Edited by: Richard Sayre, New Mexico Consortium at Los Alamos National Labs, USA
Reviewed by: Vasileios Fotopoulos, Cyprus University of Technology, Cyprus; Cordelia Bolle, Ludwig Maximilian University, Germany
*Correspondence: Ana M. Fortes
This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science
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GRAS transcription factors are involved in many processes of plant growth and development (e.g., axillary shoot meristem formation, root radial patterning, nodule morphogenesis, arbuscular development) as well as in plant disease resistance and abiotic stress responses. However, little information is available concerning this gene family in grapevine (
Transcription factors play an important role in the regulation of plant development and disease response. Among them, the plant gene family of GRAS transcription factors was defined based on nuclear localization, DNA binding and transcriptional activation features (Silverstone et al.,
Genome-wide analysis performed in nearly 30 plant species from more than 20 genera revealed that this gene family is widely distributed in the plant kingdom (Tian et al.,
The amino acid sequences of GRAS proteins are highly variable at the N-terminus, which may be responsible for the specificity of their regulatory functions (Tian et al.,
The GRAS protein family groups into eight well-known subfamilies: DELLA, HAM, LISCL, PAT1, LAS, SCR, SHR, and SCL3. However, in between 8 and 13 distinct clades can be discriminated in different studies (Huang et al.,
So far, various
Due to its economic relevance, much research in grapevine genomics has been carried out during the last decade. Among these studies, the release of the whole grapevine genome sequence in 2007 represented a breakthrough to promote its molecular genetics analysis (Jaillon et al.,
Previous comparative analysis with Chinese cabbage genome predicted 43 GRAS transcription factors in
Genes previously identified as encoding GRAS proteins in (Grimplet et al.) were blasted (blastp and tblastn) against the grapevine genome 12x.2 (
The potential coding DNA sequences (CDS) were blasted (blastx) against the NCBI public database to compare the structures with other known
Sequence information on previously reported GRAS proteins of
The alignment file between Arabidopsis and grapevine sequences was uploaded to the Jalview and UGene software for manual adjustment of the alignment and manual motif editing. Motifs identified in Tian et al. (
Expression data were retrieved from three different microarray platforms (Affymetrix Genchip (16k probesets) GrapeGen (21k probesets), Vitis Nimblegen array (29k probesets), and from our in-house RNAseq projects. Data normalization was performed on all the array of each platform (RMA normalization). After retrieving the values for the probesets corresponding to each gene, the values for the 3 or 4 replicates of the same condition were averaged to obtain a total of 256 conditions (organ, cultivar, treatment, platform). Based on expression data of the grapevine gene expression atlas (Fasoli et al.,
For further evaluation of gene expression samples corresponding to several stages of grapevine development and ripening and several abiotic and biotic stress conditions were used (Cramer et al.,
We performed a sequence comparison using the GRAS genes from 16 plant species (
Genes that were previously identified as GRAS in the grapevine genome (Grimplet et al.,
Gene models were curated using the data collected from gene structure comparisons using different databases as well as the available RNAseq data from our laboratory (Royo et al.,
Vitvi12g00665 | − | 8738265–8739902 | Vitvi11g00409 | − | 3959545–3961143 | ||
Vitvi19g00619 | − | 7772106–7773743 | Vitvi01g01509 | + | 20426662–20428254 | ||
Vitvi10g00271 | − | 2802206–2803843 | Vitvi17g01040 | − | 12688373–12689932 | ||
Vitvi19g00392 | − | 5276148–5277899 | Vitvi19g01706 | + | 23595896–23597488 | ||
+ | 19383904–19385748 | Vitvi14g01510 | − | 25316395–25317516, 25317604–25318488 | |||
Vitvi04g01696 | − | 23747087–23748793 | Vitvi07g00627 | + | 6996793–6998256 | ||
Vitvi18g01210 | + | 13411198–13412895 | Vitvi03g01226 | + | 19152243–19153571 | ||
Vitvi07g01612 | + | 21912240–21913607 | − | 115793–116261, 116647–117596 | |||
Vitvi12g00571 | + | 7509331–7510668 | Vitvi06g01133 | + | 15915179–15916597 | ||
Vitvi09g01487 | − | 910682–912319 | + | 13518884–13519177 | |||
Vitvi05g01554 | + | 23894334–23895644 | Vitvi13g01556 | − | 24957576–24959012 | ||
Vitvi07g02073 | − | 21633666–21635150 | + | ||||
Vitvi06g00491 | + | 5938487–5940601 | + | 14926630–14927997 | |||
Vitvi06g00490 | + | 5930838–5932814 | + | 3323726–3325756 | |||
Vitvi06g00492 | + | 5942791–5944119 | + | 18563606–18565456 | |||
+ | 5918887–5921169 | Vitvi19g00932 | + | 10747971–10749212 | |||
Vitvi06g00489 | + | 5925910–5928204 | Vitvi07g00418 | − | 4408615–4410429 | ||
Vitvi08g01214 | + | 14792851–14795082 | − | 1038770–1039128, 1039236–1039324, 1039452–1040602 | |||
Vitvi13g00312 | + | 3256665–3258887 | − | ||||
Vitvi13g00314 | + | 3283478–3285724 | − | ||||
Vitvi13g00311 | + | 3251727–3254009 | + | 9227520–9229388 | |||
Vitvi13g01865 | + | 3279518–3281677 | + | 22393173–22394627 | |||
+ | 3274050–3274663, 3274680–3276222 | Vitvi18g00300 | − | 3254592–3256064 | |||
+ | 3270544–3270684, 3270692–3271508, 3271694–3271929, 3271938–3272162 | Vitvi04g01247 | − | 18244582–18246198 | |||
Vitvi01g00446 | − | 4895406–4897178 | + | 5144861–5147299 | |||
+ | 14807005–14808846 | Vitvi15g00680 | − | 14397074–14399326 |
Exon/ intron structure is highly conserved amongst
For gene nomenclature, a phylogenetic tree of the GRAS protein coding genes in
Five characteristic conserved motifs were identified in the C-terminus of the GRAS proteins, namely LHRI, VHIID, LHRII, PFYRE, and SAW (summarized by subfamilies in Figure
The VHIID motif contained three units (A, B, and C). GRAS proteins could be divided into several distinct groups based on conservation of Unit A. Groups such as PAT, DELLA, and HAM presented high conservation of amino acids (VI, IX, and XIII respectively, Figure
The LHRII motif embraced units A and B. In Unit A, three regularly spaced leucine heptad repeats (LX6LX6L) could be found followed by several irregularly spaced leucine repeats. In Unit B, many GRAS proteins had a conserved LXXLL pattern (DELLA, SCL3, and LS groups) as previously described (Tian et al.,
The PFYRE motif could be divided into three units: P, FY, and RE. On the other hand, the SAW motif was composed of two units, RVER and W-W-W (Figure
In the N-terminus several units were found, in accordance with previous reports (Tian et al.,
Besides these eight known groups, five new additional groups were identified. A new
A subgroup of proteins with much similarity to the SCL group did not present VIII domain including
Based on the original phylogenetic analysis (Figure
From the alignment of predicted GRAS domain sequences we identified members containing partial GRAS domains with missing motifs (Supplementary Image
As mentioned previously we analyzed the orthologous relationships of GRAS genes in
A phylogenetic tree considering several mono and dicotyledonous species together with a sequence comparison were performed to identify genes with widely conserved functions among species (Figure
GRAS gene family has considerably evolved since the divergence of monocot and eudicot plants as determined by the orthologous relationship of GRAS genes in several species. The phylogenetic analysis of LISCL, HAM, PAT, and SCL groups revealed independent clusters with many members from only monocotyledonous species (Supplementary Image
Regarding the new
The genes
Regarding the GRAS8 subfamily, gene
GRAS genes were distributed unevenly among the nineteen chromosomes of the grapevine genome though they were mapped to all the chromosomes (Figure
Concerning LISCL genes, the tandem repetition of almost identical coding sequences (e.g.,
Tandem repeats mainly in the LISCL group were also observed in
Interestingly, the new
Therefore, segmental duplication and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family.
Three distinct approaches were performed to characterized GRAS genes expression in grapevine. First, we constructed an atlas of expression of the GRAS genes based on the absolute value of gene expression in public data. The results of this study are presented in Figure
Second, we performed a co-expression analysis based on the same original data using the relative values of expression of all the genes, centered on the average expression. The objective here was to determine expression patterns and to identify genes that were following the same pattern of expression as the GRAS genes and that could be under the same regulatory elements, or under the regulation of the GRAS gene itself. The results are presented in Table
VIT_02s0025g04000 | ||
VIT_14s0068g02000 | Ribonucleotide reductase R2 | Nucleotide metabolism. Purine metabolism |
VIT_11s0016g03750 | Myb-related protein 3R-1 (Plant c-MYB-like protein 1) | Cellular process. Cell growth and death |
VIT_18s0001g07550 | Kinesin family member 4/7/21/27 | Microtubule-driven movement |
VIT_13s0064g00560 | DNA topoisomerase, ATP-hydrolyzing | Nucleic acid metabolism. DNA metabolism |
VIT_18s0122g00550 | Cyclin-dependent kinase B2;1 | Cell growth and death; Regulation of cell cycle |
VIT_14s0108g00710 | Chromosome condensation protein | DNA metabolism. DNA replication |
VIT_11s0016g02970 | MAP kinase kinase 6 | Signaling pathway. Protein kinase. MAPK cascade |
VIT_13s0067g03250 | CENP-E like kinetochore protein | Cellular process. Cell growth and death |
VIT_13s0067g01420 | Cyclin 1b (CYC1b) | Cell growth and death; Regulation of cell cycle |
VIT_06s0004g05870 | Tubulin beta-3 chain | Microtubule organization and biogenesis |
VIT_18s0001g02060 | Cyclin A1 | Cell growth and death; Regulation of cell cycle |
VIT_07s0005g01030 | Cellulose synthase CSLD5 | Cell wall biosynthesis. Cellulose biosynthesis |
VIT_01s0010g02430 | Mitotic spindle checkpoint protein (MAD2) | MAPK cascade; Regulation of cell cycle |
VIT_12s0057g00500 | Thymidine kinase | Nucleotide metabolism. Pyrimidine metabolism |
VIT_13s0019g02710 | Rho guanyl-nucleotide exchange factor ROPGEF5 | Signaling pathway. G-protein signaling pathway |
VIT_04s0008g01080 | Calmodulin-binding region IQD6 | Calcium sensors and Signaling |
VIT_14s0068g00270 | Hydroxyproline-rich glycoprotein | Cell wall organization and biogenesis |
VIT_10s0003g05680 | CHUP1 (chloroplast unusual positioning 1) | Cytoskeleton. Actin organization and biogenesis |
VIT_04s0023g01660 | ||
VIT_12s0059g00230 | Epoxide hydrolase 2 | Epoxide hydrolase family; Biotic stress response |
VIT_12s0059g00220 | Epoxide hydrolase | Epoxide hydrolase family; Biotic stress response |
VIT_08s0007g02240 | Calcium/proton exchanger CAX3 | Electrochemical Potential-driven Transporters. Porters. Ca2+:Cation Antiporter |
VIT_05s0020g03380 | WNK1 (with no lysine (K) 1) | Signaling pathway. Circadian clock Signaling |
VIT_14s0108g01420 | DEFENSE NO death 1 | Biotic stress response. Plant-pathogen interaction |
VIT_12s0035g00970 | Evolutionarily conserved C-terminal region 11 ECT11 | RNA processing. mRNA processing. mRNA splicing |
VIT_02s0025g04120 | Calmodulin binding protein | Signaling pathway. Calcium sensors and Signaling |
VIT_04s0023g01170 | Unknown protein | Unknown |
VIT_03s0180g00140 | Acetyl xylan esterase AxeA | Unknown |
VIT_10s0003g02780 | Unknown protein | Unknown |
VIT_05s0020g00870 | UbiE/COQ5 methyltransferase | Biosynthesis of derivatives of dehydroquinic acid, shikimic acid and chorismic acid |
VIT_01s0244g00140 | Aspartate kinase | Amino acid. Glycine, serine, and threonine metabolism |
VIT_07s0005g03700 | ||
VIT_15s0046g00930 | Zinc finger (C3HC4-type ring finger) | Transcription factor. Zinc finger C3HC4 family transcription |
VIT_07s0129g00030 | ||
VIT_08s0007g04820 | Pectate lyase | Cell wall catabolism. Pectin catabolism |
VIT_07s0129g01070 | Leucine-rich repeat protein kinase | Signaling. Signaling pathway. Protein kinase |
VIT_02s0025g02700 | Glutaredoxin family protein | Response to stimulus. Stress response. Abiotic stress |
VIT_18s0001g09920 | Cyclin delta-3 (CYCD3_1) | Cytokinin-mediated Signaling pathway |
VIT_12s0059g01900 | Unknown protein | Unknown |
VIT_01s0026g01420 | Wall-associated kinase 4 | Signaling. Signaling pathway. Protein kinase |
VIT_01s0137g00720 | Lipase GDSL | Unclear |
VIT_07s0005g00740 | Endo-1,4-beta-glucanase | Cell wall catabolism. Cellulose catabolism |
VIT_09s0002g00450 | Subtilase | Subtilase-mediated proteolysis |
VIT_05s0077g02270 | Unknown protein | Unknown |
VIT_18s0001g07340 | Aspartic proteinase nepenthesin-1 precursor | Proteolysis. Peptidase-mediated proteolysis |
VIT_03s0038g02180 | Glycosyl hydrolase family 10 protein | Cell wall catabolism. Xylan catabolism |
VIT_14s0030g01870 | NIMA protein kinase | Signaling. Signaling pathway. Protein kinase |
VIT_01s0010g01660 | Receptor protein kinase | Signaling. Signaling pathway. Protein kinase |
VIT_08s0056g00050 | ||
VIT_18s0001g10380 | Heat shock transcription factor B4 | HSP-mediated protein folding; Temperature stress response |
VIT_09s0002g01540 | Unknown protein | Unknown |
VIT_04s0044g01100 | Invertase/pectin methylesterase inhibitor | Cell wall organization and biogenesis |
VIT_11s0016g04630 | ||
VIT_08s0007g02760 | IAA-amino acid hydrolase 1 (ILR1) | Auxin activation by conjugation hydrolysis |
VIT_13s0019g01780 | ||
VIT_10s0003g02350 | SRG1 (senescence-related gene 1) oxidoreductase | Unclear |
VIT_13s0019g01810 | ||
VIT_07s0005g05640 | Unknown protein | Unknown |
VIT_18s0001g03310 | ||
VIT_13s0067g01190 | Cellulase | Cell wall catabolism. Cellulose catabolism |
VIT_03s0088g00890 | Pathogenesis related protein 1 precursor [ |
Jasmonate-mediated Signaling pathway; Biotic stress response. Plant-pathogen interaction |
VIT_05s0094g01310 | Polygalacturonase GH28 | Cell wall modification. Pectin modification |
VIT_10s0092g00070 | Taxane 13-alpha-hydroxylase | Diterpenoid biosynthesis |
VIT_08s0105g00170 | Dof zinc finger protein DOF3.5 | C2C2-DOF family transcription factor |
VIT_05s0124g00210 | Peptidase S26A, signal peptidase I | Proteolysis. Peptidase-mediated proteolysis |
VIT_05s0062g00690 | Heat shock protein 81-2 (HSP81-2) | HSP-mediated protein folding; Biotic stress response. Plant-pathogen interaction |
VIT_15s0021g01590 | RKL1 (Receptor-like kinase 1) | Signaling. Signaling pathway. Protein kinase |
VIT_03s0091g00890 | Endoxylanase | Cell wall organization and biogenesis |
VIT_12s0055g00980 | Peroxidase precursor | Phenylalanine biosynthesis; Abiotic stress response. Oxidative stress response |
Third, we mined public expression data to identify the behavior of
Out of the 52 genes analyzed, six were not detected in any analyzed tissue. The rest of the genes mostly showed a general pattern; they were either highly expressed or lightly expressed in all tissues considered. Nevertheless, about one third of the genes showed some tissue-specific expression. Pollen stands out as a different tissue in terms of GRAS genes expression. Differential expression of some GRAS genes among different tissues was previously shown for tomato and Populus (Liu and Widmer,
Expression studies of
Concerning SHR subfamily,
Members of the LISCL subfamily showed distinctive expression patterns.
Interestingly,
Genes
The gene
Three SCL3 genes (
In this subfamily,
Genes
Expression of genes belonging to these new subfamilies was low. For some of them, their possible expression could not be confirmed (
The SCL26 genes showed a reduced expression level in various tissues. Most notably
This subfamily is present in all tissues with notable lower values in pollen (Figure
The availability of sequenced genomes, expression data and associated bioinformatics tools enable the study of the genomic information to predict the putative function of a gene family in developmental processes and in stress response. In general, transcription regulators belonging to the same taxonomic group exhibit common evolutionary origins and specific conserved motifs related to molecular functions, making their genome-wide analysis an effective and practical method to predict unknown protein functions.
We have performed an exhaustive analysis of
The grapevine
We have also identified duplicated grapevine genes such as
The exon-intron organization analysis showed that 88.46% (46 out of 52) of
GRAS proteins have also been involved in axillary meristem development. Knock-out Arabidopsis plants for
In grapevine,
Many
DELLA genes presented a wide range of expression patterns among tissues consistent with their role as negative regulators of gibberellin signal transduction (Peng et al.,
The rice DLT gene modulates brassinosteroid-related gene expression (Tong et al.,
As previously mentioned, expression of GRAS genes in pollen tissue differed from other tissues.
Several GRAS genes (
Several grapevine
Altogether, these observations could suggest the relevance of
Several GRAS proteins have been associated with a role in stress signaling (reviewed by Bolle,
Other grapevine
The Arabidopsis GRAS protein SCL14 was shown to be essential for the activation of stress-inducible promoters (Fode et al.,
The
Altogether, the expression of several grapevine
GRAS transcription factors have been characterized in several species and were proven to be involved in diverse developmental processes and stress responses. However, their involvement in fruit ripening is only now starting to be disclosed. Grape berry development and ripening could be under control of
AF and JG designed the study. JG, PA, RT, and AF analyzed the data. AF wrote the manuscript with valuable input from JG and JM. All the authors revised and approved the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding was provided by the Portuguese Foundation for Science and Technology (SFRH/BPD/100928/2014, UID/MULTI/04046/2013 and PEst-OE/BIA/UI4046/2014) and is integrated in the COST (European Cooperation in Science and Technology) Action FA1106 “Quality fruit.” JG was supported by the Ramon y Cajal program (RYC-2011-07791) and the AGL2014-59171-R project from the Spanish MINECO.
The Supplementary Material for this article can be found online at: