Adaptation of Puromycin-Associated Nascent Chain Proteomics to Arabidopsis thaliana and Pisum sativum

Translation is a biological function present in all forms of life that allows for the production of proteins from strands of messenger RNA (mRNA). In plants, such as Arabidopsis thaliana or Pisum sativum (garden pea), translation is tightly regulated because of the plants’ sessile nature and need to adapt to a variety of stresses. PuNCH‐P is a proteomic technique that offers a snapshot of the translational status of cells in both a noninvasive and time sensitive manner. It was previously developed for mammalian tissues but had not been adapted to plants. PuNCH‐P delivers an estimate of the rate of protein production using a technique complementary to ribosome footprinting or polysome loading. This method circumvents the need for in‐vivo labeling such as in SILAC or BONCAT methods, and also avoids high sequencing costs associated with ribo‐seq methods. Nascent peptides attached to ribosomes are isolated from a frozen tissue sample by centrifugation through a sucrose cushion. These peptides are then C‐terminally labelled with biotin‐puromycin. The incorporation of biotin allows for downstream peptide visualization on an immunoblot (Western Blot) using streptavidin‐HRP as a detection antibody. Peptides may also be captured with streptavidin coated beads. Here, we validate the PuNCH‐P method for Arabidopsis and peas. It is well known that heat shock causes an overall decrease in the translational activity of plants due to mechanisms such as the sequestering of cytoplasmic mRNAs into stress granules and the shift to translating heat shock proteins (HSPs). Here we present methods to efficiently label, capture and detect biotin‐labeled nascent peptides from plants. With heat treatment as a proof of concept, we show that puromycylation recapitulates the shift in the patterns of translation expected from polysome profiling. These data set the stage for the subsequent proteomic analysis of nascent peptides in plants. Support or Funding Information NSF MCB 1546402