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Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.
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The putative two‐pore Ca2+ channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co‐localizes with the K+‐selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca2+‐activated slow vacuolar (SV) currents, which were increased in AtTPC1 ‐over‐expressing Arabidopsis compared to the wild‐type. A Ca2+‐insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1‐2 knockout plants. The kinetics of ABA‐ and CO2‐induced stomatal closure were similar in wild‐type and tpc1‐2 knockout plants, excluding a role of SV channels in guard‐cell signalling in response to these physiological stimuli. ABA‐, K+‐, and Ca2+‐dependent root growth phenotypes were not changed in tpc1‐2 compared to wild‐type plants. Given the permeability of SV channels to mono‐ and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca2+ entry into the cytosol. Ca2+ responses as measured in aequorin‐expressing wild‐type, tpc1‐2 knockout and TPC1 ‐over‐expressing plants disprove a contribution of TPC1 to any of the stimulus‐induced Ca2+ signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca2+ concentration and biotic factors (elf18, flg22). In good agreement, stimulus‐ and Ca2+‐dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization‐activated Ca2+‐permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca2+ homeostasis.
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