Systematical Characterization of the Cotton Di19 Gene Family and the Role of GhDi19-3 and GhDi19-4 as Two Negative Regulators in Response to Salt Stress

Drought-induced 19 (Di19) protein is a Cys2/His2 (C2H2) type zinc-finger protein, which plays a crucial role in plant development and in response to abiotic stress. This study systematically investigated the characteristics of the GhDi19 gene family, including the member number, gene structure, chromosomal distribution, promoter cis-elements, and expression profiles. Transcriptomic analysis indicated that some GhDi19s were up-regulated under heat and salt stress. Particularly, two nuclear localized proteins, GhDi19-3 and GhDi19-4, were identified as being in potential salt stress responsive roles. GhDi19-3 and GhDi19-4 decreased sensitivity under salt stress through virus-induced gene silencing (VIGS), and showed significantly lower levels of H2O2, malondialdehyde (MDA), and peroxidase (POD) as well as significantly increased superoxide dismutase (SOD) activity. This suggested that their abilities were improved to effectively reduce the reactive oxygen species (ROS) damage. Furthermore, certain calcium signaling and abscisic acid (ABA)-responsive gene expression levels showed up- and down-regulation changes in target gene-silenced plants, suggesting that GhDi19-3 and GhDi19-4 were involved in calcium signaling and ABA signaling pathways in response to salt stress. In conclusion, GhDi19-3 and GhDi19-4, two negative transcription factors, were found to be responsive to salt stress through calcium signaling and ABA signaling pathways.


Introduction
Adverse abiotic environmental factors, including drought, soil salinity, and heat and cold during crop development, limit the production area of crops and adversely affect both crop productivity and quality [1]. Hence, investigating the mechanism of plants adaptation toward stress cues and strategies to cope with adverse environmental factors is critical for maintaining crop production and food security [2]. Plants have developed and formed various levels of stress response mechanism and physiological mechanisms as well as other adaptive strategies to deal with abiotic environmental threats and relay this information to trigger appropriate physiological and cellular responses. At present, there have been two types of proteins reported to participate in the regulatory process of stress conditions [3]. The first type is the functional proteins involved in the direct regulation of stress, including osmotic regulators, ion channel proteins, antioxidant protection enzymes, etc., and the second type is the regulatory proteins, including transcription factors, plant protein phosphatases, and other signaling proteins. Transcription factors, including TCP [4], NAC [5], and C2H2 type zinc-finger proteins [6,7], are identified in varied abiotic stress signals and hormone regulatory pathways. Di19 proteins involved in plant abiotic stress belong to a small zinc-finger protein transcription factor gene family in plants. Di19 proteins are comprised two unusual, conserved domains, the Di19_zinc-binding domain and the Di19_C terminal domain [8]. Di19 proteins have been reported in Arabidopsis [9], cotton [10], rice [8], soybean [11], maize [12], moso bamboo [13], and other species.
At present, Arabidopsis is the most indepth studied plant of the Di19 gene family [14]. Previous studies have revealed that there are seven AtDi19 genes (AtDi19-1-to AtDi19-7) in the model plant Arabidopsis, which could be induced to express under stress. For example, the expression of AtDi19-1 and AtDi19-3 is regulated under drought stress, while the expression of AtDi19-2 and AtDi19-4 is regulated by high salt stress [8,14]. A previous study showed that the Atdi19-1 mutant exhibited a hypersensitive phenotype under drought stress [15]. AtDi19-1 up-regulated the expression level of pathogenesis-related (PR1, PR2, and PR5) genes through binding to the TACA (A/G) T element in PR1, PR2, and PR5 gene promoters under drought stress [15]. While AtDi19-3 has also been able to bind to the TACA (A/G) T element and the loss of the AtDi19-3 function in plants has led to an enhanced resistance to drought, salinity, and ABA stress. Compared to the wild-type, Atdi19-3-overexpressed plants have been shown to be more sensitive to drought, salinity, and ABA [16]. Most AtDi19 gene family members have been shown to interact with and be phosphorylated by calcium signal-related protein kinases to perform their functions [8]. In vitro, AtDi19-1 has interacted with AtCPK11/CDPK2 [17], and its transactivation activity was enhanced by AtCPK11/CDPK2 [15]. AtDi19-2 can be strongly phosphorylated by AtCPK16 [18]. AtDi19-3 has interacted with and was phosphorylated by the calcineurin B-like interacting protein kinase 11 (CIPK11) protein, which partially mediated in drought stress response through the regulating of AtDi19-3 [19]. In addition, Di19-3 has been shown to interact with AtIAA14, which affects the lateral root development in Arabidopsis [20]. AtDi19-7 (also known as HRB1) expression level was mediated by light signal and was involved in the red-light response mediated by phytochrome B and the blue-light response mediated by cryptochrome [21]. AtDi19-7 protein also interacts with the F-box protein LKP2 in the light signaling pathway [22]. In short, Di19 protein has been demonstrated to have important functions in plant development and abiotic stress response.
Di19 proteins also play an unusual role in coping with adversity in important food and cash crops. Previous studies have shown that overexpression of the TaDi19A gene in Arabidopsis plant leads to the reduced tolerance to salt stress, ABA, and mannitol [23]. TaDi19A gene transcription also regulates ABA and SOS signaling networks in stress response [23]. OsDi19-3 and OsDi19-4 gene expression levels are induced by dehydration and salt stress in rice [8]. Overexpression of OsDi19-4 has enhanced the scavenging activity of reactive oxygen species (ROS) and has improved the ability of rice to resist drought stress, whereas, the OsDi19-4 knockout lines have been found to be less sensitive to ABA treatment [8,24]. Interaction between OsCDPK14 and OsDi19-4, was found to be responsible for the phosphorylation of OsDi19-4, which was further improved after ABA treatment [24]. Further, OsDi19-4 was shown to directly bind to two ABA-responsive gene promoters, namely OsASPG1 and OsNAC18, and to participate in their expression [24]. This suggested that OsDi19-4 functions downstream of OsCDPK14 to positively promote ABA responses through regulating ABA-responsive gene expression in rice [24]. Additionally, ABAinduced OsDi19-1, together with OsSCP46 and other unknown proteins, function as a protein complex in the proteolysis during seed development [25]. ZmDi19-1 expression is also induced by various abiotic stresses in maize and has been shown to recognize and bind to the TACA (A/G) T element [12]. Ectopic expression of ZmDi19-1 in Arabidopsis affected downstream stress-related gene expression and improved its salt tolerance [12]. Soybean GmDi19-5 interacts with GmLEA3.1 and the sensitivity to abiotic stress of its transgenic plants were improved, where GmDi19-5 acted as a negative regulator in abiotic stress response through ABA and SOS signaling pathways [11]. In addition, PheDi19-8 protein interacts with the PheCDPK22 protein and has also been shown to play a role in drought stress response in moso bamboo [13]. These results suggest that different Di19 proteins perform different functions when plants are responding to abiotic stress.
In addition to being an important natural fiber material for the textile industry, cotton is also a pioneer crop in the restoration of saline-alkali soil. A previous study has indicated that the GhDi19-1 and GhDi19-2 up-regulated expressions in cotton are induced by salt and drought stress [10]. Although ectopic expression of GhDi19-1 and GhDi19-2 in Arabidopsis have conferred high sensitivity to salt and ABA [10,26], the mechanisms underlying the involvement of GhDi19s in stress responses have remained unknown. Due to the pivotal roles of Di19 proteins in plant stress responses, the Di19 gene family in cotton and the functions and regulatory networks of Di19-3 and Di19-4 genes in abiotic stress are systematically investigated in this study. Our investigation provides a theoretical basis for understanding the regulatory mechanism of Di19s in cotton under salt stress and enriches the genetic resources for the breeding of stress resistance cotton.

Plant Materials and Growth Conditions
Upland cotton (Gossypium hirsutum cv. Zhongmiansuo100) seeds were soaked in warm sterile water for 12 h after removing the lint using sulfuric acid, and then were sown on the germination plate and incubated at 28 • C for 3 days. Seedlings were transferred to small pots of the nutrient soil mixed with vermiculite (the ratio of nutrient soil and vermiculite was 3:1 (V:V)) and were grown in the greenhouse (at 23 • C, 16 h light/8 h dark cycle) of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences (China, Anyang). Three-week-old tobacco (N. benthamiana) seedlings from the same growth conditions of 23 • C, 16 h light/8 h dark cycle were used in the subcellular localization analysis of target genes.
Using the BLASTP program, amino acid sequences of AtDi19s were used as a query sequence, and genes with the e-values less than 1e −5 were selected in the protein database of the three cotton species and other plant species as candidate genes. Interproscan 5 (http://www.ebi.ac.uk/interpro/, accessed on 17 December 2021) [30] and SMART (http://smart.embl.de/, accessed on 17 December 2021) online tools [31] were used, the Di19_zinc-binding domain (IPR008598) and the Di19_C terminal domain (IPR027935) were retrieved and used as the query sequences [9,10], and ultimately Di19 genes were identified in ten plant species. Sequence alignment was performed using DNAMAN while phylogenetic trees were generated using MEGA 7.0 [32].

Sequence Characteristics and Chromosome Distribution of Di19s
Molecular weights and theoretical isoelectric points (pIs) of Di19 proteins were analyzed using ExPASy (http://web.expasy.org/protparam/, accessed on 27 December 2021). Prediction of subcellular localization was conducted using the CELLO v.2.5 server [33]. Protein 3D structure of GhDi19-3 and GhDi19-4 was predicted by the SWISS-MODEL server (http://swissmodel.expasy.org, accessed on 16 February 2022) [34]. MapChart software [35] was used to map the Di19 genes and the cotton chromosomes and GSDS software was used to visualize the exons and introns of GhDi19s [36]. The PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 9 December 2021) was used to predict the cis-elements in the promoter regions (2000 bp) of Di19 genes. Conserved motif and domain were examined using the MEME Suite [37] and TBtools software [38].
The extraction of total RNA and the reverse transcription were prepared according to the previous methods [41]. qRT-PCR primers for GhDi19s and the associated networkrelated genes were shown in Table S1. GhUBQ7 was selected as an internal reference gene. qRT-PCR was run on a Bio-Rad 7500 fast fluorescence quantitative PCR platform following the operation protocol. Each treatment had three biological replicates, and the relative expression levels of the genes were analyzed using the 2 −∆∆Ct method [42].

Protein Association Network of GhDi19s
STRING software (https://string-db.org/, accessed on 24 February 2022) [41] was used to illustrate the association network of GhDi19 proteins based on the orthologs in Arabidopsis and G. raimondii with a high confidence parameter set at 0.7 and 0.4 thresholds, respectively.

Subcellular Localization of GhDi19-3 and GhDi19-4 in Tobacco
To determine the subcellular localization of GhDi19-3 and GhDi19-4, CDS of GhDi19-3 and GhDi19-4 were cloned into P-super1300 vector to generate C-terminally fused green fluorescent protein (GFP) constructs 35S:: GhDi19-3-GFP and 35S:: GhDi19-4-GFP, respectively, using a one-step cloning kit (Vazyme Biotech Ltd., Nanjing, China). Vector construction primers are listed in Table S1. These vectors were firstly transformed into the Agrobacterium tumefaciens strain GV3101 and were then infiltrated into the tobacco leaves. After two days, the green fluorescence of the leaf cells was detected under a confocal microscope (Carl Zeiss LSM710, Oberkochen, Germany).

VIGS Vector Construction and Treatments
The VIGS system was generated following the protocol of Shaban et al. [43]. The 213 bp and 396 bp sequence fragments of GhDi19-3 and GhDi19-4 were cloned from G. hirsutum cv. Zhongmiansuo 100, respectively, and were then cloned into the TRV: 00 plasmids, to generate the TRV: GhDi19-3 and TRV: GhDi19-4 vectors using the one-step cloning kit (Vazyme Biotech Ltd., Nanjing, China). TRV: GhDi19-3, TRV: GhDi19-4, and TRV1 constructs were transformed into the A. tumefaciens strain GV3101 by electroporation. Primers for vector construction were listed in Table S1. When GhCLA1 gene-silenced (positive control) plants showed the albinism phenotype, it indicated that the silencing mechanism had been successfully induced in the positive control. Then the VIGS efficiency of GhDi19-3 and GhDi19-4 was verified by qRT-PCR. TRV: 00, TRV: GhDi19-3, and TRV: GhDi19-4, plants were subjected to mock treatment (water) and 400 mM NaCl was used as salt stress treatments for up to ten days. The experiment was repeated three times, and forty plants were used for each treatment.

Detection and Analysis of Salt Stress-Related Physiological Parameters
Physiological parameters, including malondialdehyde (MDA) content, superoxide dismutase (SOD), peroxidase (POD), and H 2 O 2 activities, were detected in the control and GhDi19-3and GhDi19-4-silenced cotton plants under salt treatment. The H 2 O 2 content was estimated using the protocol of the H 2 O 2 quantification assay kit (Solarbio, Beijing, China). About 0.1 g of the leaf was ground and blended in a 1 mL lysate. Then the homogenate was centrifuged at 4 • C for 10 min at 8000× g. The collected supernatant was collected and then mixed with the same amount of H 2 O 2 detection reagent and then homogenized by vortexing. Absorbance was measured at 415 nm, and then using the standard curve as the reference to calculate the content of H 2 O 2 . The MDA content, POD, and SOD activities were quantified from 0.1 g cotton leaves using MDA, POD, and SOD assay kits from Solarbio Science and Technology Co., Ltd., Beijing, China. Tissue extracts were prepared according to the manufacturer's instructions in the relevant kits, as previously described [43].
General biochemical and physical characteristics of Di19 gene in cotton and other plant species are given in Table S2. The size of GhDi19 proteins ranged from 210 to 239 amino acids, with theoretical pIs ranges from 4.47 to 6.23, and molecular weights ranges from 23.86 to 27.02 kDa (Table S2). Analysis of subcellular localization predicted that 16 GhDi19s were localized in the nucleus (Table S2).

Phylogenetics, Sequence Structure, and Conserved Motif of the Di19s
To investigate the evolutionary relationships among Di19 proteins, 89 Di19 proteins from 11 plant species were analyzed and a phylogenetic tree was constructed ( Figure 1, Table S2). These proteins were divided into five different subfamilies, named as I, II, III, IV, and V ( Figure 1). In subgroups I, II and III, the Di19 protein from dicotyledonous plants was clustered, while the other two subgroups (IV and V) contained Di19 proteins from both dicotyledonous and monocotyledonous plants ( Figure 1). The Di19 proteins in subfamily III had the least number, with only six proteins, while the subgroup IV was the largest, with 31 Di19 proteins ( Figure 1). Furthermore, phylogenetic analysis showed that upland cotton had undergone a significant gene expansion due to the presence of almost more than double the number of Di19 genes compared with all other plant species except for G. max. These results indicated that the Di19 proteins were conserved during species evolution. from both dicotyledonous and monocotyledonous plants ( Figure 1). The Di19 proteins in subfamily III had the least number, with only six proteins, while the subgroup IV was the largest, with 31 Di19 proteins ( Figure 1). Furthermore, phylogenetic analysis showed that upland cotton had undergone a significant gene expansion due to the presence of almost more than double the number of Di19 genes compared with all other plant species except for G. max. These results indicated that the Di19 proteins were conserved during species evolution. To further investigate the sequences of GhDi19 genes, the exon and intron structure of GhDi19 genes ( Figure 2) was investigated. It was found that all 16 GhDi19 members shared similar structures with five exons and four introns ( Figure 2B). Based on the results of a motif analysis of GhDi19 proteins using MEME suite, a total of ten conserved motifs were identified. As expected, the GhDi19 proteins from the same subgroup had a similar conserved motif distribution pattern, while the pattern differed between different subgroups (Figure 2A,C). Subgroup V (GhDi19-9 and GhDi19-15) and subgroup IV (GhDi19-7 and GhDi19-13) contained the least motifs (only seven motifs), while subgroup II (GhDi19-5, GhDi19-8, GhDi19-11, and GhDi19-14) contained the most motifs (nine motifs) ( Figure 2C). In subgroup I, GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 had only one motif (Motif 10) less compared with GhDI19-6 and GhDi19-12, indicating that these conserved motifs may specify the unique functions of different subgroups of Di19. In general, these conserved structures and motifs reflected the phylogenetic tree analysis results. Further, conserved domains of GhDi19s were demonstrated by multiple To further investigate the sequences of GhDi19 genes, the exon and intron structure of GhDi19 genes (Figure 2) was investigated. It was found that all 16 GhDi19 members shared similar structures with five exons and four introns ( Figure 2B). Based on the results of a motif analysis of GhDi19 proteins using MEME suite, a total of ten conserved motifs were identified. As expected, the GhDi19 proteins from the same subgroup had a similar conserved motif distribution pattern, while the pattern differed between different subgroups (Figure 2A,C). Subgroup V (GhDi19-9 and GhDi19-15) and subgroup IV (GhDi19-7 and GhDi19-13) contained the least motifs (only seven motifs), while subgroup II (GhDi19-5, GhDi19-8, GhDi19-11, and GhDi19-14) contained the most motifs (nine motifs) ( Figure 2C). In subgroup I, GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 had only one motif (Motif 10) less compared with GhDI19-6 and GhDi19-12, indicating that these conserved motifs may specify the unique functions of different subgroups of Di19. In general, these conserved structures and motifs reflected the phylogenetic tree analysis results. Further, conserved domains of GhDi19s were demonstrated by multiple protein sequence alignment of GhDi19s. Almost all proteins contained the Di19_zinc-binding domain (IPR008598) and the Di19_C terminal domain (IPR027935) ( Figure 2D). The Di19_zinc-binding domain composed of two unusual strictly conserved Cys2/His2 zinc-finger domains ( Figure 2D). Additionally, all GhDi19 proteins had the peptide of nuclear localization signal (NLS) ( Figure 2D). GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 had the highest protein identities of 63.68%, 62.33%, 64.13%, and 61.43%, respectively, with AtDi19-3 among the seven AtDi19 genes. Further analysis of the 3D structure of those four GhDi19 proteins using the SWISS-MODEL [34] showed that they had highly similar structures to AtDi19-3 ( Figure 2E-H) suggesting potential function similarity.

Conserved Cis-Elements and Expression Profiles of GhDi19 Genes
Cis-elements related to plant growth and development, abiotic or biotic stress response, and phytohormone responses were found in this study (Figure 3). Among the cis-elements related to plant growth and development, the number of light-responsive elements were the majority, including Box-4, as well as certain cis-elements involved in zein metabolism regulation, cis-elements related to meristem expression, cis-elements involved in endosperm expression, and so on ( Figure 3, Table S3). Among the cis-elements related to phytohormone response, those related to ABA (such as ABRE and AAGAA-motif), auxin (such as AuxR-core, MYB, and TGA-element), gibberellins (such as GARE-motif and TATC-box), ethylene (such as ERE), MeJA (such as TGACG-motif

Conserved Cis-Elements and Expression Profiles of GhDi19 Genes
Cis-elements related to plant growth and development, abiotic or biotic stress response, and phytohormone responses were found in this study (Figure 3). Among the cis-elements related to plant growth and development, the number of light-responsive elements were the majority, including Box-4, as well as certain cis-elements involved in zein metabolism regulation, cis-elements related to meristem expression, cis-elements involved in endosperm expression, and so on (Figure 3, Table S3). Among the cis-elements related to phytohormone response, those related to ABA (such as ABRE and AAGAA-motif), auxin (such as AuxR-core, MYB, and TGA-element), gibberellins (such as GARE-motif and TATC-box), ethylene (such as ERE), MeJA (such as TGACG-motif and CGTCA-motif), and salicylic acid (such as TCA-element) were mainly included. Among them, the number of auxin-responsive elements (such as MYB) was the largest suggesting that GhDi19 genes may also have important functions in the auxin signal pathway. Further, the number of ARE, MYC, and STRE cis-elements were the largest of the abiotic stress-related cis-elements of GhDi19s (Table S3), indicating that certain GhDi19 genes participated in the stress response, which is consistent with previous studies [10,14,26]. and CGTCA-motif), and salicylic acid (such as TCA-element) were mainly included. Among them, the number of auxin-responsive elements (such as MYB) was the largest suggesting that GhDi19 genes may also have important functions in the auxin signal pathway. Further, the number of ARE, MYC, and STRE cis-elements were the largest of the abiotic stress-related cis-elements of GhDi19s (Table S3), indicating that certain GhDi19 genes participated in the stress response, which is consistent with previous studies [10,14,26]. To understand the role of cotton Di19 proteins under abiotic stress, the transcriptome data of 16 GhDi19s under different stress conditions, including hot, cold, NaCl, and polyethylene glycol (PEG), were analyzed ( Figure 3, Table S4). Compared to the control, the expression of GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 genes increased significantly under heat stress conditions, reaching the highest level after 3-12 h. Under cold stress conditions, expression levels of GhDi19-1, GhDi19-3, GhDi19-7, GhDi19-13, GhDi19-9, and GhDi19-15 were significantly increased compared to the control. These results showed that GhDi19s respond to temperature stress. Under salt stress conditions, the expression levels of GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 genes showed a drastic increase of expression at 1 h after salt stress and reduced afterwards. This suggested that these GhDi19 genes were related in response to salt stress. However, under drought treatments, most GhDi19s expression had no significant changes compared with the control. Based on these results, it was concluded that the GhDi19s were differentially regulated under abiotic stress (hot and salt stress) in cotton.
In addition, the transcriptome of GhDi19 genes were analyzed in different tissues ( Figure S2, Table S5). Most GhDi19 genes exhibited a constitutive expression pattern. GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 genes were mainly expressed in the ovules (0, 1, and 3 days post anthesis (DPA)) and the fiber tissue at the elongation stage (10, 20, and 25 DPA), while GhDi19-10 was predominantly expressed in the fiber tissue of 10 DPA. It was speculated that these GhDi19s were involved in the ovule's development and fiber elongation, especially the GhDi19-10 which possibly was a candidate gene for regulation of fiber elongation in cotton.
Based on the above results, we demonstrated that GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4, had important functions in cotton, either in response to stress conditions To understand the role of cotton Di19 proteins under abiotic stress, the transcriptome data of 16 GhDi19s under different stress conditions, including hot, cold, NaCl, and polyethylene glycol (PEG), were analyzed ( Figure 3, Table S4). Compared to the control, the expression of GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 genes increased significantly under heat stress conditions, reaching the highest level after 3-12 h. Under cold stress conditions, expression levels of GhDi19-1, GhDi19-3, GhDi19-7, GhDi19-13, GhDi19-9, and GhDi19-15 were significantly increased compared to the control. These results showed that GhDi19s respond to temperature stress. Under salt stress conditions, the expression levels of GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 genes showed a drastic increase of expression at 1 h after salt stress and reduced afterwards. This suggested that these GhDi19 genes were related in response to salt stress. However, under drought treatments, most GhDi19s expression had no significant changes compared with the control. Based on these results, it was concluded that the GhDi19s were differentially regulated under abiotic stress (hot and salt stress) in cotton.
In addition, the transcriptome of GhDi19 genes were analyzed in different tissues ( Figure S2, Table S5). Most GhDi19 genes exhibited a constitutive expression pattern. GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4 genes were mainly expressed in the ovules (0, 1, and 3 days post anthesis (DPA)) and the fiber tissue at the elongation stage (10, 20, and 25 DPA), while GhDi19-10 was predominantly expressed in the fiber tissue of 10 DPA. It was speculated that these GhDi19s were involved in the ovule's development and fiber elongation, especially the GhDi19-10 which possibly was a candidate gene for regulation of fiber elongation in cotton.
Based on the above results, we demonstrated that GhDi19-1, GhDi19-2, GhDi19-3, and GhDi19-4, had important functions in cotton, either in response to stress conditions or to regulate the development of cotton fiber (Figure 3 and Figure S2). However, the function of the two genes GhDi19-1 and GhDi19-2 has been relatively well documented [10,26]. Therefore, we focused on the other two, GhDi19-3 and GhDi19-4, which were used as candidate genes for further functional verification of their potential roles.

Protein Association Network
Based on the association network of Di19s, the functions of cotton Di19 genes were speculated upon (Figure 4). Overall, we found that the response to the abiotic stim-Antioxidants 2022, 11, 2225 9 of 18 ulus signaling pathway (NOG09218) in the network center ( Figure 4A) and other signaling pathways related to abiotic stress, including the stress-induced protein Di19, Cterminal (NOG54298) and Zn-finger protein (KOG3173) pathways [8,14], suggesting a certain connection between the Di19 members and the abiotic stress signaling pathway. Plant calmodulin-binding domain (NOG11332) and short calmodulin-binding motif with conserved IIe and Gln residues (NOG263047) association pathways [8,14,18,19] involved in response to abiotic stimuli were found in the association network, indicating that certain Di19 members were also regulated in the abiotic stress response through interaction with the CDPK proteins. Interestingly, through a multiple sequences search, it was found that GhDi19-3 and GhDi19-4 were associated with the CDPK proteins (Gorai.001G200300.1, Gorai.005G074300.1, Gorai.009G290200.1, and Gorai.012G045700.1) (Figure 4B), which were also believed to be special regulators of the plant hormone ABA signaling pathway and that CDPKs performed important functions in response to ABA through triggering downstream related factors [8,14,18,19,45]. Further, GhDi19-3 and GhDi19-4 associated with GhPP7 (Gorai.001G200300.1) (Figure 4B), were thought to be participate in plant photoreceptor regulation that regulated photomorphogenic development.

Subcellular Localization of GhDi19-3 and GhDi19-4 Proteins
Through the tobacco transient expression system, it was shown that the GhDi19-3 and GhDi19-4 proteins were exclusively localized in the nucleus, whereas the GFP signal of the control expression vector was found in both the nucleus and the plasma membrane ( Figure 5). This result together with the presence of the conserved NLS domain in GhDi19-3 and GhDi19-4 proteins ( Figure 2D) suggested that GhDi19-3 and GhDi19-4 were nuclear proteins.

Subcellular Localization of GhDi19-3 and GhDi19-4 Proteins
Through the tobacco transient expression system, it was shown that the GhDi19-3 and GhDi19-4 proteins were exclusively localized in the nucleus, whereas the GFP signal of the control expression vector was found in both the nucleus and the plasma mem-brane ( Figure 5). This result together with the presence of the conserved NLS domain in GhDi19-3 and GhDi19-4 proteins ( Figure 2D) suggested that GhDi19-3 and GhDi19-4 were nuclear proteins.

Silencing of GhDi19-3 and GhDi19-4 Increases the Tolerance of Cotton
Based on the GhDi19s promoter analysis and transcriptome dat ent abiotic stressors, it was believed that GhDi19s had a potential role Using the VIGS system, we transiently knocked down the expressio GhDi19-4 in TRV: GhDi19-3 and TRV: GhDi19-4, respectively. After te induction, the positive control (TRV: GhCLA1) plants displayed an ( Figure 6A). The transcript levels of GhDi19-3 and GhDi19-4 were f cantly decreased two weeks after VIGS ( Figure 6B). This suggested and GhDi19-4 were successfully silenced in cotton plants.

Silencing of GhDi19-3 and GhDi19-4 Increases the Tolerance of Cotton to Salt Stress
Based on the GhDi19s promoter analysis and transcriptome data analysis in different abiotic stressors, it was believed that GhDi19s had a potential role in stress responses. Using the VIGS system, we transiently knocked down the expression of GhDi19-3 and GhDi19-4 in TRV: GhDi19-3 and TRV: GhDi19-4, respectively. After ten days of the VIGS induction, the positive control (TRV: GhCLA1) plants displayed an albino phenotype ( Figure 6A). The transcript levels of GhDi19-3 and GhDi19-4 were found to be significantly decreased two weeks after VIGS ( Figure 6B). This suggested that both GhDi19-3 and GhDi19-4 were successfully silenced in cotton plants. Salt stress treatment was applied on the GhDi19-3and GhDi19-4-silenced and control plants to evaluate the functions of GhDi19-3 and GhDi19-4 in salt stress response. No phenotypic differences were observed between the control and target gene-silenced (TRV: GhDi19-3, TRV: GhDi19-4) plants in mock treatment after ten days. However, the plants of the knock down of GhDi19-3 and GhDi19-4 showed a salt-insensitive phenotype under salt stress, while the control showed mild plant wilting and leaf yellowing phenotypes ( Figure 6C). Plants accumulated excessive amounts of ROS (mainly O 2 − and H 2 O 2 ) in leaf cells when subjected to adverse environmental influences such as abiotic stress, causing oxidative damage to cells, disrupting cell membrane structure, and damaging plant cells and tissues. This ultimately affected plant metabolism and signal transduction [46]. MDA, POD, and SOD are known as the antioxidants that play important roles in stress responses and oxidative stress tolerance [47,48]. To investigate the regulation mechanism underlining the improved salt stress tolerance of the gene-silenced cotton plants, relevant physiological parameters, including H 2 O 2 , POD, MDA, and SOD contents, were analyzed in the target gene-silenced and control plants under different treatments ( Figure 6D-G). Compared to the mock treatment, H 2 O 2 contents in the leaves of the control plants were significantly induced under salt stress, while the H 2 O 2 levels in leaves of TRV: GhDi19-3 and TRV: GhDi19-4 plants were significantly lower compared to the control ( Figure 6D). Similar to the trend of H 2 O 2 , changes in leaves between mock treatment and salt treatment were also found in the comparison of POD activities. However, under mock and salt stress conditions, POD activities in the control plants were significantly higher than both genesilenced (TRV: GhDi19-3, TRV: GhDi19-4) plants ( Figure 6E). Under mock treatment, MDA levels in the gene-silenced (TRV: GhDi19-3, TRV: GhDi19-4) plants were significantly higher compared to the control. However, MDA levels increased after salt stress treatment, but were significantly lower compared to the control ( Figure 6F). SOD activities in the genesilenced (TRV: GhDi19-3, TRV: GhDi19-4) plants were higher compared to the control in both mock and salt stress treatment ( Figure 6G). Together the results suggest that the improved salt stress tolerance of the GhDi19-3 and GhDi19-4 gene-silenced plants could be caused by the enhanced ROS-scavenging activity.

Expression Profiling of Calcium Signaling-Related and ABA-Responsive Genes in Simulated Salt Stress Treatment of Control and GhDi19-3-and GhDi19-4-Silenced Cotton Plants
Simulated salt stress treatment of the control and GhDi19-3and GhDi19-4-silenced cotton plants revealed that the sensitivity to salt stress was decreased in GhDi19-3and GhDi19-4-silenced cotton plants. To further explore the regulation mechanism to explain the phenotype of genes involved in calcium signaling and ABA signaling pathways, and based on previous studies [8,10,14,15,18,19,45,49] and the protein association network results, calcium signaling related genes (GhCDPK2-1A, GhCDPK2-1D, GhCDPK2-2A, GhCDPK2-2D, GhCIPK11-A, and GhCIPK11-D) and ABA-responsive genes (GhABI5, GhRD29B-A, and GhRD29B-D) expression profiling was analyzed to monitor ABA and calcium signaling pathways in cotton.
Almost all genes were strongly induced in both the control and the GhDi19-3 and GhDi19-4 gene-silenced cotton plants under salt treatment after ten days (Figure 7). Transcript levels of four calcium-dependent protein kinase genes, including GhCDPK2-1A, GhCDPK2-1D, GhCDPK2-2A, and GhCDPK2-2D, were lower in GhDi19-3and GhDi19-4silenced cotton plants compared to the TRV: 00 plant under salt treatment. Under mock treatment, the expressions of GhCDPK2-1A and GhCDPK2-1D in the silenced plants were lower compared to the control, with a significant difference was observed only between GhDi19-3-silenced plants and the control. However, the expressions of GhCDPK2-2A and GhCDPK2-2D in the silenced cotton plants were higher compared to the control, where only GhCDPK2-2A expression showed a significant difference between GhDi19-4-silenced plants and the control. An exception was found in the expression of GhCIPK11-D, which had no significant difference between the GhDi19-4-silenced cotton plants and the control. The expressions of two CIPKs, GhCIPK11-A and GhCIPK11-D, in GhDi19-3and GhDi19-4-silenced cotton plants before and after salt stress treatment were substantially lower compared to the control. Similarly, three ABA-responsive genes, including GhABI5, GhRD29B-A, and GhRD29B-D, expression levels were also substantially lower in GhDi19-3and GhDi19-4-silenced cotton plants under salt treatment compared to the control. However, under mock treatment, it was surprising to observe that the mRNA levels of GhRD29B-A and GhRD29B-D were hardly detected between the control and GhDi19-3and GhDi19-4silenced cotton plants under mock treatment. Above results indicated that both GhDi19-3 and GhDi19-4, as two negative regulators, may participate in plant calcium signaling and ABA signaling pathways.
lenced plants were lower compared to the control, with a significant difference was observed only between GhDi19-3-silenced plants and the control. However, the expressions of GhCDPK2-2A and GhCDPK2-2D in the silenced cotton plants were higher compared to the control, where only GhCDPK2-2A expression showed a significant difference between GhDi19-4-silenced plants and the control. An exception was found in the expression of GhCIPK11-D, which had no significant difference between the GhDi19-4-silenced cotton plants and the control. The expressions of two CIPKs, GhCIPK11-A and GhCIPK11-D, in GhDi19-3-and GhDi19-4-silenced cotton plants before and after salt stress treatment were substantially lower compared to the control. Similarly, three ABA-responsive genes, including GhABI5, GhRD29B-A, and GhRD29B-D, expression levels were also substantially lower in GhDi19-3-and GhDi19-4-silenced cotton plants under salt treatment compared to the control. However, under mock treatment, it was surprising to observe that the mRNA levels of GhRD29B-A and GhRD29B-D were hardly detected between the control and GhDi19-3-and GhDi19-4-silenced cotton plants under mock treatment. Above results indicated that both GhDi19-3 and GhDi19-4, as two negative regulators, may participate in plant calcium signaling and ABA signaling pathways.

Discussion
Previous studies on the potential functions of Di19s in response to biotic or abiotic stress mainly focused on certain plant species [8,[10][11][12][13][14][15][16]24]. However, these previous investigations were not extended to cotton species. In this study, we found that the Di19

Discussion
Previous studies on the potential functions of Di19s in response to biotic or abiotic stress mainly focused on certain plant species [8,[10][11][12][13][14][15][16]24]. However, these previous investigations were not extended to cotton species. In this study, we found that the Di19 protein was conserved in plants. Sixteen GhDi19 proteins were identified in upland cotton and clustered into five subgroups. All GhDi19s had a similar sequence structure which composed of five exons and four introns. Furthermore, all GhDi19s contained two zincfinger domains and one NLS domain. Due to the presence of NLS, these Di19 proteins were localized in the nucleus. GhDi19-3 and GhDi19-4 genes encoding Di19 proteins were identified in cotton in addition to previously reported GhDi19-1 and GhDi19-2. Similar, to previously reported studies where GhDi19-1 and GhDi19-2 were found localized in the nucleus [10,26], subcellular localization of GhDi19-3 and GhDi19-4 suggested that both GhDi19 proteins were localized in the nucleus. These results laid the foundation to investigate the evolution of the Di19s in different species and further study the Di19 gene family functions, especially in the pioneering of growing cotton in saline-alkali land.
Soil salinity is a major abiotic stress around the world, which seriously affects plant growth and productivity [1,50]. As salt-tolerant crop, corresponding stress response mechanisms in cotton have been widely investigated [50][51][52]. The transcriptome data show that both GhDi19-3 and GhDi19-4 have similar expression dynamics to the previously reported results of GhDi19-1 and GhDi19-2 under salt stress, indicating potential functions in salt stress response. Abiotic stress-related cis-elements, including MBS, STRE, MYC, etc., were identified in GhDi19-3 and GhDi19-4 promoter regions. It was speculated that the sensitivity of GhDi19-3 and GhDi19-4 to salt stress may be regulated by abiotic stress-related cis-elements in these gene's upstream promoter. Interestingly, the cotton plants with a reduction of GhDi19-3 and GhDi19-4 expression by the VIGS system have accelerated the tolerance to salt stress. Hence, GhDi19-3 and GhDi19-4 are two candidate genes for enhancing the salt-tolerance of cotton in the future.
Previous studies have demonstrated that AtDi19-3 was involved in plant responses to drought and high salt stress conditions [16] and functions as a negative regulator under drought stress by interacting with AtCIPK11 [19]. In addition, AtDi19-3 interacts with IAA14 in the auxin signal transduction [20], indicating that AtD19-3 have important functions in regulating plant responses to stress as well as to hormone signals. In this study, bioinformatics analysis indicated that GhDi19-3 and GhDi19-4 have the highest identify to the At19-3 protein of the AtDi19 gene family, and we speculate that GhDi19-3, GhDi19-4, and AtDi19-3 had the same function in response to stress. AtDi19-3 expression was induced under salt stress, and its mutant exhibits enhanced salt tolerance [14,16]. In our study, results of the simulated salt stress treatment of the control and GhDi19-3and GhDi19-4-silenced cotton plants, similar to the phenotypic changes of Atdi19-3, showed a decreased sensitivity to salt stress. This suggested that GhDi19-3 and GhDi19-4 are negative regulators in salt stress response. Further, it suggests that GhDi19-1 and GhDi19-2 are from the same evolutionary branch as GhDi19-3 and GhDi19-4, which through phosphorylation of serine residue regulate the stress response of cotton through mediating the ABA signal transduction [10,26]. The results of physiological parameters measured in the simulated salt stress treatment of the control and GhDi19-3and GhDi19-4-silenced cotton plants showed that silencing of GhDi19-3 and GhDi19-4 genes regulated the scavenging ability of ROS, thereby enhancing plants tolerance to salt stress. The expression patterns of the proteins regulatory pathways and their related genes in the simulated salt stress treatment of the control and GhDi19-3and GhDi19-4-silenced cotton plants further showed that certain calcium signaling and ABA-responsive genes are up-and down-regulated under salt stress treatment. Both GhDi19-3 and GhDi19-4 may participate in calcium signaling and ABA signaling pathways in response to salt stress. Additionally, we speculate that GhDi19-3 and GhDi19-4 may have redundant functions similar to GhDi19-1 and GhDi19-2, which need to be verified in the future. Based on the above results, our study provided a model to visually describe the elucidated mechanisms of GhDi19-3 and GhDi19-4 genes in response to salt stress ( Figure 8). GhDi19-3 and GhDi19-4, as two negative regulatory transcription factors, respond to salt stress through calcium signaling and ABA signaling pathways.  , which then resulted in their abilities improving to effectively reduce the ROS damage when cotton plants were subject to salt stress. Taken together, GhDi19-3 and GhDi19-4, as two negative regulatory transcription factors, responded to salt stress by being involved in calcium signaling and ABA signaling pathways.

Conclusions
In this study, 82 Di19 proteins have been identified from 10 plant species, and the characteristics of the GhDi19 gene family from three cotton species, including the analysis of the gene number, gene structure, chromosomal distribution, promoter cis-elements, and expression profiles have been systematically investigated and analyzed. GhDi19 members have shared similar conserved structures of five exons and four introns. The GhDi19 proteins have five highly conserved motifs. All GhDi19 proteins contain the Di19_zinc-binding domain, the Di19_C terminal domain, and a nuclear localization signal.
Moreover, the cis-elements related to abiotic stress such as ARE, MYC, and STRE have been found at the promoter of Di19 genes. Transcriptomic analysis of Di19 genes in different tissues and their responses to abiotic stresses indicate that certain GhDi19s may play a significant role in the growth and development of upland cotton. Among them, GhDi19-3 and GhDi19-4 are up-regulated under salt stress. GhDi19-3 and GhDi19-4 are used as candidate proteins to further verify their potential roles in salt stress response. Subcellular localization indicates that the two GhDi19s are nuclear-localized proteins. GhDi19-3 and GhDi19-4 have decreased the sensitivity under salt stress through VIGS, and have showed significantly lower levels of H 2 O 2 , MDA, and POD as well as significantly increased SOD activity, this suggests that the tolerance to salt stress was improved by reducing the ROS damage. Further, certain calcium signaling and ABA-responsive gene expression levels shows up-and down-regulation changes in target gene-silenced plants, suggesting that GhDi19-3 and GhDi19-4 may be involved in calcium signaling and ABA signaling pathways in response to salt stress. Overall, our findings indicate that GhDi19-3 and GhDi19-4, as two negative regulatory transcription factors, respond to salt stress by being involved in calcium signaling and ABA signaling pathways. The comprehensive understanding of the physiological and molecular mechanisms of GhDi19-3 and GhDi19-4 can serve as an important genetic resource for the improvement in salinity stress tolerance and the yield of cotton.
Supplementary Materials: The online version contains Supplementary Material available at: https: //www.mdpi.com/article/10.3390/antiox11112225/s1, Figure S1. Chromosomal localization of Di19s on G. arboreum (A), G. raimondii (B) and G. hirsutum (C); Figure S2. Expression profile of GhDi19s in different tissues of G. hirsutum. Table S1, The primers used in this study. Table S2, Information of Di19s in different plant species. Table S3, Analysis conserved cis-elements of GhDi19 gene promoters. Table S4, The transcriptomic data analysis of GhDi19s in different stresses. Table S5, The transcriptomic data analysis of GhDi19s in different tissues.