PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5#-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5#-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B 6 pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5#-phosphate than the wild type, reflected in an increase in total vitamin B 6 observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B 6 de novo pathway enzyme PDX1, correlating with increased total B 6 levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B 6 observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.
Morphological characteristics and vegetative compatibility groups (VCGs) of 486 isolates of Glomerella cingulata, Colletotrichum gloeosporioides, and C. acutatum collected from apple leaves with Glomerella leaf spot (GLS) symptoms and fruit with bitter rot symptoms in the United States and Brazil were studied. From this collection, 155 isolates of G. cingulata (93 from fruit, 61 from leaves, and 1 from buds), 42 isolates of C. gloeosporioides from fruit, and 14 isolates of C. acutatum (10 from fruit and 4 from leaves) were studied using mitochondrial (mt)DNA restriction fragment length polymorphism (RFLP) haplotypes. A subset of 24 isolates was studied by examining the sequence of a 200-bp intron of the glyceraldehyde 3-phosphate dehydrogenase (GDPH) nuclear gene. In addition, 98 isolates were tested for pathogenicity on leaves of cvs. Gala and Golden Delicious in the greenhouse, and 24 isolates were tested for pathogenicity on fruit of cv. Gala in growth chambers. In total, 238 and 225 isolates of G. cingulata were separated into four distinct morphological types and six VCGs, respectively. Five morphological types and six VCGs were identified among 74 and 36 isolates of C. gloeosporioides, respectively. Three morphological types and four VCGs were identified among 74 and 23 isolates of C. acutatum, respectively. Seven different mtDNA RFLP haplotypes were observed within isolates of G. cingulata, two within isolates of C. gloeosporioides, and two within isolates of C. acutatum. Phylogenetic trees, inferred based on maximum likelihood and maximum parsimony methods using the intron sequence, produced similar topologies. Each species was separated into distinct groups. All isolates tested were pathogenic on fruit, though only isolates with specific VCGs and haplotypes were pathogenic to leaves. Vegetative compatibility was a better tool than molecular characters for distinguishing isolates of G. cingulata pathogenic on both leaves and fruit from the ones pathogenic only on fruit. Isolates of G. cingulata capable of causing both GLS and bitter rot were included in haplotypes and groups based on the sequence analysis of the 200-bp intron that also included isolates capable of causing bitter rot only. Additionally, isolates of G. cingulata from the United States and Brazil which cause GLS were included in different haplotypes and sequence analysis groups. Therefore, one hypothesis is that isolates of G. cingulata from the United States capable of causing both GLS on foliage and bitter rot on fruit may have arisen independently of Brazilian isolates of G. cingulata capable of causing both GLS and bitter rot, and the two groups of isolates may represent distinct populations.
Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.
In August 1998, severe leaf spot, resulting in extensive defoliation, was observed on cv. Gala apple trees in two orchards in eastern Tennessee. Symptoms were similar to those reported in Brazil for Glomerella leaf spot on Gala (1), and Glomerella cingulata (Stoneman) Spauld. & H. Schrenk was observed fruiting in lesions. Single-ascospore isolates were obtained by placing individual perithecia from different lesions on microscope slides in a drop of sterilized distilled water. Perithecia were crushed, and the spore and mycelial suspension was distributed on the surface of petri dishes containing acid-water agar (AWA). Petri dishes were incubated at 24°C in light for 24 h. Germinated ascospores were transferred to petri dishes containing AWA. Cultures were transferred to potato dextrose agar and grown at 24°C in light for 14 days to induce sporulation. Four isolates (TN-1A, TN-1B, TN-2A, and TN-2B) were selected for pathogenicity tests. Three virulent isolates from Brazil (FK6, R-11, and 2VGE) also were included. Trees of apple cvs. Gala and Golden Delicious were placed in humidity chambers before inoculation. After 24 h, shoots on three trees of each cultivar were sprayed with an aqueous spore suspension of each isolate (1× 105 spores per ml) and maintained at 100% relative humidity (RH) and ≈22°C for 2 days. Shoots on three trees sprayed with sterilized distilled water and maintained at 100% RH served as a control. All isolates from Brazil and isolates TN-1A and TN-1B caused symptoms characteristic of Glomerella leaf spot on both cultivars after 2 days. Five days after inoculation disease severity on each leaf was visually rated on a scale of 0 to 5 (where 0 = no lesions and 5 = 25 to 50% of leaf surface covered with lesions). FK6 and R-11 were the most aggressive isolates on both cultivars. 2VGE and TN-1B were the least aggressive isolates. Reference: (1) T. B. Sutton and R.M. Sanhueza. Plant Dis. 82:267, 1998.
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