800.1	609	0.04249	0.11411	0.37237	YES	0.00460089
LEA2	CotAD_17062	Cotton_A_14370	732	0.0099	0.02648	0.37402	YES	0.0618224
LEA2	CotAD_02652	Cotton_A_02370	636	0.01256	0.03311	0.37934	YES	0.101339
LEA2	CotAD_19214	CotAD_35514	543	0.00955	0.02509	0.38065	YES	0.19023
LEA2	CotAD_21731	Cotton_A_14370	732	0.00899	0.02354	0.38192	YES	0.138838
LEA2	CotAD_13584	Cotton_A_01845	750	0.00878	0.02294	0.38264	YES	0.139381
LEA2	CotAD_17101	CotAD_31535	666	0.01576	0.04026	0.3915	YES	0.077316
LEA2	CotAD_35091	CotAD_60435	753	0.03016	0.07689	0.39221	YES	0.0144267
LEA2	CotAD_20308	CotAD_70003	573	0.00915	0.02291	0.39918	YES	0.206152
LEA2	CotAD_50359	CotAD_66538	633	0.01677	0.04094	0.40958	YES	0.0891624
LEA2	CotAD_01700	Cotton_A_07036	780	0.01551	0.03701	0.41916	YES	0.0752732
LEA2	CotAD_02652	CotAD_14147	636	0.00835	0.01974	0.42281	YES	0.226532
LEA2	CotAD_35513	Cotton_A_30890	651	0.02193	0.05102	0.42978	YES	0.0738291
LEA2	CotAD_35514	Cotton_A_30889	543	0.00716	0.01659	0.43135	YES	0.312651
LEA2	CotAD_28872	Gorai.005G203000.1	720	0.01233	0.02762	0.44641	YES	0.170613
LEA2	CotAD_56699	Cotton_A_38534	639	0.02021	0.04493	0.44988	YES	0.106618
LEA2	CotAD_01700	CotAD_09578	780	0.01202	0.02634	0.45642	YES	0.151105
LEA2	CotAD_40972	Cotton_A_29659	591	0.96659	2.0709	0.46675	YES	0.00123143
LEA2	CotAD_40972	CotAD_38978	591	0.96025	2.04193	0.47026	YES	0.00125135
LEA2	CotAD_17101	Gorai.006G150200.1	666	0.01977	0.04018	0.49197	YES	0.209339
LEA2	CotAD_50359	Cotton_A_33548	633	0.01678	0.03388	0.49528	YES	0.175709
LEA2	CotAD_74713	Cotton_A_33548	633	0.01678	0.03388	0.49528	YES	0.175709
LEA2	CotAD_03649	CotAD_31344	960	0.01103	0.02214	0.49798	YES	0.177084
LEA2	CotAD_13584	CotAD_20020	750	0.00878	0.01711	0.51348	YES	0.287642
LEA2	CotAD_13115	Cotton_A_31059	576	0.0207	0.0379	0.54625	YES	0.312514
LEA2	CotAD_19214	Gorai.010G176400.1	543	0.00955	0.01664	0.57418	YES	0.403293
LEA2	CotAD_20308	Cotton_A_17625	573	0.01375	0.02296	0.59881	YES	0.347235
LEA2	CotAD_25271	CotAD_48769	405	0.00647	0.01063	0.6094	YES	0.539117
LEA2	CotAD_12681	Cotton_A_08212	432	0.03121	0.04928	0.63319	YES	0.35887
LEA2	CotAD_19623	CotAD_36999	282	0.03296	0.04771	0.69081	YES	0.631725
LEA2	CotAD_23646	Cotton_A_27282	609	0.02587	0.03738	0.69204	YES	0.542393
LEA2	Cotton_A_13471	CotAD_17103	180.94	2.32397	1.11323	0.77822	YES	837
LEA2	CotAD_53438	CotAD_68189	618	0.02341	0.02898	0.80786	YES	0.519399
LEA2	CotAD_56696	Cotton_A_38535	630	0.01838	0.02269	0.80979	YES	0.670475
LEA2	CotAD_44941	Cotton_A_17986	720	0.01233	0.01369	0.90056	YES	0.874489
LEA2	CotAD_22539	Cotton_A_25195	408	1.23265	1.24112	0.99317	YES	1
LEA2	CotAD_28872	CotAD_44941	720	0.0141	0.01369	1.03042	NO	0.900519
LEA2	CotAD_17103	Cotton_A_13471	837	2.58712	2.32397	1.11323	NO	0.778217
LEA2	CotAD_13827	Cotton_A_18645	1104	2.12092	1.89653	1.11832	NO	0.642563
LEA2	CotAD_10044	Cotton_A_09473	1902	0.00274	0.00228	1.20458	NO	0.731531
LEA2	Cotton_A_31083	CotAD_35069	939	2.2748	1.83858	1.23726	NO	0.447623
LEA2	CotAD_30219	Gorai.006G104100.1	597	0.00884	0.00707	1.25015	NO	0.743557
LEA2	CotAD_03649	Cotton_A_08663	960	0.00549	0.00437	1.25606	NO	0.744588
LEA2	CotAD_11658	Cotton_A_40499	789	0.02309	0.01751	1.3191	NO	0.985982
LEA2	CotAD_12375	CotAD_42408	597	2.42062	1.68288	1.43838	NO	0.288342
LEA2	CotAD_35091	Cotton_A_24371	699	3.50309	1.61186	2.17333	NO	0.036477

Cis element prediction in LEA2 proteins

Transcription factors (TFs) and cis-acting regulatory elements contained in stress-responsive promoter regions function not only as molecular switches for gene expression, but also as terminal points of signal transduction in the signaling processes (). The cis-regulatory promoters are located on the upstream of genes and functions as binding sites for transcription factors (TFs) which play essential functions in determining the tissue-specificity or stress-responsive expression patterns of the genes (Yamaguchi-Shinozaki and Shinozaki 2005). For better understanding of the potential roles of the LEA2 genes, 1000 bp regions upstream of the transcriptional start site were extracted and used in the identification of cis-regulatory promoters and other important motifs. Abiotic stress-related cis-elements were found in the putative promoters of LEA2 genes in upland cotton, G. hirsutum, (Figure 2) and (Supplementary table S5). For instance, MYBCORE, is known to have a functional role in drought and regulation of flavonoid biosynthesis (Solano ). ABRELATERD1, ABRE-like sequence and ACGTATERD1 are responsive to dehydration (Simpson ). ACGTATERD1 is associated to early responsive to dehydration (Simpson ). The presence of the stress promoter elements strongly supported the possible role of upland cotton LEA2 proteins in enhancing drought tolerance in cotton. The high proportion of cis promoter elements in LEA2 proteins, could possibly explain why genes encoding LEA proteins are highly expressed under abiotic stress, as was found in the root tissues of Arabidopsis under drought stress (Dalal ; Candat ). It is also important to mention that various transcription factors (TFs) and cis-acting regulatory elements contained in stress-responsive promoter regions function not only as molecular switches for gene expression, but also as terminal points of signal transduction in the signaling processes (Yamaguchi-Shinozaki and Shinozaki 2005).

Figure 2

Average number of the cis-elements in promoter region of upland cotton G. hirsutum LEA2 genes. The cis-elements were analyzed in the 1 kb upstream promoter region of translation start site using the PLACE database.

Prediction of LEA genes targeted by miRNAs

Drought is a recurring climate feature in most parts of the world (Kang ). The sessile nature of the plants, has made the plants to developed their own defense systems to cope up with perennial and erratic adverse climatic conditions (Bartwal ). One of the defense mechanisms used by the plants toward the effect of drought stress is the reprogramming of gene expression by microRNAs (Ferdous ). The small RNAs (miRNAs) are known as the small noncoding RNAs with approximately 22 nucleotides length. The miRNAs are mainly involved in the regulation of genes at post-transcriptional levels in a range of organisms (Grivna ). Large groups of small RNAs have been reported as regulators in plant adaptation to abiotic stresses (Xie ). To get more information on the LEA2 genes functions, we determined the prediction of miRNAs targets on LEA2 genes by the use of psRNATarget, the same as been applied for other functional genes in cotton (Dai and Zhao 2011). Out of 157 upland cotton LEA2 genes, 63 genes were found to be targeted by 48 miRNAs, representing 40% of all the LEA2 genes (Supplementary Table S6). The highest levels of target was detected for the following genes with more than 6 miRNAs, CotAD_00799 being targeted by ghr-miR2948-5p, ghr-miR7492a, ghr-miR7492b, ghr-miR7492c, ghr-miR7494 and ghr-miR7510b. CotAD_19205 targeted by ghr-miR390a, ghr-miR390b, ghr-miR390c, ghr-miR7492a, ghr-miR7492b and ghr-miR7492c. CotAD_31936 targeted by ghr-miR7492a, ghr-miR7492b, ghr-miR7492c, ghr-miR827a, ghr-miR827b and ghr-miR827c. CotAD_32487 targeted by ghr-miR156a, ghr-miR156b, ghr-miR156d, ghr-miR7507 and ghr-miR7509. CotAD_33143 targeted by ghr-miR2948-5p, ghr-miR482a, ghr-miR7492a, ghr-miR7492b, ghr-miR7492c and ghr-miR7510b. CotAD_41925 targeted ghr-miR396a, ghr-miR396b, ghr-miR7492a, ghr-miR7492b, ghr-miR7492c, ghr-miR827a, ghr-miR827b and ghr-miR827c. The rest of the genes were either targeted by 1 or 5 miRNAs. The high number of miRNAs targeting LEA2 genes could possibly have direct or indirect correlation to their stress tolerance levels to abiotic stress more so drought. Some specific miRNAs had high level of target to various genes such as ghr-miR164 (4 genes), ghr-miR2949a-3p (4 genes), ghr-miR2950 (8 genes), ghr-miR7492a (10 genes), ghr-miR7492b (10 genes), ghr-miR7492c (10 genes), ghr-miR7504a (5 genes), ghr-miR7507 (5 genes), ghr-miR7510a (6 genes), ghr-miR7510b (10 genes), ghr-miR827b (4 genes) and lastly ghr-miR827c (4 genes). It has been found that miRNAs might be playing a role in response to drought and salinity stresses through targeting a series of stress-related genes.

The plant specific transcriptome factors such as NAC gene family have been found to have varied functional roles in plant growth and development (Pereira-Santana ), myeloblastosis (MYB) is highly correlated to various stress factors (Ambawat ). The detection of some the LEA2 genes being targeted by specific miRNA linked to mitogen-activated protein kinase (MAPK), N-acetyl-L-cysteine (NAC) and myeloblastosis (MYB) provided a stronger indication of the significance contributions of the LEA2s in enhancing drought tolerance in plants. The micro/small RNAs mediated post-transcriptional processes have been linked to response to water deficit condition. Plant miRNAs are involved in multi-complex and arrays of processes, including but not limited to response to stress, nutrient limitation, development, pattern formation, flowering time, hormone regulation, and even self-regulation of the miRNA biogenesis pathway (Yamaguchi-Shinozaki and Shinozaki 2005). It is important to note that most of the miRNA target genes encode transcription factors, which place miRNAs at the focal point of gene regulatory networks. Moreover, the availability of genome-wide characterization of cotton miRNA genes enabled us to perform the prediction of the miRNA targets involved in drought response.

Expression Patterns of LEA2 Genes in Different Tissues of Upland cotton as determined Through RNA sequence

Analysis of the RNA expression profile provides an indicator of the functional role of the genes in the plant. We therefore carried the RNA expression analysis (RPKM > 1) in various tissues of the cotton plant, out of the entire 157 LEA2 genes in upland cotton, G. hirsutum, 117 (75%) of all the LEA2 genes showed differential expression in various tissues, such as the leaves, roots, stem, petal, pistil, stamen, torus and calycle (Figure 3). Based on their expression profiling, the genes were clustered into three broad groups. Group 1 members with 29 genes were highly up regulated under drought and salt conditions. Under salt and drought stress, CotAD_33321, CotAD_41571, CotAD_11876, CotAD_24498 and CotAD_59405 showed the highest expression levels, Similarly CotAD_11876, CotAD_24498 and CotAD_59405 were equally significantly up regulated in all the tissues tested. A total of 23 genes were highly up regulated in 5 tissues, which provided a strong evidence of the functional role of the LEA2 genes in enhancing stress tolerance in plants. Majority of the analyzed genes, showed relatively lower expression levels in the root tissues, but CotAD_11876, CotAD_59405 and CotAD_24498 exhibited significant higher expression levels, with expression values of more than 2. A unique observation was made, among the moderately up regulated genes in the roots, the genes exhibited significant up regulation in the calyx. The up regulation of these genes in the reproductive tissues could be an indication of their functional role in the fiber development process.

Figure 3

Expression profile analysis of LEA2 genes in 5 upland cotton tissues. The LEA2 genes expressed (RPKM > 1) in leaf, stem, root, calyx and petal were represented according to their tissue specificity: (A): LEA2 genes RNA seq. expression profile under drought and salt stress. (B): LEA2 expression in the 8 different tissues and (C): Venn diagram quantification and common genes expressed among the 5 tissues.

In the validation of the expression profile of the LEA2 genes under drought stress condition, CotAD_24498, CotAD_21924, CotAD_20020 and CotAD_59405 were highly up regulated in root, stem and roots tissues under drought stress condition. However, the expression levels were much higher in G. tomentosum as opposed to G. hirsutum, suggesting that, these genes could be the key genes.

qRT-PCR Expression profiling of the LEA2 genes in leaf, stem and roots of upland cotton

Based on the results obtained from the RNA sequence data, 48 genes were selected for qRT-PCR validation. Two cotton genotypes were used, G hirsutum an elite cultivar, majorly grown around the world; it covers more than 90% the cotton growing regions in China but susceptible to drought stress condition. The second plant used was the G. tomentosum, wild cotton, native to the Hawaiian island, it is known for its high ability to tolerate salinity and drought stress conditions. The two cotton plants were grown in the greenhouse, and at three leaf stage, were exposed to drought for a period of 14 days. The roots stem and leaves were obtained for RNA extraction and qRT-PCR analysis. In the analysis of qRT-PCR profiling of various tissues, the results indicated high variability in transcript abundance of LEA2 genes in upland cotton (Figure 4). In G. tomentosum and G. hirsutum, majority of these genes showed relatively high expression in the root and leaf, except in stem. Leaves and roots are the main plant organs affected by drought stress (Alexandersson ). The plant leaf is the site for photosynthesis; drought stress might possibly be the cause of excess release of reactive oxygen species (ROS). ROS are toxic to the plants, the genes with high expression in the leaves, could perhaps be involved in the ubiquitin of the ROS, thus preventing the damage and maintain the normal functions of the photosynthetic cells. The high osmotic potential generated in the cytoplasm of guard cells during stomatal opening could probably lead to accumulation of LEA2s in leaf tissue. Increased osmotic potential within the guard cells necessitates mass flow of water into the guard cells, leading to its turgidity and thus opening of the stomatal pore, but during drought stress, the osmotic potential is never offset, and thus dehydration stress on the nucleus. The LEA2s increased accumulation within the leaf tissues, could be due to maintaining structural integrity and preventing the membranes from dehydration stress. The finding is consistent to proposed functions of the LEA genes, which is the protective role during abiotic stresses (Nylander ). The roots are the connection point between the water reservoir and the plants. High up regulation of LEA2 genes in the roots indicated that these genes could be involved in the water balance in the roots. Increased or high up regulation of LEA2s in the roots, further augment the primary role of LEA genes in plants, the protective function, roots are the very first plant organs to be affected by drought stress.

Figure 4

Venn den diagram of differential expressions of LEA2 genes in different plants tissues. A. tissues of G. hirsutum and B. tissues of G. tomentosum.

Expression profiles of LEA2 genes Under drought treatment in G. hirsutum and G. tomentosum

Gene expression profile provides vital information of the roles played by the genes in plants (Movahedi ). In order to determine the expression pattern of the LEA2 genes in tolerant and non-tolerant upland cotton genotypes, we carried the qRT-PCR validation of 48 LEA2 genes in leaves, roots and stem tissues. The 48 genes were selected based on the RNA sequence expression profile, 24 genes were up regulated while the other half were down regulated. The samples for qRT-PCR were collected at 0, 7 and 14th day of stress exposure, in which 0 day (control) was used as the reference point. More genes were up regulated in all the tissues of the drought tolerant genotype, G. tomentosum as compared to the drought sensitive genotype, G. hirsutum (Figure 5). The result obtained denotes that the drought resistant genotype have the potential to mobilize more drought related genes, when exposed to drought tolerance as opposed to the less tolerant genotypes, thus the higher expression levels, similar results were obtained in the expression for cold tolerance genes in Arabidopsis with varying tolerance levels, more genes were up regulated in the cold tolerant and in the cold susceptible genotype (Hannah ).

Figure 5

Differential expression of upland cotton LEA2 genes under drought stress. The heat map was visualized using Mev.exe program. (Showed by log2 values) under control and in treated samples for 7 and 14 days after drought treatment (i) G. tomentosum and (ii) G. hirsutum. Red–up regulated, green-down regulated and black–no expression. Red box indicate the cloned gene.

The up regulation of LEA2 genes under drought stress, could possibly explain their protective role in plants tissues under dehydration stress. For instance, HVA1, a LEA gene from barley (Hordeum vulgare L) was found to confer drought stress in transgenic rice (Babu ). Interestingly, some phylogenetic LEA2 gene pairs, orthologous genes were found to have differential expression pattern in either of the cotton genotypes (Figure 6), for instance, CotAD_71431 and CotAD_51205 exhibited varied expression pattern under drought and salt stress conditions as evident in the RNA expression analysis. The result suggests that even if these genes are cladded together; they could have developed different biological function over time. Orthologous genes are members of the genes with a common evolutionary origin and share greater percentage of sequence similarity (Nehrt ). According to the expression pattern of LEA2 genes in different tissues, it would be interesting to functionally characterize these genes in upland cotton, G. hirsutum. Majority of the LEA2 genes showed higher expression level in leaf and root tissues, which indicated the functional conservation of the gene sub family. The variation in expression between G. hirsutum and G. tomentosum could be due to broad changes in environmental conditions, G. tomentosum exhibits divergence signals that are associated with directionally selected traits and are functionally related to stress responses. These results suggest that stress adaptation in G. tomentosum might have involved the evolution of protein-coding sequences and thus these genes can be introgressed in to elite upland cotton, in order to boost their performance in the current face of declining fresh water and precipitation.

Figure 6

Quantitative PCR analysis of the selected LEA2 genes. Abbreviations: 7d-7 days and 14d-14 days of stress. Gh–G. hirsutum and Gt–G. tomentosum. Y-axis: relative expression (2-ΔΔCT. The enclosure indicated the cloned gene.

qRT-PCR Analysis of the Transformed Gene in Upland Cotton Tissues

Based on the expression analysis of the LEA2 genes in the various tissues of G. tomentosum (drought susceptible) and G. hirsutum (drought susceptible). We identified a single gene with significant expression in the various tissues and transformed the gene into the model plant, A. thaliana (Colombia ecotype-0). The gene CotAD_24498 was analyzed in various tissues of the upland cotton, G. hirsutum. This was carried out in order to determine its relative abundance within the plant. We found that the gene was more abundantly expressed in the reproductive tissues, more specifically in the petal and stamen (Figure 7A). In addition, we further carried out treatment on cotton seedlings after three true leaves stage under drought stress (PEG6000_15%) the samples for RNA extraction and qRT-PCR analysis were obtained from leaf, root and leaves at intervals of 0 h, 3 hr, 6 hr, 12 hr and 24 hr of post stress treatment. In all the three tissues, 6 hr marked the peak up-regulation of the gene, and then a gradual decline was observed with increase in time of stress exposure. The gene exhibited a significant up regulation in the root as compare to leaf and stem tissues (Figure 7B). We successfully transformed 9 lines with overexpressed gene CotAD_24498 (Figure 7C), out the nine (9) lines, three (3) lines showed the highest level of overexpression and were further used in the investigation of the potential of the gene in the transgenic lines under drought stress conditions (Figure 7D).

Figure 7

The qRT-PCR analysis of the expression of the cloned gene CotAD_24498 (A) Total RNA isolated from various tissue of cotton plant under normal conditions; (B) Total RNA extracted from drought-stressed cotton seedlings; (C) Polymerase chain reaction (PCR) analysis performed to check 630bp coding sequence (CDS) integration in transformed T1 generation, number 1–10 transgenic lines, 11 positive control (pWM101- CotAD_24498 and 12 is the negative control (wild-type, WT). (D) The transcripts expression levels of the CotAD_24498 of T2 transgenic lines analyzed through qRT-PCR.

Overexpression of CotAD_24498 in plants promote root growth and confers tolerance to drought stress tolerance

Increased primary root growth and overall plant fresh biomass are indicators of tolerance to various abiotic stresses in which plants are exposed to (Verslues ; Jisha ). We sought to investigate the response of the transgenic lines and the wilt type to drought stress condition in relation to primary root length elongation and fresh biomass accumulation. The transgenic lines showed enhanced performance with relatively increased primary root growth and with higher fresh biomass increment compared to the wild type under drought stress condition. The drought stress was imposed by exposing the transgenic lines to different concentrations of mannitol 0 mM, 100 mM, 200 mM and 300 mM for a period of six (6) days. Under osmotic stress, highest level of root length assays and fresh biomass accumulations was observed at 100 mM of mannitol concentration (Figure 8B). The transgenic lines had significantly higher primary root length and fresh biomass accumulation (Figure 8C), an indication that the photosynthetic processes were not impaired by the drought stress as compared to the wilt type.

Figure 8

Overexpression of CotAD_24498 enhances root growth and drought stress tolerance in Arabidopsis transgenic lines (A) CotAD_24498 overexpressing and WT plants were grown vertically in 0.5 Murashige and Skoog (MS) medium supplemented with 0, 100, 200 and 300 mM mannitol and incubated for 6 days. (B). Root elongation comparisons on 0.5 MS put at normal and osmotic stress for 6 days. The seedlings were scored and photographed after 6 days post germination. (C). Quantitative determination of fresh weight biomass of wild-type (WT) and both transgenic lines (L2, L3 and L3) after 6 days post germination at normal and drought stress condition. In (B, C,), each experiment was repeated three times. Bar indicates standard error (SE). Different letters indicate significant differences between wild-type and transgenic lines (ANOVA; P < 0.05). CK: normal conditions.

Transcripts Investigation of Drought Stress-Responsive Genes

The root appears to be the most relevant organ for breeding drought stress tolerance (Henry 2013). Underlying the ABA-mediated stress responses is the transcriptional regulation of stress-responsive gene expression (Giraudat ). Numerous genes have been reported that are up-regulated under stress conditions in vegetative tissues, these include a class of genes known as LEA genes, which are expressed abundantly in developing seed under normal conditions, osmolyte biosynthetic genes, and genes of general cellular metabolism. We undertook to check the expression of two known abiotic stress responsive genes on the transgenic lines (L2, L3 and L4) and the wild types when the plants are exposed to drought condition. The result showed that the stress responsive genes were highly up-regulated in the transgenic lines as opposed to the wild type (Figure 9). The result obtained was in agreement to the result obtained when the various LEA2 genes were analyzed through qRT-PCR on the tissues obtained from two upland cotton genotypes. More genes were found to be up regulated on the various tissues of the more tolerant genotype as opposed to the less tolerant. Constitutive expression of RD29A and ABF4 demonstrated enhanced drought tolerance in the transgenic Arabidopsis plants.

Figure 9

Expression levels of drought stress-responsive genes (ABF4 and RD29A) in transgenic lines and wild-type. Arabidopsis ACTIN2 was used as the reference gene mean values with ± SD. * P < 0.05 as calculated by Student’s t-test.

Oxidants and antioxidant determination in the transgenic lines

In order to understand the role of the transformed LEA2 genes in the transgenic lines in relation to drought stress. We carried out the analysis of the various oxidants and antioxidants measurements in the leaves of the transgenic lines and the wild type. The levels of oxidants were significantly reduced in the transgenic lines compared to the wild type (Figure 10A-B). When plants are exposed to drought the level of ROS increases, which results into oxidative stress. MDA concentration provides a measure on the damage caused on the membrane lipids due to oxidative stress (Jain ). The significant reduction in MDA and H2O2 in the leaf tissues of the transgenic lines showed that the transformed gene had a regulatory role in controlling various biological pathways geared toward detoxification of the reactive oxygen species in the cells. In addition, we quantified the levels of various antioxidants, SOD, POD and CAT. In all the three antioxidants, there was significant increased levels in the transgenic lines (L1, L2 and L3) compared to the wild type (Figure 10 C-D). The increased levels of the antioxidants showed that the transgenic lines had a higher ability to tolerant drought stress compared to the wild types. The results obtained in this research, correlates to previous findings, in which drought stressed wheat plants were found to have higher accumulation of oxidants levels (Luna ). More tolerant plants genotypes have ability to induct more of the antioxidants such as the CAT, POD and SOD in order to scavenge on the excess ROS and other deleterious molecules released by the cells due to stress condition (Bian and Jiang 2009).

Figure 10

determination of the oxidants and antioxidants in the transgenic lines under stress condition (A) Determination of hydrogen peroxide (H2O2) accumulation in leaves of wild-type (WT) and both transgenic lines (L2, L3, and L4) after 8-days drought stress (B) Determination of MDA accumulation in leaves of wild-type (WT) and both transgenic lines (L2, L3, and L4) after 8-days drought stress; (C) Catalase (CAT) activity, (D) peroxidase (POD) activity and (E) superoxide dismutase (SOD) activity. Data are means ± SE calculated from three replicates. Different letters indicate a significant difference between the WT and both transgenic lines (ANOVA; P < 0.05).

Conclusions

In this study, the identification, phylogenetic relationships, miRNA targets, cis promoter analysis, GO functional annotation and exon/intron structures of LEA2 genes family members were evaluated in upland cotton, Gossypium hirsutum, and the tissue expression pattern of the two tetraploid cotton species, G. hirsutum (drought sensitive) and G. tomentosum (drought tolerant) were detected under drought stress. The abundance of LEA2 genes and unique gene structure reported in this work provide a solid foundation for future research to understand the evolution of LEA2 gene family and the potential functional role of the 157 LEA2 genes in plants under drought stress condition. Since the discovery of LEA genes, little work has been reported on LEA genes as a whole in upland cotton. The transformation and expression analysis of the transformed LEA2 gene indicated that the LEA2 genes have a profound role in enhancing drought stress tolerance. The transgenic lines L2, L3 and L4 exhibited superior performance compared to the wild type. The roots were significantly longer than the wild type under drought stress condition; similarly, the levels of oxidants in the levels were significantly reduced while the antioxidants levels were higher in the leaves of the transgenic lines compared to the wild type. An indication that the transgenic plants had a higher capacity to regulate the oxidative stress as opposed to the wild type (WT). The genes could be promoting growth of the root cells under limited water condition. Primary root growth is linked to drought stress tolerance; due to increased surface area of the roots thus improving its ability maximally absorb any little moisture available. Deep or extensive root growth is a trait known for most of the xerophytic plants (Brunner ).