Effects of the overexpression of NADP-MALIC ENZYME in plastids of Arabidopsis thaliana in the context of a biotechnological approach to enhance CO2 assimilation

VERÓNICA MAURINO; MARIANA SAIGO; HOLGER FAHNENSTICH; MARÍA I. ZANOR; CARLOS ANDREO; ALISDAIR FERNI; ULF INGO FLÜGGE

Potsdam, Alemania

Simposio; 3rd International Symposium "Signals, Sensing and Plant Primary Metabolism" of the Collaborative Research Center SFB 429; 2006

Collaborative Research Center SFB 429

In order to release and re-fix CO2 directly within the chloroplast and thereby reduce the oxygenase activity of Rubisco, we started a biotechnological approach to introduce a complete glycolate catabolic cycle into the C3-plant Arabidopsis thaliana. For this purpose, three new activities are required inside the plastids. As a result of this cycle, one molecule of glycolate is converted by oxidation into two molecules of CO2. To analyse transformants expressing the whole pathway, single transformants were first characterized. In this report, analysis of plants expressing the maize C4 NADP-malic enzyme (NADP-ME; EC 1.1.1.40) driven by the CaMV 35S promoter is presented. NADP-ME catalyses the convertion of malate into pyruvate and CO2. Overexpressing lines (MEm) 2, 4 and 5, which accumulated NADP-ME activity 13 to 30-fold higher than the wild-type showed only one transgene insertion. In these lines, a 62 kDa protein was recognized by antibodies raised against maize C4 NADP-ME. The plastidic localization of the overexpressed enzyme was confirmed by immunoassays in leaf sections of all MEm plants. The transformants grow normally under greenhouse conditions but had a higher area/weight ratio. Chlorophyll b content was lower in the transformants, resulting in a rise of chlorophyll a/b ratio. CO2 assimilation rates of transgenic lines were lower than the wild-type at all PFDs tested. In contrast, the rate of photosynthetic electron transport resembled in all cases the wild-type, indicating the necessity of alternative electron acceptors. Enhanced photoinhibition was detected after 3 h under 700 mE only in line 5, which also showed a pale green leaf colour. PSII operating efficiency was lower and the NPQ values were significantly higher in all MEm lines. The NADP-MDH activation state was measured as an indirect indicator of the redox status of the chloroplasts. All transgenic lines contained GSH-GSSG levels lower than the wild-type, with unchanged GSH/GSSG ratios in relation to the wild-type. Interestingly, 3 h after high light treatment, the GSH-GSSG levels of the transgenic lines increased nearly three times with a higher increase in the reduced fraction. Similar but less pronounced effects were found for ascorbate. A metabolite profiling study by GC-MS was conducted on samples collected 3 h after the end of the dark period. In all transgenic lines, malate contents were drastically reduced (13-26% of the wild-type). The levels of fumarate, 2-oxoglutarate and citrate were also decreased. These observations suggest a reduction of the tricarboxylic acid cycle activity probably due to a depletion of malate caused by the high NADP-ME activity in the chloroplasts. Aspartate and threonine contents were also decreased whereas pyruvate and the three-carbon amino acids alanine, isoleucine and valine contents were increased. GC-MS also revealed low levels of glutamine but higher levels of glutamate and GABA. Contents of free sugars and sugar-P were decreased. Interestingly, dark-induced senescence of intact plants progressed more rapidly in the mutants compared to the wild type. While four week-old wild-type plants became pale green five days after the onset of darkness, MEm plants showed this characteristic already after two days with severe yellowing after three days in darkness.