Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

Abstract

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.

STCH4/REIL2 Confers Cold Stress Tolerance in Arabidopsis by Promoting rRNA Processing and CBF Protein Translation

“…To further investigate protein translation during cold treatment, we examined de novo protein synthesis under low temperatures by labeling newly synthesized proteins with L-Azidohomoalanine (AHA), as previously described (Glenn et al, 2017), and performed mass spectrometry. We identified a total of 3,652 proteins with the AHA tag in both Col-0 and stch4-1 mutant plants, and 1,570 showed a significant change in abundance between stch4-1 and Col-0.…”

“…Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in plant BONCAT was performed as described in a previous report (Glenn et al, 2017). In brief, to label the newly synthesized proteins, AHA was pulsed into 1-week-old seedlings by submerging the seedlings in the AHA-containing medium for 2 min.…”

Visualization of giant virus particles using BONCAT labeling and STED microscopy

“…Although AHA and its alkyne analog L-homopropargylglycine (HPG) have been mostly used in methionine auxotrophic organisms to increase the efficiency of incorporation (Wisse et al , 2017;Zhang et al , 2017;Saleh et al , 2019), its use has recently been extended to the analysis of natural bacterial communities, resulting in good incorporation in active methionine prototrophic bacteria (Hatzenpichler et al , 2014;Leizeaga et al , 2017;Couradeau et al , 2019). In addition, four wild-type prototrophic eukaryotic organisms were reported to incorporate AHA or HPG: Danio rerio (Hinz et al , 2012), Caenorhabditis elegans (Ullrich et al , 2014), Arabidiopsis thaliana (Glenn et al , 2017) and the coccolithophore Emiliania huxleyi (Pasulka et al , 2018).…”

A Plant Biologist’s Toolbox to Study Translation

“…A modern adaptation of radiolabeling is the use of synthetic methionine analogs (for example, homopropargylglycine or azidohomoalanine) that can be detected using fluorescence and a “click” chemistry reaction ( Tom Dieck et al, 2012 ). This method, termed f l u orescent n on c anonical a mino acid t agging (FUNCAT, Figure 1A ), has been utilized in tissue culture and in live organisms, but to date, has not been widely explored in plant systems ( Glenn et al, 2017 ). However, this technique still requires extensive sample manipulation, detection is limited by the methionine content of a given protein, and the poorly characterized toxicity of these amino acid analogs in different species presents a challenge for the adaptation of FUNCAT in plant systems.…”

“…BONCAT employs a “click” chemistry reaction to attach a tag (e.g., biotin ) to the noncanonical amino acid, which would then allow purification of the tagged proteins (e.g., via streptavidin beads). BONCAT has been reported in only one Arabidopsis study ( Glenn et al, 2017 ), implying that MS-based identification of nascent proteins may be an under-utilized technique for plant translational experiments. A combination of these two approaches, termed qua ntitative n on c anonical a mino acid t agging (QuaNCAT, Figure 1D ), aims to provide better translational data by overcoming the limited labeling achieved by a SILAC pulse and the lack of quantitative data obtained with label-free BONCAT ( Howden et al, 2013 ).…”