Transcriptional reprogramming of nucleotide metabolism in response to altered pyrimidine availability in Arabidopsis seedlings.

In Arabidopsis seedlings, inhibition of aspartate transcarbamoylase (ATC) and de novo pyrimidine synthesis resulted in pyrimidine starvation and developmental arrest a few days after germination. Synthesis of pyrimidine nucleotides by salvaging of exogenous uridine (Urd) restored normal seedling growth and development. We used this experimental system and transcriptional profiling to investigate genome-wide responses to changes in pyrimidine availability. Gene expression changes at different times after Urd supplementation of pyrimidine-starved seedlings were mapped to major pathways of nucleotide metabolism, in order to better understand potential coordination of pathway activities, at the level of transcription. Repression of de novo synthesis genes and induction of intracellular and extracellular salvaging genes were early and sustained responses to pyrimidine limitation. Since de novo synthesis is energetically more costly than salvaging, this may reflect a reduced energy status of the seedlings, as has been shown in recent studies for seedlings growing under pyrimidine limitation. The unexpected induction of pyrimidine catabolism genes under pyrimidine starvation may result from induction of nucleoside hydrolase NSH1 and repression of genes in the plastid salvaging pathway, diverting uracil (Ura) to catabolism. Identification of pyrimidine-responsive transcription factors with enriched binding sites in highly coexpressed genes of nucleotide metabolism and modeling of potential transcription regulatory networks provided new insights into possible transcriptional control of key enzymes and transporters that regulate nucleotide homeostasis in plants.

Constitutive expression of a pea apyrase, psNTP9, increases seed yield in field-grown soybean.

To address the demand for food by a rapidly growing human population, agricultural scientists have carried out both plant breeding and genetic engineering research. Previously, we reported that the constitutive expression of a pea apyrase (Nucleoside triphosphate, diphosphohydrolase) gene, psNTP9, under the control of the CaMV35S promoter, resulted in soybean plants with an expanded root system architecture, enhanced drought resistance and increased seed yield when they are grown in greenhouses under controlled conditions. Here, we report that psNTP9-expressing soybean lines also show significantly enhanced seed yields when grown in multiple different field conditions at multiple field sites, including when the gene is introgressed into elite germplasm. The transgenic lines have higher leaf chlorophyll and soluble protein contents and decreased stomatal density and cuticle permeability, traits that increase water use efficiency and likely contribute to the increased seed yields of field-grown plants. These altered properties are explained, in part, by genome-wide gene expression changes induced by the transgene.

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

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.

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 beta-alanine, CO(2) and NH(3) through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and beta-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-(14)C]-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-(14)C]-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.

Expression and functional analysis of aspartate transcarbamoylase and role of de novo pyrimidine synthesis in regulation of growth and development in Arabidopsis.

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) catalyzes the committed step in the de novo synthesis of uridine-5'-monophosphate (UMP), from which all other pyrimidine nucleotides are made. In Arabidopsis, ATCase is encoded by a single PYRB gene, whose expression was regulated by tissue pyrimidine availability. RT-PCR and PYRB:GUS expression profiles showed markedly increased expression of PYRB in root tissues during the first 5days after germination, as seed pyrimidine reserves were exhausted and de novo synthesis was required to support new growth. Growth of seedlings in the presence of the ATCase inhibitor N-(phosphonacetyl)-l-aspartate (PALA) resulted in complete developmental arrest at the day 5 stage, which was reversible upon addition of exogenous uracil. Arabidopsis RNAi lines exhibiting 70-95% reductions in PYRB transcript and ATCase protein levels had delayed growth and development, produced smaller plants with reduced root to shoot biomass ratios, few flowers, and siliques that produced smaller seeds with greatly reduced viability, compared with wild type plants. The severity of the phenotype was correlated with the extent of PYRB silencing and was reversible by pyrimidine addition. These results suggest that de novo synthesis is required, although minimal activities, supplemented by efficient salvaging pathway activities, are able to meet metabolic demands for pyrimdines during growth and development. Coordinate changes in expression of salvage and catabolic pathway genes in RNAi plants indicate that pyrimidine metabolism responds dynamically to changes in tissue pyrimidine availability.

Genes, enzymes and regulation of arginine biosynthesis in plants.

Arabidopsis genes encoding enzymes for each of the eight steps in L-arginine (Arg) synthesis were identified, based upon sequence homologies with orthologs from other organisms. Except for N-acetylglutamate synthase (NAGS; EC 2.3.1.1), which is encoded by two genes, all remaining enzymes are encoded by single genes. Targeting predictions for these enzymes, based upon their deduced sequences, and subcellular fractionation studies, suggest that most enzymes of Arg synthesis reside within the plastid. Synthesis of the L-ornthine (Orn) intermediate in this pathway from L-glutamate occurs as a series of acetylated intermediates, as in most other organisms. An N-acetylornithine:glutamate acetyltransferase (NAOGAcT; EC 2.3.1.35) facilitates recycling of the acetyl moiety during Orn formation (cyclic pathway). A putative N-acetylornithine deacetylase (NAOD; EC 3.5.1.16), which participates in the "linear" pathway for Orn synthesis in some organisms, was also identified. Previous biochemical studies have indicated that allosteric regulation of the first and, especially, the second steps in Orn synthesis (NAGS; N-acetylglutamate kinase (NAGK), EC 2.7.2.8) by the Arg end-product are the major sites of metabolic control of the pathway in organisms using the cyclic pathway. Gene expression profiling for pathway enzymes further suggests that NAGS, NAGK, NAOGAcT and NAOD are coordinately regulated in response to changes in Arg demand during plant growth and development. Synthesis of Arg from Orn is further coordinated with pyrimidine nucleotide synthesis, at the level of allocation of the common carbamoyl-P intermediate.

Effects of phosphate limitation on expression of genes involved in pyrimidine synthesis and salvaging in Arabidopsis.

Arabidopsis seedlings grown for 14 d without phosphate (P) exhibited stunted growth and other visible symptoms associated with P deficiency. RNA contents in shoots decreased nearly 90%, relative to controls. In shoots, expression of Pht1;2, encoding an inducible high-affinity phosphate transporter, increased threefold, compared with controls, and served as a molecular marker for P limitation. Transcript levels for five enzymes (aspartate transcarbamoylase, ATCase, EC 2.1.3.2; carbamoyl phosphate synthetase, CPSase, EC 6.3.5.5); UMP synthase, EC 2.4.1.10, EC 4.1.1.23; uracil phosphoribosyltransferase, UPRTase, EC 2.4.2.9; UMP kinase, EC 2.7.1.14) increased 2-10-fold in response to P starvation in shoots. These enzymes, which utilize phosphorylated intermediates at putative regulated steps in de novo synthesis and salvaging pathways leading to UMP and pyrimidine nucleotide formation, appear to be coordinately regulated, at the level of gene expression. This response may facilitate pyrimidine nucleotide synthesis under P limitation in this plant. Expression of P-dependent and P-independent phosphoribosyl pyrophosphate (PRPP) synthases (PRS2 and PRS3, respectively) which provide PRPP, the phosphoribosyl donor in UMP synthesis via both de novo and salvaging pathways, was differentially regulated in response to P limitation. PRS2 mRNA levels increased twofold in roots and shoots of P-starved plants, while PRS3 was constitutively-expressed. PRS3 may play a novel role in providing PRPP to cellular metabolism under low P availability.

UPS1 and UPS2 from Arabidopsis mediate high affinity transport of uracil and 5-fluorouracil.

Salvage pathways play an important role in providing nucleobases to cells, which are unable to synthesize sufficient amounts for their needs. Cellular uptake systems for pyrimidines have been described, but in higher eukaryotes, transporters for thymine and uracil have not been identified. Two plant transporters, AtUPS1 and PvUPS1, were recently identified as transporters for allantoin in Arabidopsis and French bean, respectively. However, Arabidopsis, in contrast to tropical legumes, uses mainly amino acids for long distance transport. Allantoin transport has not been described in the Brassicaceae. Thus, the physiological substrates of ureide permease (UPS) transporters in Arabidopsis may be compounds structurally related to allantoin. A detailed analysis of the substrate specificities of two members of the AtUPS family shows that AtUPS1 and AtUPS2 mediate high affinity uracil and 5-fluorouracil (a toxic uracil analogue) transport when expressed in yeast and Xenopus oocytes. Consistent with a function during germination and early seedling development, AtUPS1 expression is transiently induced during the early stages of seedling development followed by up-regulation of AtUPS2 expression. Arabidopsis ups2 insertion mutants and ups1 lines, in which transcript levels were reduced by post-transcriptional gene silencing, are more tolerant to 5-fluorouracil as compared with wild type plants. The results suggest that in Arabidopsis UPS transporters are the main transporters for uracil and potentially other nucleobases, whereas during evolution legumes may have taken advantage of the low selectivity of UPS proteins for long distance transport of allantoin.