April 9, 2015

González-Cabanelas et al. "The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana"

Diego González-Cabanelas, Louwrance P. Wright, Christian Paetz, Nawaporn Onkokesung, Jonathan Gershenzon, Manuel Rodríguez-Concepción and Michael A. Phillips*
The Plant Journal, Volume 82, Issue 1, pages 122–137, April 2015 DOI:10.1111/tpj.12798

Today I would like to highlight a publication of my own, as self-serving as that might be. This is the product of a Master's project carried out by my former student Diego González-Cabanelas, who has since gone to a PhD program at the Max Planck Institute of  Chemical Ecology (Jena, Germany). In this report, we describe a metabolic "shunt" funneling carbon flux away from the main isoprenoid precursor pathway in the chloroplast when flux is elevated. That's pretty important information if you are trying to metabolically engineer this pathway and don't understand why so much of the expected flux seems to be disappearing when we use the standard 'upregulation of structural genes' approach. An unusual cyclic diphosphate intermediate in the pathway has been recently alleged to have signaling properties, activating defense gene expression in the nucleus. Naturally, this would require this metabolite to physically leave the confines of the plastid and interact with other factors in the cytosol, nucleus, or elsewhere to control gene expression. It is also quite strange that a metabolic intermediate in a pathway would be selected to play such a different parallel role in defense signaling, so the actual story may be much more complex. Due to our limited knowledge of how isoprenoid biosynthesis is regulated at the molecular level, we are still making early discoveries into how this biosynthetic network is controlled. I like to think this is one of them.

One of our first steps in characterizing the separate lives led by 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP) was to show that under some conditions, a separate pool of this intermediate becomes isolated from the main pool used to make isoprenoid precursors in the plastid. This second pool can then be available for signaling or altering cellular metabolism in some way so as to activate cellular defenses, which in this case turns out be the salicylic acid pathway related to defense against microbial infections and some phloem feeding insect attacks. Plants have several distinct defense pathways which prime its metabolism against attackers as varied as viruses, arthropods, and bacteria. In the case of MEcDP, only the salicylic acid pathway is activated. Our study confirmed prior indications of this and went further to provide proof that some MEcDP leaves the plastid  when it builds up to high concentrations there, forming a second pool somewhere else. We found that this occurred either when a downstream step is blocked through mutagenesis or when a rate-controlling upstream step's activity is dialed up. This provides a series of new targets to learn more about how this defense signaling system using MEcDP might work. For instance, we can now look for stress conditions that downregulate the enzyme which consumes MEcDP to corroborate a role of concentration-dependent export as a means of activating this stress signal. It also provides an opportunity to improve engineering efforts since we now know about a potential source of loses we were previously ignorant of.

The other main novelty of our study was to shown that MEcDP that leaves the plastid, for whatever reason, winds up being dephosphorylated and converted to glycosides. This is a common phenomenon in plants for the deactivation of spent phytohormones and in the sequestration of stray metabolites alike. However, the addition to glycosyl residues to the dephosphorylated carbon skeleton of MEcDP (denoted simply as ME) follows a regiospecific pattern we observed for other hemiterpene glycosides, so it is quite possible this represents a specific conversion rather than just a general detox mechanism. Now that additional downstream metabolites for MEcDP in this branch pathway have been discovered, we can search for the responsible enzymes and better define the subcellular compartment where these conversions occur. For instance, it could all happen in the cytosol, which would keep the possibility of a signaling role for MEcDP alive. This would mean that after some biotic stress, caused by infection or aphid attack, MEcDP levels might rise in the plastid and become exported out to the cytosol. There, some could enter the nucleus and induce gene defense gene expression through some mechanism we could in principle test for. But after activating gene expression, it probably isn't very helpful to the cell to have this alarm signal floating around, so it should be unsurprising that some recycling mechanism exists to eliminate the signal and neutralize it so it isn't shifting cellular metabolism to defense long after the threat has been dealt with. That would explain the specific glycosylation/deactivation of ME we observed. On the other hand, this metabolite could just as easily be sent to some other compartment, such as the peroxisome, the ER, or the vacuole. It is too early to speculate how this might occur, but teasing apart these molecular defense systems teach us a lot about how to manage pests in agriculture more efficiently and sustainably. We now have a series of well defined targets for future investigations, thanks to Diego's and Lawrie's hard work and dedication to characterize the chemical steps of this branch pathway which connects isoprenoid biosynthesis to plant defensive signaling.

From the paper:


2-C-Methyl-d-erythritol-2,4-cyclodiphosphate (MEcDP) is an intermediate of the plastid-localized 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co-factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds-3 mutant, defective in the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2-C-methyl-d-erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds-3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds-3 mutant also showed enhanced resistance to the phloem-feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP-mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds-3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.

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