Large-scale analysis of mRNA translation states during sucrose starvation in arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control.

Sucrose starvation of Arabidopsis (Arabidopsis thaliana) cell culture was used to identify translationally regulated genes by DNA microarray analysis. Cells were starved by subculture without sucrose, and total and polysomal RNA was extracted between 6 and 48 h. Probes were derived from both RNA populations and used to screen oligonucleotide microarrays. Out of 25,607 screened genes, 224 were found to be differentially accumulated in polysomal RNA following starvation and 21 were found to be invariant in polysomal RNA while their total RNA abundance was modified. Most of the mRNA appears to be translationally repressed (183/245 genes), which is consistent with a general decrease in metabolic activities during starvation. The parallel transcriptional analysis identifies 268 regulated genes. Comparison of transcriptional and translational gene lists highlights the importance of translational regulation (mostly repression) affecting genes involved in cell cycle and cell growth, these being overrepresented in translationally regulated genes, providing a molecular framework for the arrest of cell proliferation following starvation. Starvation-induced translational control also affects chromatin regulation genes, such as the HD1 histone deacetylase, and the level of histone H4 acetylation was found to increase during starvation. This suggests that regulation of the global nuclear transcriptional activity might be linked to cytoplasmic translational regulations.

Molecular characterization of L2 lipoxygenase from maize embryos.

We investigated the expression and accumulation pattern of lipoxygenase isoforms throughout the maize plant life. Two forms of lipoxygenase L1 and L2 have been identified as acidic proteins of 100 kDa (pI 6.4) and 90 kDa (pI 5.5-5.7) which accumulate in dry embryos and in various organs of maize seedlings. In young embryos, only the L2 form was detected and accumulation of L2 mRNA decreased during embryo development. Identification of lipoxygenases from in vivo and in vitro synthesized proteins indicates that similar levels of both L1 and L2 forms accumulated during treatment with abscisic acid, (ABA) gibberellic acid (GA3) and jasmonic acid (JA). However, differences in the activity of both enzymes were detected. By using an antiserum directed against purified L2 we isolated and characterized a partial cDNA clone of maize embryos encoding a lipoxygenase. The deduced amino acid sequence of L2 cDNA shares 78% identity with the rice L2 protein, and 51-56% identity with lipoxygenases from the dicotyledonous plants soybean and Arabidopsis. DNA blot analysis indicated that maize contains a family of lipoxygenase genes which are presently being characterized.

Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings.

Oxalate oxidase activity was detected in situ during the development of barley seedlings. The presence of germin-like oxalate oxidase was confirmed by immunoblotting using an antibody directed against wheat germin produced in Escherichia coli, which is shown to cross-react with barley (Hordeum vulgare) oxalate oxidase and by enzymatic assay after electrophoresis of the protein extracts on polyacrylamide gels. In 3-d-old barley seedlings, oxalate oxidase is localized in the epidermal cells of the mature region of primary roots and in the coleorhiza. After 10 d of growth, the activity is detectable only in the coleorhiza. Moreover, we show that oxalate oxidase is induced in barley leaves during infection by the fungus Erysiphe graminis f. sp. hordei but not by wounding. Thus, oxalate oxidase is a new class of proteins that responds to pathogen attack. We propose that oxalate oxidase could have a role in plant defense through the production of H2O2.

Genotypic variation of quantitative trait loci controlling in vitro androgenesis in maize.

A quantitative trait loci (QTL) analysis for androgenetic capability has been conducted on three different crosses in maize, including very high and nonresponding lines for androgenesis. The doubled haploid lines derived by anther culture from the crosses DH5 x DH7, A188 x DH7, and R6 x DH99 showed a range of 0-70%, 0-40%, and 0-50% androgenetic responding anthers, respectively. The genotypic heritability of means for this trait is close to 0.90 for A188 x DH7 and 0.78 for R6 x DH99. The QTL analysis involved in each population the mapping of more than 100 loci covering a large part of the genome with reasonably spaced markers averaging 12 cM. Different measurements describing the androgenetic process were studied: AC, percentage of responding anthers; ELS, number of androgenetic embryos produced per 100 plated anthers; PLE, number of plantlets regenerated per 100 embryos; PLA, number of plantlets per 100 plated anthers. In each cross, three to four QTLs were found for AC, explaining 30-40% of the phenotypic variation. The QTL detected for PLA was also strong QTL for AC or ELS. This agrees with the observation that these last two traits are good predictors for final plantlet yield. The QTLs found were specific, although the same line DH7 was used in two crosses and DH99 derived from DH5 and DH7 in the third cross. These results suggest that the transfer of the androgenetic capabilities in elite germplasm will still involve a phenotypic evaluation of the androgenetic performances. A backcross-assisted selection based only on the genotype at the QTL is probably possible but only within the crosses used for this QTL analysis.

Identification of barley oxalate oxidase as a germin-like protein.

A barley oxalate oxidase was purified to homogeneity and the N-terminal sequences of the protein and of two peptides generated by CNBr cleavage of this protein were determined. Searches for similarities in data bank revealed that the sequences are highly homologous to the amino-acid sequence of a wheat protein, germin, which is synthesized de novo during germination. The similarity of the two proteins was confirmed by showing that anti-oxalate oxidase antibodies strongly recognize germin produced in Escherichia coli. We show that like germin, oxalate oxidase is glycosylated, resistant to SDS denaturation, heat stable, and protease resistant. Moreover, oxalate oxidase activity is strongly induced during germination of barley embryos resulting from an accumulation of the protein. Thus, we conclude that barley oxalate oxidase is a germin-like protein.

Expression of Chloroplast and Mitochondrial Genes during Microsporogenesis in Maize.

Mitochondrial and plastid gene expression has been examined during maize (Zea mays) microsporogenesis. Accumulation of transcripts was found for three mitochondrial genes studied (cob, atp6, and atp9) at the mid-term of pollen development. In contrast, these mitochondrial transcripts were undetectable in mature pollen. Southern and DNA gel blot experiments showed that the copy number of mitochondrial genes was amplified in microspores at stages preceding the accumulation of these transcripts. Plastid transcripts of the photosynthetic psbA and rbcL genes could not be detected after the two mitoses, whereas precursors of the 16S rRNA are detected at low levels.

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum.

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45 degrees C. Activities of both enzymes were inhibited by Hg, Zn, Cu, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn and Co. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.

Subfamilies of histone H3 and H4 genes are located on most, possibly all of the chromosomes in maize.

It has been previously shown that in the genome of maize the multiple copies of the histone H3 and H4 multigenic families are organized into eight to ten subfamilies each containing a variable number of copies. Each subfamily is characterized by a specific proximal environment and thus can be revealed by blot-hybridization with its specific 5' probe. Restriction fragment length polymorphism (RFLP) combined with monosomic analysis was used to localize several H3 and H4 subfamilies on maize chromosomes. H3 and H4 genes were found to be located on most, possibly all of the chromosomes, revealing a remarkably dispersed organization of these multigenic families.

Regulation of the maize rab17 gene promoter in transgenic heterologous systems.

The maize rab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardell et al., Plant Mol Biol 14 (1990) 423-432). Here we demonstrate that 5' upstream sequences of the rab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5' upstream fragment of rab17 (-1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused to rab17 promoter deletions indicate that a 300 bp DNA fragment (-351/-102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (-219/-102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.

Nucleotide sequence and characterization of a maize cytoplasmic ribosomal protein S11 cDNA.

We isolated a Zea mays cDNA encoding the 40S subunit cytoplasmic ribosomal protein S11. The nucleotide sequence was determined and the derived amino acid sequence compared to the corresponding Arabidopsis thaliana protein showing an homology of 90%. This ribosomal protein is encoded by a small multigene family of at least two members. The mRNA steady-state level is about one order of magnitude higher in rapidly growing parts of the plant such as the roots and shoots of seedlings compared to fully expanded leaf tissue.

Production of Cell Wall-Degrading Enzymes by the Phytopathogenic Fungus Sclerotinia sclerotiorum.

The range of polysaccharide-degrading enzymes and glycosidases formed by the phytopathogenic fungus Sclerotinia sclerotiorum was monitored following growth on 16 carbohydrate substrates. Endo- and exoenzymes capable of degrading cellulosic, hemicellulosic, and pectinolytic polysaccharides were secreted. Pectinolytic activities were produced constitutively on all of the substrates tested. Cellulolytic enzymes were not induced in simple sugar (i.e., glucose or xylose) media. Polysaccharide growth substrates and cellulase inducers increased all of the enzyme activities tested. Gel filtration analysis revealed the appearance of new molecular forms of pectinase, beta-xylosidase, and cellobiosidase during induction on pectin and carboxymethyl cellulose media.

Identification of an RFLP marker tightly linked to theHt1 gene in maize.

We have identified tight linkage of an RFLP marker to theHt1 gene of maize that confers resistance to the fungal pathogenHelminthosporium turcicum race 1. This was accomplished by the use of four pairs of near isogenic lines (NILs; B73, A619, W153R, and CM105), each differing by the presence or the absence of the geneHt1. SinceHt1 maps to chromosome 2, 26 clones already mapped to this chromosome were labeled and probed against Southern blots of these NILs DNA digested with three restriction enzymes:EcoRI,BamHI, andHindIII. Six markers exhibited an RFLP for at least one pair of NILs. Presumptive linkage was further tested by analyzing the segregation of five of the six markers (one was monomorphic in the cross studied) and resistance toH. turcicum race 1 on 95 F2 individuals from the cross DF20 × LH146Ht. The results indicate a tight linkage between one of the DNA markers,UMC150B, and theHt1 gene.

Production of isolated somatic embryos from sunflower thin cell layers.

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.

Transformed calli obtained by direct gene transfer into sunflower protoplasts.

Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 10(6) treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.

Callus and embryoid formation from protoplasts of Helianthus annuus.

Sunflower hypocotyl protoplasts have been isolated and cultured. Optimum plating density for cell division and colony formation was in the range of 5 to 7×10(4) cells/mi in an agarose medium supplemented with BAP (1 mg/l) and NAA (1 mg/l). Plating efficiency was 60% after 21 days of culture. In the resultant culture a mixed population of calli and embryoids was observed. Thirty seven percent of the cell clusters exhibited a developmental pattern similar to an embryoid. Many stages of embryogenesis were observed in the same cultures.

Amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit from Euglena.

The amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (SSU) from Euglena has been established by alignment of the sequence of peptides obtained by cleavage with chymotrypsin, trypsin, Staphylococcus aureus protease or formic acid. The Euglena SSU has 138 amino acids and thus represents longest SSU sequence described so far. Homology is only 41% with cyanobacteria SSU and about 51% with higher plant SSU, whereas it is around 75% between higher plants. The largest homologous portion between all the known SSU sequences is localized in the second half and covers about 20 amino acids. The phylogenetic tree based on known SSU sequences has been established and the rate of amino acid substitution for SSU is estimated to be about 1.35×10(-9) per year and per site. Despite heterogeneity in amino acid sequence, we found that the overall secondary structure is fairly well conserved.

Comparison between the organization of nuclear ribosomal DNA unit of Euglena gracilis Z and var. Bacillaris.

We have characterized the nuclear rDNA unit of Euglena gracilis var. bacillaris and compared it to that of the Z strain. We have localized restriction sites for Eco R1, Sal 1, Sma 1, Hind III, Bam H1 and Bgl II on this unit as well as the coding region for 20 S and 25 S rRNAs. For both strains, results suggest an homogeneity of the 11.6 kbp rDNA units. Comparison between strains shows differences characterized by two additional Sal 1 sites in bacillaris and the likely methylation of one Sma 1 site in Z. Both differences are localized in a non-coding region of the rDNA unit. Analyses of 18 Euglena strains from various origins confirm these differences and allow easy recognition of bacillaris and Z type strains.