Ascorbic acid (AA) protects plants against abiotic stress. Previous studies suggested that this antioxidant is also involved in the control of flowering. To decipher how AA influences flowering time, we studied the four AA-deficient Arabidopsis (Arabidopsis thaliana) mutants vtc1-1, vtc2-1, vtc3-1, and vtc4-1 when grown under short and long days. These mutants flowered and senesced before the wild type irrespective of the photoperiod, a response that cannot simply be attributed to slightly elevated oxidative stress in the mutants. Transcript profiling of various flowering pathway genes revealed a correlation of altered mRNA levels and flowering time. For example, circadian clock and photoperiodic pathway genes were significantly higher in the vtc mutants than in the wild type under both short and long days, a result that is consistent with the early-flowering phenotype of the mutants. In contrast, when the AA content was artificially increased, flowering was delayed, which correlated with lower mRNA levels of circadian clock and photoperiodic pathway genes compared with plants treated with water. Similar observations were made for the autonomous pathway. Genetic analyses demonstrated that various photoperiodic and autonomous pathway mutants are epistatic to the vtc1-1 mutant. In conclusion, our transcript and genetic analyses suggest that AA acts upstream of the photoperiodic and autonomous pathways.

The ascorbic acid (AA)-deficient Arabidopsis thaliana vtc1-1 mutant exhibits increased resistance to the virulent bacterial pathogen Pseudomonas syringae. This response correlates with heightened levels of salicylic acid (SA), which induces antimicrobial pathogenesis-related (PR) proteins. To determine if SA-mediated, enhanced disease resistance is a general phenomenon of AA deficiency, to elucidate the signal that stimulates SA synthesis, and to identify the biosynthetic pathway through which SA accumulates, we studied the four AA-deficient vtc1-1, vtc2-1, vtc3-1, and vtc4-1 mutants. We also studied double mutants defective in the AA-biosynthetic gene VTC1 and the SA signaling pathway genes PAD4, EDS5, and NPR1, respectively. All vtc mutants were more resistant to P. syringae than the wild type. With the exception of vtc4-1, this correlated with constitutively upregulated H(2)O(2), SA, and messenger RNA levels of PR genes. Double mutants exhibited decreased SA levels and enhanced susceptibility to P. syringae compared with the wild type, suggesting that vtc1-1 requires functional PAD4, EDS5, and NPR1 for SA biosynthesis and pathogen resistance. We suggest that AA deficiency causes constitutive priming through a buildup of H(2)O(2) that stimulates SA accumulation, conferring enhanced disease resistance in vtc1-1, vtc2-1, and vtc3-1, whereas vtc4-1 might be sensitized to H(2)O(2) and SA production after infection.

Summary Here we describe an in vitro primary culture system for C. elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions.

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