Ethylene-controlled Induction of Phenylalanine Ammonia-lyase in Citrus Fruit Peel

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cellfree extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-0-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.Several Citrus spp. (Rutaceae) contain bitter tasting flavanone-disaccharides (18). They are used as food additives for their flavor or can be converted by hydrogenation into the extremely sweet dihydrochalcone derivatives (18). Little information is available on the biosynthetic pathways to flavanone-glycosides in Citrus (16). Chalcone-synthase is a key enzyme in the flavonoid pathway in several plants (13), but has not previously been reported in Citrus tissues. Chalconesynthase catalyzes the condensation of p-coumaryl-CoA with three molecules of malonyl-CoA yielding naringenin-chalcone (20). The substrate specificity of a 'naringin-chalconecyclase' activity, from immature grapefruit fruit (25), suggested that the flavonoid pathways in Citrus, might be different from the more studied flavonoid pathways of other plants (13,14) (Fig. 1). They interpreted their results (25) as suggesting that glucosylation and rhamnosylation occurred during chalcone formation in Citrus, as the aglycones were not ' Supported by a Minerva foundation travel grant. Present address:

contrasting

confidence: 40%

Flavanone Glycoside Biosynthesis in Citrus

Lewinsohn

Britsch²,

Mazur³

et al. 1989

“…Ehylene induces the appearance of PAL activity in citrus flavedo (15). The wound-ethylene produced from flavedo discs induced strong PAL activity in the disc tissue within 24 hr (0.36 + 0.02 ,umol cinnamic acid/g fresh wt/hr).…”

Section: Resultsmentioning

confidence: 99%

“…Sugars were visualized by spraying with 0.1 M p-anisidine and 0.1 M phthalic acid dissolved in 96% ethanol and heating at 100 C for 10 min (I 1). Phenylalanine ammonia lyase (PAL)3 activity was determined according to Riov et al (15).…”

Section: Methodsmentioning

confidence: 99%

Differential Counteraction of Ethylene Effects by Gibberellin A₃ and N₆-Benzyladenine in Senescing Citrus Peel

Goldschmidt¹,

Aharoni²,

Eilati³

et al. 1977

Treatment of mature citrus fruit (Citrus sinensis) with ethylene induced rapid chlorophyHl destruction, a rise in respiration, a release of free amino acids, aa accumulation of reducing sugars, and an appearance of phenylalanine ammonia lyase activity. Gibberellin A3 (GA3) and N6-benzyladenine (BA) opposed the effects of ethylene on chlorophyll, amino acids, and to a lesser extent, reducing sugar levels. The ethyleneinduced respiratory rise was only slightly modified by GA3 and BA. Phenylalanine ammonia lyase activity was not affected by GA3.The antagonism between ethylene and the senescence-delaying regulators GA3 and BA seems to operate mainly within the chloroplast, but might not be confined to this compartment. The accumulation of reducing sugars exhibits the antagonism although it is not apparently related to the chloroplast. Scott and Leopold (16) were the first to draw attention to the opposing effects of ethylene and GA3. Citrus rind maturation served as one of the most evident examples since it was known by then that GA3 delays the senescent color changes of citrus rind whereas ethylene accelerates them. Cytokinins also delay citrus rind coloration, in accordance with their universal senescence-delaying effects (8, 10). Opposing effects of ethylene and GA3 or BA have been reported also for the coloration of ripening tomatoes (7) and bananas (17,18). The respiratory behavior of these climacteric fruits has not, however, been altered by GA3 treatments (7,17).In the present study, we examined the ability of GA3 and BA to counteract ethylene effects associated with various cellular compartments in senescing citrus rind. MATERIALS AND METHODSDetached mature green Shamouti orange (Citrus sinensis L. Osbeck) fruits were used, unless otherwise specified. Fruits were dipped twice, for 30 sec each, in GA3, BA, or control solutions containing 5% ethanol + 0.02% Tween-20. The ethylene-releasing chemical, 2-chloroethylphosphonic acid (Ethephon), was applied to the fruit calyx as a 25-,ul droplet of 1% solution (9). GA3 and BA were applied to fruits immediately after harvest. Ethephon or ethylene gas was usually supplied 2 to 4 hr later, after the fruits had dried from the GA3 and BA solutions.Rind color changes were measured by a Hunter color differ- quantitative estimation of color development (3). According to this method, the intensity of green color is represented by negative values which rise gradually and pass to the positive range during development of yellow and orange colors. For the assay of CO2 and ethylene, samples of four fruits were placed in 2-liter glass jars at 25 C. Duplicate samples were run for each treatment. The jars were continuously ventilated (100 ml/min) with water-saturated air, with or without 12 ,ul/l ethylene. Jars were sealed for 1 hr prior to removal of jar atmosphere samples for determination of CO2 and ethylene evolution. CO2 was determined by a Packard gas chromatograph equipped with a Poropak Q column and a thermal conductivity detector. Ethylene was determined by a Packa...

“…3 and 5). If ethylene had a role in conjugation, ethylene evolution should have preceded the rise in NAAsp (12,29). However, in the case of pea roots auxin-induced ethylene evolution and growth inhibition occur simultaneously (12).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Naphthaleneacetic Acid Conjugation

Gören¹,

Bukovac²

1973

Formation of naphthaleneacetic acid-glucose (NAGlu) in detached leaves, floating on a-naphthaleneacetic acid-1-V4C (NAA, 0.05 microcurie per milliliter, 3.1 ,uM)-buffer solution (phosphate-citrate, pH 4.2) began immediately while there was a 2-to 4-hour lag before NAA-asparatate (NAAsp) could be detected. Subsequent increase in the NAAsp conjugate reflected a decrease in free NAA to 1 to 2 % of the total radioactivity taken up. Pretreatment with 31 gM 'C-NAA for 18 hours doubled NAAsp formation after transfer for 4 hours to '4C-NAA. Pretreatment with ethylene, as ethephon (up to 400 milligrams per liter) or ethylene gas (10 microliters per liter), did not induce NAAsp formation. In the presence of NAA, ethylene had no effect on NAA conjugation. Similarly, CO2 (5%) did not modify the formation of the conjugates. Rhizobitoxine (1.87 uM) inhibited NAA-induced ethylene production but did not prevent NAA-induced formation of NAAsp. We concluded that the conjugation of NAA with aspartic acid is not mediated by ethylene.