Source: https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-015-0552-z
Timestamp: 2019-04-20 13:02:38+00:00

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Calcium-dependent protein kinases (CDPKs) play vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. Little is known about the CDPK gene family in grapevine.
In this study, we performed a genome-wide analysis of the 12X grape genome (Vitis vinifera) and identified nineteen CDPK genes. Comparison of the structures of grape CDPK genes allowed us to examine their functional conservation and differentiation. Segmentally duplicated grape CDPK genes showed high structural conservation and contributed to gene family expansion. Additional comparisons between grape and Arabidopsis thaliana demonstrated that several grape CDPK genes occured in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grapevine and Arabidopsis. Phylogenetic analysis divided the grape CDPK genes into four groups. Furthermore, we examined the expression of the corresponding nineteen homologous CDPK genes in the Chinese wild grape (Vitis pseudoreticulata) under various conditions, including biotic stress, abiotic stress, and hormone treatments. The expression profiles derived from reverse transcription and quantitative PCR suggested that a large number of VpCDPKs responded to various stimuli on the transcriptional level, indicating their versatile roles in the responses to biotic and abiotic stresses. Moreover, we examined the subcellular localization of VpCDPKs by transiently expressing six VpCDPK-GFP fusion proteins in Arabidopsis mesophyll protoplasts; this revealed high variability consistent with potential functional differences.
Taken as a whole, our data provide significant insights into the evolution and function of grape CDPKs and a framework for future investigation of grape CDPK genes.
Calcium (Ca2+), a universal second messenger in eukaryotes, mediates stimulus–response coupling in the regulation of diverse cellular functions [1, 2]. Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. The three main classes of Ca2+ sensors identified in plants are: CaMs (calmodulins) and CaM-like proteins, CBLs (calcineurin B-like proteins), and CDPKs (calcium-dependent protein kinases) [3–5]. The CDPKs, also termed CPKs, consist of a variable N-terminal domain, a conserved serine/threonine kinase domain, an auto-inhibitory junction region, and a C-terminal regulatory CaM-like domain . Unlike the other Ca2+ sensors, CDPKs have both Ca2+ sensing and responding activities due to their unique, CaM-like domain and protein kinase domain, which convert upstream Ca2+ signals into downstream phosphorylation events and cellular responses [7, 8].
Genome-wide analysis, together with comparative genomics, provides an effective way to understand the structures and functions of members of a gene family, using the insights gained from evolutionary relationships and experimental data. The original, systematic report on the CDPK gene family in Arabidopsis thaliana identified 34 CDPK gene family members , and was followed by research in rice (Oryza sativa)  and wheat (Triticum aestivum) . Recently, genome-wide analyses of the CDPK gene family have been reported in maize (Zea mays L)  and poplar (Populus trichocarpa) . Meanwhile, more and more investigations of CDPK genes have also involved horticultural plants, such as alfalfa , potato , strawberry , and tomato .
Work in Arabidopsis showed that CDPKs function in immune and stress signaling, growth and development, and hormone responses. AtCPKs play vital parts in immune signaling pathways; for example, AtCPK1 activates NADPH oxidase, resulting in an oxidative burst , and phosphorylates PAL (Phenylalanine ammonia-lyase) resulting in accumulation of salicylic acid (SA) . AtCPK4/5/6/11 phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, which participate in NLR-dependent restriction of pathogen growth. In addition, AtCPK5 activates RBOH to induce a reactive oxygen species (ROS) burst [20, 21]. CPKs also function in the response to abiotic stress. For example, AtCPKs help to enhance drought tolerance by responding to abscisic acid (ABA), leading to induction of expression of genes such as AtCPK4/11 , and stomatal closure via induction of AtCPK3/6 . AtCPK4/11  and AtCPK23  trigger plant salt tolerance via controlling of K+ channels. In hormone signaling, studies have systematically examined ABA [25, 26], and a few studies have examined MeJA , SA , and ethylene . The rich sequence and functional information from Arabidopsis enables us to extrapolate the functions of the orthologous genes in other species.
Grapevine is one of the most important fruit crops in the world. However, most cultivated grapevine varieties (Vitis vinifera) are susceptible to many pathogens and are sensitive to abiotic stresses. By contrast, Chinese wild grapevine (Vitis pseudoreticulata) accession Baihe-35-1 has demonstrated resistance to multiple diseases and to various environmental stresses [29, 30]. Previous examination of the CDPK gene family in Vitis vinifera identified 17 members , and in Vitis amurensis isolated 13 members . However, prior work focused on evolutionary relationships with only a few transcriptional analyses in some tissues and developmental stages. In this study, we employed bioinformatics and publicly available data to identify and analyse grape CDPK genes on a genome-wide scale in Vitis pseudoreticulata, identifying two more members. Furthermore, we measured the expression of the CDPK genes (VpCDPKs) in Chinese wild grapevine Vitis pseudoreticulata accession Baihe-35-1 in response to various biotic and abiotic stresses as well as multiple phytohormone treatments. In addition, we showed that the VpCDPKs have different subcellular localizations when transiently expressed in Arabidopsis mesophyll protoplasts. More significantly, comparing Arabidopsis and grapevine CDPK gene structures, evolution, and experimental data provides insights on the functions of VpCDPKs. Our results provide a set of potential candidate CDPK genes for future genetic modification of pathogen resistance and stress tolerance in grapevine.
We identified CDPK genes by searching the Pfam database and obtained the HMM (Hidden Markov Model) profiles of protein kinase domain Pkinase (PF00069) and EF-hand domain EF-hand_7 (PF13499). Then we used BLAST-P to search the 12X grapevine (V. vinifera) genome using Pkinase and EF-hand HMM profiles. We also performed BLAST-P searches at NCBI using full-length amino acid of putative grape CDPK genes. After that, we identified a bunch of candidates (data not shown). By removing incomplete gene sequences, transcripts of the same genes, and redundant sequences, we identified nineteen non-redundant CDPK genes in the grape genome (Table 1). Among those with alternative splice variants, we selected the longest variant for further analysis. The sequences of the nineteen grape CDPKs were submitted to InterPro and SMART databases to confirm their Pkinase domains and EF-hand domains. Previous work identified seventeen CDPK genes in grapevine . The seventeen genes were named based on their distributions and relative linear orders on the respective chromosomes. On this basis, we added two members (GSVIVT01025745001 and GSVIVT01027353001) and named them VvCDPK18 and VvCDPK19 according to their chromosomal locations. The nineteen putative CDPK genes mapped on eleven grape chromosomes (Fig. 1). Among them, eight chromosomes possess one CDPK gene, three possess two CDPK genes, and Chr. 8 possesses five CDPK genes.
Characteristics of the nineteen CDPK genes are shown in Table 1. The lengths of the gene sequences vary widely, from 3.103 to 21.654 kb, but the numbers of predicted amino acids are relatively similar for most genes, around 520 to 580, except VvCDPK16 with 626 amino acids. Fifteen of the nineteen CDPKs are predicted to be palmitoylated and eight of the nineteen CDPKs are predicted to be myristoylated. The predicted proteins for six CDPK genes have both N-terminal myristoylation sites and palmitoylation sites. Nine CDPK genes encode proteins with only palmitoylation sites and two (VvCDPK3 and VvCDPK7) have only myristoylation sites. Besides these, VvCDPK16 and VvCDPK19 have neither myristoylation nor palmitoylation sites. Typical CDPKs have four EF-hands, but not necessarily [9, 33]. The seventeen previously identified CDPKs were predicted to each have four EF-hands. In addition, the two new members, VvCDPK18 and VvCDPK19 were predicted to have two EF-hands and three EF-hands, respectively.
The predicted protein sequences of the nineteen grape CDPKs contain the four typical CDPK domains, including the N-terminal variable domain, the protein kinase domain, the junction domain, and the calmodulin-like domain (data not shown). The intron-exon organization can indicate the evolutionary relationships within multi-gene families . As shown in Fig. 2, grape CDPK genes can be divided into four groups (I-IV) on the basis of the Arabidopsis classification . Most of the grape CDPK genes have seven or eight exons, six or seven introns, and clear intron phase patterns. Group I members have seven exons, except VvCDPK16 with ten exons, and their intron phases, with respect to the open reading frame, occur in the same pattern, with type-2 intron phases at the first three positions from the beginning and type-0 intron phases at the last three positions. All of the Group II members contain eight exons and also share the same intron phase pattern. Compared with the Group I members, Group II members have an additional exon. Group III can be divided into two subgroups. Subgroup 1 shares the same intron-exon organization as most of the Group I members, such as VvCDPK10 and VvCDPK14, but subgroup 2 members, such as VvCDPK4, VvCDPK5, and VvCDPK11, have one more exon at the first position and one more type-0 phase intron. Compared with the other three groups, Group IV has a complicated intron-exon organization with differing numbers of exons and intron phases. Clustering the intron-exon structures of the nineteen CDPK genes by an unrooted phylogenetic tree suggests a connection between intron-exon structures and evolutionary relationships.
Gene duplication and divergence are important in gene family expansion  and in the evolution of novel functions . Grapevine has undergone whole-genome duplications during its evolutionary history . To examine the effect of duplications on the CDPK gene family, we obtained tandem duplication and segmental duplication gene pairs from PGDD (Plant Genome Duplication Database) and visualized them using Circos. In this study, we identified two segmental duplication pairs of grape CDPK genes (VvCDPK5 and VvCDPK11, VvCDPK12 and VvCDPK17) (Fig. 3) but did not find tandem duplication events among the grape CDPK genes.
To further explore the origin and evolution of grape CDPK genes, we investigated the syntenic relationship between grapevine and Arabidopsis. The synteny analysis showed that grape CDPK genes can be divided into two types (Fig. 3, Additional file 1. The first type of syntenic genes has a single grape gene that corresponds to a single Arabidopsis gene, including VvCDPK2-AT4G35310 (AtCPK5), VvCDPK4-AT2G31500 (AtCPK24), VvCDPK5-AT5G12480 (AtCPK7), VvCDPK10-AT3G51850 (AtCPK13), VvCDPK15-AT1G76040 (AtCPK29), and VvCDPK17-AT3G20410 (AtCPK9). The second type has a single grape CDPK gene that corresponds to multiple Arabidopsis genes, including VvCDPK3-AT2G17890 (AtCPK16)/AT4G36070 (AtCPK18)/ AT5G66210 (AtCPK28), VvCDPK11-AT2G41860 (AtCPK14)/ AT3G57530 (AtCPK32), VvCDPK12-AT1G61950 (AtCPK19)/ AT3G20410 (AtCPK9)/ AT4G04695 (AtCPK31)/ AT4G21940 (AtCPK15), and VvCDPK14-AT1G74740 (AtCPK30)/ AT1G18890 (AtCPK10). These results provide insights that will assist in prediction of the functions of grape CDPKs.
To investigate the evolutionary relationships and functional associations, we constructed a neighbor-joining tree using the full-length amino acid sequences of CDPKs from grape, Arabidopsis, rice, maize, and poplar (Fig. 4, Additional file 2). The phylogenetic analysis indicated that nineteen VvCDPKs can be divided into four groups. Of the four groups, three groups appear to be well-defined, except Group IV, according to the distributions of branches. Most of the CDPK genes from monocots (rice and maize) clustered into one sub-branch, as did the CDPK genes from the eudicots (Arabidopsis, poplar and grape). Also, grape CDPK genes clustered more often with poplar CDPK genes than with Arabidopsis CDPK genes. For the four Groups of VvCDPKs, Group I contains six VvCDPK genes. Five of the six genes have homologs in Arabidopsis or poplar, or both, except VvCDPK2, which clustered with ZmCPK6 and OsCPK6. Group II contains five grape CDPK genes and clustered into two subgroups. Among these genes, VvCDPK12 and VvCDPK17 show high sequence similarity with each other. Group III contains five grape CDPK genes and Group IV, the smallest group, contains three grape CDPK genes. These phylogenetic relationships suggest evolutionary conservation of the basal architecture of the CDPK family.
Most CDPKs possess either N-myristoylation sites, S-palmitoylation sites, or both. These acylation sites are believed to be involved in targeting to membranes . Because those acylation sites are predicted to be present in grape CDPKs (Table 1), to determine if grape CDPKs localize to membranes, we cloned six VpCDPKs from Chinese wild Vitis pseudoreticulata accession Baihe-35-1 and assessed the subcellular localizations of the encoded VpCDPKs by transient expression assays in Arabidopsis protoplasts, using translational fusions to GFP.
As shown in Fig. 5, five genes (VpCDPK2, VpCDPK3, VpCDPK5, VpCDPK10, and VpCDPK11) expressed VpCDPK-GFP fusion proteins in transformed Arabidopsis protoplasts. VpCDPK3-GFP, VpCDPK10-GFP, and VpCDPK11-GFP only localized on the plasma membrane. VpCDPK5-GFP localized on the plasma membrane and in the nucleus, and VpCDPK2-GFP localized in the nucleus and cytosol.
Unlike the other CDPKs, VpCDPK9 localized to four places (Fig. 6): (i) VpCDPK9 localized on some kind of plastids that could not be identified. Considering the closest gene in Arabidopsis is AtCPK1, which localizes to lipid bodies and peroxisomes [19, 38], the plastids that VpCDPK9-GFP localized in may well include lipid bodies and peroxisomes. (ii) VpCDPK9-GFP localized to the biomembrane system, most likely on the endoplasmic reticulum (ER), as well as on vesicles. (iii) VpCDPK9-GFP also showed extra fluorescence in the cytosol. (iv) We also detected VpCDPK9-GFP in the nucleus. The complexity of VpCDPK9 subcellular localization suggests its functional diversity and variety.
Increasingly evidence shows that plant CDPKs function in responses to biotic and abiotic stress and signal transduction [9, 33]. To further test how grape CDPK genes respond to various stresses, we measured the expression of grape CDPK genes in Chinese wild grape (Vitis pseudoreticulata) accession Baihe-35-1.
CDPKs act as vital sensors and responders in immune signaling . In this study, we used RT-qPCR to perform a time course analysis of the transcript levels of VpCDPKs following inoculation of Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1 with Erysiphe necator, the causal agent of grapevine powdery mildew (Fig. 7-a and Additional file 3). Five of the grape CDPK genes were up-regulated, including VpCDPK6, VpCDPK9, VpCDPK14, VpCDPK16, and VpCDPK19, with transcript levels that increased up to 3.0-fold (p < 0.05) but differed in response time and degree. For example, VpCDPK9 transcript rapidly increased in abundance and reached a peak of 5.3-fold at 96 h post inoculation (hpi) (Fig. 7-b). Similarly, VpCDPK14 transcript also rapidly increased and remained at high levels (Additional file 3). VpCDPK6 (Additional file 3) and VpCDPK19 (Fig. 7-b) shared similar expression patterns, with a slow increase in transcript levels to a peak and gradual plateau. VpCDPK16 (Fig. 7-b) transcript abundance increased to nearly 3.3-fold, nearly instantly at 72 hpi, and remained at high levels. By contrast, several CDPK genes were down-regulated. For instance, VpCDPK1 and VpCDPK18 (Additional file 3) transcript levels decreased to 0.3- to 0.5-fold. These results demonstrate that several CDPK genes responded to powdery mildew inoculation in Chinese wild grapevine V. pseudoreticulata accession Baihe-35-1, suggesting their vital roles in immune signaling and responses.
Previous work showed that CDPKs participate in abiotic stress responses, including responses to salt and osmotic stress [22, 39] and to temperature stress . In this study, we used NaCl treatment, and incubation at low or high temperature (4 °C or 42 °C) to understand how VpCDPKs responded to these abiotic stresses on the transcriptional level. Our expression data show consistency with the expression of CDPK genes in Vitis vinifera  and Vitis amurensis .
As shown in Fig. 8 (see also Additional files 4, 5, 6), almost all nineteen CDPK genes were up- or down-regulated to at least one abiotic stress treatment, but some responded rather slightly. As a whole, the VpCDPK genes responded to NaCl treatment much more strongly than to temperature stress (Fig. 8-a). VpCDPK6 and VpCDPK9 transcript abundance increased in all three treatments, but none of the CDPK genes showed continuously low transcript levels in all treatments.
After NaCl treatment (Fig. 8 and Additional file 4), twelve of the nineteen CDPK genes were up-regulated, and six genes (VpCDPK3, VpCDPK9, VpCDPK14, VpCDPK16, VpCDPK17, and VpCDPK19) responded strongly with transcript levels increasing to 5.0- to 16-fold higher than the control sample (p < 0.05). VpCDPK9, the most rapidly responding gene, reached a peak of nearly 10-fold at 0.5 h post treatment (hpt) and rapidly decreased to 3.9-fold at 2 hpt, before its transcript levels increased from 3.9-fold to 9.7-fold at 2 h to 12 hpt (Fig. 8-b). The homologous gene of VpCDPK9 in Vitis amurensis, VaCPK1, was also highly up-regulated under NaCl treatment . In addition, VpCDPK17 showed a peak of 16.3-fold at 8 hpt and increased transcript abundance of more than 9.0-fold from 2 to 48 hpt (Fig. 8-b), similar with the expression pattern of its homologs, VaCPK9  and VvCPK17 . We noted that the margins of treated leaves became dry from the outside to the inside and the plants had dry-dead leaves after 48 hpt, which might relate to the rapid down-regulation of VpCDPK14 at 48 hpt. By contrast, VpCDPK5 (Fig. 8-b) and VpCDPK8 (Additional file 4) transcript abundance decreased after 2 hpt, indicating their probable negative regulatory roles. The CDPK genes that responded rapidly and strongly most likely participate in the NaCl stress response.
For 4 °C treatments (Fig. 8 and Additional file 5), only VpCDPK6, VpCDPK9 and VpCDPK16 (Fig. 8-a) showed up-regulation (p < 0.05). VpCDPK6 was rapidly up-regulated, reached a peak of 12.4-fold at 2 hpt, and then declined to lower than 2.0- fold (Fig. 8-b). Moreover, VpCDPK16 had a peak of 7.7-fold at 8 hpt but then fluctuated around 1.0-fold during the rest of the treatment time (Fig. 8-b). The VpCDPK genes with decreased transcript abundance might not participate in the signaling response to cold stress but their low activities might result in pervasive down-regulation of transcription. However, VpCDPK8 (Fig. 8-b) was an exception, as its transcript levels decreased rapidly at 2 hpt and remained at a steady low level. Intriguingly, VaCPK20, as the homolog of VpCDPK8 in Vitis amurensis, showed significant up-regulation under 10 °C treatment .
We also measured the transcript levels of VpCDPK genes in response to 42 °C treatment. We found that five CDPK genes (VpCDPK4, VpCDPK6, VpCDPK9, VpCDPK10, and VpCDPK19) significantly responded to 42 °C treatment (the peaks are over than 3.0- fold, p < 0.05); of these, VpCDPK6 (Fig. 8-b) had a strong response at 2 hpt, with its transcript abundance increasing to 14.6-fold and remaining at a high level. The transcript abundance of VpCDPK9 (Fig. 8-b) and VpCDPK19 (Additional file 6) increased, similar to VpCDPK6 (Fig. 8-b), although VpCDPK6 transcripts were almost undetectable at 48 hpt. In particular, VpCDPK4 (Fig. 8-b) responded to 42 °C treatment rapidly and intensely, reaching a peak of more than 8.3-fold and then returning to nearly normal transcript levels at 4 hpt. Taken together, our data showed that expression of VpCDPKs responded to low and high temperatures (4 °C and 42 °C) suggest that the VpCDPKs may play key roles in the response to temperature stress.
Plant hormones such as ABA, SA, MeJA, and ethylene have well-established roles in modulating plant signaling networks . In this study, hormone treatments resulted in a wide variety of changes in the transcript levels of VpCDPK genes (Fig. 9 and Additional files 7, 8, 9, 10).
An increasing body of evidence has shown that CDPKs regulate ABA-mediated signal transduction in plants [22, 23, 41]. Our experimental data suggest that 16 of the 19 VpCDPK genes respond to ABA treatment (Fig. 9 and Additional file 7). Among them, eight VpCDPK genes (VpCDPK1, 3, 4, 5, 10, 16, 17, 19) showed significant up-regulation in response to ABA (the peaks are over than 3.0- fold, p < 0.05). However, VpCDPK1 (Fig. 9-b) transcript levels rapidly increased up to 8.3-fold at 4 hpt and then decreased to lower than 0.3-fold at 8 hpt. Interestingly, the majority of the responsive CDPK genes showed a similar pattern, with their transcript abundance increasing rapidly to a peak at 4 hpt and remained at high levels until 12 hpt.
Similar to the ABA response, 13 of the 19 VpCDPK genes also showed positive regulation under SA treatment (Fig. 9 and Additional file 8). Six genes (VpCDPK3, 5, 14, 16, 17, 19) were significantly up-regulated (the peaks are over than 3.0-fold, p < 0.05), exhibiting a steady or gradual increase from 2 to 12 or 2 to 24 hpt. Interestingly, the expression of VpCDPK6, VpCDPK9, VpCDPK14, and VpCDPK16 was also induced after inoculation with powdery mildew pathogen (Fig. 7). For instance, VpCDPK9 showed positive regulation by inoculation of powdery mildew and peaked (~5.5-fold) at 96 hpi (Fig. 7-b), while under SA treatment, VpCDPK9 was also up-regulated and peaked (~2.8-fold) at 12 hpt (Additional file 8). The expression pattern after SA treatment and powdery mildew inoculation, suggests the vital roles of grape CDPK genes in immune signaling.
In contrast with ABA and SA, the VpCDPKs showed more limited responses to MeJA treatment. A total of ten genes (VpCDPK1, 2, 3, 4, 5, 6, 8, 9, 17, 19) were up-regulated; and among them, only three VpCDPK genes (VpCDPK5, 9, 17) showed distinct up-regulation (the peaks are over than 3.0-fold, p < 0.05) (Fig. 9 and Additional file 9). In particular, their transcript abundance peaked at 4 or 8 hpt, except for VpCDPK9 (Fig. 9-b), which strongly and rapidly responded to the treatment from 0.5 to 12 hpt with MeJA and peaked at 0.5 hpt. That might indicate its functional significance in the MeJA signaling pathway.
The expression changes caused by ethylene treatment (Fig. 9 and Additional file 10) were distinct from those caused by the other hormone treatments. For ethylene treatment, only a handful of VpCDPK genes (VpCDPK2, 9, 13, 16, 17, 19) were up-regulated (p < 0.05), while more genes showed down-regulation. For example, two up-regulated genes, VpCDPK2 (Additional file 10) and VpCDPK9 (Fig. 9-b), their transcript abundance increased rapidly at 0.5 hpt, but then increased only slightly or returned to normal transcript levels, respectively. Chinese wild grapevine Baihe-35-1 seems to be very sensitive to ethylene treatment and almost all the leaves of Baihe-35-1 plants fell at 48 hpt, so we had to use the fallen leaves for RNA extraction. VpCDPK6, VpCDPK7, VpCDPK8, VpCDPK11, and VpCDPK18 had decreased transcript abundance lower than 0.5- fold during the treatment time, suggesting their potential negative regulatory functions in plant responses to ethylene signaling.
Previously, Chen  investigated the CDPK gene family in grapevine and identified 17 members, which were predicted to have the typical CDPK gene structure with a Ser/Thr protein kinase domain and four EF-hands. Considering the complexity and variability in the calmodulin-like domain, we performed BLAST-P searches in NCBI using the putative grape CDPK genes predicted by Pkinase (PF00069) and EF-hand_7 (PF13499) HMM profiles, and identified the previously discovered 17 CDPKs and two more members (Fig. 1). Compared with Arabidopsis CDPKs  and rice CDPKs , we found that GSVIVT01025745001 (LOC100246588 in NCBI, designated here as VvCDPK18) possesses a similar domain organization to AtCPK25 and OsCPK30, which are predicted to have no EF-hand with default parameters in Pfam but are predicted to have EF-hands in PROSITE and InterPro. In addition, the phylogenetic tree showing that GSVIVT01025745001 (VvCDPK18) and OsCPK30 are homologous genes provides more evidence (Fig. 4). Therefore, we considered GSVIVT01025745001 (VvCDPK18) as members of the grape CDPK gene family and the same with GSVIVT01027353001 (LOC100250591 in NCBI, designated here as VvCDPK19). It is worth to mention that a homolog of VvCDPK19 in Vitis amurensis, named as VaCPK25, has been identified as a CDPK gene . Both of these two genes (VpCDPK19 and VaCPK25) are same in gene length and phylogenetic relationship. Consider that, VpCDPK19 can also be identified as a CDPK gene. These two new members of the grape CDPK gene family were designated VvCDPK18 and VvCDPK19 on the basis of their chromosomal locations.
The structural conservation and divergence of grape CDPK genes led to gene family expansion and functional conservation or differentiation. Structural characteristics such as acylation sites and intron-exon structures show the details of gene family expansion and divergence.
Most of CDPKs possess acylation sites, including N-myristoylation sites and S-palmitoylation sites, which are generally believed to function in membrane targeting . In Arabidopsis, 21 AtCPKs have both myristoylation and palmitoylation sites and all of them target exclusively to membranes, especially to the plasma membrane, although several have not been determined . Among our identified nineteen grape CDPK genes, six contain both myristoylation and palmitoylation sites, nine contain only palmitoylation sites, two contain only myristoylation sites, and two do not contain any acylation site (Table 1). We tested the subcellular localization of six VpCDPK proteins. VpCDPK2, VpCDPK5, VpCDPK10, and VpCDPK11 have palmitoylation sites while VpCDPK3 has a myristoylation site (Table 1) and they all localized on the plasma membrane (Fig. 5), consistent with results in Arabidopsis .
Three pivotal mechanisms contribute to gene family evolution and expansion: exon/intron gain or loss, exonization/ pseudo-exonization, and insertion /deletion . Previous work reported that grape CDPK genes have a single origin and can be dated back to green algae, before plants colonized the land . For the four groups in Fig. 2, Group IV was the earliest one that expanded from the evolutionary branch . As a result, it has the longest evolutionary history, leading to complex intron-exon organization. Both Group II and Group III originated from Group I in evolutionary history, and show similar structural divergence. All of the members of Group II contain one more exon and a phase-2 intron at the sixth position, indicating that there was an intron insertion in the last exon, which contributed to the generation of Group II and expansion of the VvCDPK family. Group III can be separated into two subgroups in evolutionary history, as reflected in their intron-exon structures. One subgroup was similar to Group I in intron-exon structures and the other one contains one more exon and a phase-0 intron at the first position, suggesting that there was an intron insertion in the first exon. Our result is consistent with the previous work that can be reflected in the origin and evolutionary history of VvCPKs . The intron-exon divergence was closely related to the evolutionary relationship of the grape CDPK family and might result in functional diversity.
To study the evolutionary relationships among different VvCDPK genes and the history of the VvCDPK gene family, as well as to further study their gene function, we investigated gene duplication events, syntenic regions, and phylogenetic relationships among the VvCDPK genes.
Segmental duplications and tandem duplications are the main mechanisms leading to gene family expansion . These processes may lead to functional redundancy, sub-functionalization and neo-functionalization. No tandem duplication involving VvCDPK genes was discovered in the grapevine genome, but two segmental duplications were found, VvCDPK5-VvCDPK11 and VvCDPK12-VvCDPK17 (Fig. 3, Additional file 1). Previously, Chen identified three segmental duplications . Except for VvCDPK5-VvCDPK11 and VvCDPK12-VvCDPK17, they also identified triplet-duplicated genes, VvCPK8-VvCPK9-VvCPK13. However, the method they used was not clearly presented, so we cannot follow their method. Anyway, both of Chen and us indeed identified the two duplicated gene pairs, VvCDPK5-VvCDPK11 and VvCDPK12-VvCDPK17. These two duplicated gene pairs are quite similar in gene length, acylation sites, and intron-exon organization (Table 1, Fig. 2). Furthermore, they possess the closest phylogenetic relationship among grape CDPK genes (Fig. 4). However, despite the fact VvCDPK12 and VvCDPK17 are phylogenetically closest among CDPKs from other species, these two genes have different syntenic genes in Arabidopsis (Fig. 3, Additional file 1), which means the duplication events happened before the evolutionary divergence of Arabidopsis and grapevine. Both VvCDPK5 and VvCDPK11 showed closer phylogenetic relationships with Arabidopsis CDPK genes than that with each other, indicating that this duplication event might also have happened before the divergence of Arabidopsis and grapevine. From what has been discussed above, VvCDPK5-VvCDPK11 and VvCDPK12-VvCDPK17 likely have different functions but still possess potential functional connections and similarities.
Comparative genomics approaches structure genomes into syntenic blocks that exhibit conserved features across the genomes . The synteny analysis provides evolutionary and functional connections between grape and Arabidopsis syntenic genes. Furthermore, a large number of syntenic relationships suggest that some of the grape CDPK genes arose before the divergence of the Arabidopsis and grapevine lineages. Ten grape CDPK genes were found to have syntenic relationships with Arabidopsis genes (Fig. 3) All of these VvCDPK genes show close phylogenetic relationships with the corresponding AtCDPK genes (Fig. 4), suggesting their potential functional similarities. Interestingly, all of the grape CDPK genes in Group III (Fig. 4) have syntenic genes in Arabidopsis, suggesting that Group III CDPK genes are relatively conserved over evolutionary history.
Analysis of the phylogenetic tree revealed that CDPK homologs among several monocots and eudicots clustered into four distinct groups, which correspond to the clades formerly identified in green plants . Previously, Hamel  examined the CDPK families from green algae to land plants to demonstrate that CDPK families are conserved among land plants, whereas CDPKs from green algae have continued to evolve independently. Supporting this, our data suggest that CDPKs from monocots and eudicots cluster separately (Fig. 4) and evolutionary relationships of the four clades are relatively conserved. The previous work  also constructed a phylogenetic tree that gives the consistent results and conclusions.
Comparative genomics provides an effective way to understand the structure and function of genomes by translating knowledge gained from model species to the species of interest. Combining synteny analysis with phylogenetic analysis provides new insights for further investigating the functions of the grape CDPK genes by comparing orthologous genes between two species, in this case, between grapevine and Arabidopsis. Our study provided substantial evidence to help predict the functional conservation or divergence of CDPK genes between grapevine and Arabidopsis.
Chinese wild grape (Vitis pseudoreticulata) accession Baihe-35-1, compared with the sequenced Vitis vinifera, reported to have remarkable resistance to both biotic and abiotic stress [29, 30]. In addition, Vitis amurensis is also a wild grapevine species with a high level of resistance to multiple stresses . Our expression profile of VpCDPK genes showed consistency with that reported in Vitis vinifera  and Vitis amurensis . For example, the homologous CDPK genes, VpCDPK3/VvCPK3/VaCPK16 and VpCDPK17/VvCDPK17/VaCPK17 showed distinct up-regulation under salt stress treatment [31, 32] (Fig. 8-b). However, there are also some differences on genes expression, e.g., VaCPKs respond to cold stress much significantly than VpCDPKs, probably because of Vitis amurensis shows strong capacity of cold-resistance.
Two duplicated gene pairs, VpCDPK5/VpCDPK11 and VpCDPK12/VpCDPK17, as discussed above, provide experimental evidence to support our predictions and views. VpCDPK5 and VpCDPK11 each have particular syntenic genes in Arabidopsis showing a complicated evolutionary history. In addition, the experimental data showed that these two genes have distinct expression patterns under a range of treatments. For example, VpCDPK5 was obviously up-regulated under hormone treatments (except ethylene), while VpCDPK11 was only slightly up-regulated under ABA treatment (Fig. 9 and Additional files 7, 8, 9, 10). Also, for abiotic stress treatments, VpCDPK5 was down-regulated under NaCl and 42 °C treatments, but VpCDPK11 transcripts remained at constant levels (Fig. 8 and Additional files 4, 6). Furthermore, subcellular localization analysis demonstrated that VpCDPK5-GFP localized on plasma membrane and in the nucleus, but VpCDPK11-GFP only localized on plasma membrane (Fig. 5). The aforementioned information indicated that VpCDPK5 and VpCDPK11 might well have undergone neo-functionalization. Moreover, VvCDPK11 in V. vinifera cv. Corvina expressed in almost all of the organs, whereas VvCDPK5 only expressed in pollen . However, we tested VpCDPK5 in the Chinese wild grape V. pseudoreticulata responded to hormone treatments (Fig. 9) in leaves, suggesting that VpCDPK5 was not only transcribed in pollen. For VpCDPK11, its low expression under multiple treatments (Figs. 7, 8, 9) and high expression in almost all tissues  indicates that its function most likely relates to housekeeping genes. The other duplicated gene pair, VpCDPK12 and VpCDPK17 were quite different. The transcript levels of these two genes showed similar tendencies under abiotic stress, ABA and SA treatments (Figs. 8, 9 and Additional files 4, 5, 6, 7, 8), however, VpCDPK17 seemed to respond much strongly than VpCDPK12. Based on Chen , VvCDPK17 had high transcript levels in almost all tissues while VvCDPK12 had low transcript levels. These data suggest that the duplicated gene pair VpCDPK12/VpCDPK17 probably have undergone sub-functionalization and VpCDPK12 tends to be non-functional.
It is worth noting that VpCDPK9 is unique in the grape CDPK family. For one thing, it showed an unusual expression pattern under various treatments, being strongly up-regulated, except for 4 °C treatment (Fig. 7, 8, 9). Its transcript abundance increased to a high level during most or all of the treatment (Figs. 7, 8, 9). Subcellular localization also demonstrated its considerable differences with the other grape CDPKs we examined. The other VpCDPKs have only one pattern of localization (Fig. 5), while VpCDPK9 shows four patterns, including in some kind of plastids, in biomembranes, in the cytosol, or in the nucleus (Fig. 6). Testing why VpCDPK9 has four patterns of localization, and the relationship between its localization and function, will require more experimental data. The genes that are phylogenetically close to VpCDPK9 in Arabidopsis are AtCPK1 and AtCPK2 (Fig. 4). VpCDPK9 has structural and functional commonalities with AtCPK1. For example, AtCPK1 expression is rapidly induced by fungal elicitors and loss-of-function mutants of AtCPK1 exhibit higher susceptibility to pathogen infection . Ectopic expression of AtCPK1 enhanced NADPH oxidase activity and the oxidative burst in tomato protoplasts . Cold-stress can also induce AtCPK1 transcripts via phosphoprotein signals . Our data also showed that VpCDPK9 not only responded to powdery mildew infection and abiotic stress, but also responded to hormone treatments (Figs. 7, 8, 9). These results are consistent with the expression profile of AtCPK1. Future work will be directed toward identification of potential defense components that may be directly or indirectly regulated by VpCDPK9 in grapevine Baihe-35-1.
VpCDPK2, whose Arabidopsis homolog is AtCPK5, is found in the chromosomal region syntenic with AtCPK5, and plays positive regulatory roles in various treatments (Figs. 7, 8, 9). Results on AtCPK5 show high consistency with our results on VpCDPK2. (i) AtCPK5 was reported to localize to the plasma membrane and the nucleus [47, 48], consistent with our data showing that VpCDPK2 also localized to the plasma membrane and nucleus (Fig. 5). (ii) AtCPK5 can localize to the nucleus where it interacts with and phosphorylates WRKY8, 28, and 48 to activate defense genes , while VpCDPK2 also localized in the nucleus (Fig. 5), suggesting it has similar functions with AtCPK5. (iii) AtCPK5 also phosphorylates RBOHD in vivo, resulting in H2O2 production and leading to cell death [21, 49], involving in immune signaling. VpCDPK2 responded to powdery mildew inoculation with up-regulated transcript levels (Fig. 7), indicating its potential function in immune signaling and that VpCDPK2 most likely participates in the analogous pathways to those mentioned above for Arabidopsis. These structural and locational similarities provide indications of functional consistency.
VpCDPK10 has a syntenic gene in Arabidopsis, AtCPK13. Further analysis provides valuable comparisons of these two genes and helps predict the function of VpCDPK10. In our study, VpCDPK10 localized on the plasma membrane (Fig. 5), and AtCPK13 was also reported to localize on the plasma membrane . VpCDPK10 expression did not respond to stress treatments but was distinctly up-regulated in response to ABA treatment (Fig. 8). AtCPK13 specifically inhibits KAT1 and KAT2 shaker channels , which participate in ABA-induced stomatal movements by affecting CDPK phosphorylation . These insights indicate that VpCDPK10 plays important roles in ABA- and Ca2+-mediated stomatal regulation.
Another intriguing gene, VpCDPK16, is phylogenetically close to AtCPK4 and AtCPK11 (Fig. 4), which are activated by ABA and phosphorylate the C-terminus of ACS6 in ethylene biosynthesis . Meanwhile, AtCPK4 and AtCPK11 can phosphorylate the ABA-responsive transcription factors ABF1 and ABF4 and lead to salt insensitivity in seed germination and decrease tolerance of seedlings to salt stress . Sustained AtCPK4 and AtCPK11 activation directly phosphorylated WRKY transcription factors involved in immune signaling . Expression patterns suggest that VpCDPK16 responded to powdery mildew inoculation as well as NaCl, ABA, and SA treatments (Figs. 7, 8, 9), showing high functional consistency with AtCPK4 and AtCPK11. This evidence indicates that VpCDPK16 might participate in similar pathways as AtCPK4 and AtCPK11 in Arabidopsis.
In addition to stress, CDPK genes also play important roles in growth and development process. On this regard, plant hormones are mainly responsible. For instance, several plant hormones may play central roles in the control of ripening in the grape berry . As a support, Kühn et al.  investigated the aminocyclopropane-1-carboxylate (ACC) synthases genes in first developed grape berries, which pathway is related to ethylene signaling. For CDPK genes, several genes including AtCPK16 , NtCDPK2  and LeCDPK2  were reported to phosphorylate ACC synthases to trigger ethylene biosynthesis and accumulation. Coincidence is that VpCDPK9 and VpCDPK13, as homologs of NtCDPK2 and LeCDPK2, are positively regulated under ethylene treatment, indicating their possible functions involved in ethylene signaling. However, CDPKs functions in plants growth and development process still need more experimental evidence to deeply understand their biological roles and the pathways they involved in.
So far, little systematic analysis of the CDPK family has been reported in grapevine, and the functions of most CDPK genes remain unclear. However, accumulating evidence indicates that CDPKs play important roles in response to a broad variety of abiotic and biotic stresses and biological processes. In this paper, genome-wide identification, evolutionary, and expression analyses were carried out to provide a framework for further analysis of grape CDPK genes in defining their biological functions and pathways during stress responses as well as growth and development. Expression profiles showed grape CDPK genes respond to various stresses and hormone treatments; moreover, analysis of CDPK subcellular localization gave evidence as to their functions. Comparisons of the grape and Arabidopsis genomes and expression profiles provide novel insights into the functions of less well-studied genes according to their better-understood homologs. By prediction and experimental data, we speculated that CDPK gene family might participate in responses to pathogen, cold, heat and salt stress, and the related biological processes might covering the regulation of gene expression, control of the ion channel, regulation of the enzyme activity, mediation of the cross-talk between signaling pathways and so on. These observations may lay the foundation for future functional analysis of grape CDPK genes to unravel their biological roles.
To identify the CDPK genes in grapevine, we downloaded the Hidden Markov Model (HMM) profiles of the core protein kinase domain (PF00069) and EF-hand domain (EF-hand_7, PF13499) from Pfam database (http://pfam.xfam.org/). Then we performed a BLAST-P search in the Grape Genome Database (12X) (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/) using the HMM profiles as queries with e-value of 0.01. We also performed BLAST-P searches at NCBI using full-length amino acid sequences of the primarily identified grape CDPK genes and chose the candidates of e-value lower than 1e−60. All putative CDPK genes were manually verified with the InterProScan program (http://www.ebi.ac.uk/Tools/pfa/iprscan/) to confirm their completeness and existence of the core domains. Among those with alternative splice variants, we selected the longest variant for further analysis. Sequences of Arabidopsis, rice, maize, and poplar CDPK genes were obtained from the Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/), rice genome database in TIGR (http://rice.tigr.org), maize genome database (http://www.maizesequence.org/index.html), and Phytozome (http://www.phytozome.net/), respectively.
Grape CDPK genes were mapped to chromosomes by identifying their chromosomal locations, as obtained from the Grape Genome Database (12 X) (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/) and NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). The segmental and tandem duplication regions, as well as chromosomal location, were established using PLAZA v3.0 Dicots (http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v3_dicots/). For synteny analysis, synteny blocks within the grape genome and between grape and Arabidopsis genomes were downloaded from the Plant Genome Duplication Database and visualized using Circos (http://circos.ca/).
Myristoylation and palmitoylation sites were predicted by Myristoylator (http://web.expasy.org/myristoylator/) and CSS-Palm 3.0 (http://csspalm.biocuckoo.org/), respectively. The intron-exon organization analysis was carried out using GSDS 2.0 (http://gsds.cbi.pku.edu.cn/) by alignment of the cDNA sequences with their corresponding genomic DNA sequences, and the results were consistent with the phylogenetic analysis. Multiple alignments of the identified grape CDPK amino acid sequences were performed using ClustalX. The phylogenetic tree was constructed with MEGA5.0 using the Neighbor-Joining method and the bootstrap test carried out with 1,000 replicates .
The Chinese wild grapevine V. pseudoreticulata accession Baihe-35-1 was grown in the grapevine germplasm resources greenhouse of Northwest A&F University in China, at temperatures of 22 to 27 °C, relative humidity of 60 to 80 %, and without supplemental lighting. When shoots of vines were 40–50 cm in length, the third and fourth fully expanded young grapevine leaves beneath the apex were selected for treatments. Plants of Arabidopsis thaliana ecotype Col-0 were grown at 22 °C, 75 % relative humidity, and under short-day (8 h light at 125 μmol · m−2 · s−1, 16 h dark) conditions for 4 to 5 weeks before transformation.
A grapevine powdery mildew (Erysiphe necator) isolate NAFU1 (GenBank accession no. KJ539202) was collected from a vineyard in Northwest China, and maintained on the leaves of V. vinifera cv. Thompson seedless, which was soil-grown in pots. The pathogen was sub-cultured onto fresh leaves every twenty days. The leaves of Baihe-35-1 were inoculated by touching the adaxial epidermis with sporulating colonies on the surface of the ‘Thompson Seedless’ infected leaves. Plants were then incubated in the greenhouse. Inoculated leaves were collected at 0, 6, 24, 48, 72, 96, 120, and 144 h post-inoculation (hpi). Inoculations were repeated three times.
For salt stress treatments, four-month-old soil-grown plants were irrigated with 300 mM NaCl. Treated leaves were collected at 0, 0.5, 2, 4, 8, 12, 24, and 48 h post-treatment (hpt). For cold treatment, plants were first grown at 22 °C, and then transferred to 4 °C, and treated leaves were collected at 0, 0.5, 2, 4, 8, 12, 24, and 48 h post-treatment (hpt). For high temperature treatment, plants were first grown at 22 °C, and then transferred to 42 °C, and treated leaves were collected at 0, 0.5, 2, 4, 8, 12, 24, and 48 h post-treatment (hpt). The leaves of Baihe-35-1 were sprayed with solution of 0.1 mM abscisic acid (ABA), 1 mM salicylic acid (SA), 0.1 mM methyl jasmonate (MeJA), or 0.5 g/L ethephon (Eth), and then collected for RNA isolation. The treated leaves were collected at 0, 2, 4, 8, 12, 24, and 48 h post-treatment (hpt). Another set of control plants were similarly treated with distilled water. All plants were treated in the light and three independent experiments were performed.
Total RNA of grape leaves was extracted using the E.Z.N.A. Plant RNA Kit (Omega, Guangzhou, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 2 μg total RNA using PrimeScript RTase (Takara, Dalian, China). Quantitative PCR (qPCR) was carried out using SYBR green (Takara, Dalian, China) on an IQ5 real time PCR machine (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Thermal cycling consisted of a hold at 95 °C for 30 s, followed by 40 cycles of 95 °C for 30 s and 58 °C for 30 s. After amplification, samples were kept at 50 °C for 1 min and the temperature was raised gradually by 0.5 °C every 10 s to perform the melt-curve analysis. Grape VpActin (accession no. AY680701) was amplified as an internal control. All reactions were performed in triplicate in each experiment and three biological repeats were conducted. Primers used for RT-qPCR are listed in Additional file 11. Each relative expression level was analyzed with IQ5 software using the Normalized Expression method (2-△△CT method). Expressional data consist of three replicated treatments and controls, which were calculated as 2-log-based values and were divided by the control.
The statistical analysis was performed with the SPSS 19.0 software. The data were showed as mean value ± SD. We examined the homoscedasticity of our data by F-test. The significance of the differential expression between treatments and controls (0 hpt) was verified by performing Student’s t-test. p < 0.05 and p < 0.01 were taken as statistically significant or highly significant, respectively. The biological significance of RPKM was set as a fold change greater than 2-fold or less than 0.5-fold.
The predicted full-length coding sequences of grape VpCDPK genes, including VpCDPK2, VpCDPK3, VpCDPK5, VpCDPK9, VpCDPK10, and VpCDPK11, were amplified by high-fidelity Taq HS-mediated PCR from cDNA of the Chinese wild grapevine V. pseudoreticulata accession Baihe-35-1 leaves. The amplified PCR products were digested with SalI and XhoI and fused in-frame with GFP in the SalI and XhoI site of the pBI221 vector containing the CaMV 35S promoter (Clontech, Beijing, China) resulting in plasmids pVpCDPKs-GFP. Primers used for cloning genes and for constructing vectors are shown in Additional file 11.
For transient expression of VpCDPKs-GFP in Arabidopsis mesophyll protoplasts, DNA of the corresponding pVpCDPK-GFP plasmids was transformed into Col-0 leaves using a previously described method . After transformation, Col-0 leaves were kept in darkness at room temperature for 16–18 h before examination by fluorescence microscopy. Images were acquired using an Olympus BX-51 inverted fluorescence microscope (Olympus, Japan). The image data were processed using Adobe Photoshop (Mountain View, CA, USA). All transient expression assays were repeated at least three times.
Phylogenetic data (alignments and phylogenetic trees) supporting the results of this article have been deposited in TreeBASE respository and is available under the URL http://purl.org/phylo/treebase/phylows/study/TB2:S17752.
Sequence data of the isolated VpCDPK genes in this article can be found in GenBank (http://www.ncbi.nlm.nih.gov) under the accessions of KR153945- KR153946 and KR153948- KR153951.
Kai Zhang and Yong-Tao Han contributed equally to this work.
The authors are grateful to Dr. Wen-ming Wang (Sichuan Agricultural University) for critical review of this manuscript and to Dr. Guang-lin Li (Shaanxi Normal University) for preparing the phylogenetic data profiles deposited to TreeBASE. We also thank Dr. Jennifer Mach for her useful comments and language editing. The authors would also like to thank the anonymous reviewers for comments on the manuscript. This work was supported by the National Natural Science Foundation of China (Grant No. 31372022, 31071772), and the program for Innovative Research Team of Grape Germplasm Resources and Breeding (2013KCT-25).
Additional file 1: The syntenic relationships among grape and Arabidopsis CDPK genes.
Additional file 2: The CDPKs amino acid sequences used to construct the phylogenetic tree.
Additional file 3: Detailed expression profiling of the remaining 15 VpCDPK genes during powdery mildew infection. Detailed expression levels were measured by RT-qPCR. Grape Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 4: Detailed expression profiling of the remaining 15 VpCDPK genes under NaCl treatment. Detailed expression levels were measured by RT-qPCR. Grape Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 5: Detailed expression profiling of the remaining 15 VpCDPK genes under 4°C treatment. Detailed expression levels were measured by RT-qPCR. Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 6: Detailed expression profiling of the remaining 15 VpCDPK genes under 42°C treatment. Detailed expression levels were measured by RT-qPCR. Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 7: Detailed expression profiling of the rest remaining VpCDPK genes under ABA treatment. Detailed expression levels were measured by RT-qPCR. Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 8: Detailed expression profiling of the remaining 15 VpCDPK genes under SA treatment. Detailed expression levels were measured by RT-qPCR. Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 10: Detailed expression profiling of the remaining 15 VpCDPK genes under ethylene treatment. Detailed expression levels were measured by RT-qPCR. Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. The data were showed as mean value ± SD. * and ** represent statistically significant (p<0.05) or highly significant (p<0.01), respectively.
Additional file 11: Primers used in this study.
YQW, KZ and YTH conceived and designed the research. KZ and YZ carried out bioinformatics analyses. YTH performed all treatments and RT-qPCR tests, with assistance of YRG and YFM. FLZ, YH and KZ carried out subcellular localization experiments. KZ and YTH analysed and interpreted the data, and performed statistical analysis. KZ draft the manuscript and YQW revised it. YJW contributed with consultation. All authors read and approved the final manuscript.

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