Tree peony (Paeonia suffruticosa) is one of the most important ornamental plants grown in many parts of the world. The phenomenon of successive secondary flowering (autumn-flowering) is commonly observed in many horticultural plants including tree peony. Varying levels of hormones and sugars are thought to influence autumn-flowering tree peony. Therefore, the objective of this study was to evaluate the quantitative changes in the endogenous hormones and sugars levels in the bud samples of 'Ao Shuang' cultivar of tree peony during the flowering period of autumn in comparison with that of spring (2010). Endogenous hormones (IAA, ABA and GA3) and carbohydrates (sucrose, reducing sugars and starch) levels were determined for both seasons. Both hormone and carbohydrate levels showed significant different patterns in spring season plants from that in autumn season plants. Sucrose had the highest concentration in both seasons followed by reducing sugars and starch. Increase in sucrose and reducing sugar contents in autumn season plants was accompanied by decrease in starch concentration in autumn season plants. Whereas, similar fluctuating patterns was observed for those of spring season plants. High concentrations of IAA, GA3 and low contents of ABA were observed in autumn season tree peony and may have a positive effect on the flowering process. Thus, the results suggest that quantitative changes in endogenous hormones and carbohydrates may have a direct influence on the flowering mechanism of autumn flowering tree peony.
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The tree peony (Paeonia suffruticosa), native to China, and one of the most magnificent, most beautiful, and most interesting of all plants (Wister, 1995), has come into its own as an important garden plant in many countries of Asia, America, Europe and Australia. It is an excellent ornamental plant that has been playing an important social, economic and medicinal role in the lives of the Chinese people. Respected as "the King of flowers" in China with a special cultural symbol of peace and happiness, prosperity and development, power and wealth over many other common plants, the tree peony has been extensively cultivated in China. Although having abundant species and cultivars of tree peony, China is rare in successive secondary flowering cultivars that flower twice or more in the same year. If these rare resources are utilized by forcing them to re-flower in autumn will bring benefits in the floriculture industry.
The development of flowering controlling mechanism in tree peony cultivation is very important because the plant growing in the field flowers only once a year with short flowering period. Due to the high demand and high price value associated with this plant, this phenomenon is undesirable for growers. In a production system of tree peony with at least 3-5 years before producing flowers, with a single bud for both leaves and flower production and where each planting is normally harvested only once a year, plant quality as well as flower quantity is of overriding importance for economic reasons and optimal strategies should be ensured in order to obtain maximum yield. This creates opportunities to probe into the flowering physiology of the plant under cultivation in order to enhance its production. Understanding the physiological mechanism of inducing successive secondary flowering in tree peony will be indispensable in horticultural industry.
Several physiological changes occur in the shoot apex that specifically commits the apical meristem to produce flowers. The developmental changes that bring about flowering include endogenous factors, such as sugar changes, genes and hormones, and external factors, such as day length and temperature. Therefore, flowering, which represents the expression of reproductive phase, often depends on specific environmental and internal developmental signal. The transition from juvenile to adult phase often proceeds in response to environmental factors. These factors exert inductive effect by consequently causing changes in sugar and hormone levels (Zeevart, 1979). This phase change is a critical stage in plant reproductive development because it can be affected by changes in hormone and sugar levels consequently leading to seed and flower abortion and fruit abscission.
Tree peony naturally experiences a period of cool temperature before the buds grow out to produce shoots and flowers. Usually, flower bud initiation begins in the perennial 'crown' in later summer, as the leaves begin to senesce, then flower bud development continues till the plant enters dormancy (Byrne and Halevy, 1986). These buds will hardly develop further until a period of cool temperature has been experienced (Byrne and Halevy, 1986; Fulton et al., 2001; Halevy et al., 2002; Kamenetsky et al., 2003). As long as sufficient period of cold temperatures have been accumulated to break dormancy, then buds grow out to produce shoots and flowers during the warmer temperatures of spring (Kamenetsky et al., 2003). This phenomenon is different for some peony cultivar such as 'Ao Shuang' which can also bloom in summer in the absence of cool temperature.
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Peony, like many other flowering plants requires hormones for the growth and normal maintenance of their physiological and biochemical processes. As organic substances that regulate plant's growth, hormones play a central role in several plant developmental processes and promote a number of desirable effects such as embryogenesis, lateral root development, vascular differentiation, apical dominance, climate responses, and flower development (Friml, 2003; Katia and Gilberto, 2004; Ana et al., 2004 cited by Liu et al., 2008). As a result, hormones are believed to strongly participate in flowering mechanism in most perennial plants including in tree peony.
Nevertheless, the effect of these hormones varies with environmental conditions at different period during the season (Koshita et al, 1999). For instance, according to Altman and Goren (1972), IAA delays summer bud sprouting, while GA enhances it and ABA completely inhibit it. Of recent time, cytokinin profiles in different plant organs have been reported to differ consistently with seasons. The level of cytokinin has been reported to be minimum in mid-June and maximum in late summer in apical buds of Abies nordmanniana. Subapical buds showed the same June minimum but peaked in mid autumn at a much lower level (Rasmussen et al., 2009). In addition, an inhibitory effect of exogenous IAA and ABA, and endogenous IAA were reported in Pharbitis nil by wijayanti et al (1997) in which exogenous GA induce flowering.
Besides hormones, plant growth and development are also influenced by nutrient availability. Numerous studies have shown that sucrose, glucose and fructose are the major assimilate required by most plants. Sucrose is the main carbohydrate transported in the plants that provides energy and carbon skeletons needed for the synthesis of compounds like amino acids, lipid and secondary metabolites. It also plays a major role in plant metabolism as an important storage sugar and the main form of reduced carbon translocated from leaves (source) to developing growth centre (sink) in plant (Heldt and Heldt 2005).
Flowering is usually associated with sugar changes. The alteration in the availability of soluble sugars, such as sucrose, can help regulate many different processes. Parker (1962); Li et al (1965); Sauter and Kloth (1987); Nelson and Dickson (1981) have reported sugars accumulation in woody plant tissues in response to seasonal variation. For instance, maximum sugar content in winter has been reported and as plants deacclimate in the spring, a decrease in sugar content was observed (Parker 1962; Nelson and Dickson, 1981; Fege and Brown, 1984; Bonicel et al., 1987).
Research on the relationship between hormonal changes and floral bud development may be of importance for the formulation of protocol for a year-round flowering programme in tree peony. Although few research have reported on the autumn flowering mechanism of tree peony (Jiang et al., 2007; Li, 1998; Xiao et al, 2001), changes in endogenous hormones has not been documented and no studies have included a comparison of endogenous hormones and sugars between spring season and autumn season flowering tree peony species. Therefore, this research was conducted to determining the dynamic changes in endogenous hormones and sugar levels in flowering mechanism between spring-flowering tree peony that flowers after going through cold period and autumn-flowering tree peony that grows on the same field but flowers in the absence of cold period during the year. Information from this research will help in setting up a protocol by which re-blooming ('out-of-season' technology) can be enhanced and hence scheduling of the crop in flowering for specific marketing dates at different times of the year will be promoted.
Materials and methods
The experiment was conducted at the Beijing Forestry University nursery, Jiu Feng during the spring (2010) in comparison to the same cultivar naturally grown in the field in summer of 2010. Eighteen plants of 5 years old tree peony, Ao Shuang cv. with similar growth vigor were chosen for investigation in February 2010 and August 2010 for the spring and summer season, respectively.
Prior to sample collection, agronomic practices such as earthening, fertilization, watering, pruning of feeble branches etc were carried out. Leaves defoliation was manually exercised in late August before dormancy-releasing treatment. The dormancy-releasing agent used was GA3 at 500 ppm applied at one dose on developed buds using a painting brush. A stainless clean 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 and upon taken to the laboratory, these samples were immediately dipped in liquid nitrogen (N2) and stored at -80OC until plant hormone extraction and analysis.
Sampling for plant hormone analysis
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Bud samples were collected before and after leaf defoliation and then after GA3 application. Sample collection after GA3 treatment, targeted bud developmental stages I, II, III, IV, V, VI, VII and VIII according to Cheng et al. (2001).
Hormonal extraction and analysis
The extraction of endogenous hormones was conducted as described by Chen (1991), with slight modifications. The quantity of endogenous levels of ABA, GA3 and IAA was determined using fresh tissue (0.5g) of the bud. The plant tissue was grind with antioxidant (copper) and 10 ml of 80% cool methanol until it becomes homogenate and transferred into a test tube. Small amount of PVP was added into the homogenate, then spin the mixture on shaker for 10 min. and incubated at 4 OC overnight. The supernatant was transferred into 10ml tube the next morning and spin at 6000 rpm for 20 min. The residue was washed and re-extracted twice more with 2 ml of cold methanol for another 12 hours, and centrifuged under the same conditions as mentioned above and discarded the dust. The combine extracts, up on adding 2-3 drops of NH3, were evaporated (35-40OC) to the aqueous phase in a rotary evaporator. The aqueous phase was then dissolved by adding some distilled H2O followed by the separation of the mixture into two equal parts. One part of the mixture was adjusted to pH 2.5-3.0 with 1N HCl and then extracted three times with equal volumes of ethyl acetate. The combine ethyl acetate fraction was evaporated to dryness. The dry part was diluted with 1ml of 3% methanol and 97% 0.1 M HAc for the determination of acidic hormones such as IAA, GA3 and ABA.
Determination of hormone
Hormonal determination was carried out by using the HPLC method, Agilent 1100 chromatography, C18 tube (250*4.6 mm) matrix contest. 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 speed flood is 1 ml/min, contest with "outer-standard method" and the standard sample used is Sigma production.
Soluble sugar extraction and analysis
Fresh leaf material (1g) was grind in 20 ml of distil water and extracted in water bath at 80OC for 30 min. The suspension was centrifuged for 10 min. at 6000 rpm. The supernatant and extracted residues were collected for the determination of sucrose and reducing sugars, and starch, respectively. Reducing sugars was determined calorimetrically using dinitrosalicylic acid; sucrose and starch was determined by anthrone reagent method with glucose as a standard using the colorimetry of anthrone method as modified for determination of non-reducing sugars (Xue and Xia, 1985). The absorbance was determined by spectrophotometer.
All the statistical analyses were conducted using Statistical Package for social Sciences (SPSS). The mean values of the various targeted hormones and carbohydrates were taken and a one-way ANOVA was used to determine the significant between treatments at P < 0.05 among the treatments.
Hormonal contents in the buds
A seasonal comparison of GA3 level in buds is presented in fig. 1. GA3 contents in both spring and autumn plants showed a peak each, corresponding to bud swelling stage and shoot development stage, respectively. After that, in each case, the trend of changes in GA3 levels showed similar pattern for both season plants with slight increase observed at stage 8 for autumn season plants.
IAA levels in the buds of spring season plants showed two peaks corresponding to leaf emerging (stage 3) and blooming (stage 8) stages, whereas IAA levels of autumn season plant showed three peaks corresponding to bud swelling (stage 1), shoot developing (stage 4) and flower bud anthesis (stage 7) stages. Interestingly, the high levels of IAA in autumn season plants occurred at the stages where it is low in the spring season plants.
There was a difference in the ABA content between the spring season plants and the autumn plants at the initial stages of growth development. After that there was no seasonal difference in the ABA content between the two plants. The ABA content exhibited a high level at the initial stages of development in spring plants than in autumn plants but dropped sharply at stage 3 and maintained similar trend as that of the autumn plants throughout the growth stages, with a slight increase at growth stage 5 in autumn plants.
Carbohydrate contents in the buds
A quantitative difference in the change patterns among developmental stages in contents of carbohydrate were observed between spring and autumn season plants (Fig 4, 5, 6). Sucrose and reducing sugar levels in spring season plants showed initial increase, then fall as buds sprout (stage 2) and shoot emerges (stage 3), rise again as flower buds starts to develop (stages 4 & 5), then fall again as flower buds open with a sharp rise at blooming. Whereas that of autumn season plants showed an initial decrease then gradually increase as buds sprout and maintain a stable change patterns throughout the investigation period (Fig. 3 & 4). Moreover, starch in spring season buds showed initial decrease then after that showed similar change patterns as that of sucrose and reducing sugars of spring season plants (Fig. 6). For autumn season plants, starch content showed an initial increase then subsequently reduced with the flush of new vegetative growth and decline until flower bud formation, at which time starch reached seasonal lows (stages 3, 4 & 5) (Fig 6). Under both temperature regimes, sucrose proved to be the major sugar followed by reducing sugars and starch. There were no consistent differences in change patterns of sucrose, reducing sugars and starch in spring season plants whereas in autumn season plants, a consistent differences in change pattern among the stages was observed between sucrose and reducing sugar on one hand and starch on the other hand (Fig. 4, 5, & 6).
Plants growth and development is based on the production of cells in the meristems and the ensuing elongation of these newly formed cells (Clark, 1997; Cosgrove, 1997). Plant hormones are known to affect both cell division and cell elongation. Therefore, there is a possibility that hormones regulate cell division and cell growth in plants, directly or indirectly (Zeevart, 1976; Friml, 2003; Katia and Gilberto, 2004). Our result showed a significant different pattern in the concentrations of endogenous ABA at the initial stages of growth (b4 - stage 3) (fig. 1) in spring season plants from that of the autumn season plants. After that, ABA concentrations showed similar change pattern at the remaining stages in both season plants. It is possible that the different pattern was due to the opposite temperature regimes experienced at the initial stages of growth. At the time of significance different level of ABA, temperature for spring season plants was very low (February-March) and that for autumn season plants was high (August-September). Because different levels of ABA concentration were observed during the initial stages of bud development, the stages at which morphological establishment of the flower bud occurs, where normal structures and development of flowering are set up while abortion associated with flowering rate is determined to a larger extent in tree peony (Cheng, 2001), it is possible that endogenous ABA might be one of the key factor responsible for bud development and subsequent flowering in autumn season tree peony. Meaning that, the low concentration of ABA in autumn season tree peony probably resulted in autumn flowering. The high level of ABA content at the initial stages of growth in the spring season plants is not surprising as the plant buds were dormant or had just been released from winter dormancy and dormancy has been reported to be associated with high level of ABA (Dunstone, 1988; Djilianov et al., 1994; Kim et al., 1994; Yamazaki et al., 1995; 1999a, b, 2002 Bhargava 1997). This result suggests that the biosynthesis/catabolism or import/export of ABA is affected by autumn season condition especially at the early stages (August-September) of bud development of tree peony. Our results are consistent with the results by Altman and Goren (1972) which suggested that ABA completely inhibit summer bud sprouting but are inconsistent with the results of Southwick and Davenport (1987b) which concluded that endogenous ABA might not play a major role in flower bud formation that was controlled by drought and chilling treatments. The inhibitory effect of ABA in the autumn season tree peony also agrees with Wijiayanti et al (1997) and Marumo et al (1990). However, Harada et al (1971) and Nakayama and Hashimoto (1973) reported a promotive effect of ABA on flowering.
In this study, high contents of IAA was observed at two peaks synchronizing with leaf emergence (stage 3) and blooming (stage 8) stages in spring season plants whereas, three peaks coinciding with bud swelling (stage 1), leaf developing (stage 4) and flower bud opening (stage 7) stages was observed in autumn season plants (Fig. 2). Because the autumn season plants showed three peaks of IAA coinciding with stages at which the spring season plants showed a comparatively low level of IAA, it is possible that endogenous IAA might have been influenced by the autumn temperature condition. This result showed there may be a correlation between endogenous IAA and flower bud formation, bud outgrowth, and flower bud anthesis of tree peony and acted on flower bud development and its subsequent flowering. The significant increase in endogenous IAA level during bud outgrowth has also been suggested in other species of plants (Pilate et al., 1989; Gocal et al., 1991; Koshita and Takahara, 2004; Liu et al., 2008).
IAA proved to be the major growth-promotion hormone in the autumn season plants since it showed high levels at stages where cell division and cell elongation during plant development are active. This suggests that IAA plays the key role of inducing and promoting cell division and cell elongation in autumn season plants. The high content of IAA at these stages is not surprising as it is known for its high nutrient attraction ability (Koutinas et al., 2010) and that nutrients are highly needed for developmental, physiological and metabolic processes in plants (Hartmann et al., 1966; Priestly, 1977; Golomp and Goldschmidt, 1981). This is important since the early, fast development of the leaf primordial and of young leaves during the early growth stage, is a prerequisite of flower bud initiation. For the counterpart spring season plants, IAA peaks were only shown at leaf emergence and blooming stages.
It is surprising to note that the change patterns of IAA level in spring season plants and autumn season plants were opposite almost throughout the growth stages. The concentrations of IAA increased in autumn season plants at the same stages as a decrease in IAA concentrations in spring season plants was observed. The same change patterns were observed in the concentrations of IAA and GA3, and IAA and ABA in autumn season plants. It means that the raised IAA content was followed by a fall in the GA3 and ABA contents in the autumn season plants (compare Fig. 1, 2 & 3). Possibly, there may be an opposite functional relationship between these hormones and the maintenance of high IAA/GA3 and IAA/ABA has correlation with autumn flowering for which further study is suggested.
A significant different pattern in GA3 levels was observed in buds of autumn season plants from that of spring season plants. GA3 contents in buds of spring season plants showed a peak synchronizing with bud swelling stage (stage 1), whereas that of autumn season plants coincided with complete leaf emergence stage (stage 3) (figure 3). After that both maintained the same change trend with autumn season plants being the predominant concentrations. Interestingly, the level of GA3 in the spring season plants was low at the stages of high level in autumn season plants and vice-versa. The high level of GA3 in autumn season plants coincided with the stage at which leaf has completely emerged and flower buds can be partially or visibly seen in tree peony, suggesting that high levels of GA3 might have influence on flower bud formation in autumn tree peony plant. Although previous studies have suggested an inhibitory effect ( 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 effect (Garner and Armitage, 1996) of GA on flower bud formation, our results corroborate with the results of Luckwill (1974, 1980) which attributed flower bud formation in apple and pear to the presence of GA and that its absence was the fundamental factor for the lack of flower buds in alternately bearing cultivars of these plants. Studies by others have also attributed flowering to high level of GA (Vlahos, 1991; Rebers et al., 1994; wijayanti et al., 1997; Nadia et al., 2006; Liu et al., 2008), which may be attributed to the growth-promotion effect of GA in stimulating and accelerating cell division and cell elongation (Hartmann et al., 1990).
Carbohydrate composition changed in tree peony bud tissues. A seasonal interchange in the levels of sucrose, reducing sugar and starch between autumn season plants and spring season plants was observed. This interchanging pattern was also observed between sucrose and reducing sugars, and starch of autumn season plants. The concentrations of these carbohydrates in autumn season plants increased at the same stages as a decrease was observed for that of spring season plants. The concentrations of sucrose and reducing sugars in bud tissues were high at the initial growth stages in spring season plants than in autumn season plant but leveled as buds sprout (stage 2) and shoot emerges (stage 3) and then continued to be inversely related to each other throughout the investigation period with a marked difference at flower opening stage (stage 7) (Fig. 4, 5 & 6). Different temperature regimes experienced by both plants may have influenced the concentrations of these sugars. The decrease in starch levels was also accompanied by increased levels of sucrose and reducing sugars in autumn plants, a pattern that has also been reported by Ashworth et al (1993); Mohammadkhani and Heidari (2008).The increase in sugar concentrations may be as a result of the degradation of starch (Fischer and Höll, 1991), suggesting that starch may have an important impact on the accumulation of sugars in plant cells. In spring season plants, a similar fluctuating change patterns were observed in the concentrations of sucrose, reducing sugar and starch. This means that spring temperature condition induced accumulation of sucrose, reducing sugar and starch among developmental stages in a rise-and-fall pattern, whereas autumn temperature condition induced a stable accumulation and degradation of sucrose and reducing, and starch, respectively. Though difference in the change patterns of carbohydrates between spring and autumn season plants was observed, interestingly, the contents of sucrose, reducing sugar at bud sprouting and leaf emergence stages (stages 2 and 3) and that of starch at bud sprouting stage (stage 2) were equal in both season plants (Fig. 4, 5 & 6) . This suggests that carbohydrate hydrolysis is a key factor during bud sprouting and shoot development. The high and low stable interchanging level patterns in autumn season plants between sucrose and reducing sugar, and starch during the growth period as a result probably, of autumn temperature regimes might be responsible for autumn flowering in tree peony. Starch content in autumn season plants subsequently reduced with the flush of new vegetative growth, presumably as a result of the utilization of storage carbohydrate reserves to support shoot and flower bud development and further declined until flower bud formation at which time starch level reached seasonal minimum (Fig 6). Our results are inconsistence with Liu et al (2008) which concluded that sugar appeared to be irrelevant to flowering of abnormal chestnut.
In both season plants, sucrose proved to be the major sugar followed by reducing sugars and starch. Our results corroborate with Mitthiesen and Stoller (1978); Ashworth et al (1993); Spencer et al (2001); Mohammadkhani and Heidari (2008). This suggests that sucrose is the main carbohydrate transported in the plants that provides energy and carbon skeletons needed for the synthesis of compounds like amino acids, lipid and secondary metabolites.
In this study, different patterns in the changes in the endogenous hormones (IAA, GA3 and ABA) and carbohydrate (Sucrose, reducing sugars and starch) levels among the developmental stages were observed between spring and autumn season plants. Endogenous hormones and sugars have a major influence on the autumn-flowering. Autumn temperature conditions induced the accumulation of IAA, GA3, sucrose and reducing sugar and also the degradation of ABA and starch that subsequently induced flowering in autumn season plants of tree peony. The results suggest that autumn season plant's developmental stages has great influence on the accumulation of IAA, GA3, sucrose and reducing sugars. Furthermore, the quantitative changes in endogenous hormones and carbohydrates may be influenced by seasonal variation that may regulate different flowering processes.
This work was supported by the Government of Sierra Leone and the Government of PR China through the Chinese Scholarship Council (CSC) (No. 2007694T10). We greatly acknowledge ------ for her technical support.
Fig. 1. Comparison of ABA content between autumn flowering and spring flowering tree peony (ABA-S: ABA content in spring plants, ABA-A: ABA content in autumn plants, b4: before deleting leaves, S1-S8: Stage 1- 8).
Fig. 2. Comparison of IAA content between autumn flowering and spring flowering tree peony (IAA-S: IAA content in spring plants, IAA-A: IAA content in autumn plants, b4: before deleting leaves, S1-S8: Stage 1- 8).
Fig. 3. Comparison of GA3 content between autumn flowering and spring flowering tree peony (GA3-S: GA3 content in spring plants, GA3-A: GA3 content in autumn plants, b4: before deleting leaves, S1-S8: Stage 1- 8).
Fig. 4. Comparison of sucrose content between autumn flowering and spring flowering tree peony (GA3-S: GA3 content in spring plants, GA3-A: GA3 content in autumn plants, b4: before deleting leaves, S1-S8: Stage 1- 8).
Fig. 5. Comparison of reducing sugar content between spring flowering and autumn flowering tree peony (GA3-S: GA3 content in spring plants, GA3-A: GA3 content in autumn plants, b4: before deleting leaves, S1-S8: Stage 1- 8).
Fig. 6. Comparison of starch content between spring flowering and autumn flowering tree peony (GA3-S: GA3 content in spring plants, GA3-A: GA3 content in autumn plants, b4: before deleting leaves, S1-S8: Stage 1- 8).