Abstract: Tree peony (Paeonia suffruticosa) native to China is one of the most widely cultivated ornamental plants in the world and grows to flower normally in late spring. Successive secondary flowering in autumn season is critical for some ornamental plant production, including tree peony. Varying levels of hormones and sugars are thought to influence flowering in plants. This study analyzed quantitative changes in endogenous hormone (indole-3-acetic acid (IAA), abscisic acid (ABA) & gibberellic acid (GA3)) and sugars (sucrose, reducing sugars & starch) levels in bud samples of 'Ao-Shuang' tree peony cultivar during autumn and spring flowering period. Hormone and carbohydrate levels in spring and autumn flowering buds were notably different. The developmental stages of autumn flowering bud (AFB) not only accumulate IAA, GA3, sucrose and reducing sugars, but also degrade ABA and starch that probably contributed to induce flowering. For both seasons, sucrose concentration was highest then followed by reducing sugars and starch. Whereas sucrose and reducing sugar content increased in autumn flowering bud, that of starch decreased. Spring flowering bud (SFB) showed similar trends for sucrose, reducing sugar and starch. The results suggest that 'Ao Shuang' probably blooms in autumn because of lack of dormancy due to low ABA. Although quantitative changes in endogenous hormones and carbohydrates could be influenced by seasonal variations, the observed mix could enhance tree peony flowering mechanism.
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Keywords: Autumn/spring flowering; hormone; ornamental plant; sugar; tree peony.
Paeonia suffruticosa tree peony is native to China and is a magnificent, beautiful and attractive ornamental plant (Wister, 1995). As an excellent ornamental plant with socio-economic, cultural and medicinal connotations in China, it has made its way into the fabrics of modern society in this country and is also widely cultivated in the continents of Asia, America, Europe and Australia (Cheng, 2007).
Nicknamed as the "King of flowers", tree peony culturally symbolizes peace, happiness, prosperity, development, power and wealth in China. Because of its vastly rich cultural connotation, it is extensively cultivated in China. In spite of the fact that there are abundant tree peony species and cultivars, China largely lacks successive secondary flowering cultivars that flower two or more times in a year (Zhang, Unpublished). Hence special technique that promotes induction of autumn or even winter flowering could bring tremendous benefits to the horticultural industry in China.
The study of flowering mechanisms in tree peony cultivation is important because the field plants flower not only once per year, but also have short flowering periods. Even so, the plant normally takes 3-5 years to start flowering and the leaves and flowers share the same bud. Driven by economic benefits, the quality and quantity of flowers are therefore overridingly important in tree peony cultivation. The high demands and price values associated with tree peony flower make research on this ornamental plant even more desirable. Therefore, understanding the physiological mechanisms of successive secondary flowering is a promising alternative in the horticultural industry of tree peony.
Several endogenous (e.g., sugars, genes & hormones) and exogenous (e.g., day length & temperature) factors influence flower production in apical meristems (Zeevart, 1979; Pallardy, 2008). The transition from juvenile to adult phases is heavily influenced by changes in environmental factors. These factors in turn influence sugar and hormone levels in many plants (Zeevart, 1979), including tree peony plants. Tree peonies require a period of cool temperature prior to bud growth and flower production as done in herbaceous peonies, another group of ornamental peonies used in the flower industry (Byrne & Halevy, 1986). The flower buds grow out on perennial crowns in later summer, followed by leaf senescence and bud dormancy. Bud development then only resumes after winter cold vernalization (Byrne & Halevy, 1986; Fulton et al., 2001). But some tree peony cultivars such as Ao-Shuang, however, do not follow this routine and therefore also blossom in autumn without cold vernalization (Cheng and Zhao, 2008). This makes it a very special cultivar for horticultural use and provides competitive values in tree peony production.
Peony, like any other flowering plant, requires hormones for growth and normal maintenance of physiological processes. As growth regulators, hormones are critical in plant developmental processes as they regulate root development, vascular differentiation, response to climate, apical dominance and flower development (Katia & Gilberto, 2004; Ana et al., 2004). The effects of hormones, however, changes with environmental conditions in different seasons of the year (Koshita et al, 1999). For instance, Altman & Goren (1972) noted that IAA, GA and ABA respectively delays, enhances and inhibits summer bud sprouting. Recent studies have shown that cytokinin profiles of different plant organs change with season (Rasmussen et al., 2009). Cytokinin levels in apical buds of Abies nordmanniana are lowest in mid-June and highest in late summer.
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Like hormones, plant growth and development are also influenced by available nutrients. Studies have shown that sucrose, glucose and fructose constitute the main assimilates of most plants (Katovich et al., 1998 and Pallardy (2008). According to Pallardy (2008) sucrose is the main form of plant carbohydrate for the energy and carbon skeletons used in the synthesis of amino acids, lipids and metabolites. Flowering too is associated with changes in sugar levels. Soluble sugar levels can be altered to regulate plant growth processes.
Therefore, understanding hormonal and carbohydrate changes and the associated floral developments could rationally transform tree peony flower production. As of date, there is relatively little research on autumn flowering of tree peonies (Jiang et al., 2007; Zhang, 2004). Hardly is the effect of changes in endogenous hormones on tree peony flower production documented in the literature. Least documented are endogenous hormone and sugar variations in spring- and autumn-flowering tree peonies. The task of this research was to determine the dynamics of endogenous hormone and sugar levels in flowering Ao-Shuang tree peony cultivar in autumn in comparison with that of spring season. The result will elucidate the physiological causes of peony flowering in autumn and provide the basis of production technology for peony successive secondary blooming.
Materials and methods
The research was conducted at Jiufeng peony collection base of Beijing Forestry University in spring and summer of 2009 and 2010. A total of 20 5-year-old plants of P. suffruticosa 'Ao-Shuang' cultivar with similar growth vigor, for each season, were selected for investigation in the study. Samples collection was done in late Feb-mid May and mid Aug-late Sept. of 2009 and 2010 corresponding with spring and summer seasons in Beijing (Fig.2).
For autumn samplings, leaf deletion was manually done in mid August, before applying dormancy releasing treatment. The dormancy releasing solution, GA3, was applied at 500 mg/L per dose on developed buds using a paintbrush, five days after leaf deletion. Spring plants went through the winter cold to break dormancy. Bud samples were collected before (BD) and after (AD) leaf deletion, and then after dormancy release. Sample collection after dormancy release targeted stages I to VIII of bud development as described by Cheng et al. (2001)(stage 1- bud swelling; stage 2- bud sprouting; stage 3- shoot emerging; stage 4- shoot elongation; stage 5- leaflet extending; stage 6- flower bud enlarging; stage 7- color appearance; stage 8- flowering).
A clean stainless knife was used to harvest sample buds from the plants and then rinsed with distilled water to minimize surface contamination. The collected bud samples were immediately placed in an ice box, conveyed to the laboratory, dipped into liquid nitrogen (N2), and stored at -80 °C until everything was set for analyzing hormones and soluble sugars.
Hormone extraction and analysis
With only slight modifications, endogenous hormones extraction was conducted as described by Chen et al (1991). The levels of ABA, IAA and GA3 were determined using fresh bud tissues (0.5 g). The plant tissues were ground in antioxidant (copper) and 10 ml of 80% cold methanol until it was completely homogeneous before transferring into the test tube. About 30 mg of polyvinylpolypyrrolidone (PVP) was added to the homogenate, the mixture then thoroughly mixed on the shaker for 10 min. and incubated at 4 OC overnight. The supernatant was transferred into 10 ml tube the next morning and spun at 6000 rpm for 20 min. The residue was washed and re-extracted with 2 ml cold methanol for 12 hrs. It was then centrifuged under the same conditions as described above before finally discarding the dust. The combined extracts, after adding 2-3 drops of NH3, were evaporated (35-40 °C) to an aqueous phase in the rotary evaporator. Thereafter, the aqueous phase was dissolved by adding distilled water, and then the mixture was adjusted to pH 2.5-3.0 with 1N HCl, and extracted three times with the same volumes of ethyl acetate. The combined ethyl acetate fraction was evaporated to dryness. The dried sample was diluted in 1.0 ml of 3% methanol and 97% 0.1 M acetic acid (HAc) to determine acidic hormones such as IAA, GA3 and ABA.
Hormonal determination was carried out using the HPLC (Agilent 1100) chromatography method. Conditions of the HPLC were as follow: capillary column (Sep-Pak C-18 cartridge, 250 x 4.6mm); mobile phase, 3% methanol and 97% 0.1 M HAc for IAA, GA3 and ABA determination; wavelength of different hormones, IAA = 280 nm, ABA = 260 nm, GA3 = 210 nm; and flow rate, 1 ml/min. Hormones were identified and quantified by comparison between retention times of the samples and standard samples (Sigma Chemical Co. USA).
Soluble sugar extraction and analysis
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Fresh leaf material (1.0 g) was ground in 20 ml of distilled water and extracted in water bath at 80 °C for 30 min. The suspension was centrifuged for 10 min at 6000 rpm. The supernatant and extractant residues were collected and used to respectively determine sucrose and reducing sugars, and starch. The reducing sugars were calorimetrically determined using dinitrosalicylic acid. The sucrose and starch were determined by anthrone reagent method with glucose as a standard, using the colorimetry anthrone method as modified for the determination of non-reducing sugars (Xue & Xia, 1985). The absorbance was then determined by spectrophotometer (TU-1901).
Statistical analyses were done using SPSS (Statistical Package for Social Scientists). The mean values of the targeted hormones and carbohydrates were taken and one-way ANOVA executed to determine in-between treatment significance at P<0.01.
Bud hormonal content
ABA: At the initial stages of growth, visible differences existed in ABA trend between spring flowering buds (SFB) and autumn flowering buds (AFB). ABA content at the initial stages of bud development was higher in SFB than in AFB (Fig. 3A). In the middle to late stages, no significant differences were noted in ABA content between both flowering buds.
IAA: A double peak was noted for IAA level in SFB, corresponding to shoot emerging (stage 3) and blooming (stage 8) stages. IAA level in AFB exhibited a triple peak, coinciding with bud swelling (stage 1), shoot elongation (stage 4) and flower bud anthesis (stage 7) stages (Fig. 3B). A more interesting observation was that high IAA levels in AFB corresponded with low IAA levels in SFB.
GA3: The level of GA3 was highest during bud swelling stage (stage 1) in SFB, and also during shoot emerging stage (stage 3) in AFB. After these stages, GA3 levels in both season buds tracked similar trends, though at a slightly higher level in AFB (Fig. 3C).
Bud carbohydrates content
Differences were also noted in the trend of carbohydrates at different developmental stages between spring and autumn flowering buds (Figs. 4). Sucrose and reducing sugars (glucose & fructose) levels increased at the early stages (AD & stage 1) of development in SFB but fell at bud sprouting (stage 2) and shoot emerging (stage 3) stages. They increased again at flower-bud development (stages 4 & 5) stages, before falling at flower bud opening stage (stage 7) and then rose sharply again at blooming stage. Autumn flowering buds , on the other hand, showed an initial drop in sucrose and reducing sugar contents, followed by a gradual increase as buds sprouted out (stage 2) and shoot emerged (stage 3). It then maintained a relatively stable trend for the rest of the growth period (Figs.4A & B).
The trend of starch in SFB more or less followed that of sucrose and reducing sugars in SFB, except for the initial stages where an opposite trend was observed (Fig. 4C). For AFB, starch level initially increased and subsequently decreased with flourishing new vegetative growth. It then continues to drop at growth stages 3, 4 & 5, reaching its lowest values during leaflet extending stage (stage 5) (Fig. 4C). Sucrose was apparently the main source of sugar in the two buds under different temperature regimes, followed by reducing sugars and starch. There were no consistent differences in change pattern of sucrose, reducing sugars and starch in SFB. In AFB, however, consistent differences existed for the different developmental stages among sucrose, reducing sugars and starch (Figs.4).
Effect of hormones on bud growth, development and flowering:
Plant growth and development is driven by meristemic cell production and subsequent elongation, which directly or indirectly is regulated by hormones (Friml, 2003; Katia & Gilberto, 2004). This study shows significant differences in the trends of endogenous ABA at the initial growth stages (BD - stage 3) between spring and autumn flowering buds (Fig. 3A). At the rest of the developmental stages, ABA change pattern follows similar trends in SFB and AFB. The different trends at the initial stages of growth could be due to the different physiological conditions within the bud as influenced by spring and autumn temperature regimes. The difference in the trend of ABA is statistically significant (p<0.01) when spring (Feb-Mar) temperature was lowest and autumn temperature (Aug-Sept) was highest. As different ABA levels were observed during the initial stages of bud development, low endogenous ABA could then be a factor influencing the development and flowering in autumn flowering bud. This is because at the initial stages where high ABA level in SFB coincided with low ABA level in AFB, SFB was either transition from endo- to eco-dormancy state or just emerging from winter dormancy, a condition that is reportedly associated with high ABA content (Djilianov et al., 1994; Rinne et al., 1994a). Based on the above, it implies that AFB could probably not have experienced dormancy, suggesting that low ABA concentration has an overall effect on autumn flowering buds, which in turn facilitates flowering. As SFB began experiencing warmer temperature (April-May) (Fig. 3A), ABA level in bud decreased (also see Barros & Neill, 1988) and showed similar pattern as that of AFB. This also suggests that a correlation exists between temperature and ABA level that possibly influences dormancy release, bud break and onset of peony re-growth. At the onset of bud breaking stage (stage 1), decreasing level of ABA synchronized with increasing level of GA3 (compare Fig. 3A & C). Possibly, there could be an inverse functional relationship between these two hormones during dormancy release in bud. These results are consistent with those of Altman & Goren (1972), who noted that ABA completely inhibits summer bud sprouting. Low ABA positive effect on autumn flowering bud is noted in this study, which is also consistent with the findings of Wijiayanti et al. (1997) and Marumo et al. (1990). However, Harada et al. (1971) and Nakayama & Hashimoto (1973) reported that ABA promotes flowering in other plants.
High levels of IAA were observed in this study at two peaks, corresponding with shoot extending (stage 3) and blooming (stage 8) stages in spring flowering buds. For autumn flowering buds, three peaks, coinciding with bud swelling (stage 1), shoot elongation (stage 4) and flower bud opening (stage 7) stages, were observed (Fig. 3B). AFB showed triple peaks for IAA, apparently coinciding with relatively low IAA level stages in SFB. This implies that endogenous IAA could be influenced by environmental conditions especially temperature. The results suggest possible correlation among endogenous IAA and flower bud formation, bud outgrowth and flower bud anthesis in AFB which in turn, may influence flower bud development and associated flowering. Significant increases in the level of endogenous IAA during bud outgrowth have also been noted in other plant species (Koshi Koshita & Takahara, 2004; Liu et al., 2008).
IAA seems to be the major growth-promoting hormone in autumn tree peony plants (also see Pallardy, 2008) as its high levels were observed at active cell division and elongation stages (stages 1, 4 & 7). This suggests that IAA may contribute to induce and promote cell division and elongation in AFB. The high IAA content at these stages is not surprising as it is related with high nutrient availability (Koutinas et al., 2010). Nutrients are needed developmental, physiological and metabolic elements by all varieties of plants (Priestly, 1977). This is important because early, rapid development of primordial or young leaves is a necessary condition for flower bud initiation. For SFB, IAA only peaked at shoot extending (stage 3) and blooming stages (stage 8) of the plant development. High IAA level at blooming stage could be attributed to the fact that spring flowering peony will continue growth after flowering, during which period the preliminary induction of the flower formation occurs within one month after full bloom (Barzilay et al., 2002). High nutrient could possibly be needed by the plant during this period since high IAA level is associated with high nutrient attraction ability (Koutinas et al., 2010).
It is interesting to note that trends of IAA in SFB and AFB were inversely correlated for almost all the growth stages. IAA levels in AFB increased at the same stages where a decrease was observed in SFB. In SFB, low IAA level at bud swelling stage (stage 1) coincided with high levels of GA3 and low ABA levels. In AFB also, high IAA levels at bud swelling stage (stage 1) coincided with low levels of both ABA and GA3. It then suggests that increase in IAA may lead to decrease in GA3 and ABA in AFB (see Figs.3B, B & C). There is apparently an inverse functional relation between these hormones which needs further study.
The levels of GA3 exhibited similar change pattern in SFB and AFB, though peaking at different bud developmental stages. For SFB, peak GA3 content coincided with bud swelling stage (stage 1). In AFB, however, peak GA3 content, coincided with full shoot emerging stage (stage 3) (Fig. 3C). For the rest of the other developmental stages, bud GA3 trends were similar for spring and autumn flowering buds though AFB GA3 levels were generally higher than SFB levels. Statistically, the difference in GA3 levels in SFB and AFB is significant at p<0.01. GA3 level increased concurrently with temperature increase at bud break in SFB, suggesting that high temperature is correlated to high GA3 level and the associated dormancy release. The relationship between increase of GA3 and dormancy release is consistent with results on study with herbaceous peony by Cheng et al (2009). Interestingly, GA3 levels in SFB were generally low for all the stages with high AFB GA3 levels. Furthermore, high AFB GA3 coincided with full shoot and flower bud emerging stage. This implies that high GA3 induces flower bud formation in AFB. Although earlier studies suggest inhibitory (Koshita et al., 1999; An et al., 2008) or no (Garner & Armitage, 1996) effects on flower bud formation, our study shows that GA3 is related with flower bud formation, a phenomenon also reported in iris (Nadia et al., 2006) and chestnut (Liu et al., 2008). This could be attributed to the growth-promotion effect of GA3, which stimulates and accelerates cell division and elongation (Hartmann et al., 1990). The result further reveals that GA3 in AFB acted more as a growth promoter rather as dormancy releasing agent, implying that probably no dormancy occurred in AFB. Reason is that high level of GA3 was observed at a stage characterized by complete bud break and rapid shoot development, increase in gibberellin and promotion of mitosis (Pallardy, 2008), whilst low GA3 level coincided with dormancy release stage (stage 1), the onset of peony re-growth (Cheng, 2009).
Effect of nutrients on bud growth, development and flowering
There were changes in carbohydrate composition in peony bud tissues. In terms of sucrose, reducing sugar and starch, spring flowering bud levels increased with decreasing levels in autumn flowering bud at different developmental stages. Sucrose and reducing sugar concentrations were high at the initial stages of growth in SFB than in AFB but however leveled out as buds sprouted (stage 2) and shoots emerged (stage 3). For the other stages, sucrose and reducing sugar concentrations were inversely related. With decreasing starch levels, the levels of sucrose and reducing sugar increased in AFB, as also reported in tropical tree species (Würth et al., 2005). Increasing sugar concentration could be driven by starch degradation (Fischer & Höll, 1991), suggesting that starch greatly influences sugar accumulation in the plants. Sucrose, reducing sugar and starch in SFB showed similar rise-fall trends. Probably the requirement for growth and development by SFB may have led to this pattern of change. In comparison, sucrose and reducing sugar increased with decreasing starch in AFB. Also AFB showed steady accumulation of sucrose and reducing sugar, and degradation of starch. The consistent inverse relationship between sucrose/reducing sugars and starch observed in AFB possibly could have contributed to induce autumn flowering.
Irrespective of the differences in the trends of carbohydrates between spring and autumn flowering bud, there were similar levels of sucrose and reducing sugar at bud sprouting and shoot emerging stages (stages 2 and 3) and also of starch at bud sprouting stage (stage 2) (see Figs. 4A, B & C) . This suggests that carbohydrate hydrolysis is a key factor of bud sprouting and shoot extension. In this study, the level of starch in AFB decreases with flourishing new vegetative parts (Newell et al., 2002; Würth et al., 2005) and bud growth (Katovich et al., 1998), possibly due to the utilization of stored carbohydrate reserves to support shoot/flower bud development (Fig. 4c), suggesting that sink activity has a major influence on use of starch reserves. Furthermore, low level of starch and high level sucrose/reducing sugar were noticed at the stages (BD-AD & AD-S1) where SFB were dormant while those of AFB were high and low respectively. This also suggests that perhaps no dormancy occurred in autumn plants as low starch and high sucrose/reducing sugar levels are reportedly associated with dormancy (Kozlowski, 1992; Chao, 2006; Pallardy, 2008).
In the Fig.4A and B, sucrose and reducing sugar levels at blooming (stage 8) rose sharply in SFB. This can be attributed to the fact that SFB completely has a distinct fate in the plant's future development from AFB. After flowering, SFB continues growth while AFB senesces as they enter cold winter period. Accumulated carbohydrate could possibly be needed by SFB since flower bud formation process starts within a month after full flowering (Barzilay et al., 2002).
For both seasons, sucrose was apparently the main sugar, followed by reducing sugars and starch. Most our findings are consistent with patterns of seasonal carbohydrate changes observed in other perennial species (Kozlowski, 1992; Katovich et al., 1998) but a par with that on chestnut by Liu et al. (2008), who conclude that sugar appears to be irrelevant in abnormal chestnut flowering. This suggests that irrespective to seasonal variation, sucrose is the main carbohydrate in tree peony which could mainly come from the transformation of stored starch. It provides energy and carbon skeleton needs in the synthesis of compounds such as amino acids, lipids and metabolites for plant growth.
Dormancy and flower bud growth in tree peony
Dormancy is a critical phenomenon that regulates the flowering period of tree peony and requires greater attention in flower production industry. In this study, the interaction of hormones and sugars appears to be involved in regulating bud dormancy and growth in tree peony. Our result shows that sucrose and reducing sugars inhibited bud growth in SFB as also reported in other plant species (Kozlowski, 1992; Katovich et al., 1998 Pallardy, 2008). The start of decrease in sucrose content coincided with increase in GA3 content (stage 1). The unsynchronized levels of GA3 and sucrose on one hand and that of GA3 and ABA on the other hand at the onset of dormancy release (stage 1), support the fact the GA3 plays an important role in bud growth resumption in SFB (Cheng et al 2009). In speculating the interaction between sucrose and GA3, sucrose possibly inhibited GA3 availability during dormancy in SFB, which requires further study. Sucrose inhibiting effect on GA3 has also been reported in root buds of Euphorbia esula (Katovich et al., 1998). The relationship between sucrose/reducing sugars and GA3 also could be responsible for regulating starch metabolism because an increased starch level was observed during dormancy break (stage 1). Our result suggests that carbohydrate may be involved in regulating dormancy status.
In investigating changes in hormone and carbohydrate levels at different developmental stages of spring and autumn flowering buds, our results show that endogenous hormones and sugars greatly influence autumn flowering. Developmental stages of autumn tree not only accumulate IAA, GA3, sucrose and reducing sugars, but also degrade ABA and starch that probably contributed to induce flowering. The results suggest that 'Ao Shuang' probably blooms in autumn because of lack of dormancy due to low ABA. Moreover, quantitative changes in endogenous hormones and carbohydrates could be influenced by seasonal variations in the developmental processes, which regulate flowering in tree peony plants.