Introduction
Cysteine-rich peptide (CRP) is a large group of proteins
including thionines, lipid transfer proteins, defensins and GASA/Snakin. Each
CRP class can be distinguished from other based on the orientation and number
of cysteine residues in the primary sequence (Oliveira-Lima
et al. 2017). Recently, CRP proteins have been reported
extensively for their functions in plant development and manipulation of
environmental stresses (Balaji and Smart 2012; Haruta
et al. 2014; Ahmad et al. 2019; Li et al. 2019; Ahmad et
al. 2020). CRP proteins constitute a large gene family and widely
distributed in plants. The GASA (Gibberellic-acid stimulated Arabidopsis) a
subfamily of CRP, is short amino acids, low molecular weights, mostly
gibberellin regulated and is widely distributed in plants. It is comprised of
three distinct domains, 18–29 residues comprising N-terminal domain, a
C-terminal domain (called GASA domain) consisting of 12 conserved cysteine
residues and an intermediate highly divergent region between C and N terminals (Herzog et al. 1995). The fact that
number and position of C-terminal conserved residues remained same throughout
the evolution in different species might suggests their key role in defining
functions of this gene family (Ben-Nissan et
al. 2004). Previous studies have reported that C-terminal GASA
domain is essential for determining antioxidant activity (Wigoda et al. 2006; Nahirnak et al.
2012b) and formation of disulfide bond during protein folding (Porto and Franco 2013).
GASA gene family plays important
roles during different processes in plant life from seed germination to
maturity. Studies based on expression pattern analysis suggest that GASA genes
have specific spatial and temporal expression pattern and most of these are
expressed in young tissues and actively growing organs (Peng et al. 2010; Nahirnak et al. 2016). These are
not only the target genes responsible for specific functions but also act as
regulatory proteins to monitor plants signaling for growth and stress responses
(Ceserani et al. 2009; Zhang et al.
2009; Sun et al. 2013). Further, these genes control plant
hormonal level and hormonal signaling network to fine tune different
physiological processes in plants (Wang et al.
2009; Rubinovich et al. 2014). Additionally, transgenic studies,
homology and expression analysis indicate substantial involvement of GASA genes
in plant developmental processes by affecting various cellular processes (Shi et al. 1992; Aubert et al. 1998; Kotilainen
et al. 1999; Fuente et al. 2006; Furukawa et al. 2006;
Nahirnak et al. 2012a). More importantly, GASA family genes have
preferential roles in floral development and regulation of floral timing (Muhammad et al. 2019). Further, GASA
genes also modulate cell elongation (Ben-Nissan
and Weiss 1996), root growth (Zimmermann et
al. 2010) and stem development (Zhang
et al. 2009) and fruits ripening (Moyano-Canete
et al. 2013) in plants. Interestingly, some GASA family members
have opposite functions related to flowering in plants, such as AtGASA4 stimulates flowering (Roxrud et al. 2007) while AtGASA5 inhibits flowering in
Arabidopsis (Zhang et al. 2009).
In addition to plant growth and
developmental functions of GASA genes, some members of this family are also
involved in regulating plants stress responses. For example, increased
anti-bacterial and anti-fungal activities were correlated with potato Snakin-1
and Snakin-2 proteins during in vivo
experiments (Segura et al. 1999; Berrocal-Lobo
et al. 2002; Almasia et al. 2008; Kovalskaya and Hammond 2009; Balaji
and Smart 2012). Similarly, CaSn
protein enhanced pepper resistance against root-knot nematode (Mao et al. 2011). In Arabidopsis, AtGASA4 and AtGASA5
substantially enhanced tolerance to heat stress by affecting BiP gene
expression and regulating SA signaling pathway, respectively (Ko et al. 2007; Zhang and Wang 2011).
Further, constitutive expression of AtGASA14
increased tolerance to salinity by restricting accumulation of reactive oxygen
species (ROS) (Sun et al. 2013).
Likewise, overexpression of FsGASA4
improves plant tolerance to abiotic stresses by enhancing SA level and induced
expressions of SA signaling pathway genes (Alonso-Ramirez
et al. 2009).
Gibberellic Acid-Stimulated
Transcript 1 (GAST1) was the first GASA gene isolated and characterized in tomato during investigation of
GA-deficient gib mutant (Shi
et al. 1992). Later, with the advancement of sequencing
technologies, more members of GASA genes were reported in diverse plant species
including Arabidopsis thaliana (Herzog et al. 1995) wheat (Zhang et al. 2017), rice (Furukawa et al. 2006), maize (Zimmermann et al. 2010), apple (Fan et al. 2017), petunia (Ben-Nissan and Weiss 1996), potato (Nahirnak et al. 2016), soybean (Ahmad et al. 2019) and grapevine (Ahmad et al. 2020). GASA family
constitutes a large number of genes in some species, for example, in soybean 37
GASA/Snakin genes were identified (Ahmad et
al. 2019), similarly 16 in potato (Nahirnak
et al. 2016), 15 in Arabidopsis (Fan
et al. 2017), 14 in grapevine have been reported (Ahmad et al. 2020). The diversity and
number of GASA/Snakin genes identified in remotely related species depicts
their significance and suggest their essential roles in life of plants.
Cotton (G. hirsutum) is a
naturally fiber and oil producing crop of huge importance for textile and oil
industry of the world. The economy of many developing countries depends on the
sustainable production of cotton. As cotton is a mesophytic plant, its growth
and yield are severally affected by both biotic and abiotic stresses (Dabbert and Gore 2014). Based on the
significance of GASA genes in regulating growth, development and responses to
different environmental stresses in multiple plant species, GASA family was
selected for systematic and comprehensive analysis in cotton. In this study, a
total 116 putative GASA genes were screened from four species of cotton. Their
phylogeny, synteny, motifs, structural features, cis-elements, spatiotemporal expressions in various tissues and
under abiotic stresses (cold, heat, salinity) was investigated in details. Our
results laid a foundation for further characterization of GASA family related
to growth, development and responses to different environmental stresses.
Materials and
Methods
Identification,
sequence analysis and properties of GASA gene family in Gossypium spp.
All the reported Arabidopsis GASA family genes were
downloaded (Fan et al. 2017).
Afterwards, BLASTP programme (Zhu et al.
2017) with e-value 1e-10 was run online to find candidate
GASA genes from four species of cotton including G. hirsutum, G. barbadense,
G. arboreum and G. raimondii using 15 Arabidopsis GASA
genes as query. Subsequently, manual and online databases including NCBI
conserved domain (https://ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al. 2015) Pfam (http://pfam.xfam.org/)
(El-Gebali et al. 2019), and SMART
(http://smart.embl-heidelberg.de/) (Letunic et
al. 2015) were accessed to confirm GASA domain of Pfam (PF02704)
from all the members of GASA family. Further, physio-chemical properties of
GASA genes including theoretical molecular weight, protein length and
isoelectric point were determined through ExPASy web tool
(http://web.expasy.org/) (Bjellqvist et al.
1994).
Chromosomal
mapping, synteny and duplication analysis
Chromosomal positions of GASA genes were
plotted on cotton chromosomes using CIRCOS software. Based on the gene
position, the distribution of each GASA gene was analyzed. Further, homologous
genes among G. hirsutum, G. arboreum and G. raimondii were
screened using BLASTP program with similarity > 80% and alignment percentage
> 80% compared to total length of proteins (Yang
et al. 2008). The sequences of orthologous genes were aligned
using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clusalo/). Subsequently,
aligned sequences were submitted to PAL2NAL software (http://www.bork.embl.de/pal2nal/)
(Suyama et al. 2006) for determination
of synonymous (Ks), non-synonymous (Ka) substitution rates and their ratios
among duplicated gene pairs. Separately, GhGASA
genes were also mapped on chromosomes using Mapchart software (Version 2.0) (Voorrips 2002).
Phylogenetic tree
construction
The phylogenetic tree between GhGASA, GbGASA, GaGASA and GrGASA and Arabidopsis GASA genes was constructed using their protein
sequences. Firstly, multiple sequence alignment of protein sequences was
generated using ClustalW software (Thompson et
al. 2002). Later, this alignment file was submitted in MEGA 6.0
software (Tamura et al. 2013) for
generation of phylogenetic tree. Neighbor-joining (NJ) method was adopted with
parameters as follows, bootstrap values: 1000 replicate, pairwise deletion,
Poisson correction and uniform rates.
Gene structure,
motifs prediction and cis-elements
analysis
Coding DNA sequence and corresponding Genomic sequence
of each GhGASA gene was applied in
online tool Gene Structure Display Server (GSDS, V.2) (http://gsds.cbi.pku.edu.cn/)
(Hu et al. 2015) to construct gene
model showing organization and number of exon-introns. The conserved motifs in GhGASA genes were predicted using MEME
suits (Multiple Expectation Maximization for Motif Elicitation) (Bailey et al. 2015). Further,
annotations of these motifs were obtained from Pfam database (http://pfam.xfam.org/)
(El-Gebali et al. 2019). 1.5 kb
upstream 5’ flanking region of each GhGASA
gene was retrieved from CottonFGD website (https://cottonfgd.org/analyze/) (Zhu et al. 2017) and subsequently subjected to
Plant-CARE program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)
(Lescot et al. 2002) for
prediction of potential cis-acting
elements.
Abiotic stress
treatments
Seeds of upland cotton (G. hirsutum cv YZ1) were
soaked in water and put at 28°C for one night. Next day drained out the excess
moisture and put back at 28°C in incubators to induce germination, seeds with
good growth potential were selected and planted into soil pots filled with
commercially available sterilized composted soil under controlled conditions in
growth chamber. The temperature, humidity and photoperiod cycle was set as 28°C,
60%, 16 h light/8 h dark period, respectively. Uniform cotton seedlings at 3–4
leaf stage were selected and subsequently subjected to salt (200 mmol/L NaCl)
and heat stress (38°C) for 48 h. Alternatively, control seedlings were watered
normally and used as mock. Leaf samples were collected after 72 h from both
control and salt treated plants, immediately kept in liquid nitrogen and stored
at -80°C for subsequent RNA extraction.
Expression pattern
analysis
The plant total RNA from all samples was extracted using
the method as reported earlier by Tu et al.
(2007). After dilution,
RNA was reverse transcribed into cDNA using the reverse transcription kit
(Promega, U.S.A.). qRT-PCR was run as reported earlier by Xu et al. (2014) with thermal cycles
was as follows, 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. The 2-∆∆Ct
method was adopted to compare the gene expression values and GhUBQ7 (DQ116441) gene (Xu et al. 2014) served as an internal
control. Primers used for expression analysis are enlisted in Table S10.
For expression pattern analysis
of GhGASA genes, publically available
transcriptomic data related to different tissues (roots, stems, leaves, petals,
ovules, seeds) and under multiple stresses including cold, heat, PEG and
salinity were retrieved from Cotton FGD (https://cottonfgd.org/) (Zhu et al. 2017). Afterwards, Genesis
software (Version 1.0) was used to normalize expression values and generation
of heatmap (Sturn et al. 2002).
Results
Identification and
characterization of GASA genes in Gossypium
spp.
The genome-wide identification of GASA gene family in
four species of cotton (G. hirsutum, G. barbadense, G. arboreum
and G. raimondii) was carried out through using the Arabidopsis GASA domain as query in BLASTP search against the
corresponding genome of four cotton species. As a result, total 116 GASA genes
were obtained from the studied four Gossypium
species (Table S1). The numbers of putative GASA genes were as 40 in G. hirsutum, 32 in G. barbadense, 22 in G. arboreum
and 22 in G. raimondii. These identified genes were further manually checked
using NCBI conserved
domain, SMART and
Pfam databases. All the genes possessed the putative GASA domain as reported in
previous studies (Fan et al. 2017; Zhang
and Wang 2017; Ahmad et al. 2020). Further, these genes were
named based on their chromosomal locations. Multiple sequence alignment showed
that all these GASA family genes harbored 12 conserved cysteine residues, a
characteristic motif of GASA gene family (Fig. S2). This motif is responsible
for maintaining stability and structure and of GASA proteins (Betz 1993; Darby and Creighton 1995).
Additionally, protein properties including length, molecular weights and
isoelectric points of all the predicted GASA family members were analyzed using
ExPASy program (Table S2).
A circos plot was created between
two diploid species (G. arboreum, G. raimondii) and
subgenomes of allotetraploid specie (G.
hirsutum) to visualize the
chromosomal distribution and syntenic relationship of GASA family genes. As
shown in Fig. 1, all the GASA genes of studied species are evenly distributed
on chromosomes. As GaGASA, GrGASA and GhGASA genes were found to be
distributed on 10 chromosomes of A, D, and At and Dt subgenomes,
respectively (Table S3). The distribution was uneven, with chromosomes A05 and
A09 harbored 6 and 4 GhGASA genes
while chromosomes A06, D05, D07, D09 and A07, D03, D04, D06 possess 3 and 2 GhGASA genes, respectively. Other
chromosomes A02, A03, A04, A10, A11, A12, D02, D10, D11, and D12 contained
single GhGASA gene (Fig. S1).
Additionally, 40 and 42 duplicated gene pairs were identified from At to A2 and
Dt to D5, respectively and members of each duplicated gene pairs exhibited
great similarity to each other (Table S4).
Additional duplication analysis
among GhGASA genes found 14
duplicated gene pairs and all of these pair experienced segmental duplications
(Table S5). Further, All the duplicated GhGASA
genes experienced purifying selection except two duplicated genes pairs (GhGASA10-GhGASA31 and GhGASA20-GhGASA39) that undergoes positive
selection (Table S5).
Phylogenetic
clustering and Structural properties of GASA genes in cotton
For estimation of phylogenetic relationship among
members of GASA family, a phylogenetic tree was constructed using amino acids
sequences of model plant Arabidopsis and four cotton species. According to
phylogenetic tree, GASA genes of cotton and Arabidopsis were clustered into
three groups (G1, G2 and G3) based on their homology and protein structures
(Fig. 2). Group 3 contained highest number of GASA family members 51, while
group 1 and 2 possess 41 and 39 GASA genes, respectively.
The evolutionary relationship
based on genes structural diversity was considered an important component in
the study of multigene families. To study the structural similarity or
differences among putative GhGASA
genes, an exon-intron map was constructed (Fig. 3A). Results revealed variation
in number of exons among GhGASA genes
that fluctuated from 2 to 5, with highest number of introns and exons was found
in GhGASA17 (introns: 4, exons: 5).
Expectedly, the number and composition of exon-intron between closely related
members was same within the same group. More variations in number of exons and
introns were observed among group 3 members. All members of group 2 contained
one intron and 2 exons, while all members of group 1 include 3 introns and 4
exons, with the exceptions of GhGASA19
and GhGASA38 that contained 2 introns
and 3 exons (Fig. 3). To get some perceptible information about paralogous GhGASA genes in phylogenetic tree, we
analyzed their exon-intron structures. Interestingly, most of the paralogous GhGASA genes harbored same number and
orientation of exons-introns. For example, GhGASA1/30 possessed three exons and two introns
but some variations were observed in intron length. Probably, these relations
have formed the size and structures of putative GASA genes in cotton.
To further explore the
structures and features of GhGASA
gene family, MEME server was accessed for finding distinctive or similar motifs
in GhGASA genes. Ten distinct motifs
of different length were found among 40 GhGASA
genes (Fig. 3B) and annotated using SMART and Pfam servers. Only motif 1 was
found to be the representative of GASA domain, while the functions of other
motifs were unknown (Table S6). Normally, most of the closely related members
harbored similar motifs within the same group, proposing their similar function.
For example, GhGASA7/12, GhGASA14/34, GhGASA16/36, GhGASA9/25
and GhGASA3/26 shared similar motifs.
In detail investigation found that only motif 3 was present in all GhGASA genes, while motifs 4, 6, 7, 8
were absent in all members of group 1 and 2. Moreover, some motifs were found
to be specific to special GhGASA
members, such as motif 10 was only found in GhGASA1
and GhGASA24. However, it is not
known whether these motifs confer unique functions to these GhGASA genes or not.
Promoter analysis
of GhGASA genes
Cis-elements are considered to
participate in controlling expression of genes. To explore the probable
association of these cis-elements
with the expression or functions of GhGASA
genes, 1.5 kb promoter region of each GhGASA
genes was extracted and analyzed using PLANTCARE website. A diversity of cis-elements responsive to growth,
development, stress, light and phytohormones were identified. Among 40 GhGASA genes, light responsive elements
were predominant (57%), followed by hormones responsive (19%), growth and
development related (14%) and environmental stress responsive (10%) (Fig. 4A
and Table S7).
%20181-190_files/image002.png)
Fig. 1:
Synteny analysis of GASA gene family
Syntenic association between two
diploid species (G. arboreum, G. raimondii) and one
tetraploid specie (G. hirsutum) was generated through circos
program. Different chromosomes of selected species are marked with different
colors
%20181-190_files/image004.png)
Fig. 2: Phylogenetic
relationship of GASA genes from Arabidopsis
and Gossypium species
Phylogenetic tree was constructed by MEGA 6.0 software using
neighbor-joining (NJ) method with 1000 replicates. The GASA genes from G. hirsutum,
G. raimondii, G. arboreum, G. barbadense and Arabidopsis were marked with different
colors and shapes. Group1, Group 2 and Group 3 were indicated in green, red and
blue colors, respectively
%20181-190_files/image006.jpg)
Fig. 4:
Promoter region analysis of GhGASA
genes
A. Percentage of cis-acting elements in the promoter regions of GhGASA genes, different colors corresponds to percentage of
specific cis-elements category. B.
presence of cis-elements in promoter
region of each GhGASA gene was shown
in column and in different colors
The pattern of cis-elements
varied among GhGASA genes.
Surprisingly, GhGASA11 harbored only
one site for auxin from hormones responsive elements but possess highest number
of low temperature responsive cis-elements.
Moreover, GhGASA9 possessed highest
number of gibberellin responsive sites among other GhGASA genes (Fig. 4B and Table S7).
Differential tissue-specific
expression of GhGASA genes in upland
cotton
To investigate the expression pattern of GhGASA genes in %20181-190_files/image008.png)
different tissues of cotton, a heatmap was generated.
Results revealed that all the GhGASA
genes were expressed in at least one tissue, except GhGASA19 and GhGASA29
(Fig. 5 and Table S8). However, three genes (GhGASA17, GhGASA20, and GhGASA35) expressed in all the seven
tissues at all the time points [Fragments per kilobase of transcript per
million mapped reads (FPKM ≥1)]. Moreover, four genes (GhGASA10, GhGASA17, GhGASA20, and GhGASA35)
highly expressed in roots (FPKM ≥20) and six
%20181-190_files/image010.jpg)
Fig. 5:
Expression patterns of GhGASA genes
in various tissues of upland cotton.
The RNA-seq data related to organ-specific expression were accessed from
CottonFGD (https://cottonfgd.org/) and Genesis software package was
used for generation of heatmaps. DPA (days post anthesis), SW0 (sowing in water
0 h), SW5 (sowing in water 5 h)
genes (GhGASA4,
GhGASA9, GhGASA27, GhGASA28, GhGASA30 and GhGASA32) were found to be dominantly induced in leaves (FPKM
≥5). While, four genes (GhGASA13,
GhGASA17, GhGASA31 and GhGASA39)
were highly induced in petals (FPKM ≥51) and five genes (GhGASA6, GhGASA8, GhGASA14, GhGASA24, and GhGASA34 in ovules (FPKM ≥2.7). Expression of some genes
found to be specific to single tissue, such as GhGASA11& GhGASA12
only expressed in fiber (20 DPA).
Differential
expression of GhGASA genes under
multiple abiotic stresses
Considering important functions of GASA genes against
various environmental stresses in a number of plant species, firstly, we thoroughly
investigated the expression pattern of GhGASA
genes using published transcriptomic data of cotton treated with heat, cold,
polyethylene glycol (PEG) and salt (NaCl). From the results, it was observed
that all GhGASA genes showed altered
expression under one or more stress conditions except five GhGASA genes (GhGASA5, GhGASA19, GhGASA23, GhGASA29, GhGASA40) that were not expressed under
any stress condition (Fig. 6 and Table S9). Comparing four treatments, more
numbers of genes were differentially expressed against salt and cold stresses
as compared with heat and PEG. In response to cold stress, seven GhGASA genes highly induced during 3 h
time period and GhGASA6/16 had the
highest expression. Under heat stress, five genes showed increased expression
at 12 h time period, with GhGASA7 had
the highest transcript abundance. Similarly, under salt stress seven GhGASA genes were highly expressed at 12
h time period (treatment RPKM/control RPKM ≥ 2.5) with highest expression
noted for GhGASA26. Interestingly,
some genes only expressed under specific stress condition, such as GhGASA7/21 and GhGASA6/16 only expressed in response to heat (12 h) and cold (3 h)
(treatment RPKM/control RPKM ≥ 3). This specific expression pattern might
support their involvement in modulating these stress responses in cotton. Secondly,
to validate expression profile of GhGASA
genes obtained through transcriptomic data, we choose eleven GhGASA genes based on their higher
expression under salt or heat stress for further analysis through qRT-PCR.
Similar to transcriptomic profile of GhGASA
genes, most of the selected genes were up-regulated under salt stress as
compared to heat stress. Four GhGASA
genes including GhGASA18, GhGASA22, GhGASA36, and GhGASA37
were highly expressed under salt stress. Expression of three genes including GhGASA20, GhGASA33, and GhGASA39 was more under heat stress as
compared to salt stress. However, three genes GhGAS24, GhGASA30 and GhGASA32 were down-regulated both under
salt and heat stress.
Discussion
Earlier studies comprehensively showed the roles of GASA
genes in monitoring plant developmental processes and responses to various
stress responses in various plant species (Nahirnak
et al. 2012b; Zhang and Wang 2017). However, previously it was
not focused to identify and characterize GASA genes in cotton. In this study, a
total of 116 putative GASA genes were identified from four species of cotton.
Multiple sequence alignment of all these genes highlighted the existence of 12
amino acids conserved cysteine residues, a motif considered essential for
maintaining structure and functions of GASA genes in different plant species (Betz 1993; Darby and Creighton 1995; Silverstein et
al. 2007; Muhammad et al. 2019). In G. hirsutum, 40 GASA
genes were identified, which is relatively a larger gene family among other
three species of cotton (G. barbadense, G. arboreum and G. raimondii)
and other reported plant species (Fan et al.
2017; Ahmad et al. 2019). Interestingly, all the GaGASA, GrGASA, and GhGASA are
evenly distributed on 10 chromosomes of A, D and At and Dt subgenomes,
respectively.
Gene duplication is an important
mean for functional diversification, evolution and expansion of gene family (Kong et al. 2007). There are three ways
through these duplication events takes in plants. Segmental duplications
prevails when distribution of duplicated gene pairs are on different
chromosomes, while tandem duplication occurs when duplicated genes are located
on the same chromosomes. Duplication analysis among GhGASA genes revealed that segmental duplication is the leading
cause for expansion of GhGASA gene
family. Further, synonymous (Ks), non-synonymous (Ka) substitution rates and
their ratio were computed to investigate the duplication mechanism of GhGASA genes after being diverged from
their ancestor. The Ka/Ks ratio equal to 1 shows neutral selection, while
greater than 1 and less than 1 indicates positive and purifying selection,
respectively (Hurst et al. 2002).
Interestingly, in our study most of the duplicated GhGASA genes experienced purifying selection mechanism. Additional
systemic analysis is needed to explore further insights in the evolution of
cotton GASA gene family.
%20181-190_files/image012.jpg)
Fig. 6:
Expression pattern of GhGASA genes
against different abiotic stresses in upland cotton
A. Transcriptomic data related to heat, cold, PEG and
drought were accessed from CottonFGD website (https://cottonfgd.org/),
normalization and visualization of these data were performed using Genesis
software package. B. Expression pattern of selected GhGASA genes under salt and heat stress by qRT-PCR. Columns
represent the mean of three biological repeats and error bars represents the
standard deviation
In phylogenetic tree, all the
GASA genes were distributed in three groups (G1, G2 and G3) based on their
homology and structures. G3 contained more number of GASA genes than others.
Similar kind of grouping among GASA family genes were also observed in other
reported plant species (Zimmermann et al.
2010). Gene structure analysis showed that most of GhGASA genes within specific group harbored similar exons-introns
number and organization corresponding to their conserved functionality.
However, some variations were observed among group 3 members that indicate
their functional diversity. In terms of exons-introns numbers, G2 was found to
be more conserved than others, suggesting that other groups might gain or lost
exons during evolutionary process leading to differences in structures. Similar
trend of exons-introns conservation among G2 members was also noted in previous
study (Ahmad et al. 2020). To
further explore GhGASA genes in
detail, we analyzed their motifs distribution. Expectedly, most of closely
related GhGASA genes with in specific
groups constitute similar motifs. Moreover, some specific motifs were found to
be associated with some special GhGASA
genes. However, it is not known whether these motifs confer unique functions to
these GhGASA genes or not. In any
case, conservation of these motifs within and between groups further supports
the results of phylogenetic tree. Cis-elements
present in gene promoter normally regulate gene expression and confers unique
functionality to genes (Biłas et al.
2016). To study important roles of GhGASA
genes under changing environmental conditions and various physiological
processes of plants, we thoroughly investigated the promoter regions of each GhGASA gene. Mainly four types of
elements were observed including growth, development-related, light, hormones
and environmental stress-responsive elements. The diversity of cis-elements in GhGASA genes agree with the reported multidimensional functions of
GASA genes in different plants (Oliveira-Lima et
al. 2017; An et al. 2018; Li et al. 2019; Muhammad et al.
2019).
Expression pattern of genes
provide useful clues for their functional characterization. Previously, it has
been noted that GASA genes have spatiotemporal specificity in various plant
species which might be due their probable involvement in diverse hormone
signaling pathways (Wang et al. 2009;
Zhang and Wang 2017). In present study, expression analysis of GhGASA genes in various tissues of
cotton showed that maximum number of GhGASA
genes induced in roots and ovules, signifying their important roles in
development of these tissues. Moreover, considering the transcript abundance of
GhGASA genes in cotton and the
published role of their orthologs in Arabidopsis
helped us to predict their probable functions. For example, GhGASA35 along with higher expression in
roots also strongly induced in seeds (SW5) (Fig. 5; Table S9) and its artholog AtGASA4 in Arabidopsis regulates seed germination (Roxrud et al. 2007; Rubinovich and Weiss 2010). Similarly, GhGASA2 did not expressed in any tissue
except seed (SW0) and its orthologous gene AtGASA10
reported to have important roles in seed germinations (Trapalis et al. 2017). This suggests that might be GhGASA35 and GhGASA2 have related roles in cotton as AtGASA4 and AtGASA10 in Arabidopsis. Nevertheless, this trend
was not consistent to all GhGASA
genes, signifying that these GASA genes might be undergone functional
diversification across species.
The quality and yield of cotton
is substantially affected by multiple abiotic stresses during the development
of plant (Hassan et al. 2020).
Therefore, we comprehensively studied the transcript abundance of GhGASA genes under several environmental
cues including heat, cold, PEG and salt stress using published transcriptomic
data. The results showed that most of GhGASA
genes changed their expression under one or more stress conditions.
Additionally, more numbers of GhGASA
genes were highly induced under cold and salt stresses as compared with heat
and PEG stresses, suggesting their probable function in monitoring these
stresses in cotton. Further, qRT-PCR analysis of selected GhGASA genes under salt and heat stress supported the results of
transcriptomic profile. Moreover, by comparing GhGASA genes in response to stress conditions and the existence of
relevant cis-elements in promoter
regions of those genes further support their effectiveness against these
stresses. For example, GhGASA26 was
highly induced under salt stress (12 h) and contain maximum number of stress
responsive elements in its promoter region. Interestingly, some GhGASA genes only induced under specific
stress treatment, such as GhGASA7/21
and GhGASA6/16 only expressed in
response to heat (12 h) and cold (3 h) (treatment RPKM/control RPKM ≥ 3).
This specific expression of selective genes might further support their
importance in regulating these stress responses.
Conclusion
In silico analysis of cotton genomes enabled us to investigate GASA
family genes in details. By adopting systematic approach consisting of
conserved domain analysis, chromosomal distribution patterns, synteny,
phylogeny, exons-introns organization and motifs division analysis,
comprehensive characteristic features of GASA genes in cotton were elucidated.
Promoter region analysis of GhGASA
genes supported their involvement in a variety of biological functions in
plants. Moreover, spatiotemporal tissue specific expression of GhGASA in upland cotton showed that most
of GhGASA genes were induced in
leaves and ovules than other. Additionally, qRT-PCR and transcriptomic
expression profiles of GhGASA genes
under various abiotic stress factors suggested that most of GhGASA genes have the capacity to
regulate tolerance against multiple abiotic stresses. In short, the present
genome-wide investigation of GhGASA
gene family provides potential information for future more focused studies
regarding in-depth characterization of GASA genes in cotton.
Acknowledgement
This study is a part of Interim Placement of Fresh Ph.D.
program granted by Higher Education Commission of Pakistan.
Author Contributions
MS designed and wrote manuscript. AK, EN, MS, UA, AR
helped in performing experiments and analyzing data. WM and MT helped in
revising manuscript. AQ gave suggestions to improve the experimental work.
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