Differential Contribution of Malic Enzymes during Soybean and Castor Seeds Maturation

Abstract

Malic enzymes (ME) catalyze the decarboxylation of malate generating pyruvate, CO2 and NADH or NADPH. In some organisms it has been established that ME is involved in lipids biosynthesis supplying carbon skeletons and reducing power. In this work we studied the MEs of soybean and castor, metabolically different oilseeds. The comparison of enzymatic activities, transcript profiles and organic acid contents suggest different metabolic strategies operating in soybean embryo and castor endosperm in order to generate precursors for lipid biosynthesis. In castor, the malate accumulation pattern agrees with a central role of this metabolite in the provision of carbon to plastids, where the biosynthesis of fatty acids occurs. In this regard, the genome of castor possesses a single gene encoding a putative plastidic NADP-ME, whose expression level is high when lipid deposition is active. On the other hand, NAD-ME showed an important contribution to the maturation of soybean embryos, perhaps driving the carbon relocation from mitochondria to plastids to support the fatty acids synthesis in the last stages of seed filling. These findings provide new insights into intermediary metabolism in oilseeds and provide new biotechnological targets to improve oil yields.

Fig 1. Maturing soybean and castor seeds collected for this work.

The samples span the period of lipids biosynthesis already established in [15; 16].

Fig 2. Phylogenetic tree of NADP-ME.

(A). Protein sequences were aligned using Mega 5.10 and the phylogenetic tree was constructed by the neighbor joining method. The numbers indicate the statistical significance of each branch obtained by bootstrap analysis. Relative level of the transcripts of NADP-ME genes in maturing soybean embryos (B, D and F) and castor bean endosperms (C, E and G) determined by qRT-PCR. The functionality of primers for GmNADP-ME1.2 and 1.3 was verified using soybean genomic DNA. The primers for GmNADP-ME2.2 also anneals with the splicing version GmNADP-ME2.2spl. The actin genes Glyma04g39380 and Rco30206.m000761 were used as reference in soybean and castor, respectively. The values are the average of at least two independent experiments ± SD. For each transcript, values with the same letter are not significantly different (p < 0.05).

Fig 3. Phylogenetic tree of NAD-ME.

(A). Protein sequences were aligned using Mega 5.10 and the phylogenetic tree was constructed by the neighbor joining method. The numbers indicate the statistical significance of each branch obtained by bootstrap analysis. Relative level of the transcripts of NAD-ME genes in maturing soybean embryos (B and D) and castor bean endosperms (C and E) determined by qRT-PCR. The actin genes Glyma04g39380 and Rco30206.m000761 were used as reference in soybean and castor, respectively. The values are the average of at least two independent experiments ± SD. For each transcript, values with the same letter are not significantly different (p < 0.05).

Fig 4

NADP-ME (A and B), NAD-ME (C and D) and NAD-MDH (E and F) activities in soybean embryos and castor endosperms. NAD(P)-ME/NAD-MDH ratios (G and H). One unit (U) is defined as the amount of enzyme that catalyzes the formation of 1 μmol of NAD(P)H min^-1 under the specified conditions. FW: fresh weight. The values are the average of two technical duplicates from two independent samples ± SD. For each activity, values with the same letter are not significantly different (p < 0.05). In G and H, the asterisk denotes significant differences (p < 0.05) between the two ratios for each stage and for each species.

Fig 5

Organic acid content relative to stage R5.5 in soybean embryos (A) and stage III in castor endosperms (B) determined by GC-MS analysis. The peak areas were normalized according to the ones of the ribitol standard and the moles of each compound were determined using calibration curves. Each value was then relativized according to the fresh weight (FW) of each sample (S2 Fig). To facilitate the comparison between organic acids, the values were further relativized to the first stage analyzed in each species. The values are the average of three independent experiments ± SD.

Fig 6. Proposed model for the generation of precursors for lipid biosynthesis in maturing soybean and castor bean.

PEP, phosphoenolpyruvate; NADP-ME, NADP-dependent malic enzyme; NAD-ME, NAD-dependent malic enzyme.