| Maize(Zea mays L.) is a very important cultivated crop. The yield and quality for maize is affected by the attack of pathogens and herbivores, which threatens the food security. The phytoalexins synthesized as a kind of specific small molecule secondary metabolite when plants get infected by pathogens. The first kind of phytoalexins found in maize is kauralexins, with effective roles in suppressing the growth of pathogens and the antifeedant activity for European corn borer larva. Kuaralexin is synthesized through a sequential catalytic reaction from GGPP to ent-isokaurene by copalyl diphosphate synthase (CPS) and kaurene synthase-like(KSL) to form the kaurene skeleton, and then further modified by P450. The CPS involved in kauralexins biosynthesis was identified to be An2, while the KSL is still unknown. Moreover, biosynthesis of the plant hormone GA also needs the participation of kaurene synthase(KS). The maize dwarf-5(d5) mutant, which shows a drawf phenotype, was found to lose the function to produce ent-kaurene which acts as a precursor in GA biosynthesis in maize. But the relavant mutant genes in d5 is still uncharacterized. Up to now, no KS was identified in maize, thus blocks the study and application of kauralexins. Meanwhile, the accumulation of kauralexins is highly associated to the expression level of its biosynthesis genes. So the analysis for the effective cis-elements in An2 promoter is necessary, which lays a foundation for the transcriptional regulation study of An2.In this study, we firstly searched the potential KS(L)s genes from the maize whole genome, and the precdicted the fuctions for candidate genes by phylogenetic analysis Among these genes, we focused on ZmTPS1, ZmKSL3 and ZmKSL5 for their closer relationship to other cereal KSs. By the recombinant biochemical analysis in Escherichia Coli and transient expression in Nicotiana benthamiana, the biochemical functions of the three synthase were clarified. The subcellular localizations of them were also observed to further verified the relevant fuctions. After that, we detected the expression of the three genes in the d5 mutant by RT-PCR, in this way we identified the ent-kaurene synthase involved in GAs biosynthesis. In addition, expression patterns of these three genes in different treatment were analyzed by qRT-PCR and compared with that of An2, which has a known role in kauralexins biosynthesis. Finally, the activity region of An2 promoter was characterized by particle bombardment in maize callus. The main conculsions are as follows:1. Base on the gene annotation, we found six potential ent-kaurene synthase from maize whole genome, namely ZmKSL1 to ZmKSL5 and ZmTPS1 respectively. All these KS(L)s and other cereal KS(L)s as well as AtKS and CmKS were used to produce a phylogenetic tree from their animo-acid sequence. Phylogentic analysis reveals that ZmKSL3 has a closer relationship with KSs from other plant in GAs biosynthesis, while the ZmKSL5 and ZmTPS1 are close to TaKSL5-1/2, which also do not contain the y domain. Furthermore, we found that the positions of these three genes on chromosome 2 form a tandem array, suggesting that the tandem duplication occurred during evolution.2. Each of the three genes was co-expressed with An2 in E.coli, the GC-MS results reveals that all of them have the ability for using ent-CPP to form ent-kaurene. In the same way, the identification of sesqueiterpene activety of the three genes was also measured. No relevant product(s) were detected for ZmKSL5 and ZmKSL3, while a few sesqueiterpene was dectected in the product from ZmTPS1, which is consistent with the previously report. The three genes were then introduced into N. bethamiana by Agrobacterium and resulted in detection of ent-kaurene, demonstrating that the in vivo fuctions of the all three are ent-kaurene synthases. The further subcellular localizations of the three genes were observed by laser scanning confocal microscopy and resulted in a plastid localization, affirmed their KS activity.3. RT-PCR results demonstrated that only ZmKSL3 was not dectected in d5 mutant, which displays the dwarf phenotype. Besides, ZmKSL3 is specifically and highly expressed in activity growing tissues, indicating the role for ZmKSL3 in GAs biosynthesis. By cloning and sequencing the ZmKSL3 genome sequence in d5 mutant, we found that a 100bp deletion in 5'UTR close to the start codon. The deletion seuquence contains a CAAT box, loss of which presumably explain the loss of ZmKSL3 expression in d5. By contrast, in the available Uniform Mu mutants of ZmTPS1 and ZmKSL5, although in both mutant the expression levels were reduced for the two genes, the height of them shows no difference compared to the wild type. In vitro expression for ZmTPS1 and ZmKSL5 cloned from d5 revealed that both of them can synthesize ent-kaurene, explaining the appearance of ent-kaurene dectected from d5 in previous study.4. The expression patterns for the three genes in different treatment were analyzed by qRT-PCR with a comparison to the previously described An2. Firstly, ZmKSL3 is induced in leaves infected by Fusarium graminearum and roots treated by ABA. The relevant cis-element found in ZmKSL3 gene promoter may account for the induction. On the other hand, an elevate transcription levels of ZmKSL5 and ZmTPS1 are showed in all treatment including leaves infected by F. graminearum or treat by MeJA+ EP or MeJA alone and roots treated by ABA. The results of ZmTPSl and ZmKSL5 are partly corresponds to that of An2, indicating the role for both genes involved in kauralexins biosynthesis.5. By predicted online, we found 2 W-box elements in An2 promoter which are probablybe responsible for the induction of An2 by the inoculation of F. graminearum. So the function analysis for which w-box are actually in charge of this induction by particle bombardment in maize callus resulted in the only detectable activity for the first w-box. The promoter fragments doesn't show any activity when neither without the first W-box nor mutant the first W-box. Hence make it clear that the first W-box is the active one for the response to F. graminearum or MeJA+EP treatment. This result suggests a potential target to regulate the expression of An2 as well as the accumulation of kauralexins.Conclusion:In this study, we identified three KS(L) genes, with one participated in GA biosynthesis and served as the mutant KS genes in d5, while the other two were suggested to be responsible for the biosynthesis of kauralexins with their inducibility by the infection of pathogens as well as the treatment of plant hormones. Besides, the three genes form a tandem array in chromosome 2, indicating the occurrences of gene duplication and function variation during evolution. The activity analysis of An2 promoter characterized that the W-box is a target cis-element, which provides the foundation for the metabolism regulation study of kauralexin. |
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