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We have investigated the expression of nuclear-encoded chloroplast proteins that are not associated with chlorophyll (the lumenal 33-kDa and 23-kDa polypeptides of the oxygen-evolving system of photosystem 11, plastocyanin and the Rieske Fe/S protein) by comparing mRNA-accumulation rates with those of the corresponding proteins during illumination of etiolated tobacco seedlings. Using subcellular fractionation, pulsekhase, Northern and Western techniques, we found that the biogenesis and stability of these proteins are regulated both translationally, as well as post-translationally, including the efficiency of mRNA uptake into polysomes, processes that operate between translation and assembly or monitor the status (soluble and membrane-attached) of a terminally processed polypeptide. Polypeptide synthesis is generally not limited by mRNA amounts. For instance, steady-state transcript levels may increase 10-fold during illumination, while those associated with polycomes increase only 2-3-fold without measurable influence on the rate of protein synthesis. The 23-kDa and Rieske polypeptides are predominantely membrane associated, but plastocyanin and the 33-kDa polypeptide are distributed among both soluble and membrane-associated protein fractions. Plastocyanin appears to be comparably stable in both forms. However, for the 33-kDa polypcptide, only the membrane-attached form is stable (> 8 h) and only this pool increases upon illumination. Its soluble form is rapidly degraded with a half-life of approximately 1 h under the chosen conditions. Our findings probably reflect part of a more general regulatory principle operating in the differentiation and maintenance of subcellular structure.Thylakoid membranes of higher plants consist of approximately 70 polypcptide species, the majority of which are assembled in five major multisubunit complexes, the photosystems I and 11, the cytochrome complex, the ATP synthase and the NAD(P)H dehydrogenase. Most of the genes that code for these polypeptides have been isolated during the past decadc (summarized in Hallick, 1989, andHemnann et al., 1991); approximately half of them originate in plastids, the remaining in the nucleus. The dual genetic origin and a vast difference of 2-3 orders of magnitude in genc-copy number between nucleus and plastids implies that the synthcsis and assembly of thylakoid polypeptides must be highly coordinated in time and space, with regard to stoichiometry, and in response to external stimuli.Light plays a crucial role among the various factors that influence the biosynthesis of the photosynthetic membranc and appears to operate at various levels. The light-dependent synthesis of polypeptides made in plastids is generally controlled post-transcriptionally (Herrmann et al

Homologous cDNA clones of the nuclear-codes 23- and 33-kDa polypeptides of the oxygen-evolving system, as well as the Rieske iron/sulfur protein, were expressed in antisense or sense orientation under the control of the 35S RNA cauliflower mosaic virus promoter in transgenic tobacco (Nicotiana tabacum, L.) in order to modulate stationary concentrations of transcripts for studying relationships between transcript and protein levels. In all instances, in individuals that contained as little as 10% or less of the transcripts of untransformed plants, the corresponding protein levels were not notably altered. This indicated that the mRNA levels for components under study are not rate-limiting for protein accumulation and that severely reduced amounts of these transcripts still allow normal plant development. Overexpression of the 23-kDa polypeptide by the corresponding cDNA integrated in sense orientation resulted in both higher mRNA and protein levels without detectable enhancement of oxygen evolution. At least some of the excess protein was found in the soluble fraction and not associated with the photosystem-II reaction center. An attempt has been made to reconcile the varied responses obtained using these approaches with different expression patterns.

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