Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

The mycorrhiza-specific ammonium transporter ZmAMT3;1 mediates mycorrhiza-dependent nitrogen uptake in maize roots.

Most plant species can form symbioses with arbuscular mycorrhizal fungi (AMFs), which may enhance the host plant's acquisition of soil nutrients. In contrast to phosphorus nutrition, the molecular mechanism of mycorrhizal nitrogen (N) uptake remains largely unknown, and its physiological relevance is unclear. Here, we identified a gene encoding an AMF-inducible ammonium transporter, ZmAMT3;1, in maize (Zea mays) roots. ZmAMT3;1 was specifically expressed in arbuscule-containing cortical cells and the encoded protein was localized at the peri-arbuscular membrane. Functional analysis in yeast and Xenopus oocytes indicated that ZmAMT3;1 mediated high-affinity ammonium transport, with the substrate NH4+ being accessed, but likely translocating uncharged NH3. Phosphorylation of ZmAMT3;1 at the C-terminus suppressed transport activity. Using ZmAMT3;1-RNAi transgenic maize lines grown in compartmented pot experiments, we demonstrated that substantial quantities of N were transferred from AMF to plants, and 68%-74% of this capacity was conferred by ZmAMT3;1. Under field conditions, the ZmAMT3;1-dependent mycorrhizal N pathway contributed >30% of postsilking N uptake. Furthermore, AMFs downregulated ZmAMT1;1a and ZmAMT1;3 protein abundance and transport activities expressed in the root epidermis, suggesting a trade-off between mycorrhizal and direct root N-uptake pathways. Taken together, our results provide a comprehensive understanding of mycorrhiza-dependent N uptake in maize and present a promising approach to improve N-acquisition efficiency via plant-microbe interactions.

The LaCLE35 peptide modifies rootlet density and length in cluster roots of white lupin.

White lupin (lupinus albus L.) forms special bottlebrush-like root structures called cluster roots (CR) when phosphorus is low, to remobilise sparingly soluble phosphates in the soil. The molecular mechanisms that control the CR formation remain unknown. Root development in other plants is regulated by CLE  (CLAVATA3/ EMBRYO SURROUNDING REGION (ESR)-RELATED) peptides, which provide more precise control mechanisms than common phytohormones. This makes these peptides interesting candidates to be involved in CR formation, where fine tuning to environmental factors is required. In this study we present an analysis of CLE peptides in white lupin. The peptides LaCLE35 (RGVHy PSGANPLHN) and LaCLE55 (RRVHy PSCHy PDPLHN) reduced root growth and altered CR in hydroponically cultured white lupins. We demonstrate that rootlet density and rootlet length were locally, but not systemically, impaired by exogenously applied CLE35. The peptide was identified in the xylem sap. The inhibitory effect of CLE35 on root growth was attributed to arrested cell elongation in root tips. Taken together, CLE peptides affect both rootlet density and rootlet length, which are two critical factors for CR formation, and may be involved in fine tuning this peculiar root structure that is present in a few crops and many Proteaceae species, under low phosphorus availability.

Phosphoregulation in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1 and kinase HPCAL1 in Arabidopsis.

NRT2.1, the major high affinity nitrate transporter in roots, can be phosphorylated at five different sites within the N- and the C-terminus. Here, we characterized the functional relationship of two N-terminal phosphorylation sites, S21 and S28, in Arabidopsis. Based on a site-specific correlation network, we identified a receptor kinase (HPCAL1, AT5G49770), phosphorylating NRT2.1 at S21 and resulting in active nitrate uptake. HPCAL1 itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, HPCAL1 was found to interact with the N-terminus of NRT2.1, mainly when S28 was dephosphorylated. Phosphorylation of NRT2.1 at S21 resulted in a reduced interaction of NRT2.1 with its activator NAR2.1, but nitrate transport activity remained. By contrast, phosphorylated NRT2.1 at S28 enhanced the interaction with NAR2.1, but reduced the interaction with HPCAL1. Here we identified HPCAL1 as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21.

A systems-biology approach identifies co-expression modules in response to low phosphate supply in maize lines of different breeding history.

Plants cope with low phosphorus availability by adjusting growth and metabolism through transcriptomic and proteomic adaptations. We hypothesize that selected genotypes with distinct phosphorous (P) use efficiency covering the breeding history of European Flint heterotic pool provide a tool to reveal general and genotype-specific molecular responses to P limitation. We reconstructed protein and gene co-expression networks by weighted correlation network analysis and related these to phosphate deficiency-induced traits. In roots, low phosphate supply resulted in a decreasing abundance of proteins in the oxidative pentose phosphate pathway and a negative correlation with root and shoot phosphate content. We observed an increase in abundance and positive correlation with root and shoot phosphate content for proteins in sucrose biosynthesis, lipid metabolism, respiration and RNA processing. Purple acid phosphatases, superoxide dismutase and phenylalanine ammonia lyase were identified as being upregulated under low phosphate in all genotypes. Overall, correlations between protein and mRNA abundance changes were limited, with ribosomal proteins and the ubiquitin protein degradation pathway exclusively responding with protein abundance changes. Carbohydrate, phospho- and sulfo-lipid metabolism showed abundance changes at the protein and mRNA levels. These partially non-overlapping proteomic and transcriptomic adjustments to low phosphate suggest sugar and lipid metabolism as metabolic processes associated with improved P use efficiency specifically in Founder Flint lines. We identified a mitogen-activated protein kinase-kinase as a potential genotype-specific regulator of sucrose metabolism at low phosphate in Founder Flint line EP1. We conclude that, during breedingt of Elite Flint lines, regulation of primary metabolism has changed to result in a distinct low phosphate response in Founder lines.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

Calcium-Dependent Protein Kinase CPK1 Controls Cell Death by In Vivo Phosphorylation of Senescence Master Regulator ORE1.

Calcium-regulated protein kinases are key components of intracellular signaling in plants that mediate rapid stress-induced responses to changes in the environment. To identify in vivo phosphorylation substrates of CALCIUM-DEPENDENT PROTEIN KINASE1 (CPK1), we analyzed the conditional expression of constitutively active CPK1 in conjunction with in vivo phosphoproteomics. We identified Arabidopsis (Arabidopsis thaliana) ORESARA1 (ORE1), the developmental master regulator of senescence, as a direct CPK1 phosphorylation substrate. CPK1 phosphorylates ORE1 at a hotspot within an intrinsically disordered region. This augments transcriptional activation by ORE1 of its downstream target gene BIFUNCTIONAL NUCLEASE1 (BFN1). Plants that overexpress ORE1, but not an ORE1 variant lacking the CPK1 phosphorylation hotspot, promote early senescence. Furthermore, ORE1 is required for enhanced cell death induced by CPK1 signaling. Our data validate the use of conditional expression of an active enzyme combined with phosphoproteomics to decipher specific kinase target proteins of low abundance, of transient phosphorylation, or in yet-undescribed biological contexts. Here, we have identified that senescence is not just under molecular surveillance manifested by stringent gene regulatory control over ORE1 In addition, the decision to die is superimposed by an additional layer of control toward ORE1 via its posttranslational modification linked to the calcium-regulatory network through CPK1.

Combining analyses of metabolite profiles and phosphorus fractions to explore high phosphorus utilization efficiency in maize.

Phosphorus (P) limitation is a significant factor restricting crop production in agricultural systems, and enhancing the internal P utilization efficiency (PUE) of crops plays an important role in ensuring sustainable P use in agriculture. To better understand how P is remobilized to affect crop growth, we first screened P-efficient (B73 and GEMS50) and P-inefficient (Liao5114) maize genotypes at the same shoot P content, and then analyzed P pools and performed non-targeted metabolomic analyses to explore changes in cellular P fractions and metabolites in maize genotypes with contrasting PUE. We show that lipid P and nucleic acid P concentrations were significantly lower in lower leaves of P-efficient genotypes, and these P pools were remobilized to a major extent in P-efficient genotypes. Broad metabolic alterations were evident in leaves of P-efficient maize genotypes, particularly affecting products of phospholipid turnover and phosphorylated compounds, and the shikimate biosynthesis pathway. Taken together, our results suggest that P-efficient genotypes have a high capacity to remobilize lipid P and nucleic acid P and promote the shikimate pathway towards efficient P utilization in maize.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

Plasma membrane calcineurin B-like calcium-ion sensor proteins function in regulating primary root growth and nitrate uptake by affecting global phosphorylation patterns and microdomain protein distribution.

The collective function of calcineurin B-like (CBL) calcium ion (Ca2+ ) sensors and CBL-interacting protein kinases (CIPKs) in decoding plasma-membrane-initiated Ca2+ signals to convey developmental and adaptive responses to fluctuating nitrate availability remained to be determined. Here, we generated a cbl-quintuple mutant in Arabidopsis thaliana devoid of these Ca2+ sensors at the plasma membrane and performed comparative phenotyping, nitrate flux determination, phosphoproteome analyses, and studies of membrane domain protein distribution in response to low and high nitrate availability. We observed that CBL proteins exert multifaceted regulation of primary and lateral root growth and nitrate fluxes. Accordingly, we found that loss of plasma membrane Ca2+ sensor function simultaneously affected protein phosphorylation of numerous membrane proteins, including several nitrate transporters, proton pumps, and aquaporins, as well as their distribution within plasma membrane microdomains, and identified a specific phosphorylation and domain distribution pattern during distinct phases of low and high nitrate responses. Collectively, these analyses reveal a central and coordinative function of CBL-CIPK-mediated signaling in conveying plant adaptation to fluctuating nitrate availability and identify a crucial role of Ca2+ signaling in regulating the composition and dynamics of plasma membrane microdomains.

The Systemin Signaling Cascade As Derived from Time Course Analyses of the Systemin-responsive Phosphoproteome.

Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.

Sucrose-induced Receptor Kinase 1 is Modulated by an Interacting Kinase with Short Extracellular Domain.

Sucrose as a product of photosynthesis is the major carbohydrate translocated from photosynthetic leaves to growing nonphotosynthetic organs such as roots and seeds. These growing tissues, besides carbohydrate supply, require uptake of water through aquaporins to enhance cell expansion during growth. Previous work revealed Sucrose Induced Receptor Kinase, SIRK1, to control aquaporin activity via phosphorylation in response to external sucrose stimulation. Here, we present the regulatory role of AT3G02880 (QSK1), a receptor kinase with a short external domain, in modulation of SIRK1 activity. Our results suggest that SIRK1 autophosphorylates at Ser-744 after sucrose treatment. Autophosphorylated SIRK1 then interacts with and transphosphorylates QSK1 and QSK2. Upon interaction with QSK1, SIRK1 phosphorylates aquaporins at their regulatory C-terminal phosphorylation sites. Consequently, in root protoplast swelling assays, the qsk1qsk2 mutant showed reduced water influx rates under iso-osmotic sucrose stimulation, confirming an involvement in the same signaling pathway as the receptor kinase SIRK1. Large-scale phosphoproteomics comparing single mutant sirk1, qsk1, and double mutant sirk1 qsk1 revealed that aquaporins were regulated by phosphorylation depending on an activated receptor kinase complex of SIRK1, as well as QSK1. QSK1 thereby acts as a coreceptor stabilizing and enhancing SIRK1 activity and recruiting substrate proteins, such as aquaporins.

Plasma Membrane-Associated Receptor-like Kinases Relocalize to Plasmodesmata in Response to Osmotic Stress.

Plasmodesmata act as key elements in intercellular communication, coordinating processes related to plant growth, development, and responses to environmental stresses. While many of the developmental, biotic, and abiotic signals are primarily perceived at the plasma membrane (PM) by receptor proteins, plasmodesmata also cluster receptor-like activities; whether these two pathways interact is currently unknown. Here, we show that specific PM-located Leu-rich-repeat receptor-like-kinases, Qian Shou kinase (QSK1) and inflorescence meristem kinase2, which under optimal growth conditions are absent from plasmodesmata, rapidly relocate and cluster to the pores in response to osmotic stress. This process is remarkably fast, is not a general feature of PM-associated proteins, and is independent of sterol and sphingolipid membrane composition. Focusing on QSK1, previously reported to be involved in stress responses, we show that relocalization in response to mannitol depends on QSK1 phosphorylation. Loss-of-function mutation in QSK1 results in delayed lateral root (LR) development, and the mutant is affected in the root response to mannitol stress. Callose-mediated plasmodesmata regulation is known to regulate LR development. We found that callose levels are reduced in the qsk1 mutant background with a root phenotype resembling ectopic expression of PdBG1, an enzyme that degrades callose at the pores. Both the LR and callose phenotypes can be complemented by expression of wild-type and phosphomimic QSK1 variants, but not by phosphodead QSK1 mutant, which fails to relocalize at plasmodesmata. Together, the data indicate that reorganization of receptor-like-kinases to plasmodesmata is important for the regulation of callose and LR development as part of the plant response to osmotic stress.

High light intensity aggravates latent manganese deficiency in maize.

Manganese (Mn) plays an important role in the oxygen-evolving complex, where energy from light absorption is used for water splitting. Although changes in light intensity and Mn status can interfere with the functionality of the photosynthetic apparatus, the interaction between these two factors and the underlying mechanisms remain largely unknown. Here, maize seedlings were grown hydroponically and exposed to two different light intensities under Mn-sufficient or -deficient conditions. No visual Mn deficiency symptoms appeared even though the foliar Mn concentration in the Mn-deficient treatments was reduced to 2 µg g-1. However, the maximum quantum yield efficiency of PSII and the net photosynthetic rate declined significantly, indicating latent Mn deficiency. The reduction in photosynthetic performance by Mn depletion was further aggravated when plants were exposed to high light intensity. Integrated transcriptomic and proteomic analyses showed that a considerable number of genes encoding proteins in the photosynthetic apparatus were only suppressed by a combination of Mn deficiency and high light, thus indicating interactions between changes in Mn nutritional status and light intensity. We conclude that high light intensity aggravates latent Mn deficiency in maize by interfering with the abundance of PSII proteins.

Global Identification of Protein Complexes within the Membrane Proteome of Arabidopsis Roots Using a SEC-MS Approach.

Biological processes consist of several consecutive and interacting steps as, for example, in signal transduction cascades or metabolic reaction chains. These processes are regulated by protein-protein interactions and the formation of larger protein complexes, which also occur within biological membranes. To gain a large-scale overview of complex-forming proteins and the composition of such complexes within the cellular membranes of Arabidopsis roots, we use the combination of size-exclusion chromatography and mass spectrometry. First, we identified complex-forming proteins by a retention shift analysis relative to expected retention times of monomeric proteins during size-exclusion chromatography. In a second step we predicted complex composition through pairwise correlation of elution profiles. As result we present an interactome of 963 proteins within cellular membranes of Arabidopsis roots. Identification of complex-forming proteins was highly robust between two independently grown root proteomes. The protein complex composition derived from pairwise correlations of coeluting proteins reproducibly identified stable protein complexes (ribosomes, proteasome, mitochondrial respiratory chain supercomplexes) but showed higher variance between replicates regarding transient interactions (e.g., interactions with kinases) within membrane protein complexes.

Venus flytrap carnivorous lifestyle builds on herbivore defense strategies.

Although the concept of botanical carnivory has been known since Darwin's time, the molecular mechanisms that allow animal feeding remain unknown, primarily due to a complete lack of genomic information. Here, we show that the transcriptomic landscape of the Dionaea trap is dramatically shifted toward signal transduction and nutrient transport upon insect feeding, with touch hormone signaling and protein secretion prevailing. At the same time, a massive induction of general defense responses is accompanied by the repression of cell death-related genes/processes. We hypothesize that the carnivory syndrome of Dionaea evolved by exaptation of ancient defense pathways, replacing cell death with nutrient acquisition.

Ammonium and nitrate regulate NH4+ uptake activity of Arabidopsis ammonium transporter AtAMT1;3 via phosphorylation at multiple C-terminal sites.

In plants, nutrient transporters require tight regulation to ensure optimal uptake in complex environments. The activities of many nutrient transporters are post-translationally regulated by reversible phosphorylation, allowing rapid adaptation to variable environmental conditions. Here, we show that the Arabidopsis root epidermis-expressed ammonium transporter AtAMT1;3 was dynamically (de-)phosphorylated at multiple sites in the cytosolic C-terminal region (CTR) responding to ammonium and nitrate signals. Under ammonium resupply rapid phosphorylation of a Thr residue (T464) in the conserved part of the CTR (CTRC) effectively inhibited AtAMT1;3-dependent NH4+ uptake. Moreover, phosphorylation of Thr (T494), one of three phosphorylation sites in the non-conserved part of the CTR (CRTNC), moderately decreased the NH4+ transport activity of AtAMT1;3, as deduced from functional analysis of phospho-mimic mutants in yeast, oocytes, and transgenic Arabidopsis. Double phospho-mutants indicated a role of T494 in fine-tuning the NH4+ transport activity when T464 was non-phosphorylated. Transient dephosphorylation of T494 with nitrate resupply closely paralleled a transient increase in ammonium uptake. These results suggest that T464 phosphorylation at the CTRC acts as a prime switch to prevent excess ammonium influx, while T494 phosphorylation at the CTRNC fine tunes ammonium uptake in response to nitrate. This provides a sophisticated regulatory mechanism for plant ammonium transporters to achieve optimal ammonium uptake in response to various nitrogen forms.

Physiological and Proteomic Evidence for the Interactive Effects of Post-Anthesis Heat Stress and Elevated CO2 on Wheat.

Elevated CO2 promotes leaf photosynthesis and improves crop grain yield. However, as a major anthropogenic greenhouse gas, CO2 contributes to more frequent and severe heat stress, which threatens crop productivity. The combined effects of elevated CO2 and heat stress are complex, and the underlying mechanisms are poorly understood. In the present study, the effects of elevated CO2 and high-temperature on foliar physiological traits and the proteome of spring wheat grown under two CO2 concentrations (380 and 550 µmol mol-1 ) and two temperature conditions (ambient and post-anthesis heat stress) are examined. Elevated CO2 increases leaf photosynthetic traits, biomass, and grain yield, while heat stress depresses photosynthesis and yield. Temperature-induced impacts on chlorophyll content and grain yield are not significantly different under the two CO2 concentrations. Analysis of the leaf proteome reveals that proteins involved in photosynthesis as well as antioxidant and protein synthesis pathways are significantly downregulated due to the combination of elevated CO2 and heat stress. Correspondingly, plants treated with elevated CO2 and heat stress exhibit decreased green leaf area, photosynthetic rate, antioxidant enzyme activities, and 1000-kernel weight. The present study demonstrates that future post-anthesis heat episodes will diminish the positive effects of elevated CO2 and negatively impact wheat production.

Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day-1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate.

Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

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 mis-targeted 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-3ß and ap-4ß knock-out mutants. In ap-3ß 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-4ß mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3ß and ap-4ß 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.