In 1971 Bradbeer and Stocking followed up a previous investigation of the development of the activities of the photosynthetic carbon cycle enzymes in greening primary leaves of Phaseolus vulgaris (Bradbeer, 1969, Bradbeer et al., 1969) by making a similar study of Zea mays. The objectives were to gain an understanding of the development of photosynthesis in greening leaves and to elucidate the relative contributions of protein synthesis and enzyme activation. Soon after completing our experiments we were fortunate to be able to resolve a number of questions relating to the properties and regulation of the glyceraldehyde-phosphate dehydrogenases of green leaves at an informal meeting held during the Stresa Photosynthesis Congress at which the contributions from the laboratories of M. Gibbs, P. Pupillo and H. Ziegler were particularly important. As a result it was established for C3 plants that phosphoribulokinase [EC 2.7.1.19] possessed a similar regulatory mechanism to that of the chloroplastic glyceraldehyde-phosphate deydrogenase [EC 1.2.1.13] (Bradbeer, 1973 and Wara-Aswapati et al., 1980) and it was shown that both protein synthesis and enzyme activation were responsible for the increases of activities which occurred during greening (Bradbeer, 1976). The regulation and development of these enzymes in Zea mays proved to be somewhat different from that of C3 plants (Wara-Aswapati, 1973; Lin, Stocking, 1980, Bradbeer et al., 1981) and our recent effort to examine this aspect of regulation in Zea mays (Rtlffer-Turner, Bradbeer, 1983) has enabled us to make a comprehensive report of this work.

C4 plants differ from C3 plants in leaf anatomy in that two distinct cell types cooperate to carry out photosynthesis and in that these cells possess structurally and functionally dimorphic chloroplasts. The situation is further complicated by the occurrence of three distinct varieties of C4 photosynthesis. The state of knowledge with respect to chloroplast development in the most investigated C4 species, Zea mays, is reviewed. Fine structural dimorphism is relatively easy to investigate while the development of functional dimorphism is more difficult to understand. It is, however, an important fundamental question as to how nuclear and plastid genomes are expressed differently in adjacent cells so as to produce this dimorphism.

The coordination of the synthesis of the large and small subunits of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was studied in young light-grown barley (Hordeum vulgare L. var. UC566) leaves. Since a barley leaf is a continuum of different aged cells with the youngest cells at the base and the oldest at the tip, developmental changes could be investigated by comparing different leaf regions. The rate of total cytoplasmic protein synthesis increased to a maximum before the rate of total organelle protein synthesis. The different positions of the maxima suggested that the synthesis of the small RuBPCase subunit on cytoplasmic ribosomes and the large RuBPCase subunit on chloroplast ribosomes might not be coupled during barley leaf development. However, measurements of the amounts and rates of synthesis of the subunits showed that they were coupled. Although the amounts of the RuBPCase subunits increased from the younger to the older leaf regions, the subunits were present in an equimolar ratio. While the rates of synthesis of both subunits increased to a maximum in a midleaf region and then declined, the ratio of the rates remained constant. That the subunit amounts remained equimolar and the synthetic rates proportional while total RuBPCase synthesis was changing indicated that the synthesis of the subunits was closely coordinated during leaf development. A close coordination was also supported by the kinetics of the inhibition of subunit synthesis in the presence of cycloheximide.

The electrophoretic mobility of mature spinach (Spinacia oleracea L. var. Americana) chloroplasts sampled over a 7-month period was between -2.03 and -2.45 micrometers per second per volt per centimeter when suspended in a solution containing 1 millimolar CaCl(2). The surface charge density of EDTA-treated chloroplasts was calculated to be -7,400 electrostatic units per square centimeter representing, on the average, one electronic charge per 645 square Angstroms. Electrophoretic mobility increases during plastid maturation. Calcium, but not magnesium, generally stabilized the envelope of isolated plastids against small increases in surface charge that occur with time in the absence of calcium. Pronase caused a sharp, but temporary, decrease in the electrophoretic mobility of chloroplasts. This was interpreted as representing a transient binding of pronase to the envelope surface during proteolysis. No -SH groups were detected on the surface of the plastid envelope. Inasmuch as the isoelectric point of intact chloroplasts was found to be at pH 4.5, it is likely that the major part of the total surface charge results from the presence of exposed carboxyl groups of intrinsic envelope proteins that are not readily hydrolyzed by mild pronase treatment.

Pronase, cationic ferritin, and ferritin‐conjugated plant lectins were used to study the chloroplast envelope. Negative charges (binding cationic ferritin) are fairly uniformly distributed over the envelope surfaces in contact with the hyaloplasm and are not appreciably altered by mild pronase treatment of isolated plastids. All surfaces of stroma‐free thylakoids previously exposed to the stroma uniformly bind cationic ferritin. RicinII‐ferritin binding to the membranes of the chloroplast envelope indicates that galactolipids are distributed in the outer membrane in such a way that their galactose moieties are exposed on the envelope surface. In addition, the outer surface of the inner membrane (the intermembrane face) contains uniformly distributed galactose which binds ricinII when this membrane is exposed to the reaction medium. Isolated vesicles of the chloroplast envelope bind ricinII, while isolated envelope vesicles as well as the envelopes of intact chloroplasts failed to bind concanavalin A. Thylakoid surfaces showed minor binding of ricinII as well as concanavalin A.

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10(-5)m and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions.

Subjecting isolated spinach chloroplasts to mild proteolysis (10-minute incubation at 20 C in 500 micrograms per milliliter pronase) caused chloroplast clumping but did not affect their integrity as measured by their ability to carry out light stimulated, glycerate-3-P-dependent O(2) evolution. Transmission electron microscopy revealed no detectable differences between the control and treated plastids. Mild proteolysis inactivated exogenously added pyruvate kinase and should be a useful technique in certain enzyme distribution studies.

The relations between leaf age and polyribosome levels were studied with dark-and light-grown maize (Zea mays L.) seedlings. In general, polyribosome levels decline with the period of growth in darkness. Light induces an increase in the polyribosome level in dark-grown seedlings. The response can be detected after 30 min exposure to light. Seven or eight-day-old dark-grown corn seedlings, used in the present study, have high levels of polyribosomes when greened in the light. This is indicative of healthy seedlings, competent in protein synthesis. The polyribosome levels in iron deficient maize plants were significantly different from plants grown under complete nutrient solution, while there is no significant difference among plants suffering different degrees of iron deficiency.

A rapid and simple procedure was used for chemical dehydration of plant tissue during sample preparation for light and electron microscopy. Chemically fixed tissues were washed with distilled water and then rapidly dehydrated with either 2,2‐dimethoxypropane or 2,2‐diethoxypropane for 15 minutes. Light microscopic observation of paraffin‐embedded tissue or tissue embedded in Spurr's plastic showed excellent preservation. Electron microscopic examination of plastic‐embedded tissue showed well maintained ultrastructural morphology. The dehydration procedure was also successfully applied to plant tissue destined for examination in a scanning electron microscope.

The use of microautoradiography at the electron microscopic level indicates that the vacuole is the site of accumulation of the cyanogenic glucoside of Sorghum bicolor. When a specific biosynthetic precursor of dhurrin, p-hydroxy[3,5-(3)H]phenylacetaldoxime, was used, 90% of the tritium label was recovered in the vacuoles of tissue prepared for microautoradiography. l-[3,5-(3)H]Tyrosine and d-[1-(3)H(N)]glucose, nonspecific precursors of dhurrin, of differing solubilities and biosynthetic capacity, were also fed to the shoots. The data obtained from these controls indicated that the high recovery of label in the vacuole of aldoxime-fed shoots was not indicative simply of the size of the vacuole, nor was it a result of movement of labeled compounds during preparation of the tissue for electron microscopy. The problem of movement of these labeled compounds during dehydration of tissue was dramatically reduced by chemical dehydration in 2,2-dimethoxypropane in less than 30 minutes rather than with routine dehydration in acetone or alcohol series for 24 hours.

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.

AbstractThe mechanism of the light‐stimulated absorption of nitrate by Wolffia arrhiza was studied. The nitrate‐absorption mechanism in ammonium‐grown plants is stimulated by the presence of nitrate. In a manner similar to the absorption of many other ions, the absorption of nitrate follows a typical biphasic pattern in relation to external nitrate concentration. Mechanism 1 is effective at nitrate concentrations up to 0.5 to 0.75 mM and mechanism 2 becomes operative at higher nitrate levels.Light stimulates the absorption of nitrate independently of the effect of light on the reduction of nitrate. The effects of uncouplers, inhibitors, and light of wavelengths of 700 nm or longer indicate that nitrate absorption by Wolffia cells is reduced when non‐cyclic electron transport is blocked. It is postulated that under this condition, ATP in the chloroplast (produced by cyclic photophosphorylation) may be less readily transported across the chloroplast envelope than when non‐cyclic electron transport is proceeding.

Chloroplasts contain the enzyme glutamine synthetase. Formation of glutamine by isolated chloroplasts is light-dependent and requires an intact outer envelope. Addition of exogenous glutamic acid, as well as nitrogen donors such as nitrite or ammonium, stimulate the synthesis of this amide. Photosynthetic generation of ATP satisfies the light requirement of glutamine synthesis. The process is supported by cyclic as well as noncyclic photophosphorylation.

AbstractChloroplasts from spinach were fixed in glutaraldehyde and extracted with three different lipid solvents, after which the lipid composition was analyzed. Studies were also made with the electron microscope. In cold dry acetone, which removes 75 % of lipids, the basic structure of the membranes is unchanged. Acetone with 10 % water removes 89 % of the lipids and a mixture of chloroform with methanol removes 93 % of the lipids, both solvents leaving nearly unrecognizable membrane structures. The relationship between lipid composition and membrane structure is discussed.

AbstractFreshly isolated spinach (Spinacea oleracea) chloroplasts were treated with 4 different neutral salt solutions at concentrations of 1 and 2 M for ultrastructural study. Each salt induces a specific pattern of structural disorganization. In KCl, there is principally swelling of the fret‐canal and end‐granal‐compartment loculi. With KI, there is removal of green pigment, loss of some stainability, swelling, and separation of the individual membranes of the granal partition layers. Sodium trichloracetate causes apparent end‐to‐end fusion of granal stacks and their curvature; and in KSCN there is a great volume increase, related to the separation of the membranes of the partition region and extensive swelling of all the loculi.This work has been rendered possible through a grant from the National Science Foundation (GB 12906).

ABSTRACT Ferricyanide was used as a Hill oxidant to localize the site of photoreduction in chloroplast lamellae. The ferrocyanide formed on illumination was complexed with copper ions to form insoluble, electron-dense precipitates of Cu ferrocyanide, which can be easily seen in unstained preparations in the electron microscope. Control experiments showed no precipitates in the dark, at zero time, in the absence of ferricyanide, or on addition of sodium ascorbate. Discrete precipitates of Cu ferrocyanide were seen on both stroma and grana lamellae. It is concluded that both stroma and grana lamellae have photosystem II activity necessary for reduction of ferricyanide.

This chapter discusses the isolation of chloroplasts from nonaqueous method. The method of grinding is critical because the purity of the chloroplast preparation is dependent on a separation of the dried cytoplasm from the chloroplasts. Grinding must be done in nonaqueous solvents precooled to about –10%. A purer preparation of chloroplasts is obtained by grinding the freeze-dried leaves in a hexane-carbon tetrachloride mixture of a density equal to the heaviest density to be used in the gradient. In this case, the ground and filtered sample are placed at the bottom of the centrifuge tube and the density gradient is layered on top of it. The chloroplasts move up through the density gradient during the centrifugation and are washed continuously during this process. To obtain the purest chloroplast preparation, the chloroplasts isolated by the density gradient centrifugation are resuspended in a light hexane-carbon tetrachloride mixture (60:40 v/v) and separated from small particles by repeated (8–10 times) short-term centrifugations (70 seconds at 3500 rpm) in this medium.

Nitrate reductase was not found to be present in or associated with partially purified, intact chloroplasts aqueously isolated from Wolffia arrhiza. Such chloroplasts are capable of using nitrite but not nitrate as an electron acceptor during light-stimulated electron transport in the absence of additional cytoplasmic components. When nitrite acts as an electron acceptor under these conditions, on the average 1.5 moles of oxygen are evolved per mole of nitrite reduced by the chloroplasts, indicating a probable reduction of nitrite to ammonia. Chloroplasts ruptured by osmotic shock fail to reduce nitrite in the absence of additional components.