The Regulation Of Isoprenoid Biosynthesis By A Key Enzyme And Intermediates From The 2-C-methyl-D-Erythritol-4-phosphate(MEP) Pathway In Arabidopsis Thaliana

Isoprenoids,also known as terpenoids,are all derived from the same five-carbon building blocks,isopentenyl diphosphate(IDP)and its isomer dimethylallyl diphosphate(DMADP).The IDP and DMADP building blocks give rise to a very diverse group of compounds and occur in all living organisms.Isoprenoids have important functions in primary metabolism.In secondary metabolism,an enormous variety of isoprenoid structures play a multitude of roles in defence against enemies,attraction of mutualists and internal signalling.In addition,the commercial value of some isoprenoids as pigments(carotenoids),industrial materials(resins and rubbers),flavour and fragrances(monoterpenes),drugs(taxol,artemisinin),and even biofuels hold much potential for industry.Isoprenoid biosynthesis is unusual in the fact that two completely different metabolic routes are responsible for the biosynthesis of the five-carbon precursors,IDP and DMADP.Before the 2-C-methyl-D-erythitol 4-phosphate(MEP)pathway was discovered in the 1990s,the biosynthesis of isoprenoids was thought to be only through the mevalonate(MVA)pathway.Most organisms use only one of the two pathways for the biosynthesis of their isoprenoid precursors.By contrast,green algae,plants,and some microorganisms use both pathways.Whereas the MVA pathway occurs in the cytosol and involves sequential additions of acetyl-CoA units followed by reduction and phosphorylation to produce IDP and DMADP,the MEP pathway is localized in the chloroplast and starts with the synthesis of 1-deoxy-D-xylulose 5-phosphate(DXP)by condensation of glyceraldehyde 3-phosphate and pyruvate via DXP synthase(DXS).DXP is then reduced to MEP that is converted into 2-C-methyl-D-erythritol-2,4-cyclodiphosphate(MEcDP)in three steps.Finally,MEcDP is successively reduced to 1-hydroxy-2-methyl 2-(E)-butenyl 4-diphosphate(HMBDP),catalysed by HMBDP synthase(HDS),and then to a mixture of IDP and DMADP catalysed by HMBDP reductase(HDR).The importance of compartmentation in plant isoprenoid biosynthesis is highlighted by the occurrence of two pathways,as well as the recent discovery that the MEP pathway derived intermediate,MEcDP,is involved in complex signalling cascades from plastids to the nucleus.Earlier research also showed that HDS,the enzyme that metabolizes MEcDP,is very sensitive to increased reactive oxygen species.Under stress conditions that cause the formation of reactive oxygen species,an enormous increase in MEcDP levels is observed.Furthermore,over-expressing DXS,the first enzyme of the MEP pathway,resulted in increased pathway flux accompanied with an efflux of MEcDP out of the MEP pathway.Consequently the compartmentation of MEcDP seems to be important in the regulation of the MEP pathway with additional effects on cell signalling.So the research of the compartmentation of the MEP pathway and how the flux through this important biosynthetic pathway is controlled in will ultimately lead to improve metabolic engineering strategies for producing economically important natural isoprenoid compounds.A transcriptomic profile contributed by Xiao et al.(Xiao et al.,2012)was used in our study.This dataset were obtained from the wild-type control and the mutant plants designated as constitutively expressing hydroperoxide lyase(HPL)(mutant cehl)which encodes HDS.This work investigated bioinformatic analysis of this dataset and the compartmentation of the MEP pathway with emphasis on the subcellular distribution of MEcDP.Additionally the effect of HDS on MEcDP distribution and pathway flux was studied.(1)A total of 762 different expressed genes(DEGs)including 620 up-regulated and 142 down-regulated genes were screened.KEGG pathway enrichment analysis revealed that DEGs were mainly associated with biosynthesis and metabolism-related pathways.It was shown that stilbenoid,diarylheptanoid,and gingerol biosynthesis was the most significantly enriched pathway.(2)To determine the compartmentation of the interm ediates we performed non-aqueous fractionation on wild type Arabidopsis thaliana rosette leaves.With non-aqueous fractionation compounds are enriched into different density zones on a density gradient,which in turns depends on the density of the plant cell organelle they originate from.This revealed that MEcDP also occurs outside the chloroplast,with 22.0 ± 6.4%occurring in the cytosol and 2.5 ± 1.1%occurring in the vacuole.This is the first direct experimental proof that MEcDP is exported out of the chloroplast.The compartmentation results of the first and last metabolites of the MEP pathway were much more unexpected.Only about half the amount of DXP occurs in the chloroplast(49.7± 5.6%)while the rest are distributed within the cytosol(30.3 ± 3.3%)and vacuole(20.0 ± 3.3%).IDP and DMADP,in contrast,occur only in the chloroplast.Since the MVA pathway in the cytosol also produces IDP and DMADP,this indicates that the MVA is not significantly active when our samples were harvested during the middle of the day.(3)To determine the effect of HDS on the MEP pathway,enzyme levels were altered using over-expressed transgenic Arabidopsis thaliana plant lines as well as the csb3 mutant plant with highly reduced HDS activity.Since HDS contains a[4Fe-4S]cluster that is sensitive to oxidation,no methodology is currently available to measure HDS activity in crude plant extracts.It was,however,shown by others that there is a direct correlation between transcript levels and HDS protein content,and transcript level was therefore used as an indication of HDS levels.We then looked at the correlation between the transcript levels of HDS in the different lines and the flux in the MEP pathway.The flux in the different plant lines was determined by measuring the incorporation of 13C into DXP over time,after fumigating plants with air containing 13CO2.Since the substrates of the MEP pathway are derived from freshly assimilated CO2,13C is rapidly incorporated into pathway intermediates.The inability of increased HDS levels to affect flux revealed that HDS does not control flux in wild type Arabidopsis thaliana.(4)Using our new knowledge on the compartmentation of three of the MEP pathway intermediates,we investigated the effect of HDS on the ratio of the MEcDP pools within and without the chloroplasts.After long term 13CO2 labelling,when label incorporation into intermediates are at its maximum,an apparent reduction of label relative to expected levels will be dependent on the size of external metabolic pools.Since non-aqueous fractionation showed that IDP and DMADP only occurs in the chloroplast,their maximum label incorporation amounts can be used as the labelling levels expected in the linear MEP pathway.This revealed that the ratio between the fraction of MEcDP outside and inside the MEP pathway decreased significantly with increased HDS transcripts in the transgenic lines.This effect is however too weak to have an effect on the ultimate end products of the MEP pathway(mostly the carotenoids and chlorophylls).In conclusion,this research was the first to determine the compartmentation of the three most abundant intermediates of the MEP pathway.This confirmed the presence of MEcDP in the cytosol,but unexpectedly showed that DXP also occurs in the cytosol and the vacuole.In addition IDP and DMADP were only detected in the chloroplast,implying that the MVA pathway is not active under light.Lastly,although HDS did not influence the flux in wild type plants,it did have a significant effect on the efflux of MEcDP out of the chloroplast.