Genome-wide identification and analysis of mitogen activated protein kinase kinase kinase gene family in grapevine (Vitis vinifera)
© Wang et al.; licensee BioMed Central Ltd. 2014
Received: 29 April 2014
Accepted: 4 August 2014
Published: 27 August 2014
Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize.
In this study, we performed a genome-wide survey and identified 45 MAPKKK genes in the grapevine genome. Chromosome location, phylogeny, gene structure and conserved protein motifs of MAPKKK family in grapevine have been analyzed to support the prediction of these genes. In the phylogenetic analysis, MAPKKK genes of grapevine have been classified into three subgroups as described for Arabidopsis, named MEKK, ZIK and RAF, also confirmed in grapevine by the analysis of conserved motifs and exon-intron organizations. By analyzing expression profiles of MAPKKK genes in grapevine microarray databases, we highlighted the modulation of different MAPKKKs in different organs and distinct developmental stages. Furthermore, we experimentally investigated the expression profiles of 45 grape MAPKKK genes in response to biotic (powdery mildew) and abiotic stress (drought), as well as to hormone (salicylic acid, ethylene) and hydrogen peroxide treatments, and identified several candidate MAPKKK genes that might play an important role in biotic and abiotic responses in grapevine, for further functional characterization.
This is the first comprehensive experimental survey of the grapevine MAPKKK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses, and the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPK cascade in grapevine.
KeywordsGrapevine Mitogen-activated protein kinase kinase kinase (MAPKKK) Gene family Phylogenetic analysis Expression analysis Stresses
Plants are constantly confronted by various pathogenic and environmental stresses that challenge their survival. To deal with stresses, plants have evolved a variety of biochemical and physiological mechanisms. Stress-activated molecular pathways include multiple inter-linked regulatory networks such as protein kinase signaling cascades that can efficiently transduce input signals into suitable outputs . The best characterized protein-kinase-based amplification cascades rely on the mitogen activated protein kinases (MAPKs), which are conserved components of signal transduction in all eukaryotic organisms . The MAPK cascades rapidly transduce stress signals into various appropriate intracellular responses . The basic MAPK cascades are composed of three classes of protein kinases: MAPK kinase kinase (MAPKKK/MAP3K), MAP kinase (MAPKK/MKK) and MAPK (MAPK/MPK). MAPKKKs are the first component of the cascades that activate MAPKKs by phosphorylating two amino acids in the S/T-XXXXX-S/T (x represents any amino acid) motif of the MAPKK activation loop, and then MAPKKs become dual-specificity kinases that activate the downstream MAPK through double phosphorylation of the T-X-Y motif in the activation loop (T-loop) ,. The activated MAPK leads to the phosphorylation of transcription factors and other signaling components that regulate the expression of downstream target genes .
So far, different members in MAPK cascades have been identified and characterized by functional genomics approach in a variety of plant species, including Arabidopsis, tobacco, rice, alfalfa and poplar. Arabidopsis thaliana genome contains 80 MAPKKKs, 10 MAPKKs and 20 MAPKs ,, whereas the rice genome contains 75 MAPKKKs, 8 MAPKKs and 17 MAPKs ,. Compared with MAPKs and MAPKKs, MAPKKKs act at the top of MAPK cascades with much greater numbers and show more complexity and sequence diversity. According to characteristic sequence motifs, MAPKKKs are divided into three groups in higher plants: the MEKK-like subfamily, ZIK subfamily and Raf-like subfamily. Compared to ZIK subfamily and RAF-like subfamily, MEKK subfamily members have a less conserved protein structure . The RAF and ZIK subfamily proteins have a C-terminal kinase domain (KD) and a long N-terminal regulatory domain (RD) that might function in scaffolding to recruit MAPKKs and MAPKs ,.
In plants, MAPK cascades have been implicated in the signaling pathways related to various stresses, ethylene signaling, innate immunity and defense responses -. In Arabidopsis, the cascade MEKK1-MKK4/5-MPK3/6-WRKY22/WRKY29 plays an important role in plant innate immunity . Investigations in alfalfa (Medicago sativa) have indicated that OMTK1, a MAPKKK, was activated by hydrogen peroxide (H2O2) . Two well-studied MAPKKK are CTR1 (Constitutive Triple Response 1) and EDR1 (Enhanced Disease Resistance 1) of A. thaliana, both belonging to the RAF-like subfamily. The CTR1 multigene family encodes an essential negative regulator for ethylene-induced gene expression in Arabidopsis, while EDR1 was shown to be a negative regulator in salicylic acid-inducible defense responses  with edr1 mutants showing increased resistance to powdery mildew . In addition, it was reported that AtRaf5 mutant exhibited an enhanced tolerance to salt in Arabidopsis. Over-expression of Os-MAPKKK6 increased the tolerance to dehydration stress through ROS scavenging in rice . In contrast to several reports on MAPKKKs in Arabidopsis and rice, research on MAPKKKs in grapevine is still very limited.
Grapevine (Vitis vinifera L.) is one of the most economically valuable and most widely grown fruit crops in the world. Sequencing of the highly homozygous grapevine PN40024 genome  provides a great opportunity for analysis of the grapevine genome and gene family evolution. Previously we have validated 12 grapevine MAPK gene by gene isolation and expression . To further understand how the MAPK cascade operates in grapevine and their internal relationships, we surveyed the gene family of MAPKKKs, the top of the MAPK cascade in the grapevine genome. Fourty-five grapevine MAPKKK genes were identified by a detailed bioinformatics analysis, annotated and named according to their sequence similarity with Arabidopsis genes, as established by the grapevine scientific community ( personal communication) and their chromosomal position and gene structure were determined. In addition, we analyzed their transcript profiles in different organs and developmental stages using published microarray data. Finally, we examined their expression patterns in response to different stresses using quantitative real time polymerase chain reaction (qRT-PCR). These results indicate that the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPKKK-MAPKK-MAPK cascade in grapevine.
Results and discussion
Identification of MAPKKK family in grapevine and construction of a phylogenetic tree
Characteristics of MAPKKKs of grapevine
Gene length (bp)
Aminoacid length (AA)
When one or more genes in grapevine matched more than one gene in Arabidopsis, a new name was attributed consisting of the common MAPKKK term and an increasing numbering. The detailed information on the VviMAPKKK genes identified in the present study is listed in Table 1 and Additional file 1, including nomenclature, accession numbers, chromosomal localizations, gene length, number of amino acid in the protein, isoelectric point (PI) and molecular weight (MW). These genes were distributed over almost all chromosomes, except chromosome 9. The gene length ranged from 1,678 bp (VviMAPKKK34) to 52,549 bp (VviMAPKKK6). The open reading frames (ORFs) encoded polypeptides ranging from 211 AA (VviMAPKKK46) to 1425 AA (VviMAPKKK51). The predicted molecular masses ranged from 23.84 to 155.43 kD and isoelectric point value ranged 4.76-10.32 (Table 1). According to the present study, the number of grapevine MAPKKK genes is significantly smaller than those of Arabidopsis MAPKKKs (80)  and rice MAPKKKs (75) .
Phylogenetic analysis of VviMAPKKK genes
To investigate the evolutionary relationships between MAPKKK members in grapevine and Arabidopsis, and also to assign a name to grapevine MAPKKK genes (see below) a phylogenetic tree was constructed from alignments of the full coding sequences of all 125 MAPKKK genes (45 from grapevine and 80 from Arabidopsis, Additional file 2) with the procedure and parameters described in Materials and Methods (Figure 1). Based on the phylogenetic tree, grapevine MAPKKK were classified into the same corresponding categories in Arabidopsis, which include MEKK-like, RAF and ZIK subfamilies. There were 9 VviMAPKKKs and 21 AtMAPKKKs in the MEKK subfamily, only 9 VviMAPKKKs and 11 AtZIKs in ZIK subfamily, while 27 VviMAPKKKs and 48 AtRAFs grouped in the RAF subfamily (Figure 1).
In the three clades, there were many grapevine MAPKKKs clustering together, suggesting that these homologous genes may have derived from multiple duplications after the speciation of grape during the evolution. Moreover, many grapevine MAPKKK genes have their clear orthologues in the Arabidopsis genome, which suggests that these genes might be conserved for some specific functions in the two species. Interestingly, one grapevine gene, VviMAPKKK29, stands outside the main branches and was included in the ZIK family with a bootstrap values of 71%, just above the threshold of 70% established for the phylogenetic analysis (Figure 1).
Chromosomal location of VviMAPKKK genes
Gene structural analysis of VviMAPKKK genes
As shown in Figure 3 and Table S2 (Additional file 3), the number of introns in VviMAPKKK genes was highly variable, ranging from 4 to 16 introns, whereas two genes (VviMAPKKK47 and VviMAPKKK34) had only one intron. The large variation in structures of VviMAPKKK genes suggests that the grapevine genome has changed significantly during its long evolutionary history. However, a certain degree of similarity could be observed among subgroups, supporting evolutionary relationships among members of each clade. The majority of genes in the ZIK subfamily contain 6–7 introns, genes in the MEKK subfamily mostly ranged between 8 and 10 introns, the RAF subfamily showed a variable exon number but with a majority of genes ranging between 12 and 15 introns, often with very long introns. Within this frame, paralogous gene pairs generally shared highly similar exon-intron structures (Figure 3). Collectively, the divergent gene structures between the different phylogenetic subgroups suggest that duplication events of MAPKKK genes might have occurred in ancient times and that offspring genes evolved into diverse exon/intron structures, possibly to accomplish different functions in the grapevine genome.
Analysis of conserved domains among VviMAPKKKs
The pattern of amino acid residues found in many subdomains is conserved among the family members . All VviMAPKKK genes grouped under MEKK, ZIK and RAF subfamilies were further analyzed for the presence of specific signatures. Nine VviMAPKKKs and 21 AtMAPKKKs which belong to MEKK subfamily share the conserved signature motif G (T/S) Px (W/Y/F) MAPEV, as revealed by the amino acid sequence analysis of the protein kinase domain (Additional file 4: Figure S1A). Presence of this signature in 8 out of 9 VviMAPKKK further confirmed their grouping into the MEKK subfamily, while VviMAPKKK28 showed a substitution of the methionine residue with a threonine. The ZIK subfamily consists of 9 VviMAPKKKs and 11 AtZIKs. The characteristic feature of this subfamily consists of a conserved signature GTPEFMAPE (L/V) Y across all grapevine members (Additional file 4: Figure S1B). No additional kinase domains were identified in the grapevine MEKK or ZIK subfamilies, except for VviMAPKKK27 (Additional file 5: Table S3). One exception is VviMAPKKK29, which clusters together with ZIK-encoding genes at the nucleotide level (Figure 1) with a bootstrap values just above the threshold of 70%, but the alignment of the corresponding predicted protein with other grapevine MAPKKKs at the aminoacid level revealed the presence of a slightly modified RAF domain instead of the typical ZIK domain. For this reason it was simply named VviMAPKKK29, without any reference to subfamily.
The RAF subfamily is the largest of the 3 clades of MAPKKKs. Twenty-seven and 48 MAPKKKs were grouped in the RAF subfamily in grapevine and Arabidopsis, respectively. Multiple alignments of the kinase domains revealed the presence of the RAF specific signature GTxx (W/Y) MAPE in almost all grapevine MAPKKK proteins, with only slight variations in VviMAPKKK38 and 40 (Additional file 6: Figure S2). Moreover, the majority of proteins in the RAF subfamily contained additional protein domains (Additional file 2: Table S3), the most frequent one being the EDR1 domain (7 proteins) followed by the PB1 domain (5 proteins), and other additional domains with lower frequencies. Interestingly, two “stress/fungal response” domains were detected in the sequence of VviMPKKK29, which shows a relevant divergence from other members of both clades as already mentioned.
Among the components of the kinase cascade in plants, only a few MAPKKK genes have been characterized. It was shown that AtMAPKKK1 and AtMAPKKK2 played important roles in plant innate immunity ,. MAPKKK1 in Arabidopsis was found to be responsible for oxidative stress and to be involved in negative regulation of hormone signaling . It was reported that OMTK1, a MAPKKK from M. sativa, regulates oxidative stress signaling . Recently, the Arabidopsis AtZIK4 protein WNK1 (At3g04910) was demonstrated to phosphorylate the putative circadian clock component APRR3 in vitro and might be involved in the control of circadian rhythms by regulating its biological activity, suggesting a different function from that of other MAPKKKs . Two of the best-studied RAF-like MAPKKKs in Arabidopsis, CTR1 [AtRAF1] and EDR1 [AtRAF2], act as negative regulators in ethylene-induced gene expression , and in response to powdery mildew attack , respectively. However, neither CTR1 nor EDR1 have been confirmed to participate in a classic MAPK cascade . Among those genes, only EDR1 has a clear orthologue in grapevine (VviMAPKKK60 [VviRAF2]) and can be an interesting candidate to ascertain its possible analogous functions in this species, while other characterized Arabidopsis MAP3Ks show different degrees of similarity with several grapevine genes.
Expression profiles of VviMAPKKK genes in different developmental stages and tissues
The most peculiar expression of VviMAPKKK was in pollen samples where most genes showed a up- or down-regulation, in comparison to other organs. The clustering of VviMAPKKK s according to their expression profile (Figure 4) revealed that the expression of a group of genes was much higher in young tissues and organs than in ripening or senescing ones, suggesting that these VviMAPKKK are mostly related to signal transduction during development in metabolically active tissues. The decreased transcript levels of VviMAPKKK genes in Cluster A were especially evident in post-withering stages, in which berries are left to natural dehydration for about 3 months. On the opposite, VviMAPKKK transcripts in Cluster B showed a higher level in later stages of grape development and during withering, suggesting that this set of genes may be responsive to dehydration and putatively involved in the deep transcriptomic and metabolic changes controlling biosynthesis of secondary metabolites responsible for the typical aromas of wines. This information can be important for further dissection of the signal transduction pathways operating in the transition from vegetative to reproductive stages  and in the regulation of the biosynthesis of aromatic compounds in the berry, in which different groups of MAPKKK may be involved.
It should be noted that the clustering of expression profiles does not reflect phylogenetic similarities. We only found similar expression profiles for the couples of MAPKKKs 22/23, 4/26, 56/57, and 42/47. In general, genes within the 3 clades of MEKK, ZIK and RAF or even paralogous genes may have very different expression profiles and possibly serve different functions in each organ and stage. This could have resulted from post-duplication diversifications, including subfunctionalization, neofunctionalization, or sub-neofunctionalization . These results provide a basis for further investigations on the function of VviMAPKKK genes in grapevine developmental biology.
Expression profiles of VviMAPKKK genes in response to biotic and abiotic stresses
Only a limited number of genes in MAPKKK family have been functionally characterized in Arabidopsis and even less in other species . Among those characterized genes, some were shown to be involved in the response to biotic and abiotic stresses ,-. In particular, two members of Arabidopsis RAF-like MAPKKKs with a function in plant defense were characterized: CTR1 [AtRAF1], negatively regulating ethylene responses , and EDR1 [AtRAF2], acting as a negative regulator of disease resistance and ethylene-induced senescence in Arabidopsis. Gene expression patterns usually act as indicators of gene function. In the present study, we investigated the expression patterns of all VviMAPKKK genes by semi-quantitative real-time RT-PCR in response to biotic (powdery mildew) and abiotic (drought) stress conditions, as well as in response to hormones (SA, ETH) and H2O2 treatments. Powdery mildew caused by the biotrophic ascomycete Erysiphe necator Schw. adversely affects vine growth, berry quality and grape production worldwide . Salicylic acid (SA), ethylene (ETH) and hydrogen peroxide (H2O2) play central roles in biotic stress signaling upon pathogen infection. SA and ETH are signal molecules implicated in plant defense responses to pathogens ,. H2O2 is an important ROS and a critical signaling molecule in cascades leading to plant responses to pathogens and abiotic stress factors . Treated samples were collected in all cases at 6 time points, that is 4, 8, 12, 24, 48 and 72 hours post-treatment (hpt), except for samples subjected to drought stress, which were collected after 4, 8 and 12 days (dpt).
Only a few genes diverge from these 3 main groups: VviMAPKKK47, which is almost invariably repressed, and VviMAPKKK38, which is strongly induced by E. necator, by SA and by H2O2 at the same time points of 4 and 24 h post-treatment.
Examining each stress condition separately, it can be observed that E. necator caused a strong increase of transcripts of most genes in cluster A (VviMAPKKK46,50, 32, 39, 34) and additionally of VviMAPKKK31 and 38; in particular, VviMAPKKK50 showed the highest transcript abundance, between 6 and 27-fold the control (Figures 5 and 10). A few genes (VviMAPKKK4, 54 and 51) are significantly down regulated by powdery mildew infection, especially VviMAPKKK54, while other genes are variably but slightly modulated. It can be observed however that many VviMAPKKK transcripts showed a decreased abundance at the very early time point (4hpt), a trend to a more or less pronounced increase afterwards, and a new decrease at the last collection time (72 hpt). This observation might correlate with the full establishment of infection and a possible down-regulation of defense responses, as it was reported in barley that powdery mildew can induce susceptibility in infected cells . Although elucidating the exact roles of these VviMAPKKK genes in pathogen interactions requires further functional analysis, our findings provide the first gene-family-wide survey on the expression patterns of specific grapevine MAPKKK in pathological conditions, and these highly up- and down-regulated genes can be candidate genes for future investigations.
Salicylic acid and ethylene were chosen to investigate transcriptional responses of VviMAPKKKs to hormone treatments. Regarding the response to SA, Figures 6 and 10 shows a general picture of VviMAPKKKs down regulation for most genes at most time points, especially in Cluster A, in which VviMAPKKK34 and 46 show a decreased transcript abundance of more than 20 fold at 12 hpt, but increased afterwards, especially at 48 hpt. Clear increases could be detected for VviMAPKKK60 and 64 at 12 hpt and for VviMAPKKK34 and 50 at 48 hpt. Response to ethylene was striking for some VviMAPKKK genes (examples: VviMAPKKK39, with an increase above 10-fold at 8 hpt; VviMAPKKK60, induced by 9-fold at 12 hpt; VviMAPKKK34 and 46 with fold change values between 3 and 7 (Figure 7). The same VviMAPKKK60 and 64, responsive to SA at 12hpt, were also responsive to ethylene, especially at early time points, as well as VviMAPKKK52 and the paralogous couple VviMAPKKK 22/23. VviMAPKKK60 is the grape orthologue of Arabidopsis EDR1 gene. EDR1 exerts its negative control at a point of cross talk between ethylene and salicylic acid signaling . Therefore it seems interesting that treatments with both SA and ETH may induce an increase of VviMAPKKKK60 at early time points, possibly as a regulatory mechanism to keep a balance between the two pathways. SA and ETH are involved in different signal transduction pathways and their action is often considered antagonistic, but may also cooperate in regulating defense responses . Other MAPKKK genes were mostly down regulated by ethylene along the whole time course, such as VviMAPKKK30 and 36 (Figure 7). In some cases we observed a very similar expression profile in response to these two treatments, such as for VviMAPKKK30, 36 and 61, all down regulated and for VviMAPKKK27, 40 and 52 with variable but similar profiles along the time course of the experiments.
To further investigate the roles of VviMAPKKK genes, their expression levels were measured in response to the defense signaling compound H2O2. Several transcripts reacted strongly to this treatment along the whole experiment (Figures 8 and 10), especially VviMAPKKK39, which increased up to 50 fold at 24 hpt, and VviMAPKKK34, 46 and 50, with relevant increases at several collection times. A slight increase, of about 3-fold (24 hpt) was also observed for VviMAPKKK22, which shows a high degree of sequence similarity with Arabidopsis AtMAPKKK1 [MEKK1], involved in the regulation of reactive oxygen species (ROS) homeostasis . The level of this grapevine transcript was down regulated by E. necator infection and induced by ethylene. Since plant genomes generally contain a relatively large number of MAPKKK genes, this differential regulation of MAPKKK gene expression may be a mechanism by which stress responses are fine tuned, although additional work is needed to confirm this hypothesis.
To analyze the responsiveness of VviMAPKKK to abiotic stress, drought treatments were performed. Drought is a major environmental factor limiting productivity and distribution of plants . When grapevine plants were subjected to drought stress, almost all VviMAPKKK genes displayed significantly increased expression levels especially at 8 days post-treatment (Figures 9 and 10). Among all performed treatments, drought is the one which caused the strongest effect, with many VviMAPKKK transcripts increasing more than 10 fold, and 4 transcripts (VviMAPKKK22, 23, 51, and 54) showing a more than 20-fold increased expression (Figure 9). Down regulation, although rare, was also very strong in the case VviMAPKKK46, (up to 30-fold less than control). For several unregulated genes, the increase was sustained also in the last collection time of 12 dpt. The first collection time (4 days) did not reveal a significant modulation, suggesting that plants could maintain more or less stable mRNA levels in the first days after watering suspension, except for VviMAPKKK24, 34 and 46, strongly and early down regulated. Therefore, in comparison to other stresses, drought determines a peculiar response of strong transcriptional activation on most VviMAPKKKs, especially at 8 d after drought treatments, suggesting that grapevine MAPKKKs are very likely playing roles in response to drought stress. The involvement of MAPKKK in drought resistance was seldom investigated. Among the few examples there are a RAF-Like MAPKKK gene DSM1 with a function in drought and oxidative stresses signaling in rice  and a tobacco MAPKKK (NPK1) which enhanced drought tolerance in transgenic maize . Our data provide the first insight into the possible involvement of grapevine MAPKKKs in this type of stress. However, more research is needed to determine the specific functions of the MAPKKK family by additional biological experiments, particularly to investigate why such a high number of VviMAPKKKs genes are responsive to drought.
So far, MAPKKKs have only been investigated in some plant species including Arabidopsis, rice and maize, while no systematic analysis has been conducted in grapevine. In the present study we identified 45 MAPKKK coding genes in the V. vinifera genome, which were grouped into three subfamilies - MEKK, ZIK and RAF- and named according to their sequence similarity to Arabidopsis genes. The exon/intron structure, phylogeny and conserved domains strongly supported their identity as members of each subfamily. Furthermore, by exploring a previously published microarray analysis, we provided information about the expression profiles of all VviMAPKKK genes across different tissues and developmental stages. Finally, we experimentally determined the expression profiles of all grapevine MAPKKK genes in response to biotic and abiotic stress conditions, as well as hormone and H2O2 treatments. In conclusion, our work provides an inventory of VviMAPKKK genes potentially involved in environmental stresses, an initial insight into this important gene family and a number of possibly stress-related candidates for future functional analysis. This information provides a framework to unravel the biological roles of the VviMAPKKK genes family in grape and their regulatory mechanism, particularly their apparently wide implication in drought responses.
Identification of MAPKKK gene family in grapevine
MAPKKK protein sequences of Arabidopsis were used as query to search against the Proteome databases of V. vinifera (12X, V1) (http://genomes.cribi.unipd.it/grape) , Vitis-URGI (http://urgi.versailles.inra.fr/Species/Vitis) and NCBI databases (http://www.ncbi.nlm.nih.gov/) using the BLASTP program with e-values > 1E-5. The Protein family (Pfam) database (http://pfam.sanger.ac.uk/) was used to identify their protein domains using HMMER3.0. MAPKKK gene models were only accepted if they displayed the consensus sequences of dual-specificity protein kinases. Then, the online software SMART (http://smart.embl-heidelberg.de/) was used to further confirm the predicted MAPKKK protein sequences . To increase efficiency of the search, we also investigated the results of a recently published paper  reporting a comparison of gene predictions between the 8× and the 12X genome coverages. Three additional MAPKKK genes were identified in the 8X genome, which are not present in the 12X version, but we could not find evidence that these 3 genes are actually expresses in the ESTs databases, and therefore they were not included in the family.
Phylogenetic and motif analysis of the MAPKKK gene family in grapevine
The phylogenetic tree was constructed with the web service Phylogeny.fr (http://www.phylogeny.fr/) following the rules defined by the Grapevine Super Nomenclature Committee (; Grimplet J. personal communication); bootstrap values below 70% were collapsed. The Arabidopsis nomenclature used following the MAPKKK nomenclature reported in the TAIR database (https://www.arabidopsis.org/) in which MEKK genes were named MAPKKK while RAF and ZIK genes were annotated maintaining the subfamily name. Multiple-sequence alignments of computationally predicted MAPKKK proteins belonging to each group of both Arabidopsis and grapevine (including characteristic sequence motifs) were performed using ClustalX program (version 1.83)  and GeneDoc (http://www.nrbsc.org/gfx/genedoc/). The PFAM database (http://pfam.sanger.ac.uk/) was used for identification of additional conserved motifs outside the MAPKKK domain. The protein sequences of Arabidopsis MAPKKKs were obtained from the TAIR (http://www.arabidopsis.org/) database.
Gene structure, chromosomal location and gene duplication of grapevine MAPKKK genes
The information on MAPKKK genes in the grapevine genome, including accession number, chromosomal location, open reading frame (ORF) length, molecular masses, isoelectric point value (pI) and exon-intron structure were retrieved from the grapevine database (http://genomes.cribi.unipd.it/grape/). Gene duplication events of MAPKKK genes in grapevine were investigated based on three criteria: 1) The alignment length covered >90% of the longer gene; 2) The aligned region had an identity >90%; 3) Only one duplication event was counted for tightly linked genes . GSDS (Gene Structure Display Server, http://gsds.cbi.pku.edu.cn/) was exploited to illustrate exon-intron organization of MAPKKK genes .
Microarray data analysis of VviMAPKKK genes
To understand the spatial and temporal expression patterns of MAPKKK genes during the grapevine life cycle, the expression profiles of the MAPKKK genes was analyzed based on published high-throughput microarray data . In the data sets, a total of 54 grapevine samples were included (bud, inflorescence, carpel, petal, pollen, berry, withering berry, leaf, root, seed, seedling, rachis, stem, and tendril), covering most organs at several developmental stages. The expression data were transformed in log2 values. The heat map was made with software MeV4.8 (http://www.tm4.org/mev/).
Plant materials and stress treatments
PN40024 plants (V. vinifera inbred line of Pinot noir, sequenced genotype) were kindly provided by Dr. Anne-Françoise Adam-Blondon, INRA, France, and maintained in vitro on 1/2 MS medium supplied with 0.3 mg/L Indole 3-butyric acid (IBA, Sigma, USA), under a 16/8 h photoperiod (100 μmol m−2 s−1) at 25°C in the growing chamber. Five-week-old plants were used in all treatments.
For treatments with salicylic acid (SA, Sigma, USA), ethylene (ETH) (Ethephon, Sigma, USA) and hydrogen peroxide (H2O2, Sigma, USA), plants with fully expanded six to eight leaves per tissue-culture container (240 mL) were sprayed with 5 mM SA, 5 mM ethephon (as an ethylene donor) and 10 mM H2O2. All the chemicals were purchased from Sigma and dissolved in sterile distilled water. The samples (the second to fourth leaf counted from the top) were harvested at 4, 8, 12, 24, 48 and 72 h post-treatment. Each collected sample contained independent biological replicates (three independent treated plants) and three corresponding controls. For powdery mildew infection, a local strain of Erysiphe necator Schw. was maintained on PN40024 in a greenhouse. Young leaves of similar developmental stages were inoculated with E. necator by gently pressing and tapping conidia from infected leaves on healthy ones. The second to fourth leaves were sampled at 4, 8, 12, 24, 48 and 72 h post-inoculation. Each collected sample contained independent biological replicates (three independent treated plants) and three corresponding controls. For drought treatments, in vitro plants were acclimated to pots filled with a mixture of soil and sand (1:1) in the greenhouse until they grew to a length of about 40 cm with 14 leaves. The plants were watered thoroughly first and then not watered. The sixth leaves were collected at 4, 8 and 12 days after watering interruption, immediately frozen in liquid nitrogen and stored at −70°C until analysis. Every treated sample had a corresponding regularly-watered control. For each point, three independent biological replications (three independent plants) were sampled.
Total RNA isolation and qRT-PCR expression analysis
Total RNA was extracted from the collected samples according to described previously with some modifications . The concentration and purity of RNAs were examined by measuring optical density (OD) absorption ratio at 260 and 280 nm in a One Drop™ OD-1000 spectrophotometer (Thermo Fisher Scientific, USA). The RNA integrity was checked by electrophoresis on 1.0% agarose gels stained with ethidium bromide (EB). The first-strand cDNA templates were synthesized from1 μg total RNA using PrimeScritpt RT reagent Kit (TaKaRa, Japan) following the manufacturer’s instructions.
The expressions of VviMAPKKKs were examined by qRT-PCR using a SYBR Green method on an ABI 7300 Real-time PCR System (Applied Biosystems). Primers were designed by Beacon Designer 7.0 software (Premier Biosoft International, USA), based on the 3′-untranslated region and the 3′ terminal sequences of the coding region according to the predicted mRNA sequence. The amplification product of each reaction was about 200 bp. The reaction mix (total volume of 20 μL) contained: 10 μL SYBR® Premix Ex Taq™, 0.2 μL of each primer, 1 μL of template and 8.6 μL ddH2O. The PCR conditions were: pre-denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 20 s, 60°C for 20 s, and 72°C for 43 s. Grapevine actin gene (actin-101- like, VIT_12s0178g00200) was used as the internal normalize, which was previously shown to be a suitable internal standard . The relative gene expression level was calculated according to the 2-ΔΔCt method, where ΔΔCt = (Cttarget gene - Ctactin) treatment - (Cttarget gene - Ctactin) control,. To visualize the relative expression levels data are presented as the mean fold changes between treated and control samples at each time point ± standard deviations (SDs). Mean values and standard deviations (SDs) were obtained from three biological replicates, each with three technical replicates. The gene specific primers are listed in Table S1 (Additional file 9).
Statistical analyses were performed using the software SPSS version 13.0 (Chicago, IL) and Excel. All results were indicated as means ± standard deviations (SDs) based on Duncan’s multiple range test. P < 0.05 and P < 0.01 were taken as statistically significant or highly significant, respectively.
Availability of supporting data
Here we are with the supporting data (including sequence data, microarray data and expression data) as additional files.
This research was supported partly by the Ministry of Education-Nanjing Agricultural University Project # KYZ201311.
We are grateful to Dr. Jérôme Grimplet (Instituto de Ciencias de la Vid y del Vino, CSIC, Universidad de La Rioja, Gobierno de La Rioja, Logroño, 26006, Spain) for sharing unpublished information and kind support in nomenclature assignment.
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