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    Topic review

    EXO70 Gene Family in Cotton

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    The EXO70 gene is a vital component of the exocytosis complex and participates in biological processes ranging from plant cell division to polar growth. There are many EXO70 genes in plants and their functions are extensive, but little is known about the EXO70 gene family in cotton. Here, we analyzed four cotton sequence databases, identified 165 EXO70 genes, and divided them into eight subgroups (EXO70A–EXO70H) based on their phylogenetic relationships. EXO70A had the most exons (≥11), whereas the other seven each had only one or two exons. 

    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][14][15][16][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][19][20][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][28][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].

    2. 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 CottonFGD database, namely, G. hirsutum, G. barbadense, G. arboretum, and G. raimondii. There were 27, 26, 55, and 57 genes in G. arboretum, G. raimondii, G. barbadense, and G. 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][33] and their EXO70s could be divided into eight categories. Relative to the monocotyledon rice, the dicotyledons lacked four EXO70 categories, such as EXO70I–EXO70L [34]. 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).
    Figure 1. Numbers and phylogenetic relationships of EXO70 family genes in Arabidopsis, rice, upland cotton, sea island cotton, Asian cotton, Raymond cotton, and tobacco. (A) Numbers of EXO70 family genes in Arabidopsis, rice, upland cotton, sea island cotton, Asian cotton, Raymond cotton, and tobacco. At: Arabidopsis thaliana; Nt: Nicotiana tabacum; Os: Oryza sativa; Ga: G. arboretum; Gr: G. raimondii; Gb: G. barbadense; Gh: G. hirsutum. (B) Quantitative statistics for each subgroup of the EXO70 family genes in upland cotton, sea island cotton, Asian cotton, and Raymond cotton. (C) Phylogenetic analysis of EXO70 family genes in Arabidopsis, rice, upland cotton, sea island cotton, Asian cotton, Raymond cotton, and tobacco.
    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 was ≥9, and the number of EXO70 in rice A group was ≥12. Therefore, this phenomenon is not unique to cotton EXO70s, but is conserved in plants. 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 [35] (Table 1).
    Table 1. Nomenclature and analysis of physicochemical properties of EXO70 family genes in G. hirsutum.
    Gene ID Name Chromosome Start End Exon Number Protein Length (aa) Molecular Weight (kDa) Isoelectric Point Subcellular Location
    Gh_A10G1765 GhEXO70A1-A A10 92,079,304 92,086,988 12 650 73.432 8.308 Cell membrane
    Gh_D10G2039 GhEXO70A1-D D10 56,152,841 56,160,513 12 650 73.429 8.131 Cell membrane
    Gh_A09G1270 GhEXO70A2-A A09 64,875,878 64,879,803 11 640 72.863 8.901 Cell membrane
    Gh_D09G1272 GhEXO70A2-D D09 39,824,276 39,828,245 11 644 73.467 9.329 Cell membrane, cytoplasm
    Gh_A03G0212 GhEXO70B-A A03 3,226,810 3,228,732 1 640 72.955 4.951 Cell membrane, cytoplasm
    Gh_D03G1369 GhEXO70B-D D03 42,280,554 42,282,476 1 640 72.962 4.914 Cell membrane, cytoplasm
    Gh_A01G1064 GhEXO70C1-A A01 36,760,999 36,763,005 1 668 77.345 8.781 Cell membrane, cytoplasm
    Gh_D01G1124 GhEXO70C1-D D01 23,935,803 23,937,809 1 668 76.941 8.482 Cell membrane, cytoplasm
    Gh_A10G0625 GhEXO70C2-A A10 9,989,979 9,992,177 1 732 84.71 4.555 Nucleus
    Gh_D10G0774 GhEXO70C2-D D10 9,213,316 9,215,523 1 735 85.065 4.537 Nucleus
    Gh_A04G0860 GhEXO70C3-A A04 55,909,662 55,911,515 1 617 70.763 4.975 Cell membrane, cytoplasm
    Gh_D04G1359 GhEXO70C3-D D04 44,235,768 44,237,621 1 617 70.799 5.024 Cell membrane, cytoplasm
    Gh_A09G0369 GhEXO70C4-A A09 20,318,531 20,320,444 1 637 73.612 5.53 Cell membrane, cytoplasm
    Gh_D09G0388 GhEXO70C4-D D09 14,114,761 14,116,674 1 637 73.682 5.35 Cell membrane, cytoplasm
    Gh_A05G2929 GhEXO70C5-A A05 70,962,860 70,964,782 1 640 73.595 6.664 Cell membrane, cytoplasm
    Gh_D04G0713 GhEXO70C5-D D04 14,452,693 14,454,615 1 640 73.566 6.384 Cell membrane, cytoplasm
    Gh_A10G2233 GhEXO70D1-A scaffold2452_A10 2396 4279 1 627 71.136 5.36 Cell membrane, cytoplasm
    Gh_D10G0529 GhEXO70D1-D D10 5,107,684 5,109,567 1 627 71.084 5.278 Cell membrane, cytoplasm
    Gh_A05G1157 GhEXO70D2-A A05 11,706,409 11,708,259 1 616 69.599 5.35 Cell membrane
    Gh_D05G1334 GhEXO70D2-D D05 11,742,012 11,743,850 1 612 69.264 4.972 Cell membrane, cytoplasm
    Gh_A09G0090 GhEXO70E1-A A09 2,303,006 2,304,805 1 599 69.086 4.988 Cell membrane, cytoplasm
    Gh_D09G0087 GhEXO70E1-D D09 2,312,361 2,314,160 1 599 69.308 5.089 Cell membrane, cytoplasm
    Gh_D01G1051 GhEXO70E2-D D01 19,584,687 19,585,379 2 134 15.208 6.674 Cell membrane
    Gh_A05G3215 GhEXO70E3-A A05 84,043,718 84,045,679 1 653 74.613 4.761 Cell membrane
    Gh_D04G0392 GhEXO70E3-D D04 6,206,299 6,208,260 1 653 74.511 4.731 Cell membrane
    Gh_D12G0327 GhEXO70E4-D D12 4,666,459 4,667,427 1 322 36.173 4.875 Cell membrane
    Gh_A09G2154 GhEXO70E5-A A09 74,578,905 74,580,839 1 644 73.351 5.784 Cell membrane, cytoplasm
    Gh_D09G2359 GhEXO70E5-D D09 50,553,799 50,555,733 1 644 73.402 6.24 Cell membrane, cytoplasm
    Gh_A12G2651 GhEXO70E6-A scaffold3396_A12 4667 6610 1 647 73.345 5.788 Cell membrane
    Gh_D12G1810 GhEXO70E6-D D12 50,610,382 50,612,325 1 647 73.122 5.417 Cell membrane
    Gh_A03G0449 GhEXO70F1-A A03 9,703,926 9,705,884 1 652 73.754 4.614 Cell membrane, cytoplasm
    Gh_D03G1089 GhEXO70F1-D D03 36,373,153 36,375,111 1 652 73.79 4.587 Cell membrane
    Gh_A12G1712 GhEXO70F2-A A12 78,884,906 78,886,861 2 593 67.361 4.566 Cell membrane
    Gh_D12G1873 GhEXO70F2-D D12 51,339,965 51,341,920 2 593 67.36 4.589 Cell membrane, cytoplasm
    Gh_A13G1576 GhEXO70G1-A A13 74,625,245 74,627,293 1 682 77.054 8.387 Cell membrane, cytoplasm
    Gh_D13G1935 GhEXO70G1-D D13 54,742,538 54,744,586 1 682 76.932 8.152 Cell membrane, cytoplasm
    Gh_A05G0971 GhEXO70G2-A A05 9,685,126 9,687,123 1 665 74.839 6.629 Cell membrane, nucleus
    Gh_D05G1080 GhEXO70G2-D D05 9,208,639 9,210,636 1 665 74.79 6.457 Cell membrane, nucleus
    Gh_A13G1577 GhEXO70G3-A A13 74,629,925 74,631,973 1 682 77.006 8.013 Cell membrane, cytoplasm
    Gh_D13G1936 GhEXO70G3-D D13 54,747,144 54,749,192 1 682 77.129 8.008 Cell membrane, cytoplasm
    Gh_A05G1829 GhEXO70G4-A A05 19,148,304 19,150,892 2 705 80.991 6.269 Cell membrane, nucleus
    Gh_D05G2026 GhEXO70G4-D D05 18,583,197 18,585,799 2 706 81.133 6.088 Cell membrane
    Gh_A05G2577 GhEXO70H1-A A05 36,616,771 36,618,594 1 607 68.055 7.626 Cell membrane
    Gh_D05G2864 GhEXO70H1-D D05 32,263,469 32,264,524 1 351 39.036 8.216 Cell membrane, nucleus
    Gh_A04G0671 GhEXO70H2-A A04 45,359,837 45,362,111 2 621 70.248 5.721 Cell membrane, cytoplasm
    Gh_D04G1136 GhEXO70H2-D D04 37,216,953 37,218,701 1 582 65.703 5.697 Cell membrane, cytoplasm
    Gh_A11G2905 GhEXO70H3-A A11 92,971,783 92,973,285 1 500 56.067 7.521 Cell membrane, nucleus
    Gh_D11G3290 GhEXO70H3-D D11 65,820,199 65,822,067 1 622 69.832 7.178 Cell membrane, cytoplasm
    Gh_A01G1870 GhEXO70H4-A A01 98,692,379 98,694,289 1 636 71.827 7.493 Cell membrane, cytoplasm
    Gh_D01G2127 GhEXO70H4-D D01 60,327,492 60,329,402 1 636 72.148 7.783 Cell membrane, cytoplasm
    Gh_A07G0865 GhEXO70H5-A A07 15,194,291 15,196,123 1 610 69.021 6.068 Cell membrane, cytoplasm
    Gh_D07G0937 GhEXO70H5-D D07 12,444,178 12,446,010 1 610 68.925 6.316 Cell membrane, cytoplasm
    Gh_A05G0839 GhEXO70H6-A A05 8,379,960 8,381,828 1 622 70.171 5.757 Cell membrane
    Gh_D05G3898 GhEXO70H6-D scaffold4075_D05 141,419 143,287 1 622 70.049 5.361 Cell membrane, cytoplasm
    Gh_A03G0316 GhEXO70H7-A A03 5,680,420 5,682,288 1 622 70.772 5.209 Cell membrane, cytoplasm
    Gh_D03G1262 GhEXO70H7-D D03 40,093,224 40,095,089 1 621 70.603 5.889 Cell membrane, cytoplasm
    Gh_A11G2904 GhEXO70H8-A A11 92,966,319 92,968,183 2 568 63.859 8.556 Cell membrane, cytoplasm

    3. Chromosome Distribution Analysis of EXO70 in the Cotton Genome

    Diploid G. arboretum, G. raimondii, and Arabidopsis have 27, 26, and 23 EXO70 genes, respectively, while diploid rice has 47. Tetraploid G. hirsutum and G. barbadense have 57 and 55 EXO70 genes, respectively. The 27 EXO70 genes of G. arboretum are located on chromosomes 1–2, 4–5, 7, and 9–13, respectively (Figure 2C). The 26 EXO70 genes of G. raimondii are located on chromosomes 1–3 and 6–12, respectively (Figure 2D). The 57 EXO70 genes of G. 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 G. 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).
    Figure 2. Chromosome distributions of EXO70 in upland cotton, sea island cotton, Asian cotton, and Raymond cotton. (A) Chromosome distribution map of EXO70 in upland cotton. (B) Chromosome distribution map of EXO70 in sea island cotton. (C) Chromosome distribution map of EXO70 in Asian cotton. (D) Chromosome distribution map of EXO70 in Raymond cotton.
    Statistical analysis of the EXO70 gene distributions on the chromosomes revealed that there were relatively more EXO70 genes on chromosomes 5 and 9 in G. arboretum, G. barbadense, and G. hirsutum, but no EXO70 genes on chromosome 6 or 8. The EXO70 gene on chromosome 9 was distributed in G. raimondii, but that which was on chromosome 5 was not distributed. The EXO70 gene distributions on chromosomes 6 and 8 of G. raimondii (four and two, respectively) were the opposite of those for the other three cotton species (Figure 2; Table 2).
    Table 2. Number of EXO70s in each chromosome of different cotton species.
    Chromosome Ga (27) Gr (26) Gb (55) Gh (57) Total
    A D A D A D
    Chr.1 3 1 2 2 2 3 13
    Chr.2 1 2 0 0 0 0 3
    Chr.3 0 3 3 3 3 3 15
    Chr.4 4 0 2 4 2 4 16
    Chr.5 5 0 7 5 7 4 28
    Chr.6 0 4 0 0 0 0 4
    Chr.7 1 1 1 1 1 1 6
    Chr.8 0 2 0 0 0 0 2
    Chr.9 4 6 4 4 4 4 26
    Chr.10 3 1 3 3 2 3 15
    Chr.11 2 3 2 0 2 1 10
    Chr.12 2 3 2 3 1 3 14
    Chr.13 2 0 1 1 2 2 8
    total 27 26 27 26 26 28 160
    unknown 0 0 1 1 2 1 5
    The number of EXO70 genes in tetraploid cotton was nearly twice that in diploid cotton. The diploid cotton species (G. arboretum and G. 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 species and equal to the sum of the number in the allodiploid species (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.
    Table 3. Numbers of EXO70s in each subgroup of different cotton varieties.
    Subgroup G. arboretum G. raimondii G. barbadense G. hirsutum
    A 2 2 4 4
    B 1 1 2 2
    C 4 5 10 10
    D 2 2 4 4
    E 4 4 9 10
    F 2 2 4 4
    G 4 3 7 8
    H 8 7 15 15

    4. Analysis of EXO70 Gene Structure in G. hirsutum

    G. 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 GhEXO70G1GhEXO70G3 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).
    Figure 3. EXO70 family motif, domain, and gene structure in upland cotton. (A) Phylogenetic tree and motif of GhEXO70 proteins. (B) The conserved domains in GhEXO70 proteins. (C) Gene structure of the GhEXO70 family.
    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 10 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, and 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 Conserved Domain Database (CCD) 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. For the prediction of the transmembrane domain of Arabidopsis EXO70, the results showed that AtEXO70C1, AtEXO70C2, AtEXO70H5, AtEXO70H8, and AtEXO70A3 have transmembrane domains, but they are not obvious, and the other EXO70s have no transmembrane domains. 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, and OsEXO70L1 have a transmembrane domain. In addition, OsEXO70A4 has a more obvious transmembrane domain at the C-terminus, and none of the other rice EXO70s has a transmembrane domain. 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.

    5. Analysis of EXO70 Gene Expression Patterns in G. arboretum and G. 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 CottonFGD and Cottongen (https://www.cottongen.org/, accessed on 22 March 2021) databases to clarify the spatiotemporal expression characteristics of the EXO70 gene. In G. hirsutum and G. arboretum, the EXO70 gene is commonly expressed in the roots, stems, leaves, flowers, fibers, and ovules and has spatiotemporal properties (Figure 4). GhEXO70A1-A, GhEXO70A1-D, GhEXO70B-A, GhEXO70B-D, GhEXO70D1-A, GhEXO70E1-A, GhEXO70E6-A, GhEXO70F2-D, and other genes in G. 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 G. 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 4. EXO70 family gene expression patterns in different tissues and organs of upland cotton.
    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.

    6. EXO70 Gene Transcription Regulation Analysis

    Spatiotemporal gene expression is regulated mainly by transcription factors (TFs) and epigenetics [36]. 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/, accessed on 20 April 2021) to analyze the cis-elements in the promoter region. A total of 1081 cis-elements were predicted in the 57 GhEXO70 gene promoter regions. Of these, 10 and 11 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. It shows that this phenomenon is not unique to cotton EXO70, but is conserved in plants.
    Figure 5. Cis-acting elements in GhEXO70 promoter. (A) Cis-elements involved in phytohormones were predicted. ABRE: cis-acting regulatory element involved in abscisic acid response. AuxRR-core: cis-acting regulatory element involved in auxin response. CGTCA-motif: cis-acting regulatory element involved in methyl jasmonate (MeJA) response. GARE-motif: gibberellin response element. TGACG-motif: cis-acting regulatory element involved in MeJA response. TGA-element: auxin response element. ERE: cis-acting ethylene response element. P-box: gibberellin response element. (B) Predicted cis-elements involved in environmental stress response. GC-motif: enhancer-like elements involved in specific hypoxia induction. LTR: cis-acting elements involved in low temperature response. MBS: MYB binding sites related to drought induction. STRE: stress response elements. TC-rich repetitive sequences: cis-acting elements involved in defense and stress responses. WUN-motif: wound response elements. MYC: cis-acting elements involved in drought stress. W box: cis-acting elements involved in sugar metabolism and plant defense signals. DRE core: dehydration response element. ARE: cis-acting regulatory element for anaerobic induction.

    7. 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 [37][38][39]. 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).
    Figure 6. GhEXO70A1-A expression and subcellular localization analyses. (A) GhEXO70A1-A expression analyses in various upland cotton tissues. (BI) Subcellular GhEXO70A1-A localization in tobacco. GFP: green fluorescence. Chloroplast: chloroplast spontaneous red fluorescence. Merge: green and red fluorescence and bright field fusion. (BE): 35S-GFP empty vector as control. (FI): 35S-GhEXO70A1-A-GFP vector located in plasma membrane. Bar: 10 μm. Data are means ± SD for three replicates.
    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.

    8. 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.
    Figure 7. Y2H analysis of interactions among GhEXO70A1-A and other exocyst subunits. GhEXO70A1-A is connected to PGBK-T7 carrier. Other subunits of secretory complex are connected to PGAD-T7 carrier. BD: PGBK-T7 empty vector. AD: PGAD-T7 empty vector.

    9. 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. 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.
    Figure 8. Transcriptome sequencing of differences in cotton transcriptome expression after GhEXO70A1-A gene silencing. (A) GhEXO70A1-A expression levels after virus-induced gene silencing (VIGS). Relative GhEXO70A1-A expression levels in plants numbered 4, 6, 9, 10, 12, and 16 significantly decreased. (B) Correlation analyses of transcriptome samples. (C) Differential gene volcano map in transcriptome. (D) Differential gene heat map in transcriptome. (E) KEGG functional enrichment dot plot of DEGs. (F) GSEA diagram showing changes in photosynthesis antenna proteins after EXO70A1 gene silencing. (G) GSEA diagram showing changes in photosynthetic pathway after EXO70A1 gene silencing. (H) GSEA diagram showing changes in circadian rhythm pathway after EXO70A1 gene silencing. Data are means of three replicates ± SD. ** p < 0.01.
    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 3264 upregulated and 1103 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 (Figure 8F–H). 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.
    Table 4. DEG function pathway enrichment.
    KEGG ID Description Gene Ratio Bg Ratio p Value Up Down
    ghi00196 Photosynthesis antenna proteins 33/877 53/11,853 1.32 × 10−24 33 0
    ghi00940 Phenylpropanoid biosynthesis 61/877 285/11,853 1.91 × 10−14 50 11
    ghi00941 Flavonoid biosynthesis 33/877 101/11,853 9.05 × 10−14 32 1
    ghi00500 Starch and sucrose metabolism 55/877 324/11,853 4.27 × 10−9 45 10
    ghi04712 Circadian rhythm—plant 28/877 127/11,853 1.26 × 10−7 25 3
    ghi00073 Cutin, suberine, and wax biosynthesis 17/877 64/11,853 2.56 × 10−6 15 2
    ghi00100 Steroid biosynthesis 19/877 84/11,853 8.89 × 10−6 18 1
    ghi00909 Sesquiterpenoid and triterpenoid biosynthesis 15/877 58/11,853 1.41 × 10−5 11 4
    ghi00480 Glutathione metabolism 33/877 249/11,853 0.00076 20 13
    ghi00460 Cyanoamino acid metabolism 16/877 91/11,853 0.00095 16 0
    ghi00195 Photosynthesis 20/877 138/11,853 0.002806 20 0
    ghi00966 Glucosinolate biosynthesis 7/877 29/11,853 0.004374 1 6
    ghi00670 One carbon pool by folate 10/877 56/11,853 0.007353 10 0

    This entry is adapted from 10.3390/genes12101594


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