Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process

Autophagy in plant cells is induced by nutrient starvation. Initially, double membrane-bound organelles, termed autophagosomes, enclose a portion of cytoplasm, and then fuse with a vacuole or lysosome to give an autolysosome. Autolysosomes can be visualized by incubating cells in the presence of a membrane-permeable cysteine protease inhibitor. The inhibitor presumably decreases proteolytic degradation of the autolysosome contents that are composed of portions of cytoplasm enclosed by the membrane originating from the inner membrane of autophagosomes, and allows them to accumulate. The origin of membranes that give rise to autophagosomes and autolysosomes is unknown. Here we use an acidotropic fluorescent dye, LysoTracker Red, to label autolysosomes specifically. We demonstrate that autolysosome membranes are marked by the presence of alpha-tonoplast intrinsic protein (alpha-TIP) but not by gamma-TIP or delta-TIP. The identification of a TIP specifically associated with membranes derived from an autophagic process may help our understanding of how plant cells generate and maintain functionally distinct types of vacuoles.     

Evaluation of the Edman degradation product of vancomycin bonded to core‐shell particles as a new HPLC chiral stationary phase

A modified macrocyclic glycopeptide‐based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide‐based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide‐based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core‐shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography‐mass spectrometry with EDP was performed in approximately 3 minutes. Other highlights include simultaneous liquid chromatography separations of rac‐amphetamine and rac‐methamphetamine with VancoShell, rac‐pseudoephedrine and rac‐ephedrine with NicoShell, and rac‐dichlorprop and rac‐haloxyfop with TeicoShell.     

Caleosins: Ca2+-binding proteins associated with lipid bodies

We have previously identified a rice gene encoding a 27 kDa protein with a single Ca²⁺-binding EF-hand and a putative membrane anchor. We report here similar genes termed caleosins, CLO, in other plants and fungi; they comprise a multigene family of at least five members in Arabidopsis (AtClo1–5). Northern hybridization demonstrated that AtClo2–4 mRNAs levels were low in various tissues, while AtClo1 mRNA levels were high in developing embryos and mature seeds. Analysis of transgenic Arabidopsis plants expressing the GUS reporter under control of the AtClo1 promoter showed strong levels of expression in developing embryos and also in root tip cells. Antibodies raised against AtCLO1 were used to detect caleosin in cellular fractions of Arabidopsis and rapeseed. This indicated that caleosins are a novel class of lipid body proteins, which may also be associated with an ER subdomain.     

Tonoplast intrinsic protein isoforms as markers for vacuolar functions

Plant cell vacuoles may have storage or lytic functions, but biochemical markers specific for the tonoplasts of functionally distinct vacuoles are poorly defined. Here, we use antipeptide antibodies specific for the tonoplast intrinsic proteins alpha-TIP, gamma-TIP, and delta-TIP in confocal immunofluorescence experiments to test the hypothesis that different TIP isoforms may define different vacuole functions. Organelles labeled with these antibodies were also labeled with antipyrophosphatase antibodies, demonstrating that regardless of their size, they had the expected characteristics of vacuoles. Our results demonstrate that the storage vacuole tonoplast contains delta-TIP, protein storage vacuoles containing seed-type storage proteins are marked by alpha- and delta- or alpha- and delta- plus gamma-TIP, whereas vacuoles storing vegetative storage proteins and pigments are marked by delta-TIP alone or delta- plus gamma-TIP. In contrast, those marked by gamma-TIP alone have characteristics of lytic vacuoles, and results from other researchers indicate that alpha-TIP alone is a marker for autophagic vacuoles. In root tips, relatively undifferentiated cells that contain vacuoles labeled separately for each of the three TIPs have been identified. These results argue that plant cells have the ability to generate and maintain three separate vacuole organelles, with each being marked by a different TIP, and that the functional diversity of the vacuolar system may be generated from different combinations of the three basic types.     

Functional characterization of ice plant SKD1, an AAA-type ATPase associated with the endoplasmic reticulum-Golgi network, and its role in adaptation to salt stress

A salt-induced gene mcSKD1 (suppressor of K+ transport growth defect) able to facilitate K+ uptake has previously been identified from the halophyte ice plant (Mesembryanthemum crystallinum). The sequence of mcSKD1 is homologous to vacuolar protein sorting 4, an ATPase associated with a variety of cellular activities-type ATPase that participates in the sorting of vacuolar proteins into multivesicular bodies in yeast (Saccharomyces cerevisiae). Recombinant mcSKD1 exhibited ATP hydrolytic activities in vitro with a half-maximal rate at an ATP concentration of 1.25 mm. Point mutations on active site residues abolished its ATPase activity. ADP is both a product and a strong inhibitor of the reaction. ADP-binding form of mcSDK1 greatly reduced its catalytic activity. The mcSKD1 protein accumulated ubiquitously in both vegetative and reproductive parts of plants. Highest accumulation was observed in cells actively engaging in the secretory processes, such as bladder cells of leaf epidermis. Membrane fractionation and double-labeling immunofluorescence showed the predominant localization of mcSKD1 in the endoplasmic reticulum-Golgi network. Immunoelectron microscopy identified the formation of mcSKD1 proteins into small aggregates in the cytosol and associated with membrane continuum within the endomembrane compartments. These results indicated that this ATPase participates in the endoplasmic reticulum-Golgi mediated protein sorting machinery for both housekeeping function and compartmentalization of excess Na+ under high salinity.     

Microspectrophotometry of mitochondrial cytochrome P-450 in single adrenal cells

Synopsis The cytochrome P-450 is known to be a key enzymic component in steroid hydroxylation. Biochemically it is localized in adrenal cells, both in the mitochondrial and in the microsomal fractions. Owing to its characteristic light absorptivity, attempts were made at its localization and measurement in a microspectrophotometric system. Specific difference spectra were obtained in the cytoplasm of some cells from an adrenal cell suspension before and after saturation with carbon monoxide. Scanning at 450 nm showed slender absorption maxima after CO saturation randomly distributed in the cytoplasm. These may be attributed to mitochondria or larger cytoplasmic functional units including mitochondria.     

Actin in mung bean mitochondria and implications for its function

Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin-green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography-tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.     

Lily cofactor-independent phosphoglycerate mutase: purification, partial sequencing, and immunolocalization

A cofactor-independent phosphoglycerate mutase (PGAM-i) was isolated to homogeneity from monocotyledonous Lilium longiflorum Thunb. Two-dimensional (2D) polyacrylamide gel electrophoresis resolved three PGAM-i forms. This enzyme was originally identified by cross-reactivity to antibodies affinity-purified from 2D gels using human vitronectin (VN). Antibody produced against a denatured protein spot from a 2D gel did not recognize VN protein, but partial protein and DNA sequencing showed similarity of the former protein to maize PGAM-i. Immunoblots from roots, styles, leaves, and anthers showed the presence of PGAM-i in all tissues examined; it was isolated predominantly from the soluble cell fraction, with some present in the insoluble cell fraction. Immunoelectron microscopy demonstrated its localization in the cytoplasm and plastids in root cells near the apical meristem. In addition, immunogold labeling detected signals from the nucleus. The immunohistochemical localization of the enzyme in the nucleus, as well as in the cytosol and plastids, indicates that lily PGAM-i might have multiple functions in the cell.Abbreviations ELISA enzyme-linked immunosorbent assay - PGAM cofactor-dependent phosphoglycerate mutase - PGAM-i cofactor independent phosphoglycerate mutase - TEM transmission electron microscopy - 2D two dimensional - VN vitronectin     

Evapotranspiration of annual and perennial biofuel crops in a variable climate

Eddy covariance measurements were made in seven fields in the Midwest USA over 4 years (including the 2012 drought year) to estimate evapotranspiration (ET) of newly established rain‐fed cellulosic and grain biofuel crops. Four of the converted fields had been managed as grasslands under the USDA's Conservation Reserve Program (CRP) for 22 years, and three had been in conventional agriculture (AGR) soybean/corn rotation prior to conversion. In 2009, all sites were planted to no‐till soybean except one CRP grassland that was left unchanged as a reference site; in 2010, three of the former CRP sites and the three former AGR sites were planted to annual (corn) and perennial (switchgrass and mixed‐prairie) grasslands. The annual ET over the 4 years ranged from 45% to 77% (mean = 60%) of the annual precipitation (848–1063 mm; November–October), with the unconverted CRP grassland having the highest ET (622–706 mm). In the fields converted to annual and perennial crops, the annual ET ranged between 480 and 639 mm despite the large variations in growing‐season precipitation and in soil water contents, which had strong effects on regional crop yields. Results suggest that in this humid temperate climate, which represents the US Corn Belt, water use by annual and perennial crops is not greatly different across years with highly variable precipitation and soil water availability. Therefore, large‐scale conversion of row crops to perennial biofuel cropping systems may not strongly alter terrestrial water balances.