The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1

Abstract

The ozone-sensitive Arabidopsis mutant vitamin c-1 (vtc1) is deficient in l-ascorbic acid (AsA) due to a mutation in GDP-Man pyrophosphorylase (Conklin et al., 1999), an enzyme involved in the AsA biosynthetic pathway (Smirnoff et al., 2001). In this study, the physiology of this AsA deficiency was initially investigated in response to biotic (virulent pathogens) stress and subsequently with regards to the onset of senescence. Infection with either virulent Pseudomonas syringae or Peronospora parasitica resulted in largely reduced bacterial and hyphal growth in the vtc1 mutant in comparison to the wild type. When vitamin c-2 (vtc2), another AsA-deficient mutant, was challenged with P. parasitica, growth of the fungus was also reduced, indicating that the two AsA-deficient mutants are more resistant to these pathogens. Induction of pathogenesis-related proteins PR-1 and PR-5 is significantly higher in vtc1 than in the wild type when challenged with virulent P. syringae. In addition, the vtc1 mutant exhibits elevated levels of some senescence-associated gene (SAG) transcripts as well as heightened salicylic acid levels. Presumably, therefore, low AsA is causing vtc1 to enter at least some stage(s) of senescence prematurely with an accompanying increase in salicylic acid levels that results in a faster induction of defense responses.

Figure 1.

Responses of the wild type and vtc1 upon infection with virulent P. syringae pv maculicola ES4326. Leaves of 5-week-old plants were inoculated with a bacterial suspension containing 10⁵ CFU (colony forming units) mL⁻¹. A, Growth curve of ES4326 in leaves of the wild type and vtc1. Mean values ± sd of three to four different plants are depicted. Similar results were obtained in four additional independent experiments. B, Western-blot analysis of wild-type and vtc1 leaves after infiltration with ES4326 was performed to demonstrate the induction of PR-1 and PR-5. Ten micrograms of total protein were loaded. To show equal loading, the blot was stained with Ponceau red to visualize the large subunit of Rubisco (RBCL). Western-blot analyses were performed two to three times with similar results. Numbers below western blots of vtc1 indicate mean fold increase of PR-1 and PR-5 protein expression, respectively, in the mutant compared to the wild type. C, Endogenous levels of free SA and SA-glucoside (the conjugated form) in 5-week-old leaves of the wild type and vtc1 not infected with ES4326 and 24 h postinoculation. Data represent means ± sd of three independent samples each. Similar results were observed in two additional experiments.

Figure 2.

Conidiophore production in wild-type, vtc1, and vtc2 plants inoculated with P. parasitica pv Noco. A, Pronounced conidiophore production (arrow) and massive hyphae spread (arrowhead) in the wild type. B, In vtc1 and C, in vtc2, conidiophore production and hyphal development were much lower than in the wild type after infection with Noco. See Table I for quantitative analyses of conidiophore production in the wild type, vtc1, and vtc2. Several leaves of individual plants (4 weeks old) were sprayed with conidiophores of Noco and analyzed 7 d after inoculation as described in “Materials and Methods.” Scale bar = 100 μm.

Figure 3.

AsA deficiency causes premature senescence in vtc1. A, Semiquantitative RT-PCR was performed on 1 μg of total RNA isolated from 5-week-old plants of the wild type and vtc1 used for bacterial infection with P. syringae ES4326. Transcription levels of cDNA fragments amplified from SAG are depicted for three independent replicates of the wild type and vtc1. B, Phenotype of wild-type and vtc1 leaf discs after dark-induced senescence. Leaf discs of 5-week-old plants were floated on water and kept in the dark for 6 d.

Figure 4.

A model describing the relationship between AsA deficiency, pathogen resistance, and premature senescence in vtc1. AsA deficiency causes the induction of signaling molecules, such as ABA, SA, and presumably other signaling factors such as JA, as indicated by the upright solid arrows. We provide evidence that elevated levels of SA result in the induction of defense responses and, therefore, in pathogen resistance of vtc1. ABA is presumably also involved in the induction of defense responses. We propose that ABA, SA, and/or other regulators cause an up-regulation of SAGs, promoting senescence and possibly contributing to pathogen resistance of vtc1.