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.     

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

Overexpression of formate dehydrogenase inArabidopsis thaliana resulted in plants tolerant to high concentrations of formate

The potential role played by formate dehydrogenase (FDH) in formate metabolism has been examined by the overexpression of FDH in Arabidopsis thaliana. Three independent transgenic lines were selected and shown to produce elevated amounts of FDH protein with a corresponding elevated FDH activity (2.5-5 fold) over wild-type (WT) plants. Under normal growth conditions, no altered phenotype was observed in these transgenic plants; in growth media supplied with formate, however, significant differences in shoot and root growth, compared to that of WT plants, were observed. WT plants were severely injured if grown in the presence of 16 mmol/L formate, while the transgenic plants were able to grow well. Formate delayed germination of both WT and transgenic seeds at concentrations above 4 mmol/L, but both types of seeds were eventually able to complete more than 95 % germination even at 32 mmol/L formate. Formate markedly inhibited primary root elongation, and its inhibitory action on WT was much stronger than on transgenic plants. Different formate salts affected root elongation similarly, indicating that the formate ion was the major factor inhibiting root growth. Sodium acetate (NaAc), an analogue of formate, also inhibited root elongation, but its action on WT and transgenic plants was the same, indicating that tolerance of transgenic plants to formate toxicity was specific. Transgenic plants showed no significant tolerance to the toxicity of two other one-carbon metabolites, methanol and formaldehyde. A role for FDH in detoxifying formate is proposed.     

Genbank accession #: AF111812

Plant Molecular Biology -     

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids—nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.     

High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production

High temperature stress (HTS), during flowering, decreases seed production in many plants. To determine the effect of a moderate HTS on flowering, fruit and seed set in Brassica napus, plants were exposed to a HTS (8/16 h dark/light, 18 degrees C night, ramped at 2 degrees C h-1, over 6 h, to 35 degrees C for 4 h, ramped at 2 degrees C h-1 back to 23 degrees C for 6 h) for 1 or 2 weeks after the initiation of flowering. Although flowering on the HTS-treated plants, during both the 1 week and 2 week HTS treatments, was equal to that of control-grown plants, fruit and seed development, as well as seed weight, were significantly reduced. Under HTS, flowers either developed into seedless, parthenocarpic fruit or aborted on the stem. At the cessation of the HTS, plants compensated for the lack of fruit and seed production by increasing the number of lateral inflorescences produced. During the HTS, pollen viability and germinability were slightly reduced. In vitro pollen tube growth at 35 degrees C, from both control pollen and pollen developed under a HTS, appeared abnormal, however, in vivo tube growth to the micropyle appeared normal. Reciprocal pollination of HTS or control pistils with HTS or control pollen indicated that the combined effects of HTS on both micro- and megagametophytes was required to knock out fruit and seed development. Expression profiles for a subset of HEAT SHOCK PROTEINs (HSP101, HSP70, HSP17.6) showed that both micro- and megagametophytes were thermosensitive despite HTS-induced expression from these genes.