Image_1_The Effect of Single and Multiple SERAT Mutants on Serine and Sulfur Metabolism.TIF

<p>The gene family of serine acetyltransferases (SERATs) constitutes an interface between the plant pathways of serine and sulfur metabolism. SERATs provide the activated precursor, O-acetylserine for the fixation of reduced sulfur into cysteine by exchanging the serine hydroxyl moiety by a sulfhydryl moiety, and subsequently all organic compounds containing reduced sulfur moieties. We investigate here, how manipulation of the SERAT interface results in metabolic alterations upstream or downstream of this boundary and the extent to which the five SERAT isoforms exert an effect on the coupled system, respectively. Serine is synthesized through three distinct pathways while cysteine biosynthesis is distributed over the three compartments cytosol, mitochondria, and plastids. As the respective mutants are viable, all necessary metabolites can obviously cross various membrane systems to compensate what would otherwise constitute a lethal failure in cysteine biosynthesis. Furthermore, given that cysteine serves as precursor for multiple pathways, cysteine biosynthesis is highly regulated at both, the enzyme and the expression level. In this study, metabolite profiles of a mutant series of the SERAT gene family displayed that levels of the downstream metabolites in sulfur metabolism were affected in correlation with the reduction levels of SERAT activities and the growth phenotypes, while levels of the upstream metabolites in serine metabolism were unchanged in the serat mutants compared to wild-type plants. These results suggest that despite of the fact that the two metabolic pathways are directly connected, there seems to be no causal link in metabolic alterations. This might be caused by the difference of their pool sizes or the tight regulation by homeostatic mechanisms that control the metabolite concentration in plant cells. Additionally, growth conditions exerted an influence on metabolic compositions.</p

Additional file 1: Table S1. of Exploring traditional aus-type rice for metabolites conferring drought tolerance

Experimental information and data on metabolite peaks. (XLSX 1006 kb

Additional file 3: Table S2. of Exploring traditional aus-type rice for metabolites conferring drought tolerance

Log2 ratios of Drought vs Well-Watered conditions per genotype and 2-way ANOVA. (XLSX 138 kb

Heat-map visualization and cluster tree representations of amino acid contents and genotypes

Data were obtained from experiments where plants were starved of sulphate for 10 d. The heat-map was generated by using log base 2-transformed fold changes. The given data represent the ratio of the determined amino acids for control and starved plants. Each amino acid is represented by a single column and each genotype by a single row. Red indicates decreased relative metabolite content whereas blue indicates increased relative contents of amino acids compared with the wild-type. Separated heat-map visualization of amino acid contents in control and mutant plants are presented in in available at online and the respective diagrams in Fig. S2.<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p

Contents of cysteine (upper row), γ-glutamylcysteine (GEC; middle row), and glutathione (GSH; lower row) are shown for plants overexpressing , , and , respectively, or down-regulated with respect to

Plants were grown for 10 weeks on soil before thiol extraction. knock-downs are represented by cross-hatched columns, overexpressing lines by white columns, and wild-type (WT) and empty-vector control lines (EV) by black columns. Values are the mean ±SD of three independent experiments. Asterisks indicate that the difference between the wild-type plants and the manipulated transgenic plants was significant using -tests ( ≤0.05).<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p

Phenotype of mature knock-down plants, a sketch depicting the location of the T-DNA insertions, and quantification of gene expression in mutant plants

(A) Wild-type plants (left in each picture) and three mutant plants (right) at 10 weeks. Each of three individually selected homozygote mutant plants is presented. The plants were grown at the same time. (B) Identification of T-DNA insertion. Boxes in black represent exons, lines represent non-coding regions, and boxes in grey indicate T-DNA insertions. (C) q-RT-PCR analysis of plants derived from the knock-down screen. RNA was extracted from 28-d-old soil-grown plants. Ratios to controls are shown.<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p

Expression analysis of , , and , respectively, in overexpressing lines from 28-d-old soil-grown plants with northern blot hybridization and q-RT-PCR, respectively

(A) Wild-type and empty-vector plants served as controls. RNA from 10 plants per treatment was extracted and 10 μg total RNA was subjected to northern blot analysis. Gene-specific probes were radioactively labelled and hybridized as described in Materials and methods. Equal loading in each track is demonstrated by comparing the amount of the 28S rRNA. Transcript sizes are given in brackets. (B) Quantification of gene expression in transgenic plants overexpressing , , and , respectively. Gene expression was assessed by q-RT-PCR as described in Materials and methods. The data represent the average relative to the gene expression in control plants.<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p

Heat map generated from amino acid measurements reflecting log base 2-transformed and normalized amino acid levels and its similarity among themselves and the genotypes

The top colour bar indicates the relative log base 2-fold changes ranging between reduced relative (red) and increased relative (blue) contents of amino acids with respect to the wild-type.<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p