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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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

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.

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.

Summary The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron‐sulfur clusters. To identify iron‐sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.

Plants can react to drought stress by anticipating flowering, an adaptive strategy for plant survival in dry climates known as drought escape (DE). In Arabidopsis, the study of DE brought to surface the involvement of abscisic acid (ABA) in controlling the floral transition. A central question concerns how and in what spatial context can ABA signals affect the floral network. In the leaf, ABA signaling affects flowering genes responsible for the production of the main florigen FLOWERING LOCUS T (FT). At the shoot apex, FD and FD-like transcription factors interact with FT and FT-like proteins to regulate ABA responses. This knowledge will help separate general and specific roles of ABA signaling with potential benefits to both biology and agriculture.

The majority of cellular processes are carried out by protein complexes. Various size fractionation methods have previously been combined with mass spectrometry to identify protein complexes. However, most of these approaches lack the quantitative information which is required to understand how changes of protein complex abundance and composition affect metabolic fluxes. In this paper we present a proof of concept approach to quantitatively study the complexome in the model plant Arabidopsis thaliana at the end of the day (ED) and the end of the night (EN). We show that size-fractionation of native protein complexes by Clear-Native-PAGE (CN-PAGE), coupled with mass spectrometry can be used to establish abundance profiles along the molecular weight gradient. Furthermore, by deconvoluting complex protein abundance profiles, we were able to drastically improve the clustering of protein profiles. To identify putative interaction partners, and ultimately protein complexes, our approach calculates the Euclidian distance between protein profile pairs. Acceptable threshold values are based on a cut-off that is optimized by a receiver-operator characteristic (ROC) curve analysis. Our approach shows low technical variation and can easily be adapted to study in the complexome in any biological system.