To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

These authors contributed equally to this work. SUMMARYPlants encounter environmental stress challenges that are distinct from those of other eukaryotes because of their relative immobility. Therefore, plants may have evolved distinct regulatory mechanisms for conserved cellular functions. Plants, like other eukaryotes, share aspects of both calcium-and calmodulin-based cellular signaling and the autophagic process of cellular renewal. Here, we report a novel function for an Arabidopsis calmodulin-related protein, CML24, and insight into ATG4-regulated autophagy. CML24 interacts with ATG4b in yeast two-hybrid, in vitro pull-down and transient tobacco cell transformation assays. Mutants with missense mutations in CML24 have aberrant ATG4 activity patterns in in vitro extract assays, altered ATG8 accumulation levels, an altered pattern of GFP-ATG8-decorated cellular structures, and altered recovery from darkness-induced starvation. Together, these results support the conclusion that CML24 affects autophagy progression through interactions with ATG4.

). † These authors contributed equally to this work. SUMMARYNitric oxide signals diverse responses in animals and plants. Whereas nitric oxide synthesis mechanisms in animals are well understood, how nitric oxide is synthesized and regulated in plants remains controversial. NOA1 is a circularly permuted GTPase that is important for chloroplast function and is implicated in nitric oxide synthesis. However, the reported consequences of a null mutation in NOA1 are inconsistent. Whereas some studies indicate that the noa1 mutant has severe reductions in nitric oxide accumulation, others report that nitric oxide levels are indistinguishable between noa1 and the wild type. Here, we identify a correlation between the reported ability of noa1 to accumulate nitric oxide with growth on sucrose-supplemented media. We report that noa1 accumulates both basal and salicylic acid-induced nitric oxide only when grown on media containing sucrose. In contrast, nitric oxide accumulation in wild type is largely insensitive to sucrose supplementation. When grown in the absence of sucrose, noa1 has low fumarate, pale green leaves, slow growth and reduced chlorophyll content. These phenotypes are consistent with a defect in chloroplast-derived photosynthate production and are largely rescued by sucrose supplementation. We conclude that NOA1 has a primary role in chloroplast function and that its effects on the accumulation of nitric oxide are likely to be indirect.

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.Tomato (Solanum lycopersicum) is an important food crop of high economic value and represents a model species for fleshy fruit physiology and ripening (Giovannoni, 2004;Mueller, 2009). The breeding history of tomato has been dominated by a focus on traits that benefit the grower, such as yield, storage characteristics, and field performance (Schuch, 1994;Giovannoni, 2006; Cong et al., 2008). As a result, there has been an unintentional loss of consumer quality traits such as flavor and nutritional value, and this has focused recent interest on the molecular genetics of such traits (Giovannoni, 2001;Causse et al., 2002 Causse et al., , 2004Fraser et al., 2009;Mounet et al., 2009;Enfissi et al., 2010; Centeno et al., 2011). The accumulation of a range of soluble metabolites is critically important for both flavor and nutrition. Tomato fruit undergo substantial changes in their metabolite content and composition during ripening (Carrari et al., 2006). Fruit flavor is influenced both by volatile and nonvolatile metabolites (Buttery et al., 1987;Goff and Klee, 2006; Carli et al., 2009). Of the nonvolatile metabolites, the balance between sugars and acidic compounds is of major importance for flavor (Tieman et al., 2012). The perceived flavor of tomato fruit is a complex is...