Glucose Transport into Spinach Chloroplasts

140

The subceflular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chhoroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADPGlucose pyropbosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosyhse) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction Chromatography of a chloroplast extract on diethylaminoethyl-ceilulose resolves the R-and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amyopectin as a substrate indicates an endolytic activity but displays properties unlike the typical a-amylase as isolated from endosperm tissue.

Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Subcellular Localization of the Starch Degradative and Biosynthetic Enzymes of Spinach Leaves

Okita

Greenberg²,

Kuhn³

et al. 1979

140

“…The chloroplast envelope is generally considered to be impermeable to sorbitol (6,7,16,17) measured by incubating parallel samples in unlabeled sorbitol and adding 3H20 and ['4C]sorbitol just prior to centrifugation to minimize any uptake of labeled sorbitol. The difference between these samples and those incubated in ['4C]sorbitol from the outset was a measure of sorbitol uptake by the chloroplasts.…”

Section: Resultsmentioning

confidence: 99%

Osmotic Adjustment by Intact Isolated Chloroplasts in Response to Osmotic Stress and Its Effect on Photosynthesis and Chloroplast Volume

Robinson

1985

Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, -1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at -1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, -2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to -1.8 megapascals and the chloroplasts had higher rates of COrdependent O2 evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20°C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless 1'4Clsorbitol was added just prior to centrifuging the chloroplasts through silicone oil. There is already some experimental evidence for osmotic adjustment by chloroplasts in response to stress. Spinach plants exposed to salt-stress have decreased osmotic potentials, yet photosynthetic capacity is not decreased (12,14), suggesting that osmotic adjustment in the chloroplasts somehow prevents the inhibition which is observed when chloroplasts are exposed to decreased osmotic potentials in vitro (2, 3, 5, 1 1). The concentration of inorganic ions increased in the leaf in response to salt stress but not in the chloroplast (12, 14) suggesting accumulation of organic solutes in the chloroplasts. Indeed, chloroplasts isolated from the salt-stressed plants had increased levels of sugars and amino acids (12) although these were not sufficient to account fully for the decrease observed in cell sap osmotic potential. Chloroplasts isolated from Mesembryanthemum grown under high salt had optimal photosynthetic rates at higher sorbitol concentrations in vitro than did chloroplasts grown in low salt (5). These observations are consistent with the accumulation of compatible solutes in chloroplasts to provide osmotic adjustment in response to salt stress.In this paper, I have investigated the possibility of inducing osmotic adjustment in chloroplasts in vitro in an attempt to determine the influence of such osmoregulation on photosynthesis. The

“…Export of carbohydrate occurs via the chloroplastic phosphate-triose phosphate translocator (9). In addition, there is evidence for a glucose transport system with low substrate affinity (25). Furthermore, a hexose phosphate transport system in the chloroplasts of Sedum (20) and Codium (24) has been described.…”

mentioning

confidence: 96%

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts

Bätz

Scheibe²,

Neuhaus³

1992