BACKGROUND
the tcp gene family, a small group of transcription factors  exclusive to higher plants, was first described in  <dig>  <cit> .
the family plays important roles in regulating diverse physiological and biological processes, including phytohormone biosynthesis and signal transduction, leaf morphogenesis and senescence, branching, flower development, pollen development and the circadian clock .
tcp proteins are characterized by a 59-amino-acid non-canonical basic-helix-loop-helix  motif that is responsible for dna binding, nuclear targeting and pair-wise protein–protein interaction  <cit> .
this domain was first identified from four proteins with critical roles in the evolution and developmental control of plant morphology: teosinte branched  <dig>  of maize , cycloidea  of snapdragon  and the proliferating cell factors  <dig> and  <dig>  of rice  .
thus the name of the tcp tf family is derived from the acronym for these proteins.
tcp genes can be divided into two subfamilies based on the homology of the tcp domains: class i  and class ii   <cit> .
tcp class i, also known as the pcf subfamily, contains rice ospcf <dig> and ospcf <dig>  whereas tcp class ii is further subdivided into the cin and cyc/tb <dig> subclades  <cit> .
the most obvious difference between the two classes is a four-amino-acid deletion in the basic region of the tcp domain of class i compared with that of class ii proteins.
moreover, the dna binding sequence for the two classes differs slightly but partly overlaps   <cit> .
accumulating evidence confirms that class i tcp proteins mainly play a role in cell growth and proliferation  <cit> , whereas the cin proteins may be involved in lateral organ development and the cyc/tb <dig> clade is mainly involved in the development of axillary meristems giving rise to either flowers or lateral shoots .
generally, the two classes of tcp genes are considered to act antagonistically on specific biological processes.
class i genes are usually assumed to promote plant growth, mainly based on the finding that ospcf1/ospcf <dig> and attcp <dig> act as transcriptional activators of pcna and cycb1; <dig> genes  <cit> .
in practice, most class i single mutants do not show conspicuous phenotypic variation, which might be because of functional redundancies.
for example, increasing evidence demonstrates that attcp <dig> and attcp <dig> function redundantly to regulate biological processes and influence plant structure.
the two genes also mediate responses of leaves and flowers to cytokinin and promotion of seed germination by gibberellin  .
more recently, attcp <dig> and attcp <dig> were shown to repress endoreduplication by directly regulating the expression of cell-cycle genes to influence cell and organ growth  <cit> .
notable plant morphological changes are observed in the tcp <dig> tcp <dig> double mutant, such as shortened internode length as well as varied leaf and sepal morphology, whereas single mutants show mild phenotypic defects  <cit> .
moreover, attcp <dig> and attcp <dig> play a positive role in a redundant manner with attcp <dig> in the control of leaf senescence, as tcp <dig> tcp <dig> and tcp <dig> tcp <dig> double mutants exhibit earlier onset of senescence in comparison with the wild type, whereas none of the single mutants exhibit accelerated senescence  <cit> .
by contrast, many phenotypic observations on mutants suggest that the class ii tcp proteins usually have preventative roles in cell growth and proliferation.
cin-type genes limit cell proliferation at the margins of the developing leaf primordium.
in snapdragon, arabidopsis and tomato cin-type mutants, leaf cells exhibit the ability to continue to divide for a longer period compared with the wild type, thus generating larger leaves of altered shape and/or with a crinkled surface  <cit> .
in addition, many tb1-type tcp genes act as axillary bud-specific regulators, such as tb <dig> of maize  <cit> , atbrc <dig> and atbrc <dig> of arabidopsis  <cit> , psbrc <dig> of pea   <cit>  and osfc1/ostb <dig> of rice  <cit> .
defects in these genes result in excessive shoot branching, which are indicative of a negative function of these tcp genes on bud activity .
in some instances, class ii tcp genes may also play positive roles in plant growth and development.
attcp <dig>  a cyc/tb <dig> subclade member, is implicated in the control of floral symmetry  <cit> .
over-expression of a dominant-negative form of tcp <dig>  tcp1-srdx, results in a dwarfed phenotype as well as defects in the longitudinal elongation of cotyledonary petioles, rosette leaves and inflorescence stems in arabidopsis  <cit> .
to date, only a small number of tcp tfs have been identified and functionally characterized in model plants such as arabidopsis and rice.
watermelon , an important cucurbit crop, is widely cultivated throughout the world.
however, little information is available on the watermelon tcp family.
in this study, a global analysis of the tcp gene family in watermelon was carried out for the first time.
twenty-seven cltcp genes were identified in the watermelon genome and a systematic analysis, including determination of chromosomal location, phylogenetic relationships, gene duplication, conserved motifs and expression pattern was performed.
plant height is an important agronomic trait of watermelon.
normally, watermelon genotypes of reduced plant height are more suitable for intensive culture and early maturation in a greenhouse.
cltcp genes involved in the regulation of plant height in watermelon were identified in this research.
RESULTS
identification of tcp genes in citrullus lanatus
to identify tcp protein-coding genes in watermelon, arabidopsis and rice tcp proteins sequences were employed as the query for a blast search against the cucurbit genomics database .
twenty-seven putative tcp tfs, which contained the conserved tcp domain, were identified .
the results of a search for watermelon tcp family members in the plant transcription factor datebase  were in agreement with the former search.
due to the lack of standard annotations designated to the  <dig> tcp genes in watermelon, we named the genes cltcp1a to cltcp <dig> consistent with the arabidopsis tcp proteins that showed the highest sequence similarity and following the gene nomenclature system applied to arabidopsis.
the length of the  <dig> newly identified cltcp tfs ranged from  <dig> to  <dig> amino acids with an average of  <dig>  amino acids.
other characteristics of the cltcp tfs, including molecular weight , isoelectric point , type and chromosome location, are listed in table  <dig>  the cltcp tfs can be classified into the two tcp classes based on the differences within their tcp domains:  <dig> of the tfs belong to class i because of the presence of a four-amino-acid deletion in the basic domain relative to the other tfs; the  <dig> class ii cltcp tfs can be further clustered into the cin subclass and the cyc/tb <dig> subclass .
the genomic location of each cltcp in watermelon is shown in fig.
<dig>  the  <dig> cltcp genes were mapped to nine chromosomes.
moreover, based on a neighbor-joining  phylogenetic tree constructed from the full-length amino acid sequences, a putative orthologous relationship between the  <dig> cltcp tfs and  <dig> attcp tfs was established .
the number of tcp genes in watermelon is similar to that in arabidopsis, which is in strong agreement with the fact that the number of protein-coding genes in the watermelon genome   <cit>  approximates that in arabidopsis   <cit> .
a number of arabidopsis tcp genes have more than one counterpart in the watermelon genome, which might be a result of differential gene expansion in watermelon and arabidopsis after their divergence from a common ancestor.table  <dig> tcp gene family in citrullus lanatus
cltcp1a
cltcp1b
cltcp2a
cltcp2b
cltcp3
cltcp4
cltcp5
cltcp7
cltcp8
cltcp9
cltcp10a
cltcp10b
cltcp11
cltcp12a
cltcp12b
cltcp13
cltcp14a
cltcp14b
cltcp15
cltcp16
cltcp17
cltcp18a
cltcp18b
cltcp19
cltcp20a
cltcp20b
cltcp21
aa amino acid, mw molecular weight, pi isoelectric point, chr chromosome
fig.
<dig> visualization of the tcp maps linkage groups.
a circos diagram illustrates the relative positions of tcp genes.
the genes are plotted against their linked counterpart chromosomes.
chromosomal locations were determined according to chromosomal location information gathered from tair  and cucurbit genomics database .
the map was obtained using circos software
phylogenetic analysis and conserved motifs
to evaluate the phylogenetic relationships among the tcp proteins in watermelon, arabidopsis and rice, an unrooted phylogenetic tree was constructed using the nj method from a multiple sequence alignment of  <dig> watermelon,  <dig> arabidopsis and  <dig> rice tcp proteins .
the tcps were divided into  <dig> subgroups, designated group a to group k, according to their sequence features within and outside the tcp domain.
the tcps in groups a, b and c belonged to the class ii subfamily cin-type, group d belonged to the class ii cyc/tb1-type, whereas the remainder of the tcps belonged to the class i subfamily .
the tcp genes from the three species were distributed in almost all clades, which indicated that the tcp family diversified before the divergence of these plants.
notably, rice tcp was absent in group e and a similar result was observed for sorghum bicolor .
this finding implies that this clade may have been lost in rice and sorghum, or was acquired in an ancestor of arabidopsis and citrullus after the divergence of monocots and dicots.fig.
<dig> phylogenetic relationships of tcp transcription factors from watermelon, arabidopsis and rice.
the unrooted phylogenetic tree was constructed using mega  <dig>  with the neighbor-joining method.
support for the topology was assessed by means of a bootstrap analysis with  <dig> replicates
analysis of the conserved motif structure was performed to confirm the validity of the phylogenetic tree.
the r domain, an 18– <dig> residue arginine-rich motif, is absent in all class i proteins and is mainly present in cyc/tb <dig> proteins.
the mir <dig> site is only present in a subset of the cin-like genes .
in arabidopsis, mir <dig> modulates jasmonate biosynthesis, negatively regulates leaf growth, positively regulates leaf senescence and affects petal development.
these functions are dependent on post-transcriptional regulation of the mir319-targeted tcp genes   <cit> .
in the present study, five cin-type cltcp genes  contained the putative mir <dig> target site and shared the highest sequence similarity with the arabidopsis mir319-targeted tcp genes .
these findings indicated that regulation of hormone response and leaf development by mirna-targeted homologous tcp tfs may be conserved in watermelon and arabidopsis.
in addition, exon/intron structure analysis showed that most of the cltcp genes lacked an intron, with the exception of cltcp1a and cltcp12b, which contained one intron, and cltcp18a contained two introns .
interestingly, these three cltcp genes belong to the cyc/tb1-type subclade.fig.
<dig> phylogenetic analysis and conserved motifs of tcp family members in arabidopsis thaliana and citrullus lanatus.
an unrooted phylogenetic tree, showing relationships between all tcp transcription factors in a. thaliana  and c. lanatus , was constructed using mega  <dig>  with the neighbor-joining method.
support for the topology was assessed by means of a bootstrap analysis with  <dig> replicates.
class i is highlighted in blue, and class ii is highlighted in yellow.
on the right is the protein structure constructed using dog  <dig>  indicating conserved motifs: tcp domain  , r domain  .
the position of the microrna mir <dig> recognition sequence in the mrna is indicated in light purple .
the scale bar represents amino acid length
expression profiles of tcp genes in citrullus lanatus
to predict possible functions of tcp genes in watermelon, we performed semi-quantitative pcr  analysis of transcripts in different organs, including the seed, cotyledon, leaf, root, internode, shoot apical meristem , male and female flower buds, and fruit.
interestingly,expression analysis showed that every class/clade showed a characteristic expression profile.
as indicated in fig.
<dig>  most cin-type cltcp genes were not expressed or only weakly expressed in the root, flower or fruit, and were more highly expressed in the seed, cotyledon and leaf, which suggested that these genes may perform important roles in the shoot.
most cyc/tb1-type cltcp genes were relatively weakly expressed in the seed, leaf of early stage and root, but were relatively highly expressed in specific tissues.
for example, cltcp18b and cltcp12b were relatively highly expressed in the internode and sam beyond the six-leaf stage, and in the flower and fruit, whereas cltcp1a, cltcp1b and cltcp12a were only expressed in the internode and sam beyond the six-leaf stage.
these results indicated that cyc/tb1-type cltcp genes might play important roles in the development of internodes and flowers.
generally, class ii tcp genes, which function in a similar manner mainly by suppressing cell division and plant growth, exhibit tissue-specific expression pattern.
cyc/tb <dig> subclade genes have been long considered to be key players in the development of axillary meristems giving rise to either flowers or lateral shoots.
attcp <dig>  the gene most closely related to cyc, is involved in the longitudinal elongation of leaves.
the arabidopsis gain-of-function tcp1-1d mutant shows an elongated-leaf phenotype, whereas expression of a tcp1-srdx chimeric repressor gene in the wild type results in the opposite phenotype to the tcp1-1d mutant  <cit> .
moreover, mutation of the hacyc2c gene, a tcp1/cyc homolog in sunflower, promoted the developmental switch from sterile to hermaphrodite flowers  <cit> .
expression of attcp <dig> is strong in the petiole, lower portion of the inflorescence stem, and the midrib and distal region of expanding rosette leaves.
two cltcp <dig> genes, which are closely related to attcp <dig>  were strongly expressed in the internode and sam of watermelon .
this result is partly consistent with the expression pattern of attcp <dig> in arabidopsis and implies that cltcp <dig> genes may play roles in internode and inflorescence development in watermelon.
attcp <dig>  which is also known as branched <dig>  and teosinte branched1-like <dig> , acts downstream of auxin and strigolactone to coordinate axillary bud outgrowth  <cit> .
attcp <dig> also represses the floral transition of the axillary meristems by interacting with flowering locus t   <cit> .
attcp <dig>  also known as brc <dig>  exhibits a weaker or no mutant phenotype compared with attcp <dig>  <cit> .
furthermore, no interactions between the brc <dig> and ft proteins have been detected in yeast two-hybrid experiments  <cit> .
in watermelon, the expression level of cltcp12b and cltcp18b was significantly higher in the internode, sam, flower and fruit.
expression of cltcp12a was detected only in the internode and sam .
these observations suggested that these genes are likely to perform similar roles in branch and/or inflorescence development in watermelon to those of the arabidopsis homologs.
in contrast, cin-type tcp genes are considered to have originated prior to cyc/tb1-type tcps and are important for generation of the flat surface and smooth margin of the leaf.
thus, cin-type mutants usually exhibit crinkly and/or serrated leaves  <cit> .
in watermelon, expression of all cin-type tcp genes was detected in the cotyledon, leaf and sam .
this result is consistent with their predicted roles in leaf and lateral-organ development.fig.
<dig> expression patterns of citrullus lanatus tcp genes in different tissues.
the expression profile of cltcp genes in the seed, leaf, internode, shoot tip, root, flowers and fruit was obtained through semi-quantitative pcr analysis.
expression of the clyls <dig> gene was monitored as an internal control.
the phylogenetic tree of all tcp transcription factors in citrullus lanatus was constructed using mega  <dig>  with the neighbor-joining method.
support for the topology was assessed by means of a bootstrap analysis with  <dig> replicates.
s: seeds at germination; c: cotyledons; l2: leaves at two-true-leaf stage; r2: roots at two-true-leaf stage; l6: leaves at six-true-leaf stage; r6: roots at six-true-leaf stage; i: internodes; sam: shoot apical meristem; m: male flower; fm: female flower; f: immature fruit 3 hours after pollination
in contrast, most class i genes, which usually promote plant growth and cell proliferation, showed more widespread and less tissue-specific expression patterns, such as in leaf, flower, and at an early stage of fruit development .
this finding implied that these genes may play diverse regulatory roles at multiple development stages.
in arabidopsis, several important functions of class i tcp tfs have been discovered even though few phenotypic variations are observed in the single mutants.
for example: attcp <dig> is proposed to be involved in mitochondrial biogenesis  <cit> .
attcp <dig> and attcp <dig> are reported to modulate cell proliferation during seed, leaf, floral and internode development  <cit> .
attcp <dig> may also be important for endoreduplication  <cit> .
attcp <dig> plays a role in early pollen development  <cit> .
attcp <dig>  which acts upstream of attcp <dig>  controls leaf development via the jasmonate signaling pathway  <cit> .
all of these attcp genes have at least one counterpart in watermelon, implying that class i tcp in watermelon may perform similar functions.
taken together, the above-mentioned findings from model plants highlight that the tcp family performs diverse functions in multiple biological processes.
cltcp genes are likely to share conserved functions with arabidopsis homologs, as they show not only high sequence similarity but also similar expression patterns.
role of cltcp14a and cltcp <dig> in plant height
cltcp14a and cltcp <dig> are members of the class i subfamily of tcp tfs in watermelon .
these two cltcp genes are closely related to arabidopsis attcp <dig> and attcp <dig> as well as antirrhinum tcp tf tic  <cit> .
given the unavailability of a tcp-related mutant in watermelon, we examined the function of these two cltcp genes in four independent transgenic lines , which over-expressed cltcp14a or cltcp <dig> in both arabidopsis col- <dig> and tcp <dig> tcp <dig> double-mutant backgrounds.
after growth under long-day conditions for 42 days, the double-mutant seedlings showed a significant reduction in inflorescence height than that of the wild type .
no visible phenotype was identified in any single mutant, similar to the observations of kieffer et al.
<cit> .
ectopic expression of either cltcp14a or cltcp <dig> was sufficient to restore the inflorescence height and stem internodes length of tcp <dig> tcp <dig> double mutant to that of the wild type.
the p35s:cltcp14a-wt and p35s:cltcp15-wt lines exhibited an increase in inflorescence height compared with that of the wild type .
these results suggested that cltcp14a and cltcp <dig> function redundantly to control arabidopsis plant height and may play positive roles in stem internode elongation in watermelon.
scanning electron microscopy revealed that the double mutant bore excessively branched trichomes compared with those of the wild type, and that overexpression of cltcp14a or cltcp <dig> inhibited trichome branching in both backgrounds .
furthermore, ectopic expression of each watermelon tcp gene in both backgrounds increased the relative chlorophyll content in mature leaves .
these findings suggested that cltcp14a and cltcp <dig> may also be involved in leaf development.fig.
<dig> morphological effects of constitutive expression of cltcp14a and cltcp <dig> in transgenic arabidopsis.
a seedlings of the wild type , double mutant  and p35s:cltcp14a in wt and double-mutant backgrounds were grown under long-day conditions for 42 days.
b seedlings of the wt, double mutant and p35s:cltcp <dig> in wt and double-mutant backgrounds were grown under long-day conditions for 42 days.
c inflorescence height of seedlings as shown in .
d inflorescence height of seedlings as shown in .
scale bars = 3 cm.
different lower-case letters denote a significant difference in inflorescence height among genotypes .
values are means ± sd
given that ga is a regulator of plant height, we investigated whether overexpression of cltcp14a and cltcp <dig> affected ga biosynthesis and metabolism.
atko <dig> and atga2ox <dig>  which are involved in ga biosynthesis and degradation, were more weakly and more highly expressed, respectively, in the tcp <dig> tcp <dig> double mutant compared with those of the wild type.
ectopic expression of each watermelon tcp in the tcp <dig> tcp <dig> background revealed positive and negative impacts on the expression of atko <dig> and atga2ox <dig>  respectively .
however, these effects were not observed in the wild-type background.
the ga receptor, atgid1a, was slightly but significantly up-regulated in cltcp14a- and cltcp15- overexpressing arabidopsis .
these results suggested that overexpression of cltcp14a and cltcp <dig> may enhance ga accumulation and signaling in tcp <dig> tcp <dig> and the effects of these tcp genes on plant height may be associated with the ga pathway.
interestingly, the expression of all ga-related genes differed significantly between cltcp14a- and cltcp15-transgenic arabidopsis, which might reflect the higher expression level of cltcp14a compared with that of cltcp <dig> in each transgenic line .fig.
<dig> expression of gibberellic acid -related genes in transgenic arabidopsis.
the relative expression level of a the ga biosynthesis gene atko <dig>  b the ga degradation gene atga
2
ox <dig> and c the ga receptor gene atgid1a in seedlings of the wild type , double-mutant , p35s:cltcp14a and p35s:cltcp <dig> in both wt and double-mutant backgrounds was determined by quantitative rt-pcr.
expression of the clyls <dig> gene was monitored as an internal control.
different lower-case letters denote a significant difference in relative expression level .
values are means ± sd
in addition, the effects of ga and chlormequat chloride  on plant height as well as cltcp14a and cltcp <dig> expression were examined in watermelon.
the results revealed that ga and ccc were a functional enhancer and inhibitor, respectively, of watermelon plant height .
both regulators likely function by affecting internode length rather than internode number, as no differences in internode numbers were observed.
expressions of both cltcp14a and cltcp <dig> was significantly up- and down-regulated by ga and ccc treatment, respectively .
these results confirmed that cltcp14a and cltcp <dig> might positively regulate watermelon plant height and internode length via a ga-related pathway.fig.
<dig> effects of gibberellic acid  and chlormequat chloride  on plant height and expression of cltcp genes in watermelon.
watermelon seedlings were treated with water , ga and ccc at the two-true-leaf stage.
a and b plant height of watermelon seedlings at the six-true-leaf stage.
scale bars = 7 cm.
different lower-case letters denote a significant difference in plant height between treatments .
values are means  ± sd.
c relative expression level of cltcp14a in watermelon seedlings 1 day after treatment.
d relative expression level of cltcp <dig> in watermelon seedlings 1 day after different treatments.
different lower-case letters denote a significant difference in relative expression level .
values are means ± sd
plant height is an important agronomic trait in watermelon, which dramatically affects planting density and fruiting position in the field.
tcp tfs, as well known cell proliferation regulators, are undoubtedly important participants in internode and plant elongation.
the present results revealed that cltcp14a and cltcp <dig> redundantly regulated internode length and plant height via a ga-related pathway in transgenic arabidopsis .
in arabidopsis, attcp <dig> and attcp <dig> are reported to regulate internode development by promoting cell proliferation, based mainly on the phenotypes observed in double-mutant and tcp14:srdx lines  <cit> .
the present results provide direct evidence for this genotype–phenotype correlation.
moreover, attcp <dig> and attcp <dig> are expressed in internodes of young inflorescence stems, young flower pedicels, cotyledons and leaf primordia  <cit> .
these results are generally consistent with the present expression analysis of cltcp genes in watermelon .
moreover, it was reported recently that attcp <dig> and attcp <dig> mediate ga-dependent activation of the cell cycle during seed germination  <cit> .
thus, we hypothesize that cltcp14a and cltcp <dig> may also act downstream of ga and promote cell proliferation during internode formation in a similar manner.
interestingly, our findings suggest that cltcp14a and cltcp <dig> might also affect ga biosynthesis and signaling , which might result from a feedback regulatory mechanism.
CONCLUSIONS
in this study,  <dig> tcp genes were identified in the watermelon genome, which were distributed on nine chromosomes with different densities.
these tcp genes were classifiable into two classes based on the similarity in tcp domain.
expression analysis showed that members of each class/clade show a similar expression pattern.
moreover, many cltcp genes showed a similar expression pattern to that of their arabidopsis homologs, which suggests that the tcp family shows conserved functions in the two species.
in addition, the function of two cltcp genes, cltcp14a and cltcp <dig>  in the regulation of internode elongation was confirmed.
ultimately, these findings will lead to potential applications for the improvement of watermelon cultivars via genetic engineering.

 the tcp gene family, a small group of transcription factors exclusive to higher plants, was first described in. the family plays important roles in regulating diverse physiological and biological processes, including phytohormone biosynthesis and signal transduction, leaf morphogenesis and senescence, branching, flower development, pollen development and the circadian clock. tcp proteins are characterized by a 59-amino-acid non-canonical basic-helix-loop-helix motif that is responsible for dna binding, nuclear targeting and pair-wise protein–protein interaction. this domain was first identified from four proteins with critical roles in the evolution and developmental control of plant morphology: teosinte branched of maize, cycloidea of snapdragon and the proliferating cell factors and of rice. thus the name of the tcp tf family is derived from the acronym for these proteins. tcp genes can be divided into two subfamilies based on the homology of the tcp domains: class i and class ii. tcp class i, also known as the pcf subfamily, contains rice ospcf and ospcf whereas tcp class ii is further subdivided into the cin and cyc/tb subclades. the most obvious difference between the two classes is a four-amino-acid deletion in the basic region of the tcp domain of class i compared with that of class ii proteins. moreover, the dna binding sequence for the two classes differs slightly but partly overlaps. accumulating evidence confirms that class i tcp proteins mainly play a role in cell growth and proliferation, whereas the cin proteins may be involved in lateral organ development and the cyc/tb clade is mainly involved in the development of axillary meristems giving rise to either flowers or lateral shoots. generally, the two classes of tcp genes are considered to act antagonistically on specific biological processes. class i genes are usually assumed to promote plant growth, mainly based on the finding that ospcf1/ospcf and attcp act as transcriptional activators of pcna and cycb1; genes. in practice, most class i single mutants do not show conspicuous phenotypic variation, which might be because of functional redundancies. for example, increasing evidence demonstrates that attcp and attcp function redundantly to regulate biological processes and influence plant structure. the two genes also mediate responses of leaves and flowers to cytokinin and promotion of seed germination by gibberellin. more recently, attcp and attcp were shown to repress endoreduplication by directly regulating the expression of cell-cycle genes to influence cell and organ growth. notable plant morphological changes are observed in the tcp tcp double mutant, such as shortened internode length as well as varied leaf and sepal morphology, whereas single mutants show mild phenotypic defects. moreover, attcp and attcp play a positive role in a redundant manner with attcp in the control of leaf senescence, as tcp tcp and tcp tcp double mutants exhibit earlier onset of senescence in comparison with the wild type, whereas none of the single mutants exhibit accelerated senescence. by contrast, many phenotypic observations on mutants suggest that the class ii tcp proteins usually have preventative roles in cell growth and proliferation. cin-type genes limit cell proliferation at the margins of the developing leaf primordium. in snapdragon, arabidopsis and tomato cin-type mutants, leaf cells exhibit the ability to continue to divide for a longer period compared with the wild type, thus generating larger leaves of altered shape and/or with a crinkled surface. in addition, many tb1-type tcp genes act as axillary bud-specific regulators, such as tb of maize, atbrc and atbrc of arabidopsis, psbrc of pea and osfc1/ostb of rice. defects in these genes result in excessive shoot branching, which are indicative of a negative function of these tcp genes on bud activity. in some instances, class ii tcp genes may also play positive roles in plant growth and development. attcp a cyc/tb subclade member, is implicated in the control of floral symmetry. over-expression of a dominant-negative form of tcp tcp1-srdx, results in a dwarfed phenotype as well as defects in the longitudinal elongation of cotyledonary petioles, rosette leaves and inflorescence stems in arabidopsis. to date, only a small number of tcp tfs have been identified and functionally characterized in model plants such as arabidopsis and rice. watermelon, an important cucurbit crop, is widely cultivated throughout the world. however, little information is available on the watermelon tcp family. in this study, a global analysis of the tcp gene family in watermelon was carried out for the first time. twenty-seven cltcp genes were identified in the watermelon genome and a systematic analysis, including determination of chromosomal location, phylogenetic relationships, gene duplication, conserved motifs and expression pattern was performed. plant height is an important agronomic trait of watermelon. normally, watermelon genotypes of reduced plant height are more suitable for intensive culture and early maturation in a greenhouse. cltcp genes involved in the regulation of plant height in watermelon were identified in this research.  identification of tcp genes in citrullus lanatus to identify tcp protein-coding genes in watermelon, arabidopsis and rice tcp proteins sequences were employed as the query for a blast search against the cucurbit genomics database. twenty-seven putative tcp tfs, which contained the conserved tcp domain, were identified. the results of a search for watermelon tcp family members in the plant transcription factor datebase were in agreement with the former search. due to the lack of standard annotations designated to the tcp genes in watermelon, we named the genes cltcp1a to cltcp consistent with the arabidopsis tcp proteins that showed the highest sequence similarity and following the gene nomenclature system applied to arabidopsis. the length of the newly identified cltcp tfs ranged from to amino acids with an average of amino acids. other characteristics of the cltcp tfs, including molecular weight, isoelectric point, type and chromosome location, are listed in table  the cltcp tfs can be classified into the two tcp classes based on the differences within their tcp domains: of the tfs belong to class i because of the presence of a four-amino-acid deletion in the basic domain relative to the other tfs; the class ii cltcp tfs can be further clustered into the cin subclass and the cyc/tb subclass. the genomic location of each cltcp in watermelon is shown in fig.  the cltcp genes were mapped to nine chromosomes. moreover, based on a neighbor-joining phylogenetic tree constructed from the full-length amino acid sequences, a putative orthologous relationship between the cltcp tfs and attcp tfs was established. the number of tcp genes in watermelon is similar to that in arabidopsis, which is in strong agreement with the fact that the number of protein-coding genes in the watermelon genome approximates that in arabidopsis. a number of arabidopsis tcp genes have more than one counterpart in the watermelon genome, which might be a result of differential gene expansion in watermelon and arabidopsis after their divergence from a common ancestor.table tcp gene family in citrullus lanatus cltcp1a cltcp1b cltcp2a cltcp2b cltcp3 cltcp4 cltcp5 cltcp7 cltcp8 cltcp9 cltcp10a cltcp10b cltcp11 cltcp12a cltcp12b cltcp13 cltcp14a cltcp14b cltcp15 cltcp16 cltcp17 cltcp18a cltcp18b cltcp19 cltcp20a cltcp20b cltcp21 aa amino acid, mw molecular weight, pi isoelectric point, chr chromosome fig. visualization of the tcp maps linkage groups. a circos diagram illustrates the relative positions of tcp genes. the genes are plotted against their linked counterpart chromosomes. chromosomal locations were determined according to chromosomal location information gathered from tair and cucurbit genomics database. the map was obtained using circos software phylogenetic analysis and conserved motifs to evaluate the phylogenetic relationships among the tcp proteins in watermelon, arabidopsis and rice, an unrooted phylogenetic tree was constructed using the nj method from a multiple sequence alignment of watermelon, arabidopsis and rice tcp proteins. the tcps were divided into subgroups, designated group a to group k, according to their sequence features within and outside the tcp domain. the tcps in groups a, b and c belonged to the class ii subfamily cin-type, group d belonged to the class ii cyc/tb1-type, whereas the remainder of the tcps belonged to the class i subfamily. the tcp genes from the three species were distributed in almost all clades, which indicated that the tcp family diversified before the divergence of these plants. notably, rice tcp was absent in group e and a similar result was observed for sorghum bicolor. this finding implies that this clade may have been lost in rice and sorghum, or was acquired in an ancestor of arabidopsis and citrullus after the divergence of monocots and dicots.fig. phylogenetic relationships of tcp transcription factors from watermelon, arabidopsis and rice. the unrooted phylogenetic tree was constructed using mega with the neighbor-joining method. support for the topology was assessed by means of a bootstrap analysis with replicates analysis of the conserved motif structure was performed to confirm the validity of the phylogenetic tree. the r domain, an 18– residue arginine-rich motif, is absent in all class i proteins and is mainly present in cyc/tb proteins. the mir site is only present in a subset of the cin-like genes. in arabidopsis, mir modulates jasmonate biosynthesis, negatively regulates leaf growth, positively regulates leaf senescence and affects petal development. these functions are dependent on post-transcriptional regulation of the mir319-targeted tcp genes. in the present study, five cin-type cltcp genes contained the putative mir target site and shared the highest sequence similarity with the arabidopsis mir319-targeted tcp genes. these findings indicated that regulation of hormone response and leaf development by mirna-targeted homologous tcp tfs may be conserved in watermelon and arabidopsis. in addition, exon/intron structure analysis showed that most of the cltcp genes lacked an intron, with the exception of cltcp1a and cltcp12b, which contained one intron, and cltcp18a contained two introns. interestingly, these three cltcp genes belong to the cyc/tb1-type subclade.fig. phylogenetic analysis and conserved motifs of tcp family members in arabidopsis thaliana and citrullus lanatus. an unrooted phylogenetic tree, showing relationships between all tcp transcription factors in a. thaliana and c. lanatus, was constructed using mega with the neighbor-joining method. support for the topology was assessed by means of a bootstrap analysis with replicates. class i is highlighted in blue, and class ii is highlighted in yellow. on the right is the protein structure constructed using dog indicating conserved motifs: tcp domain, r domain. the position of the microrna mir recognition sequence in the mrna is indicated in light purple. the scale bar represents amino acid length expression profiles of tcp genes in citrullus lanatus to predict possible functions of tcp genes in watermelon, we performed semi-quantitative pcr analysis of transcripts in different organs, including the seed, cotyledon, leaf, root, internode, shoot apical meristem, male and female flower buds, and fruit. interestingly,expression analysis showed that every class/clade showed a characteristic expression profile. as indicated in fig.  most cin-type cltcp genes were not expressed or only weakly expressed in the root, flower or fruit, and were more highly expressed in the seed, cotyledon and leaf, which suggested that these genes may perform important roles in the shoot. most cyc/tb1-type cltcp genes were relatively weakly expressed in the seed, leaf of early stage and root, but were relatively highly expressed in specific tissues. for example, cltcp18b and cltcp12b were relatively highly expressed in the internode and sam beyond the six-leaf stage, and in the flower and fruit, whereas cltcp1a, cltcp1b and cltcp12a were only expressed in the internode and sam beyond the six-leaf stage. these results indicated that cyc/tb1-type cltcp genes might play important roles in the development of internodes and flowers. generally, class ii tcp genes, which function in a similar manner mainly by suppressing cell division and plant growth, exhibit tissue-specific expression pattern. cyc/tb subclade genes have been long considered to be key players in the development of axillary meristems giving rise to either flowers or lateral shoots. attcp the gene most closely related to cyc, is involved in the longitudinal elongation of leaves. the arabidopsis gain-of-function tcp1-1d mutant shows an elongated-leaf phenotype, whereas expression of a tcp1-srdx chimeric repressor gene in the wild type results in the opposite phenotype to the tcp1-1d mutant. moreover, mutation of the hacyc2c gene, a tcp1/cyc homolog in sunflower, promoted the developmental switch from sterile to hermaphrodite flowers. expression of attcp is strong in the petiole, lower portion of the inflorescence stem, and the midrib and distal region of expanding rosette leaves. two cltcp genes, which are closely related to attcp were strongly expressed in the internode and sam of watermelon. this result is partly consistent with the expression pattern of attcp in arabidopsis and implies that cltcp genes may play roles in internode and inflorescence development in watermelon. attcp which is also known as branched and teosinte branched1-like, acts downstream of auxin and strigolactone to coordinate axillary bud outgrowth. attcp also represses the floral transition of the axillary meristems by interacting with flowering locus t. attcp also known as brc exhibits a weaker or no mutant phenotype compared with attcp. furthermore, no interactions between the brc and ft proteins have been detected in yeast two-hybrid experiments. in watermelon, the expression level of cltcp12b and cltcp18b was significantly higher in the internode, sam, flower and fruit. expression of cltcp12a was detected only in the internode and sam. these observations suggested that these genes are likely to perform similar roles in branch and/or inflorescence development in watermelon to those of the arabidopsis homologs. in contrast, cin-type tcp genes are considered to have originated prior to cyc/tb1-type tcps and are important for generation of the flat surface and smooth margin of the leaf. thus, cin-type mutants usually exhibit crinkly and/or serrated leaves. in watermelon, expression of all cin-type tcp genes was detected in the cotyledon, leaf and sam. this result is consistent with their predicted roles in leaf and lateral-organ development.fig. expression patterns of citrullus lanatus tcp genes in different tissues. the expression profile of cltcp genes in the seed, leaf, internode, shoot tip, root, flowers and fruit was obtained through semi-quantitative pcr analysis. expression of the clyls gene was monitored as an internal control. the phylogenetic tree of all tcp transcription factors in citrullus lanatus was constructed using mega with the neighbor-joining method. support for the topology was assessed by means of a bootstrap analysis with replicates. s: seeds at germination; c: cotyledons; l2: leaves at two-true-leaf stage; r2: roots at two-true-leaf stage; l6: leaves at six-true-leaf stage; r6: roots at six-true-leaf stage; i: internodes; sam: shoot apical meristem; m: male flower; fm: female flower; f: immature fruit 3 hours after pollination in contrast, most class i genes, which usually promote plant growth and cell proliferation, showed more widespread and less tissue-specific expression patterns, such as in leaf, flower, and at an early stage of fruit development. this finding implied that these genes may play diverse regulatory roles at multiple development stages. in arabidopsis, several important functions of class i tcp tfs have been discovered even though few phenotypic variations are observed in the single mutants. for example: attcp is proposed to be involved in mitochondrial biogenesis. attcp and attcp are reported to modulate cell proliferation during seed, leaf, floral and internode development. attcp may also be important for endoreduplication. attcp plays a role in early pollen development. attcp which acts upstream of attcp controls leaf development via the jasmonate signaling pathway. all of these attcp genes have at least one counterpart in watermelon, implying that class i tcp in watermelon may perform similar functions. taken together, the above-mentioned findings from model plants highlight that the tcp family performs diverse functions in multiple biological processes. cltcp genes are likely to share conserved functions with arabidopsis homologs, as they show not only high sequence similarity but also similar expression patterns. role of cltcp14a and cltcp in plant height cltcp14a and cltcp are members of the class i subfamily of tcp tfs in watermelon. these two cltcp genes are closely related to arabidopsis attcp and attcp as well as antirrhinum tcp tf tic. given the unavailability of a tcp-related mutant in watermelon, we examined the function of these two cltcp genes in four independent transgenic lines, which over-expressed cltcp14a or cltcp in both arabidopsis col- and tcp tcp double-mutant backgrounds. after growth under long-day conditions for 42 days, the double-mutant seedlings showed a significant reduction in inflorescence height than that of the wild type. no visible phenotype was identified in any single mutant, similar to the observations of kieffer et al.. ectopic expression of either cltcp14a or cltcp was sufficient to restore the inflorescence height and stem internodes length of tcp tcp double mutant to that of the wild type. the p35s:cltcp14a-wt and p35s:cltcp15-wt lines exhibited an increase in inflorescence height compared with that of the wild type. these results suggested that cltcp14a and cltcp function redundantly to control arabidopsis plant height and may play positive roles in stem internode elongation in watermelon. scanning electron microscopy revealed that the double mutant bore excessively branched trichomes compared with those of the wild type, and that overexpression of cltcp14a or cltcp inhibited trichome branching in both backgrounds. furthermore, ectopic expression of each watermelon tcp gene in both backgrounds increased the relative chlorophyll content in mature leaves. these findings suggested that cltcp14a and cltcp may also be involved in leaf development.fig. morphological effects of constitutive expression of cltcp14a and cltcp in transgenic arabidopsis. a seedlings of the wild type, double mutant and p35s:cltcp14a in wt and double-mutant backgrounds were grown under long-day conditions for 42 days. b seedlings of the wt, double mutant and p35s:cltcp in wt and double-mutant backgrounds were grown under long-day conditions for 42 days. c inflorescence height of seedlings as shown in. d inflorescence height of seedlings as shown in. scale bars = 3 cm. different lower-case letters denote a significant difference in inflorescence height among genotypes. values are means ± sd given that ga is a regulator of plant height, we investigated whether overexpression of cltcp14a and cltcp affected ga biosynthesis and metabolism. atko and atga2ox which are involved in ga biosynthesis and degradation, were more weakly and more highly expressed, respectively, in the tcp tcp double mutant compared with those of the wild type. ectopic expression of each watermelon tcp in the tcp tcp background revealed positive and negative impacts on the expression of atko and atga2ox respectively. however, these effects were not observed in the wild-type background. the ga receptor, atgid1a, was slightly but significantly up-regulated in cltcp14a- and cltcp15- overexpressing arabidopsis. these results suggested that overexpression of cltcp14a and cltcp may enhance ga accumulation and signaling in tcp tcp and the effects of these tcp genes on plant height may be associated with the ga pathway. interestingly, the expression of all ga-related genes differed significantly between cltcp14a- and cltcp15-transgenic arabidopsis, which might reflect the higher expression level of cltcp14a compared with that of cltcp in each transgenic line.fig. expression of gibberellic acid -related genes in transgenic arabidopsis. the relative expression level of a the ga biosynthesis gene atko b the ga degradation gene atga 2 ox and c the ga receptor gene atgid1a in seedlings of the wild type, double-mutant, p35s:cltcp14a and p35s:cltcp in both wt and double-mutant backgrounds was determined by quantitative rt-pcr. expression of the clyls gene was monitored as an internal control. different lower-case letters denote a significant difference in relative expression level. values are means ± sd in addition, the effects of ga and chlormequat chloride on plant height as well as cltcp14a and cltcp expression were examined in watermelon. the results revealed that ga and ccc were a functional enhancer and inhibitor, respectively, of watermelon plant height. both regulators likely function by affecting internode length rather than internode number, as no differences in internode numbers were observed. expressions of both cltcp14a and cltcp was significantly up- and down-regulated by ga and ccc treatment, respectively. these results confirmed that cltcp14a and cltcp might positively regulate watermelon plant height and internode length via a ga-related pathway.fig. effects of gibberellic acid and chlormequat chloride on plant height and expression of cltcp genes in watermelon. watermelon seedlings were treated with water, ga and ccc at the two-true-leaf stage. a and b plant height of watermelon seedlings at the six-true-leaf stage. scale bars = 7 cm. different lower-case letters denote a significant difference in plant height between treatments. values are means  ± sd. c relative expression level of cltcp14a in watermelon seedlings 1 day after treatment. d relative expression level of cltcp in watermelon seedlings 1 day after different treatments. different lower-case letters denote a significant difference in relative expression level. values are means ± sd plant height is an important agronomic trait in watermelon, which dramatically affects planting density and fruiting position in the field. tcp tfs, as well known cell proliferation regulators, are undoubtedly important participants in internode and plant elongation. the present results revealed that cltcp14a and cltcp redundantly regulated internode length and plant height via a ga-related pathway in transgenic arabidopsis. in arabidopsis, attcp and attcp are reported to regulate internode development by promoting cell proliferation, based mainly on the phenotypes observed in double-mutant and tcp14:srdx lines. the present results provide direct evidence for this genotype–phenotype correlation. moreover, attcp and attcp are expressed in internodes of young inflorescence stems, young flower pedicels, cotyledons and leaf primordia. these results are generally consistent with the present expression analysis of cltcp genes in watermelon. moreover, it was reported recently that attcp and attcp mediate ga-dependent activation of the cell cycle during seed germination. thus, we hypothesize that cltcp14a and cltcp may also act downstream of ga and promote cell proliferation during internode formation in a similar manner. interestingly, our findings suggest that cltcp14a and cltcp might also affect ga biosynthesis and signaling, which might result from a feedback regulatory mechanism.  in this study, tcp genes were identified in the watermelon genome, which were distributed on nine chromosomes with different densities. these tcp genes were classifiable into two classes based on the similarity in tcp domain. expression analysis showed that members of each class/clade show a similar expression pattern. moreover, many cltcp genes showed a similar expression pattern to that of their arabidopsis homologs, which suggests that the tcp family shows conserved functions in the two species. in addition, the function of two cltcp genes, cltcp14a and cltcp in the regulation of internode elongation was confirmed. ultimately, these findings will lead to potential applications for the improvement of watermelon cultivars via genetic engineering. 
however, no comprehensive analysis of the tcp family in watermelon has been undertaken previously. furthermore, phylogenetic and structural analyses distinguished two homology classes within the cltcp family, designated class i and class ii. the plant-specific tcp transcription factor family, which is involved in the regulation of cell growth and proliferation, performs diverse functions in multiple aspects of plant growth and development. the functions of two cltcp genes, cltcp14a and cltcp in regulating plant height were confirmed by ectopic expression in arabidopsis wild-type and ortholog mutants. the class ii genes were differentiated into two subclasses, the cin subclass and the cyc/tb subclass. this study represents the first genome-wide analysis of the watermelon tcp gene family, which provides valuable information for understanding the classification and functions of the tcp genes in watermelon.
 the plant-specific tcp transcription factor family, which is involved in the regulation of cell growth and proliferation, performs diverse functions in multiple aspects of plant growth and development. however, no comprehensive analysis of the tcp family in watermelon has been undertaken previously.  a total of watermelon tcp encoding genes distributed on nine chromosomes were identified. phylogenetic analysis clustered the genes into distinct subgroups. furthermore, phylogenetic and structural analyses distinguished two homology classes within the cltcp family, designated class i and class ii. the class ii genes were differentiated into two subclasses, the cin subclass and the cyc/tb subclass. the expression patterns of all members were determined by semi-quantitative pcr. the functions of two cltcp genes, cltcp14a and cltcp in regulating plant height were confirmed by ectopic expression in arabidopsis wild-type and ortholog mutants.  this study represents the first genome-wide analysis of the watermelon tcp gene family, which provides valuable information for understanding the classification and functions of the tcp genes in watermelon. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tcptranscription factorswatermeloninternode elongationmodern agro-industry technology research system of chinacars-26-17zhang mingfang education department research program of zhejiang provincey201329960hu zhongyuan fundamental research funds for the central universities2013qna6013hu zhongyuan http://dx.doi.org/ /501100001809national natural science foundation of china31501782hu zhongyuan zhejiang provincial natural science foundation of chinalq16c150002hu zhongyuan issue-copyright-statement© the author 2016 