4.1. Transcriptome mRNA in the Anther Development of Cotton
Anther development is a complex process, with numerous genes and various metabolic pathways being involved. In model plants like
Arabidopsis and rice, many genes that regulate the fate of somatic germ cells and the differentiation of the anther wall, as well as control the degradation of tapetum cells and microspore development during anther development, have been identified [
49,
50,
51]. In cotton, the
GhACS1 gene encodes an acyl-CoA synthetase, which is essential for normal microspore development, and highly expressed in sporogenous cells, pollen mother cells, microspores, and tapetum cells [
52]. The actin-depolymerizing factor (
GhADF7) gene may play an important role in pollen development and germination, and its transcript expression reaches a peak at flowering [
53].
GhMYB24 encodes the MYB-like transcription factor that regulates the development of the tapetum [
54].
Carbohydrates provide energy and nutrients for anther development; the sink strength of anthers is the highest in the early stages of anther development, which is intensively energy-demanding [
55] and, thus, abnormal carbohydrate metabolism can significantly damage pollen development and cause male sterility [
56,
57]. Sugar is converted to starch so as to ensure energy preservation for pollen maturation and bud germination [
57]. Zhang and his team studied the sterility of the male-sterile line 1355A of cotton, and found soluble sugar and fatty acid metabolism to play a central role in anther development. In the male sterile line 1355A, soluble sugars are decreased, and fatty acid synthesis is key for regulating normal pollen hydration and the primary component of sporopollenin, which can protect pollen from various stresses and is crucial for pollen grain development and male sterility [
58,
59]. High rates of glucose metabolism may promote fatty acid synthesis in order to promote the anther development of cotton [
59]. Researchers studying cotton (
Gossypium hirsutum) anthers under a high temperature (HT) and normal temperature (NT) indicated that HT disturbs sugar and ROS metabolism by disrupting DNA methylation, leading to microspore sterility [
60]. Flavonoids play an important role in the formation of pollen exine formation, pollen germination, and the fertility of several plants that are key branch-point genes during tetrad and uninucleate microspore periods. Some researchers who studied genic male sterility (GMS) mutant anther development indicated that flavonoid metabolism is initially activated at the tetrad stage, then suppressed at the uninucleate microspore stage, leading to male sterility and the absence of flavonoids in mature stamens [
61]. By using digital gene expression (DGE), some researchers identified many of the key genes that are required for cotton anther development and those that are mainly associated with sucrose and starch metabolism, the pentose phosphate pathway, glycolysis, and flavonoid metabolism [
61,
62].
Hormone signal transduction plays an important role in anther development, such as that involving ethylene, gibberellic acid (GA), and abscisic acid (ABA); higher amounts of ethylene may directly lead to the premature degeneration of the tapetal layer in GMS mutant anthers [
59]. GA can accelerate flowering and promote the development of female flowers; conversely, GA deficiency can cause the abnormal development of anthers [
63,
64]. In addition, high levels of ABA delayed endosperm differentiation and the lack of endosperm leads to difficulties in germination [
65]. Some researchers have shown that the MAPK signaling pathway plays a key role in the differentiation, proliferation, and apoptosis of cells [
66].
In this research, we identified 17,897 known DE mRNAs at three stages of anther development, between the CMS line C2P5A and the maintainer line C2P5B (
Table S1e). These DE mRNAs underwent GO enrichment and KEGG enriched pathway analyses, which showed that the significant terms were mostly enriched in malate synthase activity, catalytic activity, carbohydrate metabolic process, oxidoreductase activity, oxygen carrier activity, and glucan metabolic process (
Figure 3A–C and
Table S3a,c,e), and the significantly enriched pathways were flavonoid biosynthesis, plant hormone signal transduction, fatty acid metabolism, starch and sucrose metabolism, and MAPK signaling pathways in plants (
Figure 3D–F and
Table S3b,d,f). Compared to the same anther developmental stage of the maintainer line C2P5B, there were many key genes with an abnormal expression pattern in the CMS line C2P5A, which indicated that the abortion of cotton C2P5A was related to the abnormal metabolic pathway of the abnormally expressed gene regulating anther development.
4.2. lncRNA in the Anther Development of Cotton and Predicted Functions
The genomes of the eukaryotes are universally transcribed. Some RNAs are encoded into proteins, and other thousands of lncRNAs regulate key molecular and biological processes [
46]. Until now, some of the best-studied mammalian lncRNAs have associated the dysregulation of lncRNAs with reproduction, including germ cell specification, early embryo implantation and development, and reproductive hormone regulation [
67,
68], but the involvement of plant lncRNAs in reproduction is still poorly understood. In this study, we identified that the expression profiles of mRNA and lncRNA were related to cotton anther male sterility and were differentially expressed at different anther development periods. When further analyzed, the interaction network between the lncRNAs and mRNAs based on expression profiles shows that these transcriptomes may play crucial roles in the anther development of cotton.
In a previous study, we used a chromatin state map approach and RNA-seq to identify what is typically co-expressed with lncRNAs and involved in regulating their neighboring mRNA [
69,
70]. In animals, many studies have shown that lncRNAs directly regulate their neighboring genes in a
cis-acting manner, such as
lncRNA–TCONS_00175604 cis-action in dairy goat [
71], lncRNA-NEF
cis-regulating neighbor gene
FOXA2 in mice [
72], lncRNA-Six1
cis-regulating neighbor gene
Six1 in chicken [
73], lncRNA-Malat1 in mouse [
74], lncRNA–TBILA
cis-regulating
HGAL in humans [
75], and lncRNA–Jpx via both
trans- and
cis-Xist expression in mice [
76].
In plants, lncRNAs regulate many molecular functions and biological processes in various ways. LncRNAs can pair with
cis- or
trans-transcripts, translation inhibition, and gene silencing [
46]. The
Arabidopsis noncoding RNA HID1 promotes photomorphogenesis in continuous red light (CR) and acts through PIF3 [
77]. During vernalization in
Arabidopsis, antisense lncRNA
COOLAIR is associated with the
FLOWERING LOCUS C (
FLC) locus and switches of chromatin states during epigenetic regulation;
COOLAIR participates in the autonomous pathway and controls the flowering time [
8,
78,
79]; and intronic sense lncRNA
COLDAIR acts as a scaffold RNA to recruit the PRC2 complex and establish
FLC epigenetic silencing, and mediates FRIGIDA (FRI) degradation [
8,
80,
81].
Natural antisense lncRNAs
TWISTED LEAF(
TL) play a
cis-regulatory role in
OsMYB60 expression and for maintaining leaf blade flattening [
82]. Overexpressing lncRNA
LAIR upregulates the expression of the neighbor gene LRK (leucine-rich repeat receptor kinase) cluster, which increases rice grain yield [
83]. LncRNA1459 was knocked out by CRISPR/Cas9 altered tomato fruit in ripening; in these mutants, the ethylene production and lycopene accumulation were largely repressed [
84].
In cotton, previous studies have shown that lncRNAs XLOC_063105-CotAD_37096 and XLOC_115463-CotAD_12502 probably function in
cis-regulating responses to drought stress [
32]. One lncRNA, TCONS_00061835-Gh_D06G1439 (GhMYB-like), regulates cotton fiber development [
36] by complementary base pairing with the protein-coding gene, lncRNA, and the circRNA complex network, demonstrating that some lncRNAs are involved in biotic and abiotic stresses [
31,
33,
34].
In this study, we sought the
cis-target genes within a radius of 20 kbp for the lncRNAs, and identified 865 lncRNAs that might exert their functions through the predicted
cis-target genes’ 1172 mRNAs (
Table S4d). The predicted
cis-target genes underwent GO enrichment and KEGG pathway analyses, and the results provided ideas for future research. The significant GO terms were enriched in NADH dehydrogenase (ubiquinone) activity, oxidoreductase activity, photosynthetic electron transport, cytoplasm, and pectinesterase activity (
Figure 5A–C). NADH dehydrogenase (ubiquinone), oxidoreductase, and photosynthetic electron transport play a crucial role in energy production and conversion, in which the proteins are components in the mitochondrial respiratory chain. This showed that mitochondrial respiratory-related enzymes play an important role in the regulation of the CMS line C2P5A, and this result is consistent with a previous study in CMS plants [
85,
86,
87]. Pectinesterase is a wall-degrading enzyme, and abnormal expression may lead to the disruption of the cell wall structure in the CMS line C2P5A; a previous study demonstrated that pectinesterase plays a major role in the plant [
88]. In our research, the GO of the cytoplasm was significant, showing that its abnormality may be one of the key factors leading to abortion in the CMS line C2P5A compared to the maintainer line C2P5B.
Significant KEGG pathways were primarily enriched for several processes, such as fatty acid biosynthesis, flavonoid biosynthesis, glutathione metabolism, MAPK signaling pathway to the plant, oxidative phosphorylation, ubiquinone and other terpenoid quinone biosynthesis, pentose and glucuronate interconversions, and plant hormone signal transduction (
Figure 6A–C). Previous studies show that these metabolism pathways play a vital role during anther development in flowering plants, in which a disturbed metabolism pathway seriously leads to the impairment of anther development, and causes male sterility [
55,
57,
61,
62,
85,
89]. This study showed that some lncRNAs and mRNAs might play important roles in anther development, such as
cis-target gene Ghir_A09G011050.1 of LTCONS_00105434 through GO:0008137 (NADH dehydrogenase (ubiquinone) activity) and ko00190 (oxidative phosphorylation);
cis-target gene Ghir_A01G005150.1 of LTCONS_00004262 through GO:0016491 (oxidoreductase activity) and ko00941 (flavonoid biosynthesis);
cis-target gene Ghir_D05G003710.2 of LTCONS_00126105 through GO:0005975 (carbohydrate metabolic process) and ko00040 (pentose and glucuronate interconversions);
cis-target gene Ghir_A03G016640.1 of LTCONS_00085561 through GO:0016790 (catalytic activity) and ko00061 (fatty acid biosynthesis); and
cis-target gene Ghir_A12G005100.1 of LTCONS_00085561 through GO:0005524 (ATP binding) and ko04016 (MAPK signaling pathway to the plant).