Protein synthesis in chloroplasts. VIII. Differential synthesis of chloroplast proteins during spinach leaf development

Excised primary leaves of spinach (Spinacia oleracea) incorporate [35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.     

Protein synthesis in plant leaf tissue. The sites of synthesis of the major proteins.

Protein synthesis in the leaves of green pea seedlings (Pisum sativum) is examined by short term labeling with [35S]methionine and autoradiography of the labeled proteins after fractionation by sodium dodecyl sulfate-acrylamide gel electrophoresis. The two subunits of ribulose-1,5-diphosphate carboxylase and the chloroplast lamellar proteins are identified as the major proteins being synthesized. Three protein chlorophyll complexes are characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis; all three complexes are disrupted by heating to 100 degrees in sodium dodecyl sulfate solution. Studies with inhibitors of protein synthesis indicate that the large subunit of ribulos-1,5-diphosphate carboxylase is synthesized in the chloroplast, in contrast to the majority of the soluble proteins, including the small subunit of ribulose-1,5-diphosphate carboxylase, which is synthesized in the cytoplasm. PII protein, the major lamellar protein associated with photosystem II, is also synthesized on cytoplasmic ribosomes. However, many of the lamellar proteins are synthesized within the chloroplast. Integration into the lamellar system of at least one of the chloroplast-synthesized proteins is shown to be dependent on cytoplasmic protein synthesis.     

Photoperiodic sensitivity of the cotyledon and first true leaf of Xanthium

Three days after germination of Xanthium seeds, the seedlingswith only the first pair of opposite leaves attained full photoperiodicsensitivity and flowered in response to a 16-hr dark periodtreatment. The cotyledons of three- to six-day-old seedlingswere completely insensitive to inductive dark treatment. Six-day-oldseedlings could not be induced to flower with a 9-hr dark period.But with a dark period of 10 hr or longer, floral inductionoccurred and the optimum dark period was 15 hr. Developmentof the flowering apices started within 2 days after the inductivedark treatment and was completed by 9 to 11 days. ¹This work was supported by the United States Atomic EnergyCommission Contract AT (11-1)-1338. ²Present address: Plant Physiology Section, Crops Research Institute,P.O. Box 3785, Kumasi, Ghana, West Africa. (Received March 28, 1974; )     

Protein synthesis in embryonic tissues during mouse postimplantation development

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.     

Competition between isoprene emission and pigment synthesis during leaf development in aspen

In growing leaves, lack of isoprene synthase (IspS) is considered responsible for delayed isoprene emission, but competition for dimethylallyl diphosphate (DMADP), the substrate for both isoprene synthesis and prenyltransferase reactions in photosynthetic pigment and phytohormone synthesis, can also play a role. We used a kinetic approach based on post‐illumination isoprene decay and modelling DMADP consumption to estimate in vivo kinetic characteristics of IspS and prenyltransferase reactions, and to determine the share of DMADP use by different processes through leaf development in Populus tremula. Pigment synthesis rate was also estimated from pigment accumulation data and distribution of DMADP use from isoprene emission changes due to alendronate, a selective inhibitor of prenyltransferases. Development of photosynthetic activity and pigment synthesis occurred with the greatest rate in 1‐ to 5‐day‐old leaves when isoprene emission was absent. Isoprene emission commenced on days 5 and 6 and increased simultaneously with slowing down of pigment synthesis. In vivo Michaelis–Menten constant (K_m) values obtained were 265 nmol m^?2 (20 μm ) for DMADP‐consuming prenyltransferase reactions and 2560 nmol m^?2 (190 μm ) for IspS. Thus, despite decelerating pigment synthesis reactions in maturing leaves, isoprene emission in young leaves was limited by both IspS activity and competition for DMADP by prenyltransferase reactions.     

Protein synthesis by 80S ribosomes during plant development

Protein synthesis by ribosomes from the meristematic region of pea roots (0–0·3 cm) and 2-day-old corn shoots (young tissues) relative to ribosomes from matured regions of pea roots (2·0–2·5 cm) and 10-day-old corn leaves (aged tissues) was compared in the poly U-phenylalanine system. With normal polyribosome preparations, ribosomes from young tissues required approx. 16 mM Mg²⁺ while ribosomes from aged tissues required 20–22 mM Mg²⁺ for optimal activity. With monomeric ribosome preparations induced by anaerobic treatment of the seedlings, the Mg²⁺ optimum was 20–22 mM for ribosomes from both young and aged tissues. A higher level of peptidyl-tRNA in ribosomes from young tissues accounts, at least in part, for the differences in Mg²⁺ optima between ribosomes from young and aged tissues. Monomeric ribosomes were used for assaying the activity of ribosomes per se. Ribosomes from young pea root tips and ribosomes from 2-day-old corn shoots were 25–30% and 100–150% more active, respectively, than the corresponding ribosomes from aged tissues. Differences in ribosomal proteins revealed by gel electrophoresis correlated with the change in ribosomal activity. Reduced activity in the aged ribosomes was not due to RNase activity or inhibitors.     

Networks in leaf development

Shoots are characterized by indeterminate growth resulting from divisions of undifferentiated cells in the central region of the shoot apical meristem. These cells give rise to peripheral derivatives from which lateral organ initials are recruited. During initial stages of cell recruitment, the three-dimensional form of lateral organs is specified. Lateral organs such as leaves develop and differentiate along proximodistal (base-to-tip), dorsoventral (top-to bottom) and mediolateral (middle-to-margin) planes. Current findings are refining our knowledge of the genes and genetic interactions that regulate these early processes and are providing a picture of how these pathways may contribute to variation in leaf form.     

Macromolecular synthesis in oat leaf protoplasts

Oat mesophyll protoplasts isolated by 2 hr cellulysin treatmentof peeled young leaves incorporate tritiated leucine, uridineand thymidine into trichloroacetic acidinsoluble materials.Neither the protoplast-free final supernatant liquid nor protoplastsdisrupted by rapid passagethrough the tip of a Pasteur pipetteshow any incorporation activity, while intact protoplasts incubatedin the presence of penicillin and streptomycin are active. Thus,the cycloheximide and actinomycin D inhibitable incorporationprocesses appear to represent, respectively, synthesis of proteinand nucleic acids by intact protoplasts, uncomplicated by organellaror microbial contributions. Net leucine and uridine incorporation by protoplasts continueat a steady rate for about 6 hr, dien abruptly cease, whilethymidine incorporation continues linearly for at least 21 hr.Preincubation of protoplasts in leucine-free media for severalhours diminishes the duration, but not the initial rate of incorporation.The titer of protoplasts declines progressively with increasedtime of incubation. Kinetin (10^–9 to 10^–4 M) progressively inhibitsleucine incorporation, 2,4-D at concentrations higher than 10^–7M inhibits uridine incorporation, while gibberellins, abscisicacid and ethylene are without effect on any process studied. ¹ Permanent address: Department of Fruit and Vegetable Storage,.The Volcani Institute, Bet Dagan, Israel. (Received December 24, 1976; )     

Protein synthesis in imaginal disks ofPlodia interpunctella during development in vivo and in vitro

Summary Wing imaginal disks were dissected from larvae ofPlodia interpunctella (Hübner) at various stages during the larval-pupal transformation. The wing-disk proteins separated by electrophoresis and scanned with a densitometer changed quantitatively but not qualitatively during development in vivo. Treatment of wing disks in vitro with β-ecdysone resulted in a 2-fold increase in synthesis of proteins after only 2 hr incubation. The maximum rate of protein synthesis was reached 16 hr after treatment with hormone. The pattern of proteins separated by electrophoresis of wing disks that were incubated in vitro with β-ecdysone did not change qualitatively. The major features of protein synthesis during wing-disk development in vivo were similar to those observed during β-ecdysone-induced development in vitro. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA.