Deciphering transcriptional and metabolic networks associated with lysine metabolism during Arabidopsis seed development

Abstract

In order to elucidate transcriptional and metabolic networks associated with lysine (Lys) metabolism, we utilized developing Arabidopsis (Arabidopsis thaliana) seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor; however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the tricarboxylic acid cycle while largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor while suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems.

Figure 1.

A schematic diagram of the Asp family pathway. Only several enzymes are illustrated. Broken arrows represent several enzymatic steps. Abbreviations not defined in the text: AAT, Asp aminotransferase; AK, Asp kinase; CGS, cystathionine γ-synthase; TS, Thr synthase.

Figure 2.

Effects of altered Lys metabolism in the KD genotype on seed maturation and free Lys level. The developmental stages of seed maturation are marked by DAF, while mature dry seeds are designated “Dry.” A, Morphology of maturing seeds of the wild-type (WT; top) and KD (bottom) gentotypes. B, Immunoblot analysis with antibacterial DHDPS antibodies showing the level of expression of the bacterial DHPS during seed maturation in the KD genotype. C, Relative Lys level in the wild-type and KD genotypes at different stages of seed maturation. Values represent means of the response of the metabolite, expressed as peak area normalized to the internal standard ribitol as well as to dry weight. The relative metabolite levels of the wild-type and KD genotypes along the different stages of seed maturation in DAF are illustrated by white and black histograms, respectively. Error bars represent se values of four biological replicates grown together, each derived from 5 mg of isolated lyophilized seeds, bulked from at least 10 plants for each time point. [See online article for color version of this figure.]

Figure 3.

Relative contents of the four metabolites whose levels were altered significantly in maturing seeds of the KD genotype compared with the wild type. Comparable results were obtained in both growing times. The names of the metabolites are given in A to D. The relative metabolite levels in the wild-type and KD genotypes along the different stages of seed maturation in DAF are illustrated by white and black histograms, respectively. Error bars represent se. Relative values were analyzed as described in the legend of Figure 2.

Figure 4.

Network interactions between different metabolites during seed maturation in the wild-type and KD genotypes. A and B, Metabolites were grouped into five distinct communities in the wild-type genotype (A) and four distinct communities in the KD genotype (B). C, The merge of the networks visualization based on their coordinated levels during seed maturation of the KD genotype is represented by different colors for the different metabolites, each color representing a defined community in the wild-type genotype metabolic network. The edges (lines) connecting two nodes represent a significant correlation between metabolites: black when it occurs in both genotypes, blue when it occurs in the wild-type genotype and fails in the KD genotype, and red when it occurs in the KD genotype but is absent in the wild-type genotype. Metabolites abbreviations are provided in Supplemental Table S8. [See online article for color version of this figure.]

Figure 5.

MapMan RNA-protein synthesis overview maps showing differences in transcript levels between the wild-type (WT) and KD genotypes in 14-DAF (A), 16-DAF (B), and dry (C) seeds. Average transcript levels were calculated from two independent replicates of Affymetrix AtH1 GeneChips, and fold changes were calculated from the normalized gene expression data of the KD versus wild-type genotypes in 14-DAF, 16-DAF, and dry seeds. The resulting file was loaded into the MapMan Image Annotator module to generate the RNA-protein synthesis overview map (Supplemental Table S7). The normalized expression levels (in log₂) of the genes that exhibit significant changes between the wild-type and KD genotypes are available in Supplemental Table S4. [See online article for color version of this figure.]

Figure 6.

MapMan metabolism overview maps showing differences in transcript levels between the wild-type (WT) and KD genotypes in 14-DAF (A), 16-DAF (B), and dry (C) seeds. Average transcript levels were calculated from two independent replicates of Affymetrix AtH1 GeneChips, and fold changes were calculated from the normalized gene expression data of the KD versus wild-type genotypes in 14-DAF, 16-DAF, and dry seeds. The resulting file was loaded into the MapMan Image Annotator module to generate the metabolism overview map (Supplemental Table S7). The normalized expression levels (in log₂) of the genes that exhibit significant changes between the wild-type and KD genotypes are available in Supplemental Table S4. [See online article for color version of this figure.]

Figure 7.

Abiotic and biotic stress response overview maps showing differences in transcript levels between the wild-type (WT) and KD genotypes in 14-DAF (A), 16-DAF (B), and dry (C) seeds. Average transcript levels were calculated from two independent replicates of Affymetrix AtH1 GeneChips, and fold changes were calculated from the normalized gene expression data of the KD versus wild-type genotypes in 14-DAF, 16-DAF, and dry seeds. The resulting file was loaded into the MapMan Image Annotator module to generate the cell response overview map (Supplemental Table S7). The normalized expression levels (in log₂) of the genes that exhibit significant changes between the wild-type and KD genotypes are available in Supplemental Table S4. [See online article for color version of this figure.]

Figure 8.

Clustering analysis of genes whose mRNA levels were significantly different between the wild-type (WT) and KD genotypes during seed maturation. Clustering analysis was performed by comparing the average patterns of the normalized expression levels of identical groups of genes in the wild-type and KD genotypes in maturing seeds, as described in “Materials and Methods.” Error bars represent se. Seed maturation stages in DAF are provided at the bottom.