Cloning of a cDNA encoding a developmentally regulated 22 kDa polypeptide from tobacco leaf plasma membrane.

A polypeptide doublet (P18-P19, ca 22 kDa, pI 4.5) has been shown to accumulate in tobacco leaf plasma membrane in a development-dependent way, under constant environmental conditions. P18 and P19 were purified by 2D-PAGE and microsequenced. Microsequences revealed only small differences between the two polypeptides. A PCR-based cloning strategy identified a cDNA displaying a 591 bp ORF. The encoded polypeptide contained P19 specific microsequences. It was expressed in E. coli and a specific rabbit antiserum was raised. Western-blots confirmed its identification as P19. The accumulation pattern of hybridizable mRNA around the floral induction period was similar to that of P18 and P19. Searching of databases revealed no significant hits except unidentified plant ESTs. P18 and P19 are proposed as the first example of plant-specific and developmentally regulated plasma membrane proteins.

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex.

Tobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201-212, 1984).Poly(A)(+)RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)(+)RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells.Dot-blot and northern-blot hybridizations of poly(A)(+)RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304-7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)(+)RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)(+)RNA.Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613.

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration.

The frequency of incorporation of the cytokinin N(6)-[p-(3)H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 x 10(4) conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.

Incorporation of N-Benzyladenine into Messenger Poly(A)-RNA of Tobacco Cells Incubated at Stimulatory or Cytostatic Cytokinin Concentration.

Poly(A)-RNA was purified from Nicotiana tabacum cell suspension cultures grown in the presence of N(6)-benzyladenine (BA). Cells were incubated with concentrations of 0.4 micromolar BA, optimal for cell division (OPT) or 10 micromolar BA, a cytostatic concentration (OVD), or without cytokinin (CTL) as a control. After 55 hours, total RNA was extracted from the cells and poly(A)-RNA was purified by oligo(dT)-cellulose binding. Similar yields of poly(A)-RNA were obtained for OPT or CTL cell samples; the compared recovery from the OVD cell samples was reduced by more than half. Poly(A)-RNA extracted from OPT or OVD cells contained BA nucleotide, inserted at the respective frequencies of 78 and 506 micromoles per mole conventional nucleotide. BA was inserted into the transcript as well as into the poly(A) segments. No qualitative or quantitative difference was observed between the in vitro translation activities of poly(A)-RNA extracted from OPT or CTL cells. The electrophoretic analysis of translation products from OVD mRNA showed a deficiency in proteins of molecular weight over 50 kilodaltons. This deficiency may be explained by a change in the coding properties of OVD mRNA rather than by a deficiency of the high molecular weight components in the mRNA population.