- NSF-PAR ID:
- 10309340
- Date Published:
- Journal Name:
- The Plant Journal
- ISSN:
- 0960-7412
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Summary l ‐Tyrosine is an essential aromatic amino acid required for the synthesis of proteins and a diverse array of plant natural products; however, little is known on how the levels of tyrosine are controlledin planta and linked to overall growth and development. Most plants synthesize tyrosine by TyrA arogenate dehydrogenases, which are strongly feedback‐inhibited by tyrosine and encoded byTyrA1 andTyrA2 genes inArabidopsis thaliana . While TyrA enzymes have been extensively characterized at biochemical levels, theirin planta functions remain uncertain. Here we found thatTyrA1 suppression reduces seed yield due to impaired anther dehiscence, whereasTyrA2 knockout leads to slow growth with reticulate leaves. Thetyra2 mutant phenotypes were exacerbated byTyrA1 suppression and rescued by the expression ofTyrA2 ,TyrA1 or tyrosine feeding. Low‐light conditions synchronized thetyra2 and wild‐type growth, and ameliorated thetyra2 leaf reticulation. After shifting to normal light,tyra2 transiently decreased tyrosine and subsequently increased aspartate before the appearance of the leaf phenotypes. Overexpression of the deregulated TyrA enzymes led to hyper‐accumulation of tyrosine, which was also accompanied by elevated aspartate and reticulate leaves. These results revealed that TyrA1 and TyrA2 have distinct and overlapping functions in flower and leaf development, respectively, and that imbalance of tyrosine, caused by altered TyrA activity and regulation, impacts growth and development of Arabidopsis. The findings provide critical bases for improving the production of tyrosine and its derived natural products, and further elucidating the coordinated metabolic and physiological processes to maintain tyrosine levels in plants. -
ABSTRACT Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache ( ca lcium channels and che motaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpV PR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans , which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpV PR . Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpV PR were in the micromolar range. An McpV PR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction. IMPORTANCE Legumes share a unique association with certain soil-dwelling bacteria known broadly as rhizobia. Through concerted interorganismal communication, a legume allows intracellular infection by its cognate rhizobial species. The plant then forms an organ, the root nodule, dedicated to housing and supplying fixed carbon and nutrients to the bacteria. In return, the engulfed rhizobia, differentiated into bacteroids, fix atmospheric N 2 into ammonium for the plant host. This interplay is of great benefit to the cultivation of legumes, such as alfalfa and soybeans, and is initiated by chemotaxis to the host plant. This study on carboxylate chemotaxis contributes to the understanding of rhizobial survival and competition in the rhizosphere and aids the development of commercial inoculants.more » « less
-
Abstract l ‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrAa), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrAp), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterizedTnt1 ‐transposon mutants ofMtPDH1 (pdh1 ) inMedicago truncatula to investigate PDH function. Thepdh1 mutants lackedPDH transcript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence thatMtPDH1 is responsible for the PDH activity detected inM. truncatula . Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity ofpdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism. -
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 . -
Summary This work revisits a publication by Bean
et al. (2018) that reports seven amino acid substitutions are essential for the evolution ofl ‐DOPA 4,5‐dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Beanet al. (2018).Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Bean
et al. (2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Beanet al. (2018) to re‐observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2‐mut3 variant.Multiple
in vivo assays, in bothSaccharomyces cerevisiae andNicotiana benthamiana , did not result in visible DODA activity in BvDODAα2‐mut3, with betalain production always 10‐fold below BvDODAα1.In vitro assays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2‐mut3 proteins, explaining their differing performancein vivo .In summary, we were unable to replicate the
in vivo analyses of Beanet al. (2018 ), and our quantitativein vivo andin vitro analyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Beanet al. (2018 ).