Glycinebetaine was determined in leaves and in isolated chloroplasts of spinach (Spinacia oleracea) by nuclear magnetic resonance spectroscopy. Some leakage of glycinebetaine from the chloroplasts occurred during the isolation so the concentration in chloroplasts in vivo could be up to 1.5 times higher than that measured in isolated chloroplasts. It was demonstrated that any contamination of the chloroplast preparations by glycinebetaine originating from other cellular compartments or from broken chloroplasts would have amounted to less than 10% of the measured values. Leaf osmotic potential of salt-stressed plants was -2.09 MPa compared to -0.91 MPa in non-stressed controls. This was accompanied by a sixfold increase in glycinebetaine content in the leaf but the levels of choline and proline were not increased. In chloroplasts isolated from control leaves the calculated glycinebetaine concentration was 26 mM which was 10-fold higher than the concentration in the leaf as a whole but only contributed 7% of the osmotic potential of the chloroplast. Chloroplasts from salt-stressed plants contained up to 300 mM glycinebetaine which was 20 times the concentration in the leaf as a whole. The glycinebetaine concentration in chloroplasts from salt-stressed leaves was equivalent to an osmotic potential of -0.75 MPa and this contributed 36% of the osmotic potential of the chloroplast and 64% of the decrease in osmotic potential induced by salt stress. At least 30-40% of the total leaf glycinebetaine was localized in the chloroplast. The results demonstrate that glycinebetaine accumulates in chloroplasts to provide osmotic adjustment during salt stress and provide support for the hypothesis that glycinebetaine is a compatible cytoplasmic solute which may be preferentially located in the cytoplasm of cells.

Severe sapburn occurs in mango fruit of the cultivar Kensington when sap contacts the fruit, resulting in browning and then blackening of the skin. Both the sap and skin of mango fruit contained considerable polyphenol oxidase (PPO) activity. The sap enzyme was not activated by SDS, was inhibited by hexadecyltrimethylammonium bromide, and was active with both para- and ortho-diphenol substrates. The skin enzyme was activated by SDS, was inhibited by salicylhydroxamic acid and polyvinylpyrrolidone, and was active only with ortho-diphenol substrates. These properties suggest that the sap PPO is a laccase-type enzyme (EC 1.10.3.2) whereas the skin contains the more common catechol oxidase-type PPO (EC 1.10.3.1). The skin enzyme had a temperature optimum at 30�C but the sap enzyme had maximum PPO activity at 75�C. Both enzymes were relatively thermostable, requiring more than 15 min at 80�C for 50% loss of activity. It is concluded that browning of mango skin induced by the sap is predominantly catalysed by PPO in the skin and that this is unlikely to be prevented by heat treatment of the fruit.

Damage caused to the skin of mango fruit by contact with sap exuded from the cut or broken pedicel reduces consumer acceptance and storage life of the fruit. Mangoes of the Kensington cultivar are particularly susceptible to sapburn injury. On centrifugation, the fruit sap separated into two phases. Skin damage was caused predominantly by the upper non-aqueous phase. A major component of this phase was terpinolene which gave symptoms indistinguishable from sapburn injury when applied to the fruit surface. The same type of damage could be induced by the application of synthetic terpinolene when applied undiluted, diluted in hexane or as an aqueous emulsion. Non-volatile sap components separated by distillation were not damaging to mango skin. Sap exuded from the mango leaf petioles also contained terpinolene, but its concentration was less than 1% of the concentration in pedicel sap and this sap was not damaging to the fruit skin.The Florida cultivar Irwin is less susceptible to sapburn injury and the predominant terpene in its sap was identified as car-3-ene. When applied to Kensington skin, car-3-ene caused significantly less damage than terpinolene. We conclude that the primary cause of mango sapburn is entry of volatile components of the sap such as terpinolene through the lenticels, resulting in tissue damage and subsequent enzymic browning.

Fruit of the variegated grapevine mutant Bruce's Sport is known to dry to a lighter colour than other seedless varieties. The biochemical basis for this decreased browning capacity was investigated. Bruce's Sport grapes had similar levels of phenolic compounds to Sultana H5. Activity of the enzyme polyphenol oxidase (PPO EC 1.10.3.1) in mature berries of Bruce's Sport was only 20-30% of that in Sultana H5. No evidence of inhibitors or activators of PPO was found when berry extracts were mixed. PPO activity on a fresh weight basis was highest in the grape seed traces, intermediate in the skin, and lowest in the pulp in both varieties. In each tissue type, however, PPO activity in Bruce's Sport was less than 25% of that in Sultana H5. On a fresh weight basis, PPO activity in Sultana H5 berries was high at fruit set then declined as the berry developed. PPO activity per berry increased from fruit set until veraison, then remained constant. PPO activity showed similar changes during development of Bruce's Sport berries but was lower than in Sultana H5 at all stages. The Bruce's Sport grapes were variegated and the green regions of skin had similar PPO activity to Sultana H5 skin while the white regions had very low activity. Only the green regions of skin of Bruce's Sport grapes stained for PPO activity with endogenous or exogenously applied phenolic substrates. The decreased browning in this grapevine mutant apparently results from decreased levels of PPO activity in the white regions of the berry, possibly arising from a disruption in chloroplast development.

The concentration of inorganic orthophosphate (Pi) was determined in the stroma of isolated chloroplasts during photosynthesis under Pi-saturated and Pi-limited conditions. Pi was determined calorimetrically or by high performance liquid chromatography of extracts of chloroplasts labelled with 32Pi. When chloroplasts were illuminated in the absence of added Pi, photosynthesis soon declined due to Pi-depletion. After 5 min in the light, photosynthesis had declined to 2% of the maximum rate. At this point, stromal Pi was estimated to be 1.4 mM by the colorimetric method and 0.2 mM by 32P chromatography. Using the colorimetric method, Pi equivalent to approximately 1 mM in the stroma was found to be associated with thylakoid membranes isolated from chloroplasts, irrespective of the Pi content of the intact chloroplasts. This was considered to be a non-metabolic pool of Pi. During steady- state photosynthesis with optimal concentrations of Pi added to the reaction medium, the stromal Pi concentration was estimated to be 2.6 mM and 1.6 mM with the colorimetric and 32P methods, respectively. Measurement of stromal 32Pi in chloroplasts illuminated with varying concentrations of 32Pi in the reaction medium suggested that photosynthesis was saturated at stromal Pi concentrations above 2.0-2.5 mM. Photophosphorylation by thylakoid membranes was saturated at Pi concentrations above 1.2-1.5 mM. It is concluded that, during photosynthesis in isolated chloroplasts in the presence of an optimal supply of Pi from the reaction medium, the stromal Pi concentration is just above that required to saturate photophosphorylation. Any decrease in the supply of Pi from the medium results in a rapid decrease in stromal Pi to the point where photophosphorylation may become Pi-limited, decreasing the rate of CO2 fixation.