Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana . As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana , we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea , Capsella rubella , Thellungiella salsuginea , Thellungiella halophila , Solanum tuberosum , Capsicum annuum , and N. benthamiana . Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein–protein interactions in the sucrose biosynthesis pathway in Arabidopsis .

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Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identities of the protein kinases/phosphatases involved in this mechanism and their roles in the regulation of the tricarboxylic acid (TCA) cycle remain unclear. In this study, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) along with pyruvate dehydrogenase kinase (PDK). Protein-protein interaction analysis, quantitative phosphoproteomics, and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex (PDC), while PP2c63 nonspecifically regulates PDC. When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants, protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC, while Sal2 only indirectly affects TCA cycle enzymes. Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle, and that altered metabolism in the sal2 pp2c63 double mutant compromises plant growth. These results are discussed in the context of current models of the control of respiration in plants.

Enzyme‐enzyme interactions can be discovered by affinity purification mass spectrometry (AP‐MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP‐MS. The success of AP‐MS depends on the levels and stability of the bait protein, the stability of the protein‐protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in‐gel‐digestion AP‐MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics‐based in‐solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP‐MS experiment constitutes a single sample for LC‐MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold‐change abundance (FC‐A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co‐sublocalization of “bait” and “prey,” we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC‐A values of at least four within‐biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme‐enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors. Basic Protocol 1: Gene cloning to the destination vectors Alternate Protocol: In‐Fusion or Gibson gene cloning protocol Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf Basic Protocol 3: Affinity purification of protein complexes Basic Protocol 4: On‐bead trypsin/LysC digestion and C18 column peptide desalting and concentration Basic Protocol 5: Data analysis and quality control

Proton antiport across the thylakoid membrane upregulates photosynthesis and growth in an Arabidopsis mutant with low chloroplast ATP synthase levels and high proton motive force. Footnotes: Author contributions UA designed the study together with MAS and DDS. VC performed most of the experiments with help from DH and analyzed data together with UA. BAM and BS performed MS analysis on thylakoid proteins. MAS and WT quantified thylakoid complexes spectroscopically, measured 77K chlorophyll-a fluorescence emission spectra, cyt-f redox state and performed Chl a fluorescence light response curves. EK carried out the CO2 assimilation measurement. MM, MW, FB and RH performed adenylate quantifications, SB and PJ performed the pigment analysis. UA, VC, MAS and DDS interpreted data with help from SZT.