"HAIRY CANOLA"--Arabidopsis GL3 induces a dense covering of trichomes on Brassica napus seedlings.

Transformation with the Arabidopsis bHLH gene 35S:GLABRA3 (GL3) produced novel B. napus plants with an extremely dense coverage of trichomes on seedling tissues (stems and young leaves). In contrast, trichomes were strongly induced in seedling stems and moderately induced in leaves of a hairy, purple phenotype transformed with a 2.2 kb allele of the maize anthocyanin regulator LEAF COLOUR (Lc), but only weakly induced by BOOSTER (B-Peru), the maize Lc 2.4 kb allele, or the Arabidopsis trichome MYB gene GLABRA1 (GL1). B. napus plants containing only the GL3 transgene had a greater proportion of trichomes on the adaxial leaf surface, whereas all other plant types had a greater proportion on the abaxial surface. Progeny of crosses between GL3+ and GL1+ plants resulted in trichome densities intermediate between a single-insertion GL3+ plant and a double-insertion GL3+ plant. None of the transformations stimulated trichomes on Brassica cotyledons or on non-seedling tissues. A small portion of bHLH gene-induced trichomes had a swollen terminal structure. The results suggest that trichome development in B. napus may be regulated differently from Arabidopsis. They also imply that insertion of GL3 into Brassica species under a tissue-specific promoter has strong potential for developing insect-resistant crop plants.

Non-lethal freezing effects on seed degreening in Brassica napus.

The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28-44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.

Establishment of Arabidopsis thaliana ribosomal protein RPL23A-1 as a functional homologue of Saccharomyces cerevisiae ribosomal protein L25.

Arabidopsis thaliana ribosomal protein (r-protein) RPL23A-1 shows 54% amino acid sequence identity to the Saccharomyces cerevisiae equivalent r-protein, L25. AtRPL23A-1 also shows high amino acid sequence identity to members of the L23/L25 r-protein family in other species. R-protein L25 in S. cerevisiae has been identified as a primary rRNA-binding protein that directly binds to a specific site on yeast 26S rRNA. It is translocated to the nucleolus where it binds to 26S rRNA during early large ribosome subunit assembly; this binding is thought to play an important role in ribosome assembly. The S. cerevisiae mutant strain YCR61 expresses L25 when grown on galactose, but not glucose, medium. Transformation of YCR61 with a shuttle vector containing the AtRPL23A-1 cDNA allowed transformed colonies to grow in and on glucose selection medium. R-protein AtRPL23A-1 can complement the L25 mutation, demonstrating the functional equivalence of the two r-proteins and introducing AtRPL23A-1 as the first plant member of the L23/L25 r-protein family.

Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions.

Two aquaporin genes were isolated from a cDNA library of canola (Brassica napus L.). The first aquaporin, BnPIP1 of 1094 bp, encoding a putative polypeptide of 287 amino acids with a predicted molecular mass of 30.4 kDa and a pI of 7.8, belongs to the family of plasma membrane intrinsic protein (PIPs) aquaporins. The B. napus aquaporin showed 85-94% identity to the Arabidopsis thaliana PIPs. ABA priming of seed induced high levels of BnPIP1 transcript which remained after subsequent re-drying of the seed. The second aquaporin, Bny-TIP2 of 1020 bp, encoded a putative polypeptide of 253 amino acids with a predicted molecular mass of 25.8 kDa and a pI of 5.8. Bngamma-TIP2 showed 83-90% identity to gamma-TIP genes from a variety of plant species. Bngamma-TIP2 was expressed only when radicle protrusion occurred in either untreated or primed seeds. Seeds primed with PEG or ABA germinated earlier and showed a higher final percentage of germination than unprimed seed, particularly under salt and osmotic stresses at low temperature. Transcripts of both BnPIP1 and Bngamma-TIP2 genes were present earlier during germination of primed seeds than non-primed seed. From these results, we conclude that BnPIP1 is related to the water transportation required for enzymatic metabolism of storage nutrients at the early stages of canola seed germination whereas Bngamma-TIP2 expression is related to cell growth associated with radicle protrusion. Priming induced the expression of BnPIP1 but had no effect on Bngamma-TIP2.

Molecular cloning and expression of a cDNA encoding the proliferating cell nuclear antigen from Brassica napus (oilseed rape).

A cDNA clone encoding the proliferating cell nuclear antigen (PCNA) has been isolated from a Brassica napus apical meristem cDNA library. The putative full-length cDNA contains an open reading frame of 1004 nucleotides, which predicts a protein of 263 amino acids (M(r) = 29,231). Sequence analysis has revealed that the plant PCNA exhibits 81.6% amino acid similarity with the human PCNA. Genomic Southern blot analysis indicates the presence of at least two copies of PCNA per genome. The B. napus PCNA mRNA (1.0 kb) was expressed in rapidly dividing tissues such as flower buds, apical meristems, and young leaves, while mature stem and fully expanded leaves showed significantly lower levels of PCNA transcript. The B. napus PCNA cDNA was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) in the bacterial expression vector pGEX-2T. A broad specificity monoclonal antibody raised against rabbit PCNA cross-reacted with the GST-PCNA fusion peptide but not with the GST moiety alone. This antibody also recognized the human PCNA (36 kDa) polypeptide, confirming the structural similarities between the human and plant PCNA. The high degree of structural conservation of PCNA from such diverse organisms as humans and higher plants suggests that the plant PCNA may function in a manner analogous to that found in mammals with respect to plant cell DNA replication. Such conservation suggests that PCNA is also a critical component of the plant cell DNA replication complex.

Cytoplasmic ribosomal protein S15a from Brassica napus: molecular cloning and developmental expression in mitotically active tissues.

We have isolated two cDNA clones which appear to encode the 40S ribosomal subunit protein S15a from Brassica napus (oilseed rape). The open reading frame in both clones contains 390 bases, encoding a deduced polypeptide sequence of 130 amino acids (100% homology between clones) with 76% sequence identity to the N-terminal 37 amino acids of the rat ribosomal protein S15a and 80% identity to the S24 polypeptide of yeast. Both the yeast and rapeseed proteins have a net positive charge of +9 and the rapeseed S15a protein has a molecular mass of 14778 Da compared to 14762 Da for the yeast protein. The rapeseed ribosomal protein S15a is encoded by a small multi-gene family with at least two actively transcribed members. A single transcript of ca. 1.0 kb, corresponding to ribosomal protein S15a, is abundant in actively dividing tissues such as apical meristem, flower buds and young leaves and less abundant in mature stem and fully expanded leaves.

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.

Establishment of thermotolerance in maize by exposure to stresses other than a heat shock does not require heat shock protein synthesis.

Maize (Zea mays) seedlings were pretreated prior to heat shock with either a progressive water stress of -0.25 megapascal PEG/hour from 0 to -1.25 megapascal over a 6-hour time period, or various concentrations of copper, cadmium, or zinc for 4 days. When the subsequent heat shock of 40 or 45 degrees C was administered for 3 hours, the seedlings showed an induced thermotolerance to these temperatures, which were otherwise lethal to control (water grown) seedlings. Thermotolerance was exhibited by both the root and the shoot of pretreated seedlings, even though the water and heavy metal stresses were applied only to the roots. Neither of these pretreatments had induced the synthesis of detectable levels of heat shock proteins (Hsps) at the time of heat shock. Pretreatment of seedlings with a progressive heat shock of 2 degrees C/hour from 26 to 36 degrees C, which did induce Hsps 18, 70, and 84, resulted in tolerance of a severe water stress of -1.5, -1.75, or -2.0 megapascal for 24 hours. But these seedlings producing Hsps were no better protected against water stress than those pretreated with a progressive water stress which did not produce Hsps. Hsps appear not to act as general stress proteins and their presence is not always required for the establishment of thermotolerance.