1. Introduction

Vesicle transport is an extremely important cytological process in eukaryotes. It moves proteins, lipids, and other substances between the inner membrane system and the cells, and establishes cell polarity, secretion, growth, division, and wall formation [1]. Tethering is a key step in vesicle transport. Large multi-subunit tethering complexes were first discovered in yeast [2]. Exocysts tether different vesicles to the exocytosis site required for cellular secretion [3]. They are evolutionarily conserved octameric protein complexes composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, EXO70, and EXO84 [4, 5]. EXO70 plays a key role in exocyst assembly [6]. It recruits exocysts on the target membrane and interacts with Rho protein to regulate SNARE complex assembly and activation there via SEC6. In this manner, EXO70 mediates polar exocytosis [7, 8].

The exocyst subunits are encoded by a single gene in yeast and just a few genes in metazoans. However, 23 EXO70 subunits encoded by various loci have been identified in Arabidopsis [9, 10]. The EXO70s in terrestrial plant genomes have even more copies. This phenomenon is unique to the EXO70 subunit of the exocyst [11]. In the fungal and animal genomes sequenced to date, only one EXO70 coding gene was found. Hence, multiple EXO70 gene copies are unique to higher terrestrial plants [12]. Certain EXO70 functions might have been alienated during evolution and participated in other biological processes besides membrane vesicle transport. Alternatively, various EXO70 functions are specialized and form different exocysts from other subunits that participate in specific membrane vesicle transport processes in the organization, carrier substrate, or transport link [12]. The expression profiles of the 23 members of the Arabidopsis EXO70 family have been analyzed. Expression of this gene family has the following characteristics: spatiotemporal expression specificity at the cell and tissue levels; no constitutive expression; and specific expression in dividing, growing, differentiating, and secreting cells [12]. Plant EXO70 gene family members participate at the transcriptional level in the biological processes of different cell types via cell- and tissue-specific expression patterns.

EXO70 is an important part of the secretory complex mediating exocytosis, and it regulates neurite growth in animal cells, epithelial cell polarity, and cell movement and morphogenesis [13-17]. In plants, EXO70 regulates pollen tube elongation and polarization, root hair growth, cell wall material deposition, cell plate activation and maturation, defense and autophagy, and so on [10, 18-21]. Defects in AtEXO70C2 gene function affect pollen tube growth, which results in significant male-specific transmission defects in Arabidopsis [22]. EXO70H4- and PMR4-dependent corpus callosum deposition in trichomes is necessary for cell wall silicification [23]. EXO70B1 knockdown resulted in impaired light-induced stomatal opening [24]. AtEXO70B1 and AtEXO70B2 regulate FLS2 to participate in plant immune response[25]. AtEXO70D regulates cytokinin sensitivity by mediating the selective autophagy of Type-A ARR protein, thereby maintaining cell homeostasis and normal plant growth and development[26]. OsEXO70A1, OsEXO70L2, and AtEXO70A1 affect tracheary element (TE) development [27-29]. Hence, the roles of EXO70 in plant organ development have undergone differentiation.

Cotton is a major global economic crop. It is a source of seed, fiber, oil, and medicine [30, 31]. The development of novel high-quality cotton varieties is of great commercial importance. The high copy numbers and tissue-specific functions of the EXO70 gene in plants suggest that targeting EXO70 to construct high-quality cotton is feasible. To date, however, few studies have investigated the cotton EXO70 gene. The only report is that GhEXO70B1 may respond to stressors by mediating cell autophagy [32]. Here, we conducted evolutionary analyses, systematically named cotton EXO70 gene family members, and examined the functions of GhEXO70A1-A. It is believed that the discoveries of the present work will provide theoretical and empirical references for future research on cotton EXO70.

2.Results

2.1. Identification and Analysis of the Phylogenetic Relationship of the EXO70 Gene Family in Cotton

The exocytosis complex subunits comprise mostly EXO70 gene family members. There are 23 and 47 EXO70 genes in the model dicotyledon Arabidopsis thaliana and the monocotyledon rice, respectively. Here, we identified 165 EXO70 genes among the four cotton subspecies included in the Cotton FGD database, namely, Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, and Gossypium raimondii. There were 27, 26, 55, and 57 genes in Gossypium arboretum, Gossypium raimondii, Gossypium barbadense, and Gossypium hirsutum, respectively. We also identified 48 EXO70 genes in the tobacco database (Figure 1A). A phylogenetic analysis of the evolutionary relationships of 23 Arabidopsis EXO70s, 41 rice EXO70s, 165 cotton EXO70s, and 48 tobacco EXO70s (Figure 1C) showed that cotton EXO70 resembled Arabidopsis EXO70. Both of these plants are dicotyledons [9, 41], and their EXO70s could be divided into eight categories. Relative to the monocotyledon rice, the dicotyledons lacked four EXO70 categories such as EXO70I–EXO70L [42]. Hence, the EXO70 gene may be markedly differentiated between monocotyledons and dicotyledons. Monocotyledons possess more EXO70 genes than dicotyledons. Based on the phylogenetic tree, the grouping and naming of Arabidopsis, cotton EXO70s can be divided into eight subgroups (EXO70A–EXO70H) containing 12, 6, 29, 12, 27, 12, 22, and 45 genes, respectively (Figure 1B).

According to the cotton EXO70 gene classification, we named the 57 EXO70 genes in upland cotton as GhEXO70A1–GhEXO70A2, GhEXO70B, GhEXO70C1–GhEXO70C5, GhEXO70D1–GhEXO70D2, GhEXO70E1–GhEXO70E6, GhEXO70F1–GhEXO70F2, GhEXO70G1–GhEXO70G4, and GhEXO70H1–GhEXO70H8. Groups A and D were represented by -A and -D, respectively. We predicted their genome locations, protein lengths, numbers of exons, isoelectric points, protein molecular weights, and subcellular locations. The numbers of exons widely varied among GhEXO70 genes. All four GhEXO70A genes had the most exons (≥ 11 each) (Table 1). Further analysis of the exons of the EXO70 gene in Arabidopsis and rice showed that only Group A contained more exons in Arabidopsis and rice. The number of EXO70 in Arabidopsis A group ≥9, and the number of EXO70 in rice A group ≥12, Therefore, this phenomenon is not unique to cotton EXO70s, but is conserved in plants (Table S2, Table S3). The subcellular localization prediction results showed that GhEXO70 was mostly localized in the cell membrane, cytoplasm or nucleus, which was consistent with the reported subcellular localization results of EXO70 from H. villosa[43] (Table 1).

2.2. Chromosome Distribution Analysis of EXO70 in the Cotton Genome

Diploid Gossypium arboretum, Gossypium raimondii, and Arabidopsis have 27, 26, and 23 EXO70 genes, respectively, while diploid rice has 47. Tetraploid Gossypium hirsutum and Gossypium barbadense have 57 and 55 EXO70 genes, respectively. The 27 EXO70 genes of Gossypium arboretum are located on chromosomes 1–2, 4–5, 7, and 9–13, respectively (Figure 2C). The 26 EXO70 genes of Gossypium raimondii are located on chromosomes 1–3 and 6–12 respectively (Figure 2D). The 57 EXO70 genes of Gossypium hirsutum are located on chromosomes 1, 3–5, 7, and 9–13 in groups A and D, respectively (Figure 2A). The 55 EXO70 genes of Gossypium barbadense are located on chromosomes 1, 3–5, 7, and 9–13 in group A and on chromosomes 1, 3–5, 7, 9–10, and 12–13 in group D (Figure 2B).

Statistical analysis of the EXO70 gene distributions on the chromosomes revealed that there were relatively more EXO70 genes on chromosomes 5 and 9 in Gossypium arboretum, Gossypium barbadense, and Gossypium hirsutum but no EXO70 genes on chromosome 6 or 8. The EXO70 gene on chromosome 9 was distributed in Gossypium raimondii but that which was on chromosome 5 was not distributed. The EXO70 gene distributions on chromosomes 6 and 8 of Gossypium raimondii (four and two, respectively) were the opposite of those for the other three cotton species (Figure 2; Table 2).

The number of EXO70 genes in tetraploid cotton was nearly twice that in diploid cotton. The diploid cotton species (Gossypium arboretum and Gossypium raimondii) contained two EXO70As, one EXO70B, two EXO70Ds, and two EXO70Fs whereas the tetraploid cotton species had twice these EXO70 gene copy numbers (Table 3). The numbers of EXO70Cs, EXO70Gs, and EXO70Hs in tetraploid cotton were twice those in the autodiploid and equal to the sum of the number in the allodiploid (Table 3). In polyploid cotton, then, the number of EXO70 genes increases via genome polyploidization. Most GhEXO70 genes are highly parallel in the At group and Dt subgenome. The exception is that GhEXO70E2-D and GhEXO70E4-D have no homologs in the At subgenome, while GhEXO70H8-A has no homologs in the Dt subgenome, indicating that they may be lost during evolution.

2.3. Analysis of EXO70 Gene Structure in Gossypium hirsutum.

Gossypium hirsutum is the major global cotton variety and was the focus of research attention here. Structural analysis of its 57 GhEXO70 genes showed that all of them had one or two exons except for GhEXO70A, which had 10 or 11 exons. All GhEXO70 genes with similar structures are grouped in the same clade. Moreover, the genes with closely related phylogeny in the same subgroup also had similar structures. Within the same subgroup, however, certain genes exhibited entirely different structures. GhEXO70E2-D contained two exons while the other genes within the same subgroup had only one. Similarly, GhEXO70G4 contained two exons, whereas GhEXO70G1–GhEXO70G3 each contained a single exon. GhEXO70H2-A and GhEXO70H8-A each contained two exons while the other genes within the same subgroup had only one (Figure 3).

We used MEME online software to analyze the conserved motifs in the GhEXO70 protein and study its motif composition diversity and conservation. Figure 3 shows that ten motifs (1–10) were identified, and each one was localized mainly to the C-terminal of the gene. Therefore, the C-terminal sequence of the GhEXO70 protein is highly conserved. The motif types revealed that the GhEXO70 gene members in subgroups A,B,C,D were highly conserved and included all motifs. The GhEXO70s gene members in the other subgroups presented with obvious differences in motif type distribution, and some of them were lost. GhEXO70E2-D, GhEXO70E4-D, GhEXO70H1-D, and GhEXO70G2-D contained two, three, five, and six motifs, respectively. The functions of Motifs 1–10 have not been elucidated. Nevertheless, analysis of the conserved domains via the NCBI CDD (Conserved Domain Database) disclosed that they comprise the Exo70 domain (Figure 3).

The PFam03081 domain at the C-terminus of the EXO70 protein is characteristic of the EXO70 superfamily [12], and all 165 predicted homologous clone EXO70 proteins possess it. However, the amino acid sequence lengths differed among EXO70 proteins and were in the range of 134–735 aa (average length = 618.736842105263 aa) (Table 1). It was discovered that most GhEXO70 genes lacked a transmembrane (TM) structure. Only GhEXO70E2-D might possess a transmembrane region. Therefore, it may have evolved along with eukaryote evolution (Figure S1). Prediction of the transmembrane domain of Arabidopsis EXO70, the results showed that AtEXO70C1, AtEXO70C2, AtEXO70H5, AtEXO70H8, AtEXO70A3 have transmembrane domains, but they are not obvious, and the other EXO70s have no transmembrane domains (Figure S2). The prediction results of rice EXO70 show that OsEXO70A3, OsEXO70A4, OsEXO70H1a, OsEXO70H1b, OsEXO70H2, OsEXO70H3, OsEXO70H4, OsEXO70I3, OsEXO70I4, OsEXO70L1, OsEXO6K1, OsEXO70L, OsEXO70J1, OsEXO70J1, OsEXO70J2, OsEXO70J6, OsEXO70J8, OsEXO70K1, OsEXO7K2, OsEXO70L1 have a transmembrane domain. And OsEXO70A4 has a more obvious transmembrane domain at the C-terminus, and none of the other rice EXO70s has a transmembrane domain (Figure S3). Among the prediction results of the transmembrane domain of cotton EXO70, only GhEXO70E2-D has a transmembrane domain, and the others have no transmembrane domain. Both Arabidopsis and cotton contain fewer EXO70s with transmembrane domains. As rice is a monocot, it may be evolving to have more EXO70, and there are more EXO70s with transmembrane domains.

2.4. Analysis of EXO70 Gene Expression Patterns in Gossypium arboretum and Gossypium hirsutum

Gene expression has spatiotemporal properties. The expression patterns of the various members of the EXO70 gene family may indicate the potential biological effects of these genes. We analyzed expression profile data in the Cotton FGD and Cottongen (https://www.cottongen.org/) databases to clarify the spatiotemporal expression characteristics of the EXO70 gene. In Gossypium hirsutum and Gossypium arboretum, the EXO70 gene is commonly expressed in the roots, stems, leaves, flowers, fibers, and ovules and has spatiotemporal properties (Figure 4 and Figure S4). GhEXO70A1-A, GhEXO70A1-D, GhEXO70B-A, GhEXO70B-D, GhEXO70D1-A, GhEXO70E1-A, GhEXO70E6-A, GhEXO70F2-D, and other genes in Gossypium hirsutum are generally expressed at high levels and in various tissues. The GhEXO70H3-A gene is expressed mainly in the stamens whereas the GhEXO70H5-A and GhEXO70H5-D genes are expressed mainly during the early stages of ovule development. In Gossypium arboretum, the GaEXO70A1, GaEXO70E4, GaEXO70B, GaEXO70F1, GaEXO70F2, GaEXO70D1, GaEXO70E1 genes are generally highly expressed in different tissues while GaEXO70A2 is expressed mainly in the 15D fibers (Figure S4).

Ubiquitous EXO70 expression suggests that this gene is implicated in cotton growth and development. The GaEXO70A2 gene is expressed mainly in the fibers and might participate in cotton fiber development. The GhEXO70H3-A gene is expressed mainly in the stamens and may be associated with cotton fertility. The GhEXO70H5-A and GhEXO70H5-D genes are expressed mainly in the early stages of ovule development and could be involved in cotton seed formation.

2.5. EXO70 Gene Transcription Regulation Analysis

Spatiotemporal gene expression is regulated mainly by transcription factors (TFs) and epigenetics [44]. The observed differences in spatiotemporal expression of the various EXO70 genes may be related to their promoter specificity. We intercepted the 2-kb sequence upstream of the cotton EXO70 gene start codon and used the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to analyze the cis-elements in the promoter region. A total of 1,081 cis-elements were predicted in the 57 GhEXO70 gene promoter regions. Of these, ten and eleven categories were related to phytohormones and environmental stressors, respectively. The functions of the cis-elements in phytohormone and environmental stress response are highlighted in Figure 5. Among the predicted phytohormone response elements, the ERE, ABRE, and CGTCA motifs were the most abundant. Hence, the GhEXO70 gene might respond to ethylene, abscisic acid, and methyl jasmonate (MeJA) (Figure 5A). Ten environmental stress-related elements were identified and mainly involved drought stress (MYC), stress response (STRE), and anaerobic induction (ARE) (Figure 5B). Therefore, the EXO70 gene may participate in the response to adversity. To further verify whether the above cis-acting elements are unique to cotton EXO70s, we also analyzed the EXO70s gene promoters in Arabidopsis and rice. The results indicate that the promoters of EXO70 genes in Arabidopsis and rice also contain cis-acting elements that respond to environmental stress and plant hormones (Figure S5 and Figure S6). It shows that this phenomenon is not unique to cotton EXO70, but is conserved in plants.

2.6. Expression Analysis and Subcellular Location of GhEXO70A1-A

The EXO70A1 gene is the most widely studied of all plant EXO70 genes. In Arabidopsis, AtEXO70A1 differentiates tubular molecules and regulates seed coat, root hair, stigma papillae development, and Kjeldahl band formation [45-47]. OsEXO70A1 plays important roles in vascular bundle differentiation and mineral nutrient assimilation [28]. In this study, we used GhEXO70A1-A in an experimental study on cotton EXO70 genes. We tested the GhEXO70A1-A gene expression patterns. GhEXO70A1-A was predominantly expressed in the stems, leaves, and flowers but its expression levels were low in the roots, ovules, and cotyledons (Figure 6A).

Subcellular GhEXO70A1-A protein localization predicted its roles in biological processes. Transient 35S-GhEXO70A1-A-GFP expression in tobacco produced a fluorescent signal. GhEXO70A1-A induced signals on the plasma membrane (Figure 6B). Thus, GhEXO70A1-A was localized to the endomembrane system. This discovery was consistent with the roles of EXO70s in vesicle transport.

2.7. GhEXO70A1-A Protein Interaction Analysis

We used a yeast two-hybrid (Y2H) assay to explore the interactions among GhEXO70A1-A and the other subunits of the exocytosis complex. Plasmids containing GhEXO70A1-A and the other subunits of the exocytosis complex were co-transformed into Y2H Gold cells, which can grow on SD/-Leu-Trp. However, the cells were inoculated onto SD/-Ade/-His/-Leu/-Trp medium and only GhEXO70A1-A and GhEXO84A,Gh EXO84B, GhEXO84C co-transformed cells could grow on it and express X-α-Gal activity. GhEXO70A1-A interacted with EXO84A, EXO84B, and EXO84C (Figure 7) which means that it may function as a subunit of the exocytosis complex.

2.8. VIGS Silencing of GhEXO70A1-A Causes Changes in Signaling Pathways and Gene Expression

Gene silencing is an effective method of studying gene function. To explore the functions of GhEXO70A1-A in cotton, we constructed GhEXO70A1-A gene-silenced cotton plants by virus-induced gene silencing (VIGS). qPCR demonstrated that the GhEXO70A1-A gene was successfully knocked down (Figure 8A). We then used next-generation sequencing (NGS) technology to detect any changes in the transcriptome of GhEXO70A1-A-silenced leaves. We sorted out the expression of other EXO70 genes in the NGS data, and the results are shown in the figure S7, GhEXO70B-D, GhEXO70B-A, GhEXO70E6-D, GhEXO70F1-D, GhEXO70E3-D, GhEXO70H6-D, GhEXO70E1-D, GhEXO70H6-A, GhEXO70H3-D, GhEXO70D1-D, GhEXO70E3-A, GhEXO70C1-A, GhEXO70E5-A, GhEXO70C5-A have significant changes, and there are no significant changes in other EXO70 genes. However, except for GhEXO70C1-A, GhEXO70H6-D, and GhEXO70H6-A, which decreased to 32.1%, 46.9%, and 56.6% of the control, all other genes fell to more than 60% of the control, and the fold increase was also less than 1. Although the three genes GhEXO70C1-A, GhEXO70H6-D, and GhEXO70H6-A declined slightly, their expression abundance was also very low. The above results show that the knockdown of GhEXO70A1-A by VIGS does affect the expression of other EXO70 genes, but the effect is not significant after analysis. The changes in differential genes should be mainly caused by the changes in GhEXO70A1-A.

Correlation analyses among samples disclosed significant differences between the GhEXO70A1-A-silenced (EXO70A1) and the control (VIGS-CK) groups (Figure 8B). Thus, GhEXO70A1-A silencing in cotton altered the gene expression profiles. Differentially expressed genes (DEG) were those that met the criteria of |log2(Fold Change)| ≥ 1 and P ≤ 0.05. A total of 3,264 upregulated and 1,103 downregulated genes were screened as shown in a volcano graph (Figure 8C) and a heat map (Figure 8D). Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment of the DEGs (Figure 8E) displayed 13 pathways with P < 0.01. These included photosynthesis-antenna protein, phenylpropane biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, circadian rhythm-plant, keratin, cork and wax biosynthesis, steroid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, glutathione metabolism, cyano amino acid metabolism, photosynthesis, and glucosinolate biosynthesis (Table 4).  GSEA results showed that GhEXO70A1-A was significantly related to photosynthesis-antenna protein, photosynthesis, and circadian rhythm-plants (Figs. 8F–8H). Of the 13 significantly different pathways, all except for circadian rhythm-plants were related to metabolism. Therefore, cotton leaf GhEXO70A1-A may regulate biochemical anabolism and catabolism.

3.Discussion

3.1. Evolutionary Relationships of the Cotton EXO70 Gene Family

The evolutionary relationships of the EXO70 gene family in Asian, Raymond, upland, and sea island cotton were inferred according to their total numbers, classifications, chromosome distributions, and structures. Allotetraploid cotton was crossed from its ancestors Gossypium raimondii and Gossypium arboreum about 5 million years ago [48]. There are about twice as many EXO70 genes in tetraploid cotton (57 and 55, respectively) as there are in diploid cotton. Hence, the former underwent a round of polyploidization event. Polyploidization leads to rapid, extensive genetic and epigenetic changes in the genome. It is associated with many molecular and physiological adjustments and significant gene losses in heterotetraploid cotton after domestication. In tetraploid cotton, the EXO70 gene family underwent neither large-scale amplification nor reduction according to chromosome location analyses.

Phylogenetic analyses revealed that all eight EXO70 subgroups (EXO70A–EXO70H) are represented in all four cotton species. Genes within the same subgroup had similar structures. Subgroup EXO70A consisted of numerous exons whereas there were relatively fewer in the other seven subgroups. During long-term natural selection, many EXO70 genes differentiated and evolved to contend with various stressors. A study on Arabidopsis EXO70 demonstrated that recurring gene loss was non-random. Genes involved in DNA repair were comparatively more prone to loss while those implicated in signal transduction and transcription were preferentially retained [49]. Hence, the DNA repair function appears to be absent in the cotton EXO70I subgroup. Other plant species should be examined to determine whether the EXO70I branch is unique to monocots.

3.2. Biological Processes Implicating GhEXO70A1-A

EXO70 is one of eight exocyst subunits. It participates in the movement of membrane-related substances and is vital for vesicle transport, cell secretion, growth, division, and other processes [1]. EXO70 genes are vital to plant growth, development, and metabolism. The study of EXO70 is of great significance to cotton development and metabolism as this crop is commercially important. Unlike yeast and animals, plants harbor dozens of EXO70 genes. The latest research shows that AtEXO70A1 recruits the entire complex to the cytoplasmic membrane by binding to negatively charged phospholipids, providing an important research basis for the study of plant cell polarity and morphogenesis[50]. A functional defect in the EXO70C2 gene of Arabidopsis affected pollen tube growth and led to male-specific transmission defects [22]. EXO70H4 and PMR4-dependent corpus callosum deposition in trichomes is necessary for cell wall silicification [23]. An EXO70B1 knockout mutant in Arabidopsis exhibited impaired light-induced stomatal opening [24]. An OsEXO70L2 mutation caused defects in rice root development [29]. Evidently, the EXO70 gene has been continuously amplified over the course of plant evolution and displays different physiological functions in various plant tissues and organs.

转录体测序表明，在GhEXO70A1-A棉叶被击倒后，4000多个基因的表达水平发生了显著变化。因此，GHEXO70A1-A基因沉默引发了棉叶基因表达特征的重大变化。DEG功能浓缩分析表明，棉叶中的GhEXO70A1-A功能与新陈代谢特别是光合作用密切相关。植物代谢主要受各种植物激素（如奥辛、细胞因子、茉莉酸、腹肌酸等）的调节 [51，52]。GHEXO70A1-棉花沉默诱导代谢途径的广泛改变。因此，GhEXO70A1-A损失可能会影响植物激素生物合成和/或分布。关于阿拉比多普西斯的研究表明，EXO70对于回收等离子体膜蛋白至关重要，包括奥辛埃夫勒克斯载体PIN[21，27，53]。不同植物组织中各种EXO70基因的微分功能表明，通过有针对性的EXO70基因调控，选择性地控制特定植物器官是可行的。然而，必须在这方面进行进一步的实验研究。

4.结论

在本研究中，生物信息学分析了亚洲棉花、雷蒙德棉、高糖棉和海岛棉的EXO70基因。共确定了8个类别的165个EXO70基因。进化分析表明，EXO70基因增殖模式在高原棉花和海岛棉花中几乎相同，同样的EXO70基因类型在这两个品种中都得到了高度保存。然而，EXO70基因在这些品种的繁殖经历了不同的进化。对GhEXO70A1-A的代表性研究表明，棉花中的EXO70基因可能广泛参与新陈代谢，并可能影响植物激素生物合成和/或分布。

This entry is adapted from the peer-reviewed paper 10.3390/genes12101594