NADH-GOGAT Overexpression Does Not Improve Maize (Zea mays L.) Performance Even When Pyramiding with NAD-IDH, GDH and GS.

Maize plants overexpressing NADH-GOGAT were produced in order to determine if boosting 2-Oxoglurate production used as a carbon skeleton for the biosynthesis of amino acids will improve plant biomass and kernel production. The NADH-GOGAT enzyme recycles glutamate and incorporates carbon skeletons into the ammonium assimilation pathway using the organic acid 2-Oxoglutarate as a substrate. Gene pyramiding was then conducted with NAD-IDH and NADH-GDH, two enzymes also involved in the synthesis of 2-Oxoglurate. NADH-GOGAT overexpression was detrimental for shoot biomass production but did not markedly affect kernel yield. Additional NAD-IDH and NADH-GDH activity did not improve plant performance. A decrease in kernel production was observed when NADH-GDH was pyramided to NADH-GOGAT and NAD-IDH. This decrease could not be restored even when additional cytosolic GS activity was present in the plants overexpressing the three enzymes producing 2-Oxoglutarate. Detailed leaf metabolic profiling of the different transgenic plants revealed that the NADH-GOGAT over-expressors were characterized by an accumulation of amino acids derived from glutamate and a decrease in the amount of carbohydrates further used to provide carbon skeletons for its synthesis. The study suggests that 2-Oxoglutarate synthesis is a key element acting at the interface of carbohydrate and amino acid metabolism and that its accumulation induces an imbalance of primary carbon and nitrogen metabolism that is detrimental for maize productivity.

Revisiting the Role of Ethylene and N-End Rule Pathway on Chilling-Induced Dormancy Release in Arabidopsis Seeds.

Dormant Arabidopsis (Arabidopsis thaliana) seeds do not germinate easily at temperatures higher than 10⁻15 °C. Using mutants affected in ethylene signaling (etr1, ein2 and ein4) and in the N-end-rule pathway of the proteolysis (prt6 and ate1-ate2) we have investigated the effects of cold and ethylene on dormancy alleviation. Ethylene (10⁻100 ppm) and 2⁻4 days chilling (4 °C) strongly stimulate the germination of wild type (Col-0) seeds at 25 °C. Two to four days of chilling promote the germination at 25 °C of all the mutants suggesting that release of dormancy by cold did not require ethylene and did not require the N-end-rule pathway. One mutant (etr1) that did not respond to ethylene did not respond to GA3 either. Mutants affected in the N-end rule (prt6 and ate1-ate2) did not respond to ethylene indicating that also this pathway is required for dormancy alleviation by ethylene; they germinated after chilling and in the presence of GA3. Cold can activate the ethylene signaling pathway since it induced an accumulation of ETR1, EINI4, and EIN2 transcripts, the expression of which was not affected by ethylene and GA3. Both cold followed by 10 h at 25 °C and ethylene downregulated the expression of PRT6, ATE1, ATE2, and of ABI5 involved in ABA signaling as compared to dormant seeds incubated at 25 °C. In opposite, the expression of RGA, GAI, and RGL2 encoding three DELLAs was induced at 4 °C but downregulated in the presence of ethylene.

Exploiting the Genetic Diversity of Maize Using a Combined Metabolomic, Enzyme Activity Profiling, and Metabolic Modeling Approach to Link Leaf Physiology to Kernel Yield.

A combined metabolomic, biochemical, fluxomic, and metabolic modeling approach was developed using 19 genetically distant maize (Zea mays) lines from Europe and America. Considerable differences were detected between the lines when leaf metabolic profiles and activities of the main enzymes involved in primary metabolism were compared. During grain filling, the leaf metabolic composition appeared to be a reliable marker, allowing a classification matching the genetic diversity of the lines. During the same period, there was a significant correlation between the genetic distance of the lines and the activities of enzymes involved in carbon metabolism, notably glycolysis. Although large differences were observed in terms of leaf metabolic fluxes, these variations were not tightly linked to the genome structure of the lines. Both correlation studies and metabolic network analyses allowed the description of a maize ideotype with a high grain yield potential. Such an ideotype is characterized by low accumulation of soluble amino acids and carbohydrates in the leaves and high activity of enzymes involved in the C4 photosynthetic pathway and in the biosynthesis of amino acids derived from glutamate. Chlorogenates appear to be important markers that can be used to select for maize lines that produce larger kernels.

Genetic variability of the phloem sap metabolite content of maize (Zea mays L.) during the kernel-filling period.

Using a metabolomic approach, we have quantified the metabolite composition of the phloem sap exudate of seventeen European and American lines of maize that had been previously classified into five main groups on the basis of molecular marker polymorphisms. In addition to sucrose, glutamate and aspartate, which are abundant in the phloem sap of many plant species, large quantities of aconitate and alanine were also found in the phloem sap exudates of maize. Genetic variability of the phloem sap composition was observed in the different maize lines, although there was no obvious relationship between the phloem sap composition and the five previously classified groups. However, following hierarchical clustering analysis there was a clear relationship between two of the subclusters of lines defined on the basis of the composition of the phloem sap exudate and the earliness of silking date. A comparison between the metabolite contents of the ear leaves and the phloem sap exudates of each genotype, revealed that the relative content of most of the carbon- and nitrogen-containing metabolites was similar. Correlation studies performed between the metabolite content of the phloem sap exudates and yield-related traits also revealed that for some carbohydrates such as arabitol and sucrose there was a negative or positive correlation with kernel yield and kernel weight respectively. A posititive correlation was also found between kernel number and soluble histidine.