Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa

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1 Senescence of cut carnation flowers : Ovary development and CO 2 fixation E.L. COOK and J. VAN STADEN Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa (Received 15 Sept. 1982; revised 30 Mar. 1983; accepted 8 Apr. 1983) Key words : Dianthus caryophyllus, carnation, cut flower, senescence, carbohydrates, petals, ovary, ethylene, CO 2 fixation Abstract. In the presence of ethylene, which enhances carnation flower senescence, carbohydrates contribute to ovary growth not only from the stem and calyx but also from the petals. With silver thiosulphate and ethanol treatments which delay flower senescence, the petals remain the active sink and ovary development is suppressed. Ethylene stimulated chloroplast development in the ovary wall. However, the calyx plus stem of all treatments showed the greater photosynthetic ability and transported a major portion of the synthesised products to the ovary. Introduction It is generally agreed that the carbohydrate status of the cut carnation plays an important role in regulating flower longevity [5, 6, 7, 9, 15, 16, 19, 21, 22, 23, 24, 25]. Controversy does, however, exist as to how the soluble carbohydrates in different parts of the cut flower exert their effect on the senescence process. Climacteric ethylene is a prerequisite for petal senscence, which is apparently closely linked with the movement of carbohydrates between the different components of the cut carnation flower [4, 6, 17, 20, 22, 23], and the subsequent stimulation of ovary growth [4, 22]. Due to different experimental approaches, agreement on the importance of carbohydrate movement between the senescing petals and developing ovaries has not been reached. Experiments using 14 C-sucrose led to the conclusion that with the onset of senescence a redistribution of carbohydrates from the petals towards the ovary occurred [25 ]. Using longer stemmed cut carnations together with the technique of removing specific flower parts it was questioned whether the petals contributed carbohydrates for ovary growth [17]. Thus while there is a general agreement that carbohydrates are necessary for petal longevity, and ovary development, the exact relationship and degree of transport between these flower parts remain unclear. In this investigation an attempt was made to clarify the situation. 221 Plant Growth Regulation 1 : (1982/83) Martinus NijhofflDr W. Junk Publishers, The Hague. Printed in the Netherlands

2 222 Materials and methods Plant material Flowers of Dianthus caryophyllus L. (cv White Sim) were harvested at the fully opened stage, cooled to 4 C, and then transported overnight to Pietermaritzburg. Prior to experimentation the stems were recut and immediately subjected to the following treatments : immersion in distilled water (control) ; immersion into an ethrel (2-chloroethyl phosphonic acid) solution (20 mg/l as `Ethrel') [33] ; pulsed for 10 min with a silver thiosulphate (STS) solution (4 mmol AgN0 3 with 16 mm Na2 S2 03 ) [ 27] and then immersed in distilled water ; or immersion into a 4% ethanol solution [13]. These respective treatments were selected as they either enhance or delay carnation senescence [13, 27, 331. The flowers were maintained under continuous light at 23 C and observed for irreversible petal wilting [231. At the times of harvest the fresh and dry mass of the respective flower parts were determined. Chlorophyll determination Chlorophyll was extracted from ovary tissue by homogenising with 80% acetone. After centrifugation the chlorophyll content in the supernatant was determined by recording transmittance in a Varian DMS 90 double beam spectrophotometer at 440 and 665 nm respectively. Experiments with 14 C02 To determine the extent to which various flower parts could incorporate C02, whole flowers or flowers from which the petals were removed were placed in airtight containers and exposed to 14 C0 2 (about dpm) produced by reacting Ba 14 C0 2 (specific activity 2.1 MBq mmol -1 ) with concentrated H 2 SO 4. Twenty-four h after 14 C0 2 application the flowers were removed and divided into their component parts. The respective flower parts were extracted for sugars [6]. The sugar extracts were taken to dryness and the residue resuspended in 5 ml 10% iso-propanol. Total radioactivity associated with each extract was determined by taking an aliquot (lml) and estimating the radioactivity as previously described [321. Aliquots of the remaining sugar extracts 'were streaked on TLC plates and developed with n-butanol :acetic acid : diethylether : water 9 :6 :3 :1 [11]. Sugars were tentatively identified using authentic markers with each extract. Once their chromatographic positions were known the respective Rf zones were scraped into vials and the silica gel eluted with lml 100% methanol. The radioactivity associated with these eluates was determined [321. Sucrose translocation To estimate the degree of sucrose movement from the stem material, 0.5.d 14 C-sucrose (specific activity 1.85 MBq mmol -1 ) (about cpm) was

3 TIME AFTER TREATMENT days 6 12 Figure 1. Changes in dry mass of the ovaries of intact and petalless cut carnation flowers with time. Control (- -), ethrel (- o -), STS (- A -) and ethanol (- A -). W signifies when petal wilting occurred. Vertical bars indicate standard error. injected into the stems of cut flowers just below the flower bracts. Twentyfour hours later the carnations were divided into the different flower parts and radioactivity in the dried material determined as described previously [7]. Microscopic investigations The ultrastructural development of chloroplasts in the ovaries of the different treatments was investigated by electron microscopy. One mm square sections of the ovary walls were fixed in 6% glutaraldehyde buffered at ph 7.2 with 0.05 M sodium cacodylate for 6 h. After being washed with the same buffer, the tissue was post-fixed with 2% osmium tetroxide buffered as above and again washed with the buffer. The material was then dehydrated in an alcohol series, followed by propylene oxide and embedded in araldite resin. Polymerisation lasted 48h at 70 C. Sections, ca. 700A. were cut with a glass knife on a LKB microtome and stained with uranyl acetate and lead citrate [28]. The sections were viewed with a transmission electron microscope (Jeol-100 CX). Results and discussion A preliminary experiment showed that increased stem length extended the longevity and increased the dry mass of carnation flower heads. Thus in any investigation considering the carbohydrate relationship between the ovary and petals of carnation cut flowers it is necessary to eliminate or minimise

4 FLOWER AGE days Figure 2. Radioactivity ( 14 C) detected in the sugar extracts from intact (B) and petalles (o) control-, ethrel-, STS- or ethanol-treated flowers. The flowers were exposed to a 0.03% t4 C0 2 atmosphere 24h prior to sugar extraction. Vertical bars indicate the standard error. this effect as far as possible. To achieve this, all experiments were conducted with flowers in which stems were trimmed routinely to 2 cm. In order to establish whether or not the petals of the cut carnation contribute to ovary growth during senescence, intact flowers and flowers from which the petals were removed at commencement of the experiment were subjected to treatments which both enhance and delay senescence and/or ovary growth. By manipulating ovary growth and petal longevity it was hoped to establish whether or not the petals act as a source of carbohydrate for ovary development. Application of ethrel to cut carnations enhances ovary development and accelerates petal senescence [4, 10, 20, 23]. In contrast, STS [6, 7, 27] and ethanol [13, 141 prevent ovary enlargement and extend petal longevity. This is achieved by the elimination of climacteric ethylene production [6, 7, 13, 14, 271. In both the control and ethrel-treated intact flowers ovary dry mass increased with time (Fig. 1). When the petals were removed ethrel treatment resulted in a significant decrease in ovary dry mass. This suggests that when ethylene is present the petals of the cut flower are a weaker sink than the ovary, and consequently the carbohydrates are transported towards the ovary. This is in agreement with earlier reports [7, 24, 251. In the STS and ethanol-treated intact flowers ovary dry mass did not

5 2 2 5 Figure 3. Changes in the chlorophyll content (measured at 665 nm and 440 nm) of carnation ovaries from intact and petalless flowers with time. Control (- -), ethrel (- o -), STS (- ' -) and ethanol (- -) treatment. W indicates when petal wilting occurred. Vertical bars indicate standard error. increase. Removal of the petals from these flowers did however, result in increased ovary growth (Fig. 1), implying that the ovaries could then utilise stem-derived carbohydrates more effectively. To establish the C02 fixing ability of the variously treated cut carnations, intact flowers and those from which the petals were removed were exposed to 14 C02 for 24 h at 0, 3 and 6 days after harvest. The amount of 14 C detected in the sugar extracts from each system was then determined (Fig. 2). 14 C was incorporated most efficiently by the flowers immediately after harvest (0 days). However, by days 3 and 6 there was no longer a significant difference between the different treatments in 3- and 6-day-old flowers. Thus, during the first few days after harvest, the cut carnation flower has the ability to add to its carbohydrate pool via photosynthesis ; the petalless flower being more efficient than the intact flower. Whether the 14 C incorporation by the ovary occurs directly via photosynthesis or whether carbohydrates are transported to the ovary needs clarification. In previous experiments [22] and during the present investigation it was noted that with certain treatments `greening' of the ovaries occurred. Quantifying the chlorophyll contents of the ovaries from intact flowers subjected to various treatments indicated that with STS and ethanol treatment ovary chlorophyll content over a 12 day post harvest period did not alter significantly (Fig. 3). However, in the control and ethreltreated intact flowers, the chlorophyll content of the ovaries increased. Petal removal reduced the chlorophyll levels of these latter treatments, but enhanced the chlorophyll content of STS-treated ovaries (Fig. 3). It thus appears that the control and ethrel-treated ovaries could fix 14 CO 2 themselves,

6 2 26 Figure 4. Electromicrographs showing chloroplast development in the ovary wall of cut carnation flowers. A. Chloroplasts in ovary walls 7 days after the flower had been treated with ethanol. The chioroplasts are smaller and the thylakoids (T) poorly developed. More plastoglobuli (P) are present than in the chioroplasts of the ovaries of ethrel-treated cut carnations. B. Well developed chloroplast in the ovary wall of ethrel-treated cut carnations. Thylakoids (T) are well developed and only a few plastoglobuli (P) could be observed.

7 2 2 7 Figure 5. Radioactivity ( 14 C) detected in the sugars extracted from the calyx plus stem (o), the ovary (a) and petals (when present) (a) from intact and petalless control, ethrel-, STS- and ethnol-treated flowers after being exposed for 24 h to a 0.03% 14 C0 2 atmosphere. The radioactivity detected in each flower part is expressed as a percentage of the total '4 C detected in the respective system. Vertical bars indicate the standard error. adding directly to their dry mass. Ultrastructural analysis of the chloroplast structure within the ovary wall 7 days after treatment revealed that with STS and ethanol treatment, chloroplasts remained underdeveloped (Fig. 4A), whereas in the control and ethrel treatments the higher chlorophyll content (Fig. 3) could be related to well-developed chloroplasts (Fig. 4B). It is significant that ethrel (ethylene) stimulated the development of immature chloroplasts within the ovary wall. This contrasts with the documented degradative effect of ethylene on mature chloroplasts in other tissues [1, 2, 3,

8 2 2 8 Table 1. Radioactivity ("C) that co-chromatographed with sucrose (Rf = 0.20 on TLC plates developed with n-butanol :acetic acid : diethylether : water 9 :6 :3 :1 for 4.5 h) 24 h after freshly harvested flowers were exposed to a 0.03% 14 CO 2 atmosphere. The radioactivity is expressed as a percentage of the total 14C in the extracted sugars of each sample. Treatment Radioactivity associated with sucrose (%) Control 15.2 ± 2.3 Ethrel 18.3 ± 3.0 STS 10.7 ± 5.0 Ethanol 1.3 ± 0.4 8, 26, 29, 30, 311. These data again support the fact that ovary growth which was stimulated by ethylene, could be contributed to by photosynthesis. It was therefore necessary to establish which components of the flower were responsible for CO2 fixation. To achieve this, intact and petalless carnation flowers were exposed to 14 CO 2 for 24 h. where after they were divided into petals (where present), ovary and calyx plus stem. The soluble sugars were extracted from each flower part and the radioactivity associated with the sugar extract determined. The results obtained confirmed that with age the flowers were less able to incorporate 14 C, irrespective of the treatment applied. In the control, ethreland ethanol-treated intact flowers the sugar extracts from the calyx plus stem contained the highest amount of 14 C (Fig. 5). This was followed by the ovaries and the petals which yielded the lowest 14 C levels. In the STS-treated flowers the trend differed. In this instance a higher level of 14 C was detected in the ovaries than in the calyx plus stem. Where the petals were removed the ovaries and calyx plus stem did not contain significantly different levels of 14 C (Fig. 5). Whereas it would appear that the ovaries were capable of incorporating C02, the possibility of 14 C transport between the calyx/stem and the ovaries could not be disregarded since applied 14 C-sucrose can be transported from one part of the flower to another within 24h [12, 24, 25]. Furthermore, analysis of the 14 C-labelled sugar extracts of ovaries, showed that a considerable amount of radioactivity co-chromatographed with sucrose (Table 1). It was thus necessary to establish whether isolated ovaries could fix 14 CO2. When the ovaries were excised from the treated flowers and exposed to 14 C0 2, no 14 C incorporation into the sugars occurred within the first 24h after harvest, irrespective of treatment (Table 2). After 5 days 14 C incorporation did take place. By 7 days the control and ethrel-treated ovaries, which contained more chlorophyll, were still able to fix 14 C02 whereas this ability was much reduced or lost in the STS and ethanol treatments. These results suggest that the stem and calyx were responsible for most of the 14 C02 incorporation of the intact cut flower, and that the radioactivity which was detected in the ovaries after 24h was transported towards them from this source.

9 Table 2. Radioactivity ( 14 C) detected in the extracted sugars from ovaries excised from ageing treated cut carnation flowers after being exposed for 24h to a 0.03% 14 C0 2 atmosphere. Time after harvest (days) Treatments Control ± ± 6 Ethrel ± ± 9 STS ± ± 18 Ethanol ± tooy N EE T CONTROL ETHREL ETHANOL E _o >- 50 I- it 0 Q 0 4Q FLOWER AGE days Figure 6. Radioactivity ("C) detected in the petals (o), ovary (0) and calyx plus stem (e) of treated flowers 24h after injection of "C-sucrose into the stem. The dpm's of each flower parts are expressed as a percentage of the recovered 14 C within each treated flower. Vertical bars indicate the standard error. By injecting 14 C-sucrose into the stem of the variously treated cut flowers, the possible movement of carbohydrates to the ovary and petals was monitored (Fig. 6). Initially, with the control and ethrel treatment, most of the 14 C was detected in the petals. After petal wilting, which occurred by day 5 in the control and by day 2 with ethrel treatments, the sink strength appeared to move from the petals to the ovary where the majority of the 14 C then accumulated. Finally the calyx plus stem either became the stronger sink or, due to their senescence, hindered translocation of the applied 14 C- sucrose from these tissues. With STS and ethanol treatment the calyx plus stem retained the majority of the 14 C ; yet by day 3 the petals in both these treatments became the dominant sink, the petals on STS-treated plants displaying stronger sink properties than the petals on ethanol-treated plants. Thus, carbohydrates supplied to the flower from the stem are competed for by the petals and ovaries, the relative sink strength 1 i of these two organs depending on age (Fig. 6) [19]. The presence of ethylene also appears to change the source-sink relationship of the cut carnation in such a way that the ovary becomes the major sink [7, 22]. A final experiment confirmed that the petals too are capable of contributing carbohydrates for ethylene-stimu- I

10 r F U Q 50 O 0 Q 0 0 I I I TIME days Figure 7. Radioactivity ( 3 H) detected in the labelled petals (o), ovary (a) and calyx plus stem (0) of control flowers 24 h after the injection of 3 H-sucrose into the stem. The dpm's 10 mg ' dry mass of each flower part are expressed as a percentage of the recovered 3 H within each treated flower. Vertical bars indicate the standard error. lated ovary growth (Fig. 7). Using 3 H-sucrose (to avoid the possible contamination of 14 CO 2 metabolised from 14 C-sucrose [7]), the petals of the carnation flowers were injected with 1 µl of 3 H-sucrose (specific activity 9.25 MBq mmol_ 1) in 1% ethanol 24h before analysis. Initially the radioactivity was detected mainly in the petals, with a small amount in the stem/ calyx, but, as the petals wilted, the ovary become the stronger sink. These results support previous work [7, 24, 251. By the eighth day the petals, which have senesced were unable to distribute the applied 3 H-sucrose to the remainder of the flower. At this time there would be little available carbohydrate for redistribution. The variously treated cut carnation flowers are initially all capable, to varying degress, of contributing to their soluble carbohydrate pool via photosynthesis (Fig. 2). Although the presence of ethylene stimulates the development of ovary wall chloroplasts (Fig. 4) so that by the fifth day they themselves fix 14 CO 2 (Table 2), it appears that the calyx plus stem show a far greater photosynthetic ability (Fig. 3) and transport a major portion of their fixed 14 C to the ovary. This indicates that ovary growth, which is correlated to the presence of ethylene [4, 7, 20, 22], occurs primarily in response to carbohydrates from external sources. To elicit the ovary swelling which is stimulated by ethylene, external sources of carbohydrate are necessary. These are transported from the stem [12, 19, 24] (Fig. 6) and the petals [7, 25] (Fig. 7). With ethrel treatment the ovary is therefore the dominant carbohydrate sink in the cut carnation flower. With STS and ethanol treatment the petals remain the sink whilst utilising the carbohydrates within the cut flower [6, 7, 14, 19]. Because of this the ovary appears `starved' which could account for the underdeveloped chloroplasts. Many workers have postulated that the movement of plant growth regulators within the cut flower accounts for the shifting of the carbohydrate

11 sink from the petals to the ovary [6, 10, 12, 17, 19, 21, 231. This possibility is currently being investigated Acknowledgements Mr. P.M.S. Cunningham, Florarcadia (EDMS) BPK, Heidelberg, the C.S.I.R. and the Atomic Energy Board, Pretoria are thanked for financial support. Assistance from the Electron Microscope Unit, University of Natal, Pietermaritzburg is appreciated. References 1. Aharoni N, Anderson JD and Lieberman M (1979) Production and action of ethylene in senescing leaf discs : Effects of indolacetic acid, kinetin, silver ion and carbon dioxide. PI Physiol 64 : Aharoni N and Lieberman M (1979) Ethylene as a regulator of senescence in tobacco leaf discs. PI Physiol 64 : Aharoni N, Lieberman M and Sisler HD (1979) Patterns of ethylene production in senescing leaves. PI Physiol 64 : Camprubi P and Nichols R (1979) Effects of ethylene on carnation flowers (Dianthus caryophyllus) cut at different stages of development. J Hort Sci 53 : Carpenter WJ and Dilley DR (1974) Role of sucrose in cut flower longevity. Hort Sci 9 : Dimalla GG and Van Staden J (1980) The effect of silver thiosulphate preservative on the physiology of cut carnations. 1. Influence on longevity and carbohydrate status. Z Planzenphysiol 99 : Ducasse P and Van Staden J (1981) Effect of silver thiosulphate preservative on the physiology of cut carnations. III. Regulation of ( 14 C) sucrose transport and utilization. South African J Sci 77 : Gavinlertvatana P, Read PE and Wilkins HF (1980) Control of ethylene synthesis and action by silver nitrate and rhizobitoxine in Petunia leaf sections cultured in vitro. J Amer Soc Hort Sci 105 (3) : Halevy AH and Mayak S (1974) Improvement of cut flower quality opening and longevity by pre-shipment treatments. Acta Hort 43 : Halevy AH and Mayak S (1981) Senescence and postharvest physiology of cut flowers. Part 2. Hort Rev 3 : Harbourne JB (1973) Phytochemical methods : A guide to modern techniques of plant analysis. London : Chapman and Hall. 12. Harris GP and Jeffcoat B (1972) Distribution of 14 C-labelled assimilates in flowering carnations plants. J Hort Sci 47 : Heins RED (1980) Inhibition of ethylene synthesis and senescence in carnations by ethanol. J Amer Soc Hort Sci 105 (4) : Heins RD and Blakely N (1980) Influence of ethanol on ethylene biosynthesis and flower senescence of cut carnations. Scientia Hort 13 : Laurie A (1936) A retrospect looking back ten years on floriculture research. Proc Amer Soc Hort Sci 34 : Kuc R and Workman M (1964) The relation of maturity to respiration and keeping quality of cut carnations and chrysanthemums. Proc Amer Soc Hort Sci 84 : Mayak S and Dilley DR (1976) Effect of sucrose on response of cut carnations to kinetin, ethylene and ABA. J Amer Soc Hort Sci 101 : Mor Y and Reid MS (1980) Isolated petals - a useful system for studying flower senescence. Acta Hort 113 : Mor Y, Reid MS and Kofranek AM (1980) Role of the ovary in carnation senescence. Scientia Hort 13 :

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