One particular MASP1-Adventure

Leaf discs (0.5?cm2 each) were harvested from inoculated leaves after 72?h. Four samples were made by pooling two leaf discs from different treated plants. Bacterial growth was assessed by homogenizing leaf discs in 400??L of water, plating appropriate dilutions on solid King B medium containing rifampicin and kanamycin and quantifying colony numbers after 3?d. Three allelic T-DNA mutants that carried the insertion within the cICDH coding sequence were obtained from the SALK collection (Fig.?1a). Homozygotes were readily identified by PCR analysis of genomic DNA, and analysis of total extractable ICDH activity showed that less than 10% of the wild-type ICDH capacity remained in all three lines (Fig.?1b). RT-PCR analysis confirmed the absence of cICDH expression in the mutants (Fig.?1c). Despite the drastic decrease in leaf ICDH activity, only relatively minor effects were observed AZD6738 supplier on rosette growth: all three lines were slightly smaller than Col0 (Fig.?1d,e). Growth of the mutants was also tested at high CO2 (3000??L?L?1), a condition which notably inhibits photorespiration MASP1 and represents a carbon-rich environment. Effects of the icdh mutation on growth were similar to those observed in air (i.e. a slight inhibition compared to the wild-type in the same conditions) (data not shown). cICDH function was next investigated using a non-targeted GC�CTOF�CMS metabolic profiling approach that provides a snapshot of leaf metabolites, Selleckchem PFI-2 notably compounds involved in primary metabolism. To analyse potential roles of cICDH in two metabolic conditions, the analysis was performed on wild-type and mutants grown both in air and at high CO2. In all, 76 unique metabolites could be identified and relatively quantified by GC�CTOF�CMS. A full list is given in Supporting Information Table?S2. Hierarchical clustering of all these metabolites identified two main clusters: one composed of metabolites that were more abundant at high CO2, and a smaller cluster of compounds that were more abundant in Arabidopsis leaves in air (Fig.?2a). Examination of the overall heatmap showed that the icdh mutation did not produce very dramatic effects on leaf metabolites (Fig.?2a). The data were further analysed for compounds that showed significant differences between wild-type and icdh lines using a double t-test approach. Firstly, the three wild-type samples were tested against the nine icdh samples, and all metabolites with P?>?0.05 were discounted. The remaining metabolites were further selected by retaining only those for which P?