Our previous research has demonstrated preventive effects of foliar sprays of growth regulators containing GA4+7 (ProVide or Promalin) on cold storage-induced leaf yellowing and abscission in `Stargazer' hybrid lilies. Further research was conducted to investigate the effective concentrations of Promalin and appropriate timing of promalin sprays. Lilies at “puffy bud” stage were sprayed with promalin at concentrations of 10, 25, 50 or 100 ppm (each GA4+7 and BA) just before placing them at 4 °C for 2 weeks in darkness. Promalin concentrations of 25 ppm or above completely prevented cold storage-induced leaf yellowing occurring during the poststorage evaluation phase in a simulated consumer environment, whereas 10 ppm sprays only partially prevented it. Foliar spray of Promalin (100 ppm each GA4+7 and BA) just before storage at 4 °C for 2 weeks was compared with spraying 2 or 4 weeks before cold storage. While spraying 2 weeks before storage prevented leaf yellowing to the same extent observed in plants sprayed just before cold storage, spraying 4 weeks before storage had very little preventive effect on leaf yellowing. To investigate the effectiveness of promalin sprays with different cold storage durations, puffy-bud stage plants were stored at 4 °C for 1, 2, 3, 4, or 5 weeks in darkness with or without promalin sprays (100 ppm each GA4+7 and BA) before storage. Longer cold storage durations increased the severity of leaf yellowing occurring during poststorage phase. Although promalin was able to prevent leaf yellowing completely up to 2 weeks of cold storage, beyond 3 weeks of cold-storage, effectiveness of promalin diminished with no apparent preventive effect on plants stored for 5 weeks.
Three soluble invertase isoforms from Lilium longiflorum flower buds that had been separated by DEAE-Sephacel chromatography were purified to near homogeneity by further chromatography on hydroxylapetite, Con-A sepharose, phenyl agarose, and Sephacryl S-200 gel filtration. Nondenaturing polyacrylamide gel electrophoresis (PAGE) gave a single band in all three invertases that corresponded to a band of invertase activity in a duplicate gel. The SDS-PAGE of the purified invertase I resulted in a single band with apparent relative molecular mass of 78 kDa. Invertase II and III were resolved to a similar polypeptide pattern by SDS-PAGE with three bands of 54, 52, and 24 kDa. Antiserum of tomato acid invertase cross-reacted with all three invertase protein bands. Antiserum of wheat coleoptile acid invertase cross-reacted only with 54 and 52 kDa bands of invertase II and III but did not recognize invertase I protein. Con-A peroxidase was bound to invertase I protein and all three protein bands of invertase II and III, suggesting that all proteins were glycosylated. Invertase I protein could be completely deglycosylated by incubating with peptide-N-glycosidase F to result in a peptide of 75 kDa. Invertase II and III were partially deglycosylated by peptide-N-glycosidase F resulting proteins bands of 53, 51, 50, and 22 kDa.
Easter lily flower buds at five stages of development (stage 1, 3–4 cm in length; stage 2, 6–7 cm; stage 3, 9–10 cm; stage 4, unopened buds, 13–14 cm; and stage 5, open flower 1 day after anthesis) were harvested, and flower organs were dissected for invertase assay. On a fresh weight (FW) basis, anthers had the highest soluble invertase activity (about 10-fold greater) than all other organs reaching to 15 units/g FW by the stage 2. The activity dropped to about 3 units/g FW at stage 3 and 4, and then increased up to 10 units/g FW in open flowers. Specific activity (units per mg of protein) also showed the same trend. On a specific activity basis, sepal invertase activity steadily increased during bud development, but was relatively constant on a fresh weight basis. stigma, style, and ovary, soluble invertase activity expressed on a FW and specific activity basis steadily increased as bud development. Filament soluble invertase activity on FW basis dropped at the stage 2 and 3, while specific activity steadily increased during bud development. Cell wall-bound invertase activity (released with 1 m NaCl) was present in all flower organs. However, soluble activity accounted for the most of total activity in sepal, ovary and filament (about 90%). About 75% of total activity was soluble in anther and style, whereas nearly equal amounts of soluble and cell wall activities were present in the stigma. The cell wall bound invertase activity increased throughout the bud development in sepal, stigma, style, and ovary parallel to soluble activity. Anther cell wall-bound activity fluctuated in a similar pattern as the soluble activity.
Amylolytic activities extracted from scales of tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs stored at 4 °C for 6 weeks under moist conditions were characterized. Anion exchange chromatography of enzyme extract on DEAE-Sephacel revealed three peaks of amylolytic activity. Three enzymes showed different electrophoretic mobilties on nondenaturing polyacrylamide gels. The most abundant amylase activity was purified extensively with phenyl-agarose chromatography, gel filtration on Sephacryl S-200, and chromatofocusing on polybuffer exchanger PBE 94. The purified amylase was determined to be an endoamylase based on substrate specificity and end product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55 °C when soluble starch was used as the substrate. The apparent Km value for soluble starch was 1.28 mg/ml. The inclusion of 2 mM CaCl2 in the reaction mixture resulted in a 1.4-fold increase in the enzyme activity. The presence of calcium ions also enhanced the thermo-stability of the enzyme at higher temperatures. The enzyme was able to hydrolyze soluble starch, amylose, amylopectin, and beta-limit dextrin, but it had no activity against pullulan, inulin, maltose, or p-nitrophenyl alpha-glucopyranoside. Only maltooligosaccharides, having a degree of polymerization of 7 or more, were hydrolyzed to a significant extent by the enzyme. Exhaustive hydrolysis of soluble starch with the enzyme yielded a mixture of maltose and matlooligosaccharides. This amylase activity was not inhibited by alpha- or beta-cyclodextrin upto a concentration of 10 mM. Maltose at a 50 mM concentration partially inhibited the enzyme activity, whereas glucose had no effect at that concentration.
In mature Lilium longiflorum flower buds, anther and stigma had the highest soluble acid invertase activity [3.29 and 2.31 μmol of reducing sugars (RS)/min per gram of fresh weight (FW), respectively] compared to style, ovary, petal, and filament with activities of 1.52, 1.08, 0.99 and 0.98 μmol RS/min per gram of FW, respectively. DEAE-sephacel chromatography revealed that invertase activity in petal, ovary, style, and stigma was composed exclusively of invertase II and III isoforms. Anther invertase was mainly invertase I with small amounts of invertase II and III. Filament tissue mainly had invertase II and III isoforms with a small amount of invertase I. Wall-bound invertases were extracted with 1.0 m NaCl. Anthers had the highest wall-bound invertase activity (4.42 μmol RS/min per gram of FW) followed by stigma (0.42 μmol RS/min per gram of FW). Other tissues had low wall-bound invertase activity (<0.1 μmol RS/min per gram FW). For further purification, the binding of soluble invertases to nine different reactive dyes was investigated. Invertase I was bound to Reactive Green 5, Reactive Green 19, and Reactive Red 120 columns and was eluted with 0.5 m NaCl, resulting in increase in specific activity ≈10-fold with ≈70% recovery. Invertase II and III bound only to Reactive Red 120. Elution with 0.5 m NaCl resulted in an ≈6-fold increase in specific activity.
The effects of Promalin® [PROM; 100 mg·L–1 each of GA4+7 and benzyladenine (BA)] sprays on leaf chlorosis and plant height during greenhouse production of ancymidol-treated (two 0.5-mg drenches per plant) Easter lilies (Lilium longiflorum Thunb. `Nellie White') were investigated. Spraying with PROM at early stages of growth [36 or 55 days after planting (DAP)] completely prevented leaf chlorosis until the puffy bud stage, and plants developed less severe postharvest leaf chlorosis after cold storage at 4 °C for 2 weeks. When PROM was sprayed on plants in which leaf chlorosis had already begun (80 DAP), further leaf chlorosis was prevented during the remaining greenhouse phase and during the postharvest phase. PROM caused significant stem elongation (23% to 52% taller than controls) when applied 36 or 55 DAP, but not when applied at 80 DAP or later. The development of flower buds was not affected by PROM treatments. Although PROM sprays applied at 55 DAP or later increased postharvest flower longevity, earlier applications did not. Chemical names used: N-(phenylmethyl)-1H-purine 6-amine (benzyladenine, BA); α-cyclopropyl-α-(p-methoxyphenyl)-5-pyrimidinemethanol (ancymidol).
Easter lily flower buds at five stages of development (stage 1, 3–4 cm in length; stage 2, 6–7 cm; stage 3, 9–10 cm; stage 4, unopened buds, 13–14 cm; and stage 5, open flower one day after anthesis) were harvested, and flower organs were dissected for carbohydrate analysis. Extracting soluble sugars in distilled water at 70°C gave the optimum yield of soluble sugars among the several extraction methods tested including 80% ethanol, and distilled water at various temperatures. Separation of the extracted soluble sugars by alkaline high performance anion exchange chromatography revealed the presence of glucose, fructose, sucrose, and two other sugars of unknown identity. Glucose and fructose concentrations increased remarkably during the flower development in sepal (about 15-fold), style (about 10-fold), and filament (about 5-fold), while sucrose levels remained constant at low concentrations. In stigma, sucrose levels increased parallel to the increase of hexose sugars during development. Ovary had high sucrose levels relative to hexoses that remained constant while hexoses increased gradually. In anther, hexose concentrations increased at the stage 2 and then dropped at stage 3 and 4. Sucrose levels were higher than hexoses in anther, and it increased from stage 1 to stage 2, then dropped at stage 3, and increased thereafter. In addition to these sugars, anthers at stages 2 and 3 had a series of late eluting oligosaccharides. These oligosaccharides could be hydrolyzed to glucose with hot 1 m H2SO4 or with amyloglucosidase.
Several experiments were conducted to find effective ways of utilizing gibberellin4+7 (GA4+7) and benzyladenine (BA) to prevent leaf chlorosis during greenhouse production of Easter lilies (Lilium longiflorum Thunb.) while minimizing the undesirable side effects on stem elongation. On an absolute concentration basis, GA4+7 was much more effective than BA in preventing leaf chlorosis. Excessive levels of GA4+7, however, tended to cause stem elongation. When applied at around the visible bud stage, if the foliage was well covered with the spray solution, 25 mg·L-1 of GA4+7 was adequate for maximum protection against leaf chlorosis. Increasing the GA4+7 concentration above 25 mg·L-1 gave no additional benefit on leaf chlorosis. Two possible modes of GA4+7 uptake during a foliar spray application (absorption through leaves and stems, and root uptake of the extra run-off) were studied in terms of their relative contribution to leaf chlorosis and stem elongation. Although both modes of uptake prevented leaf chlorosis, foliar uptake was much more effective than root uptake. However, GA4+7 taken up by the roots contributed mainly to stem elongation. When sprayed to leaves on only the lower half of the plant, a 10-mL spray of either 25 or 50 mg·L-1 of each GA4+7 and BA was enough for complete protection against leaf chlorosis. Increasing volumes had no additional benefit on leaf chlorosis, but increased the chances of unwanted stem elongation.
Case-cooled bulbs of Lilium longiflorum `Nellie White' were forced to flowering. When the tepals on the first primary flower bud split, plants were placed at 2 °C in the dark for 0, 4, or 21 days. After storage, plants were placed in a postharvest evaluation room with constant 21 °C and 18 μmol·m-2·-1 cool-white fluorescent light. Lower leaves, upper leaves, and tepals of the first primary flower from a concurrent set of plants were harvested for carbohydrate analysis using HPLC. Storage time did not affect carbohydrate levels in the lower leaf or tepal samples, but sucrose and starch levels decreased while glucose and fructose levels increased in the upper leaf tissue with increasing storage time. These changes were correlated with a decrease in postharvest longevity for the first four primary flowers. Longevity of the fifth primary flower and total postharvest life of the five primary flowers was unaffected by storage.
Ornamental geophytes comprise a large and diverse group of plants characterized by underground storage organs that serve the obvious function of reserve storage and subsequent supply during early stages of shoot growth. Relative to many agronomic and horticultural crops, the fundamental physiological bases of carbon metabolism, partitioning, and utilization in geophytes are unclear. One reason is diversity in organ morphology (bulb, corm, tuber, root, rhizome, etc.), storage carbohydrate (starch, fructan, glucomannan, etc.), and growth habit (e.g., synanthous vs. hysteranthous flowering). Knowledge of factors that control accumulation and mobilization of carbohydrate reserves may lead to manipulations that considerably improve the quality and culture of these crops. We are utilizing a variety of techniques and experimental systems to study selected internal and external controls or influences on geophyte carbohydrate metabolism and partitioning. Specific examples to be discussed include bulb storage temperature effects on starch and fructan metabolism in Tulipa, effects of carbon source and dormancy breaking treatments on starch and glucomannan metabolism in in vitro-grown Lilium bulblets, photoperiod control of fructan accumulation in Dahlia seedlings, and biochemical and molecular features of soluble and wall-bound invertases in developing Lilium longiflorum flower buds.