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The phenomenon of autumn-flowering is commonly observed in many horticultural plants. Of recent times, 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 noted as deviation from the normal where tree peony blooms without going through a cold period. This has made these cultivars very unique plants with competitive values in tree peony flower industry being that they also flower in autumn when other cultivars still continue with flower differentiation processes and subsequently enter dormancy state in winter, hence flowering only once. Although China has abundant species and cultivars of tree peony, autumn-flowering peonies are very rare. This has offer the opportunity for a comparatively study on the hormonal levels on the flowering mechanism of the plant. In this way, the quantitative dynamics of GAs and cytokinins involved in the regulation of flowering processes can be studied.
Transition of apical meristem in tree peony complete a change from vegetative to generative meristem in July to August each year after full bloom (Wang et al., 1998). Studies have also shown that initiation of flower parts begins in the renewal bud in June of herbaceous peony (Barzilay et al., 2002). Unlike other tree peony cultivars with long differentiation periods, 'Ao Shuang' and 'Cangzhi Hong' have a relatively short induction-to-differentiation cycle though flower formation processes starts at the same period as in other tree peony cultivars (Table I).
Flower bud formation is normally attained through evocation of the vegetative meristem to a productive structure. During this process various changes such as broadening of the apex meristem and morphological development occurred. Hormones, however, have been long known to play a major role in flower formation in plants. Hormones regulate various aspects of plant development including cell division, growth, morphogenesis and flowering (Pallardy 2008; Wilkie et al. 2008). For instance, works on olive (Ulger et al. 2004), peach (An et al. 2008), avocado (Garcia-Pallas et al. 2001) and sweet cherry (Lenahan et al. 2006) have reported inhibitory effect of high GA3 content on flower formation during induction, initiation and differentiation. However, although inhibitory effect of high GA3 level on flower formation in olive has been reported, a positive effect of high GA4 on floral formation has also be confirmed (Ulger et al. 2004). Furthermore, endogenous CTK level in buds has been reported to increase at the onset of floral initiation and differentiation in lychee (Chen 1991).
Despite the above, little information exists on the quantitative changes of endogenous hormones with respect to autumn flowering in tree peony. Therefore, better understanding of the mechanism underlying hormonal action could be helpful to explore the physiological causes of cultivar variations with respect to flowering mechanism in tree peony. This could not only enrich production practices of tree peony cultivars but also help to establish re-flowering technology in 'out-of-season' with controlled flowering at specific marketing dates and times of the year.
Since there is little information on the dynamics of endogenous hormones in the control of flower formation, we assessed the levels of different kinds of GA and CTK in two cultivars of tree peony, which exhibited great variation in their flowering pattern, to determine their roles in autumn flowering. This will give an insight of what type of the mentioned hormones responsible for non-autumn flowering in tree peony.
2. Materials and methods
2.1 Sample collection
A total of 24 5-year-old plants, 'Ao Shuang', 'Cangzhi Hong' (autumn flowering) and 'Louyang Hong' (non-autumn flowering) cultivars of P. suffruticosa with similar growth vigor, were selected for investigation. Samples were collected in early June to late August of 2010 at Jiufeng Peony Collection Base of Beijing Forestry University, Beijing China. Buds were collected as samples, three times in each case, with a week interval targeting floral induction, initiation and differentiation periods as described by Wang et al. (1998). Specifically, samplings were conducted in June 5th, 12th, 19th for induction; July 7th, 14th and 21st for initiation; and in August 12th, 19th, 26th for differentiation during the 2010 seasons. Buds samples were collected using clean secateurs, rinsed with distilled water and then immediately placed in an ice box. The samples were transported to the laboratory, dipped in liquid nitrogen and stored at -80OC until plant hormone extraction and analysis.
2.2 Hormone extraction, purification and quantification
With slight modifications, the extraction, purification and determination of endogenous levels of GA3, GA4, ZR and iPA was conducted by indirect ELISA technique as described by He (1993) and Yang et al. (2001). Fresh bud samples (0.5 g) were homogenized in 5 ml of 80% (v/v) methanol extraction medium containing 1 mmol.L-1 butylated hydroxytoluence (BHT) as antioxidant. About 30 mg silicon dioxide and 20 mg of polyvinylpolypyrrolidone (PVP) were added to ease grinding and remove phenol, respectively. The extracts were incubated at 4OC overnight, the next morning transferred into 10 ml test tube and centrifuged at 6000 rpm for 20 min at the same temperature. The supernatant was passed through a C18 Sep-Pak cartridge (Waters Corp., Milford, MA) and dried in N2. The residues were dissolved in 0.01 mol. L-1 of phosphate buffer saline (PBS) (pH 7.5) for the determination of ZR, iPA, GA3 and GA4 levels. The mouse monoclonal antigens and antibodies against GAs, ZR and iPA and IgG-horseradish peroxidase used were produced at the Phytohormones Research Institute (China Agricultural University). Briefly, microtitration plates (Nunc) were coated with synthetic GA3, GA4, ZR, iPA or ABA-ovalbumin conjugates in 50mmol.L-1 of NaHCO3 buffer solution (pH 9.6) and kept overnight at 37°C. For the purpose of blocking nonspecific binding, 10 mg ml-1 of ovalbumin solution was added to each well. After incubation at 37°C for 30 min, standard GAs, ZR and iPA, samples and antibodies were added and incubated for another 45min at 37°C. Horseradish peroxidase-labelled goat anti-rabbit immunoglobulin was then added to each well and incubated for 1 h at 37°C. Afterwards, the buffered enzyme substrate (orthophenylenediamino) was added and the enzyme reaction was conducted in the dark at 37 °C for 15 min then stopped by using 3 mol. L-1 H2SO4. The ELISA Recorder (Model DG-3022 A; Huadong Electron Tube Factory, Shanghai, China) was used to measure the optical density of each well at A490 nm using Calculation of the enzyme. GAs, ZR, and IPA contents were calculated following Weiler et al. (1981). The results are the means Â± SE of three replication. In this investigation, the percentage recovery of each hormone was calculated by adding known quantity of standard hormone to a split extract. All percentage recoveries were > 90% and all the sample extract dilution curves paralleled the standard curves, indicating the non existence of nonspecific inhibitors in the extracts.
2.3. Statistical analysis
All statistical analyses were done using Statistical Package for Social Scientists (SPSS). The means of the target hormones were taken and one-way ANOVA executed to determine the level of significance at P<0.05.
3.1. Bud hormonal content
The comparison of hormonal levels at developmental stages between AFPs and NAFP are presented in figure 1. The level of ZR exhibited marked differences between AFPs and NAFP during floral initiation phase. During this phase, ZR levels in AFPs were lower than that in NAFP. However, no significant difference in ZR levels existed between AFPs and NAFP during induction and differentiation stages (Fig. 1A).
Consistent differences in iPA levels existed during flower formation phases between AFPs and NAFP (Fig. 1B). The level of iPA in AFPs were generally higher than in NAFP. Such differences were more pronounced and significant (P<0.05) during flower initiation phase. For floral induction and differentiation phases, though differences in iPA levels existed between AFPs and NAFP, they are insignificant at P<0.05.
A marked difference in GA3 and GA4 levels existed between AFPs and NAFP during induction and initiation phases (Fig. 1C & D). However, during differentiation phase, no marked difference was observed AFPs and NAFP. GA3 and GA4 levels were higher in NAFP than in AFPs. Such differences were significant (P<0.05) during induction for GA4 and during flower induction and initiation phases for GA3.
An insignificant increase in ZR, iPA, GA3 and GA4 levels in AFP over NAFP was observed during flower differentiation phase.
Flowering processes in plants are generally regulated by the interactions of endogenous hormones in plant tissues. Various aspects of plant development such as cell division, growth, morphogenesis and flowering are influenced by hormones (Pallardy, 2008; Wilkie et al., 2008). In this study, a marked difference in ZR levels existed between AFPs and NAFP during the initiation period of flower formation. ZR levels in AFPs were comparatively lower during floral initiation period than in NAFP (Fig. 1A). Such difference during initiation phase is significant at P<0.05. In this experiment, since a marked difference in ZR levels existed between AFPs and NAFP during initiation phase, it suggests that ZR is directly involved in the control of flower formation in tree peonies. Low ZR levels in AFPs coincided with the period of the beginning of the second flower organogenesis and histological transformation of apical meristem to new flower buds (Barzilay et al., 2002; Koutinas et al., 2010). This implies that lower ZR levels in AFPs probably facilitated tree peony apex evocation and flowering. Concurrently, high level of ZR was observed during floral initiation phase. The high ZR level during this period in NAFP buds apparently inhibited flower initiation and the related non autumn-flowering. However, this hypothesis of ZR playing floral repressor role in tree peony needs to be confirmed in further studies. Our results are consistent with the results by Liu et al. (2008), in which they suggested that lower level of ZR resulted in flowering in the abnormal chestnut tree. The results further corroborate with that of Chang et al. (1999), which reported lower ZR level in Polianthes tuberose during initiation period. However, the results of this study disagree with those of Bernier et al. (1993), who concluded that ZR not only acted as a major component of floral stimulus in Sinapis alba, but also affected processes such as cell division and vegetative bud and root formation.
A marked difference in the levels of iPA exhibited between AFPs and NAFP. IPA levels were higher in AFPs than in NAFP and the difference in change was significant (P<0.05) during initiation phase (Fig. 1). The significant increase in iPA levels in AFP coincided with the period of morphological developments, apical meristems transformation to new flower buds and active mitosis (Koutinas et al., 2010). During the period of initiation, a marked increment in iPA level was detected in AFPs, which synchronized with the start of the second period of floral primordial formation. It implies that increase in cytokinin, especially iPA is associated with early events of floral transition in tree peony. Interestingly, the same period coincided with the lower level of iPA in NAFP. This suggests that, changes in iPA concentration had a direct effect on flower bud formation, which in turn probably influenced second flowering in AFP. Thus, this study shows potential correlation between high iPA levels during initiation and flower formation in AFP, a phenomenon also reported by Jiang et al. (2010), and Jacqmard et al. (2002) on works with Chrysanthemum morifolium 'Jingyun' and Sinapis alba, respectively. Increase in iPA level during initiation phase is not surprising as it occurred during a period characterized by possible rapid cell division (Wilkie et al., 2008; Koutinas et al., 2010). Since cytokinins are generally associated with the acceleration of cell division, it is possible that the observed high level of iPA during floral initiation is a pre-requisite for the formation of floral organs.
Generally, gibberellins are known to regulate floral formation in many plants (Pharis and King, 1985). GA3 plays many roles in influencing plant growth, cell division, elongation and flowering. High GA3 is reported to inhibit flower formation during induction and differentiation stages in olive (Ulger et al., 2004), peach (An et al., 2008), and also during the flower initiation in avocado (Salazar-Garcia and Lovatt, 1998), peach (Garcia-Pallas et al., 2001) and sweet cherry (Lenahan et al., 2006). Furthermore, lower GA3 level during induction and initiation was reported to promote flower formation in lychee (Chen, 1990), citrus (Koshita et al., 1999) and mango (Wilkie et al., 2008). In this study, both endogenous GA3 and GA4 exhibited lower levels during floral induction and initiation period in AFPs than in NAFP. Lower GA3 and GA4 levels coincided with the beginning of the second period of flower organ development (sepal, petal, stamen and pistil primordial) and histological transformation of apical meristem (Barzilay et al., 2002; Koutinas et al., 2010). The decrease in both GA3 and GA4 levels in AFPs during floral induction and initiation phases suggests that lower GA3 and GA4 levels seems to be relevant for floral induction and initiation in tree peony. However, during floral induction and initiation phases in NAFP, both GA3 and GA4 levels were comparatively higher. Higher levels of GA3 and GA4 during these phases possibly inhibited flower formation and associated non autumn flowering in NAFP. The results of this study are inconsistent with those by Cheng et al. (2004) and Kawabata et al. (2009) where they respectively attributed positive effect of gibberellin to petala and stamens development in Arabidopis and flowering in Eustoma grantiflorum. Furthermore, our results disagree with those on Olea europaea (Ulger et al., 2004), who noted a positive effect of high GA4 level on flower formation.
The high GA3 and GA4 contents during floral induction and initiation in NAFP are not entirely surprising because the plants had just bloomed in spring season and had developing fruits. Fruits and seeds are rich sources of GAs and the rate at which this hormone is exported from seeds to buds influences flower initiation (Pharis and King, 1995). High GA3 and GA4 contents in NAFP may be attributed to their rapid diffusion out of developing fruits to the meristematic zone to inhibit flower initiation (Pharis and King, 1995). GA may have inhibited floral formation in NAFP by deterring the development of nodal buds, bud appendages and sizes. It may have also delay or retard bud transition from vegetative to floral phase; and vital bud release time (Bertelsen et al., 2002; Wilkie et al., 2008).
Endogenous hormones levels showed marked variation between autumn flowering and non-autumn flowering plants throughout the flower formation phases. Therefore, an inference can be drawn that ZR, iPA, GA3 and GA4 greatly contributed to the variation of flowering pattern between AFP and NAFP. Both ZR and iPA contents increased in response to floral initiation, whereas those of GA3 and GA4 decreased. These findings therefore, suggest that probably the interactions among ZR, iPA, GA3 and GA4 during induction and initiation phases resulted to flower formation in AFP.