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Next generation sequencing unravels the biosynthetic ability of spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics.

Jin J, Panicker D, Wang Q, Kim MJ, Liu J, Yin JL, Wong L, Jang IC, Chua NH, Sarojam R - BMC Plant Biol. (2014)

Bottom Line: A significant number of these unigenes remained unannotated or encoded hypothetical proteins.We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT.Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT's dynamic metabolic activities which will help towards pathway engineering.

Results: Spearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases.

Conclusions: The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint.

No MeSH data available.


Expression level of unigenes involved in MEP pathway. The number in green represents the expression level of a particular unigene in PGT (log2 of estimate abundance of transcripts by RSEM value). The number in red represents the fold change in expression level when compared to leaf-PGT (log2 fold change between PGT and leaf-PGT). In cases of enzymes with more than one unigene, the unigene with the highest abundance was taken into consideration. The number in brackets represents the number of unigenes identified for each enzyme in the pathway. DXS: 1-deoxy-D-xylulose-5-phosphate (DXP) synthase; DXR: DXP reductoisomerase, MCT:MEP cytidyltransferase, CMK:4-(cytidine 5- diphospho)-2-C-methyl-D-erythritol kinase MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-2,4cPP) synthase, HDS: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPP) synthase, HDR: HMBPP reductase, IPPI : Isopentenyl diphosphate (IPP,C5) Delta-isomerase.
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Fig5: Expression level of unigenes involved in MEP pathway. The number in green represents the expression level of a particular unigene in PGT (log2 of estimate abundance of transcripts by RSEM value). The number in red represents the fold change in expression level when compared to leaf-PGT (log2 fold change between PGT and leaf-PGT). In cases of enzymes with more than one unigene, the unigene with the highest abundance was taken into consideration. The number in brackets represents the number of unigenes identified for each enzyme in the pathway. DXS: 1-deoxy-D-xylulose-5-phosphate (DXP) synthase; DXR: DXP reductoisomerase, MCT:MEP cytidyltransferase, CMK:4-(cytidine 5- diphospho)-2-C-methyl-D-erythritol kinase MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-2,4cPP) synthase, HDS: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPP) synthase, HDR: HMBPP reductase, IPPI : Isopentenyl diphosphate (IPP,C5) Delta-isomerase.

Mentions: The building blocks for all different classes of terpenes produced by plants are C5 units of isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP). They are generated either by plastidial MEP or cytoplasmic MVA pathway. The MEP pathway requires seven enzymes to synthesize IPP and DMAPP from pyruvate and glyceraldehyde 3 phosphates which feed the monoterpene pathway [33]. From peppermint EST studies it has been proposed that the active pathway for the formation of IPP/DMAPP in the PGT is the MEP pathway. This is consistent with our analysis too where MEP pathway transcripts were more abundant in PGT than MVA. High expression of MEP pathway transcripts correlates well with the production of monoterpenes in PGT. It has been reported that 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of this pathway is important for the overall regulation of the pathway [34]. Multiple DXS genes have been found in plants like Zea mays, Medicago truncatula, Oryza sativa, Ginkgo biloba and Pinus densiflora and Picea abies [35-40]. In all these plants, two or three candidate DXS genes have been reported. From our data we were able to identify 2 different 1-deoxy-D-xylulose-5-phosphate synthase (DXS) unigenes showing different levels of abundance in PGT. The number of genes coding for each MEP pathway enzyme varies from plant to plant [33,41]. Presence of multiple genes with differential tissue-specific expression levels might contribute towards the regulation of the MEP pathway, in different organs of the plant. FigureĀ 5 shows the number of unigenes identified for each enzyme of the MEP pathway and their RNA seq expression levels. In cases of enzymes with more than one unigene, the unigene with the highest abundance in PGT was taken into consideration. Their expression was further validated by qRT-PCR (Additional file 4). From our RNA seq data and qRT-PCR analysis, DXR and MCT transcript levels were low when compared to levels of other enzymes in MEP pathway. This might suggest that possibly these two enzymes are the rate limiting steps of this pathway. A possible option to explore in future will be to enhance the expression level of various rate limiting steps to enhance the production of terpenes.Figure 5


Next generation sequencing unravels the biosynthetic ability of spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics.

Jin J, Panicker D, Wang Q, Kim MJ, Liu J, Yin JL, Wong L, Jang IC, Chua NH, Sarojam R - BMC Plant Biol. (2014)

Expression level of unigenes involved in MEP pathway. The number in green represents the expression level of a particular unigene in PGT (log2 of estimate abundance of transcripts by RSEM value). The number in red represents the fold change in expression level when compared to leaf-PGT (log2 fold change between PGT and leaf-PGT). In cases of enzymes with more than one unigene, the unigene with the highest abundance was taken into consideration. The number in brackets represents the number of unigenes identified for each enzyme in the pathway. DXS: 1-deoxy-D-xylulose-5-phosphate (DXP) synthase; DXR: DXP reductoisomerase, MCT:MEP cytidyltransferase, CMK:4-(cytidine 5- diphospho)-2-C-methyl-D-erythritol kinase MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-2,4cPP) synthase, HDS: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPP) synthase, HDR: HMBPP reductase, IPPI : Isopentenyl diphosphate (IPP,C5) Delta-isomerase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232691&req=5

Fig5: Expression level of unigenes involved in MEP pathway. The number in green represents the expression level of a particular unigene in PGT (log2 of estimate abundance of transcripts by RSEM value). The number in red represents the fold change in expression level when compared to leaf-PGT (log2 fold change between PGT and leaf-PGT). In cases of enzymes with more than one unigene, the unigene with the highest abundance was taken into consideration. The number in brackets represents the number of unigenes identified for each enzyme in the pathway. DXS: 1-deoxy-D-xylulose-5-phosphate (DXP) synthase; DXR: DXP reductoisomerase, MCT:MEP cytidyltransferase, CMK:4-(cytidine 5- diphospho)-2-C-methyl-D-erythritol kinase MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-2,4cPP) synthase, HDS: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPP) synthase, HDR: HMBPP reductase, IPPI : Isopentenyl diphosphate (IPP,C5) Delta-isomerase.
Mentions: The building blocks for all different classes of terpenes produced by plants are C5 units of isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP). They are generated either by plastidial MEP or cytoplasmic MVA pathway. The MEP pathway requires seven enzymes to synthesize IPP and DMAPP from pyruvate and glyceraldehyde 3 phosphates which feed the monoterpene pathway [33]. From peppermint EST studies it has been proposed that the active pathway for the formation of IPP/DMAPP in the PGT is the MEP pathway. This is consistent with our analysis too where MEP pathway transcripts were more abundant in PGT than MVA. High expression of MEP pathway transcripts correlates well with the production of monoterpenes in PGT. It has been reported that 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of this pathway is important for the overall regulation of the pathway [34]. Multiple DXS genes have been found in plants like Zea mays, Medicago truncatula, Oryza sativa, Ginkgo biloba and Pinus densiflora and Picea abies [35-40]. In all these plants, two or three candidate DXS genes have been reported. From our data we were able to identify 2 different 1-deoxy-D-xylulose-5-phosphate synthase (DXS) unigenes showing different levels of abundance in PGT. The number of genes coding for each MEP pathway enzyme varies from plant to plant [33,41]. Presence of multiple genes with differential tissue-specific expression levels might contribute towards the regulation of the MEP pathway, in different organs of the plant. FigureĀ 5 shows the number of unigenes identified for each enzyme of the MEP pathway and their RNA seq expression levels. In cases of enzymes with more than one unigene, the unigene with the highest abundance in PGT was taken into consideration. Their expression was further validated by qRT-PCR (Additional file 4). From our RNA seq data and qRT-PCR analysis, DXR and MCT transcript levels were low when compared to levels of other enzymes in MEP pathway. This might suggest that possibly these two enzymes are the rate limiting steps of this pathway. A possible option to explore in future will be to enhance the expression level of various rate limiting steps to enhance the production of terpenes.Figure 5

Bottom Line: A significant number of these unigenes remained unannotated or encoded hypothetical proteins.We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT.Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT's dynamic metabolic activities which will help towards pathway engineering.

Results: Spearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases.

Conclusions: The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint.

No MeSH data available.