Evolutionary and functional relationship between the biosynthetic pathways towards leucine and aliphatic glucosinolates in plants
Final Report Abstract
In the now completed funding period several branched-chain aminotransferases (BCAT) and the isopropylmalate isomerase (IPMI) were successfully characterized in Arabidopsis thaliana. This model species encodes the six transcribed genes for BCAT1 to 6. Beside BCAT4 and BCAT3, which had been investigated in the first funding period, now BCAT1, BCAT2, BCAT5 and BCAT6 were studied in detail. The new analyses showed that BCAT1 and BCAT2 localize to mitochondria. Expression of BCAT1 is almost completely independent from environmental conditions, whereas BCAT2 mRNA levels increased 250-fold after cultivating plants for 16h in the dark, which induced a carbohydrate starvation in plants. Metabolite studies of mutants plants grown under different conditions revealed that both proteins are involved in degradation of Val, Leu and Ile. BCAT5 most likely has a function in amino acid metabolism that differs from its highly similar counterpart BCAT3. But similar to this protein, BCAT5 might influence Met chain elongation when BCAT4 is absent. Both BCAT6 and BCAT4 localize to the cytosol. BCAT6 is most likely active in Met chain elongation, but its function seems to be less important in comparison to BCAT4. The investigation of the small subunits of the isopropylmalate isomerase (IPMI SSU1 to 3) revealed several unexpected results. An artifical microRNA-induced knockdown of IPMI SSU1 provoked a strong morphological phenotype. The plants exhibit stunted growth with abnormal leaf tissue patterning and flattened chloroplasts with almost no starch. Metabolite studies demonstrated that IPMI SSU1 is required for Leu biosynthesis but is also involved in the first cycle of Met chain elongation, the initial part of the biosynthesis of aliphatic glucosinolates. Strikingly, the knockdown mutant accumulates more Leu than wild-type plants. In contrast to IPMI SSU1, IPMI SSU2 and IPMI SSU3 seem to be exclusively involved in Met chain elongation. Detailed expression studies with full length protein transcriptional/translational fusions to various fluorescent proteins provide interesting insights into the spatial separation of the different IPMI heterodimers. In above ground tissues IPMI SSU1 is always found in leucoplasts of epidermal cell layers excluding large parts of the green tissues from the biosynthesis of Leu and the first cycle of Met elongation. In contrast, IPMI SSU2 and IPMI SSU3 were always found together in chloroplasts of cells in the periphery of the phloem bu t also in cells located next to the inward looking boundary of the xylem. In roots, there is also a strict separation of IPMI SSU1 and IPMI SSU2/IPMI SSU3 at the cellular level. But despite this spatial separation of the small IPMI subunits and thus of the corresponding mature heterodimers the dual function of IPMI SSU1 suggests that there is no clear cut separation of primary and specialized metabolism in Arabidopsis and probably other Brassica species.
Publications
- (2014). The small subunit 1 of the Arabidopsis isopropylmalate isomerase is required for normal growth and development and the early stages of glucosinolate formation. PLoS ONE 9, e91071
Imhof, J., Huber, F., Reichelt, M., Gershenzon, J., Wiegreffe, C., Lächler, K., and Binder, S.
(See online at https://doi.org/10.1371/journal.pone.0091071) - (2015). The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism. Plant Mol. Biol.
Lächler, K., Imhof, J., Reichelt, M., Gershenzon, J. and Binder, S.
(See online at https://doi.org/10.1007/s11103-015-0312-3)
