Genome-Wide Identification of Two-Component Signal Transduction System Genes in Melon ( Cucumis melon L . )

Two-component system (TCS) is responsible for cytokinin signaling, which plays critical roles in plant development and physiological process. This system is generally composed of two signaling factors, a histidine kinase (HK) and a response regulator (RR) that is associated with a histidine phosphotransfer (HP) protein. In this study, we performed systematic investigation on TCS genes in melon (Cucumis melon L.). We identified 44 TCS genes in melon, including 18 HK(L)s (9 HKs and 9 HKLs), 5 HPs (4 authentic and 1 pseudo), and 21 RRs (7 Type-A, 8 Type-B, and 6 pseudo). The classification and structure of these melon TCS members were introduced in detail as well. Our results provided new insights into the characteristics of the melon TCS genes and might benefit their functional study in future.


Identification of the Putative Melon TCS Genes
Previously cucumber and watermelon TCS members have been successfully identified by using Arabidopsis TCS genes [13].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 [14].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 together with position information in melon genome were obtained from Cu curbit Genomics Database (http://cucurbitgenomics.org/organism/3).The transmembrane domains of melon TCS proteins were analyzed by TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/).

Phylogenetic Analysis and Gene Structure Construction
Phylogenetic analysis of the full-length protein sequences was conducted using MEGA5 [15].The evolutionary history was inferred using the Neighbor-Joining method with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates) [16] [17].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/).
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).3), a positive regulators in CK signaling [26], 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 [26].CmPHP1 exhibited the longest CDS and  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://www.cbs.dtu.dk/services/TMHMM/). 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 [27].
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  participating in a negative feedback loop to reduce the plant sensitivity to cytokinins [28] [25].
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 [29] [30] [31] [32] [33].
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.

Figure 1 .
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.

Figure 2 .
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.

Table 1 .
Summary of the TCS gene number identified in plants.

Table 2 .
Features of HK genes in melon.

Table 3 .
Features of HP genes in melon.

Table 4 .
Features of RR genes in melon.