Abstract: Tree peony (Paeonia suffruticosa) is one of the most widely cultivated ornamental plants in the world. 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 analyses quantitative changes in endogenous hormone (IAA, ABA & GA3) and sugar (sucrose, reducing sugars & starch) levels in bud samples of 'Ao-Shuang' tree peony cultivar during autumn and spring flowering period. The levels of hormones and carbohydrates in spring and autumn flowing were notably different. For both seasons, sucrose concentration was highest then followed by reducing sugars and starch. Whereas sucrose and reducing sugar content increased in autumn, that of starch decreased. Spring season plants showed similar trends for sucrose, reducing sugar and starch. High concentrations of IAA, GA3 were observed along with low concentrations of ABA in autumn flowering tree peony. Although quantitative changes in endogenous hormones and carbohydrates could variously influence tree peony flowering, 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). Tree peony has made its way into the fabrics of modern society as one of the most important garden plants. It is widely cultivated in the continents of Asia, America, Europe and Australia (Cheng, 2007). Tree peony is an excellent ornamental plant with socio-economic, cultural and medicinal connotations in China.
Nicknamed as the "King of flowers" in China, it culturally symbolizes peace, happiness, prosperity, development, power and wealth in China. Because of its vastly rich cultural connotation, tree peony is extensively cultivated in China. In spite of the fact that there are abundant tree peony species and cultivars in China, the country largely lacks successive secondary flowering cultivars that flower two or more times in the year (Zhang, Unpublished). Hence special technique to overcome problems like delay of sprouting due to dormancy and occurrence of flower bud abortion (that induces autumn or even winter flowering) could bring tremendous benefits to the industry of horticulture in China.
The study of flowering control and development mechanisms in tree peony cultivation is important because the field plants flower not only once in the year, but also have short flowering periods. The high demands and price values associated with tree peony flower make research on this ornamental plant even more desirable. Tree peonies normally take 3-5 years to start flowering. Even so, leaves and flowers share the same bud and the plant is harvested only once a year. Driven by economic benefits, the quality and quantity of flowers are therefore overridingly important in tree peony cultivation. Understanding the physiological mechanisms of successive secondary flowering is therefore a promising alternative in the horticultural industry of tree peony.
Several endogenous (e.g., levels of sugars, genes & hormones) and exogenous (e.g., day length & temperature) physiological factors influence flower production in apical meristems (Zeevart, 1979; Luckwill, 1980; Koshita et al. 1999). 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, shoot and flower production. The flower buds grow out on perennial crowns in later summer, starting from leaf senescence till bud dormancy (Byrne & Halevy, 1986). Bud development then only resumes after winter cold vernalization (Byrne & Halevy, 1986; Fulton et al., 2001; Halevy et al., 2002; Kamenetsky et al., 2003). But some tree peony cultivars such as Ao-Shuang, however, do not follow this routine and therefore also blossom in autumn without cold vernalization. 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 and biochemical processes. As growth-regulating organic substances, hormones are critical in plant developmental processes. Hormones regulate root development, vascular differentiation, response to climate, apical dominance, flower development and embryogenesis (Friml, 2003; Katia & Gilberto, 2004; Ana et al., 2004; Liu et al., 2008). The effects of hormones, however, changes with environmental conditions in different periods/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. For sub-apical buds, cytokinin is lowest in June and highest in mid autumn, but with much lower levels than apical buds (Rasmussen et al., 2009). Furthermore, inhibitory effects of exogenous IAA, ABA, and endogenous IAA are reported in Pharbitis nil in which exogenous GA induces flowering (Wijayanti et al., 1997).
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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 (Ashworth et al, (1993), Spencer et al. (2001), and Mohammadkhani & Heidari (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 (Heldt & Heldt, 2005). Flowering too is associated with changes in sugar level. Soluble sugar levels can be altered to regulate plant growth processes. Accumulated sugar levels have been noted in woody plant tissues in response to seasonal variations (Li et al., 1965; Sauter & Kloth, 1987; Nelson & Dickson, 1981). For instance, while sugar increases in winter, deacclimatization leads to low levels in spring (Parker, 1962; Fege & Brown, 1984; Bonicel et al., 1987).
Understanding hormonal changes and the associated floral bud developments could rationally transform tree peony flower production. There is relatively little research on autumn flowering of tree peonies (Jiang et al., 2007; Li, 1998; Xiao et al., 2001). 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 flowering tree peonies with spring and autumn seasons. The research task of this study was to determine the dynamics of endogenous hormone and sugar levels in flowering Ao-Shuang tree peony cultivar in spring and autumn seasons.
Materials and methods
The study was conducted at Jiufeng nursery of Beijing Forestry University in the spring and autumn of 2010. A total of 20 5-year-old Ao-Shuang tree peony cultivars with similar growth vigor were selected for investigation in the study. Samples collection was done in February and August of 2010, corresponding with spring and summer seasons in Beijing. The samples were then analyzed for spring and autumn levels of selected hormones and sugars and the results compared.
Prior to sample collection, agronomic practices including earthening, fertilization, watering, pruning, etc. were carried out. Leaf defoliation was manually done in late August, before dormancy inducing treatment for autumn trial. The GA3 dormancy releasing agent was applied at 500 ppm per dose on developed buds using a paintbrush. Spring plants went through the winter cold to break dormancy. A clean stainless knife was used to harvest sample buds from the plants and then rinsed with distill water to minimize surface contamination. The collected bud samples were immediately placed in an ice box, conveyed to the laboratory, dipped in liquid nitrogen (N2), and stored at -80 °C until everything was set for hormone extraction and analysis.
Plant hormone analysis sampling
Bud samples were collected before and after leaf defoliation, 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).
Hormone extraction and analysis
With only slight modifications, extraction of endogenous hormones was conducted as described by Chen (1991). The levels of ABA, GA3 and IAA endogenous hormones 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. A small amount of PVP was added to the homogenate and 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 twice 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 the 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 equal 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. Qualitative analysis was done based on the retention time of the samples and standard samples. The quantitative analysis was done using "external standard method". The standard sample used is Sigma production (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 distil 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).
All 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.
Results and analysis
Bud hormonal content
A comparison of seasonal (spring & autumn) concentrations of bud GA3 is presented in Fig. 4. The level of GA3 was highest during bud swelling stage in spring, and also during shoot development stage in autumn. After these stages, GA3 levels in both seasons track a similar trend, though at a slightly higher level in autumn.
A double peak was noted for bud IAA level in spring, corresponding to leaf emergence (stage 3) and blooming (stage 8) stages. Bud IAA level in autumn exhibited a triple peak, corresponding to bud swelling (stage 1), shoot development (stage 4) and flower bud anthesis (stage 7) stages (Fig. 3). A more interesting observation was that high autumn IAA levels correspond with low spring IAA levels.
At the initial stages of growth, visible differences existed in ABA trend between the spring season and autumn plants. ABA content at the initial stages of development was higher in spring than in autumn plants (Fig. 2). In the middle to late stages, no significant differences were noted in ABA content between the spring and autumn plants in the study area.
Bud carbohydrate content
Differences were also noted in the trend of carbohydrate at different developmental stages between spring and autumn plants (Figs. 5, 6 & 7). There was an increasing level of sucrose and reducing sugars at the initial stages of plant development in spring. The levels of sucrose and reducing sugars fell at bud sprouting (stage 2) and shoot emergence (stage 3) stages. It increases again at flower-bud development (stages 4 & 5) stage, before falling at flower bud opening stage. At blooming stage, sucrose and reducing sugar levels rose sharply in spring. Autumn season plants, on the other hand, showed an initial drop in sucrose and reducing sugar content, followed by a gradual increase as buds sprout out into shoot (stage 2). It then maintained a relatively stable trend for the rest of the growth period (Figs. 5 & 6).
The trend in starch for spring buds more or less followed that of spring sucrose and reducing sugars, except for the initial stages where a negative trend was observed (Fig. 7). For autumn season plants, starch initially increased and subsequently dropped with flourishing new vegetative growth. It then continued to drop for the rest of the growth (stages 3, 4 & 5) stages, reaching the lowest values during flower bud for growth stage (Fig. 7). It then slightly increased at flower bud anthesis (stage 7) onto blooming (stage 8) stages. Sucrose is apparently the main source of sugar under the two temperature regimes, followed by reducing sugars and starch. There were no consistent differences in the trends of sucrose, reducing sugars and starch in spring plants. In autumn plants, however, consistent differences existed for the different developmental stages among sucrose, reducing sugars and starch (Figs. 5, 6 & 7).
Plant growth and development is driven by meristemic cell production and subsequent elongation (Clark, 1997; Cosgrove, 1997). Plant hormones affect cell division and elongation. Thus hormones directly or indirectly regulate plant cell division and growth (Zeevart, 1976; Friml, 2003; Katia & Gilberto, 2004). This study shows significant differences in the trends of endogenous ABA at the initial growth stages (b4 - stage 3) in spring and autumn tree peony plants (Fig. 2). At the rest of the developmental stages, ABA change pattern follows a similar trend in spring and autumn tree peonies. The different trends at the initial stages of growth could be due to the different 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 concentrations were observed during the initial stages of bud development, endogenous ABA could then be a factor influencing bud development and flowering in autumn tree peonies. This is because morphological developments of flower bud occur during the initial stages. Normal structures of flowering development begin at this stage, which, to a large extent, determines flower-related abortion rates in tree peonies (Cheng, 2001). It implies that this has the overall effect of low ABA concentration in autumn tree peonies, which in turn facilitates autumn flowering.
The high ABA content at initial growth stages in spring peonies is not entirely surprising. This is because plant buds are dormant or just emerging from winter dormancy; a condition that is reportedly associated with high ABA content (Dunstone, 1988; Djilianov et al., 1994; Kim et al., 1994; Yamazaki et al., 1995; 1999a, 1999b, 2002; Bhargava, 1997). As spring plants began experiencing warmer temperature (April-May) (Fig. 1), ABA level in bud decreased (also see Barros & Neill, 1988) and showed similar pattern as that of autumn plants. This suggests that a correlation exists between temperature and ABA level that probably influences dormancy release. This in turn influence bud breaking and flower bud development. Furthermore, ABA biosynthesis/catabolism or import/export could be affected by autumn temperature conditions, especially at early developmental stages (late Aug.-early Sept.) of tree peony bud. These results are consistent with those of Altman & Goren (1972), who noted that ABA completely inhibits summer bud sprouting. Our results are, however, inconsistent with those of Southwick & Davenport (1987b), who noted that endogenous ABA could not be a major player in flower bud formation under drought/chilling conditions. ABA inhibition effect on autumn tree peony flowering 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 leaf emergence (stage 3) and blooming (stage 8) stages in spring peony. For autumn peony, three peaks, coinciding with bud swelling (stage 1), leaf development (stage 4) and flower bud opening (stage 7) stages, were observed (Fig. 3). Autumn peonies have triple peaks for IAA, apparently coinciding with relatively low level IAA stages in spring peony. This implies that endogenous IAA could be influenced by autumn temperature conditions. The results suggest possible correlation among endogenous IAA and flower bud formation, bud outgrowth and flower bud anthesis in tree peonies. This in turn influences 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 (Pilate et al., 1989; Gocal et al., 1991; Koshita & Takahara, 2004; Liu et al., 2008).
IAA was a major growth-promoting hormone in autumn tree peony plants. IAA levels at cell division and elongation stage (an active stage of plant development) were high. This suggests that IAA is a key factor that induces and promotes cell division and elongation in autumn peony plants. The high IAA content at these stages is not entirely surprising because it is related with high nutrient availability (Koutinas et al., 2010). Nutrients are needed developmental, physiological and metabolic elements by all varieties of plants (Hartmann et al., 1966; Priestly, 1977). This is important because early/rapid development of primordial/young leaves is a necessary condition for flower bud initiation. For spring tree peonies, IAA only peaks at leaf emergence and blooming stages of the plant development.
It is interesting to note that trends in IAA for spring and autumn peonies were inversely correlated for almost all the growth stages. IAA levels in autumn increases at the same stages where a decrease is observed in spring. In spring, low IAA coincided with high levels of GA3 and high ABA levels. In autumn, high IAA levels coincided with low levels of both ABA and GA3. It then suggests that increase in IAA leads to decrease in GA3 and ABA in autumn peony plants (see Figs.2, 3 & 4). There was apparently an inverse functional relation between the hormones. High IAA/GA3 and IAA/ABA apparently influences autumn flowering.
The difference in the levels of GA3 in spring and autumn tree peony buds is statistically significant at p<0.01. For spring tree peony buds, peak GA3 content coincided with bud swelling stage (stage 1). Peak GA3 content, however, coincides with full leaf emergence stage (stage 3) in autumn tree peony buds (Fig. 4). For the rest of the other developmental stages, bud GA3 trends were similar for spring and autumn peonies. However, autumn GA3 values were generally slightly higher than spring values. Interestingly, GA3 levels in spring tree peonies were generally low for all the stages with high autumn GA3 values. Furthermore, high autumn GA3 coincided with the stage of full leaf and flower bud emergence (i.e., when flower buds are partially/fully visible). This suggests that high GA3 induces flower bud formation in autumn tree peonies. Although earlier studies suggest inhibitory (Davenport, 1983; Su et al., 1997; Koshita et al., 1999; Cao et al., 2000, 2001; An et al., 2008; Prat et al., 2008) or no (Garner & Armitage, 1996) effects, our study shows that GA3 is related with flower bud formation. Flower bud formation in apples and pears has been attributed to the GA3 and therefore the absence of GA3 leads to no flower bud formation in alternate cultivars of the plants (Luckwill, 1974, 1980). Other studies have also attributed flowering to high GA3 (Vlahos, 1991; Rebers et al., 1994; wijayanti et al., 1997; Nadia et al., 2006; 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).
There were changes in carbohydrate composition in peony bud tissues. In terms of sucrose, reducing sugars and starch, autumn peony bud levels increased with decreasing spring levels. Also for autumn tree peonies, sucrose and reducing sugars increased with decreasing starch. For the different developmental stages, autumn carbohydrate concentration increases with decreasing spring concentration. Peony bud tissue's sucrose and reducing sugar concentration was high in the initial stages of growth in spring than in autumn. It leveled out as buds sprout (stage 2) and shoots emerge (stage 3). For all the other stages, sucrose and reducing sugar concentrations were inversely related. The differences in concentration are highest at stage 7, which is the flower opening stage (see Figs. 5, 6 & 7).
Different temperature regimes could influence plant sugar concentration. With decreasing starch levels, the levels of sucrose and reducing sugars increased in autumn tree peonies (also see Ashworth et al., 1993; Mohammadkhani & Heidari. 2008). Increasing sugar concentration could be driven by starch degradation (Fischer & Höll, 1991). It suggests that starch greatly influences sugar accumulation in plants. Similar trends were observed for sucrose, reducing sugars and starch in spring tree peonies. It implies that spring temperatures enhance the accumulation of sucrose, reducing sugars and starch. The trend of accumulation, from the first to the last developmental stage, follows a rise-fall pattern. On the contrary, autumn temperatures induce steady accumulation of sucrose and reducing sugars, and degradation of starch.
Irrespective of the discrepancies in the trends of carbohydrate between spring and autumn tree peonies, there are similar levels of sucrose and reducing sugars at bud sprouting and leaf emergence stages (stages 2 and 3) and also of starch at bud sprouting stage (stage 2) (see Figs. 5, 6 & 7) . This suggests that carbohydrate hydrolysis is a key factor of bud sprouting and shoot development. The consistent inverse relationship between sucrose/reducing sugars and starch in autumn peonies suggests that autumn temperature regime had influence on them and could be the key driver of autumn flowering. The level of starch in autumn plants decreases with flourishing new vegetative parts, possibly due to the utilization of stored carbohydrate reserves to support shoot/flower bud development. Starch levels further declined until flower bud formation, reaching its minimum at this stage (Fig. 7). This observation is a par with that of Liu et al. (2008), who concluded that sugar appears to be irrelevant in abnormal chestnut flowering.
For both seasons, plant sucrose was apparently the main sugar, followed by reducing sugars and starch. Most of the findings of the study are consistent with those of Mitthiesen & Stoller (1978), Ashworth et al, (1993), Spencer et al. (2001), and Mohammadkhani & Heidari (2008). This suggests that sucrose is the main carbohydrate in tree peonies. It provides energy and carbon skeleton needs in the synthesis of compounds such as amino acids, lipids and metabolites.
This study has analyzed trends in endogenous hormones (IAA, GA3 and ABA) and carbohydrates (sucrose, reducing sugars and starch) at different developmental stages of spring and autumn tree peony plants. The study shows that endogenous hormones and sugars greatly influence autumn flowering. Autumn temperature conditions not only facilitate accumulation of IAA, GA3, sucrose and reducing sugars, but also degradation of ABA and starch. This in turn induces flowering in autumn tree peony plants. The results suggest that the developmental stages of autumn tree peony plants also greatly influence the accumulation of IAA, GA3, sucrose and reducing sugars. 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.
This work was funded by the Governments of Sierra Leone and Peoples Republic of China through the Chinese Scholarship Council (CSC) No. 2007694T10. We are grateful for the consistent technical support by Ms. Li.