Preventive mechanisms of gibberellin
4+7and light on
low-temperature-induced leaf senescence in
Lilium
cv.
Stargazer
Anil P. Ranwala *, William B. Miller
1Department of Horticulture,Clemson Uni6ersity,Clemson,SC29634,USA Received 10 May 1999; accepted 30 December 1999
Abstract
Liliumcv. Stargazer plants were held for 2 weeks at 4°C, either in darkness or in light (40mmol m−2s−1). Another
series of plants was held in darkness after pre-treatment with 100 mg l−1 GA
4+7. Changes in major senescence
parameters were determined during storage at 4°C and during 6 days after transferring the plants to 22°C. Foliar sprays of GA4+7or supplemental light prevented rapid leaf senescence induced by dark low-temperature storage.
During storage at 4°C, basal leaves showed no significant changes in concentrations of chlorophyll, soluble proteins, lipid peroxidation, and activity of catalase. However, shifting from 4 to 22°C induced a series of changes in basal leaves of dark-held plants including rapid loss of chlorophyll, proteolysis, increased lipid peroxidation and loss of catalase activity. Both light and GA4+7treatments prevented these changes. Total soluble carbohydrates decreased
gradually during 4°C dark storage and after transferring to 22°C. GA4+7treatments did not prevent the decline in
carbohydrate levels at 4°C, but prevented it upon transferring to 22°C. Supplemental light during 4°C storage significantly increased soluble carbohydrate concentration. The abrupt increases in metabolic activities by shifting from 4 to 22°C accompanied by oxidative stress in leaves already depleted in reserves during 4°C storage seem to induce leaf senescence in dark-held plants. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Catalase; Chlorosis; Gibberellins; Leaf senescence; Light;Lilium; Lipid peroxidation; Soluble carbohydrates; Soluble proteins; Low-temperature stress; Oxidative stress; Superoxide dismutase
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1. Introduction
Although leaf senescence is a genetically pro-grammed developmental process, it can also be triggered by many environmental factors includ-ing temperature, drought, woundinclud-ing and shadinclud-ing (Gan and Amasino, 1997). Common metabolic changes during leaf senescence are chlorophyll
* Corresponding author. Present address: Department of Floriculture and Ornamental Horticulture, 20 Plant Science Building, Cornell University, Ithaca, NY 14853, USA. Tel.:
+1-607-2554421; fax: +1-607-2559998.
E-mail address:[email protected] (A.P. Ranwala) 1Present address: Department of Floriculture and Orna-mental Horticulture, 20 Plant Science Building, Cornell Uni-versity, Ithaca, NY 14853, USA.
and protein loss and increases in lipid peroxida-tion and membrane permeability (Kar and Feier-abend, 1984; Smart, 1994; Pastori and del Rio, 1997). The relative dominance and the rate of progression of these parameters vary depending on the species, type of initiation (natural or in-duced) and physiological age of the leaf.
Low temperature exerts many adverse effects on plants including premature leaf senescence. We have been studying a commercially important postharvest leaf senescence disorder induced by dark low-temperature storage in container-grown lilies. Due to variable market demand and timing of flowering, lilies at mature flower bud stage are often stored at low temperature (ca 4°C) for short durations (ca 2 weeks) to minimize further flower development. Upon transfer to ambient tempera-ture, plants often undergo rapid leaf chlorosis and senescence from basal leaves progressing upwards. Such induced leaf senescence has been observed in both Easter (Lilium longiflorum) (Han, 1997) and hybrid (Lilium sp) lilies (Ranwala and Miller, 1998a).
In Lilium cv. Stargazer, rapid leaf senescence induced by 2 weeks of dark storage at 4°C can be effectively prevented by foliar growth regulator sprays prior to storage or by providing light (up to 40 mmol m−2 s−1 at the top of the plant) during low-temperature storage (Ranwala and Miller, 1998a). Among growth regulators tested at 100 mg l−1concentration, GA
4+7effectively pre-vented leaf senescence while GA3 or BA were not effective (Ranwala and Miller, 1998a). Similar observations have been reported for Lilium lon -giflorum (Han, 1997). However, the mechanisms by which GA4+7 or light prevents leaf senescence in lilies are not yet known.
The induction of oxidative stress under low temperature conditions has been observed in sev-eral plant species (Wise and Naylor, 1987; Prasad et al., 1994; Kerdnaimongkol et al., 1997), and may also be a major cause of low-temperature-in-duced leaf senescence in Lilium plants. Reactive oxygen species generated under oxidative stress conditions can react with cellular components leading to the disruption of cellular metabolism. Lipid peroxidation causing increased membrane permeability has been identified as a major effect
of oxidative stress. Anti-oxidative enzymes (e.g. catalase and superoxide dismutase) function as a defense system against oxidative stress by scav-enging reactive oxygen species.
The objective of this study was to compare major senescence parameters in leaves of Lilium cv. Stargazer plants held at low temperature in darkness or light or in darkness with GA4+7 pre-treatment as an initial step in understanding senescence-preventing mechanisms of light and GA4+7. We have determined concentrations of chlorophyll, soluble carbohydrate, soluble protein, lipid peroxidation products, and activities of anti-oxidative enzymes (catalase and superox-ide dismutase) in leaves during 2 weeks of 4°C storage and after their transfer to 22°C.
2. Materials and methods
2.1. Plant material
Hybrid lilies (Lilium cv. Stargazer) were grown in a greenhouse at Clemson University under conditions described earlier (Ranwala and Miller, 1998a). At the mature flower bud (puffy bud) stage, uniform healthy plants were selected for low temperature storage. Plants were held at 49 0.3°C:
1. in darkness;
2. with 24 h day−1 irradiance of 40 mmol m−2 s−1 light (measured at the top of the plants) from cool-white fluorescent lamps; or
3. in darkness, after spraying the whole plant to run-off with a solution of 100 mg l−1GA
4+7 (Abbott laboratories, North Chicago, IL, USA).The spray solution contained 0.1% Tween 20 as a surfactant, and was applied 2 h prior to the beginning of 4°C storage. After 2 weeks of 4°C storage, plants were placed in a poststorage evaluation room at 2291°C with 50 – 70% RH and 20mmol m−2s−1light (12 h day−1) at the top of the plants from cool-white fluorescent lamps.
intervals in the post-storage evaluation phase (22°C) for 6 days (days 17 and 20). Leaves were collected from the basal half of the stem during 4°C storage, and from both basal and upper parts of the stem during the post-storage phase (22°C). After collecting leaf disks for chlorophyll analysis, each leaf was cut into strips and mixed to form representative samples of leaves of a replicate plant for use in subsequent analyses of carbohy-drates, soluble proteins, anti-oxidative enzymes and lipid peroxidation products. Leaf tissues were immediately frozen in liquid nitrogen and stored at −70°C until use.
2.2. Chlorophyll
Leaf disks (0.28 cm2 each) were taken from leaves (5 disks per leaf) and chlorophyll was ex-tracted with N,N-dimethylformamide. The ab-sorbance of extracts at 647 and 664 nm was measured and total chlorophyll was estimated as described by Moran (1982). Results were ex-pressed as mg of chlorophyll g−1of fresh weight.
2.3. Soluble carbohydrates
Frozen leaf tissues were freeze-dried, and ground to a fine powder with a mortar and pestle. Soluble carbohydrates were extracted from 50 mg of dry tissue with methanol: chloroform: water (12:5:3, v:v:v) at room temperature as described by Miller and Langhans (1989). Extracted carbo-hydrates were dissolved in HPLC grade water and subjected to high-performance anion exchange chromatography (HPAE) in a Dionex DX-300 systems with a Carbopac PA-1 column. The de-tails of chromatography and estimation of the quantity of sugars were as described by Ranwala and Miller (1998b). Total soluble carbohydrates were defined as the sum of sucrose, glucose and fructose. Results were expressed as mg of carbo-hydrate g−1 of dry weight.
2.4. Lipid peroxidation products
Malondialdehyde (MDA) was determined as an indicator of lipid peroxidation. Leaf tissue (1.0 g fresh weight) was homogenized in 10 ml of 5%
(v/v) TCA and centrifuged at 20 000×g for 10 min. Malondialdehyde in the supernatant was de-termined as thiobarbituric acid-reactive sub-stances (Heath and Packer, 1968). Results were expressed as nmol of MDA g−1 of fresh weight.
2.5. Extraction and assay of anti-oxidati6e enzymes
Enzyme extractions were carried out in a cold room at 4°C or on ice. Leaf tissues were homoge-nized (10 ml buffer g−1
fresh weight) in 50 mM K-phosphate (pH 7.5) containing 1 mM DTT, 1 mM EDTA and 1% (w/v) PVPP using a Polytron (Brinkmann Instruments, Westbury, NY, USA) at full speed for three 20-s intervals. The ho-mogenate was filtered through two layers of moistened Miracloth, and the filtrate centrifuged at 26 000×g for 20 min. The supernatant was desalted with a PD-10 column (Pharmacia, Upp-sala, Sweden) equilibrated with 50 mM K-phos-phate (pH 7.5) buffer.
Catalase (EC 1.11.1.16) activity was assayed at 25°C by measuring the initial rate of decomposi-tion of H2O2using assay conditions described by Aebi (1984). The 3 ml reaction mixture contained 50 mM K-phosphate (pH 7.0) buffer, 10 mM H2O2 and 25 – 100ml enzyme extract. The decom-position of H2O2 was followed as the decline in absorbance at 240 nm after 30 s. One enzyme unit was defined as the amount of activity catalyzing the decomposition of 1 mmol of H2O2 per min. Superoxide dismutase (EC 1.15.1.1) activity was assayed at 25°C by measuring its ability to inhibit the photochemical reduction of nitro blue tetra-zolium using the assay conditions described by Dhindsa et al. (1981). Enzyme activities were ex-pressed as units g−1
of fresh weight.
2.6. Soluble protein and SDS-PAGE
SDS-PAGE was carried out in 12% (w/v) poly-acrylamide gels using the Mini-PROTEAN II Slab Cell apparatus (Bio-Rad Laboratories, Rich-mond, CA, USA) and the buffer system described by Laemmli (1970). Prior to loading, samples were diluted 1:4 (v:v) with SDS-sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 2% SDS, 5% (v/v) b-mercaptoethanol and 0.025% bromophenol blue, and heated at 95°C for 5 min. Electrophoresis was done at 200 V constant voltage for approximately 45 min. The gels were silver-stained to visualize by
proteins according to the method of Wray et al., (1981).
3. Results
3.1. Total chlorophyll
During 2 weeks of storage at 4°C, chlorophyll concentration in lower leaves was essentially un-changed in all treatments (Fig. 1A). Upon trans-ferring to 22°C, however, basal leaves of dark-held plants lost 66% of chlorophyll in 6 days, whereas light-held and GA4+7-treated basal leaves lost only 8 and 5%, respectively. At the end of 4°C storage, upper leaves of plants in all treat-ments had slightly higher chlorophyll concentra-tions than basal leaves (Fig. 1B). In all treatments, chlorophyll loss of upper leaves within 6 days of transfer to 22°C was gradual and minimal.
3.2. Soluble carbohydrates
Soluble carbohydrates in Lilium cv. Stargazer leaves were composed mainly of sucrose (ca 80%), and nearly equal amounts of glucose and fructose (ca 10% each). During 2 weeks storage at 4°C, the concentration of total soluble carbohydrates (sum of sucrose, glucose and fructose) in dark-held basal leaves decreased gradually to about 50% of the pre-storage level (Fig. 2A). GA4+7 treatments did not prevent this decline. Upon transfer to 22°C, GA4+7-treated leaves maintained carbohy-drate levels, whereas dark-held leaves without GA4+7 experienced an additional reduction in carbohydrate level. Supplemental light signifi-cantly increased soluble carbohydrate concentra-tion during 4°C storage. Upon transfer to 22°C, leaves of light-held plants rapidly lost carbohy-drates, but stabilized to levels higher than those of dark-held and GA4+7-treated leaves.
At the end of 4°C storage, upper leaves of plants in each treatment had slightly higher carbo-hydrate concentrations than basal leaves (Fig. 2B). Carbohydrate concentration decreased in up-per leaves of light-held plants at 22°C, but in-creased slightly in dark-held and GA4+7-treated plants.
Fig. 1. Changes in chlorophyll concentration in basal (A) and upper (B) leaves ofLiliumcv. Stargazer plants during 2 weeks of storage at 4°C (days 0 – 14) in darkness, irradiated with 40 mmol m−2s−1light, or in darkness with pre-storage sprays of 100 mg l−1GA
4+7, and during subsequent post-storage hold-ing for 6 days at 22°C (days 14 – 20). Each point is a mean9 SE of 4 replicates. Each replicate was a representative sample of leaves from a single plant.
Fig. 2. Changes in total soluble carbohydrate concentration in basal (A) and upper (B) leaves ofLiliumcv. Stargazer plants during 2 weeks of storage at 4°C (days 0 – 14) in darkness, irradiated with 40mmol m−2 s−1 light, or in darkness with pre-storage sprays of 100 mg l−1 GA
Fig. 3. Changes in soluble protein concentration in basal (A) and upper (B) leaves ofLiliumcv. Stargazer plants during 2 weeks of storage at 4°C (days 0 – 14) in darkness, irradiated with 40mmol m−2s−1light, or in darkness with pre-storage sprays of 100 mg l−1 GA
4+7, and during subsequent post-storage holding for 6 days at 22°C (days 14 – 20). Each point is a mean9SE of four replicates. Each replicate was a represen-tative sample of leaves from a single plant.
Fig. 5. Changes in malondialdehyde concentration in basal (A) and upper (B) leaves of Liliumcv. Stargazer plants during 2 weeks of storage at 4°C (days 0 – 14) in darkness, irradiated with 40mmol m−2s−1light, or in darkness with pre-storage sprays of 100 mg l−1 GA
4+7, and during subsequent post-storage holding for 6 days at 22°C (days 14 – 20). Each point is a mean9SE of four replicates. Each replicate was a represen-tative sample of leaves from a single plant.
Numerous low molecular weight proteins ap-peared in dark-held leaves after transferring to 22°C, however, in light-held and GA4+7-treated leaves, these proteins did not appear (Fig. 4).
3.4. Lipid peroxidation
Malondialdehyde (MDA) was determined as an indicator of lipid peroxidation in leaves. During 2 weeks of 4°C storage, MDA concentration in basal leaves of dark-held plants (with or without 3.3. Soluble protein
During 4°C storage, the concentration of solu-ble proteins did not change in basal leaves (Fig. 3A). However, after transferring to 22°C, basal leaves of dark-held plants lost soluble protein rapidly compared to light-held and GA4+7 -treated leaves. In upper leaves, protein concentra-tion decreased gradually at 22°C in all treatments (Fig. 3B).
Fig. 4. SDS-PAGE profiles of soluble proteins in basal leaves ofLiliumcv. Stargazer plants during 2 weeks storage at 4°C (days 0 – 14) in darkness, irradiated with 40mmol m−2s−1light, or in darkness with pre-storage sprays of 100 mg l−1GA
GA4+7) did not change, and light-held basal leaves showed a very slight increase (Fig. 5A). Upon transfer to 22°C, MDA concentration rapidly increased in dark-held leaves compared to light-held or GA4+7-treated leaves. No significant changes in MDA concentration were observed in upper leaves of all treatments (Fig. 5B).
3.5. Anti-oxidati6e enzymes
While catalase activity did not change in basal leaves during 4°C storage (Fig. 6A), it decreased rapidly in dark-held leaves upon transfer to 22°C (especially from day 17 to 20). In upper leaves, at 22°C, catalase activity decreased gradually in all treatments, but not to levels lower than in dark-held basal leaves (Fig. 6B).
During 4°C storage, superoxide dismutase (SOD) activity gradually decreased in dark-held basal leaves, and showed only minor fluctuations in light-held and GA4+7-treated leaves. Upon transfer to 22°C, dark-held leaves lost SOD
activ-ity gradually while light-held and GA4+7-treated leaves maintained the activity. In upper leaves, SOD activity decreased slightly in all treatments but not to levels lower than in dark-held basal leaves.
4. Discussion
Shifting the plants from 4 to 22°C triggered leaf senescence in dark-held Lilium cv. Stargazer plants. The induction of senescence was character-ized by chlorophyll loss, proteolysis, lipid peroxi-dation, and reduction in catalase activity. The abrupt increase in metabolic activities at higher temperature may quickly exhaust cellular compo-nents that had already been depleted during dark low-temperature storage. The gradual decrease in carbohydrates at 4°C storage in darkness indi-cated a depletion of cellular reserves in the leaves. Low temperature stress is known to induce oxidative stress in plants (Prasad et al., 1994; Kerdnaimongkol et al., 1997). Accumulation of reactive oxygen species (e.g. superoxide and hy-droxyl radicals) is common during low-tempera-ture storage. These species are known to induce lipid peroxidation and increase membrane perme-ability, thereby disrupting metabolic activity. Al-though lipid peroxidation (MDA accumulation) was not evident during 4°C, cells may have be-come vulnerable to attack by accumulated reac-tive oxygen species at the point of the temperature shift from 4 to 22°C. The expression of enzymes scavenging reactive oxygen species (e.g. catalase and SOD) is one of the defensive mechanisms in plants against oxidative stress (Foyer et al., 1994). We observed gradual loss of SOD activity during 4°C storage and at 22°C in dark-held leaves. Catalase activity significantly decreased in dark-held leaves upon transferring to 22°C. These changes accompanied with increases in MDA lev-els suggest that oxidative stress plays a major role in dark low-temperature induced leaf senescence in Liliumcv. Stargazer.
The physiological age of the leaf has a strong influence on the onset and rate of leaf senescence (Fischer and Feller, 1994). In our study, although all leaves were subjected to similar conditions,
Fig. 6. Changes in catalase and superoxide dismutase activities in basal (A) and upper (B) leaves ofLiliumcv. Stargazer plants during 2 weeks of storage at 4°C (days 0 – 14) in darkness, irradiated with 40mmol m−2 s−1 light, or in darkness with pre-storage sprays of 100 mg l−1 GA
leaves in the basal section of the plant initiated senescence first. Basal leaves possess lower amounts of carbohydrates compared to upper leaves as a result of the upward movement of assimilates due to high sink strength of developing buds and upper leaves. Also, basal leaves had lower levels of anti-oxidative enzyme activities compared to upper leaves at the point when plants were shifted from 4 to 22°C. These condi-tions of basal leaves may result in more vulnera-bility to low-temperature stress.
Among plant hormones, cytokinins are well known to delay leaf senescence in many species (Nooden, 1988; Smart, 1994). There is increasing evidence of leaf senescence-delaying effects of gib-berellins in some species (Hicklenton, 1991; van Doorn and van Lieburg, 1993; Han, 1995; Jordi et al., 1995). InAlstroemeria pelegrinaL. cut flower-ing stems andLilium longiflorum plants, GA4 and GA7are far more effective in delaying leaf chloro-sis than GA3or cytokinins (Jordi et al., 1995; Han 1997). Franco and Han (1997) observed reduction in respiration rates by GA3 treatments in excised L. longiflorum leaves. In that study, GA3 had no significant effect on respiration rates during 4 weeks cold storage at 2°C, but upon removal from cold storage and at 20°C, GA3-treated leaves had lower respiration rates than non-treated leaves. Our data suggest that GA4+7 has similar respiration-retarding effects in Lilium cv. Stargazer leaves since soluble carbohydrate levels were maintained in GA4+7 treated leaves upon transfer to 22°C as opposed to declining carbohy-drate levels in non-treated leaves.
Photosynthesis may be a major factor in the senescence-delaying effects of light. In our study, the most notable effect of light during 4°C storage was increased levels of soluble carbohydrates in both basal and upper leaves. Increased levels of carbohydrates have been shown to be a major factor contributing to senescence-delaying in some species. For example, leaf photosynthetic activity and increased carbohydrate pools are important in the delaying of leaf blackening by light in Protea neriifolia cut stems (McConchie et al., 1991). The effect of light quality acting through phytochrome on leaf senescence has also been suggested (Okada et al., 1992; van Doorn and van
Lieburg, 1993). van Doorn and van Lieburg (1993) suggested that chlorophyll breakdown in cut flowering branches of Alstroemeria pelegrina leaves is controlled by gibberellins and the effect of phytochrome is mediated by gibberellin synthesis.
The present study elucidated possible mecha-nisms underlying dark low-temperature induced leaf senescence in lilies. The abrupt increases in metabolic activities initiated by shifting plants from 4 to 22°C accompanied by oxidative stress in leaves already depleted in reserves during 4°C storage seem to induce leaf senescence in dark-held plants.
Acknowledgements
This research was funded by a grant from the Fred C. Gloeckner Foundation. Additional sup-port was provided by the Clemson University Ornamental Horticulture Competitive Grants Program and by the Dutch Wholesalers Associa-tion for Flowerbulbs and Nursery Stock. We gratefully acknowledge bulb donations from Dahlstrom and Watt Bulb Farms, Smith River, CA, USA, and chemical donations from Abbott Laboratories, North Chicago, IL, USA.
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