Robert D. Slocum

A functional analysis of the pyrimidine catabolic pathway in Arabidopsis

Reductive catabolism of pyrimidine nucleotides occurs via a three-step pathway in which uracil is degraded to β-alanine, CO2 and NH3 through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and β-ureidopropionase (EC 3.5.1.6, PYD3). A proposed function of this pathway, in addition to the maintenance of pyrimidine homeostasis, is the recycling of pyrimidine nitrogen to general nitrogen metabolism. PYD expression and catabolism of [2-14C]-uracil are markedly elevated in response to nitrogen limitation in plants, which can utilize uracil as a nitrogen source. PYD1, PYD2 and PYD3 knockout mutants were used for functional analysis of this pathway in Arabidopsis. pyd mutants exhibited no obvious phenotype under optimal growing conditions. pyd2 and pyd3 mutants were unable to catabolize [2-14C]-uracil or to grow on uracil as the sole nitrogen source. By contrast, catabolism of uracil was reduced by only 40% in pyd1 mutants, and pyd1 seedlings grew nearly as well as wild-type seedlings with a uracil nitrogen source. These results confirm PYD1 function and suggest the possible existence of another, as yet unknown, activity for uracil degradation to dihydrouracil in this plant. The localization of PYD-green fluorescent protein fusions in the plastid (PYD1), secretory system (PYD2) and cytosol (PYD3) suggests potentially complex metabolic regulation.

Molecular cloning and characterization of a UDP-glucose-4-epimerase gene (galE) and its expression in pea tissues

Abstract We cloned a cDNA encoding for the enzyme UDP-glucose-4-epimerase (EC 5.1.3.2; UDP-galactose-4-epimerase or ‘galactowaldenase’) in pea ( Pisum sativum L. cv. Wando). This enzyme functions in the interconversion of UDP-glucose and UDP-galactose. The open reading frame within this cDNA contains 350 amino acid residues and the polypeptide product has a predicted molecular mass of 38 994 Da. The pea enzyme exhibits extensive homology with the deduced sequences of other prokaryotic and eukaryotic UDP-glucose-4-epimerases. Northern analysis of expression of the UDP-glucose-4-epimerase gene ( galE ) in pea tissues revealed 3- and 5-fold higher mRNA levels in the leaves and roots, respectively, of 3-week old seedlings, as compared with 4-day old seedlings. GalE transcript levels did not differ significantly in the roots and leaves of light-grown versus etiolated 4-day old seedlings. Increased galE expression in the roots of older seedlings may be correlated with the copious secretion of mucigel at the root tip, as increased availability of the UDP-galactose precursor may be required to support the synthesis of this pectinaceous extracellular matrix.

Isolation and characterization of a cDNA encoding a pea ornithine transcarbamoylase (argF) and comparison with other transcarbamoylases

We used a PCR-based library screening method to isolate a 1.4 kb pea leaf cDNA encoding ornithine transcarbamoylase (OTCase). The cDNA contains a single major ORF of 375 amino acids whose deduced sequence exhibits a high degree of homology with other OTCases. The predicted molecular mass of 41 361 Da for this protein is approximately the 40 kDa size of the polypeptide that is immunoprecipitated with OTCase antibody afterin vitro translation of pea leaf mRNA.In vivo, OTCase occurs as a trimer of identical 36.5 kDa polypeptides, suggesting that this enzyme is synthesized as a cytosolic precursor protein. Southern blot analysis indicates that multiple OTCase genes occur in pea. An abundant 1.4 kb transcript is seen in northern blots of total RNA isolated from the leaves and roots of light- and dark-grown pea seedlings.

PALA-mediated pyrimidine starvation increases expression of aspartate transcarbamoylase (pyrB) in Arabidopsis seedlings

Abstract Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) catalyzes the committed step in the de novo synthesis of pyrimidine nucleotides. We investigated the effects of N -(phosphonacetyl)-L-aspartate (PALA), a transition-state analog inhibitor of ATCase, on seedling growth and development, RNA and soluble protein contents, ATCase activity and enzyme protein levels, and pyrB gene expression in Arabidopsis thaliana L. cv. “Columbia”. In vitro, PALA was a potent inhibitor of ATCase, with an apparent K i = 22 nM. After 5 d of treatment with 1 mM PALA, seedlings exhibited delayed germination, inhibition of cotyledon expansion, leaf development and root growth, and general chlorosis. Total RNA contents of these seedlings were decreased by 81% and total soluble protein contents decreased by 74%, compared with untreated control plants. Levels of pyrB mRNA increased about tenfold in PALA-treated plants, while ATCase activity and enzyme protein levels increased twofold. Plants grown on media containing a lower (0.1 mM) concentration of PALA did not exhibit significant inhibition of growth until after 9 d of treatment, but had markedly reduced RNA contents (40% of controls) and elevated pyrB mRNA levels (fourfold increase) after 12 d of treatment.

Ectopic expression of a pea apyrase enhances root system architecture and drought survival in Arabidopsis and soybean: Apyrase enhances root architecture and drought survival

Ectoapyrases (ecto-NTPDases) function to decrease levels of extracellular ATP and ADP in animals and plants. Prior studies showed that ectopic expression of a pea ectoapyrase, psNTP9, enhanced growth in Arabidopsis seedlings and that the overexpression of the two Arabidopsis apyrases most closely related to psNTP9 enhanced auxin transport and growth in Arabidopsis. These results predicted that ectopic expression of psNTP9 could promote a more extensive root system architecture (RSA) in Arabidopsis. We confirmed that transgenic Arabidopsis seedlings had longer primary roots, more lateral roots, and more and longer root hairs than wild-type plants. Because RSA influences water uptake, we tested whether the transgenic plants could tolerate osmotic stress and water deprivation better than wild-type plants, and we confirmed these properties. Transcriptomic analyses revealed gene expression changes in the transgenic plants that helped account for their enhanced RSA and improved drought tolerance. The effects of psNTP9 were not restricted to Arabidopsis, because its expression in soybeans improved the RSA, growth, and seed yield of this crop and supported higher survival in response to drought. Our results indicate that in both Arabidopsis and soybeans, the constitutive expression of psNTP9 results in a more extensive RSA and improved survival in drought stress conditions.