Changes of Carbohydrates in Pepper (Capsicum annuum L.) Flowers in Relation to Their Abscission Under Different Shading Regimes

Transcription

1 Annals of Botany 78: , 1996 Changes of Carbohydrates in Pepper (Capsicum annuum L.) Flowers in Relation to Their Abscission Under Different Shading Regimes B. ALONI*, L. KARNI, Z. ZAIDMAN and A. A. SCHAFFER Department of Vegetable Crops, Institute of Field and Garden Crops, Agricultural Research Organization, The Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel Received: 24 July 1995 Accepted: 25 January 1996 Abscission of pepper flowers is enhanced under conditions of low light and high temperature. Our study shows that pepper flowers accumulate assimilates, particularly in the ovary, during the day time, and accumulate starch, which is then metabolized in the subsequent dark period. With the exception of the petals, the ovary contains the highest total amounts of sugars and starch, compared with other flower parts and contains the highest total activity, as well as activity calculated on fresh mass basis, of sucrose synthase, in accordance with the role of this enzyme in starch biosynthesis. Low light intensity or leaf removal decreased sugar accumulation in the flower and subsequently caused flower abscission. The threshold of light intensity for daily sugar accumulation in the sink leaves was much lower than in flowers, resulting in higher daytime accumulation of sugars in the sink leaves than in the adjacent flower buds under any light intensity, suggesting a competition for assimilates between these organs. Flowers of bell pepper cv. Maor and 899 (sensitive to abscission) accumulated less soluble sugars and starch under shade than the flowers of bell pepper cv. Mazurka and of paprika cv. Lehava (less sensitive). The results suggest that the flower capacity to accumulate sugars and starch during the day is an important factor in determining flower retention and fruit set Annals of Botany Company Key words: Pepper, Capsicum annuum L., abscission, shading, pepper flowers, ovary, leaves, sugars, starch, acid invertase, sucrose synthase. INTRODUCTION In most agricultural crops, flower retention and fruit set are highly sensitive to environmental stresses. High temperature and low light conditions are known to enhance flower abscission and to affect photosynthetic rates (Gent, 1986; Shishido, Chala and Krupa, 1987; Kitroongruang et al., 1992; Havaux, 1993), assimilate partitioning (Dinar and Rudich, 1985b; Aloni, Pashkar and Karni, 1991 a) and sugar metabolism in source and sink tissues (Hawker, 1982; Dinar and Rudich, 1985a; Bhuller and Jenner, 1986; Macleod and Duffus, 1988). In addition, hormonal balance, particularly of ethylene and auxin, is important in controlling abscission (Beyer and Morgan, 1971; Wien, Turner and Yang, 1989; Ofir et al., 1993). Low light intensity enhances pepper flower abortion and thus reduces fruit yield. While fertilization is sensitive to high temperature (El- Ahmadi and Stevens, 1979; Kao, Chen and Ma, 1986; Mutters and Hall, 1992), this process may not be important in the abortion of flower buds (pre-anthesis) and fruitlets (post-fertilization). Both flower retention and fruit set depend on assimilate supply to the developing reproductive organ, as shown in many studies in which leaf area, CO enrichment, leaf removal and alterations of competing sinks were found to * For correspondence. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No E, 1995 series $ affect flower development, flower abortion and fruit set (see review by Kinet, Sachs and Bernier, 1985 and references therein). More recently it was shown that flower retention in pepper decreases under shading stress (Wien, Turner and Yang, 1989) and flower abscission was higher if shading was applied in combination with high temperature. It has been suggested (Aloni, Pashkar and Karni, 1991b) that competition for assimilates between the flowers and the adjacent young leaves may be important in determining flower retention. Only recently Turner and Wien (1994) have shown that susceptibility of pepper cultivars may be related to assimilate partitioning to the flower buds. In order to investigate the relationships between assimilate concentration and metabolism in peppers further we investigated the daily changes in carbohydrate concentration of the flowers in relation to their abscission when assimilate supply was limited by removal of source leaves or by low light intensity in pepper cultivars with different sensitivity to flower abortion. MATERIALS AND METHODS Plant material, growth conditions and shading treatments Seedlings of pepper plants of several different cultivars (Bell pepper cvs. Maor, 899, from Hazera, Israel, from Zeraim Gedera, Israel, Mazurka from Rijk Zvaan, Holland, Paprika Lehava, from The Volcani Center, Israel) were transplanted on 1 Jul into 10 l pots filled 1996 Annals of Botany Company

2 164 Aloni et al. Carbohydrate Content of Pepper Flowers Each treatment was applied to 24 plants, divided among three replicates. After the trimming treatments the rate of abscission of the primary and secondary flower buds of internode 8 was recorded. Abscission of reproductive organs was determined by counting vacant internodes on the whole plant and calculating the percentage of aborted organs. FIG. 1. Schematic drawing of leaf pruning. The arrow points to internode 8 at which the main stem flower bud ( ) was maintained. The ( ) symbolize the attached leaves. The broken line symbolizes the stem continuation. Nos 1 4 are the treatments as explained in the Materials and Methods. with a peat: perlite mixture (60:40, v v) and irrigated with commercial nutrient solution ( Shefer, Deshanim Co. Israel, N:P:K, 7:3:7 and microelements). The plants were grown in a greenhouse with minimum and maximum temperatures of 22 and 28 C, respectively. After 30 d each plant was allowed to develop two main stems, which were supported by training threads. The flowers of each internode, on both stems, were maintained with two adjacent source leaves. All the side shoots were removed. Sixty days after transplantation, pepper plants of different cultivars developed flowers, fruitlets and fruits, at different developmental stages. The mature fruits were removed in order to avoid any effects of fruit load. At that stage the plants were placed under nets of several different meshes in order to reduce the maximum photosynthetically active radiation (PAR) intensity (range µmol m s ) and different daily integrated total radiation, 2 8 MJ d. PAR was measured continuously by LI-COR (Lincoln, Nebraska, USA), LI-185B, radiometer. Each shading treatment was applied to 20 plants (5 plants 4 replicates) of each cultivar for a period of 15 d. Control plants were unshaded and received an average maximum PAR of 920 µmol m s. Photosynthesis measurements Photosynthesis, as net carbon exchange rate (CER), was determined by infra red gas analysis (IRGA) measurements (ADC The Analytical Development Co, Hoddesdon, Herts, UK). The measurements were made between 1100 and 1200 h on fully expanded leaves, 24 h from the onset of the shading treatments. Samples of sink leaves (approximately 5 cm long) were taken for sugar and starch determinations at dawn (0600 h) and dusk (1800 h) of that day. Determinations of soluble sugars and starch Five samples of leaf discs (each of 1 cm in diameter) from sink leaves 5 cm long, or of flowers or flower parts of approximately 200 mg fresh mass each, were extracted three times with 80% ethanol at 80 C. The combined extracts were evaporated to dryness and redissolved in 2 ml distilled water, from which aliquots of µl were taken for sugar determination. Reducing sugars were determined colorimetrically using dinitrosalicylic acid (Miller, 1959); sucrose was determined by using the anthrone reagent method as modified for determination of non-reducing sugars (Van Handel, 1968). Starch was determined by measuring glucose following digestion of the ethanolinsoluble residue with amyloglucosidase (Dinar, Rudich and Zamski, 1983). Total non-structural carbohydates (NSC) is the sum of soluble sugars and starch and daily accumulation of them was calculated as the difference between sugar concentrations at dawn and dusk. Calculations of significant differences at the 5% level were carried out by analysis of variance and least significant differences. Manipulations of source sink ratio Plants of cv. Mazurka were grown in an unshaded greenhouse and trimmed as described above. Leaves and flowers were trimmed at the eighth internode of each stem, as follows (see Fig. 1): (1) The primary (main stem) flower bud (3 d before anthesis) was retained but the source leaf and the side shoots at this internode were cut off. This treatment was designated as: Flower ( ). (2) The primary flower bud retained with one adjacent source leaf attached; Flower ( 1). (3) The primary flower bud retained with two adjacent source leaves attached; Flower ( 2). (4) The primary flower bud retained with two source leaves and secondary flower bud (present on the side shoot of the same internode) with its adjacent leaf attached; Flower ( 2 SF). Enzyme assays Determination of soluble acid invertase activity in pepper flowers was as described by Aloni et al. (1991a, b). In short, tissue samples of approximately 300 mg were ground with a Polytron homogenizer in 5 ml ice-cold grinding medium containing : 25 mm HEPES buffer (N-2-hydroxyethylpiperazine-N2-2-ethanesulphonic acid) ph 7 2, 5 mm MgCl,2mMDDT (DL- Dithiothreitol) and 3 mm DIDCA (diethyldithiocarbamic acid) as antioxidant. The mixture was centrifuged at g for 20 min at 4 C. Aliquots of 100 µl of the supernatant were incubated in 1 0 ml 0 1N phosphate citrate buffer, ph 5 0 and 20 mm sucrose (Km of the enzyme found to be 5 mm sucrose). The incubation was

3 Aloni et al. Carbohydrate Content of Pepper Flowers 165 carried out for 30 min at 37 C and was terminated by addition of 1 ml dinitrosalicylic acid reagent. After boiling for 5 min, the resulting sugars were determined colorimetrically as described above. Sucrose synthase activity was determined according to Schaffer, Aloni and Fogelman (1987). Following extraction as described for acid invertase the mixture was dialysed overnight in order to remove the internal sugars. The enzymatic activity was determined as sucrose breakdown on aliquots of 200 µl incubated in incubation medium containing 0 1 M phosphate-citrate buffer ph 7 0, 200 mm sucrose (with Km being 125 mm) and 5 mm UDP. After incubation at 37 C for 30 min the resulting fructose was determined by the dinitrosalicylic acid reaction. The data were expressed on fresh mass basis. RESULTS Effect of light intensity on flower abscission Table 1 shows that reduction of maximum light intensity from 920 to 200 µmol m s by shading, enhanced pepper flower abscission in several cultivars, but there were differences in their susceptibility. Paprika, cv. Lehava and bell pepper Mazurka appeared to be the least sensitive while bell pepper cvs. 899, Maor and were highly susceptible. Shading reduced CER in all the tested cultivars but it was not related to the flower abscission in these cultivars under the shading treatments. TABLE 2. The effect of pruning treatments on flower abscission, 21 d from leaf remo al and on daytime gain or loss of soluble sugars, starch and non-structural sugars (NSC, soluble sugars starch) in flowers of pepper plants c. Mazurka Pruning Flower abscission (%) sucrose Change in carbohydrates during the light period (mg per flower) reducing sugars starch total NSC Treatment Flower ( ) Flower ( 1) Flower ( 2) Flower ( 2 SF) 40 SF l.s.d. (P 0 05) Sugar was determined 24 h after the pruning treatments. and signs indicate gain and loss of sugars, respectively. Explanation for the lettering of the pruning treatments is given in the Materials and Methods section. TABLE 3. The distribution of carbohydrates (NSC is non reduced carbohydrate sucrose starch) between arious pepper flower parts (c. Mazurka ). Data are means (n 5) Flower part Reducing sugars Sucrose (mg per organ) Starch Total NSC Effect of source size The effect of source reduction was studied by removing different numbers of source leaves from the internode of given primary flower buds and by monitoring daily sugar accumulation in these flowers on the day which followed the treatment. Subsequently, the abscission rate was monitored. Table 2 shows that when a single source leaf was present, the flower at the same internode [Flower ( 1)] accumulated TABLE 1. The effect of maximum daily light intensity on photosynthesis, measured 24 h after the onset of the shading treatments, and on flower abscission (in the whole plant), determined 15 d later Cultivar Light (µmol m s ) Flower abscission (%) Photosynthesis (µmol CO m s ) Maor Mazurka Lehava l.s.d. (P 0 05) Petals Ovary Sepals Style stamen l.s.d. (P 0 05) mg sugars d. Detaching this leaf [Flower ( )] caused a loss of 1 2 mgd of sugars from this flower. When two leaves were attached [Flower ( 2)], the adjacent primary flower gained 2 9 mg sugars d on the following day. The secondary flower (SF) lost 0 5 mg in the same period. Table 2 shows that 21 d after the onset of the pruning, abscission of flowers without adjacent source leaf [Flower ( )] was 65%. In Flower ( 1) and ( 2), abscission had diminished to only 10% during that time. When a flower bud was present on the side shoot [Flower ( 2 SF)], abscission of the primary flower was increased to 40%. All the secondary flower buds were aborted 21 d after leaf pruning. Distribution of sugars in pepper flowers Flower parts differ in the amount of carbohydrates (Table 3). The petals and the ovary had the largest amounts of reducing sugars and starch. Sucrose appeared only in minute amounts in all flower parts, indicating that if translocated it was rapidly metabolized. There were differences in sugar accumulation between sink leaves and

4 166 Aloni et al. Carbohydrate Content of Pepper Flowers TABLE 4. The effect of accumulati e daily PAR, measured inside the greenhouse on the concentration and increase in concentration between dusk and dawn of total NSC (soluble starch) in sink lea es (5 cm long) and flower buds of pepper c. Mazurka Total NSC (mg g f. wt ) Light (MJ d ) Dawn Dusk Dusk Dawn (difference) Leaves l.s.d. (P 0 05) Flower buds l.s.d. (P 0 05) TABLE6. The acti ities of acid in ertase and sucrose synthase per gram fresh mass or per organ in arious parts of pepper, c. Mazurka, flowers at anthesis. Data are means (n 5) Flower part Enzymatic activity, sucrose hydrolysed Acid invertase Sucrose synthase Activity per unit (µmol g f. wt min ) fresh mass Petals Ovary Sepals Stamen style l.s.d. (P 0 05) Total activity (µmol per organ min ) per gram Petals Ovary Sepals 7 4 Stamen l.s.d. (P 0 05) flowers under different daily integrated PAR (Table 4). Sugar concentration at the end of the day was increased in 5 cm long sink leaves as light in the greenhouse increased from 1 6to8 31 MJ d. These values were typical of cloudy and sunny days, respectively, during the experiment. At a total PAR of 1 6 MJd, sugar accumulated in sink leaves during the day. On the other hand, in flowers, positive sugar balance was observed only when the daily integrated PAR was approximately 5 0 MJd. Culti ar differences The Lehava and Mazurka cultivars, which were the least sensitive to abscission (as shown in Table 1) gained sugars and starch in their flowers during the day even under shade, while the more susceptible cultivars ( Maor and 899 ) had negative daily sugar balance in their flowers under shade (Table 5). Enzymatic acti ity Acid invertase and sucrose synthase (the sucrose cleaving enzymes) are both present in the pepper flower bud. There were no significant differences in acid invertase activity between different flower parts except for the low enzymatic activity in the sepals (Table 6). On the other hand, the TABLE 5. The concentration and daily accumulation of starch and total NSC in flower buds of arious pepper culti ars in control (maximum irradiation of 920 µmol m s ) and shading (500 µmol m s ) treatments. Diff. indicates the difference between dusk and dawn alues Carbohydrate concentration (mg g f. wt ) Starch Total NSC Cultivar dawn dusk Diff. dawn dusk Diff. Maor 899 Lehava Mazurka control shade l.s.d. (P 0 05) control shade l.s.d. (P 0 05) control shade l.s.d. (P 0 05) control shade l.s.d. (P 0 05)

5 Aloni et al. Carbohydrate Content of Pepper Flowers 167 sucrose synthase concentration was greatest in the ovary, followed by stamen style and sepals with the least being in the petals. DISCUSSION Photosynthesis is an important factor in determining pepper flower abortion, since its reduction by shading enhanced flower abortion in several pepper cultivars (Table 1). However, differences in photosynthetic rates do not explain the differences in cultivar susceptibility. For example, the photosynthesis measured in cvs. Maor and 899, which are highly susceptible to shading-induced flower abscission, was greater than that measured in the less sensitive cultivars Mazurka and Lehava. This agrees with the results of Turner and Wien (1994) who found no differences in photosynthetic rates between pepper cultivars with different sensitivities to shading, and with a report by Aloni et al. (1991b) that flower abortion in pepper, caused by high temperatures, was not accompanied by changes in photosynthesis. It is suggested that assimilate allocation to the flower and its metabolism within the flower is a more important factor for its retention than photosynthesis. Nevertheless, when the availability of assimilates to the developing flower is reduced (e.g. by leaf pruning or shading) abscission is greatly enhanced. Based on leaf pruning and shading experiments, it is calculated that to set properly, a Mazurka flower has to accumulate about 1 2 mg sugars d until pollination takes place. This amount can be provided if a given flower has at least one source leaf attached at its internode and the plant is exposed to light of approximately 5 MJ d (Table 4). If irradiation is less, the daily balance of sugar accumulation in the flower becomes negative, resulting in abscission. The sink leaves, on the other hand, start to accumulate sugars at a daily light imput of only 1 6 MJd, indicating a stronger sink capacity than that of the flower bud. These results reinforce our previous suggestion that the sink leaves and the adjacent flower buds may compete for translocated assimilates (Aloni et al., 1991b) and that the sink leaves are stronger sinks than the adjacent flowers. In contradiction, Turner and Wien (1994) showed that removal of sink leaves did not affect the abscission of pepper flower buds under low light conditions. They also showed that pepper cultivars of differing susceptibility of flower abscission to low light did not differ in dry-matter partitioning between flower buds and sink leaves. However, since these studies were carried out on small numbers of cultivars and in plants subjected to a single low light treatment (30 35 µmol m s ) this hypothesis requires more tests. Within the flower, the ovary and the petals accumulate most of the incoming sugars. However, while petals accumulate reducing sugars, the ovary metabolizes a large proportion of the sugars into starch (Table 3). Walker and Hawker (1976) suggested that the sink capacity of pepper fruit is initiated only after pollination takes place and that acid invertase and sucrose synthase activities are associated with sink activity. Sun et al. (1992) suggested that sucrose synthase may serve as an indicator for sink strength in growing tomato fruits. Robinson, Hewitt and Bennett (1988) have shown that both sucrose synthase and ADPglucose pyrophosphorylase activities correlate with a transient starch accumulation found in developing tomato fruits. Our work shows that sucrose synthase activity is present predominantly in the flower ovary while acid invertase activity is similar (with the exception of the sepals) in all flower parts (Table 6), suggesting that the ovary serves as the main active sink for assimilates, possibly due to its high capacity for starch biosynthesis. The differences in cultivar susceptibility to flower abscission caused by shade (Table 1), and the differences between cultivars in daily capacity to accumulate sugar starch under shade (Table 5) indicate that pepper cultivars with differing susceptibilities to flower abscission may also differ in their capacity to metabolize sucrose and accumulate starch during the light periods until pollination takes place. The daily accumulation of starch may increase the sucrose gradient between the source leaf and the developing flower, therefore driving sucrose transport toward the flower. Sucrose synthase, located in the ovary, is a sucrose cleaving enzyme producing ADP glucose which is subsequently metabolised to starch. This enzyme may be regulatory in the pathway to starch biosynthesis, as has been previously suggested (Claussen, 1983; Claussen, Hawker and Loveys, 1985; Sung, Xu and Black, 1988). Possibly, pepper cultivars of different susceptibility to flower abscission may differ in the concentration and activity of this enzyme in the developing flower. This possibility is presently being investigated. ACKNOWLEDGEMENTS This research was supported by Grant No. US from BARD, the United States-Israel Binational Agricultural Research and Development Fund and by The Cooperative Arid Land Agricultural Research Program (CALAR II), an AID Fund, contract no: GNE-0158-G LITERATURE CITED Aloni B, Pashkar T, Karni L. 1991a. Nitrogen supply influences carbohydrate partitioning in pepper seedlings and transplant development. Journal of the American Society of Horticultural Science 116: Aloni B, Pashkar T, Karni L. 1991b. Partitioning of [14]-C sucrose and acid invertase activity in reproductive organs of pepper plants in relation to their abscission under heat stress. Annals of Botany 67: Beyer EM, Morgan PW Abscission: the role of ethylene modification of auxin transport. Plant Physiology 48: Bhullar SS, Jenner CF Effects of temperature on the conversion of sucrose to starch in the developing wheat endosperm. Australian Journal of Plant Physiology 13: Claussen W Investigation on the relationship between the distribution of assimilates and sucrose synthase activity in Solanum melongena L. 2. Distribution of assimilates and sucrose synthase activity. Zeitschrift fu r Pflanzenphysiologie 110: Claussen W, Hawker JS, Loveys BR Comparative investigation of the distribution of sucrose synthase activity and invertase activity within growing mature and old leaves of some 3-carbon photosynthetic pathway plant species. Physiologia Plantarum 65: Dinar M, Rudich J. 1985a. Effect of heat stress on assimilate metabolism in tomato flower buds. Annals of Botany 56:

6 168 Aloni et al. Carbohydrate Content of Pepper Flowers Dinar M, Rudich J. 1985b. Effect of heat stress on assimilate partitioning in tomato. Annals of Botany 56: Dinar M, Rudich J, Zamski E Effect of heat stress on carbon transport from tomato leaves. Annals of Botany 51: El-Ahmadi AB, Stevens MA Reproductive responses of heat tolerant tomatoes to high temperatures. Journal of the American Society of Horticultural Science 104: Gent MPN Carbohydrate level and growth of tomato plants: the effect of irradiance and temperature. Plant Physiology 81: Havaux M Rapid photosynthetic adaptation to heat stress triggered in potato leaves by moderately elevated temperatures. Plant Cell and En ironment 16: Hawker JS Effect of temperature on lipids, starch and enzymes of starch metabolism in grape, tomato and broad bean leaves. Phytochemistry 21: Kao CG, Chen HM, Ma LH Effect of high temperature on proline content in tomato floral buds and leaves. Journal of the American Society of Horticultural Science 111: Kinet JM, Sachs RM, Bernier G Photosynthesis, assimilate supply and utilization. In: Kinet JM, Sachs RM, Bernier G, eds. The physiology of flowering, Vol III. Boca Raton, Florida: CRC Press Inc. Kitroongruang N, Jodo S, Hisai J, Kato M Photosynthesis characteristics of melons grown under high temperatures. Journal of the Japanese Society of Horticultural Science 61: Macleod LC, Duffus CM Reduced starch content and sucrose synthase activity in developing endosperm of barley plants grown at elevated temperatures. Australian Journal of Plant Physiology 15: Miller GL Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry 3: Mutters RG, Hall AE Reproductive responses of cowpea to high temperature during different night periods. Crop Science 32: Ofir M, Gross Y, Bangeth F, Kigel J High temperature effects on pod and seed production as related to hormone levels and abscission of reproductive structure in common beans (Phaseolus ulgaris L.). Scientia Horticulturae 55: Robinson NL, Hewitt JD, Bennett AB Sink metabolism in tomato fruit. Developmental changes in carbohydrate metabolizing enzymes. Plant Physiology 87: Schaffer AA, Aloni B, Fogelman E Sucrose metabolism and accumulation in developing fruit of sweet and non-sweet genotypes of Cucumis. Phytochemistry 26: Shishido Y, Challa H, Krupa J Effects of temperature and light on the carbon budget of young cucumber plants studied by steady state feeding with CO. Journal of Experimental Botany 38: Sun J, Loboda T, Sung SS, Black CC Jr Sucrose synthase in wild tomato, Lycopersicon chmielewskii, and tomato fruit strength. Plant Physiology 98: Sung SJ, Xu DP, Black CC Purification and properties of actively filling sucrose sinks. Plant Physiology 89: Turner AD, Wien HC Dry matter assimilation and partitioning in pepper cultivars differing in susceptibility to stress-induced bud and flower abscission. Annals of Botany 73: Van Handel E Direct microdetermination of sucrose. Analytical Chemistry 22: Wien HC, Turner AD, Yang SF Hormonal basis for low light intensity induced flower bud abscission of pepper. Journal of the American Society for Horticultural Science 114: Walker RR, Hawker JS Effect of pollination on carbohydrate metabolism in young fruits of Citrullus lanatus and Capsicum annuum. Phytochemistry 15: