GhAAO2 was observed responding to NaHCO3 stress in cotton compared to AAO family genes

Background Abscisic acid (ABA) is an important stress hormone, the changes of abscisic acid content can alter plant tolerance to stress, abscisic acid is crucial for studying plant responses to abiotic stress. The abscisic acid aldehyde oxidase (AAO) plays a vital role in the final step in the synthesis of abscisic acid, therefore, understanding the function of AAO gene family is of great significance for plants to response to abiotic stresses. Result In this study, 6, 8, 4 and 4 AAO genes were identified in four cotton species. According to the structural characteristics of genes and the traits of phylogenetic tree, we divided the AAO gene family into 4 clades. Gene structure analysis showed that the AAO gene family was relatively conservative. The analysis of cis-elements showed that most AAO genes contained cis-elements related to light response and plant hormones. Tissue specificity analysis under NaHCO3 stress showed that GhAAO2 gene was differentially expressed in both roots and leaves. After GhAAO2 gene silencing, the degree of wilting of seedlings was lighter than that of the control group, indicating that GhAAO2 could respond to NaHCO3 stress. Conclusions In this study, the AAO gene family was analyzed by bioinformatics, the response of GhAAO gene to various abiotic stresses was preliminarily verified, and the function of the specifically expressed gene GhAAO2 was further verified. These findings provide valuable information for the study of potential candidate genes related to plant growth and stress. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03999-7

(ET) [5]. Abscisic acid belongs to isoprene (terpenoids) [6], which plays an important role in promoting plant senescence, inhibiting plant growth, regulating embryonic development, seed dormancy, root development and fruit ripening [7]. It is an important secondary metabolite and stress hormone, which plays a vital role in abiotic stress. When plants are subjected to heat, salt, drought, cold and herbicides, the ABA levels in plants would be affected [8]. In the adversity environment, plants could synthesize abscisic acid, which can reduce the leaves expansion, induce the accumulation of proline, soluble sugar accumulation, promote the active oxygen metabolism, reduce the accumulation of malondialdehyde, regulate stomatal movement [9], which can strengthen the ability of plant resistance to adversity [10]. Abscisic acid has great potential application value in plant growth, development and coping with abiotic stress [11].
In recent years, the research on abscisic acid has made great progress. The biochemical, molecular genetics and pharmacology methods were used to determine the synthesis pathway of abscisic acid, the enzymes needed for the synthesis of ABA and the related genes catalyzing these enzymes were analyzed. It has been suggested that there may be two ABA biosynthesis pathways in higher plants: (1) the direct pathway is the polymerization of three isopentane units into the C15 precursor farnesyl pyrophosphate (FPP), which is composed of FPP through epoxidation and oxidation directly from 15-carbon ABA [12]; (2) In the indirect pathway, ABA is synthesized from carotenoids as precursors. Zeaxanthin epoxidase (ZEP) catalyzes cyclic zeaxanthin to form violaxanthin, which is cleaved to flavin aldehyde by 9-cis-epoxycarotenoid dioxygenase (NCED), Abscisic acid aldehyde is generated by short-chain dehydrogenase (SDR), finally, abscisic acid aldehyde is oxidized to ABA by abscisic acid aldehyde oxidase (AAO) [13]. There is now growing evidence that ABA is dominated by the C40 indirect biosynthetic pathway in higher plants, and it was confirmed that zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), short-chain alcohol dehydrogenase / reductase (SDR) and ABA aldehyde oxidase (AAO) [12] are important enzymes in the ABA synthesis pathway. AAO gene was researched on the last step of ABA biosynthesis began in the 1970s [14], ABA aldehyde oxidase present in plants was first discovered in Arabidopsis [15]. This enzyme consist a multi-component protein containing a molybdenum cofactor, two iron sulfur cluster, and flavin adenine dinucleotide (FAD) as prosthetic gene, which is composed of a multicomponent protein [16].
Arabidopsis AOα and AOβ are homodimers of A01 and A02 products, respectively, A0γ is a heterodimer of these two gene products, AOδ is encoded by A03 gene and has the properties of ABA aldehyde oxidase [15]. AOδ both catalyzes the oxidation of ABA aldehyde to ABA and catalyzes the oxidation of 3-indole aldehyde to IAA. The Km value between two substrates is different. When ABA aldehyde is the substrate, the Km value is 0.51 umol / L, which is lower than that of 3-indole aldehyde oxidation. The Km value of the reaction showed that A0δ mainly acts on ABA biosynthesis. The oxidation product was confirmed by gas-mass spectrometry (GC-MS) to be ABA, this enzyme cannot distinguish between ABA aldehyde ( +) and (-) enantiomers [15]. AAO is derived from XDH replication during the evolutionary process. Aldehyde oxidase is similar to xanthine oxidase and bisulfite oxidase, which belongs to molybdenum-flavin protein family [17]. The amino acid sequences of AAO and XDH are highly similar, but their catalytic mechanisms are obviously different. Aldehyde oxidase does not participate in electron transfer in the form of dehydrogenase during the catalytic process [18]. However, the structure of sulfite oxidase is significantly different from the aldehyde oxidase [19]. Aldehyde oxidase can catalyze the oxidation and reduction of a variety of compounds, and its substrates include aldehydes, nitroso compounds, imines and heterocyclic compounds. AAO has a broader substrate specificity than XDH [20].

Identification of AAO family members
We identified 6, 8, 4 and 4 AAO genes from the whole genome identification analysis of four cotton species (G. hirsutum, G. barbadense, G. arboreum and G. raimondii). A total of 22 AAO gene family members were identified, which renamed according to their chromosomal location distribution and Arabidopsis comparison results. The number of AAO genes in two tetraploid cotton cultivars, G. hirsutum and G. barbadense, was found to be almost twice that of the two diploid cotton cultivars G. arboreum and G. raimondii, which indicating that AAO genes have undergone amplified during evolution [21].
To clarify the evolutionary relationship between four cotton species and other closely related species, 7 species of AAO family members with close homology to cotton were identified by Blastp with evalue 1e-5, 4 from Arabidopsis thaliana, 5 from Vitis vinifera, 3 from Oryza sativa, 5 from Zea mays, 4 from Glycine max, 6 from Populus trichocarpa, and 5 from Theobroma cacao (Table  S1, Fig. 1).

Sequence and evolution analysis
To explore the evolutionary and orthologous relationship between four cotton species, 22 AAO genes were used for multiple sequence alignment. Subsequently, the phylogenetic tree constructed by using the Maximum Likelihood (ML) method and using the model as JTT + G with default parameters in MEGA software ( Fig. 2A). To understand the evolutionary and orthologous relationship of 54 AAO protein sequences (22 from Gossypium, 4 from At, 3 from Os, 6 from Pt, 5 from Vv, 5 from Tc, 5 from Zm and 4 from Gm), we constructed the phylogenetic tree with JTT + G + I model using Maximum Likelihood (ML) method (Fig. 2B). The website EvolView (https:// evolg enius. info// evolv iew-v2/# login) was used to beautify the obtained the two phylogenetic trees.
According to its branch on the evolutionary tree, we divided it into 4 subfamilies. From the evolutionary tree, we could find that AAO genes were relatively conserved in various species, and the AAO genes of the same species were all distributed on the same branch. AAO genes in cotton are closely related to Theobroma cacao, distributed on the same branch, Vitis vinifera, Populus  trichocarpa and Glycine max were distributed on the same branch, Oryza sativa and Zea mays were distributed on the same branch, Arabidopsis thaliana was independent distribution.

Chromosome distribution analysis
The chromosome distribution of AAO genes family members help us understand the specific distribution more intuitively. The results showed that AAO gene family were mainly distributed on chromosomes 05, 07, 09 in G. hirsutum A and D (Fig. 3). There were 2 AAO genes on the A chromosome and 4 AAO genes on the D chromosome. In addition, the chromosomes distribution of genes of four Gossypium were not uniform. The AAO genes in G. raimondii were located on the 01, 06, and 09 in D chromosome, it is quite different from the other three cotton species, probably because G. raimondii is wild cotton, other three cotton species were cultivated cotton, which underwent artificial selection during evolution, the incomplete genome assembly or gene loss in the evolutionary process of the cotton species could be considered. All 22 AAO genes were mapped on chromosomes, indicating that AAO genes have matured in evolution.

Analysis of conservative protein motif and gene structure
To explore the conservative structure of AAO gene family, the conserved motif of the AAO genes were identified by the online website MEME, the evolutionary relationship, motif, coding, and non-coding regions of AAO gene family members were visualized in TBtools (Fig. 4). The GBAAO8 gene lacked motif 6, motif 8 and motif 11, GhAAO2 and GBAAO4 lacked motif 6 motif 8, motif 11, motif 12, motif 10 and motif 15. The structure of the AAO genes are almost consistent, this is consistent with the finding that AAO is relatively conserved in the same species.

Analysis of promoters of AAO gene family
Through website prediction and artificial selection, 17 cis-element related stress response element in the family of AAO were predicted (Fig. 5), which contain environmental stress, plant hormone and light response. Containing most cis-elements in the branch of G. hirsutum GhAAO1 and GhAAO4 and their close relatives, cis-acting elements are involved in the regulation of gene expression. We speculate that these genes may be sensitive to the external environment, when environmental have changes a greater may impact on these genes, the expression of these genes may easily have changed. Other genes in the AAO family also contain different cis-acting elements. They may be affected by light, plant hormones, abiotic stress and other conditions, and their expression levels may difference. Therefore, AAO genes may play an important role in regulating cotton growth and defending against the external environment.

Gene duplication and collinearity analysis
It was observed that two species with a highly conservative homology relationship have common linked genes from gene collinearity [22]. Gene duplication is one of the important reasons to promote the evolution of genome and genetic system [23]. The expansion of plant gene family mainly depends on whole genome  duplication, fragment duplication and tandem duplication [24]. A total of 125 gene pairs were identified in 10 groups (Ga-Ga, Ga-GB, Ga-Gh, Ga-Gr, GB-GB, GB-Gh, GB-Gr, Gh-Gh, Gr-Gr), 31 homologous gene pairs were identified by self-comparison in Ga-Ga, GB-GB, Gh-Gh and Gr-Gr, which were predicted to be fragment duplication. 94 homologous gene pairs were identified by other combinations, which were predicted to be whole genome duplication. There were 25 pairs of GB-Gh homologous gene pairs were the most in 10 group, and 3 pairs of Gr-Gr and Ga-Ga homologous gene pairs were the least. It is speculated that fragment duplication and whole genome duplication were mainly carried out in the process of AAO genes evolution (Fig. 6).

Analysis and calculation of selection pressure (Ka / Ks) during evolution
The Ka / Ks ratio can be used as an indicator of whether genes are under positive or negative selection during evolution, The Ka / Ks ratios of Gh-Gh, Ga-Gh, Gr-Gh, Gr-Ga, Ga-Ga, GB-GB, GB-Gr and Gr-Gr were all significantly less than 1, 97.6% of which had Ka / Ks values less than 1. This indicates that the AAO gene formed by the fragment replication event and the fragment replication event mainly underwent purification selection during the evolution process. The replication event may not be seriously differentiated from the source gene, which is relatively conserved. However, in GB-Ga and Gh-GB, there were two and one gene pairs respectively have with Ka / Ks values greater than 1, 2.4% of gene pairs had Ka / Ks values greater than 1, which also indicated that natural selection changed the protein, rapidly fixed the mutation sites in the population and accelerated gene evolution (Fig. 7). These results indicated that the AAO genes have limited functional differentiation after fragment duplication and wholegenome replication. Whether they bring harmful or beneficial traits remains to be studied [25].

Expression analysis of GhAAO genes under different abiotic stresses
The RNA-seq data of different stresses was analyzed to show gene expression changes under different stresses (Fig. 8). In addition, we found that AAO genes were significantly affected by temperature and salt stress (Fig. 9), GhAAO5 almost had corresponding response under different stresses, GhAAO2 was sensitive to low temperature, the expression of GhAAO4 was significantly increased under drought stress. Overall, the response patterns of AAO genes under different stresses were significantly different. We found that the expression of AAO genes did not continuously increase or decrease under stress, possibly because plant hormones are trace amounts and they keep dynamic balance in plants [26].

Expression analysis of different tissues of AAO family genes under NaHCO 3 stress treatment
The relative expression of AAO genes in different tissues under NaHCO 3 stress was detected by qRT-PCR (Fig. 10). The sequences of primer pairs used are shown in Table S2. We found that the expression of AAO family members generally decreased under NaHCO 3 stress for 12 h, while the expression of GhAAO3 increased in stems, roots and leaves. Through SPSS analysis, the expression of AAO genes in stems showed the most significant difference, so we speculated that cotton might respond to NaHCO 3 stress by downregulating AAO gene expression. Since the differential expression of GhAAO2 gene in leaves and roots have reached a significant level, we conducted VIGS experiments on the GhAAO2 gene to verify its function.

Effect of silencing GhAAO2 on NaHCO 3 stress in cotton
After the previous gene expression pattern analysis under NaHCO 3 stress, we selected Gh_A09G0970.1 (GhAAO2) gene for further functional verification. VIGS vector pYL156: GhAAO2 was constructed to study the functions of GhAAO2 under NaHCO 3 stress. The qRT-PCR results showed that the GhAAO2 expression level of pYL156: GhAAO2 was significantly lower than the pYL156, which indicated that VIGS silencing was successful (Fig. 11). When treated with 125 mM NaHCO 3 for 12 h, we found that the phenotype of the silenced cotton was better than the negative control. The negative control plants had more severe leaves wilting and some veins appeared black ( Fig. 11). In the early stage of NaHCO 3 stress, the expression of GhAAO2 gene decreased, the cotton responded to NaHCO 3 stress possibly by adjusting the content of abscisic acid. With the increase in stress time, the damage to plants will be further aggravated. At this point, to resist the stress, the plant may close the pores and wilt the seedling to reduce water and nutrient loss. Abscisic acid can reduce the accumulation of malondialdehyde (MDA), silenced the GhAAO2 gene, the content of MDA was measured significantly increased. After treated with NaHCO 3 the content of MDA was increase too, which possibly due to the cotton were injured after NaHCO 3 treatment. Abscisic acid can promote the accumulation of proline (PRO), After silencing GhAAO2 gene, the PRO content was significantly reduced. After NaHCO 3 treatment, the PRO content of the pYL156 control group was increased, the content of PRO has reduced when treated with NaHCO 3 after GhAAO2 gene was silenced.

Discussion
Abiotic stress is one of the main factors to restrict plant growth and development, yield and quality under severe environmental conditions. Salinity and alkaline stresses are the main abiotic stresses factor affecting the plants [27]. As an important cash crop and fiber crop, cotton is widely distributed all over the world. It is a pioneer crop in saline-alkali land but faces severe biotic and abiotic stresses. Abscisic acid aldehyde oxidase is an important enzyme for the synthesis of abscisic acid, but it is rarely reported in cotton.
In this study, 6, 8, 4 and 4 AAO genes were identified in four cotton species including G. hirsutum, G. barbadense, G. arboreum, G. raimondii. The AAO family members of 7 species with close homology were identified by Blastp with e-values of 1e-5 (Blast-2.12.0 +), 4 from Arabidopsis, 5 from Vitis vinifera, 3 from Oryza sativa, 5 from Zea mays, 4 from Glycine max, 6 from Populus trichocarpa and 5 from Theobroma cacao. Studies had shown that allotetraploid cotton was formed by inter-genomic hybridization of A-genome diploids and D-genome diploids [28]. In this study, the number of AAO family genes in G. raimondii and G. hirsutum may have lost two genes during evolution.
The results showed that the number of AAO genes on the GhAt / GhDt subgenome were different. In the evolutionary analysis, the chromosomal loci of G. barbadense and G. hirsutum were similar, and the positions of most genes on their chromosomes were highly conserved. The chromosomal loci of G. barbadense, G. arboreum and G. hirsutum were similar, especially the D chromosome loci of tetraploid cotton species still showed a very conservative trend. The diploid cotton G. raimondii genes were located at a different locus from the other three cotton species, so we speculated that this may be because G. raimondii were wild and had not undergone artificial selection, while the other three cotton species are cultivated and have undergone artificial selection, so there are different loci on the chromosome. Diploid cotton species have strong conservation in the process of evolution to tetraploid cotton species.
Motifs conservative short sequences, which can be used as identification sequence, coding functional protein. Predicting the structure of the AAO genes motifs could help us to analysis the family conservative in the process of evolution, provide the basis for understanding gene function and structure of the classification. By structure analyzed we found that four cotton species almost have the same motifs, the lack of individual elements in addition to individual genes, as a whole the evolution of relationship is relatively close [29,30]. Introns in eukaryotic gene and is excised before translation, which contain a variety of non-coding RNA, also contain some genes transcription regulatory elements, which act as enhancers or silencers [31].
The cis-elements related to the stress were predicted in AAO genes, the cis-elements can provide the site of action, then cis-elements interactions with trans-acting factors, regulation of gene expression. Although the AAO genes contains cis-elements each are not identical, but all of them are included with the stress response components. Combined with the structure of the gene sequences, we found that some correlation between redundant structures and cis-element. The AAO genes contain a large number of plant hormones elements, five major plant hormones were covered, therefore, we hypothesized that inducing plant hormones related transcription factors and corresponding cis-element function, regulate the expression of AAO family genetic changes to enable plants to respond to abiotic stress [32].
Studies showed that the plants to adapt to environmental change, evolution occurs in the form of whole genome duplications, fragment duplications or tandem duplications. Dicotyledonous plants through whole genome duplication event about 130 million years ago, and cotton has experienced the whole genome duplication event in 60 million years ago, G. raimondii in evolution experienced at least two whole genome replications [33]. The A / D genome of diploid cotton was separated 5-10 Myr years ago [34]. G. hirsutum was generated from the hybridization of G. raimondii and G. arboreum, the number of chromosomes was doubled 1-2 Myr years ago [35]. However, AAO family members are not uniformly distributed across chromosomes, according to the calculation of selection pressure, most AAO genes had undergone purification selection and were relatively conserved.
When plants are subjected to abiotic stress, many signal transduction pathways are activated, a series of adaptation mechanisms are stimulated [36]. The plants respond to stress through changes in cell and physiology [37]. QRT-PCR results of AAO genes in different tissues under NaHCO 3 stress show that AAO gene family responds to NaHCO 3 stress by down-regulating the expression genes. Moreover, we found that significant differential expression of GhAAO2 occurred in leaves and roots. In addition, we also silenced GhAAO2 gene by VIGS, and the silenced GhAAO2 plants showed better tolerance under NaHCO 3 stress compared with the negative control plants, which also confirmed that this gene was indeed a negative regulatory gene under NaHCO 3 stress.
Aldehyde oxidase gene belongs to a multi-gene family, and the number of AAO genes varies in different species. Through the study of the evolutionary relationship of AAO genes, it is believed that the occurrence of gene duplication and gene repression events have led to the differences in plants, vertebrates and insects. The generation of aldehyde oxidase isoforms is generally considered to be the ancestor of all AAO genes. AAO genes originally evolved from the XDH genes through at least two independent gene duplication events. The physiological function of aldehyde oxidase was first studied in plants. Plant AAO genes are thought to play a catalytic role in the synthesis of abscisic acid and indoleacetic acid, thereby participating in many aspects of plant growth, development, and environmental adaptation. The researchers found different numbers of aldehyde oxidase genes in different plant species separately and performed an evolutionary analysis of them, finding that the genes were clustered together. However, amino acid residue similarity between AAO genes within species is higher than between species. This suggests that it is possible that all plant aldehyde oxidase genes originated from the same ancestor and evolved independently in the form of gene duplication [38]. In plants, it participates in the synthesis of abscisic acid and indoleacetic acid, which in turn regulates plant growth, development and adaptation to the environment [39]. In mammals, aldehyde oxidase studies have focused on drug and exogenous metabolism [40].
In plants, guard cells can sense environment information from the leaves, including abiotic stresses and biotic stresses, abscisic acid signaling plays a vital role in the face of these stresses. Ion channels are key signaling elements mediated by abscisic acid in stomatal movement. In guard cells, abscisic acid can regulate cell ion flux, mediate stomatal closure, reduce water loss, maintain physiological and metabolic balance in plants under stress conditions. AAO can regulate the synthesis of ABA in roots and leaves, thereby alleviating the damage to plants. Osmotic stress can cause the expression of AAO [41]. ABA synthesis may be regulated by NCED and AAO in drought [42], ABA plays an important role in salt stress signal transduction, under salt stress, the activity of AAO in roots and leaves of Pisum sativum was enhanced [43]. Relevant reports have found that the level of AAO mRNA in roots of Arabidopsis thaliana under salt stress is also significantly increased [44]. AAO activity was detected in the roots of Lolium perenne and Hordeum vulgare under salt and ammonium treatment, the change was more obvious in the roots than in the leaves [45].
According to the expression of AAO genes in different tissues under NaHCO 3 stress, the expression pattern of AAO genes under different abiotic stress, and the types of cis-elements contained, GhAAO2 was selected for further study. The wilting degree of seedlings after silencing GhAAO2 gene was found to be less severe than that of the negative control. We speculated that GhAAO2 might play an important role in responding to NaHCO 3 stress (Fig. 12). The change of ion concentration in plant root environment under NaHCO 3 stress, large accumulation of ions could reduce soil water potential, which made plants suffer osmotic stress [46], root cells could not take advantage of the water potential difference to absorb water from the soil would cause physiological drought of plants, which affect the growth and development of plants. It was found that abscisic acid could regulate the synthesis of osmotic regulatory substances proline and betaine, relieved the damage from high pH, maintained the stability of cell membrane structure, Abscisic acid also activates Ca 2+ channels in guard cells. Regulating the movement of ions in and out of cells alters the turgor pressure surrounding cells, thus inhibiting stomatal open or close [47].

Conclusion
This study comprehensive analyzed the structure and chromosome distribution of the AAO genes, and found the AAO genes keep conservative in the process of evolution, cis-element analysis that found a lot of stress response elements, we analyzed the different tissues response NaHCO 3 stress and AAO genes response of different stresses. We selected the GhAAO2 gene for further function verification, which confirmed that this gene could regulates NaHCO 3 stress. These results enrich our understanding of cotton AAO genes and provide reference for the study of candidate genes related to stress response.

Identification of AAO family members
The Hidden Markov Model (HMM) profile of PF02738 was downloaded from the Pfam (https:// pfam. xfam. org/) website, all the possible members of AAO gene family were retrieved by using hmmer (version 3.3.1) (http:// www. hmmer. org/) [50] and BLAST (Basic Local Alignment Search Tool). Using NCBI Batch Web CD-Search Tool (https:// www. ncbi. nlm. nih. gov/ Struc ture/ cdd/ wrpsb. cgi) to screen the genes with default parameters which most probably belongs to AAO gene family. We renamed the identified AAO family members GhAAO1-GhAAO6 (Gh stands for G. hirsutum, AAO stands for abscisic acid aldehyde oxidase, and 1-6 are serial numbers).

Phylogenetic analysis and multiple sequences alignments
In order to study the evolutionary relationship between AAO genes in four cotton species, the AAO genes were provided in MEGA software (version 7.0) [51], analyzing the multiple sequence alignment by using ClustalW algorithm. Subsequently, the phylogenetic tree was constructed by using the Maximum Likelihood (ML) method in MEGA software [52]. The Blastp soft was used to obtain homologous genes of other species (Theobroma cacao (Tc), Arabidopsis thaliana (At), Oryza sativa (Os), Populus trichocarpa (Pt), Vitis vinifera (Vv), Glycine max (Gm), Zea mays (Zm)) with the parameters evalue 1e-5. Then we used MEGA7.0 software to multiple sequences alignments [53], constructed the phylogenetic tree with Maximum Likelihood (ML) method [54]. Online website EvolView (https:// evolg enius. info// evolv iew-v2/# login) was used to beautify the obtained phylogenetic tree.

Chromosomal distribution of AAO genes from four Gossypium species
To study the chromosomal distribution of AAO gene family members in four cotton species, physical positions of chromosomal locations from four cotton species including G. hirsutum, G. arboreum, G. raimondii, and G. barbadense were visualized by TBtools software [55]. Genome annotation file, coding sequences of four cotton species were downloaded from CottonFGD [48].

Analysis of the conserved protein motifs and gene structure
Multiple Em for Motif Elicitation (https:// meme-suite. org/ meme/) was applied to analyze AAO genes conserved motifs [56]. MAST file predicted in MEME website, phylogenetic tree analysis of NWK file, GFF3 genome annotation file of Gossypium were put into these files in TBtools software to visualized the evolutionary relationship, gene structure, and conserved motifs of GhAAO genes.

Analysis of AAO gene family members' promoter regions
The promoter sequences 2 kb upstream of the start codon of GhAAO genes were downloaded from the Cot-tonFGD, using PlantCARE (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/) to extract cis-acting elements related to stress response in the promoter region of GhAAO genes.

Collinearity analysis of AAO genes in four Gossypium species
To investigate the collinearity and analyze the syntenic relationship among AAO genes of four cotton species, the collinear and homologous chromosomal regions among four cotton species visualized in the circos in TBtools [55]. Diagram was illustrated by using the genome annotation file and complete genome sequences of these cotton species by MCScanX tool [57].

Calculation of selection pressure
CDS sequences of the AAO genes in the four cotton species were downloaded from CottonFGD. 125 pairs of gene pairs obtained from 10 combinations of collinear were used to calculate the ratio of the number of nonsynonymous substitutions to the number of synonymous substitutions, the MEGAX comparison method was used to identify the selection pressure to analyze the selection pressure in the evolution of the genes [28].

Differentially expressed genes under different abiotic Stresses
The RNA-Seq data (PRJNA490626) of cotton under salt, PEG, cold, and heat stress was downloaded from the online website (http:// grand. crica as. com. cn/ page/ tools/ expre ssion Visua lizat ion), the heat map along with phylogenetic tree was generated through TBtools software to analyze these genes in the expression level under different stresses [58].

Expression analysis of different tissues of AAO family genes under NaHCO 3 stress treatment
According to the previous exploration of the conditions of NaHCO 3 stressed cotton in our laboratory, we finally chose 125 mM NaHCO 3 to treat cotton at the three leaves and one heart stage for 12 h (water treatment was the control). The roots, stems and leaves were taken, and the RNA was extracted, then reverse transcribed into cDNA for fluorescence quantitative experiment [59]. The AAO genes of different tissues were analyzed under the NaHCO 3 stress.

VIGS and alkaline treatment
The 300 bp silencing fragment was selected from the online website (https:// vigs. solge nomics. net/), the purified fragment was inserted into the pYL156 vector fragment. The Agrobacterium carrying pYL156 (Blank control, empty vector), pYL156-GhAAO2 and PDS (Positive control) were mixed with pYL192 (Helper Bacteria) were injected in cotyledons of Zhong 9807. After 24 h in the dark, then cultured them normally, when the leaves turned white in the Positive control, indicating that the experiment was successful. When cotton grew to the three leaves and one heart phase, treated with 125 mM NaHCO 3 . Phenotypes were observed and samples were taken for quantitative fluorescence experiments to further determine whether genes were silenced.