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Phytochemical investigation of the leaves of Paullinia pinnata L. (Sapindaceae) resulted in the isolation of the two new flavone glycosides characterized as diosmetin-7-O-(2' '-O-beta-D-apiofuranosyl-6' '-acetyl-beta-D-glucopyranoside) (1) and tricetin-4'-O-methyl-7-O-(2' '-O-beta-D-apiofuranosyl-6' '-acetyl-beta-D-glucopyranoside) (2).

Changes in net carbon assimilation and water status were studied during leaf development in the deciduous, tropical species Brachystegia spiciformis Benth. In this upland savanna African tree, bud-burst and leaf development occur approximately two months before the rainy season. The newly formed leaves synthesize anthocyanin until the fully expanded leaves of the whole canopy are red. This foliage is referred to as "spring flush" foliage. Subsequently, the anthocyanins are metabolized and the pre-rain leaves become green. Carbon dioxide assimilation exhibited a bimodal diurnal pattern and was similar for pre-rain green leaves and fully expanded flushing leaves, although pre-rain green leaves showed a net uptake of carbon throughout the daylight period, whereas flushing leaves exhibited only brief periods of net photosynthesis in the morning and early afternoon. Measurements of leaf water potential and relative water content showed a diurnal pattern with considerable variation throughout the day. Leaf water potential and relative water content values decreased soon after sunrise reaching a minimum at a time corresponding to the afternoon peak in CO(2) assimilation. Stomatal conductance was closely related to transpiration rate in both flushing and pre-rain green leaves, although flushing leaves had lower stomatal conductances than pre-rain green leaves. Pre-rain green leaves exhibited a compensation irradiance of approximately 180 micro mol m(-2) s(-1), whereas flushing leaves had positive net photosynthesis only at PPFDs greater than 300 micro mol m(-2) s(-1). Rate of photosynthesis (expressed per leaf area or chlorophyll unit) increased as anthocyanin concentration decreased, although the photosynthetic rate continued to increase long after the leaf anthocyanins had been degraded to low, visually undetectable amounts. Post-rain green leaves had chlorophyll concentrations, transpiration rates and stomatal conductances similar to those of pre-rain green leaves; however, photosynthetic rates in post-rain leaves were more than three times higher. Thus, during the early stages of the spring flush, carbon asimilation rates of the flushing leaves were inversely related to leaf anthocyanin concentrations. In pre-rain green leaves, photosynthesis was limited by other non-stomatal factors.

Cotton (Gossypium hirsutum L.) embryos excised from bolls 38-43 d after anthesis and cultured in vitro for 4 d on a nutrient agar medium containing 3.8 μM abscisic acid (ABA) developed enzyme activity and accumulated insoluble protein, neutral lipid, and dry weight similar to embryos maturing on the plant. Inclusion of ABA in the medium prevented precosious germination and allowed continued increases in catalase, malate dehydrogenase, citrate synthase, aspartate aminotransferase, and β-oxidation enzyme activities as well as de-novo synthesis of malate synthase. Isocitrate lyase activity was not detectable in ABA-cultured embryos nor normally-developed embryos. Omission of sucrose from the medium resulted in near-doubling of the development of malate synthase activity, with minimal effects on the other enzyme activities. Addition of Actinomycin D, cordycepin, or cycloheximide to ABA-containing cultures did not overcome the observed inhibition of germination, but severely reduced both the appearance of new malate synthase activity and further production of other related enzyme activities. Thus, development of these enzyme activities in the presence of ABA appears dependent on transcription and translation, while inhibition of germination by ABA at this stage of development is not sensitive to the RNA- and protein-synthesis inhibitors. The results indicate that ABA does not prevent vivipary by suppressing translation of m-RNAs coding for isocitrate lyase and its companion enzymes, as previously proposed.

Glyoxysomes isolated from endosperm and cotyledons of germinated fatty seeds have been extensively characterized (12,22). Recent emphasis, however, has been on the biogenesis of glyoxysome, especially in castor bean endosperm (2, 10, 14, 15). The collective studies have supported the concept that glyoxysomes are derived directly from the ER by a process of vesiculation. Synthesis of the limiting membranes with their constituent enzymes and initial segregation of matrix proteins are believed to occur in the ER prior to detachment and formation of separate organelles (2).In this paper we examined the events of glyoxysome (microbody) proliferation and enzyme development in a new system, viz. embryonic cotton cotyledons. This system has afforded the opportunity to study the critical stages of microbody ontogeny without the complications of large amounts of intracellular storage lipid and protein encountered with dry or early germinated seeds. We have ultrastructurally documented the microbody-ER association that has been inferred in the above cited studies. Furthermore, we have evidence that marker glyoxylate-cycle enzymes are not regulated in a coordinated fashion, a finding not previously noted in seed germination studies.' Supported by National Science Foundation Grant PCM74-01442, A04. MATERIALS AND METHODSGrowth and Selection of Plants. Cotton plants, Gossypium hirsutum L. cv. Deltapine 16, were grown under greenhouse conditions; flowers were tagged at anthesis to determine the age of the developing boils. Ovules removed from bolls were selected mainly on the basis of a standard degree of seed coat sclerification (Fig. 1). Embryos were excised from these ovules and selected on the basis of age in DAA,2 gross morphology (Fig. 1), and fresh wt. In some experiments, untagged field plants were used and material was selected on the basis of all parameters except age. Seeds were presoaked and germinated on Petri plates as described previously (18).Homogenization and Centrifugation Procedures. To determine total enzyme activities, embryos and seeds were homogenized thoroughly in 100 mm K-phosphate (pH 6.9) with 3 mM MgC12 (containing 3 mm DTT in experiments testing for isocitrate lyase activity) with a Potter-Elvehjem motor-driven Teflon homogenizer, then centrifuged at 30,900g for 20 min. The volume (ml) of grinding medium/embryo varied with the age examined ranging from 1:4 at 22 DAA to 1:1 at 50 DAA; seeds were done at 1:1. The supernatant was used directly for assays. For differential centrifugation experiments, embryos were chopped with razor blades attached to an electric knife in grinding medium containing 20 mm K-phosphate (pH 6.9), 2.5% Ficoll, 0.5 M sucrose, and 3 mM EDTA. The resulting homogenate was then filtered through three layers of Miracloth into cold centrifuge tubes and spun at 270g for 10 min in a Beckman JA-20 rotor to remove cell debris, followed by two additional spins of l0,800g for 30 min to obtain a microbody pellet, and 150,000g for 60 min with a Beckman TI-50 rotor in a Sorvall OTD-...