Functional analysis of the final steps of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing.
Isoprenoid biosynthesis in plant plastids occurs via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. We used tobacco rattle virus (TRV) to posttranscriptionally silence the expression of the last two enzymes of this pathway, the IspG-encoded (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and the IspH-encoded isopentenyl/dimethylallyl diphosphate synthase (IDDS), as well as isopentenyl/dimethylallyl diphosphate isomerase (IDI), the enzyme that interconverts IPP and DMAPP. TRV-IspG and TRV-IspH infected Nicotiana benthamiana plants had albino leaves that contained less than 4% of the chlorophyll and carotenoid pigments of control leaves. We applied [(13)C]DXP and [(14)C]DXP to silenced leaves and found that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate accumulated in plants blocked at HDS while DXP, (E)-4-hydroxy-3-methylbut-2-enyl phosphate and (E)-2-methylbut-2-ene-1,4-diol accumulated in IDDS-blocked plants. Albino leaves from IspG- and IspH-silenced plants displayed a disorganized palisade mesophyll, reduced cuticle, fewer plastids, and disrupted thylakoid membranes. These findings demonstrate the participation of HDS and IDDS in the DXP pathway in plants, and support the view that plastid isoprenoid biosynthesis is metabolically and physically segregated from the mevalonate pathway. IDI-silenced plants had mottled white-pale green leaves with disrupted tissue and plastid structure, and showed an 80% reduction in pigments compared to controls. IPP pyrophosphatase activity was higher in chloroplasts isolated from IDI-silenced plants than in control plant chloroplasts. We suggest that a low level of isoprenoid biosynthesis via the DXP pathway can occur without IDI but that this enzyme is required for full function of the DXP pathway.
Page
Jonathan E
JE
Leibniz-Institut für Pflanzenbiochemie, 06120 Halle, Germany. jon.page@nrc-cnrc.gc.ca
Hause
Gerd
G
Raschke
Maja
M
Gao
Wenyun
W
Schmidt
Jürgen
J
Zenk
Meinhart H
MH
Kutchan
Toni M
TM
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D019748:Carbon-Carbon Double Bond Isomerases / Q000235:genetics* / Q000378:metabolism; D002338:Carotenoids / Q000032:analysis; D002734:Chlorophyll / Q000032:analysis; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing / Q000502:physiology*; D008969:Molecular Sequence Data; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism*; D010641:Phenotype; D018515:Plant Leaves / Q000235:genetics / Q000821:virology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics* / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000254:growth & development
C109460:1-deoxylulose 5-phosphate; D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D018076:DNA, Complementary; D010428:Pentosephosphates; D010940:Plant Proteins; D013729:Terpenes; D002734:Chlorophyll; D002338:Carotenoids; D019883:Alkyl and Aryl Transferases; C488494:(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase, Nicotiana benthamiana; C488495:isopentenyl-dimethylallyl diphosphate synthase, Nicotiana benthamiana; D019748:Carbon-Carbon Double Bond Isomerases; C488497:isopentenyl-dimethylallyl diphosphate isomerase, Nicotiana benthamiana