Plastid Development in Pisum sativum Leaves during Greening

Abstract

ABSTRACIWhen isolated pea plastids are incubated with translation products of poly(A') mRNA they specifically import precursor molecules of plastid polypeptides. Etioplasts and chloroplasts import the same polypeptides from identical translation products, and, the imported polypeptides can be well resolved by two-dimensional gel electrophoresis. Therefore, the posttranslational uptake system using isolated chloroplasts can monitor changes in the abundance of translatable plastid-targeted messages. Poly(A') mRNA was isolated from peas at various times during greenig and analyzed by this technique. (a) After 48 hours of illumination of dark-gown plants, the relative portion of npelear eitcoded messages for plastid targeted proteins had increased by a factor of 2. The percentage of polypeptides recovered in the stroma fraction increased from about 50 to 65%. (b) More than 140 imported polypeptide species could be detected in fluorograms of two-dimensional gels, most of which could be identified throughout the time course ofgreening. At least 37 imported polypeptides decreased and 36, increased in relative abundance during greening of darkgreen plants. (c) In most cases, where differences In translatable messages were seen between dark-and light-grown plants, they were accompanied by parallel changes in polypeptide abundance.Most plastid polypeptides are encoded by nuclear genes. However, experimental data regarding the regulation of polypeptide and transcript abundance during greening is limited to a few polypeptides. Our understanding of the regulation of the two most prominent nuclear encoded chloroplast polypeptides, the SSU3 and LHCP, has increased during the last years due to the work of several research groups. As with all nuclear encoded plastid proteins whose amino acid sequences have been described, SSU and LHCP are synthesized as precursor molecules on cytoplasmic ribosomnes and their primary translation products possess aminoterminal extensions (7) In this line of consecutive events, well established for these two polypeptides, several different points of regulatory control can be easily imagined: regulation at the level of transcription; RNA stability; regulation of translation; precursor stability in the cytosol; rate and specificity ofuptake; processing ofthe imported precursors; assembly in the presence ofthe corresponding factors; and rate of degradation inside the plastid (5,12,22).In

In-vitro transport of chloroplast proteins in a homologousEuglena system with particular reference to plastid leucyl-tRNA synthetase

“…Our observations demonstrate and confirm results from other laboratories that the reprogramming of protein synthesis of dark-grown cells upon illumination is in general not determined by qualitative changes in the spectrum of abundant mRNAs: post-transcriptional (translational) control seems to play an important role in the regulation of light-induced chloroplast differentiation (Cannons and Merrett 1983;McCarthy and Schwartzbach 1984;Tobin and Silverthorne 1985;Monroy et al 1987;Javed and Merrett 1987;Berry et al 1988). This fact does not lower the importance of transcriptional control (Tobin and Silverthorne 1985;Krauspe et al 1987), but the significance of post-transcriptional control has become more obvious in recent years because of improved methods of in-vitro-translation and improved in-vitro transport systems (Dietz and Bogorad 1987;Keegstra 1989).…”

“…plastids and mitochondria (Dietz and Bogorad 1987). By coupling in-vitro translation to post-translational uptake into chloroplasts, mRNAs for nuclear-coded plastid proteins can be recognised and quantified via corresponding precursor polypeptides which are targeted to the organelles.…”

“…In order to provide further information about the control of expression of nuclear-encoded chloroplast proteins in Euglena, we tried to approach this problem by using a protein-transport assay into isolated organelles as an indicator system for monotoring mRNA changes (Dietz and Bogorad 1987). Owing to the protein-species-specific import into chloroplasts, certain invitro-synthesized polypeptides can be recognised as plasrid-targeted proteins.…”

Lawrence Bogorad (1921–2003), a pioneer in photosynthesis research: a tribute

“…The group was also at the forefront of using 'functional genomics' approaches (or their precursors) to understand fundamental problems in photosynthesis research. Mutant approaches have been discussed, but others included global transcript profiling experiments, e.g., to identify coordinately regulated genes during chloroplast biogenesis (Rodermel and Bogorad 1985), and analyses of the plastid proteome during greening, using techniques of 2-D gel electrophoresis (Dietz and Bogorad 1987). …”

Plastid Development in Pisum sativum Leaves during Greening

“…7b) Table I. The transcriptional-translational origin of 36 of the 100 polypeptides can be traced back by comparing stained twodimensional gels of plastid polypeptides with autoradiographed gels of in organello translation products and with autoradiographed gels of polypeptides which had been synthesized from poly(A+) mRNA preparations in a cell-free system and which had been posttranslationally imported by chloroplasts in vitro (for the detailed description see [5]). this relatively low percentage of polypeptides which can be identified needs some explanation (18): (a) Only methionine-containing polypeptides can be detected.…”