Alteration of organic acid metabolism in Arabidopsis overexpressing the maize C4NADP-malic enzyme causes accelerated senescence during extended darkness

The full- length cDNA encoding the maize ( Zea mays) C 4 NADP- malic enzyme was expressed in Arabidopsis ( Arabidopsis thaliana) under the control of the cauliflower mosaic virus 35S promoter. Homozygous transgenic plants ( MEm) were isolated with activities ranging from 6- to 33- fold of those found in the wild type. The transformants did not show any differences in morphology and development when grown in long days; however, dark- induced senescence progressed more rapidly in MEm plants compared to the wild type. Interestingly, senescence could be retarded in the transgenic lines by exogenously supplying glucose, sucrose, or malate, suggesting that the lack of a readily mobilized carbon source is likely to be the initial factor leading to the premature induction of senescence in MEm plants. A comprehensive metabolic profiling on whole rosettes allowed determination of approximately 80 metabolites during a diurnal cycle as well as following dark- induced senescence and during metabolic complementation assays. MEm plants showed no differences in the accumulation and degradation of carbohydrates with respect to the wild type in all conditions tested, but accumulated lower levels of intermediates used as respiratory substrates, prominently malate and fumarate. The data indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark- induced senescence encountered in MEm plants. Thus, in prolonged darkness these metabolites are consumed faster than in the wild type and, as a consequence, MEm plants enter irreversible senescence more rapidly. In addition, the data revealed that both malate and fumarate are important forms of fixed carbon that can be rapidly metabolized under stress conditions in Arabidopsis.