Text by Ülo Niinemets
Isoprenoids constitute a versatile pathway of compounds that play major roles as integral components of foliage photosynthetic apparatus (chlorophylls and carotenoids) and as volatile infochemicals and molecules improving plant stress resistance via their antioxidant and membrane-stabilizing properties. Plants synthesize isoprenoids via two pathways, the mevalonic acid dependent pathway that is located in cytosol and methyl-erythritol 4-phosphate/1-deoxy-xylulose 5-phosphate (MEP/DOXP) pathway that is located in chloroplast. Both pathways produce isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) as the end-products. In the cytosol, IDP and DMADP are used to produce sesquiterpenoids (C15) and triterpenoids (C30), while in the plastids, DMADP is used to produce isoprene (C5) in isoprene-emitting species, and IDP and DMADP are used to produce mono- (C10), di- (C20) and tetraterpenes (C40, e.g. carotenoids).
So far, there is still limited information on the simultaneous regulation of the two pathways, in particular, on the degree to which the end-products of the two pathways can be exchanged. In isoprene-emitting species, a very large proportion of DMADP is used for synthesis of isoprene, but it is unclear to what extent synthesis of isoprene and larger isoprenoids compete for MEP/DOXP pathway end-products. A number of previous studies have demonstrated that the rate of isoprene synthesis depends on DMADP pool size, and thus, an increase in DMADP pool size is likely to enhance isoprene emission. On the other hand, it has recently been demonstrated that DMADP and IDP inhibit the first reaction of the MEP/DOXP pathway, and thus, their accumulation could result in feedback inhibition of the pathway flux (Banerjee et al. 2013). Such inhibition can be particularly relevant in isoprene-emitting species where MEP/DOXP pathway is particularly active and DMADP pool size undergoes large changes during the day due to changes in light intensity (Rasulov et al. 2009).
In this study, we have used two bisphosphonate inhibitors, pyrophosphate analogs, alendronate and zoledronate. Both alendronate and zoledronate are human drugs that inhibit isoprenoid synthesis in osteoclasts in bone tissue and are used to treat osteoporosis and bone cancer. In plants, they inhibit the prenyltransferase reactions in both cytosol and plastids and thus, are expected to inhibit synthesis of all isoprenoids other than isoprene. Thus, we expected that bisphosphonate application leads to enhanced pool sizes of DMADP and IDP and enhanced isoprene emissions until the feedback limitation sets in. In addition, we used fosmidomycin, an inhibitor that blocks one of the initial steps of the MEP/DOXP pathway, alone or in combination with bisphosphonates to gain insight into the possible contribution of cytosolic mevalonate-dependent pathway to plastidic isoprenoid synthesis.
We indeed observed that bisphosphonate-inhibition resulted in extensive plastidic pools of DMADP. However, evidence of feedback-inhibition of MEP/DOXP pathway was limited. In fact, the intermediates upstream of DMADP strongly accumulated such that DMADP concentration varied much less than expected. The contribution of mevalonate-dependent pathway intermediates to isoprene emission was significant under combined bisphosphonate/fosmidomycin treatment, but the overall isoprene flux that cytosolic intermediates could support was small. These results suggested that the capacity to accumulate metabolic intermediates upstream of DOXP/MEP pathway intermediates is very large. This large pathway elasticity implies that IDP and DMADP concentrations are maintained at levels low enough such that the feedback inhibition only seldom sets in under physiological conditions. We conclude that in isoprene-emitting species, the DOXP/MEP pathway can extremely elastically respond to differences in the rate of production of pathway intermediates and in differences in metabolic pull generated for instance by different stresses.
Full citation: Rasulov, B., Talts, E., Kannaste, A., & Niinemets, U. (2015). Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen. Plant physiology, pp-00470. (link to full text)