In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro.     

Transmitting Tissae and Pollen Tube Growth in Sweet Pepper

Fine structural events in the mature stylar transmitting tissue ofsweet pepper (Capsicum frutescens L. var. grossum Bally) have been investigated byelectron microscope with regard to pollen tribe grwoth. The transmitting tissue consists of parenchymatous cells with large intercellular spaces filled with an electron densesubstance. The pollen tubes grow through the intercellular spaces in the intercellular substance. Cells of this tissue are rich in organelles, especially the rough and tubular ER, and numerous lomasomes near the plasma membrane. It is demonstrated that they function as secretory cells. On the other hand, the fact that the transmitting tissue contains many large amyloplastids with several starch grains in the cytoplasm and numerous globular protein bodies in vacuoles, indicates that the transmitting tissue mayhave some nutritive value for the growth of pollen tubes. The results obtained from this observation are in agreement with those of mostspecies reported by other authors and support the conclusion that the transmitting tissue is not a collenchyma tissue and the nature of intercellular substances is essentially same as that of the middle lamella.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

Characterization of cDNAs for stylar transmitting tissue-specific proline-rich proteins in tobacco

The pistil of flowers is a specialized organ which contains the female gametophytes and provides the structures necessary for pollination and fertilization. Pollen deposited on the stigmatic surface of a compatible plant germinates a pollen tube which penetrates the stigmatic papillae and grows intercellularly through the style towards the ovules in the ovary. Pollen tube growth is largely restricted to the transmitting tissue in the style. Therefore the stylar transmitting tissue is extremely important for the migration of the pollen cell towards the ovary. We have isolated two related cDNAs, transmitting tissue-specific (TTS)-1 and TTS-2, derived from two proline-rich protein (PRP)-encoding mRNAs that accumulate specifically in the transmitting tissue of tobacco. The deduced PRP sequences share similarities with proline-rich cell wall glycoproteins found in a variety of plants. TTS-1 and TTS-2 mRNAs are induced in very young floral buds, accumulate most abundantly during the later stages of flower development when style elongation is the most rapid, and remain at relatively high levels at anthesis. These mRNAs become undetectable in maturing green fruits. In situ hybridization shows that TTS-1 and TTS-2 mRNA accumulation is restricted to the transmitting tissue of the style. The possible roles that these transmitting tissue-specific PRPs may play in maintaining the structural integrity of the style or in the function of this organ is discussed.     

VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.     

Two differentially regulated class II chitinases from parsley

Two distinct cDNA clones, PcCHI1 and PcCHI2, with high sequence similarity to plant chitinases were isolated from parsley (Petroselinum crispum), expressed in Escherichia coli, and the encoded proteins functionally identified as endochitinases. Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development. Infection of parsley leaf buds with Phytophthora sojae resulted in the rapid, transient and highly localized accumulation of PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 mRNA and protein were systemically induced at later infection stages. Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells. In uninfected plants, PcCHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth. Localization of PcCHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue. In addition to such distinct roles of PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.     

Direct evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots

An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.     

Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.     

Temporal and transient expression of olive enoyl-ACP reductase gene during flower and fruit development.

Enoyl-ACP reductase is a catalytic component of the fatty acid synthetase (FAS) type II system in plants that is involved in the de novo fatty acid biosynthesis in plastids. A cDNA encoding an enoyl-ACP reductase responsible for the removal of the trans-unsaturated double bonds to form saturated acyl-ACP has been isolated from a library made from ripening fruits of Olea europaea L. The predicted protein contains 393 amino acid residues including a consensus chloroplast specific transit peptide. A strong homology was observed when olive enoyl-ACP reductase aligned with other plant sequences. Southern hybridization analysis revealed that enoyl-ACP reductase is encoded by a single gene in olives. Northern hybridization showed a transient expression of the enoyl-ACP reductase (ENR) gene at early stages of drupe (5-7 weeks after flowering, WAF), embryo and endosperm (13-16 WAF) while in mesocarp (13-19 WAF) the expression remained at high levels. In situ hybridization showed particularly prominent expression in the palisade and vascular tissue of young leaves, the tapetum, developing pollen grains and vascular tissue of anthers and to less extent in the embryo sac and transmitting tissue of the carpel. The distinctive spatial and temporal regulation of the ENR gene is consistent with major roles, not only in thylakoid membrane formation and fatty acid deposition, but also in the provision of precursor molecules for the biosynthesis of oxilipins that are important in plant tissues involved in transportation and reproduction.     

A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation factor-1 alpha.

Higher plant proteins immunologically related to the animal substrate adhesion molecule vitronectin have recently been observed and implicated in a variety of biological processes, such as plasma membrane-cell wall adhesion, pollen tube extension, and bacterium-plant interaction. We provide evidence that, similar to vitronectin, one of these proteins, PVN1 (plant vitronectin-like 1), isolated from 428 mM NaCl-adapted tobacco cells binds to glass surfaces an heparin. PVN1 was isolated by glass bead affinity chromatography. Isolated PVN1 has adhesive activity based on results from a baby hamster kidney cell-spreading assay. This plant adhesion protein was detected in all tissues examined but was most abundant in roots and salt-adapted cultured cells. Immunogold labeling indicated that PVN1 is localized in the cell wall of cortical and transmitting tissue cells of pollinated mature styles. A partial amino acid sequence of PVN1 revealed no similarity with vitronectin but, instead, was nearly identical to the translational elongation factor-1 alpha (EF-1 alpha). A clone isolated by screening a tobacco cDNA expression library with anti-PVN1 encoded a protein with greater than 93% identity to sequences of EF-1 alpha from plants of numerous species. Immunological cross-reactivity between tobacco PVN1 and EF-1 alpha as well as the reaction between the EF-1 alpha antibody and the 65- and 75-kD vitronectin-like proteins of a fucoidal alga supported the conclusion that the plant extracellular adhesion protein PVN1 is related to EF-1 alpha.     

Adipocyte triglyceride lipase expression in human obesity

We have investigated the gene and protein expression of adipose triglyceride lipase (ATGL) and triglyceride (TG) lipase activity from subcutaneous and visceral adipose tissue of lean and obese subjects. Visceral and subcutaneous adipose tissue was obtained from 16 age-matched lean and obese subjects during abdominal surgery. Tissues were analyzed for mRNA expression of lipolytic enzymes by real-time quantitative PCR. ATGL protein content was assessed by Western blot and TG lipase activity by radiometric assessment. Subcutaneous and visceral adipose tissue of obese subjects had elevated mRNA expression of PNPLA2 (ATGL) and other lipases including PNPLA3, PNPLA4, CES1, and LYPLAL1 (P < 0.05). Surprisingly, ATGL protein expression and TG lipase activity were reduced in subcutaneous adipose tissue of obese subjects. Immunoprecipitation of ATGL reduced total TG lipase activity in adipose lysates by 70% in obese and 83% in lean subjects. No significant differences in the ATGL activator CGI-58 mRNA levels (ABHD5) were associated with obesity. These data demonstrate that ATGL is important for efficient TG lipase activity in humans. They also demonstrate reduced ATGL protein expression and TG lipase activity despite increased mRNA expression of ATGL and other novel lipolytic enzymes in obesity. The lack of correlation between ATGL protein content and in vitro TG lipase activity indicates that small decrements in ATGL protein expression are not responsible for the reduction in TG lipase activity observed here in obesity, and that posttranslational modifications may be important.     

A Simple Method for In Vivo Expression Studies of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.     

Pollination induces mRNA poly(A) tail-shortening and cell deterioration in flower transmitting tissue

Pollination induces many physiological responses in the flower, including deterioration and death in specific pistil cell types. It is shown here that within the style of tobacco, pollination-induced cell deterioration was restricted to the transmitting tissue while the surrounding cortical tissue was not affected. It was distinct from general senescence since exogenously applying the senescence-inducing hormone ethylene, or its precursor aminocyclopropane-1-carboxylic acid (ACC), to the flower or the pistil induced overall deterioration in the entire flower. Furthermore, both pollen tube growth and ethylene action were needed for the entire spectrum of cellular changes associated with this pollination-induced transmitting tissue deterioration process. It is also shown that pollination-induced mRNA poly(A) tail-shortening for at least three major classes of transmitting tissue-specific mRNAs. As is commonly observed for poly(A) tail-shortened mRNAs, the levels of two of these three mRNA classes decline after pollination. On the other hand, the third class of mRNAs, transmitting tissue-specific (TTs) mRNAs, was maintained at a very high level subsequent to pollination, even after substantial poly(A)-tail shortening. TTS mRNAs encode a pollen tube growth-promoting and -attracting protein needed for optimal in vivo pollen tube growth. The specific preservation of TTS mRNAs in the deteriorating transmitting tissue cells suggests that these cells can distinguish molecules needed in the pollinated styles from those that are dispensable, and protect them from degradation. It is suggested that the pollination-induced mRNA poly(A) tail-shortening and cell death are programmed processes suited to the post-pollination transmitting tissue environment. Results showing that ACC is a candidate signal molecule for the pollination-induced mRNA-shortening which is accentuated by ethylene and mediated via a protein phosphorylation-dependent signal transduction pathway are also presented.     

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.     

Arabidopsis genes encoding mitochondrial type II NAD(P)H dehydrogenases have different evolutionary origin and show distinct responses to light

In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.     

Cyclic AMP as an intraspheroidal differentiation signal in Volvox carteri

The action of the macromolecular inducer glycoprotein on sexual reproduction in the green alga Volvox carteri can be modified by altering the external (intraspheroidal) cAMP concentration. Direct proof for the presence of cAMP in the spheroids is given. Protein binding assay and HPLC-fluorimetric analysis independently demonstrate the existence of cAMP in the matrix, cells, and culture medium. Its concentration is higher in sexual cultures, pointing to a transmitting function in sex induction. The presence in the matrix of other members of a protein phosphorylation system suggests an induction-specific signal cascade in this plant.