Gel‐based and gel‐free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response

Abstract

Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense.

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity

“…Sugar esters are also acetylated via acetyl-coA produced together with CO 2 from pyruvate by the pyruvate decarboxylase. These three enzymes have been identified in previous proteomic analyses of tobacco trichomes (Van Cutsem et al, 2011;Sallets et al, 2014). Thus, active terpene and sugar ester synthesis releases large amounts of CO 2 in trichome cells, generating a CO 2 -rich environment similar to that found in algae or C4 plants and allowing for a Rubisco enzyme with lower affinity and higher catalytic activity.…”

“…Glyceraldehyde-3-phosphate is a direct product of the Calvin cycle. Pyruvate might be generated together with CO 2 from malate by the chloroplast NADP+-malic enzyme, which has been identified in proteomic analyses of tobacco trichomes (Van Cutsem et al, 2011;Sallets et al, 2014). Acyl acids of sugar esters are derived from Thr and the pyruvate pathways of branched-chain amino acid metabolism (Kandra et al, 1990).…”

“…For MS analysis, 500 mg of soluble proteins from tall trichomes were electrophoresed and stained with colloidal blue. Spots of interest were manually excised from the gel and digested with trypsin, and MALDI-MS/MS analysis was performed as previously described (Van Cutsem et al, 2011). The acquired MS/MS spectra were used to search the EST database of NCBI (July 5, 2012).…”

“…For analysis of the different plant tissues, stem, leaf, and root materials were taken just prior to the onset of flowering. Trichomes were removed from frozen leaves and stems as described by Van Cutsem et al (2011). For analysis of seedlings, seeds were sterilized for 1 min in 70% ethanol and 5 min in 50% v/v commercial bleach.…”

Preventing False Negatives for Histochemical Detection of Phenolics and Lignins in PEG-Embedded Plant Tissues

“…30 In addition, as less stressed plants produce less lignins and polyphenols, studies on the effects of distinct stresses in agricultural plants are demanded. 19,20,31 Consequently, cheaper and faster ways of diagnosing such kinds of substances in plant structures may be useful for monitoring the distribution of phenolics and lignins in plants.…”