Identification and characterization ofCucurbita Linn.YABBY transcription factor
YABBY proteins of Cucurbita moschata, Cucurbita maxima, and Cucurbita pepo were identified by the local BLASTp program using six AtYABBY protein sequences as query sequences. All candidate protein sequences were confirmed by the NCBI CD-search. Finally, 11, 12, and 11 YABBY proteins were identified in Cucurbita moschata, Cucurbita maxima, and Cucurbita pepo, respectively. The YABBY genes were named according to the naming of the AtYABBY genes and the position on the chromosome (from the first chromosome to the last chromosome, from the top position to the end position of one chromosome) (Table 1).
The amino acid sequence of YABBY was analyzed by the ExPASy proteomics server. The results showed that the coding regions of 11 CmoYABBYs ranged from 510 bp (CmoYAB2) to 930 bp (CmoINOa) (Table 1). The number of translated amino acids ranged from 169 aa to 309 aa, and the MW of protein ranged from 18889.49 Da to 35478.2 Da. The PI is between 6.29 (CmoYAB5b) and 9.99 (CmoCRCb). In Cucurbita maxima, the coding regions of 12 CmaYABBY genes ranged from 537 bp (CmaYAB5b) to 1179 bp (CmaINOa) (Table 1). The number of translated amino acids ranged from 178 aa to 392 aa, and the MW of protein ranged from 19479.12 Da to 43636.39 Da. The PI is between 6.37 (CmaINOb) and 9.32 (CmaYAB2). In Cucurbita pepo, the coding regions of 11 CpeYABBY genes ranged from 471 bp (CpeINOb) to 1110 bp (CpeYAB1a) (Table 1). The number of translated amino acids ranged from 156 aa to 369 aa, and the MW of protein ranged from 17044.7 Da to 40999.44 Da. The PI is between 6.06 (CpeYAB5a) and 9.76 (CpeYAB1b). Based on the physical and chemical characteristics of the YABBY gene in three cultivars of Cucurbita Linn., it was found that they had similar characteristics, and they had the properties of basic protein (Table 1). The predicted subcellular localization results showed that 34 Cucurbita Linn.YABBY genes were all located to the nucleus (Table 1), which accorded with the characteristics of transcription factors.
Phylogenetic relationship of YABBY in three cultivars of Cucurbita Linn.
In order to analyze the evolutionary relationship of YABBY proteins, we constructed a phylogenetic tree with 6 AtYABBYs, 11 CmoYABBYs, 12 CmaYABBYs and 11 CpeYABBYs (Fig. 1). All YABBYs were divided into five subfamilies (YAB1/YAB3, YAB2, INO, CRC and YAB5) according to the identity of amino acid sequences. Each subfamily contained AtYABBY, CmoYABBY, CmaYABBY and CpeYABBY proteins. The YAB1/YAB3 subfamily consisted of 2 CmoYABBYs, 3 CmaYABBYs, 3 CpeYABBYs, 2 AtYABBYs (AtYAB1 and AtYAB3); YAB2 subfamily contained 4 proteins: CmoYAB2, CmaYAB2, CpeYAB2 and AtYAB2; YAB5 subfamily contained 4 CmoYABBYs, 4 CmaYABBYs, 4 CpeYABBYs and AtYAB5; INO subfamily contained 2 CmoYABBYs, 2 CmaYABBYs, 2 CpeYABBYs and AtINO; CRC subfamily contained 2 CmoYABBYs, 2 CmaYABBYs, 1 CpeYABBYs and AtCRC proteins.
Gene structures and motif composition of YABBY gene family in three cultivars of Cucurbita Linn.
The gene structure can provide valuable information for the phylogenetic relationship of YABBY in three cultivars of Cucurbita Linn.. Based on the phylogenetic tree, the exon-intron structure and conserved motifs of 34 YABBYs were analyzed by TBtools (Fig. 2B). The classification pattern of exon-intron was consistent with the phylogenetic tree (Fig. 1; Fig. 2A). All YABBYs from three cultivars of Cucurbita Linn. contain introns, and the number of exons was 5-12. The number of exons and the length of introns in one branch were similar. For instance, in the YAB2 branch, all YABBY genes contained six exons. What’s more, from the phylogenetic analysis, the closer the homology was, the more similar the structure was, such as CmoYAB1a_CmaYAB1a, CmoYAB5b_CmaYAB5b, and CmoYAB1b_CmaYAB1b (Fig. 2B). Through multiple sequence alignment, it was found that YABBY protein contained a conserved YABBY domain at the C-terminal, and most YABBY proteins contained a conserved C2C2 domain (Fig. S1). Ten conserved motifs of YABBYs were searched and identified by MEME online tools (Fig. 2C; Fig. S2). Motif analysis showed that motifs 1 and 2 existed in all YABBYs proteins. In addition to CpeYAB1b protein, motif 3 existed in all other proteins. Motif 4 existed in all other proteins except CpeYAB5d. These results indicated that the domain and motif of YABBYs were highly conserved. What’s more, motif 6 only existed in YAB5 subfamily, motif 9 only existed in a branch of YAB5 subfamily; motif 8 only existed in some genes of INO and YAB5 subfamily, and motif 10 only existed in some genes of YAB5 and CRC subfamily (Fig. 2C), indicating that these genes may have special functions.
Distribution, gene duplication and collinearity of YABBY transcription factors in three cultivars of Cucurbita Linn.
According to the genome sequence of Cucurbita, the chromosome position of YABBY in each cultivar of Cucurbita Linn. was determined (Fig. 3A). Eleven CmoYABBYs were located on 8 of the 21 chromosomes, and there were 2 CmoYABBYs on chromosomes Cmo_chr02, Cmo_chr04, and Cmo_chr05, respectively. Among the 12 CmaYABBYs, 11 CmaYABBYs, were similar to CmoYABBYs, and CmaYB3 gene were located on chromosome Cma_chr06. Eleven CpeYABBYs were located on 9 of 21 chromosomes. Except for 2 CpeYABBYs on chromosomes CP4.1LG05 and CP4.1LG11, respectively, there was one CpeYABBYs on the other chromosomes (CP4.1LG01, CP4.1LG04, CP4.1LG08, CP4.1LG09, CP4.1LG13, and CP4.1LG18), respectively (Fig. 3A).
According to the amino acid sequence identity > 80% and gene alignment coverage > 0.75, we found three duplicated gene pairs (CmoYAB1a-CmoYAB1b; CmoYAB5a-CmoYAB5d and CmoYAB5c-CmoYAB5b) in CmoYABBYs, two duplicated gene pairs (CmaYAB1b-CmaYAB1a and CmaYAB5d-CmaYAB5a) in CmaYABBYs, and one duplicated gene pairs (CpeYAB5b-CpeYAB5c) in CpeYABBYs, with all Ka/Ks < 1.0, indicating that these duplicated gene pairs mainly underwent purification selection, and the divergence time was 6.64~16.47 (MYA) (Table 2).
Synteny relationship of YABBY genes among Arabidopsis and three cultivars of Cucurbita Linn. was also analyzed. Eight, eight and six collinear genes of AtYABBYs were found in Cucurbita moschata,Cucurbita maxima and Cucurbita pepo, respectively (Fig. 3B). Based on the collinearity analysis of YABBYs among Cucurbita moschata, Cucurbita maxima, and Cucurbita pepo, it was found that there were 30 pairs of collinear genes between CmoYABBYs and CpeYABBYs, 29 pairs of collinear genes between CmoYABBYs and CmaYABBYs and 26 pairs of collinear genes between CmaYABBYs and CpeYABBYs (Fig. 3B; Table S1).
Cis-acting elements in Cucurbita Linn. YABBY gene promoters
To understand the transcriptional regulation of the YABBYs in three cultivars of Cucurbita Linn., we extracted the 2.0 kb sequence before the translation initiation site (ATG) and predicted the cis-elements on the PlantCARE server. Fig. 4A showed the position of the cis-acting element on the promoter. It is worth noting that five kinds of plant hormone-responsive cis-acting elements were identified, including abscisic acid response element, methyl jasmonate response element, gibberellin response element, salicylic acid response element and auxin response element (Fig. 4A; Fig. 4B). Thirty-four YABBY gene promoters contained at least two plant hormone response element. CpeINOb and CmaINOb contained the most (21) plant hormone response elements. Among them, there were 10 and 12 elements involved in methyl jasmonate, respectively (Fig. 4B). Additionally, we found that 74% (25) of the YABBYs contain elements that participate in the growth and development of plants, including 19 genes involved in meristem expression, and 3 genes (CmoYAB1b, CmaYAB1b, and CpeYAB1b) involved in seed-specific regulation (Fig. 4C). The YABBYs may also respond to abiotic stresses such as defense and stress, anaerobic, drought-induced and low temperature (Fig. 4D). CmaINOb contained the highest number (9) of abiotic stress elements, and CmoYAB5a contained 5 elements involved in anaerobic induction. These data suggestted that YABBY may be involved in plant hormone response, abiotic stress, and plant growth and development through a complex mechanism.
The expression profiles of CmoYABBYs and CmaYABBYs in different tissues
According to RNA-seq data (BioProject: PRJNA385310), the expression profiles of CmoYABBYs and CmaYABBYs in root, stem, leaf and fruit were obtained. In general, CmoYABBY and CmaYABBY genes were mainly expressed in roots and leaves (Fig. 5). In Cucurbita maxima, CmoYAB1a, CmoYAB5a, CmoYAB5a, and CmoYAB5a were mainly expressed in leaves. The expression of CmoYAB1b in the root, leaf, and fruit was higher than that in the stem. The relative expression of CmoINOa in four tissues was higher than other genes. CmoYAB2 was mainly highly expressed in root and stem; The remaining CmoYABBY genes have a low expression (Fig. 5A). In Cucurbita maxima, CmaYAB1b, CmaYAB5a, CmaYAB3, CmaINOa, CmaYAB5c, and CmaYAB1a were mainly expressed in leaves. The expression of CmaYAB2 in stems, leaves, and fruits was higher than that in roots. CmaYAB5b was highly expressed in all tissues. The relative expression levels of other CmaYABBY genes were relatively low (Fig. 5B). Based on the above analysis, we speculated that CmoYABBYs and CmaYABBYs have tissue specificity.
Expression profiles of CpeYABBYs in fruit mesocarp and seed
To study the expression patterns of CpeYABBYs in seed and fruit mesocarp, the Cucurbita pepo cultivar, "Sweet REBA" was used and the published transcriptome data (BioProject: PRJNA339848)  was naalysed. It showed that all CpeYABBYs (except CpeCRC) were highly expressed in the seed at 20 days after pollination (DAP). Meanwhile, CpeYAB5c, CpeYAB5a, CpeYAB1a, CpeYAB1b, CpeINOa, CpeINOb and CpeYAB2 were highly expressed in the fruit mesocarp at 20 days after pollination (DAP). CpeYAB2 had a relatively high relative expression profile at each developmental stage in both seed and fruit mesocarp (Fig. 6).
Expression patterns of CmoYABBYs in leaf mesophyll and leaf vein under salt stress
To determine the expression pattern of the YABBY gene in Cucurbita moschata (salt-sensitive type) under salt stress, the RNA-seq data (BioProject: PRJNA464060) was analyzed. The results showed that the expression of all CmoYABBYs (except for CmoCRCa was unchanged) in leaf veins was inhibited under salt stress, while all CmoYABBYs in leaf mesophyll were induced. For instance,, the expression level of CmoYAB1a in the leaf vein under salt stress was significantly reduced compared with the control treatment, while the relative expression of CmoYAB1a in leaf mesophyll under salt stress was 2.29 times higher than the control treatment (Fig. 7).
Expression patterns of CmoYABBYs in root tip under salt stress
Tissue location analysis showed that all CmoYABBYs were mainly expressed in roots and leaves (Fig. 5A). In the leaves, the expression level of CmoYABBYs in the leaf vein was significantly inhibited under salt stress, while the expression level of CmoYABBYs in the leaf mesophyll was significantly induced under salt stress. However, the response of CmoYABBY in the root to salt stress was not clear. For further determine the response of CmoYABBYs in root to salt stress, the qRT-PCR data was analyzed. The results showed that most of the CmoYABBYs had low expression levels in root tips, but CmoCRCb, CmoINOb and CmoYAB5d were significantly induced under salt stress. For example, the relative expression profile of CmoINOb was 6.45 times that of the control. Therefore, we speculated that CmoINOb in root tips may play an important role in salt tolerance.