Uric acid accumulation in an Arabidopsis urate oxidase mutant impairs seedling establishment by blocking peroxisome maintenance.

Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants.

The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.

The strawberry Fra a 1 allergen is a homolog of the major birch pollen allergen Bet v 1. It is synthesized by red ripe fruits of Fragaria x ananassa while white fruits of a mutant genotype, which is known to be tolerated by individuals affected by allergy, are devoid of it. Proteomic analyses have shown that Fra a 1 is down-regulated in the tolerated white-fruited genotype along with enzymes of the anthocyanin pigment pathway. In this study, we report the spatial and temporal expression of three Fra a genes that encode different isoforms, and the transient RNAi-mediated silencing of the Fra a genes in strawberry fruits of the red-fruited cultivar Elsanta with an ihpRNA construct. As a consequence of reduced levels of Fra a mRNAs, fruits were obtained that produced significantly decreased levels of anthocyanins and upstream metabolites. This effect is consistent with the parallel down-regulation of the phenylalanine ammonia lyase (FaPAL) and to a lesser extent of the chalcone synthase (FaCHS) transcript levels also found in these fruits. In naturally occurring white-fruited genotypes of F. chiloensis and F. vesca, Fra a transcript levels are higher than those of the red-fruited varieties, likely to compensate for the low expression levels of FaPAL and FaCHS in these mutant genotypes. The results demonstrate that Fra a expression is directly linked to flavonoid biosynthesis and show that the Fra a allergen has an essential biological function in pigment formation in strawberry fruit.

Identification, cloning and expression analysis of strawberry (Fragaria x ananassa) mitochondrial citrate synthase and mitochondrial malate dehydrogenase.

Salt-extractable proteins from the cell walls of immature and ripe strawberry (Fragaria x ananassa Duch. cv. Elsanta) fruit were separated using two-dimensional polyacrylamide gel electrophoresis. Seven polypeptides (enzymes) were characterized from their N-terminal sequences: (1) glyceraldhyde-3-phosphate dehydrogenase (EC 1.2.1.12); (2) triose phosphate isomerase (TPI; EC 5.3.1.1); (3) mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37); (4) NADH glutamate dehydrogenase (EC 1.4.1.3); (5) chalcone synthase (ChS; EC 2.3.1.74); (6) mitochondrial citrate synthase (mCS; EC 4.1.3.7); and (7) UDP glucose:flavonoid 3-O-glucosyltransferase (UDPG:FGT; EC 2.4.1.91). The sequenced polypeptides identified only cytosolic proteins, two of which (ChS and UDPG:FGT) had already been identified as being up-regulated in ripening (strawberry) fruit and important contributors to ripe fruit character. Our focus was therefore diverted to the enzymes mMDH and mCS for further molecular characterization as potentially important determinants of fruit flavour via regulation of the sugar : acid balance. Citrate synthase (CS) and malate dehydrogenase (MDH) enzyme activities increased substantially during ripening, as did citrate and malate contents. The increase in CS activity is supported by western blot analysis. One strawberry mCS (Fa-mCS-I) and two mMDH (Fa-mMDH-I and -II) cDNAs were cloned that were 77, 82 and 53% identical (respectively) to sequences from other plant sources. Northern analysis showed that CS and MDH expression did not correlate with enzyme activities and these findings are discussed.

High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata.

A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.