Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Isolation of a novel RNA-binding protein and its association with a large ribonucleoprotein particle present in the nucleoplasm of tobacco cells

A cDNA encoding a protein with a consensus sequence-type RNA-binding domain (CS-RBD) has been isolated from a Nicotiana sylvestris cDNA library. The deduced protein (designated RZ-1) contains CS-RBD in its N-terminal half, arginine/aspartic acid repeats in its center and a glycine-rich C-terminal region in which a zinc finger motif of the CCHC type is present. The corresponding gene appears to be expressed constitutively in all tobacco organs. Immunocytochemical assays revealed that RZ-1 is localized in the nucleoplasm of tobacco cultured cells. Glycerol gradient fractionation of tobacco nuclear lysates showed that RZ-1 is associated with a large ribonucleoprotein particle of around 60 S in size. Nucleic acid-binding assays indicated that RZ-1 binds preferentially to poly (G) and both the CS-RBD and glycine-rich region are necessary for its binding activity. A possible role of RZ-1 is discussed.     

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO₂ fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.     

Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C, in synchronized tobacco BY-2 cells

Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5–249) and KatC(207–754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin-like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule-based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein.     

Linkage between Frl (Fusarium oxysporum f.sp. radicis-lycopersici resistance) and Tm-2 (tobacco mosaic virus resistance-2) loci in tomato (Lycopersicon esculentum)

A study was carried out on the linkage relationship between the Frl locus carrying resistance to Fusarium oxysporum f.sp. radicis-lycopersici and the Tm-2 locus carrying resistance to several races of tobacco mosaic virus in the tomato inbred line IRB-301-31. The inbred line Motelle (Frl⁺/Frl⁺, Tm-2⁺/Tm-2⁺) was crossed with the inbred line IRB-301-31 (Frl/Frl, Tm-2/Tm-l). The resulting 222 F₂ plants were selfed, and from each F₃ family groups of 15–60 seedlings were tested for resistance to either F. oxysporum f.sp. radicis-lycopersici or tobacco mosaic virus race 0. Segregation data indicated a very tight linkage between Frl and Tm-2, equal to 5.1 ± 1.07 map units.     

Alleviation of plant virus infection by humic acids

K-humates, obtained from oxihumolites, alleviate infection of tobacco with tobacco mosaic virus both in mixture with virus inoculum and by spraying of leaves before inoculation. However, applications of K-humates after inoculation did not influence the virus infectivity.