Type‐
- NSF-PAR ID:
- 10038639
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 92
- Issue:
- 1
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 82-94
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Summary II metacaspases are conserved cysteine proteases found in eukaryotes with oxygenic photosynthesis, including green plants and some algae, such asChlamydomonas andVolvox . Genetic and biochemical studies showed that some members in this protease family could be involved in oxidative stress‐induced cell death in higher plants, but their regulatory mechanisms remain unclear. Biochemically, two distinct classes of type‐II metacaspases are exemplified by AtMC 4 and AtMC 9 from Arabidopsis, with AtMC 4 activation dependent on calcium under neutralpH , whereas AtMC 9 is active only under mildly acidicpH , regardless of the availability of calcium. Here, we constructed all six possible combinations between the p20, linker, and p10 domains from AtMC 4 and AtMC 9. Our results show that calcium stimulation of AtMC 4 activity is associated with essential amino acids located in its p20 domain. In contrast, the acidicpH optimum trait is lost from AtMC 9 if one or two of its domains are replaced by that from AtMC 4, suggesting that multiple interactions between domains in AtMC 9 may be responsible for this property. Consistent with this, we found conserved ‘signature’ residues in each of the three domains that distinguish AtMC 4‐ and AtMC 9‐like classes of metacaspases. Tracing the origin of the AtMC 9 class, we found evidence for its appearance between lycophytes and gymnosperms, coincident with the evolution of more complex root archetypes in terrestrial plants. Our work suggests that the distinctive properties of the AtMC 9‐like protease could be associated with special cellular physiology in the roots of gymnosperms and angiosperms that are distinct from lycophytes. -
more » « less
Summary Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (
PAT ) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT ) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition ofPAT are currently unknown. Here we present the X‐ray crystal structures of the wild‐type and various mutants ofPAT fromArabidopsis thaliana (AtPAT ) . Steady‐state kinetic and ligand‐binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution ofPAT activity among class IbAAT enzymes. Structures of AtPAT mutants co‐crystallized with either α‐ketoglutarate or pyridoxamine 5′‐phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor ofPAT fromA. thaliana and Antirrhinum majus plants as well as the bacteriumChlorobium tepidum , uncovering a potential new effector ofPAT . -
more » « less
Summary The evolution of
l ‐DOPA 4,5‐dioxygenase activity, encoded by the geneDODA l ‐DOPA 4,5‐dioxygenase activity evolved via a single Caryophyllales‐specific neofunctionalisation event within theDODA DODA To address this, we functionally characterised 23 distinct
DODA proteins forl ‐DOPA 4,5‐dioxygenase activity, from four betalain‐pigmented and five anthocyanin‐pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updatedDODA phylogeny, we then explored the evolution ofl ‐DOPA 4,5‐dioxygenase activity.We find that low
l ‐DOPA 4,5‐dioxygenase activity is distributed across theDODA DODA l ‐DOPA 4,5‐dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro‐synteny.In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated
l ‐DOPA 4,5‐dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales. -
more » « less
Summary Malonyl‐CoA is a key intermediate in a number of metabolic processes associated with its role as a substrate in acylation and condensation reactions. These types of reactions occur in plastids, the cytosol and mitochondria, and although carboxylation of acetyl‐CoA is the known mechanism for generating the distinct plastidial and cytosolic pools, the metabolic origin of the mitochondrial malonyl‐CoA pool is still unclear. In this study we demonstrate that malonyl‐CoA synthetase encoded by the Arabidopsis
AAE 13AT 3G16170AAE 13 protein is not essential, due to the presence of a redundant malonyl‐CoA generating system provided by a cytosolic acetyl‐CoA carboxylase, the mitochondrialAAE 13 protein is essential for plant growth. Phenotypes of theaae13‐1 mutant are transgenically reversed only if the mitochondrial pre‐sequence is present in the ectopically expressedAAE 13 proteins. Theaae13‐1 mutant exhibits typical metabolic phenotypes associated with a deficiency in the mitochondrial fatty acid synthase system, namely depleted lipoylation of the H subunit of the photorespiratory enzyme glycine decarboxylase, increased accumulation of glycine and glycolate and reduced levels of sucrose. Most of these metabolic alterations, and associated morphological changes, are reversed when theaae13‐1 mutant is grown in a non‐photorespiratory condition (i.e. a 1%CO 2atmosphere), demonstrating that they are a consequence of the deficiency in photorespiration due to the inability to generate lipoic acid from mitochondrially synthesized fatty acids. -
more » « less
Summary In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexity
in vivo , we developed anin vivo lipid ‘tag and track’ method. Essentially, we probed the lipid metabolism inArabidopsis thaliana by expressing a coding sequence for a fatty acid desaturase fromPhyscomitrella patens (Δ6D). This enzyme places a double bond after the 6th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at itssn‐ 2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER ) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to ‘tag and track’ the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence ofER ‐derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this ‘tag and track’ method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions.
