A cornichon protein controls polar localization of the PINA auxin transporter in Physcomitrium patens.

Newly synthesized membrane proteins pass through the secretory pathway, starting at the endoplasmic reticulum and packaged into COPII vesicles, to continue to the Golgi apparatus before reaching their membrane of residence. It is known that cargo receptor proteins form part of the COPII complex and play a role in the recruitment of cargo proteins for their subsequent transport through the secretory pathway. The role of cornichon proteins is conserved from yeast to vertebrates, but it is poorly characterized in plants. Here, we studied the role of the two cornichon homologs in the secretory pathway of the moss Physcomitrium patens. Mutant analyses revealed that cornichon genes regulate different growth processes during the moss life cycle by controlling auxin transport, with CNIH2 functioning as a specific cargo receptor for the auxin efflux carrier PINA, with the C terminus of the receptor regulating the interaction, trafficking and membrane localization of PINA.

The C-terminus of the cargo receptor Erv14 affects COPII vesicle formation and cargo delivery.

The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. The Saccharomyces cerevisiae protein Erv14, also known as cornichon, belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (S134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14.

Ice plant root plasma membrane aquaporins are regulated by clathrin-coated vesicles in response to salt stress.

The regulation of root Plasma membrane (PM) Intrinsic Protein (PIP)-type aquaporins (AQPs) is potentially important for salinity tolerance. However, the molecular and cellular details underlying this process in halophytes remain unclear. Using free-flow electrophoresis and label-free proteomics, we report that the increased abundance of PIPs at the PM of the halophyte ice plant (Mesembryanthemum crystallinum L.) roots under salinity conditions is regulated by clathrin-coated vesicles (CCV). To understand this regulation, we analyzed several components of the M. crystallinum CCV complexes: clathrin light chain (McCLC) and subunits µ1 and µ2 of the adaptor protein (AP) complex (McAP1µ and McAP2µ). Co-localization analyses revealed the association between McPIP1;4 and McAP2µ and between McPIP2;1 and McAP1µ, observations corroborated by mbSUS assays, suggesting that AQP abundance at the PM is under the control of CCV. The ability of McPIP1;4 and McPIP2;1 to form homo- and hetero-oligomers was tested and confirmed, as well as their activity as water channels. Also, we found increased phosphorylation of McPIP2;1 only at the PM in response to salt stress. Our results indicate root PIPs from halophytes might be regulated through CCV trafficking and phosphorylation, impacting their localization, transport activity, and abundance under salinity conditions.

Plant and yeast cornichon possess a conserved acidic motif required for correct targeting of plasma membrane cargos.

The export of membrane proteins along the secretory pathway is initiated at the endoplasmic reticulum after proteins are folded and packaged inside this organelle by their recruiting into the coat complex COPII vesicles. It is proposed that cargo receptors are required for the correct transport of proteins to its target membrane, however, little is known about ER export signals for cargo receptors. Erv14/Cornichon belong to a well conserved protein family in Eukaryotes, and have been proposed to function as cargo receptors for many transmembrane proteins. Amino acid sequence alignment showed the presence of a conserved acidic motif in the C-terminal in homologues from plants and yeast. Here, we demonstrate that mutation of the C-terminal acidic motif from ScErv14 or OsCNIH1, did not alter the localization of these cargo receptors, however it modified the proper targeting of the plasma membrane transporters Nha1p, Pdr12p and Qdr2p. Our results suggest that mistargeting of these plasma membrane proteins is a consequence of a weaker interaction between the cargo receptor and cargo proteins caused by the mutation of the C-terminal acidic motif.

Membrane Proteomic Insights into the Physiology and Taxonomy of an Oleaginous Green Microalga.

Ettlia oleoabundans is a nonsequenced oleaginous green microalga. Despite the significant biotechnological interest in producing value-added compounds from the acyl lipids of this microalga, a basic understanding of the physiology and biochemistry of oleaginous microalgae is lacking, especially under nitrogen deprivation conditions known to trigger lipid accumulation. Using an RNA sequencing-based proteomics approach together with manual annotation, we are able to provide, to our knowledge, the first membrane proteome of an oleaginous microalga. This approach allowed the identification of novel proteins in E. oleoabundans, including two photoprotection-related proteins, Photosystem II Subunit S and Maintenance of Photosystem II under High Light1, which were considered exclusive to higher photosynthetic organisms, as well as Retinitis Pigmentosa Type 2-Clathrin Light Chain, a membrane protein with a novel domain architecture. Free-flow zonal electrophoresis of microalgal membranes coupled to liquid chromatography-tandem mass spectrometry proved to be a useful technique for determining the intracellular location of proteins of interest. Carbon-flow compartmentalization in E. oleoabundans was modeled using this information. Molecular phylogenetic analyses of protein markers and 18S ribosomal DNA support the reclassification of E. oleoabundans within the trebouxiophycean microalgae, rather than with the Chlorophyceae class, in which it is currently classified, indicating that it may not be closely related to the model green alga Chlamydomonas reinhardtii A detailed survey of biological processes taking place in the membranes of nitrogen-deprived E. oleoabundans, including lipid metabolism, provides insights into the basic biology of this nonmodel organism.

Erv14 cargo receptor participates in yeast salt tolerance via its interaction with the plasma-membrane Nha1 cation/proton antiporter.

The yeast Nha1p Na(+), K(+)/H(+) antiporter has a house-keeping role in pH and cation homeostasis. It is also needed to alleviate excess Na(+) or K(+) from the cytoplasm under high external concentrations of these cations. Erv14p, a putative cargo receptor for transmembrane proteins is required for trafficking of Nha1p from the endoplasmic reticulum to the plasma membrane. Sensitivity to high Na(+) concentrations of the erv14 mutant associated to the intracellular mislocalization of Nha1p-GFP, together with a lower Na(+) efflux, indicate the involvement of this mutual association to accomplish the survival of the yeast cell upon sodium stress. This observation is supported by the protein-protein interaction between Erv14p and Nha1p detected by the mating-based Split Ubiquitin System and co-immunoprecipitation assays. Our results indicate that even though Erv14p interacts with Nha1p through the TMD, the C-terminal is important not only for the efficient delivery of Nha1p to the plasma membrane but also for its dimerization to accomplish its role in yeast salt tolerance.

Cadmium and zinc activate adaptive mechanisms in Nicotiana tabacum similar to those observed in metal tolerant plants.

MAIN CONCLUSION: Tobacco germinated and grew in the presence of high concentrations of cadmium and zinc without toxic symptoms. Evidence suggests that these ions are sequestered into the vacuole by heavy metal/H + exchanger mechanisms. Heavy metal hyperaccumulation and hypertolerance are traits shared by a small set of plants which show specialized physiological and molecular adaptations allowing them to accumulate and sequester toxic metal ions. Nicotiana tabacum was used to test its potential as a metal-accumulator in a glass house experiment. Seed germination was not affected in the presence of increasing concentrations of zinc and cadmium. Juvenile and adult plants could concentrate CdCl2 and ZnSO4 to levels exceeding those in the hydroponic growth medium and maintained or increased their leaf dry weight when treated with 0.5- or 1-mM CdCl2 or 1-mM ZnSO4 for 5 days. Accumulation of heavy metals did not affect the chlorophyll and carotenoid levels, while variable effects were observed in cell sap osmolarity. Heavy metal-dependent H+ transport across the vacuole membrane was monitored using quinacrine fluorescence quenching. Cadmium- or zinc-dependent fluorescence recovery revealed that increasing concentrations of heavy metals stimulated the activities of the tonoplast Cd2+ or Zn2+/H+ exchangers. Immunodetection of the V-ATPase subunits showed that the increased proton transport by zinc was not due to changes in protein amount. MTP1 and MTP4 immunodetection and semiquantitative RT-PCR of NtMTP1, NtNRAMP1, and NtZIP1 helped to identify the genes that are likely involved in sequestration of cadmium and zinc in the leaf and root tissue. Finally, we demonstrated that cadmium and zinc treatments induced an accumulation of zinc in leaf tissues. This study shows that N. tabacum possesses a hyperaccumulation response, and thus could be used for phytoremediation purposes.

Phosphorus remobilization from rice flag leaves during grain filling: an RNA-seq study.

The physiology and molecular regulation of phosphorus (P) remobilization from vegetative tissues to grains during grain filling is poorly understood, despite the pivotal role it plays in the global P cycle. To test the hypothesis that a subset of genes involved in the P starvation response are involved in remobilization of P from flag leaves to developing grains, we conducted an RNA-seq analysis of rice flag leaves during the preremobilization phase (6 DAA) and when the leaves were acting as a P source (15 DAA). Several genes that respond to phosphate starvation, including three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a), were significantly up-regulated at 15 DAA, consistent with a role in remobilization of P from flag leaves during grain filling. A number of genes that have not been implicated in the phosphate starvation response, OsPAP26, SPX-MFS1 (a putative P transporter) and SPX-MFS2, also showed expression profiles consistent with involvement in P remobilization from senescing flag leaves. Metabolic pathway analysis using the KEGG system suggested plastid membrane lipid synthesis is a critical process during the P remobilization phase. In particular, the up-regulation of OsPLDz2 and OsSQD2 at 15 DAA suggested phospholipids were being degraded and replaced by other lipids to enable continued cellular function while liberating P for export to developing grains. Three genes associated with RNA degradation that have not previously been implicated in the P starvation response also showed expression profiles consistent with a role in P mobilization from senescing flag leaves.

Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin.

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

Cell type-specific responses to salinity - the epidermal bladder cell transcriptome of Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms.

Identification of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3.

Membrane proteins are synthesized and folded in the endoplasmic reticulum (ER), and continue their path to their site of residence along the secretory pathway. The COPII system has been identified as a key player for selecting and directing the fate of membrane and secretory cargo proteins. Selection of cargo proteins within the COPII vesicles is achieved by cargo receptors. The cornichon cargo receptor belongs to a conserved protein family found in eukaryotes that has been demonstrated to participate in the selection of integral membrane proteins as cargo for their correct targeting. Here it is demonstrated at the cellular level that rice cornichon OsCNIH1 interacts with OsHKT1;3 and, in yeast cells, enables the expression of the sodium transporter to the Golgi apparatus. Physical and functional HKT-cornichon interactions are confirmed by the mating-based split ubiquitin system, bimolecular fluorescence complementation, and Xenopus oocyte and yeast expression systems. The interaction between the two proteins occurs in the ER of plant cells and their co-expression in oocytes leads to the sequestration of the transporter in the ER. In the yeast cornichon mutant erv14, OsHKT1;3 is mistargeted, preventing the toxic effects of sodium transport in the cell observed in wild-type cells or in the erv14 mutant that co-expressed OsHKT1;3 with either OsCNIH1 or Erv14p. Identification and characterization of rice cornichon as a possible cargo receptor opens up the opportunity to improve our knowledge on membrane protein targeting in plant cells.

Progress and challenges for abiotic stress proteomics of crop plants.

Plants are continually challenged to recognize and respond to adverse changes in their environment to avoid detrimental effects on growth and development. Understanding the mechanisms that crop plants employ to resist and tolerate abiotic stress is of considerable interest for designing agriculture breeding strategies to ensure sustainable productivity. The application of proteomics technologies to advance our knowledge in crop plant abiotic stress tolerance has increased dramatically in the past few years as evidenced by the large amount of publications in this area. This is attributed to advances in various technology platforms associated with MS-based techniques as well as the accessibility of proteomics units to a wider plant research community. This review summarizes the work which has been reported for major crop plants and evaluates the findings in context of the approaches that are widely employed with the aim to encourage broadening the strategies used to increase coverage of the proteome.

Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism.

PvAMT1;1, a highly selective ammonium transporter that functions as H+/NH4(+) symporter.

One of the main forms of nitrogen assimilated by microorganisms and plants is ammonium, despite its toxicity at low millimolar concentrations. Ammonium absorption has been demonstrated to be carried out by highly selective plasma membrane-located transporters of the AMT/MEP/Rh family and characterized by the presence of a well conserved hydrophobic pore through which ammonia is proposed to move. However, uncertainties exist regarding the exact chemical species transported by these membrane proteins, which can be in the form of either hydrophobic ammonia or charged ammonium. Here, we present the characterization of PvAMT1;1 from the common bean and demonstrate that it mediates the high affinity (micromolar), rapidly saturating (1 mM) electrogenic transport of ammonium. Activity of the transporter is enhanced by low extracellular pH, and associated with this acidic pH stimulation are changes in the reversal potential and cytoplasm acidification, indicating that PvAMT1;1 functions as an H(+)/NH(4)(+) symporter. Mutation analysis of a unique histidine present in PvAMT1;1 (H125R) leads to the stimulation of ammonium transport by decreasing the K(m) value by half and by increasing the V(max) 3-fold, without affecting the pH dependence of the symporter. In contrast, mutation of the first conserved histidine within the channel modifies the properties of PvAMT1;1, increasing its K(m) and V(max) values and transforming it into a pH-independent mechanism.

Day/night regulation of aquaporins during the CAM cycle in Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum exhibits induction of Crassulacean acid metabolism (CAM) after a threshold stage of development, by exposure to long days with high light intensities or by water and salt stress. During the CAM cycle, fluctuations in carbon partitioning within the cell lead to transient drops in osmotic potential, which are likely stabilized/balanced by passive movement of water via aquaporins (AQPs). Protoplast swelling assays were used to detect changes in water permeability during the day/night cycle of CAM. To assess the role of AQPs during the same period, we followed transcript accumulation and protein abundance of four plasma membrane intrinsic proteins (PIPs) and one tonoplast intrinsic protein (TIP). CAM plants showed a persistent rhythm of specific AQP protein abundance changes throughout the day/night cycle, including changes in amount of McPIP2;1, McTIP1;2, McPIP1;4 and McPIP1;5, while the abundance of McPIP1;2 was unchanged. These protein changes did not appear to be coordinated with transcript levels for any of the AQPs analysed; however, they did occur in parrallel to alterations in water permeability, as well as variations in cell osmolarity, pinitol, glucose, fructose and phosphoenolpyruvate carboxylase (PEPc) levels measured throughout the day/night CAM cycle. Results suggest a role for AQPs in maintaining water balance during CAM and highlight the complexity of protein expression during the CAM cycle.

Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

Animal trait variation at the within-individual level: erythrocyte size variation and malaria infection in a tropical lizard.

High levels of within-individual variation (WIV) in reiterative components in plants such as leaves, flowers, and fruits have been shown to increase individual fitness by multiple mechanisms including mediating interactions with natural enemies. This relationship between WIV and fitness has been studied almost exclusively in plant systems. While animals do not exhibit conspicuous reiterative components, they have traits that can vary at the individual level such as erythrocyte size. It is currently unknown if WIV in animals can influence individual fitness by mediating the outcome of interactions with natural enemies as it has been shown in plants. To address this issue, we tested for a relationship between WIV in erythrocyte size, hemoparasite infection status, and body condition (a proxy for fitness) in a Caribbean anole lizard. We quantified the coefficient of variation of adult erythrocytes size in $n = 95$ infected and $n = 107$ non-infected lizards. We found higher degrees of erythrocyte size variation in infected lizards than in non-infected individuals. However, we found no significant relationship between infection status or erythrocyte size variation, and lizard body condition. These results suggest that higher WIV in erythrocyte size in infected lizards is not necessarily adaptive but likely a consequence of the host response to infection. Many hemoparasites destroy their host cells as part of their life cycle. To compensate, the host lizard may respond by increasing production of erythrocytes resulting in higher WIV. Our results emphasize the need to better understand the role of within-animal variation as a neglected driver or consequence of ecological and evolutionary interactions.

Ser123 is essential for the water channel activity of McPIP2;1 from Mesembryanthemum crystallinum.

The increased expression of McPIP2;1 (MipC), a root-specific aquaporin (AQP) from Mesembryanthemum crystallinum, under salt stress has suggested a role for this AQP in the salt tolerance of the plant. However, whether McPIP2;1 transports water or another solute and how its activity is regulated are so far unknown. Therefore, wild type (wt) or mutated McPIP2;1 protein was expressed in Xenopus laevis oocytes. Then, the osmotic water permeability (P(f)) of the oocytes membrane was assessed by hypotonic challenges. Selectivity of McPIP2;1 to water was determined by radiolabeled glycerol or urea uptake assays. Moreover, swelling and in vitro phosphorylation assays revealed that both water permeation and phosphorylation status of McPIP2;1 were significantly increased by the phosphorylation agonists okadaic acid (OA), phorbol myristate acetate (PMA), and 8-Br-cAMP, and markedly decreased by the inhibitory peptides PKI 14-22 and PKC 20-28, inhibitors of protein kinases A (PKA) and C (PKC), respectively. Substitution of Ser(123) or both, Ser(123) and Ser(282), abolished the water channel activity of McPIP2;1 while substitution of Ser(282) only partially inhibited it (51.9% inhibition). Despite lacking Ser(123) and/or Ser(282), the McPIP2;1 mutant forms were still phosphorylated in vitro, which suggests that phosphorylation may have a dual role on this AQP. Our results indicate that McPIP2;1 water permeability depends completely on Ser(123) and is positively regulated by PKA- and PKC-mediated phosphorylation. Regulation of the phosphorylation status of McPIP2;1 may contribute to control water transport through root cells when the plant is subjected to high salinity conditions.

Recent Advances in the Physiology of Ion Channels in Plants.

Our knowledge of plant ion channels was significantly enhanced by the first application of the patch-clamp technique to isolated guard cell protoplasts over 35 years ago. Since then, research has demonstrated the importance of ion channels in the control of gas exchange in guard cells, their role in nutrient uptake in roots, and the participation of calcium-permeable cation channels in the regulation of cell signaling affected by the intracellular concentrations of this second messenger. In recent years, through the employment of reverse genetics, mutant proteins, and heterologous expression systems, research on ion channels has identified mechanisms that modify their activity through protein-protein interactions or that result in activation and/or deactivation of ion channels through posttranslational modifications. Additional and confirmatory information on ion channel functioning has been derived from the crystallization and molecular modeling of plant proteins that, together with functional analyses, have helped to increase our knowledge of the functioning of these important membrane proteins that may eventually help to improve crop yield. Here, an update on the advances obtained in plant ion channel function during the last few years is presented.

A differential subcellular localization of two copper transporters from the COPT family suggests distinct roles in copper homeostasis in Physcomitrium patens.

The moss Physcomitrium (Physcomitrella) patens is a bryophyte that provides genetic information about the adaptation to the life on land by early Embryophytes and is a reference organism for comparative evolutionary studies in plants. Copper is an essential micronutrient for every living organism, its transport across the plasma membrane is achieved by the copper transport protein family COPT/CTR. Two genes related to the COPT family were identified in Physcomitrella patens, PpaCOPT1 and PpaCOPT2. Homology modelling of both proteins showed the presence of three putative transmembrane domains (TMD) and the Mx3M motif, constituting a potential Cu + selectivity filter present in other members of this family. Functional characterization of PpaCOPT1 and PpaCOPT2 in the yeast mutant ctr1Δctr3Δ restored its growth on medium with non-fermentable carbon sources at micromolar Cu concentrations, providing support that these two moss proteins function as high affinity Cu + transporters. Localization of PpaCOPT1 and PpaCOPT2 in yeast cells was observed at the tonoplast and plasma membrane, respectively. The heterologous expression of PpaCOPT2 in tobacco epidermal cells co-localized with the plasma membrane marker. Finally, only PpaCOPT1 was expressed in seven-day old protonema and was influenced by extracellular copper levels. This evidence suggests different roles of PpaCOPT1 and PpaCOPT2 in copper homeostasis in Physcomitrella patens.