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 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).
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. (B–I) Subcellular GhEXO70A1-A localization in tobacco. GFP: green fluorescence. Chloroplast: chloroplast spontaneous red fluorescence. Merge: green and red fluorescence and bright field fusion. (B–E): 35S-GFP empty vector as control. (F–I): 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 |