Identification of genes down-regulated in WIPK/SIPK-suppressed plants by microarray analysis
To identify genes whose expression is regulated by WIPK and SIPK, transcripts that were down-regulated in wounded leaves of WIPK/SIPK-suppressed plants were searched for using a microarray. In tobacco, the levels of ethylene emission and JA peak 3–6 h and 6–12 h after wounding, respectively [14, 15]. Therefore, total RNA was extracted from leaves at 9 h after wounding and subjected to microarray analysis. Of 43,759 oligo nucleotides probes set on the chip, 59 probes targeting 46 genes showed more than a 5-fold decrease in WIPK/SIPK-suppressed plants compared with control plants (Additional file 2: Table S1). BLASTX searches of the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were performed to predict putative functions of the target genes, and they were categorized into 14 classes according to a modified form of the classification described previously [16] (Fig. 1, Additional file 3: Table S2). Approximately half of the target genes were those involved in secondary metabolism. The second and third largest categories were “unknown” and “signal transduction”, respectively. The five genes included in the “signal transduction” category were WIPK, SIPK and Ntf4, a close homolog of SIPK whose expression is suppressed in WIPK/SIPK-suppressed plants [12]. The remaining categories contained a few genes, and their predicted functions varied, indicating that WIPK and SIPK mainly regulate the expression of the genes involved in secondary metabolism.
Among the 20 genes categorized into secondary metabolism, 15 were predicted to be involved in phytoalexin synthesis (Fig. 1, Additional file 3: Table S2). Capsidiol is a major phytoalexin in tobacco and it is produced by the actions of EAS and EAH from FPP, an intermediate in the biosynthesis of many metabolites such as sterols, sesquiterpenes, triterpenes, and ubiquinones, as well as substrates for the farnesylation of proteins (reviewed in [4]) (Additional file 1: Figure S1). Many genes encoding EAS, EAH, and their homologs were included in the list (Additional file 3: Table S2). To check the reproducibility of the microarray analysis, the transcript levels of EAS and EAH over a time course after wounding were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Expression of EAS and EAH was strongly induced by wounding, with a peak around 9–12 h after wounding, and their transcript levels were decreased in WIPK/SIPK-suppressed plants (Fig. 2a). In contrast, the transcript levels of squalene synthase (SQS), another enzyme utilizing FPP as a substrate, were not significantly affected by the silencing of WIPK and SIPK, although it was also moderately induced by wounding.
WIPK and SIPK regulate wound-induced expression of nearly all genes involved in capsidiol synthesis
EAS and EAH were shown to be induced by wounding and regulated by WIPK and SIPK; therefore, we investigated whether other genes involved in capsidiol synthesis are regulated by WIPK and SIPK and whether they are induced by wounding. IPP, a precursor of FPP, is produced through the mevalonate pathway by the actions of six enzymes, and IPP is converted to FPP by IPP isomerase (IDI) and FPP synthase (FPS) (Additional file 1: Figure S1). Transcript analysis of 11 genes encoding any one of the enzymes revealed that all the genes except for FPS2 are clearly induced by wounding (Fig. 2b). In WIPK/SIPK-suppressed plants, transcript levels of all the genes except for HMGR1 and FPS2 were significantly decreased at least at one time-point in the experiments. Notably, no genes showed WIPK/SIPK dependency at 3 h after wounding, although approximately half of the genes were already induced by wounding at this time. Additionally, in case the enzymes are encoded by two paralogous genes (AACT, HMGR, and FPS), only one of two genes showed clear WIPK/SIPK-dependency. Similar results were obtained with another line of WIPK/SIPK-suppressed plants, ruling out the possibility that this effect was caused by the introduction of the transformation vector (Additional file 4: Figure S2). These results indicated that induction by wounding of capsidiol synthesis genes is mediated by both WIPK/SIPK-dependent and -independent manners, and suggested that WIPK and SIPK regulate the expression of the specific members of gene families at relatively late time points.
IPP and dimethylallyl diphosphate, direct precursors of FPP, are produced not only in the mevalonate pathway but also in the so-called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway present in plastids (reviewed in [4]) (Additional file 1: Figure S1). Although it has been essentially considered that the two pathways function independently, some reports have indicated that interconnections exist between the pathways [17, 18]. Therefore, we investigated the transcript levels of eight genes encoding any one of seven enzymes constituting the MEP pathway (Additional file 5: Figure S3). IDI1 was considered to be involved in the conversion between IPP and dimethylallyl diphosphate produced by the MEP pathway, because it encodes a protein with a putative plastid transit peptide (AB049815). Therefore, the transcript levels of IDI1 were also investigated. In contrast to the genes of the mevalonate pathway, all genes showed no or a very weak response to wounding, and none of the genes except for IDI1 showed WIPK/SIPK dependency.
Both WIPK and SIPK are required for maximal induction of capsidiol synthesis genes
To investigate which of WIPK or SIPK is required for wound-induced expression of capsidiol synthesis genes, their transcript levels in WIPK- or SIPK-suppressed plants were quantified (Fig. 3). Although the transcript levels of the genes were generally decreased more by the silencing of SIPK than that of WIPK, single silencing of either WIPK or SIPK reduced the transcript levels of the most genes. These results suggested that WIPK and SIPK regulate the expression of capsidiol synthesis genes cooperatively, not redundantly.
Promoter analysis of EAS4
EAS is a committing enzyme for capsidiol production (Additional file 1: Figure S1). EAS4, a member of EAS gene family, is strongly induced by various forms of stress, and the responses of its promoter to pathogen-derived elicitor have been studied [19]. Therefore, EAS4 was chosen as a representative of capsidiol synthesis genes, and its promoter was analyzed to clarify how capsidiol synthesis genes are induced by wounding, and how WIPK and SIPK regulate them. Primers were designed based on database information, and an approximately 1.1-kbp EAS4 promoter region designated as 1126p was cloned (Fig. 4a). 1126p contains many sequence elements similar to the stress responsive cis-elements, but elements that mediate activation of the EAS4 promoter by elicitors have not been identified. The only functional element identified in the EAS4 promoter is a TAC-box. It was thought to function as a silencer or repressor, because the introduction of a mutation into the TAC-box increased the activity of the EAS4 promoter [20].
For the analysis of EAS4 promoter activity, we used an Agrobacterium-mediated transient expression in N. benthamiana leaves [21]. Agrobacterium cells carrying the EAS4 promoter fused to β-glucuronidase (GUS) as a reporter (EAS4p-GUS) were mixed with those carrying luciferase (LUC) driven by a Cauliflower mosaic virus 35S promoter (35Sp-LUC) as an internal control of Agrobacterium infection, and then infiltrated into the leaves. Transcript levels of GUS, LUC, and Nbactin2 were quantified by RT-qPCR, and the level of GUS transcripts was doubly normalized to those of Nbactin2 and LUC. We first confirmed that 1126p is activated by wounding. As shown in Fig. 4b, the transcript level of GUS driven by 1126p was increased by wounding about 200-fold, reflecting about 170-fold induction by wounding of the EAS transcript in tobacco (Fig. 2a). In contrast, the transcript levels of GUS driven by the 35S promoter were not increased by wounding. Next, successive 5′-deletions of the EAS4 promoter designated as 567p (− 567), 160p (− 160), 63p (− 63), and 33p (− 33), were fused to GUS to identify the regions regulating wound responsiveness of the promoter. Deletion to − 160 greatly decreased the activity of the promoter, but it was still activated by wounding more than 20-fold (Fig. 4b). Further deletion to − 63 minimized wound-induced activation of the promoter, suggesting that a region from − 160 to − 64 is important for activation by wounding of the EAS4 promoter. The promoter fragments 63p and 33p still increased transcript levels of GUS slightly in response to wounding. However, it was considered to be an experimental artifact, because a 5′-untranslated region (UTR) of EAS4 and 35S minimal promoter also showed results similar to 63p and 33p (Fig. 4d, Additional file 6: Figure S4).
To further delineate the region responsible for wound-induced activation, two deletion constructs of the 160p, 115p (− 115) and 160pΔ, were created. An internal deletion construct 160pΔ lacks a region from − 115 to − 64. As shown in Fig. 4c, both constructs were hardly activated by wounding, suggesting that both regions from − 160 to − 116 and from − 115 to − 64 are required for wound-induced activation of 160p. The importance of regions from − 160 to − 116 and from − 115 to − 64, but not a region from − 63 to − 34, was further confirmed using a gain-of-function analysis. As shown in Fig. 4d, four tandem repeats of the regions from − 160 to − 116 and from − 115 to − 64, but not the region from − 63 to − 34, conferred strong wound-responsive activity on a 35S minimal promoter.
Mutational analysis of the promoter of EAS4
To determine the regulatory elements in the region from − 160 to − 64, 10-bp substitutions were introduced into 160p (m1-m10, Fig. 5a). Substitution in any of the M2, M4, M5, M7, and M8 regions significantly decreased GUS transcript levels induced by wounding (Fig. 5b). In contrast, substitution in M1, M9, or M10 elevated GUS transcript levels induced by wounding. Without wounding, none of the substitutions affected GUS transcript levels. These results suggested that multiple wound-responsive cis-elements are present in a region from − 150 to − 81 of the EAS4 promoter.
The EAS4 promoter is activated by MEK2DD, an activator of WIPK and SIPK
Loss-of-function and gain-of-function analyses identified regions of the EAS4 promoter that are required and sufficient for activation by wounding (Figs. 4 and 5), but it was unclear if the activation is mediated by WIPK and SIPK or not. To induce activation of WIPK and SIPK specifically, we used MEK2DD, a constitutively active form of MEK2. MEK2 is an upstream MAPK kinase of WIPK and SIPK, and it directly phosphorylates and activates them [10]. As expected, the expression of MEK2DD activated the EAS4 promoter, although activation by MEK2DD was weaker than that by wounding (Fig. 6a). These results supported that the EAS4 promoter is activated by both WIPK/SIPK-dependent and -independent mechanisms.
The EAS4 promoter contains two W-box-like sequences in a region from − 410 to − 310 (Fig. 4a). The W-box is a sequence recognized by WRKY transcription factors, and recent reports have indicated that WIPK and SIPK, and their orthologs in other plant species phosphorylate WRKY transcription factors and enhance their functions [11, 22, 23]. These lines of evidence prompted us to investigate the roles of W-box-like sequences in MEK2DD-induced activation of the EAS4 promoter. Quantification of GUS transcript levels driven by a series of 5′-deletions of the EAS4 promoter showed that the W-box-like sequences are dispensable for activation by MEK2DD of the EAS4 promoter, and suggested that 160p is the shortest fragment required for activation by MEK2DD (Fig. 6a). However, activation of 160p by MEK2DD was too weak to be concluded; therefore, gain-of-function analysis was performed. As shown in Fig. 6b, tandem repeats of the regions from − 160 to − 116 and from − 115 to − 64, but not the region from − 63 to − 34, conferred MEK2DD-responsive activity on a 35S minimal promoter. Moreover, tandem repeats of a region from − 410 to − 311, which contains two W-box-like sequences, were activated by MEK2DD. These results suggested that multiple regions of the EAS4 promoter are involved in its activation by WIPK and SIPK.
Physiological roles of wound-induced expression of capsidiol synthesis genes
It has been shown that most capsidiol synthesis genes are transcriptionally induced by wounding in WIPK/SIPK-dependent and -independent mechanisms, and multiple regions of the EAS4 promoter are involved in its activation by wounding (Figs. 2, 4, and 6). These results indicated the importance of induction by wounding of capsidiol synthesis genes. However, as far as we know, no report has shown that accumulation of capsidiol is induced by wounding (similar to the majority of phytoalexins). We measured capsidiol levels in wounded tobacco leaves, but the levels were under the detection limit. Similarly, it has been reported that the accumulation of EAS protein is induced by a pathogen-derived elicitor, but scarcely by wounding in tobacco leaves [19]. We also confirmed that accumulation of EAS protein is induced by INF1, a protein elicitor secreted by Phytophthora infestans [24], but not by wounding (Fig. 7a).
Because wounding disrupts physical barriers in the leaf surfaces and causes a risk of pathogen invasion at the wound sites, it is reasonable to activate the biosynthesis of capsidiol at the wound sites during wound healing. However, it costs energy to produce capsidiol, and phytoalexins including capsidiol are toxic not only to pathogens but also to the plant themselves [25, 26]. Therefore, in case pathogens do not enter the plant during wound healing, the production of capsidiol results in loss of energy and unnecessary damage to plant tissues. These lines of evidence suggest that induction by wounding of transcript levels, not protein levels, of EAS is a preventive response against possible invasion by pathogens at wound sites. If pathogens enter the wound, plants can synthesize EAS protein quickly, which leads to a rapid production of capsidiol, whereas if pathogens are not present, plants can minimize energy loss and avoid damage to the cells by capsidiol. To test this hypothesis, we investigated whether pre-wounding increases the levels of EAS protein and capsidiol induced by INF1.
In preliminary experiments, we found that it is technically difficult to infiltrate INF1 solution into wounded sites of leaf discs. Therefore, two different methods were tested to wound the leaves. In the first method, small holes were made in the leaves by pricking with a 10-μl tip (hole method). In the other method, the leaves were crushed by holding with forceps strongly (crush method). Both methods clearly induced the expression of EAS (Additional file 7: Figure S5), and INF1 solution infiltrated relatively easily into the tissue damaged by the crush method, but not by the hole method. Therefore, leaves were wounded by the crush method, and INF1 was infiltrated into the damaged area at 9 h after wounding, at which time the accumulation of EAS transcript peaks (Figs. 2a, 3). As shown in Fig. 7b, the levels of EAS protein induced by INF1 were, as expected, increased by pre-wounding. Similarly, INF1-induced capsidiol production was enhanced by pre-wounding (Fig. 7c). At 6 h and 7.5 h after INF1 treatment, the levels of capsidiol were approximately doubled by pre-wounding. The effect of pre-wounding became less clear at 9 h after wounding, probably due to transcriptional activation by INF1 of EAS and other capsidiol synthesis genes.