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In a previous paper (9) it was demonstrated that transformation between chloroplasts and chromoplasts in citrus epicarp could be controlled in vitro by manipulating sugar and nitrogen supplies in agar media upon which pericarp segments were cultured. High concentrations of sucrose in media usually promoted degreening and inhibited regreening, whereas nitrogen as nitrate and certain amino acids acted in opposition to sucrose by inhibiting degreening and promoting regreening. In the presence ofincreasing nitrogen concentrations, the sucrose effect was much reduced and, when nitrogen supplies were sufficiently high, high concentrations of sucrose sometimes promoted regreening.In addition to promoting loss ofChl, high sugar concentrations in media increased epicarp sugar concentrations, and in media lacking nitrogen high concentrations of sugars caused a marked reduction in epicarp amino acid concentrations. In those in vitro experiments there was a significant negative correlation between Chl and the molar ratio of sugars to amino acids in the epicarp.Based on these observations, it is hypothesized that most citrus fruit degreen in response to a reduced flux in nitrogen to the fruit accompanied by increased concentrations of sugars in the epicarp, both usually induced by cool temperatures. Regreening of late season citrus fruit in the spring and summer could be attributed to renewed nitrogen flux and a reduction in sugar concentrations. Degreening of species such as C. madurensis Lour., could also be attributed to elevated sugar concentrations and reduced nitrogen content, but cool temperatures would not be the causative factor.To further examine the relationship between plastid transformations and sugar and nitrogen status, Chl, sucrose, reducing sugars, total nitrogen, and amino acids were monitored in the epicarp of C. sinensis (L.), Osbeck cv Valencia and pericarp of C. madurensis Lour. fruit degreening in situ, and in the epicarp of regreening C. sinensis fruit. Also, pericarp sections from C. sinensis fruit at different stages of development were tested for their response to sugars and nitrate in vitro. The results, reported here, suggest that accumulation ofsugars in the epicarp is indeed a major factor regulating plastid metamorphosis in citrus fruit, and that while the abundance of nitrogen is an influential factor, nitrogen flux is not a major factor in seasonal changes in plastid form. MATERIALS

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A method for reversibly regreening and degreening citrus epicarp in vitro using peel segments was developed.Peel segments from mature degreened fruit promptly regreened when kept in light upon agar medium containing low (15 millimolar) concentrations of sucrose. Higher concentrations of sucrose inhibited this regreening, but N03-and certain amino acids included in the media overcame the inhibition by sucrose. However, L-erine strongly inhibited regreening. In the presence of nitrogen, sucrose promoted regreening.Peel (14), Chrysosoplenium alternifolium and Chr. oppositifolium (29), the spathe of Zantedeschia elliottiana Eng. (14), and subepidermal tissues of Cucurbita pepo fruit (9).In addition to temperature, the degreening and regreening of citrus fruit are affected by nitrogen fertilization (18, 19), exogeneously applied gibberellins (7), and certain internal factors such as variety, rootstocks, and number of seeds per fruit (11).An in vitro method of studying this interconversion ofchromoplasts and chloroplasts in citrus peels has been developed and a preliminary report presented (15). Further in vitro studies have shown that the degreening and regreening of citrus peels can be regulated nutritionally. An abundance of nitrogen promotes the chloroplast form, whereas an abundance of sugars promotes the chromoplast form. A nutritional explanation for the degreening and regreening of certain citrus fruit is proposed. MATERIALS AND METHODSFruit ofCitrus sinensis L. Osbeck (cv Valencia) and C. paradisi Macf. (cv Marsh) were surface-sterilized by a 5-min soak in 1% (w/v) NaOCI and rinsed in sterile water. A wide section of peel was cut from the equatorial region (usually) with a razor blade and held submerged in a shallow tray of sterile water while discs were cut out with a 10-mm-diameter cork borer.Peel segments thus prepared were placed epicarp side up on 8 ml of agar media in covered 18-x 150-mm culture tubes and placed under continuous fluorescent lighting (Sylvania Cool White) of between 4 and 12 w m-2 the following morning.

Effects of zeatin on amino acid and sugar contents of detached radish (Raphanus saivus L) cotyledons were investigated to determine if accumulation of these solutes contributes to cytokinin-enhanced growth. Protein (14,18,21), and certain portions of grass leaves expand faster after treatment with either of these stimuli, as evidenced by leaf unrolling (11,23). In light-sensitive lettuce seeds, cytokinins increase the growth rate of cotyledons (5), while red light primarily enhances elongation of radicle cells (20). Cotyledons from numerous other dicots also grow faster in the presence of exogenous cytokinins (2,4,9,10,16,19), and this phenomenon has been developed as a cytokinin bioassay by Esashi and Leopold (2), Letham (9,10), and Narain and Laloraya (16). ing wall plasticity, while red light-enhanced lettuce seed germination seems to arise from an osmotic effect due to more rapid degradation of some macromolecule (15). We are not aware of data indicating that either red light or cytokinins increase wall plasticity, and our studies were to determine whether increased cell expansion (10) of excised radish cotyledons by cytokinins could be due to water uptake resulting from osmotic effects of enhanced solute accumulation. We found that both cytokinins (in the presence of light) and red light increased the reducing sugar contents of the cotyledons, and these increases were highly correlated with cotyledon growth. MATRIAILS AND METHODSRadish seeds (Raphanus sativus L. var. Early Scarlet Globe) were surface sterilized with 10% (v/v) Clorox and, after thoroughly rinsing, were germinated in darkness for 48 hr on wet paper. The smaller cotyledon cut from each selected seedling was used in all studies. Usually five cotyledons (20-35 mg fresh weight) were randomly selected for each sample, blotted gently, weighed as a group, and placed adaxial side down on Whatman No. 1 filter paper covering the bottom of a 5.5-cm Petri dish. The filter paper was previously wetted with 1 ml of 2 mm potassium phosphate buffer (pH 6.4) containing designated additives. The Petri dishes were placed on wet paper towels in glass trays covered with clear plastic film, and cotyledons were incubated at 25 C. The trays were also covered with aluminum foil for incubation in darkness. After incubation, cotyledons were blotted and reweighed.Immediately after obtaining final weights, cotyledons in each sample to be analyzed for protein and amino acids were placed in 10 ml of hot 80% (v/v) ethanol and boiled until volumes were reduced to about 2 ml. An additional 10 ml of 80% ethanol was then added, and samples were homogenized with a Brinkmann Polytron and centrifuged for 15 min at 17,300g at 4 C. Supernatant volumes were adjusted to 20 ml, and a-amino nitrogen was determined on 1-ml aliquots, as described by Yemm and Cocking (24), using glutamine as a standard. Residues from centrifugation were prepared for protein analysis by twice dissolving in 0.1 N KOH and precipitating with 10% (w/v) trichloroacetic acid. The final precipitates were...