Introduction

As the plants are sessile, they must respond quickly to damage from complex environments, including biotic (e.g., microbial and insect pathogens) and abiotic (e.g., drought, salt, and heavy metals) stresses. Among them, salt stress is a primary global environmental factor that limits plant growth and crop productivity [1], more than 6% of the world’s land area is saline (about 800 million hectares of land worldwide) [2]. Poor irrigation practices, inappropriate application of fertilizers and industrial pollution have increased salinity in the soil [3]. When the concentration of soluble salts in the soil exceeds a threshold value, saline soils were formed [4, 5]. Salt stress may adversely affect cells by causing osmotic stress [6, 7], ionic stress [8] and oxidative stress [9]. Na has been shown to be an essential nutrient required by animals [10], and excess Sodium is detrimental to animals and plants [11]. The accumulation of Na+ in plants can result in disturbed ionic dynamic balance, imbalance of potassium ion (K+)/Na+ ratio and Na+ toxicity, which could lead to secondary stresses including oxidative stress. Oxidative stress will lead to cell membrane damage, ion leakage or direct damage to proteins and other macromolecules, which in turn will result in membrane dysfunction and even cell death. Both ion stress and oxidative stress can expedite leaf senescence by degrading chlorophyll, inhibiting photosynthesis and reducing yield [12, 13].

Natural cytokinin (CK) is a plant hormone that is derived from adenine and possesses either an isoprenoid or aromatic side chain at the N6 position of the adenine ring. It can be obtained through isolation or synthesis from maize or other plant sources [14]. Therefore, CK can be categorized into isoprenoid and aromatic CK, with the former being more prevalent in plants and more abundant than the latter [15]. CK is essential for regulating plant growth, development and adaptation to environmental stresses. It is primarily synthesized in plant roots and functions as a group of compounds that stimulate cytoplasmic division. In growing seedlings, CK regulates lateral root organogenesis, root meristem size, and hypocotyl elongation [16]. Moreover, CK exerts a protective effect against plant senescence by inhibiting the breakdown of nucleic acids, proteins and other substances in the plant, while concurrently redistributing essential amino acids, hormones, inorganic salts and other compounds to other parts of the plant [17]. Since CK is a negative regulator of plant root growth and branching, CK can make plants exhibit long-term drought resistance by promoting the degradation of CK in the root system to expand the root system and increase the root to crown ratio and thus the water absorption area of the root system [12]. An experimental result showed that heat stress reduced the content of CK, and the heat resistance of plants was enhanced by exogenous treatment with CK [18].

Cytokinin oxidase/dehydrogenase (CKX) catalyzes the degradation of CK in plant tissues and is a critical negative regulator of endogenous CK content in the plant kingdom [19,20,21]. CKX was detected for the first time in crude extracts of tobacco tissues [22]. It catalyzes the cleavage of unsaturated N6 side chains of CK, such as zeatin, isopentenyl adenine or their ribosyl derivatives, resulting in the release of free adenine or free adenine nucleoside, leading to complete inactivation of CK [18]. Several studies have shown that CKX is involved in various physiological processes in a variety of plants, including CK catabolism metabolism, root structure and resistance to abiotic stresses. AtCKX overexpression in Arabidopsis induces CK deficiency which enhances salt tolerance and drought tolerance [23]. Moderate increase in CK levels by down-regulating GhCKXs expression resulted in higher fiber and seed yield in cotton [24, 25]. A CKX gene was isolated from Medicago sativa, MsCKX, and its expression was found to increase under salt stress and abscisic acid (ABA) treatment. Overexpression of the MsCKX gene increased the activity of CKX, which resulted in root expansion in transgenic Arabidopsis. Meanwhile, overexpression of MsCKX enhanced salt tolerance in transgenic plants by maintaining a high K+/Na+ ratio, enhancing the ROS scavenging activity of antioxidant enzymes and improving the expression levels of stress-related genes (ion transport proteins and H+ pumps) [26]. In rice, OsCKX11 had a role in delaying leaf senescence, increasing seed number and coordinating the regulation of the source pool, thus suggesting that CK plays an overwhelming role in leaf senescence and determining seed number [27], and disruption of OsCKX3 enhances CK content in the articular layer and also negatively regulates leaf angle [28].

Cotton (Gossypium spp.) is an extremely valuable fiber crop and oilseed crop, accounting for 35% of global fiber usage. Additionally, it is also a moderately salt tolerant crop [29]. While the impact of CKX genes on CK homeostasis and regulating growth and development in plants such as Arabidopsis thaliana and rice has been extensively studied, further research is necessary to elucidate the biological function of the CKX family in cotton. In this study, a total of 83 CKXs in four major cotton species were identified, and a systematic analysis of the CKXs was performed, including phylogenetic relationships, chromosomal localization and protein interaction networks, and their expression patterns under abiotic stresses and different tissues in cotton were also investigated. The function of GhCKX6b-Dt in salt tolerance was investigated using the virus induced gene silencing (VIGS) technique. The results of this study will contribute to future research by providing insight into the function into the phytohormone CK in cotton.

Materials and methods

Identification of CKX family members

CDS sequences and protein sequences of Gossypium hirsutum (G. hirsutum) (NAU), Gossypium barbadense (G. barbadense) (ZJU), Gossypium arboreum (G. arboreum) (CRI), and Gossypium raimondii (G. raimondii) (JGI) were obtained from the cotton database Gossypium Resource and Network Database (http://grand.cricaas.com.cn) and Cotton Functional Genomic Database (CottonFGD) (https://cottonfgd.net/) in this study. CKX protein conserved structural domains were identified by Pfam database (https://pfam.xfam.org/): FAD binding domain (PF01565) and Cytokin-bind domain (PF09265) [30]. These two Hidden Markov Models (HMM) were utilized as query files for protein screening in the HMMER (version 3.3.1) [31] to obtain candidate genes. The genes common to both screens were set as the final CKX members. CKXs were renamed according to their homology with Arabidopsis thaliana [25].

Phylogenetic analysis and sequences alignments

CKX protein sequences were downloaded from CottonFGD (https://cottonfgd.net/) for G. hirsutum (NAU), G. barbadense (ZJU), G. arboreum (CRI), and G. raimondii (JGI) and from the online database Phytozome v13 (https://phytozome-next.jgi.doe.gov/) for Arabidopsis thaliana. The software MEGA5 was used for sequence alignment, the results were analyzed to construct intraspecific (Neighbor Joining (NJ) method) [25].

Chromosomal locations of CKXs from four Gossypium species

The genome annotation files of the four Gossypium species were downloaded from the CottonFGD (https://cottonfgd.net/about/download/annotation). The software TBtools was employed to visualize the chromosome locations of CKXs of four Gossypium species [32].

Analysis of GhCKXs promoter regions and different expressions

The upstream sequence (2000 bp) preceding the start codon (ATG) of GhCKXs was obtained from the CottonFGD database (https://cottonfgd.net) and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) for the identification of cis-acting elements in the promoter of the GhCKXs. The expression levels of GhCKXs under different abiotic stresses (cold, heat, salt, and PEG) and different tissues (root, stem, leaf, torus, petal, stamen, pistil, and calycle) were shown as FPKM values from RNA-Seq data (PRJNA490626) [33]. Images containing evolutionary trees, cis-acting elements, and heat maps of expression levels were drawn for visual observation using TBtools software.

Gene ontology (GO) annotation analysis of GhCKXs

To explore the function of GhCKXs, GO annotation analysis was performed by CottonFGD (https://cottonfgd.net).

Materials, plant growth and treatments

The G. hirsutum cultivar Zhong 9807 was used as experimental material and seeds were sown on a 1:1.5 substrate of sand to vermiculite and grown in an incubator at 25 °C/23 °C with 16 h of light/8 h of darkness. The cotton seedlings were treated with 100 mmol/L NaCl solution, and samples were taken after 0, 6, 12 and 24 h, snap-frozen in liquid nitrogen and stored at − 80 °C.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Tissue (leaf) grinding and extraction of total RNA according to EasySpin plus plant RNA rapid isolation Kit (aidlab Co., Ltd, Beijing, China). RNA was reverse transcribed to cDNA using the HiScript Ill RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., LTD, Nanjing, China). qRT-PCR experiments were performed using an Applied Biosystems@7500 Fast instrument and a quantification kit (PerfectStart® Green qPCR SuperMix) (TransGene Biotech Co., LTD, Beijing, China), with the internal reference gene (GhUBQ7) as a control. 2−ΔΔCT method to calculate the relative expression of GhCKXs [34]. Primers were designed using the website (https://www.genscript.com/) and the specificity of the primers was checked at the NCBI (National Center for Biotechnology Information) (https://www.ncbi.nlm.nih.gov/) website. Gene-specific primers for qRT-PCR are listed in Additional file 1: Table S1.

Virus induced gene silencing (VIGS) experiment

GhCKX6b-Dt was significantly up-regulated at 6 h, 12 h and 24 h after treatment with 100 mmol/L concentration of NaCl solution, so this gene was selected for silencing, and then the GhCKX family function was explored. The upland cotton cultivar 9807 was cultivated in nutrient soil and subsequently placed in an incubator with a light–dark cycle of 16/8 h, ambient humidity of 50%, and temperature of 25/20 °C (light/dark). Agrobacterium tumefaciens was injected into the cotton plant when the two cotyledons were unfurled and the treatment occurred during the two leaf one heart stage of cotton. A silencing fragment of around 300 bp was designed by SGN-VIGS (https://vigs.solgenomics). The vector construction is outlined in Additional file 2: Materials and methods for a more comprehensive operating procedure for reference. The gene-specific primers for qRT-PCR are listed in Additional file 1: Table S1. The fragment was ligated into the pYL156 vector and after transformation into Agrobacterium, and pYL156: GhCKX6b-Dt, pYL156: PDS and pYL192 was cultured to OD600 = 1.2–1.5. Each mixture was injected into the lower side of cotyledons of G. hirsutum material Zhong 9807. After injection, seedlings were placed in the dark for 24 h, followed by a 16 h light/8 h dark cycle at 25/20 °C (light/dark). The cotton seedlings were treated with 100 mmol/L NaCl solution for 24 h which occurred during the two leaf one heart stage of cotton. Subsequently, the leaves were taken as samples, which were quick-frozen with liquid nitrogen and stored in a −80 ℃ refrigerator for subsequent experiments.

Determination of PRO, MDA content, SOD enzyme activity and DAB staining

0.1 g of fresh leaves were taken to determine the content or enzyme activity of each substance in the plants using Proline (PRO) Content Assay Kit (Nanjing Jiancheng Institute of Biological Engineering, A107-1-1), Malondialdehyde (MDA) Assay Kit (Nanjing Jiancheng Institute of Biological Engineering, A003-3-1) and Superoxide Dismutase (SOD) Activity Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., BC0170), respectively. Three biological replicates were available for each sample. The DAB staining method called diaminobenzidine method was used to detect the active site of peroxidase in cells [35]. Three leaves each of pYL156 and pYL156: GhCKX6b-Dt were taken after NaCl stress and placed in DAB solution, darkened for 12 h, and observed after decolorization with 95% ethanol. The dark brown polymerization products represent the reaction of DAB with hydrogen peroxide.

Gene interaction network of the GhCKX6b-Dt protein

GhCKX6b-Dt protein interaction network was analyzed by STRING database (https://string-db.org/) [36]. The Arabidopsis thaliana homolog of GhCKX6b-Dt was used to predict the interactions of GhCKX6b-Dt with other genes in cotton.

Results

Identification of CKX proteins

To investigate the precise information and potential functions of cytokinin dehydrogenase (CKX) in cotton, the putative CKXs in the cotton genome was subjected to systematic genome-wide characterization. For this purpose, Hidden Markov model (HMM) profiles were generated based on the reported protein sequences of Brassica napus [37], Vitis vinifera [30], and Medicago truncatula CKX proteins [38], respectively. Two Hidden Markov Model (HMM) profiles, FAD-binding domain (PF01565) and Cytokinin-binding domain (PF09265), were used for protein screening in the HMMER, and further analysis using the Pfam database identified 27, 28, 14, and 14 CKX members in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Then renamed the genes according to their Arabidopsis homologs (Additional file 1: Table S2) [25]. A phylogenetic tree of four Gossypium species and Arabidopsis was constructed based on the full-length amino acid sequences of 83 CKXs proteins in cotton and 7 AtCKXs proteins using the NJ method (Fig. 1A). As shown in Fig. 1B, the CKX family in cotton was clearly divided into 5 subgroups (labeled as groups I, II, III, IV and V) based on the branch in which Arabidopsis thaliana was located. The CKX proteins of the four Gossypium species were spread across subgroups, containing proteins from both diploid and allotetraploid Gossypium species in each branch, and almost twice as many CKX proteins in tetraploid cotton as in diploid cotton.

Fig. 1
figure 1

Phylogenetic analysis of CKX proteins. A Phylogenetic relationship of the 83 identified CKXs from four Gossypium species and 7 AtCKXs in Arabidopsis using MEGA 5.0 by the Neighbor-Joining (NJ) method, the tree showed 6 major phylogenetic subgroups, designated as I to VI. B Phylogenetic relationship of the 83 identified CKXs from four Gossypium species using MEGA 5.0 by the Neighbor-Joining (NJ) method, the tree showed 5 major phylogenetic subgroups, designated as I to V. The suffix “At” and “Dt” indicates the origin of genes from the cotton A subgenome or D subgenome, respectively

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Chromosomal locations of CKXs from four Gossypium species

To investigate the chromosomal distribution of CKXs, based on the Cotton Genome Database, the predicted CKXs were located to the physical locations of genes on the cotton chromosome. To study the chromosome distribution of CKX members, the predicted CKXs were mapped into the physical locations of genes on chromosomes according to the Cotton Genome Database. As shown in Fig. 2, 81 of the 83 CKXs were unequally distributed on their respective specific chromosomes, but 2 CKXs, GhCKX5a-At and GhCKX3c-Dt, were not annotated and not located on any of the chromosomes. In terms of chromosome distribution, there were 1–3 genes on each chromosome. In G. hirsutum, there were 13 genes in subgenome A and 14 genes in subgenome D. Chromosomes A02, A03, A4, A11, A12, D02, D03, D07, D11, and D12 did not have GhCKXs. In G. barbadense, there were 14 genes each in subgenome A and subgenome D. Chromosomes A02, A03, A4, A11, A12, D02, D03, D11, and D12 did not contain GbCKXs. At different from G. hirsutum, GbCKX6b-At was located on the lower part of chromosome D05 in G. barbadense; GbCKX5a-At was located on chromosome GbA06 instead of the scaffold; and GbCKX3c-Dt appeared on chromosome GbD07 instead of the scaffold. In G. arboreum, there were 14 genes irregularly arranged on their respective chromosomes, and chromosomes Chr02, Chr03, Chr11 and Chr12 had no GaCKXs distribution. In G. raimondii, there were 14 genes unevenly distributed on their respective chromosomes and no GrCKXs distribution on chromosomes Chr03, Chr05, Chr07 and Chr08. In the four Gossypium species, slight differences in the number and distribution of chromosomes were noted, so it was hypothesized that this could be due to the duplication or loss of CKX members during evolution.

Fig. 2
figure 2

Chromosome localization of CKXs from four Gossypium species. A Chromosomal location of CKXs on chromosomes in G. hirsutum A subgenome (GhAt). B Chromosomal location of CKXs on chromosomes in G. hirsutum D subgenome (GhDt). C Chromosomal location of CKXs on chromosomes in G. barbadense A subgenome (GbAt). D Chromosomal location of CKXs on chromosomes in G. barbadense D subgenome (GbDt). E Chromosomal location of CKXs on chromosomes in G. arboreum (Ga). F Chromosomal location of CKXs on chromosomes in G. raimondii (Gr). The scale of the genome size was given on the left

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Analysis of GhCKXs promoter and expression pattern

An increasing body of evidence demonstrates that cis-acting elements in gene promoters can impact gene expression and function [39]. To further analyze the transcriptional regulation and potential functions of GhCKXs, their evolutionary tree, promoter analysis and expression heat map were correlated, and potential stress-responsive cis-regulatory elements were identified using GhCKXs protein sequences, promoters in 2kb sequences upstream of the transcription start site and GhCKXs expression in the RNA-Seq database under different stresses (cold, heat, salt and PEG). As shown in Fig. 3, several light-responsive components, hormone-responsive components, and components related to abiotic stresses were identified, such as light responsiveness, auxin responsiveness, gibberellin-responsiveness, salicylic acid responsiveness, MeJA-responsiveness, abscisic acid responsiveness, wound-responsiveness, defense and stress responsiveness, meristem expression, drought-inducibility and low-temperature responsiveness and so on. The most light responsive elements were detected, with each GhCKX member containing multiple light responsive elements; secondly 18 GhCKXs contained abscisic acid responsive elements; 17 GhCKXs contained gibberellin-responsive element; 16 GhCKXs contained MeJA-responsive element; 14 GhCKXs contained auxin responsive element; 12 GhCKXs contained salicylic acid responsive element; 11 GhCKXs contained defense and stress responsive element and meristem expression element; 9 GhCKXs contained drought-inducibility element; 7 GhCKXs contained low-temperature responsive element; and GhCKXs contained wound-responsive element; 7 GhCKXs contained low-temperature responsive element; and only 2 GhCKXs contained wound-responsive element. This leads to the conclusion that GhCKXs were strongly associated with regulatory hormones and abiotic stresses.

Fig. 3
figure 3

Expression patterns and promoter analysis of the GhCKXs. A Phylogenetic relationship of GhCKXs, the 5 major phylogenetic subgroups, designated as I to V. B Cis-acting elements in promoters of GhCKXs. C Heatmap of the expression of GhCKXs under different abiotic stresses at different times of stress (cold, heat, salt and PEG)

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Meanwhile, the response of GhCKXs to various abiotic stresses was thus verified by analyzing the heat map of GhCKXs expression under different stresses (cold, heat, salt and PEG) in the RNA-Seq database. In addition, the expression levels of eight cotton tissues (root, stem, leaf, torus, petal, stamen, pistil and calycle) were analyzed using RNA-Seq data (Fig. 4). The results showed that GhCKXs were expressed in various tissues with slightly different expression patterns. GhCKX6a-At, GhCKX6a-Dt, GhCKX5c-At and GhCKX5c-Dt were highly expressed in root; GhCKX1a-Dt, GhCKX6b-Dt, GhCKX6c-At and GhCKX6c-At were highly expressed in leaves. GhCKX5b-Dt was the most highly expressed in the torus; GhCKX7b-At, GhCKX7b-Dt, GhCKX3b-At and GhCKX3b-Dt were highly expressed in the petal; GhCKX3a-At and GhCKX3c-Dt were highly expressed in the stamen; in the calycle, GhCKX5a-At, GhCKX5a-Dt and GhCKX3c-At were the most highly expressed, and GhCKX5b-At was almost not expressed. The expression of GhCKXs were lower in both stem and pistil. As with Brassica oleracea L., the diversity of expression patterns showed that CKXs have a widespread biological function in the growth and development of cotton [40].

Fig. 4
figure 4

Expression of GhCKXs in different tissues. Different tissues were represented as columns of different colors (root, stem, leaf, torus, petal, stamen, pistil and calycle), significance analysis of different tissues compared to root (*: 0.01 < p < 0.05; **: p < 0.01)

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Gene ontology (GO) annotation analysis of GhCKXs

GO enrichment consists of Molecular Function, Biological Process, and Cellular Component. GO enrichment of GhCKXs by Cotton FGD showed that GhCKXs embody the properties of genes in terms of both molecular functions and biological processes (Fig. 5). GhCKXs are the most involved in molecular functions, including catalytic activity (GO:0003824), oxidoreductase activity (GO:0016491), oxidoreductase activity, acting on CH-OH group of donors (GO:0016614), cytokinin dehydrogenase activity (GO:0019139), flavin adenine dinucleotide binding (GO:0050660) and UDP-N-acetylmuramate dehydrogenase activity (GO:0008762). GhCKXs also participates in cytokinin metabolic process (GO:0009690) and obsolete oxidation–reduction process (GO:0055114).

Fig. 5
figure 5

Gene ontology (GO) analysis of GhCKXs

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Expression of GhCKXs vis-à-vis salt stress at different durations

To verify the potential role of GhCKXs in response to salt stress, cotton seedlings were treated with 100 mmol/L NaCl solution for different time of stress. And the expression levels of GhCKXs in leaves at NaCl solution for different durations were examined by qRT-PCR (Fig. 6). It was found that except for GhCKX3a-Dt, all 26 GhCKXs responded to salt stress in leaves of seedlings subjected to different levels and the tendency of expression changes. With the change in duration of stress, the expression levels of two genes in most A\D group showed the same trend. At 6 h of 100 mmol/L NaCl stress, 19 CKXs were shown to be significantly different in expression from 0 h. At 12 h of 100 mmol/L NaCl stress, 20 CKXs were shown to be significantly different in expression from 0 h. At 24 h of 100 mmol/L NaCl stress, 24 CKXs were shown to be significantly different in expression from 0 h. The differences in the expression of 13 CKXs (GhCKX1a-At, GhCKX1a-Dt, GhCKX3a-At, GhCKX3b-At, GhCKX3c-At, GhCKX5b-Dt, GhCKX5c-Dt, GhCKX6a-Dt, GhCKX6b-Dt, GhCKX6c-At, GhCKX7a-At, GhCKX7a-Dt, and GhCKX7b-At) were significant at 6 h, 12 h, and 24 h compared with 0 h, and most of them were extremely significant. Consequently, it is speculated that CKX genes are participated in the regulation of salt stress.

Fig. 6
figure 6

Expression of GhCKXs at different durations of salt stress in leaves using qRT-PCR. Column indicate the relative expression levels of GhCKXs in leaves under 100 mmol/L NaCl stress for 0 h, 6 h, 12 h, and 24 h (*: 0.01 < p < 0.05; **: p < 0.01). The mean values were from three independent biological replicates

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Effect of silencing GhCKX6b-Dt on NaCl stress in cotton

To verify whether the CKX genes responded to salt stress, the gene GhCKX6b-Dt, whose expression level of gene was significantly up-regulated at 6 h, 12 h and 24 h after treatment with 100 mmol/L NaCl solution, was silenced. As shown (Fig. 7A), pYL156: PDS plants showed albinism, indicating successful gene silencing. After treatment with 100 mmol/L NaCl, cotton leaves lost their luster and wilted after GhCKX6b-Dt silencing compared with pYL156. The expression of GhCKX6b-Dt in cotton leaves was detected by qRT-PCR. And the results showed (Fig. 7B) that the expression of GhCKX6b-Dt was significantly decreased in the latter compared with pYL156 and pYL156: GhCKX6b-Dt, which illustrated the good effect of GhCKX6b-Dt silencing. After NaCl stress, leaves of GhCKX6b-Dt silenced plants showed dark brown spots after DAB staining, showing that GhCKX6b-Dt was more severely injured after silencing (Fig. 7C). Consistent with the above results (Fig. 7D, E), both PRO content and MDA content were highly significantly elevated in GhCKX6b-Dt silenced plants after stress compared to pYL156 plants. In contrast, the SOD activity of silenced plants after stress was significantly declined compared to pYL156 (Fig. 7F), illustrating the decline in antioxidant capacity after GhCKX6b-Dt silencing.

Fig. 7
figure 7

Effect of silencing GhCKX6b-Dt on NaCl stress in cotton. A The phenotype of cotton after GhCKX6b-Dt silencing under NaCl stress. pYL156: PDS as a positive control, pYL156 was an empty vector as control, and pYL156: GhCKX6b-Dt was GhCKX6b-Dt silenced lines. B The relative expression level of GhCKX6b-Dt under NaCl stress. C DAB staining. D PRO content of empty control and VIGS plants under NaCl stress. E MDA content of empty control and VIGS plants under NaCl stress. F SOD activity of empty control and VIGS plants under NaCl stress. *0.01 < p < 0.05, **p < 0.01

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Interaction network of GhCKX6b-Dt protein

Based on the homologous gene AtCKX6, which has the highest homology with GhCKX6b-Dt in Arabidopsis, the STRING database was used to construct an interaction network of CKX protein functions. The results showed that both AtCKX6 and Polyprenyltransferase 1 (PPT1), Glycerol-3-phosphate dehydrogenase SDP6 (SDP6), RNA dimethylallyltransferase 2 (IPT2), Adenylate dimethylallyltransferase (cytokinin synthase) (IPT1), CHASE domain containing histidine kinase protein(WOL), Peroxisomal (S)-2-hydroxy-acid oxidase GLO4 (HAOX1), Peroxisomal (S)-2-hydroxy-acid oxidase GLO3(HAOX2), Aldolase-type TIM barrel family protein (GOX2), Aldolase-type TIM barrel family protein (GOX1) and Peroxisomal (S)-2-hydroxy-acid oxidase GLO5 (GOX3) interacted with each other (Fig. 8A).

Fig. 8
figure 8

Interaction network of GhCKX6b-Dt protein and expression of related genes after GhCKX6b-Dt silencing. A The CKX6 represented the protein AtCKX6 corresponding to the protein in Arabidopsis with the highest homology to GhCKX6b-Dt. B Relative expression levels of IPT1, IPT2 and WOL in GhCKX6b-Dt silenced plants before and after NaCl stress. pYL156: PDS as a positive control, pYL156 was an empty vector as control, and pYL156: GhCKX6b-Dt was GhCKX6b-Dt silenced lines. *0.01 < p < 0.05, **p < 0.01

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After analysis, the synthesis enzymes IPT associated with CK and the signaling molecule WOL were noticed. Therefore, the expression of each gene was determined by qRT-PCR in cotton seedlings after GhCKX6b-Dt silencing. As shown in Fig. 8B, the results demonstrated that there was no significant difference in the expression of each gene in pYL156 and pYL156:GhCKX6b-Dt plants before stress. However, the differences in the expression of IPT1, IPT2 and WOL were significant after NaCl treatment, and the expression of IPT1, IPT2 and WOL were significantly increased in GhCKX6b-Dt silenced plants compared with pYL156 control plants.

Discussion

Cytokinin is an essential plant hormone that regulates various developmental and physiological processes [41, 42]. CKX, a key regulatory enzyme for the irreversible degradation of the plant hormone CK, is indispensable for maintaining CK homeostasis [43]. Although the biological functions of the CKXs have been identified in a variety of plants [27, 44, 45] and were particularly prominent in adversity stresses [46,47,48], functional studies of CKX in cotton are still limited. In this study, 27 GhCKXs, 28 GbCKXs and 14 each of GaCKXs and GrCKXs were identified. And chromosomal locations, cis-acting elements and expression patterns under different abiotic stresses were analyzed, then the CKXs functions were characterized by VIGS technique. A review of the literatures revealed that the genomes of other species closely related to cotton contain much smaller numbers of CKX members, for example, 7 CKXs in the Arabidopsis [49]; 11 CKXs in the rice [50]; 12 CKXs in the Sorghum bicolor; 11 CKXs in the Setaria italica; and 15 CKXs in the maize [51]. The CKX members has more genes in polyploid plants compared to haploids, CKX members contains 20 genes in Eleusine coracana [52]; 16 CKXs in Glycine max [53]; and more CKXs in Triticum (31) [54].

The main reason for family gene amplification is gene duplication which can diversify gene functions to facilitate rapid adaptation of organisms to different environments [55, 56]. The main source of gene duplication in eukaryotic genomes is interchromosomal duplication [57]. During the evolution of diploids to tetraploids, it can be noticed that the number of CKX genes in each branch of tetraploid cotton was almost twice as much as that of diploid cotton from the four Gossypium species evolutionary tree (Fig. 1B). However, it can be found that one gene of GhCKXs was absent in branch II, which may be due to the loss of genes during evolution. Meanwhile, as seen from the chromosome position (Fig. 2), chromosome GaA04 evolved to tetraploid Gb translocated to chromosome A05, but no gene was found on chromosome GhA05, indicating that the same translocation did not occur in G. hirsutum, so it was speculated that the loss of the gene may have occurred here. In addition, the presence of a gene on chromosome GbD07 and the absence of a gene on chromosome GhD07 suggests that the gene has been altered during the evolution of the gene. Upon scrutiny, it was found that two genes appeared in G. hirsutum that were not annotated on any chromosome, which it was possible that A06 and D07 chromosomes were the result of translocation, and there were multiple genes in the GrD group of chromosomes that did not correspond to Gh and Gb. These may be due to the translocation of CKXs through fragmentary or whole genome replication events and did not replicate in tandem during evolution of four Gossypium species, which in turn diversified the CKXs, which was consistent with the results of the Brassica oleracea L. [40].

The results of cis-acting elements in the promoter indicate (Fig. 3B) that GhCKXs are involved in light response [58], hormone response and regulation of abiotic stress and also play an active role in plant growth and development. Multiple plant hormone cis-acting elements were predicted in the promoter region of GhCKXs, such as auxin responsiveness element, gibberellin-responsiveness element, salicylic acid responsiveness element, MeJA-responsiveness element and abscisic acid responsiveness element. It was demonstrated in a number of studies that CK interacted with multiple hormones through the CKXs, thereby regulating plant growth and development [59, 60]. OsCKX4 integrated cytokinin and auxin signaling to control crown root formation in rice [44]. In addition to the promoter region of GhCKXs containing various phytohormone cis-acting elements, there were also various stress-related cis-acting elements such as wound-responsiveness element, defense and stress responsiveness element, drought-inducibility element and low-temperature responsiveness, which indicated that GhCKXs can respond to various abiotic stresses, and this is in accordance with previous reports. Reduced expression of OsCKX2, a cytokinin oxidase specific to inflorescence meristem tissue in rice, enhanced tolerance to salt stress [61]; overexpression of CKX1 in tobacco and barley improved drought tolerance and heat tolerance in plants [46, 62]. Nishiyama et al. showed that CK-deficient CKX overexpression plants (35S:CKX1-35S:CKX4) became more tolerant to salt and drought in Arabidopsis compared with WT plants [23]. Heat map analysis in this study revealed the same results (Fig. 3C), that CKX genes play an important role in response to abiotic stresses (cold, heat, salt and PEG). In addition, we found that a few GhCKXs contained meristem expression elements, and most GhCKXs were expressed in various tissues (Fig. 4), indicating that GhCKXs play a role in plant growth and development [63].

The salt tolerance mechanism is extremely complex involving ion transport, osmoregulation and oxidative stress, each of which is in turn regulated by multiple components [64,65,66]. As we all know, cytokines can regulate salt tolerance in plants [67, 68], with a positive or negative effect [68, 69]. Here, it was found that most of the GhCKXs were significantly distinct under NaCl stress during different time, indicating that GhCKXs responded to salt stress. One CKX gene, GhCKX6b-Dt, was found to respond to salt stress as revealed from RNA-Seq data, and its expression gradually increased with time and the difference was highly significant, indicating that this gene responded positively to salt stress, but negatively regulated salt stress. This’s why GhCKX6b-Dt was selected for performing virus induced gene silencing experiments. Abiotic stresses contribute to the excessive production of ROS in plant cells, leading to oxidative damage to biomolecules [70]. Damaged biomolecules include products of protein oxidation, enzyme inactivation, lipid peroxidation, increased membrane fluidity, chlorophyll degradation, nucleic acid damage, and apoptotic pathways, and these damages can affect plant growth and development [70, 71]. In this study, compared with control plants, GhCKX6b-Dt silenced plants wilted after stress, in which both PRO and MDA contents were highly significantly increased (Fig. 7D, E), demonstrating that GhCKX6b-Dt silenced plants produced excessive ROS to expose the plants to severe salt stress, and vice versa, indicating that GhCKX6b-Dt positively regulates salt stress and CK negatively regulates salt stress, which is consistent with the results of previous studies [72]. SOD is the most effective scavenger of ROS and is the first line of defense against ROS-induced damage under abiotic stresses [73]. Compared with control plants, the SOD activity of GhCKX6b-Dt silenced plants was significantly decreased after stress (Fig. 7F), presumably GhCKX6b-Dt-silenced plants failed to produce an appropriate amount of SOD to scavenge reactive oxygen species, resulting in wilting of the plants and reduced salt tolerance.

After GO analysis, the biological processes involved in the GhCKXs were revealed to be CK metabolic process and obsolete oxidation–reduction process. And the molecular functions were catalytic oxidoreductase activity (oxidoreductase activity and cytokinin dehydrogenase activity) and UDP-N-acetylmuramate dehydrogenase activity and flavin adenine dinucleotide binding, which illustrate the involvement of GhCKXs in the regulation of redox dynamics under stress. Protein interactions predicted by homologs of GhCKX6b-Dt in Arabidopsis thaliana revealed that this gene is strongly associated with CK synthase (IPT1 and IPT2), CK receptor (WOL) and redox reaction-related enzymes (GOX1, GOX2, GOX3, HOX1 and HOX2). Therefore, it was hypothesized that CKXs regulate the CK content in cotton by interacting with IPT and WOL. Seedlings after GhCKX6b-Dt silencing wilted more heavily than the negative control. It was hypothesized that GhCKX6b-Dt might play an important role in response to NaCl stress (Fig. 9). When GhCKX6b-Dt silenced plants were subjected to salt stress resulting in cellular damage, excessive ROS were produced, causing an imbalance of redox reactions in the plant, and the plants showed wilting. It has been shown in the literature that the ion stress and osmotic stress caused by salt pollution can be alleviated by osmotic regulating substances such as proline [74, 75]. Salt stress can affect CK content, and genes related to biosynthesis and signal transduction were significantly up-regulated or down-regulated, including CKXs and IPTs [76]. The results show that GhCKX6b-Dt regulates CK and thus regulates the content of proline, which enables antioxidant enzymes to clear ROS and relieve the damage of plant cells caused by salt stress [21], resisting salt stress and normalizing plant growth.

Fig. 9
figure 9

Mechanism model of GhCKX6b-Dt responding to NaCl stress in cotton. The figure was drawn using the software Figdraw

Full size image

Conclusion

In this study, the CKXs were characterized based on the results of phylogenetic relationships, gene chromosome localization and cis-acting element analysis. In addition, the expression patterns of GhCKXs were investigated under different abiotic stresses, while using VIGS technology to understand the response of GhCKXs to salt stress as a function. GhCKX6b-Dt silenced plants were found to have increased PRO and MDA contents, decreased SOD activity, and reduced ROS scavenging capacity after stress, and thus were severely injured. Combining GO analysis and the gene interaction network of GhCKX proteins, it is hypothesized that GhCKX6b-Dt alleviates salt stress by scavenging reactive oxygen species through the antioxidant system. The results revealed that GhCKX6b-Dt positively regulates salt stress, while CK negatively regulates salt stress. The results of this study provide a basis for further studies on the response of CKXs to regulate CK homeostasis and to abiotic stresses during plant development.