Comparative studies on Ureide Permeases in Arabidopsis thaliana and analysis of two alternative splice variants of AtUPS5

The recovery of free purine and pyrimidine bases and their degradation products represent alternative pathways in plant cells either to synthesize nucleotides (salvage pathways) by low energy consumption or to reuse organic nitrogen. Such recycling of metabolites often requires their uptake into the cell by specialized transport systems residing in the plasma membrane. In plants, it has been suggested that several protein families are involved in this process, but only a few transporters have so far been characterized. In this work, gene expression, substrate specificities, and transport mechanisms of members of the Ureide Permease family in Arabidopsis (AtUPS) were analyzed and compared. Promoter-GUS studies indicated that the members of the family have distinct and partially overlapping expression patterns with regard to developmental stages or tissue specific localization. In addition, two alternative splice variants of AtUPS5, a novel member of the transporter family, were identified and investigated. The abundance of both alternative mRNAs varied in different organs, while the relative amounts were comparable. AtUPS5l (longer isoform) shares similar structural prediction with AtUPS1 and AtUPS2. In contrast, AtUPS5s (shorter isoform) lacks two transmembrane domains as structural consequence of the additional splice event. When expressed in yeast, AtUPS5l mediates cellular import of cyclic purine degradation products and pyrimidines similarly to AtUPS1 and AtUPS2, but differences in transport efficiencies were observed. AtUPS5s, however, could not be shown to mediate uptake of these compounds into yeast cells and might therefore be defective or have a different function.     

Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed improved steady state amounts of untagged and tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent to heterologous expression of membrane proteins in yeast, including ER retention.     

Functional characterization of allantoinase genes from Arabidopsis and a nonureide-type legume black locust

The availability of nitrogen is a limiting factor for plant growth in most soils. Allantoin and its degradation derivatives are a group of soil heterocyclic nitrogen compounds that play an essential role in the assimilation, metabolism, transport, and storage of nitrogen in plants. Allantoinase is a key enzyme for biogenesis and degradation of these ureide compounds. Here, we describe the isolation of two functional allantoinase genes, AtALN (Arabidopsis allantoinase) and RpALN (Robinia pseudoacacia allantoinase), from Arabidopsis and black locust (Robinia pseudoacacia). The proteins encoded by those genes were predicted to have a signal peptide for the secretory pathway, which is consistent with earlier biochemical work that localized allantoinase activity to microbodies and endoplasmic reticulum (Hanks et al., 1981). Their functions were confirmed by genetic complementation of a yeast mutant (dal1) deficient in allantoin hydrolysis. The absence of nitrogen in the medium increased the expression of the genes. In Arabidopsis, the addition of allantoin to the medium as a sole source of nitrogen resulted in the up-regulation of the AtALN gene. The black locust gene (RpALN) was differentially regulated in cotyledons, axis, and hypocotyls during seed germination and seedling growth, but was not expressed in root tissues. In the trunk wood of a mature black locust tree, the RpALN gene was highly expressed in the bark/cambial region, but had no detectable expression in the sapwood or sapwood-heartwood transition zone. In addition, the gene expression in the bark/cambial region was up-regulated in spring and fall when compared with summer, suggesting its involvement in nitrogen mobilization.     

Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean

Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO(2), and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 microm manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of L-asparagine and L-aspartate but not D-asparagine. L-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum.     

PvUPS1, an allantoin transporter in nodulated roots of French bean

Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system. In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen is used for synthesis of the ureides allantoin and allantoic acid, the major long-distance transport forms of organic nitrogen in these species. The purpose of this investigation was to identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots. A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid. In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats. In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin. In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature. These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.     

Novel nitrogen sources for growth of Bacillus fastidiosus and their effect on the activity of NADP-dependent glutamate dehydrogenase

Abstract Although Bacillus fastidiosus assimilates ammonium formed internally during growth on urate, allantoin or allantoate via NADP-dependent glutamate dehydrogenase (NADP-GDH), growth on exogenous ammonium as nitrogen source has not been observed. Growth on ammonium, urea and ureidoglycolate, intermediates of the urate degradative pathway, was found to occur if the mineral growth medium containing glycerol as a carbon source was supplemented with both allantoin (0.5 mM) and brain heart infusion (BHI, 0.1%, w/v) or yeast extract. Neither allantoin nor BHI supported growth alone or in combination unless ammonium was present. NADP-GDH activity appeared to be regulated only by the extracellular concentration of allantoin or allantoate. Enzyme activity was not influenced by other nitrogen sources or the intracellular ammonium concentration.     

The effect of thiourea on ureide metabolism in Neurospora crassa

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.     

The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.     

Induction and inhibition of the allantoin permease in Saccharomyces cerevisiae.

Allantoin uptake in Saccharomyces cerevisiae is mediated by an energy-dependent, low-Km, active transport system. However, there is at present little information concerning its regulation. In view of this, we investigated the control of alloantoin transport and found that it was regulated quite differently from the other pathway components. Preincubation of appropriate mutant cultures with purified allantoate (commercial preparations contain 17% allantoin), urea, or oxalurate did not significantly increase allantoin uptake. Preincubation with allantoin, however, resulted in a 10- to 15-fold increase in the rate of allantoin accumulation. Two allantoin analogs were also found to elicit dramatic increases in allantoin uptake. Hydantoin and hydantoin acetic acid were able to induce allantoin transport to 63 and 95% of the levels observed with allantoin. Neither of these compounds was able to serve as a sole nitrogen source for S. cerevisiae, and they may be non-metabolizable inducers of the allantoin permease. The rna1 gene product appeared to be required for allantoin permease induction, suggesting that control was exerted at the level of gene expression. In addition, we have shown that allantoin uptake is not unidirectional; efflux merely occurs at a very low rate. Allantoin uptake is also transinhibited by addition of certain amino acids to the culture medium, and several models concerning the operation of such inhibition were discussed.     

Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.     

Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants.

Acquisition as well as translocation of phosphate are essential processes for plant growth. In many plants, phosphate uptake by roots and distribution within the plant are presumed to occur via a phosphate/proton cotransport mechanism. Here, we describe the isolation of two cDNAs, StPT1 and StPT2, from potato (Solanum tuberosum) that show homology to the phosphate/proton cotransporter PHO84 from the yeast Saccharomyces cerevisiae. The predicted products of both cDNAs share 35% identity with the PHO84 sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass was calculated to be 59 kD for the StPT1 protein and 58 kD for the StPT2 protein. When expressed in a PHO84-deficient yeast strain, MB192, both cDNAs complemented the mutant. Uptake of radioactive orthophosphate by the yeast mutant expressing either StPT1 or StPT2 was dependent on pH and reduced in the presence of uncouplers of oxidative phosphorylation, such as 2,4-dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. The K(m) for Pi uptake of the StPT1 and StPT2 proteins was determined to be 280 and 130 microM, respectively. StPT1 is expressed in roots, tubers, and source leaves as well as in floral organs. Deprivation of nitrogen, phosphorus, potassium, and sulfur changed spatial expression as well as the expression level of StPT1. StPT2 expression was detected mainly in root organs when plants were deprived of Pi and to a lesser extent under sulfur deprivation conditions. No expression was found under optimized nutrition conditions or when other macronutrients were lacking.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

An Oligopeptide Transporter Gene Family in Arabidopsis

We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis. These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p), Schizosaccharomyces pombe (Isp4p), and Saccharomyces cerevisiae (Opt1p and Opt2p). Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs. Two highly conserved motifs (NPG and KIPPR) were found among all OPT members. The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles. This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern. The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter. AtOPT4 expressed in S. cerevisiae mediated the uptake of KLG-[3H]L. Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth. This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants.     

Urease Is Not Essential for Ureide Degradation in Soybean

The hypothesis that soybean (Glycine max L. [Merrill]) catabolizes ureides to urea to a physiologically significant extent was tested and rejected. Urease-negative (eu3-e1/eu3-e1) plants were supported by fixed N2 or by 2 mM NH4NO3, so that xylem-borne nitrogen contained predominantly ureides (allantoin and allantoic acid) or amide amino acids, respectively. Seed nitrogen yield was equal on either nitrogen regime, although 35-d-old fixing plants accumulated about 6 times more leaf urea. In callus, lack of an active urease reduced growth on either arginine or allantoin as the sole nitrogen source, but the reduction was greater on arginine (73%) than on allantoin (39%). Furthermore, urease-negative cells accumulated 17 times more urea than urease-positive cells on arginine; for allantoin the ratio was 1.8. Urease-negative callus accumulated urea at 3% the rate of seedlings. To test whether urea accumulating in urease-negative seedlings was derived from ureides, seeds were first allowed to imbibe in 1 mM allopurinol, an inhibitor of ureide formation. Seedling ureides were decreased by 90%, but urea levels were unchanged. Thus, ureides are poor precursors of urea, which was confirmed in seedlings that converted no more than 5% of seed-absorbed [14C-ureido]allantoate to [14C]urea, whereas 40 to 70% of [14C-guanido]arginine was recovered as [14C]urea.     

Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana

A general amino acid permease cDNA ( AAP2 ) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake. Direct transport measurements in yeast show that the protein mediates uptake of l -[¹⁴C]-citrulline and l -[¹⁴C]-proline. Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e. glutamine, glutamate and asparagine. Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins. Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids. Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen. AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling. Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings.