The two cDNAs coding for the cytosolic (cyt) and the chloroplast-located (chl) Cu,Zn superoxide dismutases (SODs) of tomato (Perl-Treves et al. 1988) were cloned into respective binary vectors and mobilized into Agrobacterium strains. Potato tuber discs were infected with either of the two agrobacterial strains and cultured on selective medium containing kanaymcin. The integration of either of the cyt or the chl SOD transgenes was verified by Southern-blot hybridization. The enzymatic activity of the additional tomato chl Cu,Zn SOD could be distinguished from endogenous SOD activity since the latter isozyme migrated faster on SOD-activity gels. Several transgenic potato lines harboring either the cyt or the chl SOD genes of tomato showed elevated tolerance to the superoxide-generating herbicide paraquat (methyl viologen). After exposure of shoots to paraquat, tolerance was recorded either by scoring symptoms visually or by measurements of photosynthesis using the photoacoustic method. Root cultures from transgenic lines that harbored the additional cyt Cu,Zn SOD gene of tomato were tolerant to methyl viologen up to 10(-5) M; a lower tolerance was recorded in roots of transgenic lines that expressed the additional chl Cu,Zn SOD of tomato.
A cDNA library was derived from the poly(A)(+) RNA of young tomato leaves. The library was cloned in a λgt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5' end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)(+) RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.
The chloroplast genes coding for the 16S ribosomal RNA from several spectinomycin‐resistant Nicotiana mutants were analyzed. Two classes of mutants were identified. In one class, a G to A base transition is found at position 1140 of the tobacco‐chloroplast 16S rRNA gene, which eliminates an AatII restriction endonuclease site. This base transition is proximal to a mutation previously described for spectinomycin resistance in Escherichia coli. In the other class, a novel G to A transition is found at position 1012 of the 16S rRNA gene. Although the mutations in the two classes are 128 nucleotides apart, the secondary structure model for 16S rRNA suggests that the two mutated nucleotides are in spatial proximity on opposite sides of a conserved stem structure in the 3′ region of the molecule. Phylogenetic evidence is presented linking this conserved stem with spectinomycin resistance in chloroplasts. Perturbation of the stem is proposed to be the molecular‐genetic basis for rRNA‐dependent spectinomycin resistance.
Alternative splicing is a major contributor to genome complexity, playing a significant role in various cellular functions, including signal transduction, immunity, and development. The spliceosomal machinery is responsible for the processing of nuclear RNA. Several splicing factors associated with this complex are phosphorylated by kinases that possess a conserved LAMMER motif. We demonstrate in BY-2 tobacco cells a novel role for the LAMMER motif in the maintenance of proper subnuclear localization. Furthermore, high expression of the LAMMER kinase in Arabidopsis plants modulated the alternative splicing of specific endogenous genes and resulted in abnormal plant development and a novel transcriptome profile. A prominent feature was the upregulation of genes that play a role in protein turnover, suggesting a moderating function for these gene products in the control of alternative splicing events. Together, these results demonstrate alternative splicing modulation as a result of phosphorylation activity, providing an opportunity to study its global effect on the plasticity of plant development and gene expression at the organism level.
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