The transmembrane receptor kinase family is the largest protein kinase family in Arabidopsis, and it contains the highest fraction of proteins with yet uncharacterized functions. Here, we present functions of SIRK1, a receptor kinase that was previously identified with rapid transient phosphorylation after sucrose resupply to sucrosestarved seedlings. SIRK1 was found to be an active kinase with increasing activity in the presence of an external sucrose supply. In sirk1 T-DNA insertional mutants, the sucrose-induced phosphorylation patterns of several membrane proteins were strongly reduced; in particular, pore-gating phosphorylation sites in aquaporins were affected. SIRK1-GFP fusions were found to directly interact with aquaporins in affinity pull-down experiments on microsomal membrane vesicles. Furthermore, protoplast swelling assays of sirk1 mutants and SIRK1-GFP expressing lines confirmed a direct functional interaction of receptor kinase SIRK1 and aquaporins as substrates for phosphorylation. A lack of SIRK1 expression resulted in the failure of mutant protoplasts to control water channel activity upon changes in external sucrose concentrations. We propose that SIRK1 is involved in the regulation of sucrosespecific osmotic responses through direct interaction with and activation of an aquaporin via phosphorylation and that the duration of this response is controlled by phosphorylation-dependent receptor internalization. Molecular & Cellular Proteomics

Tannins are eco-friendly, bio-sourced, natural, and highly reactive polyphenols. In the past decades, the understanding of their versatile properties has grown substantially alongside a continuously broadening of the tannins’ application scope. In particular, recently, tannins have been increasingly investigated for their interaction with other species in order to obtain tannin-based hybrid systems that feature advanced and/or novel properties. Furthermore, in virtue of the tannins’ chemistry and their high reactivity, they either physicochemically or physically interact with a wide variety of different compounds, including metals and ceramics, as well as a number of organic species. Such hybrid or hybrid-like systems allow the preparation of various advanced nanomaterials, featuring improved performances compared to the current ones. Consequently, these diverse-shaped materials have potential use in wastewater treatment or catalysis, as well as in some novel fields such as UV-shielding, functional food packaging, and biomedicine. Since these kinds of tannin-based hybrids represent an emerging field, thus far no comprehensive overview concerning their potential as functional chemical building blocks is available. Hence, this review aims to provide a structured summary of the current state of research regarding tannin-based hybrids, detailed findings on the chemical mechanisms as well as their fields of application.

The activation of aquaporins by a receptor kinase complex of SIRK1 and QSK1 was studied in detail. Based on phosphoproteomics, pulldown studies and physiological experiments we conclude that SIRK1 may function as a main receptor which forms a complex with coreceptor QSK1. SIRK1 can autophosphorylate and then trans-phosphorylate QSK1. Phosphorylated QSK1 enhanced and stabilized the interaction with aquaporins as substrates of the receptor kinase complex.

The plasma membrane H(+) ATPase is a member of the P-ATPase family transporting H(+) from the cytosol to the extracellular space and thus energizing the plasma membrane for the uptake of ions and nutrients. As a housekeeping gene, this protein can be detected in almost every plant cell including the exclusive expression of specific isoforms in pollen grains and tubes where its activity is a prerequisite for successful germination and growth of pollen tubes. This review summarizes the current knowledge on pollen PM H(+) ATPases and hypothesizes a central role for pollen-specific isoforms of this protein in tube growth. External as well as cytosolic signals from signal transduction and metabolic pathways are integrated by the PM H(+) ATPase and directly translated to tube growth rates, allocating the PM H(+) ATPase to an essential node in the signalling network of pollen tubes in their race to the ovule.

Mass spectrometry (MS)-based large scale phosphoproteomics has facilitated the investigation of plant phosphorylation dynamics on a system-wide scale. However, generating large scale data sets for membrane phosphoproteins usually requires fractionation of samples and extended hands-on laboratory time. To overcome these limitations, we developed “ShortPhos,” an efficient and simple phosphoproteomics protocol optimized for research on plant membrane proteins. The optimized workflow allows fast and efficient identification and quantification of phosphopeptides, even from small amounts of starting plant materials. “ShortPhos” can produce label-free datasets with a high quantitative reproducibility. In addition, the “ShortPhos” protocol recovered more phosphorylation sites from membrane proteins, especially plasma membrane and vacuolar proteins, when compared to our previous workflow and other membrane-based data in the PhosPhAt 4.0 database. We applied “ShortPhos” to study kinase-substrate relationships within a nitrate-induction experiment on Arabidopsis roots. The “ShortPhos” identified significantly more known kinase-substrate relationships compared to previous phosphoproteomics workflows, producing new insights into nitrate-induced signaling pathways.

Chloroplasts and mitochondria are unique endosymbiotic cellular organelles surrounded by two membranes. Essential metabolic networking between these compartments and their hosting cells requires the exchange of a large number of biochemical pathway intermediates in a directed and coordinated fashion across their inner and outer envelope membranes. Here, we describe the identification and functional characterization of a highly specific, regulated solute channel in the outer envelope of chloroplasts, named OEP40. Loss of OEP40 function in Arabidopsis thaliana results in early flowering under cold temperature. The reconstituted recombinant OEP40 protein forms a high conductance ␤-barrel ion channel with subconductant states in planar lipid bilayers. The OEP40 channel is slightly cation-selective P K؉ /P Cl؊ ≈ 4:1 and rectifying (ı ᠬ/ı ᠭ Х 2) with a slope conductance of Ḡ max Х 690 picosiemens. The OEP40 channel has a restriction zone diameter of Х1.4 nm and is permeable for glucose, glucose 1-phosphate and glucose 6-phosphate, but not for maltose. Moreover, channel properties are regulated by trehalose 6-phosphate, which cannot permeate. Altogether, our results indicate that OEP40 is a "glucose-gate" in the outer envelope membrane of chloroplasts, facilitating selective metabolite exchange between chloroplasts and the surrounding cell.The plant cell is highly compartmentalized, containing at least seven different types of organelles that are involved in metabolism and cellular maintenance. In many cases, metabolic pathways and other cellular processes are not confined to one organelle but require the cooperation of several of them, e.g. in protein secretion or photorespiration. Metabolic networking between compartments thus requires directed and coordinated exchange of biochemical pathway intermediates across organellar membranes (1). Chloroplasts and mitochondria are unique endosymbiotic cellular organelles, which like their prokaryotic ancestors are surrounded by two membranes. The inner membrane of both organelles is derived from the bacterial plasma membrane. Surprisingly, also the outer membrane largely originated from and still resembles the outer membrane of the Gram-negative-like bacterial endosymbiont (2). In the inner chloroplast envelope membrane (IE), 5 numerous metabolite transporter proteins were identified and characterized to large detail with respect to their physiological impact and molecular mechanisms (3). These transporters are hydrophobic, polytopic, and mainly ␣-helical membrane proteins facilitating the exchange of metabolic precursors, intermediates, and final products between plastids and the cytoplasm. In contrast, the characteristic channels of the outer membranes in bacteria, chloroplasts as well as mitochondria, span the membrane in the form of ␤-strands that are organized to form a barrel-like pore structure (4). Proteins sharing this three-dimensional arrangement were implied in a variety of functions, including protein import (5). In addition, solute pores like the voltage-dependen...

The Characeae are multicellular green algae with very close relationship to land plants. Their internodal cells have been the subject of numerous (electro-)physiological studies. When exposed to light, internodal cells display alternating bands of low and high pH along their surface in order to facilitate carbon uptake required for photosynthesis. Here we investigated for the first time the subcellular membrane protein composition of acidic and alkaline regions in internodal cells of Chara australis R. Br. using MS-proteomics. The identified peptides were annotated to Chara unigenes using a custom-made Chara database generated from a transcriptome analysis and to orthologous Arabidopsis genes using TAIR (The Arabidopsis Information Resource) database. Apart from providing the first public-available, functionally-annotated sequence database for Chara australis, the proteome study, which is supported by immunodetection, identified several membrane proteins associated with acidic regions that contain a high density of specific plasma membrane (PM) invaginations, the charasomes, which locally increase the membrane area to overcome diffusion limitation in membrane transport. An increased abundance of PM H+ ATPases at charasomes is consistent with their role in the acidification of the environment, but the characean PM H+ ATPase sequence suggests a different regulation compared to higher plant PM H+ ATPases. A higher abundance of H+ co-transporters in the charasome-rich, acidic regions possibly reflects enhanced uptake of ions and nutrients. The increase in mitochondrial proteins confirms earlier findings about the accumulation of cortical mitochondria in the acidic zones. The significant enrichment of clathrin heavy chains and clathrin adaptor proteins as well as other proteins involved in trafficking indicate a higher activity of membrane transport in the charasome-rich than in charasome-poor areas. New and unexpected data, for instance the upregulation and abundance of vacuolar transporters correlating with the charasome-rich, acidic cell regions account for new perspectives in the formation of charasomes.

Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mistargeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3␤ and ap-4␤ knock-out mutants. In ap-3␤ mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4␤ mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3␤ and ap-4␤ compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking. Molecular & Cellular