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

The pyridine and adenylate nucleotide pool sizes.

<p>The concentrations of adenylates, uridinylates (A) and pyridine nucleotides (B), and some ratios (C), of coffee plants grown under low or high light (10 or 100% full sunlight, respectively). <i>n</i> = 6± SE. The means for high-light plants marked with an asterisk differ significantly from those for low-light plants (<i>P</i><0.05).</p

In High-Light-Acclimated Coffee Plants the Metabolic Machinery Is Adjusted to Avoid Oxidative Stress Rather than to Benefit from Extra Light Enhancement in Photosynthetic Yield

<div><p>Coffee (<i>Coffea arabica</i> L.) has been traditionally considered as shade-demanding, although it performs well without shade and even out-yields shaded coffee. Here we investigated how coffee plants adjust their metabolic machinery to varying light supply and whether these adjustments are supported by a reprogramming of the primary and secondary metabolism. We demonstrate that coffee plants are able to adjust its metabolic machinery to high light conditions through marked increases in its antioxidant capacity associated with enhanced consumption of reducing equivalents. Photorespiration and alternative pathways are suggested to be key players in reductant-consumption under high light conditions. We also demonstrate that both primary and secondary metabolism undergo extensive reprogramming under high light supply, including depression of the levels of intermediates of the tricarboxylic acid cycle that were accompanied by an up-regulation of a range of amino acids, sugars and sugar alcohols, polyamines and flavonoids such as kaempferol and quercetin derivatives. When taken together, the entire dataset is consistent with these metabolic alterations being primarily associated with oxidative stress avoidance rather than representing adjustments in order to facilitate the plants from utilizing the additional light to improve their photosynthetic performance.</p></div

The net CO₂ assimilation rate (<i>A</i>), maximum rate of carboxylation (<i>V</i>_cmax), maximum rate of carboxylation limited by electron transport (<i>J</i>_max), light saturation point (LSP), oxygenative (<i>J</i>_o) and carboxylative (<i>J</i>_c) electron flows as well as the <i>J</i>_o<i>/J</i>_c ratio and mitochondrial respiration rate in the light (<i>R</i>_d) of coffee plants grown under low or high light (10 or 100% full sunlight, respectively).

<p><i>A</i>, <i>V</i>_cmax, and <i>R</i>_d are expressed on area (µmol CO₂ m⁻² s⁻¹) or mass basis (µmol CO₂ g⁻¹ DW s⁻¹); <i>J</i>_max, <i>J</i>_c and <i>J</i>_o are expressed on area (µmol electrons m⁻² s⁻¹) or mass basis (µmol electrons g⁻¹ DW s⁻¹); LSP is expressed on area (µmol photons m⁻² s⁻¹) basis. <i>A</i>, <i>J</i>_c and <i>J</i>_o were measured or estimated for the prevailing light conditions and under ambient CO₂ in each treatment; <i>V</i>_cmax and <i>J</i>_max were estimated using <i>A</i>/<i>C</i>_i curves under saturating light. <i>n</i> = 6± SE. Significance: ^ns not significant, *<i>P</i><0.05, **<i>P</i><0.01.</p

The variable-to-maximum chlorophyll fluorescence ratio (<i>F</i>_v/<i>F</i>_m), non-photochemical quenching coefficient (NPQ), total antioxidant capacity and malondialdehyde concentration of coffee plants grown under low or high light (10 or 100% full sunlight, respectively).

<p><i>n</i> = 6± SE. Significance: ^ns not significant, **<i>P</i><0.01.</p

Myrosinase activity, myrosinase protein abundance and nitrilase activity in rosette leaves after extended darkness (ED).

<p>The plants were transferred to complete darkness for 3 d (<b>3d ED</b>) or 7 d (<b>7d ED</b>) after six weeks of growth under short day conditions. Myrosinase (<b>A</b>) as well as nitrilase (<b>C</b>) activity was measured photometrically (error bars show the standard deviation of five to 19 biological replicates). For protein abundance of TGG1 and TGG2 (<b>B</b>), immunoblotting and normalization to the control was performed (error bars represent the standard deviation of three biological replicates from three independently performed Western blots). Asterics indicate significant differences (Student’s T-test; P < 0.05) compared to the control.</p

The specific leaf area (SLA), total nitrogen (N_total), nitrate (NO₃⁻) and starch concentrations of coffee plants grown under low or high light (100 or 10% full sunlight, respectively).

<p><i>n</i> = 6± SE. Significance: **<i>P</i><0.01, *<i>P</i><0.05.</p

The major metabolic alterations of coffee plants in response to the light treatments.

<p>These alterations, as observed in plants grown under low or high light (10 or 100% full sunlight, respectively), are synthesized in a schematic summary wherein they are mapped onto metabolic pathways. The colors indicate the proportional content of each putatively identified metabolite among the samples, as determined by the average peak response. For the whole metabolite profiling, only 12 metabolites were identified as not significantly affected by the light treatments (marked as ‘ns’). Metabolites were determined as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094862#s2" target="_blank">Materials and Methods</a>”. <i>n</i> = 6± SE.</p

The relative secondary metabolite profile of coffee plants.

<p>The plants were grown under low light (LL) or high light (HL) (10 or 100% full sunlight, respectively). Results for LL plants were set as unit. Data are normalized with respect to the mean response calculated for the LL (to allow statistical assessment, individual plants from this set were normalized in the same way). Chlorogenic acid (3-CGA) and Rutin have been identified by standard compounds. Flavonol-3Glcs were annotated by co-elution profile of Arabidopsis leaf and tomato fruit extracts (Rohrmann <i>et al</i>. 2011; Wu <i>et al</i>. 2012). The other phenolics such as cryptochlorogenic acid (4-CGA), neochlorogenic acid (5-CGA), feruloylquinic acids (FQA), dicaffeoylquinic acids (diCQA) and mangiferin were annotated based on comparison of the tables of coffee profile (Mondolot <i>et al</i>. 2006; Alonso-Salces <i>et al</i>. 2009; Campa <i>et al</i>. 2012) Significance: ^ns not significant, *<i>P</i><0.05, **<i>P</i><0.01</p

The relative primary metabolite profile of coffee plants.

<p>The plants were grown under low light (LL) or high light (HL) (10 or 100% full sunlight, respectively). Results for LL plants were set as unit. Data are normalized with respect to the mean response calculated for the LL (to allow statistical assessment, individual plants from this set were normalized in the same way). ND  =  metabolites not detected in the LL treatment. Significance: ^ns not significant, * <i>P</i><0.05, **<i>P</i><0.01</p