Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

Summary Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.Abbreviations ATP adenosine 5-triphosphate - BCIP 5-bromo-4-chloro-3-indolyl phosphate - BSA bovine serum albumin - CCD counter-current distribution - DTT dithiothreitol - EDTA ethylene-diamine-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) - GSII 1,3--glucan synthase - HEPES N-[2-hydroxyethyl]-piperazine-N-[2-ethane sulfonic acid] - MES 2-(N-morpholino)ethane sulfonic acid - NBT p-nitro blue tetrazolium chloride - IDP inosine 5-diphosphate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - PPB potassium phosphate buffer - PM plasma membrane - PMSF phenylmethylsulfonyl fluoride - PVDF polyvinylidene difluoride - PVPP polyvinylpolypyrrolidone - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TTBS Tris-buffered saline with Tween 20 - Tris tris(hydroxymethyl) aminomethane     

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.     

Sucrose-phosphate synthase activity and yield analysis of tomato plants transformed with maize sucrose-phosphate synthase

Sucrose synthesis is a major element of the interactions between photosynthesis and plant growth and development. Tomato (Lycopersicon esculentum Mill. cv. UC82B) plants transformed with maize sucrose-phosphate synthase (SPS; EC 2.3.1.14) expressed from either a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit promoter (SSU) or the cauliflower mosaic virus 35S promoter (35S) were used to study effects of increased sucrose synthesis rates on plant growth. The plants were grown in growth chambers, field plots, and open-top chambers. The 35S plants had a 2 to 3-fold increase in young-leaf SPS activity, a 10 to 20-fold increase in young-root SPS activity and no increase in young-fruit SPS activity. The leaf SPS activity in one of the 35S lines fell to control levels by two months of age. The SSU plants had a 4 to 5-fold increase in leaf SPS activity and no significant increase in root or young-fruit SPS activity. One 35S line, which maintained high leaf SPS activity throughout development, yielded 70–80% more than controls at both normal and elevated CO₂ in open-top chambers in the field and 20–30% more than controls in two additional field trials. The other 35S line and the two SSU lines either yielded less or did not differ from controls under several growth conditions. Since only one of four transformed lines showed an increase in yield, we can not yet conclude that increased leaf SPS activity leads to increased yield. However, increased leaf SPS activity appears to result in increased fruit sugar content since all three lines with increased leaf SPS usually also had increased fruit sugars. Received: 18 November 1996 / Accepted: 22 January 1997     

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.     

Influence of differences in nutrition on important quality characteristics of some agricultural crops

Summary Over a period of several years, quality characteristics of plant products have been studied intensively. Next to variety, climate and location, the types and amounts of fertilizers used appeared to exert strong influences on quality parameters. In recent research it could be shown that especially N-, but also P-, K- and Mg-supply affected dry matter content and furthermore starch, crude protein, amino acids, nitrate, sugars and citric acid in cauliflower, lettuce, potato, spinach and tomato. Altogether the results showed that the influence of variations in nutrient supply on the quality of important compounds in usable plant parts is relatively high, but that different plants react not similarly to increasing or decreasing amounts of nutrients. These relations are of special importance when the qualitative and nutritive values of plant products are evaluated for consumption and processing purposes.     

Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplasts

Summary Deletions were made in the cauliflower mosaic virus polyadenylation sequence which was cloned downstream of the -glucuronidase gene (gus). The populations of mRNAs generated in tobacco mesophyll protoplasts by transient expression with the various constructs were analysed using a polymerase chain reaction procedure. When no deletion was present in the sequence, the mRNA appeared to be polyadenylated at two major polyadenylation sites. A deletion upstream from the AATAAA sequence made the population of polyadenylated mRNAs very heterogenous at their 3 ends. A deletion downstream of the AATAAA sequence had no effect on the choice of the site. Alternative polyadenylation sites were used when the native polyadenylation site was deleted. These results are discussed in relation to data obtained with other polyadenylation sequences from both plants and animals.     

Genome-wide identification of cytosine-5 DNA methyltransferases and demethylases in Solanum lycopersicum

Recent studies have reported that decreased level of DNA cytosine methylation in the global genome was closely related to the initiation of tomato (Solanum lycopersicum) fruit ripening. However, genome-scale analysis of cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in tomato has not been engaged. In this study, 7 C5-MTases and 3 demethylases were identified in tomato genome, which probably contributed to DNA cytosine methylation level in tomato. The 7 C5-MTases were categorized into 4 subgroups, and the 3 demethylases were classified into 2 subgroups based on phylogenetic analyses. Comprehensive analysis of their structure and genomic localization was also performed in this paper. According to online RNA-seq data, 4 S. lycopersicum C5-MTase (SlC5-MTase) genes (SlMET, SlDRM1L1, SlDRM5, SlMET3L) were expressed higher than others, and one DNA demethylase gene (SlDML) was significantly changed during tomato fruit development and ripening. Furthermore, all these five gene expressions at breaker (BK) stage changed with 1-methylcyclopropene (1-MCP) treatment, indicating that they were regulated by ethylene directly or indirectly in tomato fruit. In addition, subcellular localization analysis indicated that SlDRM1L1 and SlDRM5 located in the nucleus might have responsibility for RNA-directed DNA methylation (RdDM). Collectively, this paper provided a framework for gene discovery and functional characterization of C5-MTases and DNA demethylases in other Solanaceae species.     

Sources of resistance to downy mildew (<Emphasis Type="Italic">Peronospora parasitica</Emphasis> (Pers. (ex Fr.) Fr.) in Sicilian germplasm of cauliflower and broccoli

Downy mildew (Peronospora parasitica (Pers. ex Fr.) Fr.) is a serious disease of brassicas in several countries. Seedlings are very susceptible to this pathogen and crops require frequent fungicide treatments to reach a good marketable yield. The use of resistant cultivars can be the most economical, reliable and environmental friendly method for managing this disease. In this work 32 Sicilian landraces and 16 commercial cultivars of cauliflower and broccoli (B. oleracea) were screened for downy mildew resistance at the cotyledon stage using one P. parasitica strain from Portugal and one from Sicily (Italy). Seven-day old seedlings were inoculated by deposing a droplet of a spore suspension on the cotyledons, incubated under controlled environment and scored 7 days later using a seven-class scale of interaction phenotype (IP), which took into consideration host response and pathogen sporulation. There were no differences in virulence between the two P. parasitica isolates. Accessions ranged from very susceptible to highly resistant to downy mildew showing a variable number of resistant individuals per accession. Forty accessions were very susceptible to downy mildew and are of no interest as sources of resistance, since most of the seedlings were scored in the most susceptible IP classes. Seven accessions had intermediate resistance and included individuals that expressed some degree of resistance. Accession Cv 90 (Cavolfiore Torino) and Br 63 (Sparaceddu) showed the majority of seedlings in the resistant IP classes and may constitute valuable sources of resistance to downy mildew to be used in breeding programs.     

A new plant protein interacts with eIF3 and 60S to enhance virus‐activated translation re‐initiation

The plant viral re‐initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re‐initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ;Here, we report a new plant factor—re‐initiation supporting protein (RISP)—that enhances TAV function in re‐initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C‐terminus of ribosomal protein L24. RISP pre‐bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co‐localize in epidermal cells of infected plants, and eIF3–TAV–RISP–L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3‐bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery.