GhIMP10D, an inositol monophosphates family gene, enhances ascorbic acid and antioxidant enzyme activities to confer alkaline tolerance in Gossypium hirsutum L.

Background Inositol monophosphates (IMP) are key enzymes in the ascorbic acid (AsA) synthesis pathways, which play vital roles in regulating plant growth and development and stresses tolerance. To date, no comprehensive analysis of the expression profile of IMP genes and their functions under abiotic stress in cotton has been reported. Results In this study, the genetic characteristics, phylogenetic evolution, cis-acting elements and expression patterns of IMP gene family in cotton were systematically analyzed. A total of 28, 27, 13 and 13 IMP genes were identified in Gossypium hirsutum (G. hirsutum), Gossypium barbadense (G. barbadense), Gossypium arboreum (G. arboreum), and Gossypium raimondii (G. raimondii), respectively. Phylogenetic analysis showed that IMP family genes could cluster into 3 clades. Structure analysis of genes showed that GhIMP genes from the same subgroup had similar genetic structure and exon number. And most GhIMP family members contained hormone-related elements (abscisic acid response element, MeJA response element, gibberellin response element) and stress-related elements (low temperature response element, defense and stress response element, wound response element). After exogenous application of abscisic acid (ABA), some GhIMP genes containing ABA response elements positively responded to alkaline stress, indicating that ABA response elements played an important role in response to alkaline stress. qRT-PCR showed that most of GhIMP genes responded positively to alkaline stress, and GhIMP10D significantly upregulated under alkaline stress, with the highest up-regulated expression level. Virus-induced gene silencing (VIGS) experiment showed that compared with 156 plants, MDA content of pYL156:GhIMP10D plants increased significantly, while POD, SOD, chlorophyII and AsA content decreased significantly. Conclusions This study provides a thorough overview of the IMP gene family and presents a new perspective on the evolution of this gene family. In particular, some IMP genes may be involved in alkaline stress tolerance regulation, and GhIMP10D showed high expression levels in leaves, stems and roots under alkaline stress, and preliminary functional verification of GhIMP10D gene suggested that it may regulate tolerance to alkaline stress by regulating the activity of antioxidant enzymes and the content of AsA. This study contributes to the subsequent broader discussion of the structure and alkaline resistance of IMP genes in cotton. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04462-x.

regulation, and GhIMP10D showed high expression levels in leaves, stems and roots under alkaline stress, and preliminary functional verification of GhIMP10D gene suggested that it may regulate tolerance to alkaline stress by regulating the activity of antioxidant enzymes and the content of AsA.This study contributes to the subsequent broader discussion of the structure and alkaline resistance of IMP genes in cotton.

Background
Saline-alkaline stress seriously inhibits crops growth and yield, and it was reported that over 954 million hectares of soils in the world were poisoned by saline-alkaline conditions [1].Soil salinity is often accompanied by alkalinity because the soil tends to contain sodium carbonate (Na 2 CO 3 ) and sodium bicarbonate (NaHCO 3 ), raising the pH of the soil.Alkaline stress can cause osmotic stress and ion damage like salt stress [2], but with the additional influence of high pH.High pH environment at the rhizosphere can precipitate Fe 2+ , Mn 2+ , Ca 2+ , Mg 2+  and HPO 3 − , thus reducing the availability of mineral elements, inhibiting ion absorption, and destroying ion homeostasis [3].It was found that reactive oxygen species (ROS) and malondialdehyde (MDA) would accumulate in large quantities under alkaline stress, which in turn destroyed cell membrane structure and intracellular components [4].Therefore, excessive ROS will accumulate under stresses, causing a serious impact on plant growth and development [5].
Although alkaline stress is serious, plants also develop a series of measures to resist stresses.To eliminate harmful ROS produced by stress, plants have evolved enzymatic and non-enzymatic systems to cope with abiotic stress.The enzyme system is mainly composed of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), glutathione peroxidase (GSH-Px) and ascorbate peroxidase (APX).And reduced glutathione (GSH) and AsA are the main components of the non-enzymatic system.As an antioxidant molecule in the non-enzymatic system, AsA is a key substrate in the elimination of ROS [6][7][8].AsA can widely participate in the response to adversity stresses, such as salt stress [9,10] and drought stress [11], by regulating antioxidant defense system and activating different antioxidant enzymes.According to our previous study [12], genes in the ascorbate synthesis pathway were differentially expressed under alkaline stress, and IMP genes played a key role in the synthesis of ascorbic acid.
Phosphatases in the inositol signaling pathway can be classified into the following categories: inositol polyphosphate 5-phosphatases (5PTases), suppressor of actin (SAC) phosphatases, SAL1 phosphatase/FIERY1 (FRY1) and its homologs, inositol monophosphates (IMP), and phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-related phosphatases [13].It was also found that IMPase also catalyzed the dephosphorylation of L-galactose 1-phosphate and was a bifunctional enzyme as it participated in the biosynthesis of AsA [14].Therefore, the IMP-encoded enzyme may exhibit bifunctionality in plants.
However, with the deepening of research in recent years, more and more evidences have shown that IMP genes are involved in abiotic stress by controlling the synthesis of AsA.In tobacco, overexpression of the OsIMP gene enhanced the activities of antioxidant enzymes and thus enhanced tolerance to cold stress [15].In Cicer arietinum L., plants that overexpressed the CaIMP gene had higher AsA levels than wild type, suggesting CaIMP could improve plant tolerance to stress through AsA metabolism pathways [16].
Cotton is an important cash crop, and it is also a pioneer crop in salt-alkaline land with a certain degree of salt-alkaline tolerance, so it is an important model crop for us to study saline-alkaline stress [17].In recent years, genome sequencing including 2 diploids, G. arboreum [18], and G. raimondii [19], and 2 allotetraploid, G. hirsutum [20,21] and G. barbadense [22,23], has been completed, which provides strong support for our study of the evolution of the IMP gene family and the potential function of the IMP genes.
To obtain the evolutionary characteristics and potential function of IMP gene family in cotton, we characterized IMP gene family in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii.Then we visualized the chromosomal distribution and collinearity of four cotton species, later, gene ontology annotations, gene structures, conserved motifs, cis-acting elements of promoters and expression patterns under different stresses in G. hirsutum were also analyzed.Then, a highly expressed gene GhIMP10D induced by alkaline stress was selected, and the gene was silenced using VIGS technology to study its function.The objective of this study was to explore the evolutionary relationship of the IMP gene family and its potential function in alkaline tolerance.

Identification of IMP family genes
To identify the members of the IMP gene family in four Gossypium species, a Hidden Markov Model (HMM) profile of inositol_P domain (PF00459) from Pfam (https:// pfam.xfam.org/) as a query to search the IMP gene members in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii.Then we used Pfam and NCBI-CD search to verify the IMP genes and deleted the genes with incomplete C and N terminals manually.Online website Phytozome v13 (https:// phyto zome-next.jgi.doe.gov/) was used to compare homologous genes of other species by using PF00459 as the keyword.Finally, a total of 28, 27, 13, 13, 9, 7, 9, 7, 13, 7 and 16 IMP genes were identified in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, Arabidopsis thaliana (A.thaliana), Zea mays (Z.mays), Oryza sativa (O.sativa), Theobroma cacao (T.cacao), Populus trichocarpa (P.trichocarpa), Vitis vinifera (V.vinifera) and Glycine max (G.max), respectively (Fig. 1).To better understand the gene arrangement of the IMP gene family and the differences in the number of genes among different species, we renamed the IMP genes according to their positions on the corresponding chromosomes.Specifically, the genes on G. hirsutum were renamed GhIMP1A-GhIMP13D, and the genes on G. barbadense were renamed GbIMP1A-GbIMP13D.Detailed results of gene renaming were shown in supplementary Table S1.The two tetraploid cotton species, G. hirsutum and G. barbadense, had twice the number of IMP family genes than the two diploid cotton species G. arboreum and G. raimondii.And the number of IMP genes in G. hirsutum and G. barbadense was higher than that of other species, indicating that the IMP gene family of this two cotton species expanded massively during the process of evolution.

Phylogenetic analysis of IMP genes
To detect the evolutionary relationships of IMP proteins among four Gossypium species and other 7 species, MEGA 7 software was used to construct phylogenetic trees.It was worth noting that an unrooted phylogenetic tree between four Gossypium species was constructed based on the neighbor-joining (NJ) method with 1 000 bootstrap replicates, while a Maximum Likelihood (ML) tree was constructed between four Gossypium species and other 7 species.
According to the sequence similarity, tree topology and structural characteristics in each sequence, we divided the IMP genes into 3 clades, and each clade can be divided into different classes (Fig. 2).Among them, there were 2 classes in clade I with 87 genes, 3 in clade II with 56 genes, and 1 in clade III with 6 genes, and clade I had the most genes (Fig. 2B).The IMP genes in tetraploid cotton species were almost twice as many as diploid cotton species in each subgroup (Fig. 2A).
According to the analysis of evolutionary relationship, we can know that except clade III, the IMP genes of four cotton species in the other clades are more closely related to that of T. cacao than any other plant species, which is consistent with previous studies that cotton and cacao emerged from the same ancestor [21].

Analysis of chromosomal localization
To visualize the specific distribution of genes on chromosomes of four cotton species, we mapped the physical locations of these genes on cotton chromosomes by using TBtools software (Fig. 3).148 IMP genes were unevenly located on different specific chromosomes, of which only GhIMP1D gene was mapped on scaffold.Except for GhAt05-A05 and GbAt05-A05, the number of IMP genes on the chromosomes of the GhAt and GbAt subgenomes was the same as that of the diploid homologous chromosome A genome (G.arboretum) (Table 1), most of the genes in subgroup A were preserved during evolution, and only the genes on chromosome 05 were increased.These results showed that this may be due to segmental duplication or whole genome duplication events during evolution, and it could also be the inaccuracy of genome sequencing.

Gene duplication and collinearity analysis
Previous studies revealed that whole genome duplication, segmental duplication, and tandem duplication were the main drivers of plant gene family expansion [24].To uncover the genome-wide duplicated principle of the IMP gene family in four Gossypium species, the duplicated gene pairs of four species were acquired and filtered by running MCScanX, four duplicated gene pairs (GhIMP3A/4A, GhIMP3D/4D, GbIMP3A/4A and GbIMP3D/4D) were identified as events of tandem duplication (Fig. 4), while the rest of duplicated gene pairs were detected as segmental duplication and whole genome duplication, indicating that compared to tandem duplication, the segmental duplication played a predominant role in the evolution of IMP gene family.
G. hirsutum and G. barbadense are allotetraploid species, produced by the hybridization between diploid A and D genome species [25].To understand the evolutionary relationship between tetraploid and diploid, we further analyzed the collinear relationship among GhAt-A, GbAt-A, GhDt-D, GbDt-D (Fig. 5).There were 36, 38, 41, and 39 duplicated gene pairs in GhAt-A, GbAt-A, GhDt-D, GbDt-D, indicating that Gh and Gr had a closer relationship in the process of evolution.

Analysis of Ka/Ks
To further understand the selection pressure of the duplicated gene pairs in IMP gene family, we analyzed the Ka, Ks, and Ka/Ks ratio of 10 combined duplicate gene pairs (Gh-Gh, Gb-Gb, Ga-Ga, Gr-Gr, Gh-Gb, Ga-Gr, Ga-Gh, Ga-Gb, Gr-Gh, Gr-Gb).Among the 210 duplicated gene pairs, 5 (2.38%) pairs with Ka/Ks > 1, demonstrating that these genes may undergo the positive selection pressure.205 (97.62%) pairs with Ka/Ks < 1 (172 pairs with 0 < Ka/Ks < 0.5, 33 pairs with 0.5 < Ka/Ks < 1), based on Table 1 Comparison of chromosomes harboring number of IMP genes from different genomes and subgenomes of four Gossypium species (Ga, Gr, Gh, Gb) the Ka/Ks ratio, the IMP genes mainly underwent purifying selection (Fig. 6).

Analysis of conserved protein motifs and gene structure
To explore the genetic structure characteristics of upland cotton, we analyzed the conserved motifs and gene structures of 28 GhIMP genes in G. hirsutum by using the online website MEME, and 15 motifs were selected.In general, according to the schematic diagram (Fig. 7), we can see that different clades of GhIMP genes have different types and numbers of motifs and gene structures.Except for GhIMP6A in subgroup Ia and GhIMP6A in subgroup IIc, the types and numbers of motifs in other subgroups were almost the same.The number of motifs was different in each clade, among them, clade I had the largest number of motifs, 10-12, and the number of motifs in subgroup IIa was the least, 6.Interestingly, some motifs were found only in certain subgroups.For example, Motif 15 was found only in some subgroups (Ib, IIC, and III), Motif 12 was found only in Ib, and Motif 5 was found in all subgroups except clade III.
In addition, we also analyzed the number of exons of 28 GhIMP genes (Fig. 7C, Supplementary Table S1), and the results showed that the number of exons varied from 3 to 21, and the largest number of exons was 21, which was GhIMP5A.The number of exons in the same subgroup was generally the same, which was well reflected in the subgroups Ib, IIa and IIb, which proved that these subgroups were relatively conserved in evolution.Gene characteristics, including the protein lengths, molecular weights (MWs), isoelectric points (pIs), grand average of hydropathy, and subcellular locations were showed in Supplementary Table S1.It was observed that the protein lengths varied from 266 amino acids (GhIMP6D) to 692 amino acids (GhIMP14A), and the average number of amino acids was 382.Among them, there were 7 genes with 200-300 amino acids, accounting for 25% of the total genes, and 11 genes with 300-400 amino acids, accounting for a large percentage (39%), there are 8 genes with 400-500 amino acids, accounting for 28% of the total, and 2 genes with more than 500 amino acids, accounting for 8% of the total genes.MWs ranged from 28.672 kDa (GhIMP6D) to 75.892 kDa (GhIMP14A), with an average MWs of 41.55, and the average pIs for GhIMPs was 6.36.Prediction of the subcellular localization of GhIMP proteins revealed that most proteins function in the chloroplast and cytoplasm, but subsequent experiments were needed to verify the location of proteins.

Gene ontology (GO) annotation analysis of GhIMP genes
To study the function of GhIMP genes more intuitively, we used CottonFGD to analyze their GO annotations, which including cellular component, molecular function, and biological process.As shown in Fig. 8, GhIMP genes were categorized into 21 functional groups under main three categories.Phosphatidylinositol phosphate biosynthetic process (GO:0046854) was the most abundant biological process, with 26 genes.3'(2'),5'-bisphosphate nucleotidase activity (GO:0008441) was the most enriched molecular function, with 12 genes.

Analysis of promoters and differentially expressed genes
Promoters can interact with transcription factors to precisely regulate transcription initiation and transcription efficiency of genes, and cis-acting elements  8 Gene ontology (GO) annotations of GhIMP genes located in the promoter region were found to play vital roles in the response to abiotic stress [26].The promoters of GhIMP family genes were analyzed, and we found that most promoters contained hormonerelated elements (abscisic acid response element, MeJA response element, gibberellin response element) and stress-related elements (low temperature response element, defense and stress response element, wound response element) (Fig. 9A, B).All genes had lightresponsive elements, and each gene contained more than one light responsive element.For example, GhIMP6A contained 7 light responsive elements.16 of 28 GhIMP genes contained MeJA responsive elements, accounting for 57% of the total, and 19 (67.86%) of 28 genes contained abscisic acid (ABA) responsive elements.
Then the expression patterns of GhIMP genes under different abiotic stress including cold, hot, salt and PEG stress were analyzed (Fig. 9C).In general, different genes have different expression patterns under different stresses.The expression levels of three genes increased significantly after various stress treatments, namely GhIMP8D, GhIMP10D and GhIMP10A, while 3 genes decreased significantly, namely GhIMP7D, GhIMP6D and GhIMP6A.

qRT-PCR of GhIMP genes in response to NaHCO 3 stress
To further confirm the response of GhIMP genes to alkaline stress, 10 GhIMP genes from different clades were randomly selected for qRT-PCR to detect their expression profiles in roots, stems and leaves treated with 125 mM NaHCO 3 (Fig. 10).The treatment of the concentration of 125 mM NaHCO 3 was carried out according to our previous research results [12].The expression pattern of 5 genes was the same, namely GhIMP4A, GhIMP6A, GhIMP2D, GhIMP8D and GhIMP10D.The expressions of GhIMP6A and GhIMP10D genes increased under alkaline stress, and GhIMP10D significantly up-regulated in leaves.The remaining genes showed inconsistent expression profiles in roots, stems and leaves, suggesting that these genes may be involved in different pathways.In leaves, only the expression of GhIMP10D was significantly upregulated, and we speculated that this gene may play an important role in leaves, and we will conduct a preliminary study of its function in leaves.

Exogenous ABA can promote the expression of GhIMP genes
By analyzing the cis-acting elements of GhIMP genes (Fig. 9), we found that most GhIMP genes contained ABA responsive cis-acting elements.2 of the genes contained 3 ABA cis-acting elements, namely GhIMP6D and Fig. 9 Cis-acting element analysis and differentially expressed genes (DEGs) analysis of GhIMP genes.A Phylogenetic tree of GhIMPs.B Cis-acting elements in promoters of GhIMPs.C Heatmap of GhIMPs under cold, hot, salt, and PEG stress.The relative expression of heat map was characterized by log 2 transformed GhIMP5A.9 genes contained 2 ABA cis-acting elements, namely GhIMP1A, GhIMP2A, GhIMP5A, GhIMP14A, GhIMP3D, GhIMP5D, and GhIMP14D.To investigate the alleviating effect of ABA on alkaline stress and whether ABA can promote the expression of GhIMP genes, different concentrations of ABA (0, 10, 50 and 100 μM) were applied to cotton seedlings at three-leaf stage under NaHCO 3 treatment (Fig. 11).The results showed that the exogenous application of 50 μM ABA could alleviate the damage of alkaline stress, at the same time, the MDA contents were measured, and compared with 0 μM ABA, the MDA content in cotton seedlings with 50 μM ABA was significantly reduced.In addition, we measured 5 genes containing more ABA cis-acting elements, including GhIMP4A, GhIMP6D, GhIMP7D, GhIMP10D and GhIMP14D, and found that only GhIMP4A, GhIMP10D and GhIMP14D genes were significantly induced by ABA, and the expression of the remaining two genes did not change significantly.

Three-dimensional (3D) structure prediction of GhIMP proteins
To understand the protein structure of the GhIMP proteins, I-TASSER was used to obtained the 10 GhIMP proteins' structures.The modeled structures for the selected GhIMP proteins had 8 (GhIMP2D, GhIMP6D, GhIMP10D and GhIMP10A) to 15 (GhIMP7D) α-helices, and 10 (GhIMP8D) to 14 (GhIMP14D) were identified as β-strands (Fig. 12).The same clade had similar protein structures, such as GhIMP10D, GhIMP6D, and GhIMP10A in clade IIc, which was different from the protein structure of GhIMP14D in clade III.

Silencing GhIMP10D gene is sensitive to alkaline stress
To further investigate the function of GhIMP10D gene under alkaline stress, VIGS were performed to preliminarily investigate the gene function.Firstly, qRT-PCR was performed on pYL156 and pYL156: GhIMP10D plants when the albino phenotype was present, and the expression level of pYL156: GhIMP10D plants was significantly decreased (Fig. 14).Then, cotton seedlings were subjected to 125 mM NaHCO 3 alkaline stress treatment, and it was found that cotton seedlings with silenced GhIMP10D gene were sensitive to alkaline stress, indicating that GhIMP10D gene could positively regulate cotton alkaline tolerance.At the same time, we measured the content of ascorbic acid (AsA) and chlorophyII and found that the AsA and chlorophyII content of cotton seedlings silenced with the GhIMP10D gene was significantly lower than that of pYL156 plants.In addition, the activities of some antioxidant enzymes were also measured, and the results showed that compared with pYL56 plants, the content of SOD and POD under alkaline stress in silenced plants were decreased significantly, and the content of MDA was increased significantly.In summary, we suggested that GhIMP10D gene may positively regulate tolerance to NaHCO 3 stress by regulating the activity of antioxidant enzymes and the content of AsA to eliminate ROS.

Discussion
AsA is a kind of secondary metabolic substance that is beneficial to both plant and human health, and understanding the biosynthetic pathways of ascorbic acid and the role of key genes in the synthesis of AsA is of great significance for increasing the content of AsA in crops.At the same time, AsA has a strong ability to remove ROS, so it is of great significance to study the resistance of AsA to saline-alkaline stress.
Cotton is a standard model for studying the evolution of polyploids [27], so we used genomic data to explore the evolutionary relationship of cotton in four species.In this study, 28, 27,13 and 13 IMP genes was identified in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively.In G. hirsutum, 14 IMP genes were identified in the At subgenome, and 14 IMP genes were identified in the Dt subgenome.The numbers of IMP genes in the G. hirsutum and G. barbadense were almost the sum of those in G. arboreum and G. raimondii, which suggesting that 2 allotetraploid G. hirsutum and G. barbadense species may resulted from hybridization of the two ancestral species [21].
The expansion of gene families caused by gene duplication events is one of the main evolutionary mechanisms leading to functional diversification and speciation [28].To better understand the expansion mechanism of IMP gene family, intragenomic and intergenomic 10 duplication events (Ga-Ga, Ga-Gb, Ga-Gr, Gb-Gb, Gb-Gr, Gh-Ga, Gh-Gb, Gh-Gh, Gh-Gr and Gr-Gr) were analyzed by MCScanX (Fig. 4).Only GhIMP3A/4A, GhIMP3D/4D, GbIMP3A/4A and GbIMP3D/4D were identified as tandem duplicated gene pairs, but none of the other combinations, indicating that segmental duplication was the main driving force of IMP gene family evolution.
To illuminate the divergence after gene duplication, the Ka, Ks and Ka/Ks of duplicated gene pairs were calculated (Fig. 6A).In general, The Ka/Ks ratio could predict a gene's evolutionary background, and Ka/Ks < 1 represented that the gene had undergone purification selection, eliminating deleterious mutations and preserving important protein structures, Ka/Ks > 1 indicated that the gene had undergone positive selection, which accelerated the evolution of the gene, Ka/Ks = 1 was neutral selection, which indicated that natural selection does not lead Fig. 13 Protein interaction network of GhIMP10D.VTC4 is the corresponding name of GhIMP10D protein on String to gene mutation [29].In this study, more than 93% of the duplicated gene pairs with Ka/Ks values less than 1, indicating that the IMP gene family had undergone strong purification selection.
The increase or decrease in exon-introns can be caused by the integration and recombination of gene fragments.It was found that gene structure variation can cause the divergence of gene families in the process of evolution [24].The genes in subgroup IIc had the largest number of exons, 12 (GhIMP6D, GhIMP10D, GhIMP10A) and 21 (GhIMP5A), respectively.Motif 5 was present in all the other subgroups, but disappeared in clade III.Combined with the phylogenetic tree, we speculated that the genes of clade III may lose Motif 5 during the evolution.The presence of Motif 6 in all genes proved that it preserved as a conserved motif in the process of evolution.Motif 12 was found only in Ib, which might indicate they had acquired important functions during evolution, and this might be used as a basis to identify this subgroup, similarly, in other gene family, there was a subfamily that had only one particular motif [30].GhIMP genes of the same subfamily had similar gene structures and motif compositions (Fig. 7B, C), and high conservation of genes in the same subfamily suggested that the GhIMP genes had undergone duplication events during evolution.In summary, the differences in gene structure and motif composition among the different subfamilies suggested that their functions may be different.Approximately 90% of AsA in cells was reported to be localized in the cytoplasm, and most GhIMP genes were predicted to be localized in the cytoplasm in our study.Therefore, we hypothesized that GhIMP genes may play an important role in the regulation of AsA content in the cytoplasm.The subcellular localization of GhIMP genes need to be further verified in the future.
Cis-acting elements play an important role when plants are subjected to abiotic stresses [31].In our study, several stress-relevant cis-acting elements were identified, such as low temperature response elements, defense and stress response elements and wound response elements, which suggesting that GhIMP genes might play a critical role in response to abiotic stresses (Fig. 9B).All GhIMP family genes contained light-responsive cis-acting elements, proving that light may induce the expression of GhIMP family genes.Previous studies reported that the expression of LeIMP1 in tomato increased with the exposure to light, indicating that the IMP gene responded positively to light [32].In general, phytohormone signals will induce various adaptive responses that ultimately regulate plant physiological and biochemical processes in response to various abiotic stresses [33].ABA plays a crucial role in combating abiotic stress, it was reported that ABA signaling pathway responded positively to alkaline stress.GsSKP21 mediated the ABA signaling pathway to resist alkaline stress by changing the expression levels of ABA signaling related genes and ABA-induced genes in Glycine soja [34].It was found that ABA could enhance alkaline stress tolerance in rice by upregulating genes related to antioxidant defense and stress tolerance [35].In our study, most GhIMP genes had abscisic acid response elements, and combined with previous research, we speculated that the GhIMP genes may protect against alkaline stress by regulating the ABA signaling pathway, especially genes containing multiple abscisic acid response elements, such as GhIMP3D, GhIMP5D, GhIMP7D, GhIMP10D, GhIMP2A, GhIMP5A, GhIMP10A and GhIMP14A.In addition, MeJA-responsive cis-acting elements were also enriched in GhIMP family members, accounting for 57.14% of the total number, and almost all subgroups Ia and IIa contained this element.Moreover, several GhIMP genes also contained gibberellin responsive cis-acting elements (39.28%), most of which were concentrated in subgroups Ia and III.Previous studies showed that AsA content regulated the expression of plant defense genes and controlled plant growth and development through the hormone signaling pathway [36].Therefore, IMP genes was likely to regulate hormone signaling pathway by regulating AsA synthesis in response to stress, and relevant experiments needed to be further verified.
ABA, as an important plant hormone, plays a vital role in response to various abiotic stresses, especially drought, salt and low temperature stress [37].It was found that exogenous ABA can promote the expression of genes to resist the damage to various abiotic stresses.For example, in Grapevine, exogenous ABA can promote the expression of VviZIP gene and detoxification related genes to reduce the toxic effect caused by zinc accumulation [38], and it was found that exogenous ABA could induce the expression of galactinol synthase and raffinose synthase genes to increase tolerance to cold stress [39].In A. thaliana, ABA could upregulate the expression of ZAT6, thus positively regulates Cd accumulation and tolerance [40].In Oryza sativa, ABA could upregulate OsSMP1, and overexpression of OsSMP1 gene could improve the tolerance to CdCl 2 and CuSO 4 [41].In Nitraria tangutorum, exogenous ABA could significantly increase the transcription levels of NtFLS, NtF3H, NtF3H and NtANR genes participated in the synthesis pathway of flavonoid metabolites, while flavonoids were reported to improve tolerance to alkaline stress [42].In Jute, ABF family genes CoABF3 and CoABF7 were significantly up-regulated under salt and drought stresses after exogenous application of ABA [43].In this study, after adding different concentrations of ABA to cotton seedlings under alkaline stress, we found that exogenous ABA had a resistance to alkaline stress in cotton seedlings, which was consistent with previous studies.5 GhIMP genes with 2 or 3 ABA cis-acting elements were detected by qRT-PCR, and the expression of the 2 genes was found to be unchanged after exogenous ABA treatment, namely GhIMP6D and GhIMP7D.The expression of the 3 genes changed significantly, among which GhIMP4A and GhIMP14D were significantly downregulated after ABA treatment, and GhIMP10D was significantly upregulated.suggesting that ABA treatment could induce the expression of IMP family genes in response to alkaline stress.Combined with the phylogenetic analysis, we found that the expression patterns of IMP family genes treated with ABA was different, and the genes in clade II and III may respond positively to ABA treatment.In conclusion, ABA can increase the expression of some GhIMP genes in response to alkaline stress, which is consistent with previous studies that CaIMP gene was induced under salinity, drought and heat stress and ABA treatment in chickpea [16].
Any two proteins that are jointly involved in a specific cellular process are considered functionally related, therefore, co-expressed proteins are modulated by the same transcriptional programs and share same or similar pathways [44].In this study, we analyzed the interaction network of GhIMP10D protein.It was found that GhIMP10D protein mainly interacted with three types of proteins, namely GDP-L-galactose phosphorylase (GLDH), GDP-L-galactose phosphorylase (VTC2 and VTC5) and Inositol-3-phosphate synthase (MIPS).Previous studies reported that the GLDH and VTC played a vital role in the ascorbic acid synthesis pathway [45], and MIPS was an important enzyme in the phospholipid synthesis pathway [46].GhIMP10D protein participated in the synthesis of AsA, and it also participated in the synthesis of inositol, which was consistent with previous studies that the enzyme encoded by IMP was a bimolecular enzyme [14,47], indicating that GhIMP10D protein in cotton may also be a bimolecular protein.The specific proof needed to be verified by subsequent experiments.
Previous studies had found that IMP gene was induced by various stresses, the expression level of CaIMP was found to be increased in salt, cold, heat and dehydration stress with maximum expression with dehydration stress [16].Transcript analyses showed that Rice OsIMP gene was significantly upregulated by cold and ABA treatment [15].Similarly, IMP genes in G. hirsutum were induced by cold, hot, salt and PEG stresses, but the response of the GhIMP genes to varioust stresses was different.For example, GhIMP6A, GhIMP6D and GhIMP7D were significantly down-regulated compared with other genes.The expression levels of GhIMP8D, GhIMP10A and GhIMP10D under the four kinds of stresses were higher than that of other genes.Combined with the qRT-PCR data, we could know that the expression of GhIMP10D in leaves was the highest, so we speculated that it may play an important role in abiotic stress.
qRT-PCR results showed that GhIMP10D gene was strongly induced by alkaline stress and was differentially expressed in roots, stems and leaves, with the highest expression in leaves.Therefore, further analysis of this key gene is necessary.Previous studies showed that IMP genes were not only involved in seed development [48], AsA synthesis [14,49], but also in response to various abiotic stresses [15,50].However, there is no relevant research of IMP genes under alkaline stress.
It was found that alkaline stress was more harmful to the photosynthetic system of cotton compared with salt stress [51].In this study, we found that the chlorophyII content of both pYL156 and pYL156: GhIMP10D plants decreased significantly after alkaline stress, and the chlo-rophyII content of pYL156: GhIMP10D plants decreased significantly compared with pYL156, which was consistent with previous study.Alkaline stress could induce the production of ROS, and AsA was produced to eliminate excess ROS in the body.When the GhIMP10D gene was silenced, the content of total AsA decreased significantly.Therefore, we believed that GhIMP10D gene may regulate the response to alkaline stress by regulating the synthesis of AsA.To further verify our conjectures, we analyzed the expression levels of genes in the AsA synthesis pathway, and found that the expression levels of GhAKR and GhGLDH genes decreased significantly after GhIMP10D gene was silenced.Therefore, we believed that GhIMP10D gene regulates the response to alkaline stress by regulating the synthesis of AsA.In addition, we measured the activity of antioxidant enzymes to explore the influence of GhIMP10D gene on antioxidant enzymes.The results showed that the activities of SOD and POD decreased significantly after the silencing of GhIMP10D gene.Therefore, GhIMP10D gene can resist alkaline stress by regulating the activity of antioxidant enzymes.
In summary, an alkaline-responsive gene, GhIMP10D, was identified as playing an important role in resisting alkaline stress in cotton, and GhIMP10D might respond to alkaline stress by controlling AsA synthesis through a complex regulatory mechanism (Fig. 15).Under normal circumstances, the redox state in cotton seedlings is balanced, and GhIMP10D controls the expression of GhAKR and GhGLDH genes to keep AsA contents stable.However, when seedlings are subjected to alkaline stress, a large amount of ROS is induced in vivo, in which GhIMP10D is induced to be expressed to resist alkaline stress.At the same time, the balance of ascorbic acid in the body is also broken, and the synthesis of ascorbic acid is increased to cope with alkali stress.In addition, GhIMP10D will resist alkaline stress through two ways, firstly, GhIMP10D will control the synthesis of antioxidants to eliminate excessive ROS in vivo, on the other hand, GhIMP10D will control the expression of GhAKR and GhGLDH genes to produce a large amount of AsA to eliminate excessive ROS in the body.The results reveal the function of GhIMP10D gene in the regulation of alkaline tolerance.

Conclusions
With the deterioration of the environment, it is necessary to enhance the alkali-tolerant capability of cotton.Based on morphophysiological and bioinformatics analysis, the present study revealed that GhIMP genes had positive responses to various abiotic stresses, and cis-acting elements analysis of GhIMP genes showed that many GhIMP genes had elements associated with plant hormone and abiotic stress, of which ABA responsive elements accounted for a large proportion.Exogenous ABA was added under alkaline stress, and the results showed that ABA could induce the expression of some GhIMP genes.qRT-PCR results of GhIMP genes under alkaline stress showed that GhIMP10D was highly expressed in response to alkaline stress and ABA treatment.Silencing GhIMP10D gene in cotton made it sensitive to alkaline stress.Enhanced tolerance to alkaline stress might be because of shared role in regulating the expression of downstream GhAKR and GhGLDH genes to control the synthesis of AsA to remove ROS, in increasing the activity of antioxidant enzymes such as SOD, POD and chlo-rophyII content to eliminate ROS.

Phylogenetic analysis and sequences alignments
Multiple sequence alignment of all these IMP proteins were performed using ClustalX program in MEGA7 software.Subsequently, MEGA 7 software was used to construct a phylogenetic tree of four Gossypium species using neighbor joining (NJ) method with default parameters.A phylogenetic tree of IMP proteins in the four Gossypium species and other 7 species was constructed using the Maximum Likelihood method of MEGA7 with 1000 bootstrap replicates based on the LG + G model.Chromosomal mapping of IMP genes from four Gossypium species IMP genes of four Gossypium species were mapped on chromosomes using TBtools software with Generic Feature Format (gff ) files and gene IDs downloaded from CottonFGD [55].

Analysis of the conserved protein motifs and gene structure
Multiple Em for Motif Elicitation (MEME) website (http:// meme-suite.org/) was used to identify the conserved motifs of GhIMP proteins, and the parameters used in this study were set as follows: maximum number of different motifs is 15, and other parameters are default [56].The diagram of evolutionary relationship, gene structure, and conserved motifs of GhIMP proteins was drawn using TBtools software.

Collinearity analysis of IMP genes in four Gossypium species
To investigate the collinearity relationship among IMP genes of four Gossypium species, we analyzed the duplicated gene pairs from four cotton species G. hirsutum, G. arboreum, G. raimondii, and G. barbadense, then complete genome sequences of four Gossypium species along with genome annotation files were subjected to MCScanX software to investigate the collinearity synteny relationship [57].Finally, the diagrams were visualized using Advance Circos tool in TBtools software with chromosome length files and the genome alignment files among four Gossypium species.

Calculation of selection pressure
To assess the selection pressure of duplicated gene pairs, the nonsynonymous (Ka), the synonymous (Ks), and Ka/Ks were calculated by using Ka/Ks calculator in TBtools software.

Analysis of cis-acting element in promoters of GhIMPs
The 2000 bp DNA sequence of the upstream region of GhIMP genes were obtained from the online website Cot-tonFGD (https:// www.cotto nfgd.org/) as the promoters.The Plant CARE website (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html/) was used to predict the cisacting elements in the promoter region of GhIMP genes.Finally, we constructed a schematic diagram of phylogenetic tree and cis-acting elements in TBtools software.

Gene ontology analysis
CottonFGD was used to study the gene ontology annotations of GhIMP genes, and the annotations were based mainly on three aspects: biological process, cellular component, and molecular function.

qRT-PCR analysis under NaHCO 3 stress
To determine the response of GhIMP genes to alkaline stress, a total of 10 genes were randomly selected from each subfamily, and the relative expression levels of these 10 genes were determined by taking root, stem and leaf tissues after 125 mM NaHCO 3 treatment for 12 h.The total RNA of roots, stems and leaves were extracted using EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China).The cDNA was reverse using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China).Then qRT-PCR was performed using the Applied Biosystems@7500 Fast instrument with TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China).All operations were carried out in accordance with the manufacturer's instructions.The Actin gene was used as a control. 2 −△△CT method was used to calculate the fold change for each sample [58].

Expression patterns under different stresses
To understand the expression pattern of GhIMP genes, we analyzed it using RNA-seq data (PRJNA490626) downloaded from NCBI website (https:// www.ncbi.nlm.nih.gov/), which mainly included the expression levels under salt, PEG, cold, and heat stresses [60].In addition, we analyzed the expression pattern of GhIMP genes under alkaline stress based on published RNAseq data (GSE165472) [12].

ABA application of cotton seedlings under alkaline stress
When the cotton seedlings reached three true leaves, exogenous ABA treatment was applied to cotton seedlings to observe the alleviating effect of alkaline stress.Upland cotton material Zhong 9807 was used for further experiments, and it was obtained from Cotton Research Institute, Chinese Academy of Agricultural Sciences with the accession ID: xcy2399.ABA (Solarbio) was dissolved in a small amount of absolute ethanol and then diluted with deionized water to the different concentrations (0, 10, 50 and 100 μM).The leaves of cotton were completely soaked by spraying with different concentrations of ABA, and then dark for 12 h under alkaline stress.Cotton leaves of each treatment were sampled for the measurement of MDA and qRT-PCR at 0 h and 12 h, respectively.

Gene interaction network of GhIMP10D protein
The GhIMP10D protein interaction network was analyzed by the STRING database (https:// string-db.org/) [44], and Arabidopsis thaliana orthologs was used to predict the interaction of GhIMP10D with other proteins in cotton.

VIGS (Virus-induced gene silencing) experiment of GhIMP10D gene
The pYL156 vector was digested with BamH I and Sma I restriction enzymes, amplifying the 300 bp fragment specific to the GhIMP10D gene, ligated using ClonExpress II One Step Cloning Kit from Vazyme.After that, the constructed vector was transferred into A. tumefaciens LBA4404.Upland cotton material Zhong 9807 was used for further experiments, and it was obtained from Cotton Research Institute, Chinese Academy of Agricultural Sciences.The plant material was deposited at the gene bank of the Institute of Cotton Research, Chinese Academy of Agricultural Sciences, with the accession ID: xcy2399.The cultures, cells and the procedure of infection were handled as previously described [61].When the albino phenotype appeared, qRT-PCR experiments and physiological indicators were measured.

Measurement of ascorbic acid (AsA) and Dehydroascorbic acid (DHA) content
AsA and DHA content was determined using Ascorbic Acid (AsA) Content Assay Kit (BC1230) and Dehydroascorbic Acid (DHA) Content Assay Kit (BC1240) from Solarbio.0.1 g cotton leaves were taken and AsA content was measured according to the manufacturer's procedure, and three biological replicates were taken for each sample.

Measurement of chlorophyll, malondialdehyde (MDA) contents, peroxidase (POD) and superoxide dismutase (SOD) activity assay
The SPAD-502 PLUS measuring instrument (Konica Minolta (China) Investment Ltd) was used to detect the chlorophyl II contents in the leaves, and the same part of each leaf was taken for measurement.0.1 g cotton leaves were taken to determine MDA, POD and SOD contents using kits from Nanjing Jiancheng Bioengineering Institute (A003-3-1, A084-3-1 and A001-3-2).

Fig. 1
Fig. 1 Distribution of IMP genes among four Gossypium species and other 7 species

Fig. 2 Fig. 3
Fig. 2 Phylogenetic trees of IMP genes in four Gossypium species and other 7 species.A Phylogenetic tree of the 81 IMP genes from four Gossypium species using MEGA 7 by the Neighbor-Joining (NJ) method.B Phylogenetic tree of the 149 IMP genes from four Gossypium species, A. thaliana, Z. mays, O. sativa, T. cacao, P. trichocarpa, V. vinifera and G. max using MEGA 7 by Maximum Likelihood (ML) method

Fig. 4 Fig. 5
Fig. 4 Syntenic relationship of duplicated IMP genes pairs from four Gossypium species.Lines with different color indicated different duplicated IMP gene pairs

Fig. 6 Fig. 7
Fig. 6 Schematic diagram of non-synonymous (Ka) to synonymous (Ks) ratio in cotton.A Ka and Ks divergence values in ten different pairs.B Ka and Ks divergence values in Gh-Gh, Gb-Gb, Ga-Ga, Gr-Gr

Fig. 14
Fig. 14 VIGS experiments of the GhIMP10D gene and measurement of physiological indicators.A The true leaves of pYL156: PDS cotton showed bleaching, which proved that the VIGS system was effective.B qRT-PCR analysis of pYL156 and pYL156: GhIMP10D plants.C Phenotypes of pYL156 and pYL156: GhIMP10D cotton seedlings under alkaline stress.D-G Physiological indicators including SOD, POD, chlorophyII and MDA of pYL156 and pYL156: GhIMP10D cotton seedlings under ddH 2 O and alkaline treatment.H-J AsA, DHA and total AsA contents of pYL156 and pYL156: GhIMP10D cotton seedlings under ddH 2 O and alkaline treatment.K-M Relative expression of genes associated with the AsA synthesis pathway.Notes: SAS: 125 mM NaHCO 3

Fig. 15 A
Fig. 15 A working model of the function of GhIMP10D modulates AsA and antioxidant enzyme to eliminate ROS under alkaline stress