Identification And Functional Characterization Of Nicotiana Tabacum ABC Transporters Involved In The Transport Of Secondary Metabolites

Tobacco, Nicotiana tabacum L., is an important economic crop and a useful modelplant in research as well. During the long time interaction between plants and ecologicalenvironment, tobacco can produce many secondary metabolites with highconcentrations and variations in the growth process, such as alkaloids, phenolic andterpenoids compounds, etc, which play an important role to resist insects, enhance stresstolerance and all kind of diseases. In addition, some secondary metabolites of tobaccohave been widely applied in the fields of medicine, food and pesticides. Being a specialand important economic crop, to some extent, its value is highly related with secondarymetabolites. Hence, it is useful to conduct the research of secondary metabolicengineering and metabolic regulation in tobacco. As well known, an effective approachto regulate the secondary metabolites is to study their transport and accumulation by themean of genetic engineering. Compared to a wide variety of secondary metabolites intobacco, however, little is known about transport and accumulation of secondarymetabolites in tobacco.The ATP-binding cassette (ABC) transporters are one of the largest families oftransmembrane proteins, which are found in all organisms. ABC transporters play animportant role in the transport and accumulation of secondary metabolites, so studyingABC has become one of the most popular researches in plant metabolic engineering andsecondary metabolism regulation. In order to understand how ABC transporters regulatemechanism of transport and accumulation of secondary metabolites in the molecularlevel, in this study a series of work was carried out including systems analysis oftobacco ABC transporter proteins, and the function research of transport of secondarymetabolites. The main findings are as follows:①Based on genome and predicted genes of a cultivar tobacco (Hongda),265ABC transporters were identified in the tobacco genome, accounting forapproximately0.4%of the total genes of N.tabacum genome. This is currently thelargest ABC protein family in all known organisms. The bioinformatics analysis wasalso performed including classification, nomenclature, domain organization andsubcellular localization. The results showed that in the tobacco genome, ABCG proteinsformed the largest subfamily of ABC family with134members, and represented thelargest ABC subfamily with maximum proportion in plant genome up to now. While ABCE proteins formed the smallest subfamily with only two members.②Phylogenetic relationships of the ABC gene between the tobacco and the otherplants have been analyzed and compared. The results indicatede that ABCA subfamilyappeared to have undergone rapid divergence during evolution, and each of the tobaccoABCA gene lacked a clearly orthologous relationship to other species. ABCE geneswere highly conserved in terms of phylogenetic orthologs. Phylogenetic analysisrevealed that ABCC and ABCG subfamilies appeared to be lineage-specificallyexpanded in the tobacco. Moreover, ABC genes in most subfamilies were founded withclearly orthologous relationship to ABC genes in other plants whose functions arealready known.③Based on the data of tobacco ABC transporters，a nicotine candidate transportergene, NtWBC13was screened and cloned,, which encodes a half ABC protein of ABCGsubfamily. It was predicted as a plasma membrane protein by using membrane proteinsubcellular localization software. Semi-quantitative RT-PCR and qRT-PCR indicatedthat transcript levels of NtWBC13were reduced by nicotine, MeJA (methyl jasmonate)and wound. Multiple alignment of NtWBC13and other WBC amino acid showed thatthe amino acid sequences werehighly conserved in the motifs of Walker A, Walker Band ABC signature.④qRT-PCR was performed to examine the gene expression pattern of NtWBC13in plant organs., it indicated that NtWBC13expression was abundant in roots. NtWBC13promoter was cloned, and the results of analysis of promoter indicated that there wereseveral wound-related cis-elements and root-specific expression related elements inNtWBC13promoter, such as CATG-box (CATG), W-box (TCACC/T) andROOTMOTIFTAPOX1(ATATT). A pNtWBC13/GUS expression vector wasconstructed, and GUS signals of GUS histochemical staining in transgenic plants weredetected and predominantly observed in main root. It supposed that NtWBC13may playkey roles in the root.⑤To assess whether or not NtWBC13functioned as a nicotine transporter, weexpressed the NtWBC13in a heterologous yeast expression system. yeast cells of theNtWBC13transformant and the control were incubated in nicotine containing medium.By using HPLC to analyze nicotine content, the results showed that NtWBC13transformants accumulated more nicotine than control yeast cells under allconcentrations tested. A time course analysis showed that the nicotine content inNtWBC13transformants was consistently higher than that of control cells. Using the RNA interference (RNAi)-based approach to knock down expression of NtWBC13in N.tabacum, NtWBC13-RNAi plants showed lower root nicotine accumulation levelscompared to wild type. We examined the inhibitory effect of exogenously suppliednicotine on seed growth indicating that RNAi plants were more inhibited than thegrowth of wild type. Above results suggested that NtWBC13functioned as a nicotinetransporter might be involved in the nicotine acumination in the main root of tobacco.⑥Strigolactone is a new class of plant hormones, which have recently beendemonstrated as a shoot branching inhibitors. Based on the amino acid sequence of P.hybrida PDR1，by homology searching for the data of tobacco ABC transporters, wehave identified a homolog of PhPDR1protein, NtPDR6,and full-length CDS of thisgene was obtained through fragment cloning method.Sequence analysis of NtPDR6gene was performed by bioinformatics methods. NtPDR6encodes a typical full ABCGtransporter with a reverse domain organization NBD-TMD. Multiple alignment ofNtPDR6and other plant amino acids showed that the amino acid sequences were highlyconserved in the motifs of Walker A, Walker B and ABC signature. NtPDR6waspredicted as a plasma membrane protein by using membrane protein subcellularlocalization software. Phylogenetic analysis revealed that NtPDR6had one orthologousrelationship with P. hybrida PDR1, and NtPDR6showed a high amino-acid sequenceidentity with the PhPDR1(93%).⑦qRT-PCR was performed to examine the gene expression pattern of NtPDR6after phosphate starvation and auxin treatment. The analysis results showed thatNtPDR6transcript levels significantly increased after phosphate starvation treatment, aswell as auxin. Tissue expression pattern analysis of NtPDR6gene showed that NtPDR6was detected in all surveyed tissues (roots, stems, leaves and flowers) but preferentiallyin roots. NtPDR6promoter was cloned. The results of analysis of promoter indicatedthat there were one phosphate starvation response-related element Pho-like(GACGTGG) and four root-specific expression related elementsROOTMOTIFTAPOX1(ATATT) in NtPDR6promoter. The NtPDR6promoter-drivenGUS expression indicated that GUS activity was expressed mainly in nodes near thesoil surface, hypodermal passage cells and the elongation zone of lateral roots.Moreover, GUS signals increased by phosphate starvation treatments. The expressionpatterns of PhPDR1and NtPDR6were similar, suggesting that NtPDR6might serve asimilar function to PhPDR1.⑧After RNA interference technology was used to knock down NtPDR6 expression, the number of branches was significantly increasing in the NtPDR6knockdown plants, and the morebranches compared to control were shown over time ofthe growth. This result was consistent with the branching phenotype caused by thestrigolactone biosynthetic genes mutated or knocked down, as well as thestrigolactone transporter PhPDR1gene knocked down. This branching phenotype wasindeed caused by the lack of strigolactone, and thereby the capacity of inhibiting shootbranching has been reduced. These reslults suggested that the NtPDR6might beinvolved in the strigolactone transport, and it would play an important role in regulationof shoot branching processes in the tobacco, which provided new clues to the regulationof shoot branching.Through this study, we carried out a comprehensive analysis of the number oftobacco ABC genes, protein structure and subcellular localization, and studied themutual evolutionary relationship with the ABC genes of other plants at the genomiclevel. This will provide a theoretical and molecular referenceto understand evolutionarystatus and functional diversity of ABC genes in tobacco. Meanwhile, the functionalresearches in the transport of secondary metabolites were also performed in the tobacco.One ABC gene, NtWBC13, involved in the transport of nicotine, and another gene,NtPDR6, may be involved in the transporter of strigolactone had been cloned andstudied respectively. These results demonstrate favorable clues to study in the regulationof the secondary metabolites in tobacco. In addition, it also provides a reference for theregulation of the secondary metabolites in other plants in the future.