Conserved Aquaporins in Gossypium and Silencing of Ghpip2;7 and Ghtip2;1 Decreased Salt and Osmotic Stress Tolerances in Upland Cotton

doi:10.21203/rs.3.rs-489764/v1

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Conserved Aquaporins in Gossypium and Silencing of Ghpip2;7 and Ghtip2;1 Decreased Salt and Osmotic Stress Tolerances in Upland Cotton

https://doi.org/10.21203/rs.3.rs-489764/v1

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Aquaporins (aquaporin), membrane channel proteins, facilitate the transport of water and small molecules across intrinsic membranes and play a critical role in abiotic stresses. Here, 111, 54, and 56 candidate AQP genes were identified in Gossypium hirsutum (AD₁), G. arboreum (A₂), and G. raimondii (D₅), and classified into five subfamilies including PIP, TIP, NIP, SIP, and XIP, respectively. Some GhPIPs and GhTIPs exhibited high expression levels in drought and salt stress verified by transcriptome analysis and Quantitative Real-time PCR (qRT-PCR). The chlorophyll content, SOD activity, and POD activity were decreased in GhPIP2;7 gene-silenced plants versus the control (using blank vector) under 400 mM NaCl treatment, which indicated a negative role of GhPIP2;7 in cotton salt tolerance. Comparing with mock plants, GhTIP2;1 silenced cotton plant was more sensitive to osmotic stress. Overexpressed GhTIP2;1 plants exhibited less accumulation of H₂O₂ and MDA with higher proline contents detected under osmotic stress. Taken together these results, provide in-depth knowledge into plant response to abiotic stress and gene resource.

Plant Molecular Biology and Genetics

Agronomy

Gossypium

aquaporin

PIP

gene family

salt stress

GhPIPs and GhTIPs were induced after salt and drought stress treatments. GhPIP2;7 and GhTIP2;1 act as positive regulators in response to salt and osmotic stress in upland cotton, respectively.

Aquaporins are membrane channel proteins that facilitate the transport of water and small molecules across biological membranes in most organisms, which belong to the major intrinsic protein (MIP) superfamily (Maurel et al. 2015). Plant aquaporins are multifunctional channels, with a wide range of selectivity profiles (Baiges et al. 2002). In addition, AQPs are involved in the transportation of glycerol, urea, ammonia (NH₃), carbon dioxide (CO₂), hydrogen peroxide (H₂O₂), and metalloid such as boron and silicon (Maurel et al. 2015). The AQP family has six transmembrane domains (TM1-TM6) features connected by five loops (LA-LE), and has two NPA (Asn-Pro-Ala) motifs; ar/R filter and Froger’s position (Kaldenhoff and Fischer 2006). To date, AQP families have been revealed and characterized in many species: 35 AQP genes were identified in Arabidopsis thaliana (Johanson et al. 2001), and 47, 41, and 45 AQPs have been characterized in Solanum lycopersicum (Reuscher et al. 2013), Phaseolus vulgaris (Ariani and Gepts 2015), and Manihot esculenta (Putpeerawit et al. 2017), respectively. In addition, 43, 33, 47 and 35 AQPs have been characterized in Zea mays (Chaumont et al. 2001), Oryza sativa (Sakurai et al. 2005), banana (Hu et al. 2015), and watermelon (Zhou et al. 2019), respectively. Plant aquaporins can be divided into five subfamilies which including plasma intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs), and uncategorized X intrinsic proteins (XIPs) (Johanson et al. 2001).

Upland cotton (Gossypium hirsutum), one of the cultivated tetraploid species (2n = 52), is an important cash crop, which provides the most natural textile fiber and edible oil (Zhang et al. 2015). Up-to-date, the complete genome sequences of G. hirsutum (AD₁) TM-1, G. arboreum (A₂) Shixiya 1, and G. raimondii (D₅) have been released (Wang et al. 2012; Li et al. 2014a; Zhang et al. 2015; Chen et al. 2020). G. hirsutum consists of A sub-genome (At) and D sub-genome (Dt), originated from interspecific hybridization between the progenitors of A-genome resembling G. arboreum and D-genome resembling G. raimondii (Senchina 2003; Chen et al. 2017). Our understanding has been broadened by the release of the cotton genome and their functional gene studies with recent characterization of aquaporin genes in cotton species.

Various abiotic stresses, including cold, heat, drought, and salinity stresses, significantly reduce crop quality and production world-wide (Hossain et al. 2018; Yang and Guo 2018). In plant cells, salt stress caused an imbalance of the cellular ions resulting in ion toxicity, dehydration, and osmotic stress (Zhu 2001). Plants respond to abiotic stress by activating multiple pathways which contained the expression of genes involved in signaling cascades and transcriptional regulation, to the protection of membranes and proteins, uptake and transport of water and ions, such as aquaporins and ion carriers. Aquaporins are allocated to different subfamilies with each showing different expression patterns in response to drought stress (Zhu 2001). PIPs and TIPs are predicted as water transporters to maintain cell water balance in Arabidopsis (Wang et al. 2019). Several studies have shown that overexpression of PIPs and TIPs increased drought tolerance by decreasing transpiration rate and stomatal conductance probably (Pou et al. 2013). Whiles overexpression of PIP2;7 increased the root hydraulic conductivity (Pou et al. 2016), overexpressing of MaPIP1;1 gene improved the salt and drought tolerance by regulating primary root elongation, water up taking, and membrane stability in Arabidopsis (Xu et al. 2014). In maize, three specific isoforms (ZmPIP1;1, ZmPIP1;5 and ZmPIP2;4) were reported to transiently induced when plants regained the osmotic potential allowing water uptake (Zhu et al. 2005). TsTIP1;2 protects Thellungiella salsuginea from salinity stimuli by mediating the conduction of H₂O₂ and H₂O across the membrane which acts as a multifunctional contributor (Wang et al. 2014). In our previous reports, several aquaporin genes were found to be involved in salt stress response in cotton using RNA-seq technology (Shi et al. 2015).

In upland cotton, 71 AQP genes were identified and classified into five subfamilies as PIP (28), TIP (23), NIP (12), SIP (7), and XIP (1) based on ESTs (Expressed sequence tags). Sequences from previous research (Park et al. 2010) found 113 AQP genes, assigned to the subfamily as follow: PIP (50), TIP (26), NIP (20), SIP (11) and XIP (6) (Li et al. 2019a). It was reported that two AQP genes (GhPIP1;1 and GhTIP2;1) were down-regulated under salt stress (Braz et al. 2019). Three PIP genes (GhPIP1;1, GhPIP2;1 and GhPIP2;2) were isolated from the cotton root cDNA library, and transcriptional changes of these genes were observed under different abiotic stresses conditions (cold, salt, polyethylene glycol) (Li et al. 2009). Previous studies on the function of cotton aquaporin mainly focus on its role in fiber development and the expression pattern under abiotic stress. To fully understand the evolution of the AQP gene family and the possible function under salt and drought stresses in cotton, we deciphered the AQP genes in G. hirsutum, G. arboreum and G. raimondii upon gene structure, conserved motifs, phylogenetic relationship, chromosomal distribution, gene duplication,and assessed the expression patterns of GhAQP genes under salt and drought stress in G. hirsutum. A total of three salt stress genes (GhPIP2;2, GhPIP2;3; GhPIP2;7) and four drought stress (GhPIP1;2, GhPIP2;3; GhTIP1;1, GhTIP2;1) candidate genes were selected for further analysis. We performed virus-induced gene silencing (VIGS) to study the function of candidate genes under salt and drought stresses. GhTIP2;1 overexpressed plants were used to investigate the function of GhTIP2;1 in response to salt and drought stress. Furthermore, the transcript levels of salt stress-related genes were compared between the GhTIP2;1 overexpression (OE) Arabidopsis lines and the wild type (WT) following salt and drought treatment. These results provide helpful information for further exploring the roles of AQP gene families in the plants.

Data mining and AQPs identification in Gossypium Spp

The genome databases of G. arboreum (A2, BGI_version 1.0), G. raimondii (D5, JGI_version 2.1), and G. hirsutum acc. TM-1 (NBI_version 1.1) was obtained from the CottonGen website (Yu et al. 2014). The published data on aquaporins of Arabidopsis were obtained from TAIR (http://www.arabidopsis.org/). Aquaporin sequences of Oryza sativa and Phaseolus vulgaris were obtained from the Rice Genome Annotation Project (RGAP) databases (http://rice.plantbiology.msu.edu/) and Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html). For ease of description, G. raimondii, G. arboreum, G. hirsutum,A. thaliana, Oryza sativa, and Phaseolus vulgaris were hereafter abbreviated as Gr, Ga, Gh, At, Os, and Pv, respectively. The aquaporin genes were extracted from the whole genome sequence of the above three cotton species, and the MIP domain (Pfam accessions, PF00230) was used to build Hidden Markov Model (HMM) for searching AQPs in Gossypium using the HMMER program (Finn et al. 2011).

Structural features and promoter analysis of AQPs

The exon-intron structure was performed by Gene Structure Display Server (GSDS) tool (http://gsds.cbi.pku.edu.cn/) based on genes coding sequences and the annotated genome.

Protein sequences of AQP in G. hirsutum were submitted to an online MEME program to identify the top ten conserved motifs with default parameters (Bailey et al. 2009).

Transmembrane domains were predicted using TMHMM server v.2.0 with default parameters (http://www.cbs.dtu.dk/services/TMHMM/). Subcellular localization of aquaporins was predicted in WoLFPSORT (https://wolfpsort.hgc.jp/) (Horton et al. 2007).

The theoretical MW (molecular weight) and pI (isoelectric point) of the AQPs were predicted by ExPASy online tool (http://www.expasy.org/) (Gasteiger 2003).

The 1500 bp upstream sequences before the initiation codon (ATG) of AQPs in G. hirsutum were considered as promotors. Cis-regulatory elements of each promoter sequence were predicted against the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The cis-regulatory elements in promoter were visualized by TBtools software (http://cj-chen.github.io/tbtools/).

Phylogenetic analysis and multiple sequence alignments

The amino acid sequences of aquaporins in three cotton species, O. sativa, P. vulgaris, and Arabidopsis were used for multiple sequence alignment. The Maximum Likelihood method was applied to construct a phylogenetic tree and the reliability was obtained by bootstrapping with 1000 replicates using software MEGA 8.

Multiple sequence alignments of the aquaporins were carried out using Clustal W with default settings. The conserved residues of NPA motifs were identified manually for their corresponding amino acid residues (Johanson et al. 2001).

Chromosomal location and gene duplication analysis

The chromosomal localization of aquaporin genes was retrieved from the CottonGen website. Mapchart Version 2.3 software was used to visualize the distribution of the aquaporin genes on the chromosomes.

Gene duplication events were defined when the following conditions were fulfilled: (1) the length of aligned sequence covered more than 75% of the larger gene, (2) the identity of the aligned regions are bigger than 75%, and (3) only one duplication event was considered for tightly linked genes (Cui et al. 2017). According to the chromosomal locations of aquaporin genes, two types of gene duplication events (tandem duplication and segmental duplication) were recognized (Vision et al. 2000). The duplicated gene pairs were aligned by Clustal X program. Subsequently, non-synonymous substitution (Ka) and synonymous substitution (Ks) were calculated using DnaSP software version 5.5, and the selection pressure for each gene pair was assessed by the Ka/Ks ratio.

Expression profile analysis

The public expression profile of leaf under salt stress across time course (0, 1, 3, 6, and 12 h) of G. hirsutum TM-1 was obtained from Zhang et al. (2015). The expression data were gene-wise normalized and expression patterns were illustrated using MultiExperiment Viewer (MeV) software.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted by Hexadecyl Trimethyl Ammonium Bromide (CTAB) and precipitated by ammonium acetate method (Zhao et al. 2012). The cDNA was synthesized using PrimeScript^TM RT Reagent Synthesis Kit (TaKaRa, Dalian, China). Gene-specific primer pairs were designed by PrimerPremier software (version 5.0) based on the coding sequences of GhAQPs and stress-related genes (Table S1). GhUBQ7 and AtUBQ, were used as internal references (Table S1). Gene expression was calculated with 2^-ΔΔCt method (Livak and Schmittgen 2001). Each sample was analyzed with three technical replicates within each of the three biological duplicates.

Subcellular localization and β-Glucuronidase (GUS) histochemical staining

For the construction of the 35S::GFP-GhAQP vector, the PCR product was ligated into the BamH I site and Xba I of the pCAMBIA1300-eGFP vector driven by the cauliflower mosaic virus 35S promoter. The primers used in the subcellular localization experiments are listed in Table S2. The construct was then transformed into tobacco (Nicotiana benthamiana) K329 cultivar cells for subcellular localization analysis.

To investigate the promoter activity in different tissues, 8-day-old seedlings transferred ProGhTIP2,1::GUS were used for GUS staining. For the stress treatments, two-week-old seedlings transformed into ProGhTIP2,1::GUS were treated in ¹/₂ MS medium that was supplemented with or without 10% PEG 6000, 20% PEG 6000, and 150 mM NaCl for 24 h. GUS staining kit (Bioship company) was employed for the GUS staining. The samples were immersed in GUS histochemical staining buffers, and subsequently incubated at 37°C overnight. The samples were decolorized in 75% ethanol until the color of the negative control plants turned white. GUS activity was estimated based on the presence of blue.

Plant materials and stress treatment

The G. hirsutum cultivar GX100-2 was used for qRT-PCR and salt tolerance assay, and Zhong79 was used for qRT-PCR and drought tolerance assay. Five days old cotton seedlings with the same growth state were transferred into Hoagland liquid medium (Zhang et al. 2011) which was continuously aerated at a temperature regime of 28/20℃ with a photoperiod of 16-h light/8-h dark. At the trefoil stage of the seedlings, the expression profile of candidate genes was determined by treating half of the seedlings with salt (150 mM NaCl), and the remaining half with deionized water to serve as the control. The fresh leaves were collected at 0, 1, 3, 12, and 48 h after salt stress (Zhang et al. 2011, Su et al. 2020). These samples were frozen in liquid nitrogen immediately and stored at -80℃ for RNA isolation. Three biological repeats in each treatment were performed.

For the VIGS experiment, cotton seeds were soaked overnight in distilled water until the radicle sprouted. Seven sprouted seeds were planted in small pots filled with 1:1 (v/v) of vermiculate and nutritional soil and kept in the greenhouse at 28℃ under a 16/8 h light/dark photoperiod.

To assay, the stress tolerance in Arabidopsis, WT, and GhTIP2,1 overexpressing transgenic Arabidopsis were cultured on MS media for 4 days (22°C, 16 h light/ 8 h dark cycle). Seedlings that had no significant difference in primary root length were transferred to MS media with or without 150 mM NaCl and 15% PEG 6000, respectively for stress analysis.

The primary root length was measured on the seventh day. The experiments were carried out with three biological replicates and each replicate represents 20 seeds for each line.

VIGS analysis in cotton

Cotton leaf crumple virus (CLCrV) and tobacco rattle virus (TRV) vectors were used in the VIGS experiment under salt and osmotic stresses, respectively. The pCLCrV-fused cDNA fragment of GhChlI (magnesium chelatase subunit I) and TRV-fused cDNA fragment of GhCLA (chloroplasts alterados 1 gene) were used as a positive control to monitor the efficiency of VIGS experiments. The fragments targeting the candidate genes containing different recognition sites were amplified as a template and integrated into pCLCrVA or TRV2, then the VIGS vectors were constructed. All vectors constructed were transformed into Agrobacterium tumefaciens strain EHA105 by a heat-shock method. The EHA105 line contained pCLCrVA, pCLCrVA-genes, and TRV, TRV-genes vectors were mixed with an equal volume of Agrobacteriumtumefaciens contain pCLCrVB and TRV1, respectively, and the mixed solution was used to infiltrate plants. qRT-PCR was performed to further confirm that candidate genes had been silenced in VIGS experiments. The primers used in the qRT-PCR analysis are listed in Table S1, and the primers used in the VIGS experiments are listed in Table S2.

The cotyledons of one-week-old cotton seedlings were infiltrated with the solution containing Agrobacterium tumefaciens of pCLCrV-genes or TRV-genes according to the previous description (Gu et al. 2014, Long et al. 2020). VIGS experiments were repeated at least three times with more than three individual plants for each construct per repeat.

For salt tolerance assay, plants that infiltrated with a solution containing Agrobacterium tumefaciens of pCLCrV-GhPIPs after 10 days were watered by 400 mM NaCl solution regularly after every four days until the phenotypes appeared (Long et al. 2020).

For drought tolerance assay, cotton seedlings which silencing of candidate GhAQPs were transferred to Hoagland liquid medium containing 13% PEG6000. The phenotype was observed after 13% PEG6000 treatment for one week, and the relative water content (RWC) of leaves and roots was evaluated.

Physiological index determination

To check the chlorophyll damage from salt stress, leaves (infiltrated with a solution containing Agrobacterium tumefaciens of pCLCrV-GhPIPs after 10 days) of the same size and from the same position were picked and washed with distilled water and then were floated in the salt solution (400 mM NaCl) with the abaxial surface down. Photographs were taken after the phenotypes developed. Total chlorophyll content was calculated according to the formula described by (Arnon 1949).

To detect malondialdehyde (MDA), superoxide dismutase (SOD), and peroxidase (POD) activities, leaves were collected from the gene-silenced plants after salt stress (400 mM NaCl) or deionized water treatment for 12 days. The content of MDA and activity of SOD and POD were determined using the methods described by Ullah et al (2018). About 0.5 g fresh leaves were sampled and homogenized with a pre-cooled mortar and pestle in 5 mL 0.05 M potassium phosphate buffer (pH 7.8). The homogenate samples were centrifuged at 10000 g for 20 min at 4 ℃ and the supernatant was collected for physiological index assay. The activity of POD was determined spectrophotometrically at 470 nm using guaiacol as a substrate. The experiment was performed in 200 mM phosphate buffer (pH 6.0), 50 mM guaiacol, and 2% H₂O₂, and 2 µl of enzyme extract was added. The data was recorded after adding 2.0 ml 20% chloroacetic acid. All the above procedures of enzyme extraction were carried out at 0-4 ℃. The amount of enzyme required to cause 50% inhibition of nitro blue tetrazolium (NBT) reduction was considered as one unit of SOD activity. The SOD was measured at 560 nm by an ultraviolet spectrophotometer. The absorbance was measured using a UV-2550 UV–vis spectrophotometer (SHIMADZU). Three biological replications were performed. The enzyme assays were performed in three biological replicates. Proline was extracted and quantified as recommended by Zhao et al(Zhao et al. 2009).

Statistical analysis

The experiments were conducted with three biological replicates, and each replicate represents at least 12 plants. Each graphical plot represents the results from three repeats, and the values are displayed as the mean ± SD. Statistical significance was determined using Student’s t-tests, and P values <0.05 were considered statistically significant.

Identification and classification of AQP genes in Gossypium species

HMMER was used to identify AQP genes in Gossypium, and MIP domain (Pfam accessions, PF00230) as the model. The remaining sequences were checked for the existence of MIP domains using CDD (Conserved Domains Database). A total of 54, 56, and 111 candidate AQP genes were predicted in G. arboreum, G. raimondii, andG. hirsutum, respectively (Table S3). The number of AQPs in genome of G. hirsutum was more than that in G. arboreum (A₂) and G. raimondii (D₅). According to the prediction, upland cotton was derived from the hybridization of two progenitors with the diploid genome, and relevant chromosomes were doubled in tetraploid species. The nomenclature of AQPs was guided by phylogenetic analysis and sequence similarity. Though the genome of G. raimondii is smaller than the genome of G. arboreum, the number of AQPs in these two diploid cotton species is quite equal.

To study the origin and evolution of cotton AQP genes, the distribution of AQPs in 34 plant species was further analyzed (Fig. S1A-C). The XIP subfamily was absent in monocots and widely present in dicotyledons except for Brassica (Fig. S1B). The least amount of AQP was identified in Physcomitrella Patens (22) and Selaginella moellendorffii (18) (Fig. S1C). In monocots and eudicots which have experienced the recent genome-wide polyploidy, such as Zea mays, Gossypium, Brassica rapa, Brassica oleracea, Glycine max, Populus trichocarpa, Populus trichocarpa, and Solanum lycopersicum, the number of AQPs was significantly more than that of other species (Fig. S1C). In the allotetraploid species G. hirsutum and Brassica napus, the number of AQPs is almost equal to the sum of the two diploid ancestor species. The relationship between the number of AQP genes and the genome was identified. The results revealed that no significant correlation was identified between the number of AQPs and genome size (Fig. S1D). The number of AQP genes was positively correlated with the total number of genes in eudicots (Fig. S1E).

Phylogenetic analysis of AQP gene family

To query the evolution of AQP genes among three cotton species (G. raimondii, G. arboreum, and G. hirsutum), A. thaliana, Oryza sativa, and Phaseolus vulgaris, a Maximum Likelihood (ML) tree was constructed based on the amino acid sequences of AQPs using MEGA 7.0 (Fig. 1). The clustering data clearly showed that no AQP sequences of Arabidopsis and O. sativa were clustered into the clade of XIPs. Besides, the phylogenetic pattern of cotton AQPs was similarly to those of P. vulgaris, which clustered into five distinct subfamilies including PIPs, TIPs, NIPs, SIPs and XIPs. Compared with the other three species, AQPs from the three cotton species had a higher relative coefficient, which suggested that there is a closer relationship among the three cotton species. (Fig. 1). All the aquaporins of G. hirsutum were classified into five subfamilies consisting of 48 PIPs, 26 TIPs, 20 NIPs, 11 SIPs, and 6 XIPs (Fig. 1, Fig. 2A). The PIP subfamily which harbors 48 members was the biggest and divided into PIP1 (20 members) and PIP2 (28 members). The TIP subfamily was divided into five groups (TIP1 to TIP5), with 14 members in TIP1, six in TIP2, one in TIP3, three in TIP4, and two in TIP5. The NIP subfamily was divided into seven groups, six members in NIP1, four members in NIP7, and two in either NIP2, 3, 4, 5, and 6. The SIP subfamily was divided into SIP1 (nine members) and SIP2 (one member). Only one XIP group was identified, with six members. Three AQPs (GhPIP2,9_A, GhNIP5,1_A, GhSIP2,1_A) specifically existed in G. hirsutum, and two existed in G. arboreum (GaSIP1,4, GaSIP1,6) (Fig. 1, Table S4). The result suggesting AQPs in Gossypium are usually conserved, but the tetraploid cotton G. hirsutum had produced some specific AQP genes during the evolution and gene family duplication.

Basic characteristics and sequence analysis of AQPs

Bioinformatics analysis revealed that MW of GhAQPs ranged from 18.56 kDa to 82.36 kDa with pI ranged from 4.59 to 10.15. Comprehensive analysis of these five subfamilies indicated most members of GhPIPs were alkaline, with MW of approximately 30 kDa. However, the TIP subfamily members showed lower molecular weight. The MW of SIPs were smaller (<27 kDa) than the other four subfamilies with the exception GhSIP1,2_D (Kayum et al. 2017). The length of the aquaporins from the G. arboreum varied from 173 residues to 366 residues, with a median of 269 AAs per protein. The length of the aquaporins from the G. raimondii varied from 209 residues to 336 residues, with a median of 272 AAs per protein. The length of the aquaporins from the G. hirsutum varied from 181 residues to 759 residues, with a median of 275 AAs each protein. The predicted molecular weights and pI values f AQPs from the two diploid cotton species were within the ranges of 25.84-34.57 kDa and 4.8-10.15, respectively (Table S3).

Six TMDs (TM1-TM6), two NPA (Asn-Pro-Ala) motifs, and the ar/R filter were essential for the subjectivity of AQPs (Kaldenhoff and Fischer 2006). To detect the conservatism of AQPs, a sequence analysis of 111 putative GhAQPs was performed. Table S3 summarizes NPA motifs, the aromatic/Arg (ar/R) selectivity filter (H2, H5, LE1, and LE2), subcellular localization for the AQP protein sequences of Gossypium. Taking GhAQPs as an example, six TMDs (transmembrane domains) were identified in most of GhAQPs (70 of 111, 63.1%), except for 24 AQPs having five, two AQPs having four, two AQPs having three and 12 AQPs having seven TMDs, respectively.

NPA motifs showed the typical Asp-Pro-Ala residues in PIP and TIP subfamilies. In NIP subfamily, most of the members showed NPA motifs, but there are exceptions. For example, one GhNIP (GhNIP2,1_A) showed that proline (P) is replaced by a serine residue (S) in the first NPA motif, four GhNIPs (GhNIP5,1_A, GhNIP5,1_D, GhNIP6,1_A, GhNIP6,1_D), two GaNIPs (GaNIP6,1, GaNIP7,1) and two GrNIPs (GrNIP5,1, GrNIP6,1), showed that alanine (A) was altered by serine (S) or threonine (T) in the first NPA motif, the alanine (A) was exchanged by valine (V) in the second NPA motif. Members of SIP subfamily encoded motifs with a variable third residue in which alanine (A) was replaced by threonine (T) or leucine (L). In XIP subfamily, most of the members showed various in the third residues of the first NPA motif, in which alanine (A) was replaced by isoleucine (I) or valine (V). Especially, three members of aquaporins (GhTIP4,1_A, GhNIP7,2_D and GhXIP2,1_A) having only one NPA motif.

The ar/R selectivity filter showed various forms and family-specific sequences compared to the NPA motifs. In the PIP subfamily, the ar/R position showed conserved residues, phenylalanine (F) in H2, histidine (H) in H5, threonine (T) in LE1, and arginine (R) in LE2 (Table S3). In the TIP subfamily, the ar/R is formed by the motif of histidine/asparagine in H2, motifs of isoleucine/valine in H5, aspartate/serine in LE1, and valine/arginine/leucine in LE2. In the NIP subfamily, the ar/R position is formed by the motif of tryptophan/alanine/glycine in H2, valine/isoleucine/serine in H5, threonine/serine in LE1, and arginine in LE2. The SIP subfamily showed motifs of valine/phenylalanine/isoleucine in H2, valine/histidine in H5, threonine in LE1, and asparagine/serine/phenylalanine in LE2. The XIP subfamily showed motif of phenylalanine/valine/leucine/isoleucine in H2, valine in H5, glycine/alanine in LE1, and arginine in LE2.

Subcellular localization of GhAQPs

Subcellular localization is important for understanding the function of genes. Based on the subcellular localization prediction on the WolF PSORT tool, most PIPs are predicted to localize on the plasma membrane. TIPs were predicted to localize on the vacuolar membrane, plasma membrane as well as in cytoplasm. NIPs, SIPs, and XIPs were predicted to localize on the vacuolar membrane and plasma membrane. To determine the subcellular localization of GhAQP proteins, a C-terminal GFP fusion with GhAQPs driven by 35S promoter was constructed and the free GFP vector was used as a positive control (Fig. 5). Transient expression in tobacco leaves was performed by agroinfiltration. The GhPIP1,2, GhPIP2,3 and GhNIP5,1 were detected in the plasma membrane, GhTIP1,1 and GhTIP2,1 were detected in the vacuole membrane, while the GhSIP1,3, GhXIP1,1 and 35S-GFP signals were detected in both the nucleus and plasma membrane. It’s indicated that the subcellular localization of AQPs was consistent with the results predicted in WoLFPSORT.

Gene structure and motif analysis of AQPs in Gossypium

The exon-intron structures play crucial roles in plant evolution. To validate if the exon-intron structures influence GhAQPs evolution in cotton, we employed an online tool GSDS (Gene Structure Display Server 2.0 (gao-lab.org)) (Fig. S3). Most GhPIP subfamily members have four exons except for six members (GhPIP2,6_D, GhPIP1,2_A) which have five and ten exons, respectively. In the TIP subfamily, most members showed three exons. For an instance, GhTIP1,3_A, GhTIP1,3_D, GhTIP1,1_A, GhTIP1,1_D, GhTIP1,2_A, GhTIP1,2_D had three exons, but GhTIP4,1_D, GhTIP2,3_D had four exons. In the NIP subfamily, genes have five exons except for GhNIP5,1_A, GhNIP6,1_A, GhNIP7,1_A, GhNIP7,1_D. In the SIP subfamily, most of the genes had three exons, while GhSIP1,2_D had 12 exons and GhSIP1,2_A, GhSIP1,3_A, GhSIP1,4_D, GhSIP1,5_A, GhSIP1,5_D had only one exon. In the XIP subfamily, most of the members had three exons but GhXIP2,1_D just had one exon. The number of exons of 56 GrAQPs varied from 1 to 5 which was similar to 54 GaAQPs. In detail, most PIPs (20 GaPIPs and 20 GrPIPs) had four exons, most TIPs (10 GaTIPs and 10 GrTIPs) had three exons, most NIPs (8 GaNIPs and 8 GrNIPs) had five exons, most SIPs (3 GaSIPs and 3 GrSIPs) and XIPs (2 GaXIPs and 2 GrXIPs) had three exons. Together, these results indicated that the gene structure in each GhAQPs subfamily is conserved in Gossypium.

To investigate the functional roles of aquaporins in upland cotton, ten motifs among the 221 AQP proteins were screened by default parameters in MEME online tools (Fig. S4). Details of the ten motifs can be found in Fig. S5. Out of the ten screened motifs, eight were conserved in the PIP subfamily, four were detected in the SIP subfamily and six each were observed to be conserved in TIP, NIP, and XIP subfamilies respectively. We found that motif 1, 4, 5, and 6 appeared in all subfamilies. Motif 2, 3, 7, and 10 existed specifically in the PIP subfamily, motif 9 in the TIP subfamily, motif 8 in TIP and NIP subfamily. Moreover, we discovered that, three GhPIPs (GhPIP1,1_A/D GhPIP1,2_A/D, GhPIP1,3_A/D) lack motif 7, one (GhPIP2,13_A/D) deficient of motif 8 and one (GhPIP2,6_D) absent of motif 3, respectively. We found in Gossypium that, the structure and properties of GhAQPs in each subfamily were conserved against evolution, however, they vary between subfamilies.

Chromosomal location and duplication of AQPs in G. hirsutum

Based on the complete genome sequence of cotton, we deployed MapChart software to map the AQP genes to the chromosomes (Fig. 2). From the mapping analysis, we discovered that AQP genes are unevenly distributed on the chromosomes with the number of AQP genes on each chromosome varying from one to nine. In G. arboreum, AQP genes were found in 12 chromosomes and one scaffold except in chromosome A12. Chromosome A10 contained eight of the GaAQPs genes and was considered as having the largest number of chromosomes. In G. raimondii, AQP genes were found to be distributed on 13 chromosomes and a maximum number of nine predicted GrAQP genes were present on chromosome D11. The identified GhAQP genes were distributed randomly on 25 chromosomes and nine unmapped scaffolds. We also found that both chromosomes A10 and D10 contained the maximum number of eight AQP genes in G. hirsutum as showed in Fig. 2. There were 48 AQPs located on 12 At chromosomes, 54 AQPs on 13 Dt chromosomes, and nine located on each of the scaffolds. The number and location of GhAQPs in At sub-genome were similar to Dt sub-genome (Table S3), indicating the hybridization event by A and D diploid species. The majority of AQP genes were located on the proximate or the distal ends of the chromosomes. The physical locations of the AQP genes in the genome of Gossypium reflected the diversity and complexity of this gene family.

Genome duplication events existed in the process of plant evolution, which may result in the expansion of gene families. However, in G. hirsutum, the numbers and locations of duplicated genes were highly symmetric (Fig. 2, Table S5). A total of 115 duplicated gene pairs of AQPs that shared relatively high sequence similarities (aa identity>90%) were identified in Gossypium, indicating that AQPs were duplicated frequently. Among them, 10, 13, and 92 duplicated gene pairs were found in the genome of G. arboreum, G. raimondii, and G. hirsutum, respectively.

In G. arboreum, G. raimondii, and G. hirsutum 9, 9, and 84 gene pairs were confirmed to be segmentally duplicated genes, suggesting that segmental duplication played a crucial role in the expansion of the AQP gene family in allotetraploid cotton species G. hirsutum (Fig. S2, Table S5). Duplicated genes could undergo pseudogenization, sub-functionalization, or neo-functionalization during the progress of evolution (Cui et al. 2017). To further explore the different selective constraints on duplicated GhAQPs, the Ka/Ks ratio for 115 pairs of duplicated genes were performed (Table S5). All the values of Ka/Ks between gene pairs were less than 1, suggesting that, AQPs genes undergone strong purifying selection pressure after segmental duplication and WGD (whole genome duplication).

Expression pattern of GhAQPs under salt and osmotic stress

We found that, the expression patterns of homologous GhAQP genes on At and Dt sub-genomes were similar in salt or osmotic stress. (Fig. 4). Most GhAQPs (genes in the red box) belonging to the PIP subfamily were induced rapidly after 200 mM NaCl treatment and continued to be up-regulated until 3 h after treatment. A few GhAQPs (genes in the blue box) were induced at 3 h after salt stress and were continuously up-regulated until 12 h. Only three gene pairs (GhPIP1,9_A/D, GhPIP2,1_A/D, GhSIP1,2_A/D) were continuously up-regulated after salt stress. The expression pattern of most genes under osmotic stress and salt stress were consistent, except for GhPIP1,2_A, GhPIP1,2_A/D.

To validate the expression pattern of GhPIPs identified in upland cotton in response to salt or osmotic stress, we performed qRT-PCR (Fig. 5A). For easy description, we used GhPIP to represent the GhPIP_A/D gene pair. Among the 21 gene pairs, most of GhPIP genes were significantly induced after 150 mM NaCl treatment, while nine genes (GhPIP1,6, GhPIP1,11, GhPIP2,1, GhPIP2,11,GhPIP1,2, GhPIP1,4, GhPIP2,8, GhPIP2,10, GhPIP2,13) were not detectable. A total of ten GhPIP gene pairs (GhPIP1,3, GhPIP1,7, GhPIP1,9, GhPIP1,10, GhPIP2,2, GhPIP2,3, GhPIP2,5, GhPIP2,6, GhPIP2,7, GhPIP2,9) were up-regulated after salt stress. Among the ten up-regulated, five GhPIP genes (GhPIP1,10, GhPIP2,2, GhPIP2,3, GhPIP2,5, GhPIP2,7) showed the highest expression at 3 h, which is consistent with the RNA-seq data. Six GhPIP gene pairs (GhPIP1,1, GhPIP 1,5, GhPIP 1,8, GhPIP 2,4, GhPIP 2,12 and GhPIP 2,14) were alternately up- and down-regulated throughout the treatment time courses, however, these genes showed a radical up-regulation at 48 h. Three genes (GhPIP2,4, GhPIP2,12 GhPIP2,14) were significantly down-regulated at 12 h, while GhPIP1,8 was down-regulated at 3 h. GhPIP1,1 showed less down-regulation at 1 h and remain unchanged to 12 h, but up-regulated sharply at 48 h. GhPIP1,5 gene remains down-regulated from 1 h to 3 h, after which it was up-regulated to 48 h.

We observed four genes (GhPIP1,2, GhPIP2,3, GhTIP1,1, GhTIP2,1) were up-regulated at early stages under drought stress, and to verify their expression levels, we performed qRT-PCR (Fig. 5B). The expression of these genes reached the peak value at 2 h after osmotic stress except for GhPIP1,2, suggesting that these genes might be involved in osmotic stress response. Together this result showed that GhPIPs genes may be involved in abiotic stress in cotton

Cis-regulatory elements of GhAQP genes

Cis-regulatory sequences are linear nucleotide fragments of non-coding DNA that exist in front of the promoter region. The cis-regulatory elements of promotor have various functions depending on their types, locations, and orientation. To expound the potential function of GhPIPs, 1,500 bp upstream sequences of GhPIPs were extracted and used to predict cis-elements in promoter regions against PlantCARE database. A total of 272 cis-regulatory elements were detected in GhPIPs and GhTIPs. Of which, 85% were core promotor elements or binding sites of DNA binding protein. These elements respond to development and stress response, we found that there are 53% of motifs involved in light response, 23% involved in plant hormone-responsive, 10% involved in other-stress responsive, and 14% were other motifs whose functions were not defined. These results demonstrated that GhPIPs and GhTIPs have multiple roles in cotton developmental processes, as well as the abiotic stress response. Here, we focus on five cis-regulatory elements responding to abiotic stresses, which included ARE, a cis-acting regulatory element essential for the anaerobic induction, LTR, cis-acting element involved in low-temperature responsiveness, MBS, MYB binding site involved in drought inducibility, HD-Zip 1, motif involved in differentiation of the palisade mesophyll cells, WUN-motif, wound-responsive element (Fig. S7). GhPIP genes possessed at least one stress-response-related cis-element. We discovered that most of the GhPIP genes had an ABRE element that participated in ABA-responsive signaling pathways in response to salt stress (Fig. S3).

Silencing of GhPIP2,7 decreased salt tolerance in cotton

All plants-silenced GhChlI showed typical photobleaching phenotype in newly developed leaves, which indicated that the VIGS system was applied successfully in GX100-2 (Fig. S8). We also examined the expression levels of three genes (GhPIP2,2, GhPIP2,3, GhPIP2,7) in gene-silenced plants by qRT-PCR. The gene expression was significantly decreased in gene-silenced plants than that in mock (plants transformed in empty vector) (Fig. S8).

To further evaluate the phenotype of the gene-silenced plants, we treated with 400 mM NaCl for 12 days in the pot experiment. We observed a significant decrease in the biomass and leaf area of plants under salt stress treatment than in normal conditions. The salt injury symptoms of GhPIP2,7-silenced plants were more severe than in GhPIP2,2- and GhPIP2,3-silenced plants (Fig. 6A). Similar results were observed in 2-week-old gene-silenced and mock plants when they were subjected to 400 mM NaCl treatment. However, leaves of gene-silenced plants showed different degrees of chlorosis after 400 mM NaCl treatment for four days (Fig. 6B). We observed a significant decrease in chlorophyll content of GhPIP2,7-silenced plants after 400 mM NaCl treatment than in GhPIP2,2- and GhPIP2,3-silenced plants (Fig. 6C). MDA (Malondialdehyde) is the product of peroxidation reaction, which indicates the degree of peroxidation of the cell membrane and the strength of the stress reaction. To further understand the effect of salt stress on gene-silenced plants treated in 400 mM NaCl, we tested the MDA content, POD, and SOD activities of the leaves at the three-leaf stage of cotton (Fig. 6D-F). The salt treatment (400 mM NaCl) was dramatically increased in MDA content and POD activities in leaves, while decreased the SOD activities except for GhPIP2,2-silenced plants. The MDA content was higher in gene-silenced plants than in mock, while the trend was reversed after treating with 400 mM NaCl except for GhPIP2,7-silenced plants (Fig. 6D). The POD activity of gene-silenced plants showed no significant difference compared to the mock before salt stress but decreased significantly in mock plants after 400 mM NaCl treatment for 12 days. The POD activity in the leaves of GhPIP2,7-silenced plants was extremely and significantly down-regulated than in mock (Fig. 6E). The SOD activity of GhPIPs-silenced plants showed significant or extremely significant differences compared with mock without salt treatment, among which GhPIP2,7-silenced plants showed extremely significance difference than mock plants (Fig. 6F). The SOD activity of GhPIP2,2-silenced plants was increased after treating with 400 mM salt stress, while decreased in GhPIP2,3- and GhPIP2,7-silenced plants (Fig. 6F). The results indicated that silencing of GhPIP2,7 significantly decreased resistance to salt stress in cotton.

Overexpression of GhTIP2,1 enhanced drought tolerance in Arabidopsis

To evaluate the function of GhTIP2,1 in response to salt and osmotic stresses, one-week-old GhTIP2,1 overexpressed lines (OE1 and OE2) and wild-type (WT) seedlings were transferred to 1/2 MS medium containing 15% PEG6000 and 150 mM NaCl, and root length were measured after one week. Under control conditions, both OE and WT seedlings grew normally and had similar root lengths. Root growth was inhibited in the two OE lines and WT after NaCl or PEG treatment, but the two OE lines were less sensitive to PEG than the WT (Figure 8A and B). Additionally, we generated the transformed Arabidopsis root cells transient expressing GhTIP2,1:GFP, and the signals in cells were detected in the vacuole (Fig. 8C). The expression pattern of GhTIP2,1 was determined by analyzing transgenic plants harboring the GhTIP2,1 promoter driving the expression of the GUS reporter gene. GUS activity was strongly expressed in cotyledons, rosette leaves, and roots in the control (Figure 8D). After 10% PEG6000 treatments and 200 mM NaCl, the GUS signal was weak in the cotyledons and rosette leaves compared with the control seedlings. GUS staining was inconspicuous in the leaves when treated with 20% PEG6000. These results indicated that GhTIP2,1 was highly expressed in roots, but down-regulated in leaves after salt stress and osmotic stress.

In addition, we further evaluate the role of GhTIP2,1 under salt and osmotic stresses, we analyze the antioxidant enzyme activity in transgenic plants and WT (Fig. 8E-G). To evaluate the role of GhTIP2,1 in the oxidative stress pathway, proline, MDA, and H₂O₂ contents was measured in GhTIP2,1 transgenic plants and WT. The results showed that transgenic plants accumulated less MDA and H₂O₂than in WT under both salt and osmotic stresses. Also, the proline content was significantly higher in transgenic plants compared to WT under salt and osmotic stresses. Together, these results showed that GhTIP2,1 transgenic plant enhanced osmotic tolerance compared to WT cotton.

To study the molecular mechanism by which GhTIP2,1 contributes to abiotic response, the expression pattern of stress-responsive genes was determined using qRT-PCR (Figure 8H-J). Under control conditions, the transcription levels of stress-responsive genes, including AtNHX, AtLEA, and AtP5CS, showed no significant difference among OE lines and WT. However, the transcription of these genes in WT and OE lines was induced after 200 mM NaCl treatment. We observed that the transcription levels of these genes were substantially higher in the OE lines than in WT, indicating that GhTIP2,1 increases the tolerance to salt and osmotic stresses by upregulating the expression of stress-responsive genes.

Aquaporin is a highly diversified family in plants. Recent research in plant genome sequencing project have enabled the identification and characterization of aquaporins in Arabidopsis (Johanson et al. 2001), S. lycopersicum (Reuscher et al. 2013), P. vulgaris (Ariani and Gepts 2015), M. esculenta (Putpeerawit et al. 2017), Z. mays (Chaumont et al. 2001), O. sativa (Sakurai et al. 2005), and banana (Hu et al. 2015). These researches could be helpful in the identification of aquaporin family in cotton species.

The release of three cotton genome data allowed the identification and characterization of the aquaporin family in this study. There are four genome versions of G. hirsutum acc. TM-1 was released from different organizations, and the difference among these genome versions was mainly concentrated in duplicate genes (Zhang et al. 2015; Hu et al. 2019; Chen et al. 2020). The availability of transcriptome research and gene annotation information of NBI_V1.1 which was completed before other versions in cottonFGD and CottonGen website, we analyzed these genes and their proteins by extracting and aligning their sequences in G. hirsutum acc. TM-1 (NBI_V1.1) (Zhang et al. 2015). In total, 226 putative AQPs were identified in three cotton species. Of which, 54, 56, and 111 AQP genes were predicted in G. arboreum, G. raimondii, and G. hirsutum, respectively. 111 full-length aquaporin-coding sequences were identified in G. hirsutum, of which 48, 26, 20, 11, and 6 members belonging to the PIP, TIP, NIP, SIP, and XIP subfamily. From these identified genes, the majority of members were existed as gene pairs in At and Dt sub-genome of G. hirsutum, while a few members-only existed in one of the sub-genomes such as GhPIP1;11 (Fig. 1).

As shown in Table S4 and Table S3, 111 putative AQP genes were predicted in G. hirsutum by HMMER search in our research, while 113 were identified in previous research (Li et al. 2019a). Two GhAQP genes which consist of two PIPs (GhPIP2;4b_Dt, GhPIP2;4d_Dt) appeared in Dt sub-genome were specifically presented in Li’s research. We found that three AQP genes (GhPIP2;9_A, GhNIP5;1_A and GhSIP2;1_A) were specifically present in G. hirsutum, and two SIP genes were specifically found in G. arboretum in our study, while one (GaNIP7;1b) and two AQP genes (GrPIP2;7d, GrPIP2;8) were predicted specifically in A₂ and D₅ genome respectively which is consistence with Li’s report.

Structural conservation of AQPs in three Gossypium species

PIP subfamily was divided into PIP1 and PIP2 subgroups according to their N- and C-terminal motifs, which have different water transport activities. TIPs were divided into five groups (TIP1 to TIP5). SIPs were divided into two subgroups and NIPs divided into seven subgroups which were consistent with the previous research in cassava (Putpeerawit et al. 2017). The number of XIPs was about twice as many other diploids such as Brassica rapa (Kayum et al. 2017) and S. lycopersicum (Reuscher et al. 2013). The results on phylogeny and gene structure in upland cotton were similar to that of S. lycopersicum, P. vulgaris, and other species (Reuscher et al. 2013; Ariani and Gepts 2015). Our study revealed that the gene structure of AQPs is conserved in each subfamily in Gossypium, which provided us with additional proof to support the classification results.

AQPs have typical conserved NPA motifs and ar/R selectivity filter features, which are indispensable in determining the transport channel specificity (Maurel et al. 2015). All the members of the PIP, NIP, and SIP subfamily and most members of the TIP subfamily contained the same ar/R selectivity filter. PIPs showed typical NPA motifs and highly conserved ar/R selectivity filter (F-H-T-R), which configurate typical for water-transporting. These two motifs are highly conserved in PIPs of A. thaliana (Johanson et al. 2001), Z. mays (Chaumont et al. 2001), S. lycopersicum (Reuscher et al. 2013), and B. rapa (Kayum et al. 2017). TIPs exhibited four different forms of ar/R selectivity filter such as GhTIP1;1 (H-I-D-V), GhTIP2;2 (H-I-S-R), GhTIP3;1 (H-I-D-R) and GhTIP5;1 (N-V-S-L), which provide evidence for the variability of TIP subfamily (Sun et al. 2016). The NIP subfamily was quite divergent in NPA motifs and ar/R selectivity filter compared to other subfamilies in upland cotton suggesting that the substrates for transport were diverse (Perez et al. 2014). This finding suggested, that the domain of the PIP subfamily was more conserved than other subfamilies. Results from our subcellular localization predictions (Fig. 5), the majority members of PIP, SIP, and XIP subfamilies were predicted to be positioned on the plasma membrane in Gossypium, suggesting that they may regulate osmotic potential and water flow across this essential plant sub-cellular compartment. TIPs were mainly located in vacuolar membrane, suggesting that the TIPs may regular cellular osmosis and water homeostasis in cotton. SIPs had the smallest MW (< 27 kDa) in the five subfamilies except for GhSIP1;2_D (Table S3). The second NPA motif of SIPs was more conserved than the first in all full-length members. The ar/R filter in members of different subfamily was quite divergent, indicating their divergence solute permeability. However, the physiological role and localization of AQPs data in the public database are still limited.

Most of the AQPs included six integral TMDs in Gossypium, but there are exceptions, for example, GaPIP1;5, GrPIP1;5 and GhPIP1;5_A/D have five TMDs which also confirmed the earlier reports on AQP in other species such as B. rapa (Perez et al. 2014).

The expansion and duplication of AQPs in Gossypium

In this study, we analyzed the AQPs of G. hirsutum, G. raimondii, G. arboreum, and other 34 plant species and found that, the number of AQPs was consistent with the total gene of eudicots genomes, but it does not apply to monocots (Fig. S1). Before the formation of angiosperms, all plant genomes experienced two whole-genome replication events in which eudicots experienced a genome triploid, and monocots experienced a genome replication event (Wu et al. 2020).

Upland cotton, a natural allopolyploid, could be an excellent plant material to explore the mechanism of genome evolution and polyploidy formation. Gene duplication is an important mechanism for increasing genetic variability and creating novel genes in plants (Moore and Purugganan 2003). Previous analysis on BCCP and PLC gene evolution in Gossypium revealed that the duplicated genes evolved independently after polyploidy formation (Cui et al. 2017; Zhang et al. 2018). To further understand the duplication events, we investigated the GhAQP gene expansion mechanism. A total of 115 duplicated gene pairs were identified and most of those were distributed on different chromosomes (Fig. 2; Fig. S2; Table S5). The result demonstrated that the expansion of GhAQP genes was mainly caused by segmental duplication. The number of aquaporin coding genes in G. hirsutum was approximately twice more than the sum of G. raimondii and G. arboreum, according to the WGD (whole genome duplication) event in cotton evolution (Fig. 2, Table S5). In this study, we observed that the AQP genes in the A genome and At sub-genome had common ancestors, as well as in the D genome and Dt sub-genome, which indicated that AQP genes were highly conserved during cotton evolution. During the long history of plant evolution, genes have been exposed to different selective pressures, including positive selection, negative selection, and purifying selection (Flagel and Wendel 2009). The average Ka/Ks ratio of 115 GhAQP gene pairs was less than 1 which indicated that GhAQP genes experienced purifying selection during evolution (Table S5).

Silencing of GhPIP2;7 decreased the salt tolerance in upland cotton

Most of the homologous genes on At and Dt sub-genomes showed the same expression pattern under salt or drought stress, indicating the conserved function of GhAQP in response to abiotic stress (Fig. 4). Salt and drought stresses are major abiotic threats to plants that affect plant growth and reduce crop yield. A commonly observed plant response to salt stress is to accumulate ROS. Excess salt may become cytotoxic to the plant leading to cell membrane destruction (Zhu 2001). PIP genes play an important role in conferring abiotic stress tolerance in plants, including drought, cold, and salt (Lu et al. 2018; Wang et al. 2019). Previous reports had demonstrated that PIP genes play a vital role in response to salt stress (Table S6) and could actively regulate root and leaf hydraulics in plants (Li et al. 2014b). On the other hand, the structures of the PIP subfamily were highly conserved, indicating their probable involvement in similar biological functions in response to abiotic stress.

In our study, most of GhPIP genes or gene pairs showed transcriptional changes when exposed to salt stress (Fig. 4) which triggered our interest to investigate the expression patterns of PIP genes responsiveness to salt stress G. hirsutum leaves via qRT-PCR methods (Fig. 5). A total of ten GhPIP genes (GhPIP1;3, GhPIP1;7, GhPIP1;9, GhPIP1;10, GhPIP2;2, GhPIP2;3, GhPIP2;5, GhPIP2;6, GhPIP2;7, GhPIP2;9) were significantly induced (Log₂-based value > 1) after 150 mM NaCl treatment, suggesting that, GhPIP genes response to salt stress extensively and have potential function for improving cotton resistance to salt stress. These results were consistent with previous research which found that the expression of BvPIP2;2 was up-regulated in response to high salt stress (Skorupa-Kłaput et al. 2015). Compare with the PIP gene, the expression pattern in the root of upland cotton in the previous report (Li et al. 2019a), we found that most GhPIP genes showed similar expression patterns between At and Dt sub-genome. The corresponding gene names in two reports above are shown in Table S8. In our study, the expression of most GhPIPs was increasing at early stages after salt stress, for instance, GhPIP1;10, GhPIP2;5 and GhPIP2;7 showed the high expression after salt stress 3 h, GhPIP1;8 reached the high level at 1 h, and GhPIP2;9 was reached the peak at 12 h. (Fig. 6). The expression of GhPIPs increased rapidly under salt stress from 12 h to 24 h and reached a high expression level at 24 h except for GhPIP1;4a_At (Dt), which reached its expression peak at 48 h which explained the mechanism of leaves wilting in a short period after salt stress (Li et al. 2019a).

GhPIP2;2, GhPIP2;3, GhPIP2;7 was significantly up-regulated in response to salt stress, which was consistent with the results in Beta vulgaris (Skorupa-Kłaput et al. 2015). AtPIP2;4, AtPIP2;5 exhibited up-regulated expression to salt stress (Feng et al. 2018). Several researches showed that PIP subfamily members participated in salt stress response and our over-expression plant results (Table S7) confirmed the earlier reports. PIP2;7, which was initially referred to as Salt-Induced MIP (SIMIP) was reported to be strongly up-regulated by 150 mM NaCl treatment in two-week-old Arabidopsis seedlings (Jang et al. 2004). MsPIP2;2 confers salt tolerance by regulating antioxidant defense system-mediated ROS (reactive oxygen species) scavenging, K⁺/Na⁺ homeostasis, and stress-responsive gene expression in Arabidopsis (Li et al. 2019b). Taken together, salt stress stimuli resulted in a wide variety of PIP gene expression patterns. There was no difference in phenotype between GhPIPs-silenced plants and control under normal conditions (Fig. 6). Compared with the mock plants, the leaves of GhPIP2;7-silenced plants exhibited necrosis after salt stress including yellowing, and slight wilting (Fig. 8A-B). Also, 12 days after salt treatment, the MDA and SOD activities of the GhPIPs-silenced plants significantly increased compared to the control plants (Fig. 8D-F). POD activity and MDA content of the GhPIP2;3- and GhPIP2;7-silenced plants were increased significantly, of which GhPIP2;7-silenced plants changed more dramatically (Fig. 8D-F). We found that the HD-Zip 1 element, involved in the differentiation of palisade mesophyll cells, which existed in GhPIP1;9_A/D and GhPIP2;7_A/D. It was reported that PIP2;1 contributes to ABA-triggering stomatal closure through OST1-mediated phosphorylation (Grondin et al. 2015). Combined with the cis-elements in promoter and expression profile of GhAQPs under salt stress, GhPIP2;7 may play a positive regulatory role in response to salt stress, which affecting water transport by controlling mesophyll expansion. However, the functional roles of PIPs in response to salt tolerance in cotton still need further research.

GhTIP2;1 increases tolerance to osmotic stress by accumulating more proline and improving the Na⁺ efflux

Four genes were selected for further analysis to validate their functions involved in drought stress response using the VIGS experiment. We found that silencing of GhTIP1;1 and GhTIP2;1 resulted in wilting and yellowing of the whole plant under drought stress (Fig. 7A). The RWC in root and leaves was reduced in TRV:GhTIP1;1 and TRV:GhTIP2;1 plants under drought stress (Fig. 7C). GhTIP2;1 may be the key gene involved in drought stress response, and to further validate its roles, we over-expressed it in Arabidopsis thaliana under osmotic stress. In this study, the expression pattern of GhTIP2;1 was determined by analyzing transgenic plants harboring the GhTIP2;1 promoter driving the expression of the GUS reporter gene by Agrobacterium tumefaciens transformation. GhTIP2;1 over-expression lines grow significantly better than WT in Arabidopsis under drought stress (Fig. 8A).

Proline is an osmolyte that plays an important role in oxidative stress responses. The accumulation of H₂O₂ in the plant cell can cause oxidative damage, while its lower concentration correlates with drought tolerance (Ullah et al. 2018). The MDA level under stress conditions is an indicator of ROS destructive effects (Sharma et al. 2012). More proline accumulation and less MDA and H₂O₂ contents in transgenic plants, suggesting overexpression of GhTIP2;1 reduced the sensitivity of Arabidopsis to drought stress. NHX is Na⁺/H⁺ antiporters that maintain cellular Na⁺/K⁺ and pH homeostasis (Long et al. 2020). The upregulation of ATP5CS mainly promotes the accumulation of proline (Trovato et al. 2018). To investigate the role of GhTIP2;1 involved in osmotic stress, we analyzed the expression of three stress-related genes (AtP5CS, AtNHX, and AtLEA). The results showed that AtP5CS, AtNHX, and AtLEA genes were up-regulated in Arabidopsis overexpressing GhTIP2;1 under osmotic stress. Therefore, GhTIP2;1 may enhance the osmotic tolerance by accumulating more proline and increasing the Na⁺ efflux.

AQP, aquaporin, CLCrV, cotton leaf crumple virus, GUS, Glucuronidase, kDa, kilodalton, MDA, malondialdehyde, MW, molecular weight, NIP, nodulin 26-like intrinsic protein, NPA, Asn-Pro-Ala motifs, pI, isoelectric point, PIP, plasma intrinsic protein, POD, peroxidase, RWC, relative water content, SIP, small basic intrinsic protein, SOD, superoxide dismutase, TIP, tonoplast intrinsic protein, TMD, transmembrane domains, VIGS, virus-induced gene silencing, XIP, uncategorized X intrinsic protein, qRT-PCR, quantitative real-time PCR.

Author Contribution Statement

AHG performed bench experiments, data analysis, and manuscript preparation. JFH performed part of experiments and data analysis. BL, XKM, and MZ participated in VIGS experiments. YS and YH attended the discussion. JPH, GYS, and BMT designed the experiments and provided a research platform. JPH and YH revised the manuscript. All authors approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 31530053).

Conflict of Interest

The authors declare that they have no conflict of interest.

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FigureS1.doc
Fig. S1 Plant AQPs and species evolution
FigureS2.doc
Fig. S2. Chromosomal locations of AQPs in G. hirsutum
FigureS3.doc
Fig. S3 Phylogenetic relationship, gene structure and motifs of AQPs in Gossypium
FigureS4.doc
Fig. S4 Identification of conserved motifs of AQP proteins in G. arboreum, G. raimondii, and G. hirsutum
FigureS5.doc
Fig. S5 Sequence information of ten motifs identified by MEME
FigureS6.doc
Fig. S6 Expression patterns of candidate genes under osmotic stress
FigureS7.doc
Fig. S7 Predicted cis-elements respond to abiotic stress in promoter regions of GhAQPs
FigureS8.doc
Fig. S8 Silencing efficiency and phenotype identification of GhPIP2,2-, GhPIP2,3-, GhPIP2,7- and GhChlI-silenced plants in cotton
TableS1.xls
Table S1 Primer sequences of candidate genes used in qRT-PCR
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Table S2 Primer sequences of candidate genes in vector construction in VIGS experiment and subcellular localization
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Table S3 Comprehensive analysis and feature list of aquaporins in Gossypium genome
TableS4.doc
Table S4 Classification of aquaporins in three Gossypium species
TableS5.xls
Table S5 The identity and Ka/Ks ratio of duplicated genes in Gossypium
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Table S4 Identification of putative cis-regulatory elements in the promoters of cotton PIP genes
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Table S7 Examples of PIPs involved in salt stress response
TableS8.xls
Table S8 Comparison of aquaporin gene names between published research and present research

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Conserved Aquaporins in Gossypium and Silencing of Ghpip2;7 and Ghtip2;1 Decreased Salt and Osmotic Stress Tolerances in Upland Cotton

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