Genome-wide identification and expression analysis of the B-box transcription factor gene family in grapevine (Vitis vinifera L.)

- Genome-wide identification and expression analysis of the B-box transcription factor gene family in grapevine ( Vitis vinifera L.).
- Results: We identified a total of 25 VviBBX genes in the grapevine genome and named them according to the homology with Arabidopsis.
- Synteny analyses suggested that only segmental duplication events contributed to the expansion of the VviBBX gene family in grapevine.
- Conclusions: The results of this study establish the genome-wide analysis of the grapevine BBX family and provide a framework for understanding the biological roles of BBX genes in grapevine..
- Zinc-finger TFs are one of the most populous classes of TFs in plants, and can be classified into several families based on the number and location of characteristic amino acid sequence motifs [2].
- 1 State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China Full list of author information is available at the end of the article.
- In rice, OsBBX14 promotes photomorphogen- esis by directly binding the T/G-box cis-element of the OsHY5L1 promoter under blue light conditions [19].
- Grapevine (Vitis vinifera L.) is one of the most eco- nomically important perennial fruit crops throughout the world.
- In this study, we identified members of the BBX family in grapevine from the draft genome se- quence and gained insight into their potential func- tion based on gene and protein structure, phylogeny, synteny, subcellular localization and transcriptional activity, as well as expression during ovule develop- ment, in response to pathogen challenge and various phytohormones.
- Identification and characterization of grapevine BBX genes.
- To identify BBX genes in the grapevine genome, we employed a Hidden Markov Model (HMM)-based approach and the amino acid sequence profile of the B- box-type zinc-finger domain (Pfam.
- This resulted in the identifica- tion of 25 putative BBX genes.
- The length of the encoded proteins ranged from 127 to 469 amino acids, and their predicted molecular mass ranged from ~ 14.3 to 50.9 kDa.
- The isoelectric points of the predicted proteins ranged from ~ 4.1 to 8.7 (Table 1)..
- Phylogeny and conserved domains of the grapevine BBX proteins.
- To analyze the evolutionary relationship and potential functional divergence of the VviBBX gene family, a total of 205 BBX proteins, including 32 from Arabidopsis, 29 from tomato, 30 from rice, 64 from apple and 25 from pear, were used to construct a phylogenetic tree (Fig.
- However, an exception was found in the above clades.
- As shown in the phylogeny tree, it is evident that BBX genes of the woody plants (grapevine, apple and pear) clustered to- gether.
- And most of the grapevine BBXs also clustered together with proteins from Arabidopsis and tomato, in- stead of rice, consistent with the closer relationship of grapevine to the two eudicots..
- The conserved sequences of the B-box1 and B-box2 zinc finger domains were C-X 2 -C-X 7–8 -C-X 2 -D-X-A-X- L-C-X 2 -C-D-X 3 -H-X 2 -N-X 4 -H and C-X 2 -C-X 8 -C-X 7 -C- X 2 -C-X 4 -H(N)-X 6 – 8 -H, respectively.
- In addition, the CCT domain of twelve of the grapevine proteins with the form of R-X 5 -R-Y-X 2 -K-X 3 -R-X 3 -K-X 2 -R-Y-X 2 -R-K- X 2 -A-X 2 -R-X-R-X 2 -G-R-F-X-K was highly conserved.
- Alignment of the protein sequences revealed that the B-box1 domain was more conserved than the B-box2 as a result of five absolutely.
- Based on amino acid sequence conservation, number of B-box domains, and the presence or absence of the CCT domain, the 25 grapevine BBX proteins fell into five distinct structural classes (Table 1), which is consist- ent with previous results in Arabidopsis [4].
- Group I, comprising three of the proteins, contained two B-box domains and one CCT domain.
- The seven representa- tives of Group II also contained two B-box domains and a CCT domain, but were distinguished from Group I based on the absence of the highly conserved amino acid sequence (SANPLARR) in the B-box2 domain and VP motif amino-terminal to the CCT domain seen in Group I proteins (Additional file 4: Fig.
- To gain additional insight into the conservation and di- versification of the grapevine BBX gene family, we ana- lyzed the conserved protein motifs encoded by the genes, as well as exon-intron structures (Fig.
- (a), (b) and (c) represent the amino acid sequence alignment of the B-box1, B-box2 and CCT domain, respectively.
- 3 Characterization of grapevine BBX genes.
- a Phylogenetic analysis of BBX proteins in grapevine.
- b Distribution of conserved motifs identified in the 25 VviBBX proteins.
- c Exon-intron structure of grapevine BBX genes.
- foregoing descriptions of Holub [29], and thus only seg- mental duplication seems to have taken part in the evo- lution of the grapevine BBX gene family.
- In brief, these syntenic relationships suggest that about two-thirds of the BBX genes appeared before the divergence of grapevine and Arabidopsis..
- The approximate chromosomal locations of the BBX genes are indicated on the periphery.
- The divergence time of the segmental duplication event was calcu- lated as between ~ 77 and 110 million years ago (Mya) in grapevine alone (Additional file 6: Table S2), and between ~ 102 and 349 Mya, with an average of 178.8 Mya, in grapevine and Arabidopsis (Additional file 7: Table S3)..
- To help identify a possible function of the VviBBX genes in response to powdery mildew, we inoculated healthy plants of the powdery-mildew resistant genotype ‘Shang- 24’ with Erysiphe necator, the causative agent of grapevine powdery mildew, and monitored the expres- sion of the 25 VviBBX genes by semi-quantitative RT- PCR (Real-time polymerase chain reaction).
- 5 Synteny analysis of BBX genes between grapevine and Arabidopsis.
- Expression of VviBBX6, VviBBX19b, VviBBX21a and VviBBX25 peaked at 12 h post- inoculation, whereas VviBBX6 and VviBBX19b decreased sharply at 24 h and remained relatively stabile for the remainder of the measurement period..
- In this study, we evaluated the transcriptional response of the 25 BBX genes in plants exposed to these hormones (Fig.
- To support the results of the semi- quantitative RT-PCR analyses, expression of six, randomly-selected VviBBX genes was determined using quantitative RT-PCR (Fig.
- Expression analysis of VviBBX genes during ovule development.
- We noted most of the VviBBX genes showed different levels of expression in all organs and tissues.
- Transcripts were normalized to the expression of the ACTIN1 gene and EF1- α gene.
- c Quantitative RT-PCR analysis of expression of selected VviBBX genes after E.
- Asterisks indicate significance of the indicated differences in gene expression according to the t-test (*P<.
- Hence, we analyzed the expression of the genes in two seeded cultivars (‘Red Globe’ and ‘Kyoho’) and two seedless cultivars (‘Thompson Seedless’ and ‘Flame Seedless.
- In contrast, higher expression levels in seeded cultivars relative to seedless cultivars were observed for eight genes (VviBBX5, VviBBX6 VviBBX10, VviBBX11, VviBBX12a, VviBBX15a, VviBBX19b and VviBBX21a), suggesting that they might function in normal development of the ovule.
- Subcellular localization and transcriptional activity of the three BBX proteins.
- 7 Expression analysis of 25 VviBBX genes in two seeded cultivars.
- b Quantitative RT-PCR analysis of expression of selected VviBBX genes.
- Detailed information about these genes, including gene ID, accession numbers, structural group classification, and physiological and biochemical properties of the encoded protein are given in Table 1..
- The five Cys residues and two His residues were conserved to a greater extent than the remainder of the amino acid residues in the B-box1 do- main.
- We analyzed the conserved motifs in the grapevine BBX family genes by MEME, and only 16 motifs were.
- 8 Subcellular localization and transcriptional activity analysis of the three BBX genes.
- The majority of the VviBBX proteins in the same group showed similar motif distribution, but exceptions were found (Fig.
- 3, VviBBX genes con- taining a similar exon-intron structure clustered together in the phylogenetic tree.
- A similar observation was re- ported within the five members of the grapevine bHLH IX subfamily, VvbHLH103-VvbHLH107.
- genes likely resulting from segmental duplication were part of the same group [34].
- These observations demon- strate that segmental duplications contributed to expan- sion of the BBX gene family in grapevine.
- In this study, VquBBX15a, the same clade of AtBBX14 and AtBBX15, has the representative features of the BBX family, which was located in the nucleus (Fig.
- And we also carried out a comprehensive analysis of the BBX gene family, including phylogeny, conserved domain, motif compositions, exon-intron configurations, chromo- somal distributions, genomic synteny and expression pat- tern analysis.
- Taken together, genome-wide analysis of the VviBBX family will provide a fundamental basis for further research on the functions of BBX genes in grapevine..
- Identification and annotation of BBX genes in the grapevine genome.
- PF was used to identify putative BBX genes in the grapevine genome using HMMER3.0 [49].
- The predicted mass and charge of the BBX proteins were calculated using the ProtParam tool (http://web.expasy.org/protparam/) [53]..
- Phylogenetic and conserved domain alignments analysis Amino acid sequences of the B-box and CCT domains were aligned with DNAMAN (Version 7.0.2, Lynnon Biosoft), and sequence logos were created using Weblogo 3 (http://weblogo.threeplusone.com) [54].
- Analysis of exon-intron structure and conserved motifs Exon-intron structures of the confirmed BBX genes were determined according to the alignments of their coding sequences and genomic full-length sequences in the Grapevine Genome (12X) database ( https://www..
- TBtools software was used to draw the map of the conserved motif [58]..
- The transient expression of the fused proteins was observed by a laser scanning confocal microscope (Olym- pus FV3000, Japan) after 3 d at room temperature.
- Additional file 3: Fig.
- Alignment of the conserved domains of grapevine BBX proteins.
- Additional file 4: Fig.
- Additional file 8: Fig.
- The microarray data of the grapevine VviBBX genes in different organs, tissues and developmental stages Additional file 10: Fig.
- Alignment of the coding sequences of three cloned BBX genes.
- Primer sequences used in expression analysis of BBX genes in grapevine.
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