New insights on the role of HCN in root hair elongation through single cell proteomics.

In order to identify the possible protein targets of cyanide in the regulation of root hair, we have carried out a single cell proteomic approach to characterize the protein atlas in wild type root hair cells compared to the cas-c1 mutant cells. Root hair specific cells were obtained from isolated protoplast from root tissues of Arabidopsis wt-pCOBL9:GFP and cas-c1-pCOBL9:GFP lines. To analyze the effect of the CAS-C1 mutation, we isolated 1 x 106 root hair cells by Fluorescence-activated cell sorting (FACS) from three independent replicate of wt-pCOBL9:GFP and cas-c1-pCOBL9:GFP roots. The extracted proteins from each sample were trypsin-digested and the digested peptides were analyzed by liquid chromatography-high resolution mass spectrometry (LC-MS/MS) for protein identification. In wild type samples, we have identified 3829 unique proteins at a false discovery rate below 1% (FDR<1%), that represent almost 10% of the total Arabidopsis proteome. In protein extract of the cas-c1-pCOBL9:GFP roots samples, we have identified 3972 proteins of which 3515 were commons in both cell types, 310 were only identified y wild type and 457 only in cas-c1 mutant (Fig. 5).