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The levels of endogenous hormones [gibberellic acids (GA3), indole-3-acetic acid (IAA), cytokinin (CTK) and abscisic acid (ABA)] and carbohydrates (glucose, reducing sugar and starch) were quantified in buds of 'Ao Shuang' (autumn flowering - AFC) and 'Luoyang Hong' (non-autumn flowering - NAFC) cultivars, which manifested differences in their flowering pattern. After purifying the buds extracts, hormone and sugar levels were evaluated by high-performance liquid chromatography and spectrophotometer, respectively. The differences in flowering patterns were associated with differences in the levels of endogenous GA3, IAA, and CTK during different developmental stages. Whereas GA3 level in buds of AFC decreased in response to floral induction and initiation that of NAFC increased. Similarly, IAA level in buds of AFC increased during floral induction and initiation while that of NAFC decreased. However, the level of CTK in buds of AFC was higher during floral initiation and differentiation stages than that of NAFC. ABA, sucrose, reducing sugar and starch levels in both AFC and NAFC showed similar change pattern throughout the developmental stages. The results suggest that quantitative changes in endogenous GA3, IAA and CTK during floral formation stages may have a direct effect on the different flowering pattern in tree peony.
Key words: Autumn flowering, Flower formation, Hormones, Sugars, Tree peony
Tree peony (Paeonia suffruticosa) is extensively cultivated in gardens for its beautiful flowers, pleasant fragrance and splendid color and also for cultural reasons in China. It has a long reputation and gardeners today, in Asia, North America, Europe and Australia regard tree peony as one of the most rewarding plant to grow (Cheng, 2007).
The phenomenon of autumn flowering is commonly observed in many horticultural plants, but is rare in tree peony. Recently, some cultivars of tree peony have exhibited the habit of twice flowering in a year corresponding with spring and autumn seasons in China. Such pattern has been perceived as deviation from the normal, where tree peony blooms without going through a cold period. Although there are abundant species and cultivars of tree peony, autumn flowering cultivars are largely lacking in China. Therefore, to enrich the tree peony resources, research must focus on utilizing twice flowering cultivars to improve production and meet up with market demands.
Normally, the flower buds of tree peony start to develop on current season's growth in later summer, followed by leaf senescence and bud dormancy. Bud development then only resumes after exposure to sufficient cold and then blossoms in spring (Wang et al., 1998). However, tree peony cultivars like 'Ao-Shuang' (Cheng and Zhao, 2008) do not follow such phonological sequence instead blossom in autumn without cold vernalization, the period during which other cultivars enter into winter dormancy. Developed buds of both 'Ao Shuang' and even 'Cangzhi Hong' cultivars of tree peony when treated with exogenous GA3 (500 mg/L per dose) by our research group (unpublished data) manifested autumn flowering without cold spell, a condition uncommon to other peony cultivars such as 'Luoyang Hong'. This makes them very unique cultivars with competitive values in tree peony production and provides an opportunity for a comparative study on physiological factors like hormones and nutrients to understand the principle underlying differences in flowering pattern of this plant.
Flower formation in plants involves three stages namely, flower induction, initiation and differentiation (Wilkie et al., 2008). Flower induction is the transition of meristem development from vegetative to generative growth, resulting to flower initiation. The morphological signature of this phase is the broadening of the shoot apex, which starts within one month after full bloom in tree peony (Wang et al., 1998). Flower initiation describes the period when various histological changes, morphologically characterized as doming of the apical meristem, occur (Barzilay et al., 2002).
Like many other temperate perennial woody plants, the flower buds of tree peony complete a change from vegetative to generative meristem in July to August each year after full bloom (Wang et al., 1998). Study has also shown that initiation of flower parts begins in the renewal bud in June in herbaceous peony (Barzilay et al., 2002). Apex evocation from vegetative to flower buds is a complex process regulated by many factors including endogenous hormones (Koshita et al., 1999) and carbohydrates (Pallardy, 2008). These factors are known to greatly influence the differentiation and development of cells, tissues, and organs. Nevertheless, the effect of these hormones on flower formation varies even within the same plant species. For instance, work on chestnut (Liu et al., 2008) concluded that ABA influence on flowering varies even within a single species. Furthermore, Bernier et al. (1981) suggested that the effect of ABA on flowering is not only diverse but also species-dependent.
Although endogenous hormones have been associated with autumn flowering in tree peony (Zhang, 2004), little information exists on their quantitative changes with regards to autumn flowering in tree peony. Therefore, there is a need to assess the relationships between hormone and carbohydrate levels, and cultivar variations in flowering pattern. This could help to establish 'out-of-season' re-blooming technology (forcing culture) with controlled flowering at specific marketing dates and times of the year.
Since much work hardly exists on the dynamics of endogenous hormones and carbohydrates, and flower formation, we investigated the difference in endogenous hormone and carbohydrate levels, and flowering patterns between AFC and NAFC during flower formation period.
Materials and Methods
The experiment was conducted in two consecutive cropping seasons (2009 and 2010) on 5-year old autumn- (Ao Shuang) and non autumn- (Louyang Hong) flowering tree peony cultivars. Twenty plant samples (10 for each cultivar grown under the same field conditions) were chosen for comparison. The experiment was conducted at the Jiufeng Peony Collection Center of Beijing Forestry University in China.
Plant treatments and Sampling
Prior to sample collection, agronomic practices like earthening, fertilization and pruning were conducted. Samples were collected in June for induction; July for initiation; and in August - September for differentiation during the 2009 and 2010 cropping seasons. At least 5 apical buds were collected as sample materials, three times in each case, with a week interval targeting induction, initiation and differentiation periods as described by Wang et al. (1998). Clean secateurs was used to harvest the samples and then rinsed in distilled water. The collected bud samples were immediately placed in an ice box, transported to the laboratory, dipped in liquid nitrogen and stored at -80OC until plant hormone and sugar extraction and analysis.
Hormonal extraction and analysis
The extraction of endogenous hormones (GA3, IAA, ABA and CTK) was conducted as described by Chen et al. (1991), with only slight modifications. Fresh bud samples (0.5 g) were ground in 10 ml of 80% cold methanol extraction medium containing 1 mmol.L-1 butylated hydroxytoluence (BHT) as antioxidant until completely homogenous. The homogenate was then transferred into a test tube and about 30 mg of polyvinylpolypyrrolidone (PVP) was added then thoroughly mixed on a shaker for 10 min and incubated at 4°C overnight. The next morning, the supernatant was put into 10ml tube and centrifuged at 6,000 rpm for 20 min. The residue was washed and re-extracted in 2 ml of cold methanol for another 12 hrs, then centrifuged under the same conditions as above before discarding the residue. The combined extract, after adding 1-2 drops of ammonia (NH3), was condensed (35-40°C) to an aqueous phase using a rotary evaporator. The aqueous phase was then dissolved using distilled water and the mixture separated into two equal parts.
For the determination of GA3, IAA and ABA, the pH of one part of the mixture was adjusted to 2.5-3.0 with 1 N HCl and then extracted three times in equal volumes of ethyl acetate (HAc). The pH of other part of the mixture was adjusted to 7.5-8.0 with 1 N NH3 for the determination of CTK and also partitioned three times in equal volumes of butanol in phosphate buffer (pH 8.0). Both the ethyl acetate and butanol fractions were evaporated to dryness at 40°C and 60°C, respectively. Hormones purification was performed with 80% aqueous methanol and passage through a C18 Sep-Pak cartridge (Waters Corp., Milford, MA). The residues were collected, dissolved in methanol and dried under N2 gas. The purified extracts were dissolved in 50% methanol, filtered through a 45 µl membrane and submitted to high performance liquid chromatography (HPLC). Hormonal analysis was done on a computer-assisted Agilent HP 1100 (Agilent Technologies, CA, USA) equipped with a vacuum degasser, an auto-sampler, a quandary pump, thermostated column compartment and a diode array detector. The conditions of the HPLC were as follow: ZORBAX RX-C8 column (250 x 4.6 mm); mobile phase consisted of 3% methanol and 97% 0.1 M acetic acid for IAA, GA3 and ABA determination, and 3% ammonia and 97% pure water (pH 7.0) for CTK determinations (after filtration through a 0.45µm filter membrane); detection wavelength of different hormones (IAA = 280 nm, ABA = 260 nm, GA3 = 210 nm, CTK = 260 nm); sample quantity of 10 µl was automatically injected at a flow rate of 1 ml min-1. Hormones were quantified by comparing the peak area of the samples with those of standard samples (Sigma Chemical Co. USA).
Soluble sugar extraction and analysis
Fresh bud 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 at 6,000 rpm for 10 min. The supernatant and extractant residues were collected and used to respectively determine sucrose/reducing sugars and starch. The reducing sugars were calorimetrically determined using dinitrosalicylic acid. Sucrose and starch were determined by anthrone reagent method with glucose as the standard, using the colorimetry anthrone method as modified for the determination of non-reducing sugars (Xue and Xia, 1985). The absorbance was then determined by spectrophotometer (TU-1901).
Statistical analyses were done using the Statistical Package for Social Scientists (SPSS). The mean values of the targeted hormones and carbohydrates were taken and one-way ANOVA analysis executed to determine the treatments at p<0.05 significance level.
Morphology of tree peony bud
Buds on shoots of tree peony were divided according to their location in terminal, lateral and adventitious buds. Under normal conditions terminal and lateral buds formed on annual new growth produce flower. Tree peony has a single bud for leaf, shoot and flower production. The result showed that on 5 June, the growing points of apical buds of AFC and NAFC were small (0.5 x 0.3 cm), narrow and flat or conical. The original meristematic cells were small and well organized. By 14 July and 21 July, the apical buds became semi-circular in AFC and NAFC respectively, indicating the end of flower induction phase and the start floral initiation process. On 12 and 19 August, the apical buds for AFC became broader and dome-shaped and all the buds observed had begun morphological differentiation, with some progressed to sepal primordium differentiation. By 26 August, buds of NAFC became dome-shaped with 43% of the buds examined had begun sepal primordium differentiation whilst all those of AFC had begun petal and stamen primordium differentiation. Petal and stamen primordium differentiation in NAFC was observed on 9 September (data not shown).
Life cycle comparison between autumn flowering and non-autumn flowering tree peony
Two years of careful observation showed that AFC had a relative shorter induction-differentiation period and flower twice annually. After flowering in spring season (March- May), AFC completed induction-differentiation processes during August and flowered again in late September or early October. Comparatively, NAFC had longer flower induction-differentiation period. At the time of complete floral differentiation process in AFC that of NAFC was still in progress.
The data of this study showed marked difference in GA3 levels between AFC and NAFC during flower formation phases (Figure 1A). GA3 level in AFC was generally lower than in NAFC and the degree of difference was significant (p≤0.05) during induction and initiation periods. Like GA3, IAA level also exhibited significant difference (p≤0.05) between AFP and NAFP during induction and initiation periods (Figure 1B). Comparatively, IAA level was higher in AFC than in NAFC during induction and initiation periods. The level of CTK increased significantly in AFC during flower initiation and differentiation periods from that of NAFC. However, there existed no significant difference (p≤0.05) in the levels of ABA between AFC and NAFC. Sucrose, reducing sugar and starch also manifested similar change patterns throughout the different developmental stages in both cultivars (Figure 2A, B, C).
Life rhythm of autumn flowering and non autumn flowering cultivar
The results obtained during two years of observation permitted to identify June as the beginning of flower induction in tree peony. Within a month after full bloom, induction began in both cultivars. But marked difference in induction-differentiation period was observed. Whereas induction-differentiation period was shorter in AFC that in NAFC was longer. Thus, AFC flowered twice per year (March-May and September to October). By the time induction-differentiation was completed in NAFC, dormancy period had already set in and hence flowered once in the next spring (March-May) (see Table 1).
Effects of hormones on flower formation
Plants flowering processes are generally influenced by the interactions of endogenous hormones in plant tissues. Hormones regulate various aspects of plant development including cell division, growth, morphogenesis and flowering (Pallardy, 2008; Wilkie et al., 2008). In this study, GA3 levels showed significant increase difference between AFC and NAFC during flower formation phases (Figure 1A). GA3 levels during flower induction and initiation periods were lower in AFC than in NAFC. Low GA3 level in AFC coincided with the period of floral stimulus response by the apical meristem, induction of flower bud formation, start of flower organ development (sepal, petal, stamen and pistil primordial) and floral evocation from the vegetative to floral stage (Barzilay et al., 2002; Koutinas et al., 2010). Because endogenous GA3 level was lower during floral induction and initiation periods suggests that low GA3 could be an important requirement for floral formation in AFC. Low level of GA3 in buds during floral induction and initiation stages possibly resulted to rapid floral organs development thereby shortening the flower induction-differentiation life cycle of AFC (Table 1) that subsequently induces autumn flowering. Interestingly, higher level of GA3 was observed during these stages of flower development in NAFP. High level of GA3 in NAFC buds during these periods apparently delayed events of flower formation and transition (Bertelsen et al., 2002; Wilkie et al., 2008), resulting to the loner induction-differentiation period (Table 1) which subsequently inhibited second flowering. The results of this study show potential correlation between low GA3 levels during induction and initiation, and flower formation in AFC, a phenomenon also reported by Chen (1990), Koshita et al. (1999) and Wilkie et al. (2008) on works with lychee, citrus and mango, respectively. Accordingly, high GA3 is noted to inhibit flower formation during induction and differentiation stages in olive (Ulger et al., 2004), peach (An et al., 2008), and also during the initiation of flower in avocado (Salazar-Garcia and Lovatt, 1998), peach (Garcia-Pallas et al., 2001) and sweet cherry (Lenahan et al., 2006).
Fruits and seeds are rich sources of GA3 and the rate of its export from seeds to buds influences flower initiation (Pharis and King, 1995). Being that the plants had just bloomed in spring and had developing fruits, high GA3 in NAFC may be attributed to its rapid diffusion out of developing fruits to the meristematic zone than out of those in AFC, to inhibit flower initiation (Pharis and King, 1995). GA may have, also, inhibited floral formation by limiting the development of nodal buds, bud appendages and sizes.
Like GA3, IAA level also exhibited difference in levels between AFC and NAFC. Comparatively, IAA level was higher in AFC than in NAFC during floral induction and initiation periods (Figure 1B). Higher level of IAA synchronized with stages of floral stimulus response by apical meristem, morphological development of floral organs, apical meristems transformation to new flower buds and active mitosis at the apex (Koutinas et al., 2010). However, during the same periods, lower IAA level was detected in NAFC. This suggests that high IAA concentration probably facilitated early events of flower induction and initiation processes, which in turn shortened the induction-differentiation period (Table 1), thereby inducing autumn flowering in AFC. The results of this study corroborate with findings on Arabidopsis thaliana (Reinhardt et al., 2000) and Coffea arabica (Schuch et al., 1994) who also suggested positive effect of high IAA level on flower primordial formation. However, our results disagree with those of……
Various studies have suggested that CTK controls shoot flower bud formation (Koshita et al., 1999; Prat et al., 2008), which determines the fate of flower bud development (Rasmussen et al., 2009). According to Chen (1991), endogenous CTK levels in buds increase at the onset of floral initiation and differentiation in lychee. In this study also, a significant increase in CTK levels in buds of AFC was observed during floral initiation and differentiation when compared with those of NAFC (Figure 1C). The period of increase in CTK level apparently coincided with the organogenesis and morphological differentiation of floral organs (Wang et al., 1998; Barzilay et al., 2002), which probably facilitated these processes thereby, resulting to early initiation and differentiation of flower organs in AFC. On this basis, CTK influence on flower formation is suggested in this study. Studies on Nicotiana tabacum (Werner et al., 2001) also suggest that CTK plays a critical regulatory role during meristem morphological development. It enlarges the meristem, which in turns greatly promote flower meristem formation. Increase in CTK level during initiation and differentiation phases is not surprising as it occurred during periods of possible active cell division (Wilkie et al., 2008; Koutinas et al., 2010). In fact, CTK is reportedly associated with stimulating cell division (Pallardy, 2008). Since CTK activates cell division, possibly it contributed to initiate early flower formation and completion of induction-differentiation cycle before the on set of dormancy in AFC (Table 1).
The level of ABA in both cultivars manifested similar change patterns at the different developmental stages. Since no significant increase in the levels of ABA existed between AFC and NAFC, it implies that ABA probably has insignificant influence on flower formation to cause differences in flowering pattern of tree peony. Thus, the perceived concept of dormancy causing differences in flowering pattern in tree peony was not confirmed in this study. The results here suggest that ABA is generally unassociated with flower formation in tree peony but with dormancy induction, seed development and germination, and water stress (Walton, 1980).
Effects of carbohydrates on flower formation
Carbohydrate status is implicated in floral induction of woody trees by numerous studies. For instance, Stutte and Martin (1986) and Liu et al. (2008) respectively suggested that sugar and starch increase has no effect on flower initiation and flowering in olive and abnormal chestnut plants. In this study, irrespective of the cultivar differences, no significant increase difference was observed in carbohydrate levels between AFC and NAFC. Sucrose, reducing sugar (glucose and fructose) and starch levels in both AFC and NAFC showed similar pattern throughout the three developmental stages (Figure 2A, B, C). Since our data showed no difference in sugar levels between AFC and NAFC, we suggest that carbohydrates are not directly responsible for differences in flowering pattern of tree peony. However, our results are inconsistent with those reported on citrus (Goldschmidt and Golomb, 1982) and lychee (Menzel et al., 1995) which concluded positive effect of elevated carbohydrate contents on flower initiation. In both investigated cultivars, sucrose was the major carbohydrate followed by reducing sugar and starch. Sucrose is generally the major energy providing and skeletal carbon needed in the synthesis of compounds (amino acids, lipids and metabolites) for plant growth (Pallardy, 2008).
Our result showed different pattern in changes in the levels of endogenous hormones between AFC and NAFC with respect to developmental stages. The change in levels of GA3, IAA and CTK may contribute to the difference in flowering patterns between AFC and NAFC. ABA and sugars appeared to have no effect on difference in flowering patterns of tree peony. These findings suggest that probably the quantitative changes in endogenous GA3, IAA and CTK levels may correspond to different flowering patterns in tree peony. Therefore, autumn flowering in tree peony is attained by regulating GA3, IAA and CTK levels at different developmental stages to facilitate the completion of flower formation processes before dormancy sets in. The data of this study will be useful in the flower industry to establish re-flowering technology in 'out-of-season' with controlled flowering at specific marketing dates and times of the year (forcing culture) by applying exogenous plant growth regulators.
This work was supported by the National Science and Technology Support Program of China (2006BAD01A1801), Key Project for Forestry Science Research (2006-40), and Co-constructive Project of Beijing Education Committee (2009).