Cytokinins are essential for many of the physiological and developmental processes such as seed germination, functional root nodule establishment, lateral root development, shoot apical meristem maintenance, leaf expansion, flowering, circadian clock, nutrient mobilization, abiotic stress, and senescence    . In eukaryotes such as yeast and plant, a two-component system (TCS) has been reported for the transduction of cytokinin signal  . This TCS consists of two signaling factors, a histidine kinase (HK) gene family and a response regulator (RR) gene family . HK can sense the cytokinin signals by phosphorylating its conserved histidine residues. The phosphoryl group is then transferred to a conserved asparagine residue on the receiver (Rec) domain of an RR, which modulates the activity of concerned downstream genes directly or indirectly  . In addition, histidine phosphotransfer (HP) genes are regarded as the mediators for the transfer of the phosphoryl group between the HKs and the RRs (Figure 1)   .
Melon (Cucumis melon L.) is an economically important fruit crop that originates from Asian, with an average production during the past decade more than 29 million tons per year (FAOSTAT, 2017; http://www.fao.org/faostat/en/#home). This crop is mainly cultivated in tropical and temperate countries, especially in the Asian countries, with China leading the list  . Although melon is a eudicot of interest for its specific biological properties   , there is still no genome-wide investigation on melon TCS genes. In this study, we examined the putative TCS genes and revealed that the melon genome contained a total of 44 members. Their classification and characteristics were also analyzed systematically. Our comprehensive analysis of the TCS genes might provide a framework for future functional dissection of TCSs in melon hormone signal transduction.
2. Materials and Methods
2.1. Data Collection
Protein sequence data of Arabidopsis AHKs, AHPs and ARRs were downloaded from the NCBI databases (https://www.ncbi.nlm.nih.gov/). Genome sequence data of melon deposited in the website of Cucurbit Genomics Data (http://cucurbitgenomics.org/organism/3) were used for TCS gene identification and analysis.
2.2. Identification of the Putative Melon TCS Genes
Previously cucumber and watermelon TCS members have been successfully identified by using Arabidopsis TCS genes . So we also used Arabidopsis TCS protein sequences as queries to search for the putative counterparts in melon by BLASTP with E-value of 1e-5 . The Pfam (http://pfam.janelia.org) and SMART (http://smart.embl-heidelberg.de/) tools were used to check whether these genes contained the structural characteristics and conserved domains of TCS elements, i.e., HisKA (Histidine Kinase A phosphoacceptor) domain, HATPase (histidine kinase-like ATPase) domain, Hpt (histidine-containing phosphotransfer) domain, and Rec domain. Information of abbreviation notation used in this article was listed in Table A1. Thereafter the identity of melon TCS genes with Arabidopsis was analyzed by BLASTP against Arabidopsis databases in TAIR (http://www.arabidopsis.org/). Their CDS and protein sequences
Figure 1. Model of the two-component signal transduction pathway. Cytokinin responses (CREs), histidine-kinases (HKs), histidine phosphotransfer proteins (HPs) and type-B response regulators (RRs) work as a positive regulatory loop and transfer the cytokinin from plasma membrane (PM) to nucleus. Type-B RRs act as transcription factors to regulate some cytokinin targets, including type-A RRs. The type-A RRs can inhibit their own transcription, providing a negative feedback mechanism. H: phospho-accepting histidine residue. D: aspartate residue. P: phosphoric acid groups.
together with position information in melon genome were obtained from Cu
2.3. Phylogenetic Analysis and Gene Structure Construction
Phylogenetic analysis of the full-length protein sequences was conducted using MEGA5 . The evolutionary history was inferred using the Neighbor-Joining method with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates)  . The DNA and cDNA sequences corresponding to each predicted genes were downloaded from the melon genome database (http://cucurbitgenomics.org/organism/3), and then the gene structures were analyzed using the Gene Structure Display Server online tool (http://gsds.cbi.pku.edu.cn/).
3. Results and Discussion
The TCS signaling is widely present in higher plant, including Arabidopsis thaliana   , rice (Oryza sativa)  , lotus (Lotus japonicus)  , soybean (Glycine max)   , maize (Zea maize)  , Physcomitrella patens   , and wheat (Triticum aestivum L.)  , as well as horticultural crops such as Chinese cabbage (Brassica rapa)  , tomato (Solanum lycopersicum)  , cucumber (Cucumis sativus L.)  and watermelon (Citrullus lanatus)  (Table 1). In Arabidopsis, there are 56 TCS genes and their functions have been extensively studied  . To find the putative TCS members in melon, we performed a BLASTP search against the melon genome database by using 56 Arabidopsis TCS protein sequences. A total of 90 genes were selected as putative TCS genes including 26 HK(L)s, 6 HPs, 58 RRs in the melon genome. To confirm these putative melon TCS genes, the amino acid sequences of all 90 genes were further filtered by Pfam and SMART based on the presence of structural and conserved TCS elements. Finally, 44 typical TCS genes including 18 HK(L)s, 5 HPs, 21 RRs were identified in melon (Table 1). To better reflect the paralogous relationship, all melon TCS members were named according to their homology with Arabidopsis counterparts.
The identified 18 putative CmHK(L)s in melon were separated as 9 CmHKs and 9 CmHKLs according to the presence or absence of conserved residues required for histidine kinase activity (Figure 2(a)). Further they were classified to four distinct gene families: the typical CmHK family (four cytokinin receptor-like CmHKs, one CKI1-like CmHK, one CKI2/AHK5-like CmHK, and one AHK1-like CmHK), the ethylene response (ETR) homolog family (two ETR1-like CmHKs, one ETR2-like CmHKLs), the phytochromes (PHY) (six PHY-like CmHKLs) and the pyruvate dehydrogenase kinase (PDK) family (two PDK-like CmHKLs) (Table 2). The protein sequences of these CmHK(L)s ranged from 352 to 1261 amino acids, indicating great variations in their structures and possible functions (Table 2).
Table 1. Summary of the TCS gene number identified in plants.
Figure 2. Phylogenetic relationship and gene structure of the Histidine kinases (a), Histidine phosphotransfer proteins (b) and Response regulators (c) in melon. The phylogenetic tree was constructed based on the Neighbor-Joining method by MEGA5. Bootstrap supports from 1000 replicates were indicated at each branch. The gene structure was analyzed using the Gene Structure Display Server online tool.
Four authentic HPs and one pseudo-HP (PHP) with a pseudo-Hpt domain were identified in melon genome (Figure 2(b)). The CmHP1, CmHP2 and CmHP3 had a close relationship with AHP1 (Figure 2(b), Table 3), a positive regulators in CK signaling  , while the CmHP4 was close to AHP4 (Table 3), which was evolutionarily distinct from the other AHPs and functioned as a negative regulator in CK signaling . CmPHP1 exhibited the longest CDS and
Table 2. Features of HK genes in melon.
Note: a. Systematic names given to genes by Cucurbit Genomics Database. b. Features indicated conserved histidine-kinase (HK) domain, diverged histidine-kinase like (HKL) domain, receiver (Rec) domain, cyclases/histidine kinases associated sensory extracellular (CHASE) domain, cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, Per-ARNT-Sim (PAS) domain, and phytochrome (PHY) domain. c. Number of TM (transmembrane) from TMHMM Server v. 2.0 (http://pfam.janelia.org). d. The proteins belonged to which family in Arabidopsis, including cytokinin independent (CKI), Arabidopsis histidine-kinase (AHK), ethylene response (ETR), phytochrome (PHY), and pyruvate dehydrogenase kinase (PDK).
Table 3. Features of HP genes in melon.
Note: a. Systematic names given to genes by Cucurbit Genomics Database. b. Features included conserved histidine-containing phosphotransfer (HPt) domain and a pseudo-HPt domain lacking the histidine phosphorylation site. c. Number of TM (transmembrane) from TMHMM Server v. 2.0 http://pfam.janelia.org ). d. The proteins belonged to Arabidopsis histidine phosphotransfer (AHP) family.
amino acid sequence (Table 3) and had close relationship with Arabidopsis AHP6, which functioned as a competitor of other AHPs and played a negative role in CK responses by interfering with phosphorelay .
There were 21 protein-coding genes in the melon genome that were predicted as RRs (Table 4). Also there were 6 genes encoding RRs without the essential residues that were required for biological activity, and were thus named as pseudo-RRs (PRRs) (Table 4). Among these RRs/PRRs, we identified seven type-A RRs (CmRR1-7), each of which contained a Rec domain along with short C-terminal extension (Table 4, Figure 2(c)). Furthermore, these type-A CmRRs exhibited close relationship to their homologs, namely, ARR3, ARR5 and ARR9 in Arabidopsis (Table 4). Genetic analysis suggests that ARR3, ARR5 and ARR9 could function as negative regulators in cytokinin signaling, thus possibly
Table 4. Features of RR genes in melon.
Note: a. Systematic names given to genes by Cucurbit Genomics Database. b. Features included receiver (Rec) domain, pseudo-receiver (Pseudo-Rec) domain, Myb-like DNA binding domain and CCT (CO, COL and TOC1) motif. c. Number of TM (transmembrane) from TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/d). d. The proteins belonged to Arabidopsis response regulator (ARR) or Arabidopsis pseudo-response regulator (APRR) family.
participating in a negative feedback loop to reduce the plant sensitivity to cytokinins  .
There were eight type-B RR genes in melon, of which 6 members were transcription factors (TFs) and contained long C-terminal extensions with MYB-like DNA binding domains (Table 4). The other two type-B RRs, CmRR8 and CmRR9, only had Rec domains and their MYB-like domains might be lost during the evolution of the melon RR family (Table 4). These type-B CmRRs shared high sequence similarities to their homologs, ARR2, ARR11 and ARR12, in Arabidopsis (Table 4). It has been reported that Arabidopsis ARR2 and ARR12 play key roles in ethylene and CK signaling, respectively     .
Six melon PRRs contained highly-diverged Rec domains (Table 4) and C-terminal extensions. Intriguingly, the CCT-domain and MYB-like domain in type-B RRs were also found in CmPRR1/CmPRR6 and CmPRR2/CmPRR3, respectively. However, the exception occurred to the CmPRR4 and CmPRR5, which lacked both the CCT and the MYB-like domains (Table 4). These great divergences should be paid more attentions on in the future study.
This work was supported by National Natural Science Foundation of China (31401894 and 31501781), “Taishan Scholar” Foundation of the People’s Government of Shandong Province, and China Postdoctoral Science Foundation (2017M612741).
Table A1. Information of abbreviation notation.