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Without Abstract:

We have investigated Ca2+ activity during pollen germination and the possibility that it may be responding to a phosphoinositide signal transduction pathway, by employing inhibitors of Ca2+ channels (verapamil and TMB-8), EGTA as a Ca2+ scavenger and the inositol 1-phosphatase inhibitor lithium chloride. We have found that at least two Ca2+ pools are utilized during pollen germination. Influx of extracellular Ca2+ appears to be necessary for the germination of apple and tobacco pollen, but it does not appear to be required for the germination of potato pollen. Conversely, activation of intracellularly stored Ca2+ was necessary for optimal germination of all three pollen species. LiCI had strong effects on pollen germination. At 5 mM LiCI, pollen germination was inhibited by 78% for apple, 84% for tobacco, and 74% for potato. Li+ inhibition was overcome by the addition of Ca2+, which restores germination of all three species to 85–100% of that observed in controls, myo-lnositol also partially overcomes Li+ inhibition of pollen germination, thus providing some evidence for a link between Li+ inhibition and Ca2+ rescue, myo-lnositol rescue of Li+ inhibition was most effective for potato pollen. Chlorotetracycline (CTC) spectroscopy revealed a higher level of membrane-Ca2+ in Li + -treated pollen grains than in controls, and the short pollen tubes which did emerge did not accumulate membrane-associated Ca2+. The results suggest that Li+ inhibition may interfere with the release (activation) or partitioning of membrane-Ca2+ during pollen germination and that this Ca2+ activity may be responding, at least in part, with a phosphoinositide signal transduction pathway.

Sucrose synthetase (EC 2.4.1.13) was found in the latex of the rubber tree but the activity of sucrose phosphate synthetase (EC 2.4.1.14) was not detected. The enzyme was purified and some properties have been investigated. Examination of the kinetics of sucrose synthesis revealed Km of 0.56 mM for uridine diphosphoglucose and 3.85 mM for fructose. Mg2+ and cyanide activated sucrose synthesis but reduced the cleavage reaction. Increased pH had the same effect, the synthetic activity being higher than the activity of sucrose breakdown within the physiological levels of latex pH. In the latex of regularly tapped trees, the total enzyme activity in the direction of synthesis was about 10% or less of the total invertase activity at pH 7.0. Because of the strong limitation of invertase under natural conditions, the proportion of actual synthetase activity is, however, much higher and evidence is presented that in the latex of regularly tapped trees this activity significantly reduces carbohydrate breakdown. Some indications have been obtained that this involvement of sucrose synthetase is weakened by application of Ethrel to the bark. A reduction of its synthetic activity, accompanied by an acceleration of sucrose utilization in latex cytoplasm and by an increase of latex yield, could be observed before the treatment-induced rise of pH enhancing inver.

Treatments which increase latex yield, e. g. bark scraping, latex tapping, and bark application of 2, 4-D or 2-chloroethylphosphonic acid (CEPA) were found to enhance the activity of latex invertase. In previously untapped trees, both the introduction of tapping and the application of 2, 4-D brought about an increase in the level of invertase. In regularly tapped trees, the amount of latex invertase is several times higher than in untapped trees and evidence was obtained that its activity is regulated by the variation of latex pH. The pH of latex of the clone investigated (PR 107) was shown to vary between 6.3 and 7.1 whereas the activity of invertase, as assayed directly in the latex, has a sharp optimum at pH 7.5 and falls rapidly with the shift of pH to the acid side. There was no increase in the content of latex invertase when trees adapted to regular tapping were treated with 2, 4-D. The effect of auxin on actual invertase activity was essentially mediated through related increase of latex pH. The CEPA and bark scraping were also shown to increase latex pH in tapped trees. The treatment of the bark of tapped trees with CEPA increased the level of latex sucrose, as did auxins. Bark scraping also had a slight stimulatory effect. The Km of latex invertase as a function of pH was found to change in the same way as Vmax, being highest at pH optimum.