The effects of a cytokinin (N(6)-benzyladenine, BA) and light on plastogenesis have been studied in detached Cucurbita cotyledons using the key enzyme of photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase), as an example of a coordinated program of plastid and nucleo-cytoplasmic gene expression. Treatment of etiolated cotyledons with either BA in darkness or in light or light alone results in a marked and correlated stimulation of enzyme activity, quantity and biosynthesis (in-vivo [(14)C]leucine incorporation into immunoprecipitated enzyme protein), indicating an increase of de-novo synthesis under the influence of the two effectors. Cell-free translation of non-polyadenylated (poly(A)(-))RNA in an Escherichia coli system and total RNA in a wheat-germ system likewise demonstrate a light and hormone-dependent increase in the amounts of translatable mRNAs for the large (LS) and small subunits (SS) of RuBPCase (among other polypeptides). Hybridisation of poly(A)(-)RNA with a nick-translated LS gene of spinach RuBPCase reveals also two-to three-fold BA-or light-induced enhancement of LS mRNA content. Indications for stimulation of SS mRNA transcription are derived from inhibitor experiments with cordycepin. We conclude that the observed stimulation of de-novo RuBPCase synthesis by cytokinin and by light can be referred to the level of mRNA transcription, but it remains open whether this is a primary action. Furthermore, our results indicate that the coaction of the two exogenous factors is additive at different steps of RuBPCase formation, indicating independent actions in the causal chain between effector-signal transduction and RuBPCase gene expression.

Using solid‐phase ‘Sandwich’ immunoassays we studied DNA‐dependent RNA polymerase of spinach chloroplasts with regard to (i) polypeptide composition of the multimeric enzyme; (ii) immunological cross‐reaction with Escherichia coli RNA polymerase; (iii) sites of synthesis of polymerase polypeptides. Our main results are as follows. (i) All polypeptides of isolated chloroplast RNA polymerase (150, 145, 110, 102, 80, 75 and 38 kd) are labeled by an antibody‐linked polymerase assay (ALPA), i.e., they are immunologically related to subunits of the holoenzyme. On the other hand differences in the patterns of ‘ALPA‐reactive’ polypeptides of a crude RNA polymerase fraction and of the purified enzyme preparation indicate partial proteolytic degradation of polymerase polypeptides during purification. Thus the 80‐ and 75‐kd polypeptides, which had been previously considered as true RNA polymerase polypeptides, probably result from partial proteolytic degradation. (ii) The 150‐ and 145‐kd polypeptides show immunochemical similarities with the β and/orβ' subunits of E. coli RNA polymerase. (iii) Results from solidphase immunoassay of in vitro translated products of both chloroplast RNA and poly(A)+ (nuclear) RNA suggest that all chloroplast RNA polymerase polypeptides are coded for by the nucleus.

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Spinach DNA dependent RNA polymerase was purified from isolated chloroplasts by two different procedures. Analysis of the protein composition of the two preparations by SDS-polyacrylamide gel electrophoresis always shows six abundant polypeptides with Mr of 150, 110, 102, 80, 75 and 38 Kd and one less abundant polypeptide of 25 Kd. Some other proteins ranging from 40-70 Kd in Mr are also detected but in a minor and variable amount. The two preparations have an optimum of enzyme activity at 30°C and at 15 mM (NH4)2SO4 when tested with denatured calf thymus DNA.Binding experiments with two different nick translated fragments of spinach chloroplast DNA show that the 80 and 75 Kd polypeptides possess a strong DNA binding capacity.