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Fumarylacetoacetate hydrolase (FAH) catalyses the final step of the tyrosine degradation pathway, which is essential to animals but was of unknown importance in plants until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short-day conditions. The sscd1 mutant accumulates succinylacetone (SUAC), an abnormal metabolite caused by loss of FAH. Succinylacetone is an inhibitor of δ-aminolevulinic acid (ALA) dehydratase (ALAD), which is involved in chlorophyll (Chl) biosynthesis. In this study, we investigated whether sscd1 cell death is mediated by Chl biosynthesis and found that ALAD activity is repressed in sscd1 and that protochlorophyllide (Pchlide), an intermediate of Chl biosynthesis, accumulates at lower levels in etiolated sscd1 seedlings. However, it was interesting that Pchlide in sscd1 might increase after transfer from light to dark and that HEMA1 and CHLH are upregulated in the light-dark transition before Pchlide levels increased. Upon re-illumination after Pchlide levels had increased, reactive oxygen species marker genes, including singlet oxygen-induced genes, are upregulated, and the sscd1 cell death phenotype appears. In addition, Arabidopsis WT seedlings treated with SUAC mimic sscd1 in decline of ALAD activity and accumulation of Pchlide as well as cell death. These results demonstrate that increase in Pchlide causes cell death in sscd1 upon re-illumination and suggest that a decline in the Pchlide pool due to inhibition of ALAD activity by SUAC impairs the repression of ALA synthesis from the light-dark transition by feedback control, resulting in activation of the Chl biosynthesis pathway and accumulation of Pchlide in the dark.

Anthocyanins are widely found in plants and are responsible for the purple coloration of plants. Anthocyanin biosynthesis is induced by environmental stresses, plant hormones, sugar, and so on. Tyrosine (Tyr) is the precursor of melanin that exits in both animals and plants. However, until now it has been unknown whether Tyr induces anthocyanin biosynthesis. In this study, the seedlings of Arabidopsis thaliana were treated with exogenous Tyr and then the anthocyanin accumulation was determined. The results showed that Tyr induced anthocyanin accumulation in Arabidopsis thaliana in a dose-dependent manner. Furthermore, the expression of the late anthocyanin biosynthetic genes including DFR, LDOX, and UF3GT, and the transcription factor genes PAP1, PAP2, and EGL3 was induced by Tyr. Taken together, these results demonstrated that Tyr is able to induce anthocyanin accumulation and suggested that Tyr up-regulates transcription factors PAP1, PAP2, and EGL3, which mediates the expression of the late anthocyanin biosynthetic genes and then induces anthocyanin biosynthesis.

Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

Succinylacetone was known to be a toxic metabolite of tyrosine in human and animals caused by blockage of the final step in tyrosine degradation pathway, but its existence in plant was unclear though the metabolic disturbance of tyrosine was also found in plant. A GC-MS method for determination of succinylacetone in Arabidopsis thaliana was developed for the first time. Both oximation and silylation were applied in the derivation procedure, and a low-temperature condition before completion of oximation was found to be necessary to obtain good linearity of the calibration curve due to the thermolability of succinylacetone. The specific chromatogram pattern formed by the four isomers of succinylacetone derivatives provided a helpful feature for its identification. The detection limit of the proposed method was 0.25 ppm in A. thaliana. The recoveries were between 95.4 and 109.3 % with the coefficient of variation ranging from 4.36 to 7.81 % for intra-day assays and 6.47 to 8.52 % for inter-day assays. Application to wild-type and the short-day sensitive cell death 1 mutant of A. thaliana represented an obvious correlation between the measured amount of succinylacetone and wilting symptom, suggesting the proposed method could be a powerful tool in further study on toxicology of succinylacetone and tyrosine catabolism in plants.

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.