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Genome-wide identification, characterization and expression profiles of FORMIN gene family in cotton (Gossypium Raimondii L.)

BMC Genomic Data volume 25, Article number: 105 (2024) Cite this article

Abstract

Background

Gossypium raimondii serves as a widely used genomic model cotton species. Its genetic influence to enhance fiber quality and ability to adapt to challenging environments both contribute to increasing cotton production. The formins are a large protein family that predominately consists of FH1 and FH2 domains. The presence of the formin domains highly regulates the actin and microtubule filament in the cytoskeleton dynamics confronting various abiotic stresses such as drought, salinity, and cold temperatures.

Results

In this study, 26 formin genes were analyzed and characterized in G. raimondii and mostly were found in the nucleus and chloroplast. According to the evolutionary phylogenetic relationship, GrFH were dispersed and classified into seven different groups and shared an ancestry relationship with MtFH. The GrFH gene structure prediction revealed diverse intron-exon arrangements between groups. The FH2 conserved domain was found in all the GrFH distributed on 12 different chromosomes. Moreover, 11 pairs of GrFH transpired segmental duplication. Among them, GrFH4-GrFH7 evolved 35 million years ago (MYA) according to the evolutionary divergence time. Besides, 57 cis-acting regulatory elements (CAREs) motifs were found to play a potential role in plant growth, development, and in response to various abiotic stresses, including cold stress. The GrFH genes mostly exhibited biological processes resulting in the regulation of actin polymerization. The ERF, GATA, MYB, and LBD, major transcription factors (TFs) families in GrFH, regulated expression in abiotic stress specifically salt as well as defense against certain pathogens. The microRNA of GrFH unveiled the regulatory mechanism to regulate their gene expression in abiotic stresses such as salt and cold. One of the most economic aspects of cotton (G.raimondii) is the production of lint due to its use in manufacturing fabrics and other industrial applications. The expression profiles of GrFH in different tissues particularly during the conversion from ovule to fiber (lint), and the increased levels (up-regulation) of GrFH4, GrFH6, GrFH12, GrFH14, and GrFH26 under cold conditions, along with GrFH19 and GrFH26 in response to salt stress, indicated their potential involvement in combating these environmental challenges. Moreover, these stress-tolerant GrFH linked to cytoskeleton dynamics are essential in producing high-quality lint.

Conclusions

The findings from this study can contribute to elucidating the evolutionary and functional characterizations of formin genes and deciphering their potential role in abiotic stress such as cold and salt as well as in the future implications in wet lab.

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Background

Formins are the protein family recognized by the presence of FH1 (Formin Homology 1) and FH2 (Formin Homology 2) domains that can multimerize the actin protein in cytoskeletal dynamics through nucleating the bared end of the actin filament [1, 2]. The FH1 domain comprises polyproline interactions with the profilin-actin binding domain complex. As FH1 resides in the N terminal of the FH2 domain, FH1 transports the profilin-actin binding domain complex to the barbed ends (growing part) of the FH2 domain. The profilin induces the elongation rate of the barbed end of both the F1 and FH2 domains. The FH2 domain will polymerase the actin filament in the bared end for extension [3,4,5].

A cytoskeleton is a structure that is composed of actin filaments and microtubules, which mediate cell proliferation, and support the internal structure and movement of the cell [6]. Besides all these functions, the most crucial aspect of cytoskeleton dynamics is that upon the stress condition from the surrounding environment, it ensures cellular stability by preventing various parts of the plant such as roots and shoots, from being susceptible to stress [2]. This transpires through the alternation of cellular morphology to fight against stress and pathogens [7].

The mechanism of tolerance to the abiotic stress involves cytoskeleton dynamics, both actin filament and microtubule. The actin filament of cytoskeleton dynamics is responsible for the proper growth and advancement of the plant. But perhaps the pivotal role of actin filament, according to the genetic and pharmacological analysis, is the guarding of cell shape changes [8,9,10]. During drought stress, it is observed in Arabidopsis thaliana that stomatal opening and closing are highly monitored and regulated by the actin filament of cytoskeleton dynamics. In terms of organized and symmetrically distributed actin filament, the stomata remain open. However, the longitudinal actin filament represents the closing of the stomata. This remodeling and reshuffling of cellular changes during stress is controlled by actin-binding proteins (ABPs). ABP further consists of actin-depolymerizing factors (ADFs) that are responsible for growth and elongation of cells, nonspecific immunity to cells, as well as regulation of stomata [11,12,13].

On the other hand, another cytoskeletal dynamics, microtubules (MT), which are composed of α-tubulin and β-tubulin, play an essential role in terms of tolerance to stress. In salt stress, the polymerization and de-polymerization of MT enhance tolerance to stress. During the stress condition, various signaling molecules such as abscisic acid (ABA), cytosolic calcium ion, and reactive oxygen species (ROS) are key modulators for stress-controlling systems in plants. The presence of high-level cytosolic calcium ions in the calcium channel and the existence of ABA, a phytohormone, in the root ensuring the reconstruction of cortical MT lead to the de-polymerization of MT. Whereas ROS re-polymerizes by dismantling and rebuilding the non-uniform polymers of MT [6, 14,15,16].

As a dicot, cotton is one of the most valuable and economically lucrative plant species on the entire planet, producing fiber that is extensively utilized in the textile industry. Apart from its implementation in textile manufacturing, it serves as a model scheme in the field of cell wall biosynthesis and cellular elongation research. The D5 diploid G. raimondii is the predominant contributor of pollen [17,18,19]. According to the Food and Agriculture Organization (FAO), factors related to the environment, agroecology, and atmosphere affect the growth and cultivation of cotton [20]. Therefore, stress tolerant genes are essential for proper growth and production of quality lint.

In this study, 26 GrFH genes containing conserved formin domains were analyzed. The gene structure analysis showed similarity within the subgroups, while 11 pairs of segmental duplications were observed in 12 chromosomes. The evolutionary phylogenetic tree demonstrated a closed ancestry relationship of GrFH with MtFH. Besides, 57 cis-acting regulatory elements were found in GrFH, revealing their involvement in plant development along with response in stress conditions. ERF and GATA were the most abundant transcription factor families to bind GrFH. The protein-protein interactions showed close proximity with Arabidopsis proteins. Based on the gene ontology analysis, GrFH functions were classified into three groups, while most of them were involved in biological processes. About 96 unique microRNAs were identified to regulate gene expression in various abiotic stresses, specifically cold and salt stress. Besides, the tissue-specific expression of GrFH revealed its relations with the production of lint, which are essential for industrial and chemical applications. Moreover, certain GrFH showed resistance to abiotic stress such as cold and salt. Therefore, these stress-tolerant GrFH would maintain the production of fiber in case of a cold and salt stressed conditions.

Results

Identification and determination of physiochemical properties of GrFH (G. Raimondii Formin homology)

Among the predicted protein sequences, 26 of them were identified as FH2-containing domains, which were subsequently renamed as potential genes. For example, Gorai.001G078900 was renamed to GrFH1. The examination of the amino acid (aa) counts across the 26 GrFH proteins showed a range from 71 (GrFH20) to 2274 (GrFH10) (Table 1). According to the molecular weight measured in kilo-Dalton (kDa), GrFH20 (8283.71 kDa) had the lowest amino acid contents among the 26 encoded GrFH proteins, while GrFH10 (241075.93 kDa) had the highest amino acid contents. The pH at which a protein carries no net electrical charge (neutral state) and a protein’s pH level at which it exhibits no net electrical charge (neutral state) acts as a zwitterion ion is known as its isoelectric point (pI) [21, 22]. The pI (Isoelectric point) value varied from a range of 5.31 (GrFH3) to 9.35 (GrFH12). The result instability index showed that all the GrFH proteins were considered unstable since the score was greater than 40. Moreover, the results generated from the aliphatic index revealed that 10 GrFH proteins (GrFH1, GrFH3, GrFH4, GrFH8, GrFH13, GrFH16, GrFH20, GrFH21, GrFH23, and GrFH26) contained aliphatic index above 80, while the remaining 16 GrFH proteins ranged between 70 and 80. As far as the grand average of hydropathicity (GRAVY) was concerned, all of the GrFH proteins surpassed the negative value.

Table 1 List of 26 GrFH proteins and their basic physio-chemical characterization
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Phylogenetic analysis between GrFH, MtFH, ZmFH, OsFH, and AtFH

To demonstrate the molecular and evolutionary relationship between formin proteins, a phylogenetic tree containing 26 proteins from GrFH as candidate species, 21 from AtFH, 20 from ZmFH, 19 from MtFH, and 17 from OsFH was constructed (Fig. 1). A total of seven different categories designated as groups A, B, C, D, E, F, and G were formed from 103 formin proteins from five distinct species. GrFH were distributed in those above-mentioned groups (Supplementary Material File 1). There were 2 candidate formin proteins in each of the three groups denoted as A, E, and F. GrFH7 and GrFH22 were found in group A at the same time GrFH13 along with GrFH23 were identified in groups E, and GrFH12 and GrFH14 were found in group F. In groups B, C, and D, each one of them contained 4 formin proteins. GrFH1, GrFH9, GrFH10, and GrFH16 were found in group B, while GrFH5, GrFH17, GrFH19, and GrFH25 emerged in group C. GrFH4, GrFH15, GrFH18, and GrFH24 belonged to the group D. However, with eight GrFH proteins from group G- GrFH2, GrFH3, GrFH6, GrFH8, GrFH11, GrFH20, GrFH21, and GrFH26, it notched up rank number one. Remarkably, each formin protein species (GrFH, AtFH, ZmFH, MtFH, and OsFH) was spotted in each group at least once.

Fig. 1
figure 1

Phylogenetic relationship between candidate GrFH and AtFH, ZmFH, MtFH, and OsFH gene families. GrFH were classified into 7 groups (A, B, C, D, E, F, and G), each marked by different colors and shapes. The candidate gene GrFH was labeled by the red star. Whereas AtFH was labeled as round blue, ZmFH was labeled as violet color square, MtFH was labeled orange triangular, and OsFH was labeled as green triangular

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Conserved motif, domain and gene structure of GrFH

In conserved motifs, 20 unique motifs were analyzed in GrFH proteins (Fig. 2A). The groups that comprised 11 motifs were identified as group A (GrFH7 and GrFH22), group D (GrFH4, GrFH15, GrFH18, GrFH24), group E (GrFH13, except GrFH23, which featured an additional motif that summed to 12 motifs), group F (GrFH12, GrFH14), and some members of group G (GrFH2, GrFH6, GrFH11, GrFH21). All the group B members (GrFH1, GrFH9, GrFH10, GrFH16) contained 17 different motifs. The members of group G each possessed different motif numbers, as GrFH3, GrFH8, and GrFH26 each included six motifs. Surprisingly, GrFH20 from group G incorporated solo motif 2. The conserved domain was used to identify and investigate protein functions and evolutionary relationships. The FH2 conserved domain was found in all 26 GrFH proteins (Fig. 2B). Some of the proteins not only contained FH2 but also contained other domains. PTEN_C2 was found most frequently after FH2. On the contrary, the GrFH gene structure consisted of a number of different exons and introns (Supplementary Material File 2). Group A members, GrFH7 and GrFH22, showed the least number of exons and introns counts among the seven groups at 4 and 2, respectively (Fig. 2C). The highest number of exons were observed in groups B and C accumulating 69 and 70, respectively. GrFH1, GrFH9, and GrFH10 from Group B and GrFH17 and GrFH25 from Group C each included 17 exons and 16 introns in these two groups. Meanwhile, GrFH16 and GrFH5, GrFH19 members of group B and group C, respectively, possessed 18 exons and 17 introns. In group D, there were 25 exons and 21 introns in total. GrFH4, GrFH15, and GrFH24 each had 6 exons, while GrFH18 had 7 exons. When groups E and F exons and introns were counted, there were 16 exons and 12 introns from both groups. In the same way, GrFH12 and GrFH14 from group F and GrFH13 and GrFH23 from group E each contained 4 exons and 3 introns. Group G, the largest group, comprised a maximum of 29 exons and 21 introns. Six GrFH (GrFH2, GrFH6, GrFH8, GrFH11, GrFH21, and GrFH26) genes held with 4 exons in this largest group, whereas GrFH3 and GrFH20 featured with 3 and 2 exons correspondingly.

Fig. 2
figure 2

The motif, domain, and gene structure of GrFH genes. A The grouping and colors of the GrFH gene family members are based on the phylogenetic relationship. Each motif is illustrated by a specific-colored box aligned on the right side of the figure. Different colors indicate individual motifs. B The positions of the FH2 conserved domain is demonstrated in red color, whereas the entire protein sequence of respective GrFH is green colored. C For the color bar of GrFH gene structure, black lines represent introns, blue represents exons, and deep pink lines represent upstream/downstream

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Synonymous (Ks) and non-synonymous (Ka) substitution ratios calculation of GrFH

The Ka (nonsynonymous substitution rate) and Ks (synonymous substitution rate) values for GrFH gene pairings were evaluated (Supplementary Fig. S1). A Ka/Ks ratio that is smaller than 1 denotes purifying selection, while a higher than 1 describes positive selection. The estimated Ka value in the GrFH genes varied from 0.003824 to 0.32905, whereas the Ks value varied from 0.4263 to 0.000495 (Supplementary Material File 3). One gene pair, GrFH20-GrFH26, was going through positive selection; its Ka/Ks ratio is 7.7257807. Apart from that, the Ka/Ks ratio of other gene pairs is less than 1. For example, Ka/Ks ratio is approximately 0.771874 in GrFH6-GrFH21 along with 0.194305 in GrFH18-GrFH5, suggesting that those gene pairs have experienced purifying selection.

Evolutionary collinear relationship analysis of GrFH

The collinear relationship showed a close relationship among GrFH genes (Fig. 3). With a total of 4 collinear pairings observed in chromosomes 7 and 13, it was identified as the highest levels of duplicated collinear forming pairs. The genes on chromosome 7 were GrFH10, GrFH11, GrFH12, and GrFH13, which created collinear pairings with GrFH9 on chromosome 6, GrFH8 on chromosome 6, GrFH14 on chromosome 8, and GrFH23 on chromosome 13, respectively. The genes GrFH24, GrFH25, and GrFH26 located in chromosome 13 paired with GrFH15 from chromosome 8, GrFH1 from chromosome 1, and GrFH20 from chromosome 11, respectively. Following these, there were four additional duplicated pairings: GrFH4 on chromosome 4 paired with GrFH7 on chromosome 5, GrFH5 on chromosome 4 interacted with GrFH18 on chromosome 9, and GrFH6 on chromosome 5 linked with GrFH21 on chromosome 11 and ultimately GrFH17 on chromosome 9 formed pair GrFH19 of chr10. Despite having specific loci, four GrFH genes (GrFH2, GrFH3, GrFH16, and GrFH22) on chromosomes 1, 2, 8, and 12, respectively showed to contain no collinear pairings. But, no gene was detected on chromosome 3 which wasn’t taken into account while counting chromosomes.

Fig. 3
figure 3

The collinearity analysis of the GrFH gene family in cotton. Green color rectangles represent chromosomes 1–13 in GrFH. The dark blue colored lines linked between chromosomes represent collinear relations between them

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Evolutionary syntenic relationship analysis of GrFH

Syntenic relationships between Z. mays, O. sativa, A. thaliana, and the candidate G. raimondii formin gene were performed to provide insight into putative evolutionary relationships between the FORMIN gene in multiple species (Fig. 4). Unfortunately, no syntenic gene pairings emerged in the candidate gene GrFH with other species. Remarkably, among all the species, only the formin genes of Z. mays and O. sativa established syntenic pairing with each other. For example, chromosome 6 of ZmFH1 formed a syntenic pairing with chromosome 5 of OsFH14.

Fig. 4
figure 4

The syntenic relationship analysis of cotton and Arabidopsis, Rice, and Corn. Red color rectangles represent the GrFH chromosomes. Meanwhile, aqua-blue color rectangles represent AtFH chromosomes. Furthermore, magenta color rectangles represent OsFH chromosomes, while green color rectangles represent ZmFH chromosomes. The dark blue color represents the syntenic relationship linkage between different species

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Chromosomal localization and duplication analysis of GrFH

The duplication of genes and the formation of collinear pairings were identified as segmental types. 26 genes of GrFH were distributed across the 12 chromosomes at specific loci, and 11 gene pairs were duplicated (Supplementary Fig. S2). Chromosomal localization was carried out to figure out the gene’s extract position across chromosomes. There was at least one single gene found on chromosome 2, chromosome 10, and chromosome 12, which went by the names GrFH3, GrFH19, and GrFH22, respectively. Chromosomes 1, 4, 5, 6, 9, and 11 contained 6 pairs of genes designated as GrFH1- GrFH2, GrFH4-GrFH5, GrFH6-GrFH7, GrFH9-GrFH8, GrFH17-GrFH18, and GrFH20-GrFH21 were observed, respectively in the order of their location on a specific chromosome. The genes GrFH14, GrFH15, and GrFH16 were found sequentially on chromosome 8 in the same order as their genomic positions. Chromosome 7 and 13 each contain four genes. Specifically, chromosome 7 includes GrFH10, GrFH11, GrFH12, and GrFH13, whereas chromosome 13 includes GrFH23, GrFH24, GrFH25, and GrFH26. Notably, no genes were found on chromosome 3.

Prediction of the subcellular localization of GrFH

Subcellular localization studies were conducted to determine the organelle locations of the GrFH genes. The result showed that 10 different organelles, including the nucleus, mitochondria, chloroplast, cytoplasmic, cytoskeleton, Golgi apparatus, vacuole, endoplasmic reticulum, plasma membrane, and lastly, extracellular existed. In terms of gene presence on the particular organelle, GrFH8 and GrFH11 were found on 12 sites of chloroplast (Fig. 5A). GrFH4 and GrFH6, which are located at 10 and 11 different sites of the plasma membrane, respectively, came out as the third and fourth best, trailing only by GrFH8 and GrFH11. The bubble plot, however, represented the redundancy of a particular GrFH gene in specific organelles. For example, GrFH8 and GrFH11 were found in 12 different locations in the chloroplast. When it comes to the overall number of GrFH gene distributions in each organelle, the chloroplast and nucleus outnumbered all other organelles, with percentages of 80.76% and 76.92%, respectively (Fig. 5B). Mitochondria along with plasma membrane weren’t far behind, with percentages of 65.38% and 53.84%, respectively. The cytoskeleton proved to comprise the lowest concentration of GrFH genes. However, GrFH genes were observed in sufficient amounts in the rest of the organelles as well.

Fig. 5
figure 5

Sub-cellular localization analysis of GrFH. A The heatmap represents the sub-cellular localization analysis of GrFH. The intensity of color on the right side of the heatmap indicates the presence of protein signals corresponding to the genes. The cellular organelles include nuclear, mitochondrial, cytoplasmic, chloroplast, cytoskeletal, Golgi, vacuole, endoplasmic reticulum (ER), plasma membrane (PM), and extracellular locations. B The percentage distribution of GrFH signal across various cellular organelles is represented by a bar diagram. The percentages of protein signals appearing in different cellular organelles are shown on the left side of the diagram

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Cis-acting regulatory elements (CAREs) analysis in the promoters of GrFH

The analysis regarding the 2000 bp sequence (Supplementary Material File 4) CAREs was conducted to get a deeper comprehension of the regulatory framework driving the promoter region of cis-elements (Fig. 6). The result of the CAREs showed that 57 CARE motifs were found in GrFH (Supplementary Material File 5). Based on the functional similarities of the 57 CAREs, four particular categories were formed that is light responsiveness, tissue-specific expression, phytohormone responsiveness, and stress responsiveness. Out of these four categories, 24 motifs featuring light responsiveness were identified as the highest containing cis-acting regulatory elements.24 motifs in the light responsiveness segment included as AAAC-motif, ACE, AE-box, AT1-motif, ATC-motif, ATCT-motif, Box 4, Box II, chs-CMA1a, GA-motif, Gap-box, GATA-motif, G-Box, G-box, GT1-motif, GTGGC-motif, I-box, LAMP-element, LS7, MRE, Sp1, TCCC-motif, TCT-motif, 4 cl-CMA2b. From all these motifs of the light responsiveness group, Box 4 exhibited the most prevalent response to light. As far as genes in GrFH were concerned, GrFH14 comprised 18 Box 4 motifs. There was also a substantial amount of Box 4 motif expressed in other GrFH genes. Moreover, GT1-motif, part of a light-responsive element, was found 16 times in the GrFH4 gene. All in all, all the cis-elements representing light responsiveness played a significant role in the CARE analysis. Tissue-specific expression, which ensured 16 cis-elements, was the next largest group. The 16 motifs included a 3-AF3 binding site, A-box, ARE, AT-rich element, Box II-like sequence, CAT-box, CCAAT-box, circadian, GCN4_motif, HD-Zip 1, HD-Zip 3, Box III, MBSI, MSA-like, O2-site and RY-element. From this group, the ARE motif acting as a cis-acting regulatory element essential for the anaerobic induction was observed to be the most often occurring response in this cohort. Besides, the ARE motif was repeated 7 times in the GrFH21 gene. In comparison to ARE, other motifs in this category were less frequently noticed. Approximately 12 cis-elements were observed in the phytohormone responsiveness group. As this group regulates the hormonal response, the ABRE motif functioning as a regulator of abscisic acid was most frequently expressed. The stress responsiveness group consisting of DRE core, LTR, MBS, TC-rich repeat, and WUN-motif appeared to construct a less significant amount of expression compared to the other 3 groups.

Fig. 6
figure 6

The distribution of putative cis-acting regulatory elements GrFH is represented by a heatmap. The names of each GrFH are shown on the left side of the heatmap. The number of putative cis-acting elements for each GrFH gene is displayed on the right side of the heatmap and is represented by five different colors (black = 0, orange = 1–5, green = 6–10, blue = 11–15, and red = 16–20). Functions associated with cis-acting elements of the corresponding genes, such as light responsiveness, tissue-specific expression, phytohormone responsiveness, and stress responsiveness, are shown at the bottom of the heatmap and are labeled red, green, dark violet, and yellow respectively

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Gene ontology (GO) analysis of GrFH

GrFH genes encompassed 64 GO enrichment id that were broadly classified into three distinct groups based on gene information and functional annotation Molecular Function (MF), Cellular Component (CC), and Biological Process (BP) (Supplementary Fig. S3). Biological Process (BP) was observed as the most prominent group among these categories, accumulating 49 GO IDs. The genes GrFH4, GrFH5, GrFH7, and GrFH11 featured in biological process bearing the following GO ids (Supplementary Material File 6); GO:0007015 (p-value: 2.50E-11), GO:0030036 (p-value: 4.40E-11), GO:0030029 (p-value: 5.30E-11), GO:0007010 (p-value: 6.40E-09, GO:0071822 (p-value: 7.90E-08), GO:1902589 (p-value: 2.50E-07, GO:0022607 (p-value: 3.40E-07, GO:0043933 (p-value: 6.10E-07, GO:0044085 (p-value: 2.20E-06, GO:0006996 (p-value: 5.70E-06, GO:0016043 (p-value: 1.03E-02, GO:0071840 (p-value: 1.46E-02). GrFH4, GrFH7, and GrFH11, which were responsible for the regulation of biological processes contained GO ids as : GO:0030838 (p-value: 1.60E-09), GO:0045010 (p-value: 1.60E-09), GO:0032273 (p-value: 2.40E-09), GO:0031334 (p-value: 3.00E-09), GO:0051495 (p-value: 3.00E-09), GO:0030041 (p-value: 5.80E-09), GO:0030833 (p-value: 5.80E-09), GO:0008064 (p-value: 2.23E-06), GO:0030832 (p-value: 2.23E-06), GO:0032271 (p-value: 2.23E-06), GO:0044089 (p-value: 2.23E-06), GO:0008154(p-value: 2.23E-06), GO:0032956 (p-value: 2.23E-06), GO:0032970 (p-value: 2.23E-06), GO:0043254 (p-value: 2.23E-06), GO:0051493 (p-value: 4.34E-06), GO:0010638 (p-value: 7.11E-06), GO:0051258 (p-value: 7.45E-06), GO:0032535 (p-value:1.25E-05), GO:0090066 (p-value: 1.25E-05), GO:0051130 (p-value: 1.43E-05), GO:0044087 (p-value: 1.43E-05), GO:0033043 (p-value: 5.94E-05), GO:0043623 (p-value: 3.16E-04), GO:0051128 (p-value: 3.40E-04), GO:0006461 (p-value: 1.22E-03), GO:0070271 (p-value: 1.25E-03), GO:0034622 (p-value: 1.27E-03), GO:0065003 (p-value: 2.10E-03), GO:0048522 (p-value: 3.01E-03), GO:0048518 (p-value: 1.38E-02), GO:0065008 (p-value: 1.86E-02). Regarding the cellular component, nine GO IDs were found. The estimated range of the p-value was 4.20E-06 to 9.93 × 10−3. On the other hand, GrFH genes retained the lowest GO ID counting in the molecular function. The GO IDs were GO:0051015 (p-value: 9.07E-05), GO:0005515 (p-value: 4.18E-03), GO:0003779 (p-value: 1.51E-02), GO:0032403 (p-value: 2.68E-02), GO:0044877 (p-value: 7.95E-02), and GO:0008092 (p-value: 7.95E-02).

Transcription factors analysis of GrFH

In the analysis of transcription factors (TFs), 36 unique TFs were discovered. A total of seven groups, namely ERF, GATA, MYB, LBD, TALE, and C2H2, along with bZIP, were formed according to the similarities within the TF family (Fig. 7). ERF represented 25 of the overall 36 TFs, accounting for approximately 69.44%. Among the 25 TFs in the ERF family, Gorai.008G271200, Gorai.005G200200, Gorai.001G033200, Gorai.008G153600, Gorai.005G116500, Gorai.001G036500, and Gorai.010G048900 were most frequently featured. Besides, the TF families of GATA (Gorai.013G055000, Gorai.006G054800, and Gorai.005G065300) and MYB (Gorai.001G177100, Gorai.004G269400, Gorai.001G148500) each possessed three TFs that were considerably less prevalent than the TFs of ERF. However, the LBD family, which consisted of two TF, Gorai.007G350300 and Gorai.007G075700, was substantially more active than the GATA and MYB families. The TALE, C2H2, and bZIP TF families each contained a single TF, Gorai.010G029000, Gorai.009G128000, and Gorai.009G285000 respectively. But Gorai.009G128000 from the C2H2 TF family featured more prominently than other TF members.

Fig. 7
figure 7

A heatmap represents transcription factors (TFs) in GrFH. The 7 TFs family are ERF, GATA, MYB, LBD, TALE, C2H2, and bZIP which are colored red, sky blue, purple-blue, green, golden, purple, and orange respectively

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Regulatory network between TFs and GrFH

Prediction of the regulatory network between TFs and GrFH was performed to know about their connection. The analysis revealed that 36 TF members were found to interact with both the 26 GrFH and with themselves (Supplementary Fig. S4). The largest ERF family interacted with all of the GrFH genes except GrFH3, GrFH20, and GrFH22. The ERF family members also engaged with other TF family members. The TFs of the GATA and MYB families formed the interaction with the GrFH genes such as GrFH1, GrFH2, GrFH5, GrFH6, GrFH7, GrFH9, GrFH10, GrFH12, GrFH18, GrFH23, and GrFH25. However, LBD and C2H2 families TF members were observed to link with GrFH genes more frequently compared to MYB and TALE families TF members. bZIP, on the other hand, interacted exclusively with a single GrFH gene, GrFH20.

Prediction of potential micro-RNAs targeting GrFH

MicroRNAs (miRNAs) are the 20–24 bp long noncoding RNA [23]. The principal function of miRNA is the regulation of gene expression through the inhibition of translation and cleavage of the targeted mRNA [24, 25]. They regulate gene expression through the RNA-induced silencing complex (RISC), which is composed of the ARGONAUTE (AGO) protein [26]. So, to understand the complex regulatory mechanism of miRNA in GrFH genes, 290 mature sequences of putative miRNA belonging to 96 unique families, targeting 25 different GrFH genes, were extracted (Supplementary Material File 7). The 290 putative miRNAs targeting 25 GrFH genes were shown in the network illustration (Supplementary Fig. S5 A) and the schematic diagrams indicate the GrFH genes targeted by miRNAs (Supplementary Fig. S5 B). It was observed that 22 members of gra-miR8762 targeted the highest up to eleven distinct GrFH genes (GrFH1, GrFH2, GrFH4, GrFH5, GrFH6, GrFH9, GrFH10, GrFH13, GrFH15, GrFH18, and GrFH24). Furthermore, 19 members of gra-miR530 targeted nine different GrFH genes (GrFH1, GrFH9, GrFH10, GrFH14, GrFH15, GrFH16, GrFH17, GrFH19, and GrFH25) (Table 2); 16 members of gra-miR7494 regulated ten distinct GrFH genes (GrFH4, GrFH5, GrFH6, GrFH9, GrFH11, GrFH12, GrFH15, GrFH17, GrFH22, and GrFH25). Besides, 12 family members of gra-miR7492 controlled five GrFH genes (GrFH2, GrFH10, GrFH13, GrFH15, and GrFH17) by cleaving mRNA and translational inhibition. However, the majority of the gra-miR family targeted one gene at a particular time. For example, gra-miR399 targeted only GrFH13. Some of the gra-miR targeted two or more GrFH genes. One such instance was noticed in gra-miR7504 that targeted GrFH5, GrFH9, and GrFH24. On the contrary, GrFH1 was targeted most frequently, as much as 30 times.

Table 2 Information about abundant miRNA ID, functions, and their targeted GrFH
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Protein-protein interactions of GrFH

Arabidopsis known protein was applied to carry out protein-protein interaction network analysis of GrFH (Supplementary Material File 8). The choice of GrFH regions as STRING proteins was made depending on their higher homology with Arabidopsis proteins. Moreover, all the GrFH were linked with Arabidopsis proteins (Supplementary Fig. S6). Four GrFH proteins (GrFH1, GrFH9, GrFH10, and GrFH16) were noticed homologous with AtFH20. They were also seen forming strong relations with FH5. However, AtFH1 was identified homologous with 8 GrFH proteins (GrFH2, GrFH3, GrFH6, GrFH8, GrFH11, GrFH20, GrFH21, and GrFH26) along with bonding firm interactions with PRF1, PRF2, PRF4, ARPC5A and FH14. GrFH4, GrFH11, GrFH15, and GrFH24 were homologous with AtFH5, and the proteins interacted with PRF 1, 2, 3, and 4, FH6 as well as FH13.AtFH4 was identified as homogenous with GrFH7 and 20. Besides they discovered strong interactions between PRF 3, 4, and 5 as well as FH5. Three AtFH proteins AtFH6, AtFH11, and AtFH13 were homologous with GrFH12 and GrFH14, GrFH13 and GrFH23, and finally GrFH19 and GrFH25 respectively. They showed strong interactions with PRF1, PRF2, PRF4, PRF15, FIM5, FH1, and FH5. But GrFH17 was homologous to AtFH18 which showed interactions with T6P5.20. The broader line connecting proteins indicates the greater interaction ratio conversely. The biological roles of the GrFH proteins that strongly engage with Arabidopsis proteins could be equivalent.

Tissue-specific expression pattern analysis of GrFH

The expression of GrFH genes in various tissues, including ovule, leaf, and fiber, demonstrated that approximately 96% of GrFH were expressed in 0dpa (0-days post anthesis) ovule (Fig. 8). Likewise, 13 GrFH genes (GrFH2, GrFH4, GrFH5, GrFH6, GrFH7, GrFH10, GrFH11, GrFH14, GrFH15, GrFH17, GrFH21, GrFH22, and GrFH26) were highly expressive (Supplementary Material File 9). However, the expression patterns changed drastically in the 3dpa ovule as only GrFH6, GrFH8, and GrFH9 were highly expressed. Although the overall expression of GrFH genes in 3dpa ovule was noticed at approximately 84.6%. On the contrary, the GrFH genes were expressed more highly in vegetative tissue (mature leaf) than in reproductive tissue (0 dpa and 3 dpa ovule). In the mature leaf, the expression of all the GrFH genes was observed. GrFH1, GrFH3, GrFH18, GrFH19, GrFH20, GrFH23, and GrFH24 were noticed to express exceptionally than other genes in mature leaf. The comparison between vegetative and reproductive tissue showed that genes that are prominently expressed in vegetative tissue were shown to be expressed less frequently in reproductive tissue, and vice versa. In the formation of fiber, 10dpa fiber, and 20dpa fiber were analyzed to unveil the expression of GrFH genes. In the 10dpa fiber, about 92.3% GrFH genes were observed to express whereas about 96.15% genes were observed expressing in the 20dpa fiber. The expression patterns were quite similar between the two. However, GrFH6, GrFH12, GrFH14, and GrFH26 were more dominantly observed in 10dpa fiber than in 20dpa fiber. Few GrFH genes such as GrFH8, GrFH15, and GrFH23 in 20dpa were identified to showcase higher expression than in 10dpa fiber.

Fig. 8
figure 8

Tissue-specific expression profiles of GrFH. The bottom of the heatmap contains various tissues including 0dpa ovule (days post-anthesis), 3dpa ovule, mature leaf, 10dpa fiber, and 20dpa fiber. The intensity of the color from low to high expression (white to red color) was shown on the right side of the heatmap

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Expression profiles of GrFH under cold and salt stress

The differential expression patterns of GrFH in leaf tissues in response to several abiotic stresses demonstrated their capacity to confront abiotic stress such as cold and salt (Fig. 9). The expression rate of GrFH at 12-hour control, cold, and salt stress were similar (Supplementary Material File 10). However, certain GrFH genes were highly upregulated in either cold or salt stress. In GD5 12-hour cold stress, the upregulated genes were GrFH4, GrFH6, GrFH11, GrFH12, GrFH14, and GrFH21. Though some genes, such as GrFH1, GrFH5, GrFH16, GrFH19, and GrFH26, exhibited high expression but were subsequently down regulated. In GD5 12 h salt stress, GrFH7, GrFH10, GrFH19, and GrFH26 showed up regulation. These genes showed elevated expression compared to the control, emphasizing resistance to salt stress. Nevertheless, most of the GrFH sustained downregulation in salt stress.

Fig. 9
figure 9

Expression pattern of GrFH in various abiotic stresses (cold and salt). The bottom of the heatmap contains control and abiotic stress (cold and salt). The intensity of the color from low to high expression (white to red color) was shown on the right side of the heatmap

Full size image

Discussion

The formin protein’s FH1 and FH2 domains significantly regulate the movements of the cytoskeleton. The integrity of actin filament in cytoskeleton dynamics is altered if mutations in one or more genes encoding formin proteins occur [27, 28]. The mutation influences not just actin filaments but also microtubule activity [29, 30]. In Arabidopsis, modification of microfilament structure during abiotic stress (salt and osmotic stress) emphasized the indispensable role the cytoskeleton performs [31]. However, the molecular mechanism surrounding cytoskeleton changes during stress remains a mysterious case.

The basic physiochemical properties of 26 GrFH proteins were investigated. Subsequently, all the GrFH proteins contained an instability index greater than 40. According to a study on Caulobacter crescentus metalloprotein, proteins with an instability score greater than 40 were considered unstable [32, 33]. Therefore, GrFH proteins were considered unstable. The higher the aliphatic index, the more the thermally stable and aliphatic side chains the proteins were regarded as [34, 35]. Therefore, most of the GrFH protein is considered thermally stable. All GrFH proteins exhibited a negative value for the grand average of hydropathicity (GRAVY), indicating their hydrophilic character [36, 37].

The comprehensive phylogenetic analysis helps to discover the molecular and evolutionary basis of lineage, interactions, and diversity among distinct species [38]. Moreover, phylogenetic analysis provides the patterns and evolutionary rates of different species [39]. The summary of the phylogenetic analysis of GrFH and other species revealed that the majority of GrFH clustered with MtFH, indicating a close connection between them. However, this was not shown by ZmFH or OsFH, with GrFH exhibiting their distinct genetic feature and divergence in evolution. The motif configuration in the GrFH diverged between some groups but was generally similar within the same group showcasing functional similarity. Besides, in G. raimondii, the GrTCP gene maintained a similar motif pattern among the subfamilies [40]. Similar distribution behavior and biological relevance were revealed by the existence of the FH2 conserved domain in all GrFH. The FH2 domain mediates actin filament elongation in cytoskeleton dynamics [41, 42]. In soybean (Glycine max), 34 proteins of GmFH contained formin domains suggesting their involvement in the management of abiotic stresses such as drought, heat, and salt through regulation of the cytoskeleton [43]. Besides, gene structure consists of exon-introns, which simplifies the structural diversity within the gene family [44, 45]. Generally, the presence of a high introns number promotes post-transcriptional events like alternative splicing. The possibility of a higher and longer intron promotes high expression compared to the lower number of exons, which activate quickly [46,47,48]. Group B members, especially GrFH9, GrFH10, and GrFH11, possessed 16 introns, displayed alternative splicing among the seven different groups, whereas group C member GrFH25 featured a lengthy stretch of introns. On the other hand, group G members comprised relatively lower exons, which provided the floodgates for activating the genes early. Nonetheless, GrFH20 from group G contained only two exons, which activated more quickly than others.

In evolutionary biology and comparative genomics, the Ka/Ks ratio is essentially regarded [49]. Thus, it highlighted that except for the GrFH20-GrFH26 pair, all the other GrFH gene pairs manifested purifying selection. On the other hand, the GrFH20-GrFH26 pair, experienced positive selection, where beneficial mutations were gradually building up and signaling that the protein was adapting to new functions or stresses from the environment [50, 51]. Gene collinearity is a specific kind of synteny when gene groups are present through the chromosomes of respective genomes in usually identical patterns [52]. There were 11 collinear pairs in GrFH genes. The results of the collinear relationship suggested that various GrFH gene members in cotton experienced a close genetic connection within them. Meanwhile, no syntenic pairs were observed to describe similarities in the physical arrangement of genes across different genomes.

Chromosomal localization is required to understand the function of a gene as well as gene duplication, modification, and conservation throughout evolution [53]. The 26 GrFH genes were distributed at various locations across the 12 chromosomes. There were also 12 chromosomes in the cyclin family of rice, where 49 predicted rice cyclin genes were distributed, underlying their significance in cell cycling [54]. Gene duplication events, such as segmental, tandem, and transposition, are among the primary mechanisms that explain the expansion of gene families in a wide range of species [55]. The segmental duplication that arises from the duplication of different chromosomes was seen in all GrFH gene pairs. In cotton, segmental duplication and collinear pairs contributed to resisting the salinity stress [56].

The subcellular localization displays and represents the gene in a particular organelle to gain insight into the molecular mechanism as well as cellular compartmentalization such as the mitochondria, which act as a powerhouse, the cytoskeleton, which facilitates mobility, and the structural proteins [57,58,59]. In this study, GrFH were mostly expressed in the chloroplast and nucleus. This showed that, in appropriate circumstances, a gene may express itself in those organelles and carry vital information. Moreover, most mTERF genes were localized in chloroplasts suggesting their essential role in photosynthesis machinery in maize [60].

The initiation of regulation of gene expression depends on the external or internal condition. Thus, the regulation of genes in environmental stress is generally promoted by promoters, enhancers, or suppressors [61, 62]. The analysis of the promoter regions of GrFH, and 4 distinct groups were created based on functional similarities of the 57 motifs. Among them, most of the motifs showed a response toward light. Besides, Cold Shock Protein (CSP) in cotton species showed various functionally similar CARE motifs such as light response and cold stress management [63]. Thus, GrFH13 was seen interacting with stress-responsive motifs such as DRE core that was involved in dehydration, low-temp, and salt stresses, while GrFH21 was observed more in the LTR motif in low-temperature responsiveness. Consequently, the MYB motifs were found in the GaMATE and GrMATE genes, unveiling drought resistance ability [64]. The functionally annotated GO was categorized into molecular function, biological process, and cellular component [65, 66]. Based on the findings, the majority of GrFH played the biological role regarding the cytoskeleton actin filament organization and regulation as well as the assembly of cellular components and proteins. The 28 biological pathways were observed in Gossypium herbaceum related to the drought resistance [67].

Transcriptional factors (TFs) perform a critical role in numerous biological processes, particularly in plants, such as regulating metabolism, growth, and progression, resistance against infections caused by microbes, and responses to both biotic and abiotic stress [68, 69]. This comprised 7 independent TF families such as ERF, GATA, MYB, LBD, TALE, and C2H2, along with bZIP. Ethylene-responsive factors (ERFs) serve as crucial regulators in the development, abiotic stress responses as well as defense against pathogen and insect attacks regulated by overexpression of the AP2/ERF in plant [70]. In cotton, ERF stimulated signals for the tolerance to salt and drought stresses [71]. A family of zinc finger proteins known as GATA (GA-binding Activator Protein Transcription Activator) vital in regulating the expression of genes that bind to the GATA [72, 73]. MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs control numerous physiological and biochemical processes, including plant development, the fate of cell determination, and secondary metabolism [68]. The bZIP (basic leucine zipper) regulates a wide range of biological processes, including the proliferation of cells, cell, and tissue differentiation, flowering and seed maturation, aging, as well as abiotic stress responses such as salinity and drought [68, 74, 75]. According to the results of the TF regulatory network analysis in GrFH, it disclosed that, ERF displayed the most substantial association with GrFH and bZIP and TALE TFs exhibited the lowest association with GrFH genes. Except for GrFH3, GrFH20, and GrFH22, all the members of GrFH genes showed interactions with the ERF family.

Recently, a study on miRNA in cotton revealed that it was associated with cellular signaling such as the hormone-signaling pathway, the calcium-signaling pathway, and the reactive oxygen species (ROS) signaling mechanism at the time of fiber initiation and elongation [76]. In this study, 96 unique miRNAs were identified. Among them, gra-miR530 might inhibit the GrFH mRNA production by cleaving or halting translation. Moreover, it was found to be involved in the regulation of blast disease resistance, yield, and growth period by blocking the overexpression of certain genes in rice and regulator gene expression under salt stress in flax (Linum usitatissimum) [77,78,79]. Therefore, it might have performed similar functions in the GrFH. Another miRNA, gra-miR7494 might have cleaved the mRNA and regulated the expression of GrFH in abiotic stress such as salt and drought. In cotton species, miR7492 was involved in the control of disproportionating enzyme 2, which was critical in the conversion of maltose to glucose [80]. Thus, it was predicted that gra-miR7492 might have related roles in GrFH.

Protein-protein interactions shed light on how the direction of diversification of existing species and the regulation of cellular functions, including the transmission of signals, regulation of cell cycles, and metabolic activity, was made conceivable by the emergence of biological network connections [81, 82]. The GrFH proteins showed homology with Arabidopsis and interactions with the FH family (FH1, FH4, FH5, FH6, FH12, and FH13), PRF family (PRF1, PRF2, PRF3, PRF4, and PRF5), ARPC5A, and T14C9.40.

Gene expression reveals the activity of genes under specific conditions and identifies regulatory mechanisms that control numerous stages of development, offering essential information on gene function [83,84,85]. The GrFH genes through transcriptomic analysis provided insights about their potential expression pattern in certain tissues (ovule, leaf, and fiber). GrFH8 and GrFH9 expressed at a higher rate compared to others in the 3dpa ovule. Cottons are widely cultivated economical crops and are considered cash crops because of their ability to produce fiber [86]. Cotton fibers, units of trichomes that originated from ovular epidermal cells, consisted of four overlapping stages initiation (0–8 dpa), elongation (3–17 dpa), secondary cell wall synthesis (17–40 dpa), and maturation (40–50 dpa) to become mature fiber (lint) [87,88,89,90]. Moreover, the cytoskeleton, actin filament, and microtubule, were involved in the formation of fiber [91]. Therefore, GrFH3, GrFH12, GrFH14, and GrFH26 in 10dpa fiber and GrFH8, GrFH19, GrFH23, and GrFH26 in 20dpa fiber were observed to express highly. GrFH26, however, maintained its expression at a high rate in both 10dpa and 20dpa fiber unveiling its involvement in mature lint formation. The differential gene expression pattern of GrFH in cold and salt stress underscored the significance they performed in combating these stresses. In cold stress, six GrFH genes such as GrFH4, GrFH6, GrFH11, GrFH12, GrFH14, and GrFH21 showed up-regulation, suggesting their potential association with cold stress management. Moreover, a study on G. raimondii showed that GrPRR5.1, GrPRR5.2, and GrPRR7.2 were the up-regulator genes in cold stress. Whereas, GrHP4.2, GrHK5.2, and GrRR11 were the up-regulator genes in salt stress [92]. The upregulation of genes such as GrFH7, GrFH10, GrFH19, and GrFH26 in salt stress provided evidence of their involvement in managing salt stress. Two major organs, ovule, and fiber, are the key enforcers to produce mature lint, as well as being most sensitive to abiotic stresses [93]. Therefore, overexpression and upregulation of GrFH4, GrFH6, GrFH12, GrFH14, and GrFH26 in these organs in cold stress suggested their role in cold resistance in cotton. Moreover, GrFH19 and GrFH26 conferred resistance to salt stress in these organs. All in all, this study focused on improving breeding strategies to generate cold and salt-tolerant cotton varieties to produce quality fiber.

Methods

Database search and mining of Formin gene in G. raimondii genome

A. thaliana FORMIN DNA binding domains were used to search and retrieve the FORMIN protein sequence in the cotton (G. raimondii) genome. Phytozome v13 (https://phytozome-next.jgi.doe.gov/) was employed to extract gene sequence, reference genome, and protein sequence by using BLASTp (Protein-Basic Local Alignment Search Tool) with an expected(E) threshold value of −1, a comparison matrix (BLOSUM62), and keeping other parameters as default [94]. Further, to find out the presence of the FH2 conserved domain, SMART (Simple Modular Architecture Research Tool) (http://smart.embl-heidelberg.de/) [95], NCBI CDD (Conserved Domain Database) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [96] and PfamScan (https://www.ebi.ac.uk/Tools/pfa/pfamscan/) were used with default parameters [97]. The candidate genes were selected based on the presence of the FH2 in the predicted protein and renamed according to their sequential physical chromosome positions.

Determination of physiochemical properties of GrFH

The physicochemical properties of FORMIN proteins included with the determination of the number of A.A. residues, molecular weight (kDa), pI, instability index, aliphatic index, and GRAVY in the ProtParam online program (http://web.expasy.org/protparam/) [98].

Phylogenetic analysis between GrFH, MtFH, ZmFH, OsFH, and AtFH

The formin protein sequences of the Medicago truncatula, Zea mays, Oryza sativa, Arabidopsis thaliana, and gene Gossypium raimondii were extracted from Phytozome v13 (Supplementary Material File 11), and a phylogenetic tree between them was constructed through the MEGA11 software [99] with the ClustalW program as a sequence alignment tool [100]. The Maximum Likelihood (ML) model was set to allow more robust parameter estimates [101]. Moreover, other parameters were set as default except for a 1000 bootstrap value to support branch values and Pearson correction. The phylogenetic tree was further uploaded to iTOL v6.7.4 (https://itol.embl.de/) for proper illustration and visualization [102].

Conserved motif, domain, and gene structure analysis of GrFH

The conserved motif, domain and gene structure of GrFH were constructed in TBTOOLS v.2.010 gene structure view (advanced) [103]. For the analysis of structural motifs in GrFH, Multiple EM for Motif Elicitation (MEME) (https://meme-suite.org/meme/tools/meme) was applied, setting several motifs parameters as much as 20 and others as default [104].

Gene duplication analysis and non-synonymous (Ka) and synonymous (Ks) substitution ratio calculation of GrFH

To determine and calculate the molecular evolution rate, Ka (non-synonymous) and Ks (synonymous) substitutions were performed. For the calculation of Ka/Ks ratios of GrFH genes, CDS sequences (Supplementary Material File 12) of duplicated genes were used in the Ka/Ks calculation tool (https://services.cbu.uib.no/tools/kaks). The obtained result was further assembled and enumerated the duplication and time of divergence (measured in million years ago, MYA) by applying the T = Ks/2λ formula, where λ was equal to 6.5 × 10−9 [105]. The data was visually illustrated by TB tools v.2.010.

Evolutionary collinearity and synteny relationship analysis of GrFH

Collinearity analysis was used to explore the evolutionary relationships among homologous genes of GrFH. However, to perform the synteny analysis, evolutionary relationships between different species Z. mays, O. sativa, A. thaliana, and candidate genes G. raimondii were used to find out gene duplication between them. For the visual representation, the collinear pairs among homologous genes and syntenic pairs in different species were illustrated in TB tools v.2.010.

Analysis of chromosomal localization and duplication of GrFH

By using the Phytozome v13 and TB tools v.2.010, the information about the chromosomal length, start points, and end points of 26 GrFH was retrieved and assembled. The retrieved data was mapped and viewed by MapGene2Chrom web v2 (MG2C) web server (http://mg2c.iask.in/mg2c_v2.0/) [106]. Whereas for the chromosomal duplication, duplicated gene pairs were analyzed in the distributed chromosomes.

Prediction of the subcellular localization of GrFH

The prediction of the subcellular localization of GrFH was analyzed by using the Wolf PSORT online tool (https://wolfpsort.hgc.jp/) [107]. The collected data was displayed by RStudio version 2023.06.1 generating a comprehensive overview of the subcellular compartmentalization of different organelles present in the respective GrFH [108].

Cis-acting regulatory elements (CAREs) analysis in the promoters of GrFH

About 2000 bp from 5ʹ UTR of 26 GrFH were extracted and predicted in the plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [109]. The RStudio version 2023.06.1 was used to generate a graphical representation. The grouping of CAREs was performed according to their functional similarities.

Gene ontology analysis of GrFH

The GO enrichment data was downloaded from (PlantRegMap; http://plantregmap.gao-lab.org/binding_site_prediction.php) with an estimated threshold p-value of 0.01 and other default parameters in an attempt to classify the functions of the predicted GrFH with genomic sequences (Supplementary Material File 13) [110]. The ChiPlot (https://www.chiplot.online/) provided in-depth details about visualization and illustration of the extracted data [111].

Transcription factors analysis of GrFH

For the identification of TFs related to the GrFH, (PlantTFDB;http://plantregmap.gao-lab.org/binding_site_prediction.php) was utilized with the default parameter and threshold p-value of 1 × 10 − 4. The collected data was then further processed and visualized in the RStudio version 2023.06.1.

Regulatory network between TFs and GrFH

Cytoscape version 3.10.0, a software tool used for the analysis and graphical representation of complex networks of molecular biology interactions, was used to visualize the interactions between various transcription factors as well as interactions with GrFH [112].

Prediction of putative micro-RNAs and networks targeting GrFH

(https://mirbase.org/) [113]. The CDS sequences of GrFH were uploaded to the online psRNATarget Server18 keeping the rest of the parameters set to default (https://www.zhaolab.org/psRNATarget/analysis?function=2) [114]. Moreover, Cytoscape software version 3.10.0 was employed to generate and visualize the interaction network between the predicted miRNAs and GrFH targeting genes.

Protein-protein interactions of GrFH

The protein-protein interaction network of GrFH proteins was predicted by employing the web tool STRING version 12 with GrFH protein sequneces (Supplementary Material File 14) (https://string-db.org/) according to the A. thaliana homologous proteins [115]. The parameters of the STRING online tool were kept as network type-full STRING network; the meaning of network edges evidence; active interaction source as text mining, experiments, databases, co‑expression, neighborhood, gene fusion, co‑occurrence; minimum required interaction score was specified to medium confidence parameter (0.4); max number of interactions display 1st shell was defined to no more than 10 and 2nd shell was left blank and enabling network displayed options as 3D bubble design.

GrFH tissue-specific and differential expression patterns in various abiotic stresses

The RNA sequencing data of five tissue samples (0dpa ovule, 3dpa ovule, mature leaf from accession ID SRP009820 and 10dpa fiber and 20dpa fiber from accession ID SRP001603) [87] and abiotic stress (cold and salt from BioProject accession ID PRJNA554555) [92] were extracted from the NCBI Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra/). Further, Trimmomatic package version 0.32 was used to quality control and trim the transcriptomic data [116]. Further, the RNA seq was mapped to the reference genome G. raimondii obtained from Phytozome v13 with Bowtie2 package version 2.5.4 [117]. The sequence alignment map (SAM) files were converted to binary alignment map (BAM) files and sorted and arranged with Samtools packages version 1.20 [118]. Fragments per kilobase million (FPKM) values were computed using the RSEM package version 1.1.17 [119]. The FPKM values were converted to log2 and visualized in TB tools v.2.010 illustrating, the expression profiling of GrFH.

Conclusions

The comprehensive bioinformatics analysis of 26 GrFH genes, comprised of the FH2 domain, will shed light on their potential role in the management of various abiotic stresses such as salinity and cold. The seven distinct subfamilies of GrFH in the evolutionary phylogenetic tree provided exclusive insights about their ancient descendant with MtFH. According to the subcellular localization, the bulk amount of GrFH genes was found in the chloroplast and nucleus. Moreover, 12 chromosomes were found to locate the distinct intron-exon gene structures. Furthermore, all the GrFH gene pairs experienced purifying selection except the GrFH20-GrFH26 pair. Besides, stress-related motif was found in GrFH CARE regions. Most GrFH were associated with biological functions focusing on the actin filament organization, regulation of actin filament polymerization and de-polymerization, and cytoskeleton assembly. Major TFs, including ERF, GATA, MYB, and LBD, interacted and formed complex networks with GrFH, showing their binding and regulation attributes. The non-coding miRNA regulated the gene expression of GrFH during salinity and cold abiotic stress and in the formation of mature fiber from the ovule. Besides, certain GrFH were observed to express at a higher rate, showcasing their promising attributes in fiber formation from the ovular epidermis. Further, the upregulation of GrFH4, GrFH6, GrFH12, GrFH14, and GrFH26 under cold stress and GrFH19 and GrFH26 under salt stress provided valuable insights in confronting these challenges. Overall, the findings from this study would provide deeper knowledge and shed light on the biological significance of the FORMIN gene family in G. raimondii cotton species in managing various stresses in wet lab facilities.

Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

A.A.:

Amino Acid

kDa:

Kilo Dalton

pI:

Isoelectric point

GRAVY:

Grand average of hydropathicity

bp:

base pair

5ʹ UTR 5ʹ:

untranslated region

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Acknowledgements

The authors are very grateful to the Laboratory of Functional Genomics and Proteomics, Department of Genetic Engineering and Biotechnology, Faculty of Biological Science and Technology, Jashore University of Science and Technology, Jashore 7408 for providing the opportunity to conduct this research. The authors acknowledge and appreciate the reviewers and the members of the editorial panel for their valuable comments and critical suggestions for improving the quality of this manuscript.

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  1. Pollob Shing, Md. Abdur Rauf Sarkar and Md Shohel Ul Islam contributed equally to this work..

Authors and Affiliations

  1. Laboratory of Functional Genomics and Proteomics, Department of Genetic Engineering and Biotechnology, Faculty of Biological Science and Technology, Jashore University of Science and Technology, Jashore, 7408, Bangladesh

    Pollob Shing, Md Shohel Ul Islam, Mst. Sumaiya Khatun, Naimul Hasan, Shaikh Mizanur Rahman & Md. Abdur Rauf Sarkar

  2. Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Rajshahi, Rajshahi, 6205, Bangladesh

    Fatema Tuz Zohra

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Contributions

Conceptualization and supervision: MARSData curation: MARS, PS, MSUI, FTFormal analysis: MARS, PS, MSUI Methodology: MARS, PS, MSUI, FTVisualization: MARS, PS, MSUIWriting-original draft: MARS, PS, MSUI, FT, SK, NH, SMRWriting-review & editing: MARS, PS, MSUI, FT, SK, NH, SMR.

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Correspondence to Md. Abdur Rauf Sarkar.

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Supplementary Information

Supplementary Material 1.

Supplementary Material 2.

Supplementary Material 3.

Supplementary Material 4.

Supplementary Material 5.

Supplementary Material 6.

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Supplementary Material 9.

Supplementary Material 10.

Supplementary Material 11.

Supplementary Material 12.

Supplementary Material 13.

Supplementary Material 14.

12863_2024_1285_MOESM15_ESM.tif

Supplementary Material 15. Supplementary Fig. S1 The estimation of gene duplication time and Ka/Ks ratio in GrFH. The ratio of nonsynonymous (Ka) to synonymus (Ks) changes is represented by Ka/Ks. The time of divergence (measured in million years ago, MYA) is also represented. The different color bar represents the data range.

12863_2024_1285_MOESM16_ESM.tif

Supplementary Material 16. Supplementary Fig. S2 The chromosomal locations and duplications of GrFH. The number of distinct chromosomes is at the top of each chromosome bar. The chromosome-scale is in millions of bases (Mb), indicating the length of each chromosome on the left. Chromosome is colored magenta while blue lines indicate segmental duplications.

12863_2024_1285_MOESM17_ESM.tif

Supplementary Material 17. Supplementary Fig. S3 GrFH genes function analysis through gene ontology. On the right side of the circos plot classification of the GrFH gene functions are shown. The number of genes involved under a certain GO ID, expected value, and rich factor are shown in a distinctive color. The scaling of the –log10 p value is shown in two distinctive colors (blue and yellow).

12863_2024_1285_MOESM18_ESM.tif

Supplementary Material 18. Supplementary Fig. S4 The regulatory network between TFs and GrFH genes. The GrFHis shown in red rectangular. Whereas the TFs are shown in different colors and shapes. The 7 TF families ERF, MYB, C2H2, GATA, bZIP, LBD, and TALE are represented by a yellow diamond, dark red hexagon, magenta triangle, lime green ellipse, V shape sea green, violet parallelogram and round rectangular respectively.

12863_2024_1285_MOESM19_ESM.tif

Supplementary Material 19. Supplementary Fig. S5 Prediction of potential micro-RNAs targeting GrFH. A. Network illustration of predicted miRNA targets GrFH genes. The red rectangle represents GrFH genes while microRNA is labeled as sky blue ellipse. B. The schematic diagram indicates the GrFH genes targeted by miRNAs. The sky-blue round rectangular is shown as exons of the respective gene, green round rectangular represents UTR, straight black line represents intron and red color small round rectangular represents microRNA (miRNA).

12863_2024_1285_MOESM20_ESM.tif

Supplementary Material 20. Supplementary Fig. S6 Protein-protein interaction of GrFH based on known Arabidopsis proteins. The proteins were displayed at network nodes with the proteins in nodes, and the line colors indicate different data sources.

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Shing, P., Islam, M.S.U., Khatun, M.S. et al. Genome-wide identification, characterization and expression profiles of FORMIN gene family in cotton (Gossypium Raimondii L.). BMC Genom Data 25, 105 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12863-024-01285-z

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