Genome-wide analysis of CsWOX transcription factor gene family in cucumber (Cucumis sativus L.)

WUSCHEL-related homeobox (WOX) transcription factors are plant-specific members that characterized by the presence of a homeodomain. It has been shown that WOX members regulate several aspects of plant development, but the biological functions of this CsWOX gene family remain largely unknown in cucumber (Cucumis sativus L.). In this study, we identified and characterized 11 putative CsWOX genes in cucumber, which are also divided into three major clades (e.g., the Ancient clade, the Intermediate clade and the WUS clade). Expression pattern analysis revealed tissue-specific expression patterns of CsWOX genes, including that CsWOX9 is mainly expressed in developing fruit and also has lower expression in tip and axillary bud, which was further confirmed by in situ hybridization assay. Moreover, overexpression of CsWOX9 in Arabidopsis led to increased branches and rosette leaves, and shorter siliques. Together, these results indicated that CsWOX members may regulate cucumber growth and development.

www.nature.com/scientificreports www.nature.com/scientificreports/ Cucumber (Cucumis sativus L.) is one of the important economic vegetable crops. Many growth and development processes affect cucumber quality and yield. Although WOX members have been shown to regulate many aspects of development in several plant species, only CsWOX1 was reported to regulate early reproductive organ development in cucumber 18 . It is unknown whether CsWOX members regulate different biological processes in cucumber. Here, we identified and characterized 11 putative CsWOX genes. To investigate the potential functions of CsWOX genes in cucumber, gene expression analysis showed that these CsWOX genes have different tissue-specific expression patterns. When introduced CsWOX9 driven by the 35S promoter into the Arabidopsis, the transgenic lines exhibited shorter siliques and increased branches, consistent with its expression pattern in cucumber. Together, these results indicated that CsWOX members may regulate development in cucumber.

Name
Gene Bioinformatics analysis of csWoX family members. The amino acid sequences of CsWOX members in cucumber were obtained from the Cucumber Genome Database (http://cucurbitgenomics.org/), and then the molecular weight and isoelectric points of these members were analyzed using ExPASy Proteomics Server (https://web.expasy.org/protparam/). The gene structure analysis was performed using the online program Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) 19 . The cis-acting elements in the promoters of CsWOX genes were analyzed using the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/) 20 . The full-length sequences of CsWOX family members were aligned using ClustalW in MEGA 5.0 and this alignment was used to generate the phylogenetic tree with Neighbor-Joining (NJ) method. Poisson correction, pairwise deletion and a bootstrap were conducted with 1000 replicates 21 .
Gene expression analysis. Total RNA was extracted using a Quick RNA Isolation Kit (Waryoung, Beijing, China) and cDNAs were synthesized using TianScript II RT Kit (Tiangen Biotech, China). The qRT-PCR analysis was performed using ABI PRISM 7500 Real-Time PCR System (Applied Biosystems). The cucumber UBIQUITIN (Csa000874) and Arabidopsis ACTIN2 (AT3G18780) genes were used as internal controls. Three biological replicates were performed. Sequences of all the primers used in this study were shown in Table S2.
For gene expression pattern analysis, the cucumber root, stem, leaf, tip and tendril of 16-day-old seedlings, axillary bud from the top of the lateral branch, male and female buds at the 10 th stage of flower development, male and female flowers at anthesis, 1.5 cm fruit, 2 cm fruit and fruit on the day of blooming were harvested for RNA isolation.

Arabidopsis transformation.
To generate the CsWOX9 overexpression transgenic plants, the CsWOX9 coding sequence was cloned into the pBI121 vector. The resultant construct was introduced into Agrobacterium strain C58 by electroporation and transformed into Arabidopsis plants using the floral-dip method 24 . Primary transformants were screened on Murashige and Skoog (MS) medium with 40 mg·L −1 kanamycin. The primers used for vector construction were listed in Table S2.

Identification and phylogenetic analysis of CsWOX genes in cucumber.
To identify CsWOX members in cucumber, we performed BLASTp searches using the full-length sequences of 15 Arabidopsis AtWOX members and analyzed homeodomain using SMART software 11 . Consistent with previous study 18 , 11 putative CsWOX genes were identified in cucumber (Table 1). These genes encoded proteins ranging from 130 and 387AA (amino acids) while isoelectric point (pI) values ranging from 5.54 to 10.08, and the molecular weights from 14.66 to 43.64 kDa (Table 1). www.nature.com/scientificreports www.nature.com/scientificreports/ To explore the evolutionary relationship of cucurbit and Arabidopsis WOX members, the full-length sequences of these members were used to generate the phylogenetic tree using MEGA5.0. These cucurbit members were renamed according to the original Arabidopsis names clustered in the same clade. The WOX members in every species can be divided into three clades, which were the Ancient clade, Intermediate clade and WUS clade. The WUS clade was the largest one in the tree and also remained the largest group in Arabidopsis and each cucurbit. While the Ancient clade only contains 15 WOX members and is the smallest clade in each tested species (Figs. 1 and 2A), indicating that WOX members between Arabidopsis and cucurbit species may have a close evolutionary relationship. csWoX gene structures and conserved domains. To better understand the CsWOX gene structure in cucumber, the exon/intron organization was analyzed. The results showed that all CsWOX genes contain one to five exons (Fig. 2B). Interestingly, both CsWOX13a/13b in the Ancient clade has three exons. The exon number of CsWOX genes is variable in WUS and Intermediate clades. For example, CsWOX1a contains five exons, while CsWOX7 only has a single exon (Fig. 2B).
The WOX members are plant-specific proteins containing a conserved homeodomain 25,26 . To study whether the domain is conserved in CsWOX members, the sequences of these proteins were aligned to generate sequence logos. The alignment results showed that all the CsWOX members contain a conserved homeodomain that carrying a helix-loop-helix-turn-helix structure (Fig. 3). Consistent with previous studies 27, 28 , the representative motifs YNWFQNR, FYWFQNR and FYWFQNH also exist in homeodomains from the Ancient, Intermediate www.nature.com/scientificreports www.nature.com/scientificreports/ and WUS clades, respectively (Fig. 3B). Moreover, the CsWOX members in WUS clade also contain a WUS-box, except CsWOX7 (Fig. 3A). While CsWOX members in Ancient and Intermediate clades have no WUS-box, indicating that these CsWOX members in different clades may be involved in different biological processes in cucumber.

expression pattern of CsWOXs in cucumber.
To investigate the details of tissue-specific expression of the CsWOX genes in cucumber, the expression patterns of all 11 CsWOX genes were examined by quantitative RT-PCR (qRT-PCR) in thirteen tissues/organs, i.e., leaf, stem, tendril, tip, axillary bud, root, male bud, female bud, male flower, female flower, 1.5 cm fruit, 2 cm fruit and fruit on the day of blooming (0 day fruit). The results showed that CsWOX2 was mainly expressed in root; CsWOX3 was specifically expressed in leaf; CsWOX4 was specifically enriched in axillary bud and female flower; and CsWOX13a was highly accumulated in male and female flowers, while CsWOX1a, CsWOX1b, CsWOX7, CsWOX11, CsWOX13b and CsWUS showed no obvious organ-specific expression patterns (Fig. 4). Interestingly, CsWOX9 was mainly expressed in different developmental stages of fruit and also has lower expression in tip and axillary bud (Fig. 4G). These results suggested that CsWOX members may function in many aspects of cucumber growth and development. Values are means ± sd of three biological replicates, and significant differences between the treated seedlings and control are indicated by asterisks (*P < 0.05 and **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey post-test, three independent experiment).

Scientific RepoRtS |
(2020) 10:6216 | https://doi.org/10.1038/s41598-020-63197-z www.nature.com/scientificreports www.nature.com/scientificreports/ CsWOX gene expression in response to different hormonal treatments. Plant hormones have been shown to regulate plant development. To investigate whether CsWOX genes are regulated by plant hormones, the promoter sequences (2000 bp upstream of the start codon) of CsWOXs were analyzed by PlantCARE. Many cis-elements were detected in the CsWOX gene promoter regions including some respond to auxin, abscisic acid (ABA), gibberellin (GA), salicylic acid (SA) and Methyl jasmonate (MeJA) (Table S1). Previous studies showed that auxin and ABA regulated WOX expression in rice and Cunninghamia lanceolate 29,30 . To further confirm that the expression of CsWOX genes is also regulated by plant hormones, these members (CsWOX1b, CsWOX3 and CsWOX9) carrying auxin or/and ABA response elements in their promoters were selected for study. As expected, our results showed that the expression of CsWOX1b and CsWOX3 could be greatly increased by indole-3-acetic acid (IAA) treatment, and the transcript levels of CsWOX1b and CsWOX9 could respond rapidly to ABA treatment (Fig. 5). Together, these results suggested that some CsWOX members may be involved in different hormone-mediated development. ectopic expression of CsWOX9 affected plant architecture and fruit length in Arabidopsis. Given that CsWOX9 gene was significantly expressed in the developing fruit, it was selected for further study. in situ hybridization assay showed that CsWOX9 mRNA accumulated in the axillary meristem (AM) and the flower meristem (FM) of shoot tips (Fig. 6A,B). CsWOX9 was also expressed in carpel primordium of flower bud at stage 4, and the joint of adjacent carpals in male flower bud at stage 6 and 7 (Fig. 6C-E). In young fruit, CsWOX9 signal was specifically detected in placenta (pl) and pseudoseptum (ps) (Fig. 6F). No signal was detected upon hybridization with the sense CsWOX9 probe (Fig. 6G).
To investigate the roles of CsWOX9 in fruit development and other organ development, we further introduced the CsWOX9 driven by 35S promoter into wild-type (WT) Arabidopsis. Ten independent transgenic lines were obtained and three representative lines (OX-1, 2, 3) were chosen for further study. All the transgenic plants exhibited shorter siliques (Fig. 6H-N). The transgenic line OX-3 showed shortest silique lengths compared to other transgenic lines, consistent with their highest expression of CsWOX9 (Fig. 6L-N). Interestingly, all the three transgenic plants also displayed increased branches and rosette leaves compared to WT, a result consistent with its expression in axillary bud (Figs. 4G; 6H-K,O,P). Previous studies showed that BRANCHED 1 (BRC1) inhibits shoot branching, while BLIND (BLIND) positively regulates axillary meristem formation [31][32][33][34] . To further confirm the role of CsWOX9 in regulating axillary bud outgrowth, we detected the expression levels of AtBRC1 and AtBLIND in CsWOX9 transgenic plants. As expected, we found that the expression level of AtBRC1 was strongly reduced and AtBLIND was highly increased in three transgenic lines (Fig. 6Q-R). Taken together, these results support the notion that CsWOX members may regulate many aspects of growth and development in cucumber.

Discussion
The WOX proteins function in key developmental processes in plants [35][36][37] . However, only a few of members have been characterized in several plant species. In this study, the full length sequences of AtWOX members were used as queries for BLASTp searches, 11 CsWOX members were identified in cucumber. These CsWOX members can be also classified into three clades (e.g., the Ancient clade, the Intermediate clade and the WUS clade) (Fig. 1). As previous studies 11 , each CsWOX protein contains a homeodomain and all the members in WUS clade have a WUS-box domain except CsWOX7 (Fig. 3A). Interestingly, another previous study also identified the same CsWOX members in cucumber using the homeodomain sequences of AtWOX members for BLASTp searches 18 , indicating that the homeodomain is highly conserved in WOX members.
Expression pattern analysis of these CsWOX genes showed that a few of them were mainly expressed in specific organs, indicating that these genes may be involved in different development processes. For example, CsWOX1a mutation can lead to a mango fruit in cucumber 18 , which is consistent with that it was mainly enriched in fruit (Fig. 4A). In addition, CsWOX13a is accumulated in male and female flowers. Indeed, it has been reported that its homolog AtWOX13 regulated replum development and fruit opening in Arabidopsis 16,17 . But whether CsWOX13a regulates cucumber fruit development remains to be examined.
Our data demonstrated that overexpression of CsWOX9, a homolog of AtWOX9, resulting in short siliques. This result is consistent with its highest expression in fruit shown by both expression pattern analysis and in situ hybridization assay. But whether CsWOX9 is a regulator of fruit development in cucumber remains to be examined. Interestingly, the CsWOX9 transgenic plants also showed increased rosette leaves and branches, which is expected as its higher expression in axillary bud and axillary meristem shown by qRT-PCR and in situ hybridization. It was reported that homologs of WOX8/9 in petunia and tomato plants also contribute to inflorescence architecture by promoting the separation of lateral inflorescence meristems 38,39 . Similarly, DWARF TILLER1 (DWT1), the rice homolog of AtWOX8/9, plays important roles in balancing branch growth 40 . These results suggested that the role of WOX8/9 in regulating shoot architecture is conserved in different species. Recent studies showed that auxin plays important roles in regulating fruit length and shoot branching 32,41 . Future studies will need to unravel whether WOX9 controls branch growth by regulating auxin synthesis or/and transport.

conclusion
Together, our study identified and characterized 11 CsWOX members in cucumber. Tissue-specific expression of these CsWOX genes indicated that they may be involved in different developmental processes. When CsWOX9 delivered into the Arabidopsis, the transgenic lines exhibited shorter siliques and increased branches. These results uncovered that these CsWOX members play important roles in growth and development.