Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene family.

The five genes encoding ribulose-1,5-bisphosphate carboxylase (rbcS) from tomato are differentially expressed. Transcription of the genes is organ specific and developmentally regulated in fruit and light regulated in cotyledons and leaves. DNase I footprinting assays were used to map multiple sites of DNA-protein interaction in the promoter regions of all five genes and to determine whether the differential transcriptional activity of each gene correlated with developmental or organ-specific changes in DNA-protein interactions.

Expression dynamics of the tomato rbcS gene family during development.

The tomato rbcS gene family is composed of five genes (rbcS1, 2, 3A, 3B, and 3C) that are differentially expressed during tomato development. Nuclear run-on transcription assays and RNA analysis were used to determine the contribution of transcriptional and post-transcriptional regulation to the accumulation of mRNA from the five rbcS genes in tomato seedlings, leaves, and fruit. We found that the qualitative pattern of mRNA accumulation is regulated at the transcriptional level and that, in general, there is a correlation of rates of rbcS transcription with overall rbcS mRNA abundance in fruit and leaves.

Chloroplast mRNA 3' end processing requires a nuclear-encoded RNA-binding protein.

The protein coding regions of plastid mRNAs in higher plants are generally flanked by 3' inverted repeat sequences. In spinach chloroplast mRNAs, these inverted repeat sequences can fold into stem-loop structures and serve as signals for the correct processing of the mature mRNA 3' ends. The inverted repeat sequences are also required to stabilize 5' upstream mRNA segments, and interact with chloroplast protein in vitro.

Changes in Chloroplast mRNA Stability during Leaf Development.

During spinach leaf development, chloroplast-encoded mRNAs accumulate to different steady-state levels. Their relative transcription rates alone, however, cannot account for the changes in mRNA amount. In this study, we examined the importance of mRNA stability for the regulation of plastid mRNA accumulation using an in vivo system to measure mRNA decay in intact leaves by inhibiting transcription with actinomycin D.

Chloroplast mRNA 3' end maturation is biochemically distinct from prokaryotic mRNA processing.

We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR). In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+. In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro.

Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding.

Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B.

The chloroplast genome exists in multimeric forms.

Chloroplast DNA conformation was analyzed by pulse-field gel electrophoresis. We found that spinach leaf chloroplast DNA molecules exist in at least four distinct forms with the apparent molecular weights of monomer, dimer, trimer, and tetramer. Two-dimensional gel analysis of DNA after UV nicking and in the presence of ethidium bromide indicates that they are not isomers that differ in superhelical density.

Post-transcriptional control of plastid mRNA accumulation during adaptation of chloroplasts to different light quality environments.

The adaptation of germinating spinach seedlings to yellow and red light was studied and compared with plants grown in white light. Spinach chloroplasts isolated from cotyledons and leaves of yellow and white light-grown plants showed similar membrane structures and compositions, while chloroplasts from plants grown in red light have significant adaptive changes. Based on an equal amount of chlorophyll, these changes include a reduction in the number of photosystem I complexes, an increase of photosystem II antenna size, and an increased ratio of stacked to unstacked membranes in red light-adapted chloroplasts.

Tomato hydroxymethylglutaryl-CoA reductase is required early in fruit development but not during ripening.

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and the level of its mRNA have been determined at various stages of tomato fruit development. The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene containing compounds, such as sterols and carotenoids. A cDNA clone encoding the active site region of HMGR has been isolated from a tomato library derived from young-fruit mRNA.

Constitutive transcription and regulation of gene expression in non-photosynthetic plastids of higher plants.

The plastid genome in higher plants contains >50 genes for rRNAs, tRNAs and proteins for transcriptional and translational functions, besides the genes encoding photosynthetic proteins. Considering the totipotency of most higher plant cells and the differentiation capacity of plastids, it can be inferred that at least the genes for genetic functions must be constitutively expressed in all plant organs, including non-photosynthetic roots, to maintain a basal level of transcriptional and translational activities. To test this hypothesis, transcription, RNA accumulation and polysome formation were analyzed in root amyloplasts, and in plastids from hypocotyls and cotyledons of dark-grown spinach seedlings.

Control of plastid gene expression: 3' inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription.

We have examined the function of inverted repeat sequences found at the 3' ends of plastid DNA transcription units in higher plants, using a homologous in vitro transcription extract. The inverted repeat sequences are ineffective as transcription terminators, but serve as efficient RNA processing elements. Synthetic RNAs are processed in a 3'-5' direction by a nuclease activity present in the transcription extract, generating nearly homogeneous 3' ends distal to the inverted repeat sequence.

Diurnal mRNA fluctuations of nuclear and plastid genes in developing tomato fruits.

Steady-state transcript levels of nuclear (rbcS, cab) and plastid (rbcL, psbA) encoded photosynthesis-specific genes were determined at noon and 05.00 h in different developmental stages of tomato fruits (7-35 days after anthesis). Small alterations are observed in mRNA levels for the small subunit (ssu) and large subunit (lsu) of RuBPC/Oase and the QB-binding protein of photosystem II at these two time-points, while significant steady-state transcript level fluctuations are detectable for the light harvesting complex protein.

Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5-bisphosphate carboxylase small subunit gene family.

The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.

Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato.

We have cloned and sequenced all five members of the gene family for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato, Lycopersicon esculentum cv. VFNT LA 1221 cherry line. Two of the five genes, designated Rbcs-1 and Rbcs-2, are present as single genes at individual loci.

Plastid run-on transcription. Application to determine the transcriptional regulation of spinach plastid genes.

We have developed a spinach plastid run-on transcription system to determine the extent of transcriptional regulation of chloroplast genes during morphogenetic changes of the organelle. In contrast to transcription in a spinach chloroplast extract, which requires initiation of exogenously added genes (Gruissem, W., Greenberg, B.

Changes in Photosynthetic Capacity and Photosynthetic Protein Pattern during Tomato Fruit Ripening.

Levels of polypeptides participating in the photosynthetic light and dark reactions have been measured during fruit ripening in tomato. Photosynthetic proteins were identified by Western blot analysis with heterologous antibodies. The concentrations of proteins of photosystem (PS) I (14 kilodaltons), of PSII (47-kilodalton reaction center protein, 32-kilodalton ;Q(B) binding' protein and light harvesting complex proteins), of the photosynthetic electron transport chain (ferredoxin-NADP-oxidoreductase and plastocyanin), and of the stroma (ribulose-1,5-bisphosphate carboxylase) decrease during the ripening process.

Control of plastid gene expression during development: the limited role of transcriptional regulation.

We have analyzed the transcriptional regulation of plastid genes during chloroplast development in illuminated spinach cotyledons and during leaf formation. The RNAs encoded by plastid genes accumulate with different kinetics during the developmental transitions. Using a novel plastid run-on transcription assay we demonstrate that the transcriptional regulation of a large, diverse group of chloroplast genes is of relatively minor importance for the control of their expression.

A subpopulation of spinach chloroplast tRNA genes does not require upstream promoter elements for transcription.

We have identified a class of spinach plastid tRNA genes which do not require 5' upstream promoter elements for their expression in a chloroplast transcription system. The 5' DNA sequences flanking the trnR1 and trnS1 coding regions have little or no homology to previously characterized chloroplast promoter sequences. The deletion of the 5' DNA sequences from these genes to positions close to the start of the coding regions has little effect on their transcription in vitro.

Expression of nuclear and plastid genes for photosynthesis-specific proteins during tomato fruit development and ripening.

The expression of plastid and nuclear genes coding for photosynthesis-specific proteins has been studied during tomato fruit formation. The steady-state transcript levels for the large (rbcL) and small (rbcS) subunit of RuBPC/Oase, as well as the thylakoid membrane proteins, the 32 kD QB-binding protein of PS II (psbA), the P700 reaction center protein of PS I (psaA) and the chlorophyll a/b-binding protein (cab) vary at different time points during fruit development and ripening. Messenger RNA levels of plastid-encoded photosynthesis-specific genes (rbcL, psbA) are at least several fold higher, relative to respective nuclear-encoded genes (rbcS, cab).

Light quality regulates expression of chloroplast genes and assembly of photosynthetic membrane complexes.

The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers and the level of chloroplast reaction center gene transcripts were determined in pea plants grown under different light-quality regimes. In plants grown in light primarily absorbed by PSI ("red" light), the PSII/PSI reaction center ratio was 2-fold greater than that in plants grown in PSII-sensitizing ("yellow") light. In addition, the ratio of a PSII gene (psbB) transcript to a PSI gene (psaA) transcript was 2.