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Comprehensive identification and functional characterization of GhpPLA gene family in reproductive organ development

BMC Plant Biology volume 23, Article number: 599 (2023) Cite this article

Abstract

Background

Phospholipases As (PLAs) are acyl hydrolases that catalyze the release of free fatty acids in phospholipids and play multiple functions in plant growth and development. The three families of PLAs are: PLA1, PLA2 (sPLA), and patatin-related PLA (pPLA). The diverse functions that pPLAs play in the growth and development of a broad range of plants have been demonstrated by prior studies.

Methods

Genome-wide analysis of the pPLA gene family and screening of genes for expression verification and gene silencing verification were conducted. Additionally, pollen vitality testing, analysis of the pollen expression pattern, and the detection of POD, SOD, CAT, MDA, and H2O2 were performed.

Result

In this study, 294 pPLAs were identified from 13 plant species, including 46 GhpPLAs that were divided into three subfamilies (I-III). Expression patterns showed that the majority of GhpPLAs were preferentially expressed in the petal, pistil, anther, and ovule, among other reproductive organs. Particularly, GhpPLA23 and GhpPLA44, were found to be potentially important for the reproductive development of G. hirsutum. Functional validation was demonstrated by VIGS which showed that reduced expression levels of GhpPLA23 and GhpPLA44 in the silenced plants were associated with a decrease in pollen activity. Moreover, a substantial shift in ROS and ROS scavengers and a considerable increase in POD, CAT, SOD, and other physiological parameters was found out in these silenced plants. Our results provide plausibility to the hypothesis that GhpPLA23 and GhpPLA44 had a major developmental impact on cotton reproductive systems. These results also suggest that pPLAs are important for G. hirsutum’s reproductive development and suggest that they could be employed as potential genes for haploid induction.

Conclusions

The findings of the present research indicate that pPLA genes are essential for the development of floral organs and sperm cells in cotton. Consequently, this family might be important for the reproductive development of cotton and possibly for inducing the plant develop haploid progeny.

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Introduction

Plant reproductive development has gained a significant attention of researchers and breeders being a crucial aspect of plant life cycle. In the reproduction of flowering plants, double fertilization is an intricate vital process that serves a vital role in the production of offsprings [1]. Exploring the gene families involved in double fertilization is essential for understanding this mechanism, which is not only shared by all angiosperms but also essential for plant genetics and breeding. Pollen-specific phospholipase MTL (PLA) naturally contains frame-shifting mutations that create haploids by rearranging sperm membranes and interfering with double fertilization, it has been discovered to have a significant impact on crop development and breeding [2, 3]. It is essential to recognize and functionally describe PLA gene family in order to understand the PLA gene’s role in plant reproductive development.

Phospholipases (PLAs) are a family of ubiquitous proteins that cleaves various bonds in phospholipids to maintain membrane lipid homeostasis stability and also play an important role in signal transduction [4, 5]. PLAs are extensively dispersed and are categorized into three classes (A, C, and D) according to the particular cleavage sites that the hydrolases target [6]. Plant PLAs are classified into three families: PLA1, PLA2 (sPLA), and patatin-related PLA (pPLA). These PLAs hydrolyze either sn-1 or sn-2 acyl groups. Interestingly, pPLAs showed activity at both sn-1 and sn-2 sites, indicating the unique characteristics of pPLAs, while PLA1 and sPLA only target sn-1 and sn-2, respectively. [7] In Arabidopsis thaliana, pPLAs are divided into three subfamilies (pPLA-I、pPLA-II and pPLA-III) [7, 8]. In Arabidopsis, there is strong expression of pPLA-IIε and pPLA-IIδ in the roots and root hairs, as well as pPLA-IIδ in the leaves and cotyledons. The remaining pPLAs are primarily expressed in pollen (pPLA-I), seeds (pPLA-I, pPLA-IIβ, and pPLA-IIIα), and roots (pPLA-IIIγ and pPLA-IIIδ). Only the pPLA-IIγ is expressed in the reproductive organs [8, 9]. Various studies have also shown how pPLAs interact with phytohormones. For example, auxin significantly increases the synthesis of pPLA-IIδ and pPLA-IIIβ, whereas pPLA-I is involved in the biosynthesis of jasmonic acid [10, 11]. pPLAs were also involved in regulating the reproductive development of other plants, such as rice and maize [12].

Recently, haploids have been successfully induced using genome sequencing, genetic complementation, and gene editing methods technology of the MTL gene, which codes for a pollen-specific phospholipase of the pPLAs family [2, 3]. Similarly, haploid seeds have also been obtained by genetic mutations in Oryza sativa [13]. In the past years, mutants of TaPLA genes in wheat were successfully obtained through gene editing [14]. Furthermore, in addition to monocotyledonous plants, haploid induction has also been done in the dicotyledonous plant (Arabidopsis) [15]. These results clearly showed that pPLAs have the ability to mediate and even drive haploid development in plants.

Cotton is a significant global crop with substantial commercial value and scientific implications. In cotton, upland cotton is not only the primary source of renewable textile fiber, but also an excellent experimental system for studying polyploidy [16]. The understanding and investigation of pPLAs in dicotyledonous plants is primarily limited to Arabidopsis, despite the fact that haploids have been induced in a number of monocotyledonous plants. Moreover, the understanding of the development mechanism of pPLAs in upland cotton remains largely unknown. In this study, a systematic analysis was conducted on 13 species to investigate their collinearity, domain, 3D structures, expression patterns. Furthermore, the VIGS experiment was conducted in upland cotton to develop pPLA silenced plants, which served as a functional confirmation of the pPLA gene family. The reasonable analysis and prediction of GhpPLAs lay the foundation for further study of GhpPLAs.

Results

Identification and physiochemical characterization of pPLAs

A total of 294 pPLA genes were obtained from 13 selected species, including 21 pPLAs in Zea mays (Zm), 16 in Oryza sativa (Os), 23 in Sorghum bicolor (Sb), 23 in Vitis vinifera (Vv), 17 in Theobroma cacao (Tc), 23 in Glycine max (Gm), 13 in Amborella trichopoda (Atr), 9 in Selaginella moellendorffii (Sm), 24 in G. arboreum (Ga), 22 in G. raimondii (Gr), 46 in G. hirsutum (Gh) and 47 in G. barbadense (Gb) (Additional file 8: Fig. S1).

The genes were renamed based on their order from front to back on the chromosomes in order to more accurately characterize and identify these pPLA genes (Additional file 1: Table S1). A total of 46 pPLA genes were identified in upland cotton, and their physicochemical properties were analyzed. All of the 46 genes encoded proteins ranging from 381 (GhpPLA24 and GhpPLA45) to 1329 (GhpPLA40) amino acids, with protein PIs varying from 5.07 (GhpPLA42) to 9.04 (GhpPLA12) and MWs varying from 41.2 (GhpPLA45) kDa to 148 (GhpPLA40) kDa (Additional file 2: Table S2).

Subcellular localization prediction revealed that most GhpPLA proteins were located in the cytoplasm, with a small amount found in the nucleus and chloroplast (Additional file 2: Table S2). In subgroup I, five genes were located in the nucleus (GhpPLA27, GhpPLA40, GhpPLA18, GhpPLA14, and GhpPLA4), two in the cytoplasm (GhpPLA27 and GhpPLA36), two in the mitochondria, and one in the chloroplast. In subgroup II, all genes except for GhpPLA2, GhpPLA12, and GhpPLA26 were located in the cytoplasm. Among these three genes, GhpPLA2 and GhpPLA26 were found to be simultaneously located in the mitochondria and cytoplasm, while GhpPLA12 was located in the mitochondria, cytoplasm, and nucleus. In subgroup III-⍺, three genes were located in chloroplasts, GhpPLA11, GhpPLA20 and GhpPLA29, and two genes were located in mitochondria (GhpPLA3) and cytoplasm (GhpPLA30). Four genes in subgroup III-β were localized in both cytoplasm and chloroplast, while the remaining two genes (GhpPLA15 and GhpPLA24) were localized only in the chloroplast. In subgroup III-γ, GhpPLA23, GhpPLA28, and GhpPLA44 were located in the cytoplasm, GhpPLA41 in mitochondria, and GhpPLA19 in cytoplasm and mitochondria. These results suggested that several GhpPLAs are involved in signal transduction processes, such as auxin, pathogens, and inducers, which can activate pPLAs [17, 18].

Evolutionary relationship of pPLA in cotton and other 13 plant species

According to the clustering of the evolutionary tree and the classification of AtpPLA, this evolutionary tree was divided into three subfamilies. Subfamily III was further divided into three subgroups based on the clustering and motif similarity of the evolutionary tree, subgroup (I-III-γ) (Fig. 1A). Among them, pPLA genes from dicotyledonous and monocotyledonous plants were distributed in all five classes, in which subgroup III-β having the least number (19 genes) and subgroup II having the greatest number (75 genes). The absence of pPLA members from Amborella trichopoda in subgroups I and III-γ and Selaginella moellendorffii in subgroups II and III-α suggested that these two species may have lost genes during the evolutionary process. Arabidopsis has the lowest number, while the two allotetraploid cotton species have the highest numbers in evolutionary tree. The homologous genes of four cotton species are clustered together and closest to dicotyledonous plants (especially Arabidopsis), indicating that most of pPLAs in cotton is closely related to Arabidopsis and other dicotyledonous plants than monocotyledonous plants. As expected, a closer relationship between cotton and cacao than with other species was found in the study, pPLA genes in these two species are often phylogenetically linked [19]. Upland cotton has surprisingly undergone tremendous gene family expansion over the evolutionary process, as evidenced by the fact that its gene count in the evolutionary tree is twice or even three times higher than that of other species. These results indicate that gene duplication is the main factor in the evolution and expansion of the pPLA gene family in different species.

A phylogenetic tree (Maximum-likelihood; ML) was constructed to demonstrate the evolutionary relationship between diploid pPLAs (G. arboreum and G. raimondii) and allotetraploid cotton AtpPLAs (G. hirsutum and G. barbadense) constructed (Fig. 1B). Allotetraploid species G. hirsutum and G. barbadense were the most abundant, with ratios close to 1:1 (46 and 47, respectively), whereas diploid species G. arboreum and G. raimondii were also with ratios close to 1:1 (24 and 22, respectively). Allotetraploid cotton developed by hybridizing diploid cotton during the evolutionary process, as evidenced by the nearly two-fold increase in pPLA content in allotetraploid cotton compared to diploid cotton.

Fig. 1
figure 1

phylogenetic trees of pPLA genes. Two unrooted phylogenetic trees were constructed by the MEGA7.0 Maximum-likelihood method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. All positions with less than 90% site coverage were eliminated. (A) Phylogenetic relationship of the 294 identified pPLA genes from four cotton species and nine other plant species. (B) A phylogenetic tree of pPLA genes in four cotton species

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Chromosome mapping and collinearity analysis of pPLAs

It was shown by chromosome mapping that 24 pPLA genes were mapped on the chromosomes of G. arboreum, 22 on G. raimondii, and 47 on G. barbadense. G. hirsutum was slightly different; in addition to the 45 genes located on the chromosomes, one gene was located on the unmapped scaffold (Additional file 9: Fig. S2).

In G. arboreum, pPLAs were presented on almost every chromosome, except for Chr1, Chr7, and Chr013 (Additional file 9: Fig. S2A). Each chromosome had one to three genes however, Chr7 possessed eight pPLA genes, some of which were in tandem and might be the result of duplication occurrences. Comparably, in G. raimondii, Chr09 had six pPLAs, four of which were in tandem and most likely resulted from duplication events, while Chr01, Chr02, and Chr13 lack pPLAs. (Additional file 9: Fig. S2B).

In G. barbadense, a total of 47 GbpPLA genes were mapped on 19 chromosomes, including A2, A3, A5, A6, A8, A9, A010, A011, A012, D2, D3, D4, D5, D6, D8, D9, D010, D011 and D012 (Additional file 9: Fig. S2C). In G. hirsutum, pPLA genes were located on 18 chromosomes, including A03, A05, A06, A08, A09, A10, A11, A12, D02, D03, D04, D05, D06, D08, D09, D10, D11, and D12. Another gene (GhpPLA46) was located on an unmapped scaffold (Additional file 9: Fig. S2D). These findings indicated that the evolution of GhpPLAs was relatively mature. However, the number of genes on most chromosomes was observed to be dissimilar, indicated that pPLAs were unevenly and randomly distributed across the chromosomes of G. barbadense and G. hirsutum. This uneven distribution might have been caused by gene loss during the evolutionary process or incomplete genome assembly. Not only that, the number of pPLAs on At and Dt was close to 1:1, and the number and distribution of pPLAs on homologous chromosomes of different subgenomes were highly similar in G. hirsutum and G. barbadense. These results suggested that pPLA genes were relatively conserved in upland cotton and island cotton genomes. Notably, two tetraploid cotton sub-genomes, A and D, differed significantly from diploid cotton genomes in terms of both the number and the distribution of genes on their chromosomes. These findings imply that pPLAs might have experienced chromosomal rearrangement during cotton’s evolutionary history.

In the process of plant evolution, large fragment repetition of chromosomal regions was followed by gene loss, small-scale repetition, chromosome rearrangement, and other processes, which eventually led to the complexity of plant genomes [20]. Gene duplication mainly includes whole genome duplication (WGD), fragment duplication, and tandem duplication, which provides new genetic material for evolution and generates new gene functions, which is also an important reason for the widespread existence of gene families [21,22,23]. A total of 222 orthologous/paralogous gene pairs were obtained by collinearity analysis of four cotton species (Fig. 2; Additional file 3: Table S3). Most of these gene pairs experienced whole genome duplication (WGD), and most of them did not have tandem duplication, indicating that the amplification of cotton pPLAs mainly depended on WGD in the evolution process. These collinear pairs indicated that the evolution of cotton pPLAs is conserved. To further investigate the evolution and developmental mechanism of GhpPLAs, homo-linear analysis was performed on upland cotton and three representative species, including one dicotyledon (Arabidopsis thaliana) and two monocotyledons (Vitis vinifer and Zea mays) (Fig. 3). The results showed that there were 18 orthologous pairs in upland cotton and Arabidopsis thaliana, and a total of 10 and 29 pPLAs showed syntenic relationships with Vitis vinifer and Zea mays, respectively. This suggests that the homology of upland cotton to Vitis vinifer is higher than Arabidopsis thaliana, but lower than maize. Moreover, GhpPLAs also underwent additional genome-wide duplication due to the fact that there were 10 or more orthologous pairs between upland cotton and each species. Some GhpPLAs were colinear with Arabidopsis thaliana, maize and grape, suggested that they may play an important role in evolution. The expression patterns of orthologous pairs were similar, so pPLAs in upland cotton might have similar effects with pPLAs in different species.

Fig. 2
figure 2

The collinearity relationships of pPLA genes of four cotton species. The chromosomes of G. arboreum, G. raimondii, G. hirsutum, and G. barbadense were shown with little blue, kermesinus, orange, and brown colors, respectively

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Fig. 3
figure 3

The synteny analysis of pPLA genes between G. hirsutum and three representative plant species. The blue lines highlight the syntenic pPLA gene pairs. The specie names with the prefixes “Gh”, “Vv”, “Zm” and “At” indicate G. hirsutum, Vitis vinifera, Zea mays, and Arabidopsis thaliana, respectively

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Conserved motif and structural analysis of GhpPLAs

Conserved domains, motifs, and gene structure analysis were carried out, as these are directly linked with the function of a gene family (Fig. 4). Structural analysis demonstrated that different subgroups contained different numbers of introns and exons. Each gene in subgroup I contained more than 10 exons and more than 9 introns, and upland cotton genes (GhpPLA18, GhpPLA36, GhpPLA14, GhpPLA31 and GhpPLA4) had the highest number of exon and intron, with a total of 18 exons and 17 introns. Whereas, subgroup III-α contained only two exons and one intron, and subgroup III-γ contained up to six exons. Subgroup II basically contained 7 exons and 6 introns (Fig. 4D). The number of exons in different subfamilies varied, but the number of exons in the same subfamily was almost identical and had the same gene structure. This fully indicted that genes from different subfamilies have different biological functions, while genes from the same subfamily have a certain degree of conservation.

A total of 15 conserved motifs (named as Motif 1-Motif 15) were predicted in upland cotton (Fig. 4B), among which subgroup I contained 10 conserved motifs, Subgroup III-α contained 8 conserved motifs, and subgroup III-γ contained 9 conserved motifs. Subgroup III-γ and subgroup II had the largest number of conservative motifs, with a total of 11. The pPLA proteins of cotton, Arabidopsis and maize all contained a conserved ‘GXSXG’ typical serine hydrolase motif (named Motif 1) that catalyzes the dimer of serine and aspartic acid (Fig. 5A). Apart from ‘GXSXG’, all contained a conserved ‘DGG’ motif (named Motif 6) which was known as an enzyme active site and it was localized in the first one of the five Ca2+ binding β-rolls that formed the so-called C-terminal repeat domain [24] (Fig. 5C). However, in addition to subgroup I, the other subgroups also contained the ‘DGGGXRG’ motif (named Motif4), which were binding sites for phosphate anion element (Fig. 5B).

Fig. 4
figure 4

The gene structure, conserved motifs and domains in pPLA genes between Arabidopsis and four cotton species. (A) The ML phylogenetic tree was constructed based on the full-length sequences of Arabidopsis, Maize and four cotton species pPLA proteins using MEGA 7.0 software. Different subfamilies are highlighted in different colors. (B) Conserved motif of Arabidopsis, Maize and four cotton species. Different colored squares represent different motifs, named motif1-motif15. (C) The conserved domains in Arabidopsis, maize and four cotton species pPLA proteins. (D) The exon-intron structure of Arabidopsis, maize, and four cotton species pPLA genes. Blue for exons; black lines for introns

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Homology modeling of ertiary 3D protein structure of GhpPLA

The evaluation of the projected 3D configurations was conducted by assessing their GMQE score and the highest percentage of amino acids in the favored area of the Ramachandran plot. Among the templates considered, 1OXW.1B was deemed the most appropriate due to its substantial sequence similarity (43%) and GMQE score (0.72) with the query proteins. The target sequence had a length of 396 residues, while the template had a coverage range of 16–392 residues. More than 90% of amino acid residues were present in the Ramachandran plot (Fig. 5D), which can be considered as a high-quality protein model [25]. As depicted in Fig. 7B, the structure model of GhpPLAs revealed that they were primarily comprised of α-helices with a minor number of β-sheets. These proteins exhibited an α/β class protein fold with around three layers, as described in previous research [26]. Additionally, the catalytic Ser-Asp dyad was presented, with the serine residue located within the highly conserved lipase motif (GXSXG) [27]. Its folded topology and active site structure were closely related to the catalytic domain structure of human cPLA2α [28, 29]. By importing the PDB file into Prankweb, five ligand binding sites were predicted, two of which were more conservative. Based on the sequence analysis of each ligand binding site, pocket1 was identified as the true ligand binding site with a ser-catalyzed residue (Fig. 5F).

Fig. 5
figure 5

Sequences logos of conserved motifs and 3D structure of GhpPLA proteins. (A) Motif 1. (B) Motif 4. (C) Motif 6. (D) Ramachandran plot for predicting 3D structure of GhpPLA proteins. (E) Prediction of the 3D structure of GhpPLA proteins. The green arrow represents the active site. (F) Prediction of binding sites of GhpPLA proteins ligand. The blue areas represent ligand binding sites

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Promoter cis-element analysis of GhpPLAs

A cis-regulating element refers to a sequence within a gene sequence that plays a role in regulating gene expression. These elements can participate in the transcriptional regulation of a dynamic network of gene activities that control various biological processes [30]. In the case of GhpPLA, a total of 61 cis-regulating elements were identified, which were classified into three categories: hormone-acting elements, stress-acting elements, and growth and development-acting elements (as shown in Figs. 6 and 7, and detailed in Additional file 4: Table S4).

The pPLA genes of upland cotton contained 266 hormone cis-regulating elements, 29 growth cis-regulating elements and 309 abiotic stress cis-regulating elements (Figs. 6 and 7B; Additional file 4: Table S4). Auxin-related components (47.37%) were the most prevalent among phytohormone-related action elements. MYB, MYB-like sequence, TGA-element, AuxRR-core and MYB recognition site. Salicylic acid responsiveness elements (TCA-element and SARE), gibberellin-responsive elements (P-box and GARE-motif), MeJA-responsiveness elements (CGTCA-motif and TGACG-motif) and abscisic acid responsiveness elements (ABRE and AAGAA-motif) were counted as 6.77%, 4.89%, 18.8% and 22.18%, respectively. Conclusionary, there were more auxin reaction elements and abscisic acid reaction elements, indicated that most pPLA genes might be involved in the reproductive development and the senescence process of plants.

The growth and development-acting elements had the highest number of light-responsive elements, which made up approximately 92.41% of the total number of identified cis-acting elements (Figs. 6 and 7B; Additional file 4: Table S4). ATC-motif, GATA-motif, G-box, AE-box, GT1-motif, ATCT-motif, GATA-motif, GA-motif, Box4, chs-CMA1a, MRE, Sp1, ACE, 3-AF1 binding site, I-box, TCCC-motif, LAMP-element and Box II all belong to the light-responsive elements. It was hypothesized that the expression of GhpPLAs might be regulated by light. Additionally, specific response elements such as CAT-box, GCN4-motif, MSA-like, O2-site, RY-element, and circadian were also identified in relatively small numbers. For instance, there was only one circadian response element and one RY-element present in GhpPLAs. The MSA-like response element was also limited to only one, and they were only located in GhpPLA44, GhpPLA13, and GhpPLA2. Based on these findings, it can be speculated that GhpPLA44 might be regulated by day-night alternation, while GhpPLA13 might be specifically regulated in seeds, and GhpPLA2 by the cell cycle.

The most biotic or abiotic stress elements were dehydration-responsive elements, accounting for 44.34% of the total, with a total of 137 elements, such as Myb, MYC, Myb-binding site and DRE core (Figs. 6 and 7B; Additional file 4: Table S4). Secondly, the wound reactive element (W-box, WRE3, WUN-motif) and anaerobic inducible element including ARE were 63 and 61 respectively, which were close to 1:1. The reactivity element involved in low temperature responsiveness was LTR. Some elements can be involved in drought induction, for example, MBS. The element involved in defense and stress response were TC-rich repeats. The least number of anoxic-specific inducibility elements were GC-motif. The expression of GhpPLA genes might be primarily influenced by light, auxin, and dehydration responses, and they might play a role in controlling the process of upland cotton reproductive development, according to predictions derived from the investigation of these reaction elements.

Fig. 6
figure 6

The number statistics of GhpPLAs cis-regulating elements in the promoter

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Fig. 7
figure 7

Analysis of GhpPLA genes promoter and its expression pattern in different tissues. (A) Phylogenetic tree of GhpPLAs. (B)Cis-elements in promoters of GhpPLAs. (C) Expression pattern of GhpPLAs in different tissues

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Expression patterns of GhpPLA and qRT-PCR analysis

The heat map of the GhpPLA expression level was constructed using GhpPLA gene expression data that was available on the RNA-seq database (Fig. 7C; Additional file 5: Table S5). Heat map clearly showed that only GhpPLA7 was specifically expressed in cotton fiber, which might be important for the growth of cotton fiber. In addition, GhpPLA23 and GhpPLA44 were specifically expressed in anthers, pistils, and petals, which were probably related to the reproductive development of cotton. The remaining genes were expressed in vegetative organs and reproductive organs, or only specifically expressed in vegetative organs. The previous phylogenetic tree results suggested that the induced haploid gene GRMZM2G471240 (renamed ZmpPLA2) in maize belongs to subgroup III- γ. Five upland cotton genes (GhpPLA23, GhpPLA44, GhpPLA41, GhpPLA19, and GhpPLA28) with the closest genetic relationship to this gene and a gene GhpPLA7 specifically expressed in fibers were selected for qRT-PCR analysis.

Expression analysis elucidated that GhpPLA7 was expressed exclusively in roots and fibers, with a particularly high expression in roots and 24 DPA fibers (Fig. 8, Additional file 6: Table S6). This observation suggested that GhpPLA7 might play a crucial role in regulating the development of cotton fibers. It was shown in Fig. 8 that the expression level of GhpPLA19 in ovules at 0 DPA was slightly higher than other tissues, but the database showed that the expression level of each tissue was not high, which might be due to the selection of G. hirsutum cultivar Zhongmiansuo 100 and the selection of G. hirsutum cultivar TM-1 in the database, so the findings were little inconsistent. Similarly, in the public database, GhpPLA28 only expressed in leaves, while qRT-PCR analysis showed that GhpPLA28 was also expressed in anthers. This might be due to subtle differences between different cotton varieties, but it has no impact on later analysis and experimental research. Among them, GhpPLA41 was specifically expressed in the roots, and this gene might not affect the reproductive and developmental processes of cotton. Furthermore, expression analysis showed that GhpPLA23 and GhpPLA44 were specifically expressed in pollen and petals on the day of flowering, and these two genes might be the breakthrough in inducing haploid production.

Fig. 8
figure 8

The expression of pPLAs in different tissues in G. hirsutum

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Development of GhpPLA23 and GhpPLA44 silent plants via VIGS

Based on the analysis of cis-acting elements and expression patterns of GhpPLA, it was speculated that GhpPLA gene family might have potential functions in cotton reproductive development. Therefore, GhpPLA23 and GhpPLA44 were selected as candidate genes for further functional studies to prove this inference. The gene-silenced cotton plants were grown in a greenhouse until maturity, the plant heights of the positive plants carrying CLCrV: PDS, the negative control plants, and the two plants with genes silencing were basically the same, indicating that GhpPLA23 and GhpPLA44 didn’t affected cotton plant height after being silenced (Fig. 9A). However, it was observed that the petals and stigmas were obviously smaller (Fig. 9C and D). In addition, it was detected that the silencing of these two genes not only led to a significant reduction in the number of anthers but also caused a substantial decrease in the amount of released pollen (Fig. 9C). These findings suggested that the silencing of GhpPLA23 and GhpPLA44 has a considerable impact on the development of cotton floral organs, particularly on anthers and pollen, which will ultimately affect the reproductive developmental process of upland cotton. The qRT-PCR results demonstrated that the yield of pollen in plants subjected to VIGS was substantially lower compared to the control plants (Fig. 9B). All these findings indicated the significant impact of GhpPLA23 and GhpPLA44 on pollen development, thereby provided further evidence for their role in the reproductive development of upland cotton.

Effect of GhpPLA23 GhpPLA44 and silencing on pollen activity and physiological index

The control plant group showed almost 100% pollen viability with only one or two inactive pollen grains, while the VIGS plants exhibited a decrease of approximately 3.6% in pollen viability, indicating a significant decline trend (Fig. 9E, F, G and H, Additional file 7: Table S7). This observation also indicated, GhpPLA23 and GhpPLA44 might have an impact on pollen viability and development that indirectly affects the pollen fertilization. By detecting POD, SOD, CAT, H2O2, and MDA in pollen, it was observed that level of CAT, POD, SOD, and MDA in pollen increased dramatically in the silenced plants, although H2O2 showed a negative trend (Fig. 9). In maize, it has been confirmed that knocking out ZmPLA1 lead to the ROS outburst, which lead to DNA damage in sperm cells and cause induction of haploids containing only the maternal genome. Therefore, the significant changes in ROS and ROS scavengers in cotton after gene silencing of GhpPLA23 and GhpPLA44 may also be caused by the outbreak of ROS after gene silencing, which is consistent with research in maize [31]. These two genes play an important role in sperm cell development and fertilization in upland cotton, but respective mechanisms need further exploration and verification.

Fig. 9
figure 9

Phenotypic observation and changes of pollen viability in WT plants and plants with VIGS vectors. (A) Height comparison of VIGS vector containing plants. (B) Changes of pollen expression after VIGS. (C) Comparison of flower size difference. (D) Comparison of stigma size difference. (E) Pollen vitality of CLCrV: 00. (F) Pollen vitality of CLCrV: GhpPLA23. (G) Pollen vitality of CLCrV: GhpPLA44. (H) Pollen non-viable rate in plants with VIGS vectors. (I) Changes of CAT activity in pollen of plants with VIGS vectors. (G) Changes of POD activity in pollen of plants VIGS vectors. (K) Changes of SOD activity in pollen of plants with VIGS vectors. (L) Changes of MDA activity in pollen of plants with VIGS vectors. (M) Changes of H2O2 activity in pollen of plants with VIGS vectors. *** represent significant difference at the level of p < 0.001. ** represent significant difference at the level of p < 0.01

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Interaction network of GhpPLA proteins

The protein-protein interaction network is composed of individual proteins through interactions with each other [32]. To gain insight into the function of pPLA proteins, STRING data was used to construct and analyze protein interaction network based on Arabidopsis homologous sequences using protein family and multi-sequence search (Fig. 10). Through the in-depth study of Arabidopsis, the function of GhpPLA proteins can be speculated. By searching the protein families (Fig. 10A), Patatin-like phospholipase/acyl hydrolase (COG3621) was found in the center, and other pathways, such as diacylglycerol choline phosphotransferase activity (COG5050) [33], which can reduce seed metabolism. The pathway of FFAT motifs (COG5066) [34] targets cytoplasmic proteins to the surface of the endoplasmic reticulum as well as the nuclear membrane, suggested GhpPLAs might be involved in some signal transduction processes. Ca2+ dependent lipid-binding protein (COG5038) was found around the interaction network, suggested that some GhpPLAs also participate in pollen germination and pollen tube growth through this signaling pathway [35,36,37].

By the method of multiple sequences search (Fig. 10B), the interaction between GhpPLAs was fully demonstrated. The results showed that PLP8, PLP9 (GhpPLA45, GhpPLA43, GhpPLA37, GhpPLA24, GhpPLA22, GhpPLA15) and PLP1 (GhpPLA4, GhpPLA14, GhpPLA16, GhpPLA17, GhpPLA18, GhpPLA19, GhpPLA21, GhpPLA31, GhpPLA36, GhpPLA38, GhpPLA39, GhpPLA40, GhpPLA41, GhpPLA42) directly interacted with NLM1, suggested that the expression levels of PLP8 and PLP9 might be decreased due to ABA, NaCl and drought stress induction. Interaction of PLP3 (GhpPLA27) with SDP6 indicated that PLP3 was involved in storage lipid catabolism.

Fig. 10
figure 10

Interaction network of pPLA proteins. (A) Interaction network of pPLA proteins families. (B) Interaction network of GhpPLA proteins with other proteins. The red letters represent GhpPLA proteins

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Discussion

Patatins refer to proteins found in potato tubers that possess acyl hydrolysis activity [38]. The patatin catalytic domain is present in various organisms including plants, animals, bacteria, etc., and can effectively catalyze the hydrolysis of both phospholipid sn-1 and sn-2, thereby releasing free fatty acids simultaneously. According to the genome-wide identification of 13 species, a total of 294 pPLA genes were identified and classified into three distinct subfamilies. Notably, allotetraploid cotton had the greatest number of pPLA genes compared to the other studied species. Previous studies have identified 10 pPLA genes in upland cotton [39]. However, this current study has conducted a more comprehensive analysis and identified a total of 46 pPLA genes, including those identified in previous studies. Moreover, the study has also verified the functions of candidate genes in reproductive development. Out of the 46 pPLA genes identified in upland cotton, 24 genes were in At genome, 22 genes in the Dt genome, and one gene was located on the unmapped stent, indicating that the evolution of the pPLA gene family was relatively conservative and mature in upland cotton. Compared to other species and diploid cotton, upland cotton has 2–3 times more pPLA genes, which might be attributed to chromosome doubling and genome amplification in allotetraploid cotton. According to a genome-wide duplication investigation, G. arboreum and G. raimondii underwent genome-wide duplication specific to cotton 1.6 million years ago. Another genome-wide duplication occurred between 13 and 20 million years ago, and upland cotton originated between 1 and 2 million years ago [40,41,42,43]. The complexity and functional expansion of cotton pPLAs were mainly caused by gene duplication. During the evolution of GhpPLAs, random distribution and uneven number of GhpPLAs on chromosomes were caused by gene loss, gene increase, chromosome rearrangement, and incomplete genome assembly.

pPLAs have a specific patatin domain, mainly PLP1, PLP2, PLP3, PLP5, PLP6, PLP7 and PLP9 in upland cotton. The conserved motif analysis showed that all upland cotton contained a conserved ‘GXSXG’ typical serine hydrolase motif [27] and a conserved ‘DGG’ motif, in which ser residue in ‘GXSXG’ was the catalytic central residue, which catalyze the dimer of serine and aspartic acid. All these results indicated the evolutionary conservation of GhpPLAs. The gene structure of GhpPLAs showed that the number of exons were different in different subfamilies and the same subfamily had a similar gene structure, indicated that the biological functions of different subfamilies might differ, and the biological functions of the same subfamily were likely to be similar. The similarities and differences in GhpPLAs domains, conserved motifs, and gene structures might be related to conserved and sub-functionalization, mainly due to gene duplication during evolution [43]. Subcellular localization predicts that GhpPLAs are mainly located in the cytoplasm, which may be involved in plant signal transduction, such as auxin, pathogens, and other inducers [17, 18]. Moreover, through the analysis of homeopathic elements in the upstream 2000 bp region of the promoter, it has shown that pPLAs maybe responsive to auxin, abscisic acid, light, and drought, indicating that these elements are important for pPLA gene regulation. It was speculated that GhpPLAs may regulate the reproductive development process of upland cotton through the effect of auxin. The protein interaction network showed that pPLA proteins acted directly with diacylglycerol choline phosphotransferase activity (COG5050) [33], FFAT motifs (COG5066) [34], and Ca2+ dependent lipid-binding protein (COG5038) [35,36,37], suggested that pPLAs might be involved in seed lipid storage and plant signal transduction. Moreover, it was reported that Ca2+ dependent lipid-binding protein was involved in pollen germination and pollen tube growth [36], so GhpPLAs might play a role in pollen and thus affect the reproductive development of upland cotton.

Silencing of GhpPLA23 and GhpPLA44 in upland cotton has been shown to have a significant effect. It has observed that silencing of GhpPLA23 and GhpPLA44 caused the smaller the petals, stigmas, and anthers. And the amount of viable pollen has also been significantly reduced. This further supports the hypothesis that GhpPLAs play a crucial role in the reproductive development of upland cotton. In addition, the expression of pollen has a significant decreased trend compared with the control. Through the observation of the pollen vitality in plants after VIGS, it has shown that there are certain level of non-viable pollen. All of the evidence points to the possible role of GhpPLA23 and GhpPLA44 during upland cotton’s reproductive development. In addition, ROS has been were greatly impacted in the GhpPLA23 and GhpPLA44 silenced plants. The pollen’s POD, H2O2, CAT, MDA, and SOD levels all differed dramatically in GhpPLA23 and GhpPLA44 silenced plants compared with control [31]. Moreover, studies have proved that haploid induced by pPLA were because of ROS explosion, and the sperm cell DNA rupture finally produced the haploid containing only the maternal genome [44]. Therefore, GhpPLA23 and GhpPLA44 are likely to be the key genes for the production of haploid for upland cotton.

Haploid breeding plays an important role in crop breeding. Previous studies have found some candidate genes responsible for the haploid, such as CENH3 [45, 46], DMP [47,48,49] and PLD3 [50]. MATL, also known as ZmPLA1 (renamed ZmpPLA2 in this article) belongs to the pPLA gene family and can induce maize haploid [11, 12]. In recent years, researchers have successfully developed rice haploids by knocking out MATL homologous genes in rice and wheat, indicating that pPLA gene mutations is linked with the haploid induction in monocotyledons [13, 14]. Additionally, pPLAs are also involved in plant growth and development regulation in Arabidopsis thaliana [15]. In this study, several genes closely related to ZmPLA1 were analyzed systematically. It was found that GhpPLA23 and GhpPLA44 were specifically expressed in pollen and petals, and the silencing of these two genes significantly affected the size of cotton petals and stigma, the number and expression of pollen and various physiological indicators. It is hypothesized that, GhpPLA23 and GhpPLA44, could serve as potential candidate genes for future haploid breeding of cotton. However, it is important to note that whether these genes can induce haploids requires further investigation. Currently, this study confirmed that GhpPLAs play a role in the reproductive development of upland cotton, and further research is needed to explore their potential applications in haploid breeding.

Conclusions

In this study identification and phylogenetic analysis of cotton, as well as, the analysis of the chromosome location, conservative motif, gene replication events, protein structure, and interaction network of 46 genes in upland cotton were carried out to explore the role of pPLAs in haploid induction. These genes were classified into three subgroups, leading to a deeper understanding of pPLA. The expression analysis of GhpPLA23 and GhpPLA44 and the results of VIGS proved that these two genes play an important role in the reproductive development of upland cotton, which laid a foundation for the study of pPLAs in the reproductive process of upland cotton and the induction of haploid.

Materials and methods

Databases

The genome information of four cotton species, including G. arboreum (ZJU, version 1.0), G. raimondii (ZJU, version 2.0), G. hirsutum (ZJU, version 1.0) and G. barbadense (ZJU, version 1.0), were downloaded from CottonFGD (http://cottonfgd.org) [51]. The genome information of other nine species, including Arabidopsis thaliana (TAIR, version 10), Zea mays (Ensembl-18_2010_01), Amborella trichopoda (version 1.0), Glycine max (version 1.0), Oryza sativa (version 7.0), Sorghum bicolor (version 3.1.1), Selaginella moellendorffii (version 1.0), Theobroma cacao (version 2.1) and Vitis vinifera (version 2.1), were acquired from phytozome (https://phytozome-next.jgi.doe.gov/ accessed on 22 November 2020) [52].

Identification of pPLA family members

The genome annotation data (in gff3 format) and gene sequence data (in Fasta format) for four cotton species were downloaded and converted to protein information using TBtools [53]. The amino acid sequences of AtpPLAs were used as query sequences to search for the pPLA genes in four cotton protein databases using the blast program. Subsequently, Pfam (http://pfam.xfam.org/) [54] and SMART (http://smart.embl-heidelberg.de/) [55] were used to screen candidate sequences containing patatin domain (PF01734). Similar method was used to extract the pPLA sequences of other nine species, that were used to construct evolutionary tree.

The physical and chemical properties of GhpPLA proteins, such as amino acid length, molecular weight, and isoelectric point, were analyzed using the ExPasy website (https://web.expasy.org/compute_pi/) [56] and CottonFGD. The subcellular location of candidate GhpPLA genes was predicted using the CELLO v2.5 server [57].

Evolutionary relationship analysis among different species

ClustalW was used for sequence alignment in MEGA7.0 software [58], and a phylogenetic tree was constructed after sequence alignment, using the Maximum Likelihood (ML) method with 1000 bootstrap replications. To find out the genetic relationship between G. hirsutum and other species, two phylogenetic evolution trees were constructed. The first tree included four cotton species, Arabidopsis, and Z. mays, while the second tree encompassed G. hirsutum, A. thaliana, Z. mays, and seven additional species (A. trichopoda, G. max, O. sativa, S. bicolor, S. moellendorffii, T. cacao, V. vinifera).

Chromosome mapping and collinearity analysis of pPLAs

Chromosome mapping of pPLAs in four cotton species was performed by TBtools software using gene annotation information (Gff3 format) and genome assembly sequences depending on (Fasta format) downloaded from CottonFGD data. The protein sequences of the four cotton species were used as query sequences to perform a self-blast using blastp in TBtools. The results of the blastp analysis were used to create a collinear atlas using Advanced Circos in TBtools. Blast analysis was performed between G. hirsutum and three different species (A. thaliana, Z. mays and V. vinifera) using One Step MCScanX and constructed figures by Text Merge for MCScanX.

Analysis of the conserved protein motifs and gene structure

The MEME website (https://meme-suite.org/meme/tools/meme) [59] was utilized to detect conserved motifs in pPLAs gene sequences of four cotton species, with a p-value lower than 1e − 5, and the results were saved as a MAST file. The Batch Web CD-Search Tool in NCBI [60] was used to predict conserved domains in gene sequences, with an e-value lower than 0.01, and the hit data file was saved. TBtools was then used to combine the conserved motifs, conserved domains, phylogenetic tree, and intron/exon structure, by utilizing the nwk file, MAST file, hit data file, and gff genome files.

Homology modeling of the 3D structure of GhpPLA proteins

The 3D crystal structure of GhpPLAs is still unknown, so the computer-based prediction was carried out through a homology modeling approach using the SWISS-MODEL website (https://swissmodel.expasy.org/) [61]. GhpPLA23, which was closely related to ZmPLA1, was selected for homology modeling. A template having the highest sequence identity and coverage to the GhpPLAs was selected from the PDB with ID: 1OXw.1B. Its sequence similarity reached 48.15%, and the model was evaluated as 0.72, showing high reliability. PDB files of the model were downloaded from SWISS-MODEL and used as input files into PrankWeb (https://prankweb.cz/) [62] to predict the active sites of protein 3D structure.

Promoter Cis-element analysis and digital expression analysis of GhpPLAs

TBtools software was used to analyze 2000 bp DNA sequences in the upstream region of pPLA genes in upland cotton. Then the cis-regulatory elements of the promoter region from GhpPLAs were predicted through the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [63]. After that, cis-regulation elements were screened and classified, including phytohormone, plants growth and abiotic stress. To investigate the expression patterns of GhpPLAs across diverse tissues, RNA-Seq data (PRJNA490626) was acquired from the Cotton Omics Database (http://cotton.zju.edu.cn/). The quantification of gene expression was conducted using the fragments per kilobase of exon per million mapped (FPKM) approach. Subsequently, TBtools software was utilized to create a heat map, which was supplemented with a phylogenetic tree and cis-elements to provide additional information on the genes.

qRT-PCR analysis of pPLAs

Six GhpPLAs were selected for qRT-PCR analysis, including GhpPLA7, GhpPLA19, GhpPLA23, GhpPLA28, GhpPLA41, and GhpPLA44. Different tissues were selected for qRT-PCR analysis, including sepal, bracts, petal, and anther (flowers bud size < 3 mm, 4–5 mm, 5–8 mm and > 8 mm [64, 65]). Pollen, ovule and fiber were collected from the experimental field of G. hirsutum cultivar Zhongmiansuo 100. Roots, stems and leaves were collected at the seedling stage of G. hirsutum cultivar Zhongmiansuo 100. Total RNA was extracted from these tissues using RN38-EASYspin-Plus Plant RNA Kit (AidlabCo., LTD, Beijing, China). The extracted RNA was reversely transcribed with PrimeScript™ RT reagent Kit (Takara Biomedical Technology Co., LTD, Beijing, China). Specific primers were designed for the six selected genes using qPrimerDB-qPCR primer Database websites (Additional file 6: Table S6) [66]. RT-qPCR analysis was performed using Bio-Rad 7500 fast fluorescence quantitative PCR platform with SYBR® Premix Ex Taq™ (Takara Biomedical Technology Co., LTD, Beijing, China). The experiments were independently repeated three times, and the 2–ΔΔCt method was used to calculate the relative expression levels of GhpPLAs.

CLCrV vector construction of GhpPLA23 and GhpPLA44

The specific sequence of 247 bp was selected from the CDS of the two candidate genes for the primer design of the two gene fragments. Two insertion fragments CLCrV: GhpPLA23 and CLCrV: GhpPLA44 were amplified by PCR using upland cotton pollen as a template. The vector was digested using ascI and speI as restriction sites and the restricted fragments were ligated with the digested CLCrV vector. The connected plasmids were transferred into GV3101 through transformation, the OD value of shaken strains was adjusted to 1.0 by suspension, and the cotton with two cotyledons were transformed after two hours. Transformed plants were incubated darkly in a greenhouse at 23 °C for 24 h and the yellowing phenotype was successfully observed after two weeks, indicating that VIGS was successful. After five or six months of plant growth, the flowers were picked, RNA was extracted from pollen, and qRT-PCR experiment was performed on the Bio-Rad 7500 fast fluorescence quantitative PCR platform using SYBR® Premix Ex Taq™ (Takara Biomedical Technology Co., LTD, Beijing, China). The experiments were independently repeated three times, and the 2–ΔΔCt method was used to calculate the relative expression levels of GhpPLA23 and GhpPLA44.

Determination of physiological parameters of VIGS plants and observation of pollen activity

The freshly picked pollens were fixed with Carnot fixative, and then washed three times with 95% alcohol and 75% alcohol, and then washed three times with ddH2O. The washed pollens were dripped into the slide and stained with Alexander’s staining solution. After 4 h, the pollen viability was observed by microscope, and pollen observation of each plant was repeated three times. The pollens of the control and silenced plants were collected, cold-shocked in liquid nitrogen and stored at -80℃ for later use. Catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), malonyl dialdehyde (MDA), and H2O2 activities were determined using the kits (Solarbio, Beijing, China).

Interaction network of the pPLA proteins

STRING website (https://cn.string-db.org/) [67] was used to construct protein interaction networks for the whole family and pPLAs of A.thaliana respectively with a confidence parameter set at 0.15 threshold.

Data Availability

The source data underlying the graphs in the main figures are available in Supplementary information. Sequence data from this work can be found in Cotton FGD (https://cottonfgd.org/) and Phytozome (https://phytozome-next.jgi.doe.gov/).

Abbreviations

CAT:

Catalase

SOD:

Superoxide dismutase

POD:

Peroxidase

MDA:

Malonyl dialdehyde

PLAs:

Phospholipases as

pPLA:

Patatin-related PLA

JA:

Jasmonic acid

Zm:

Zea mays

Os:

Oryza sativa

Sb:

Sorghum bicolor

Vv:

Vitis vinifera

Tc:

Theobroma cacao

Gm:

Glycine max

Atr:

Amborella trichopoda

Sm:

Selaginella moellendorffii

Ga:

G. arboreum

Gr:

G. raimondii

Gh:

G. hirsutum

Gb:

G. barbadense

At:

Arabidopsis thaliana

qRT-PCR:

quantitative real-time PCR

WGD:

Whole genome duplication

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Acknowledgements

We thank Dr. Javaria Tabusam (Institute of Cotton Research, CAAS, China) for critical reading and professional editing of the manuscript.

Funding

This work was supported by National Engineering Research Center of Cotton Biology Breeding and Industrial Technology / Institute of Cotton Research of CAAS (NERC010115), Science and Technology Plan Project of Anyang City (2023C01NY019), and Hainan Yazhou Bay Seed Lab (B21HJ0206).

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Author notes

  1. Mingyang Wang and Dingyan Tian contributed equally to this work.

Authors and Affiliations

  1. National Engineering Research Center of Cotton Biology Breeding and Industrial Technology, Institute of Cotton Research, Chinese Academy of Agricultural Science, Anyang, Henan, 455000, China

    Mingyang Wang, Dingyan Tian & Yongshan Zhang

  2. Zhengzhou Research Base, State Key Laboratory of Cotton Biology, Zhengzhou University, Zhengzhou, Henan, 450001, China

    Tengyu Li, Chenlei Wang, Lanxin Wu, Zhenyu Mei, Wei Chen, Jinbo Yao, Yan Li, Shouhong Zhu & Yongshan Zhang

  3. College of Agronomy, Tarim University, Alar, Xinjiang, 843300, China

    Jingwen Pan & Jinwei Liu

  4. College of Advanced Agricultural Science, Zhejiang A&F University, Hangzhou, Zhejiang, 311300, China

    Kun Luo

  5. College of Life Sciences, Hebei University, Baoding, Hebei, 071002, China

    Fuxin Wang

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  1. Mingyang Wang
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Contributions

Writing the original draft, Mingyang Wang, Dingyan Tian; Formal analysis, Mingyang Wang，Jingwen Pan, Tengyu Li; Validation, Chenlei Wang, Lanxin Wu, Kun Luo; Methodology, Zhenyu Mei, Jinwei Liu, Wei Chen, Jinbo Yao and Yan Li; Concept of study, Supervision and revised the manuscript, Fuxin Wang, Shouhong Zhu and Yongshan Zhang. All authors have read and approved the final manuscript and they declare that they have no competing interests.

Corresponding authors

Correspondence to Fuxin Wang, Shouhong Zhu or Yongshan Zhang.

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Ethics approval and consent to participate

All the cotton material was collected from the Institute of Cotton Research, Chinese Academy of Agricultural Sciences, which is publicly available for non-commercial purpose. All experimental studies on plants were complied with relevant institutional, national, and international guidelines and legislation.

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Wang, M., Tian, D., Li, T. et al. Comprehensive identification and functional characterization of GhpPLA gene family in reproductive organ development. BMC Plant Biol 23, 599 (2023). https://doi.org/10.1186/s12870-023-04590-4

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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