cDNA, from Hevea brasiliensis latex, encoding 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Natural rubber from Hevea brasiliensis is synthesized by enzymic polymerization of isopentenyl diphosphate (IDP) units. This has been proposed to occur inside the latex vessel in a thread-like tubular reticulum, connecting rubber particles to single- (lutoid) and double- (Frey-Wyssling, FW) membrane-bound organelles. We show that a membrane-free preparation from FW particles converted [¹⁴C] glucose into radio labeled prenyl products and this was more efficiently inhibited by fosmidomycin, the MEP pathway inhibitor, than mevilonin, the inhibitor used to block the MVA pathway. This implicated the alternative plastid associated MEP pathway for IDP synthesis. We then identified a cDNA clone (Hbdxr) from a Hevea rubber latex cDNA library, encoding for 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), a key enzyme of the MEP pathway for the IDP biosynthesis. Sequence analysis and the deduced amino acid sequence had >80% homology to other plant DXR enzymes with an ORF consisting of 1413 bp capable of encoding a 50.97 kDa polypeptide. A highly conserved binding site for NADPH was identified and an N-terminal transit peptide with a putative conserved cleavage site. The Hbdxr-gfp gene, transformed into Arabidopsis cells was located in the chloroplasts, thus Hbdxr may be expressed and localized in the FW plastids. The levels of Hbdxr mRNA detected in young latex containing tissues, inflorescence and seedling stems, were higher than those found in the latex from the tree and were barely detectable in the mature leaves. We therefore suggest that one function of the FW particles could be to supply IDP for rubber biosynthesis through the tubular thread-like reticulum.