Involvement of endogenous plant hormones (IAA, ABA, GAs) in leaves and flower bud formation of satsuma mandarin (Citrus unshiu Marc.

Transcription

1 Scientia Horticulturae 79 (1999) 185±194 Involvement of endogenous plant hormones (IAA, ABA, GAs) in leaves and flower bud formation of satsuma mandarin (Citrus unshiu Marc.) Yoshiko Koshita a,*,1, Toshio Takahara a, Tatsushi Ogata a, Akihiko Goto b a Department of Citriculture, National Institute of Fruit Tree Science, Kuchinotsu, Nagasaki , Japan b National Institute of Fruit Tree Science, Fujimoto 2-1, Tsukuba, Ibaraki 305, Japan Accepted 10 August 1998 Abstract Plant hormones (IAA, ABA, GA 1/3,GA 4/7 ) contents in the leaves of satsuma mandarin trees (Citrus unshiu Marc. `Okitsu') were measured to elucidate the relationship between endogenous plant hormones and flower buds formation. The ringing treatment to lateral branch in October enhanced the percentage of leafless inflorescence and number of flowering shoots in the following spring. While, more vegetative spring shoots were formed on bearing shoots which were left the fruit load in the previous autumn to winter. GA 1/3 were the dominantly detected GAs in the leaves. Their content was higher in the leaves of the bearing shoots than in that of the vegetative shoot but was not affected by the ringing treatment. Ringing increased the percentage of leafless inflorescence and the contents of IAA and ABA in December. These results suggested that flower bud formation was inhibited by GA 1/3 in October, and that high IAA and ABA content in December also promote the percentage of leafless inflorescence and the number of flower buds per node. The relationship between endogenous plant hormones and flower bud formation was discussed. # 1999 Elsevier Science B.V. All rights reserved. Keywords: GAs; IAA; ABA; Ringing; Bearing branch; Vegetative branch; Flower bud formation Abbreviations: IAA, indole acetic acid; ABA, abscisic acid; GA, gibberellic acid; EtOAc, ethyl acetate; MeOH, methanol; MeCN, acetonitrile * Corresponding author. Tel.: ; fax: ; koshita@akt.affrc.go.jp 1 Present address: Persimmon and Grape Research Center, National Institute of Fruit Tree Science, Akitsu Hiroshima, , Japan /99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S (98)00209-X

2 186 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185± Introduction Maintaining stable fruit production each year is essential for growers. Citrus trees, however, often bear fruits only in alternate years because of their property that flowers do not form on a bearing shoots the following spring. Several techniques for regulating the flower formation which is imperative for stable production have been developed in citrus. Goldschmidt et al. (1985) and Iwahori et al. (1990) reported that flower bud formation was enhanced by ringing of the main branch. Water stress also induced floral bud formation (Nir et al., 1972; Southwick and Davenport, 1986, 1987a, b). While, the number of flower buds can be reduced by GA spray (Hirose, 1968; Monselise and Halevy, 1964; Goldschmidt and Monseile, 1972; Lenz and Karnatz, 1975; Guardiola et al., 1982; Davenport, 1983). Takahara et al. (1990) showed that the effect of the GA treatment was promoted when the GA was mixed with a machine oil emulsion. Investigation of the relationship between flower bud formation and endogenous plant hormone contents is valuable to develop a technique for effectively controlling the amount of flowers in spring as well as to elucidate the physiological role of plant hormones in tissue. Although many researches have been conducted on endogenous GA contents and flower bud formation, no consistent results have yet been realized. Southwick and Davenport (1987a, b) reported that there was no evidence supporting the flower inducing role of ABA and GAs with water stress or chilling treatment. Takagi and Suzuki (1985) determined the GA content during the blooming period, showing that it is higher in leaves of the preceeding shoot which produced only a vegetative shoots than in those of the shoots producing generative shoots. These contradictory results might be the result of one or more of the following: the changes in GA content were determined by different methods and with extracts of differing purity; plant hormones were determined at different periods, not throughout the season of flower formation and differentiation. Still more, the relation to other plant hormones, e.g. IAA, ABA, and cytokinins, was not simultaneously investigated. Therefore, the aim of this study is to clarify the relationship between flower bud formation and plant hormones (IAA, ABA, GA 1/3,GA 4/7 ) contents under flower bud inductive or suppressive condition measuring by the reliable hormone analysis method. 2. Materials and methods 2.1. Plant material Four trees of 25-year-old satsuma mandarin grafted on to Poncirus trifoliate rootstocks grown in an orchard of the Department of Citriculture of the National

3 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185± Institute of Fruit Tree Science, Kuchinotsu, Nagasaki, Japan were used in the experiment. Eight lateral branches of about 1 cm in diameter and consisting only of vegetative shoots were chosen per tree. Four of these lateral branches were ringed by peeling the bark with a knife on October 12. About 60 fruit bearing shoots per a tree were chosen and fruits were left on the shoots until December to impose the fruit load to the shoots, though other fruits were harvested on November The leaves on ringed and untreated vegetative branch were picked from four branches of each tree and the leaves from four to five bearing shoots were taken in each tree. These samples were immediately dipped in liquid N 2 and were stored at 808C until analysis Assessment of floral and foliar formation The degree of floral and foliar formations on vegetative shoots with or without the ringing treatment and on bearing shoots of the previous year were assessed in May, Twenty shoots of each treatment per tree were evaluated. Spring shoots were assessed and buds were classified into three types: vegetative type, leafless inflorescence and leafy inflorescence Extraction and purification procedure 13 C 6 IAA and d6aba were added to each sample of plant material as internal standards. The plant material was homogenized with 80% aqueous acetone with 100 mg l l BHT. The homogenates were filtered, and the filtrate was concentrated to an aqueous residue in vacuo, adjusted to ph 8.0, and applied to a column of polyvinylpolypyrrolidone (PVPP). The eluate was adjusted to ph 2.5 with 6 N H 2 SO 4 and extracted three times with EtOAc. The EtOAc extracts were combined and extracted three times with saturated NaHCO 3. The NaHCO 3 extracts were combined, adjusted to ph 2.5 with 6 N H 2 SO 4, and extracted three times with EtOAc. The combined EtOAc extracts was applied to a column of anhydrous sodium sulfate and then evaporated to dryness in vacuo. The residue was dissolved in 80% aqueous MeOH and pre-purified on a Sep-pak (C18) cartridge eluted with 80% MeOH. The eluate was evaporated to dryness in vacuo and the residue was dissolved in MeOH and dried under N 2 gas Reverse-phase high-performance liquid chromatography (HPLC) The purified extracts were dissolved in 50% aqueous MeCN containing 0.5% CH 3 COOH, applied to the HPLC. Conditions of HPLC were as follows; column ODS (PEGASIL ODS, Senshu Pak, mm i.d.); temperature 408C; flow rate 1.5 ml min 1 ; solvent A 5% MeCN with 0.5% CH 3 COOH; solvent B 80%

4 188 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185±194 MeCN with 0.5% CH 3 COOH; gradient profile 0±5 min 0% of B, 5±50 min 0% to 33% of B, 50±70 min 33% to 100% of B. Under these conditions, the retention times of the plant hormones were IAA 34 min, ABA 44 min, GA 1/3 29 min, and GA 4/7 59 min. The fractions of IAA, ABA, GA 1/3,GA 4/7 were collected and dried Quantitative analysis by GC/MS IAA and ABA samples intended for GC/MS analysis were methylated with ethereal diazomethane. After evaporation, 10 ml of N-methyl-N-TMS-trifluoloacetamide (MSTFA) was added to the methylated IAA sample and the mixture was heated at 808C for 15 min. Samples diluted by EtOAc were injected into gas chromatography (GC) equipped with mass spectrometry (MS) (QP-5000, Shimadzu Inc., Kyoto, Japan) using the split-less technique. The column was fused silica capillary column (CBP 1.25 m 0.22 mm i.d mm film thickness, Shimadzu). Injector temperature was 2508C. The column temperature was maintained at 1208C for 2 min, then was increased at 208C min 1 up to 2808C Quantification of GAs The monoclonal antibody against GA 4 was kindly provided by Dr. Yamaguchi (the University of Tokyo), and the GA 4 tracer by Dr. Kusaba (National Institute of Fruit Tree Science, Tsukuba, Japan). The procedure for ELISA was basically the same as that reported by Atzorn and Weiler (1983). Assays were performed in triplicate for each fraction. Cross-reactivities of the GAs were examined according to the report by Weiler and Zenk (1976). 3. Results 3.1. Floral and foliar formation pattern in the following spring Flowering and sprouting patterns were significantly different depending on the ringing treatment and the fruit load of the previous year (Table 1). Almost all the buds on the vegetative shoots sprouted flowers and fruit bearing shoots sprouted leaves. The ringed treatment enhanced the ratio of leafless inflorescence in the vegetative branches and did not affect much on percentage of flowering node in the vegetative branches. Moreover, flowering potential was enhanced by ringing. The average number of flower buds per node was 2.4 in the ringed shoot and 1.2 in the untreated shoot (Table 2).

5 Table 1 The effect of ringing and fruit load in autumn on flowering characteristics in the following spring in vegetative and bearing branch of satsuma mandarin trees Plant materials Percentage of flowering node per total node Percentage of vegetative node per total node Percentage of leafless inflorescence per total node Percentage of leafy inflorescence per total node Vegetative branches (ringed) a Vegetative branches (untreated) Fruit bearing branches a Values are mean SE (n ˆ 4). Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185± Table 2 The effect of ringing and fruit load in autumn on number of inflorescence per node in the following spring in vegetative and bearing branch of satsuma mandarin trees Plant Materials Number of inflorescence per node Number of vegetative shoots Leafless Leafy Total Vegetative branches (ringed) a Vegetative branches (untreated) Fruit bearing branches a Values are mean SE (n ˆ 4) IAA contents in the leaves There were no significant differences in IAA contents between vegetative and bearing shoots in October (Fig. 1(A)). The content in leaves on ringed shoots remarkably increased from October to December, then decreased from December to February of the next year. IAA contents in the fruit bearing shoots didn't show seasonal change throughout the measurement period. IAA content in non-ringed vegetative shoots gradually increased with time ABA content in the leaves ABA content in all the treatment increased from October to December, then decreased from December to February of the next year (Fig. 1(B)). The ringing treatment remarkably enhanced the content from October to February GAs content in the leaves GA 1/3 contents in the leaves was considerably higher than GA 4/7 throughout the period of measurement (Fig. 2(A), (B)). The GA 1/3 level in bearing shoots leaves

6 190 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185±194 Fig. 1. Differences of IAA (A) and ABA (B) contents in bearing and vegetative shoot leaves. Data points represent the means of four trees determinations SE. Bars represent the SE and were not shown when smaller than the data symbol. was three times higher than in vegetative shoots in October, then drastically decreased from October to December (Fig. 2(A)). Significant difference could not be found in February. GA 4/7 content was slightly higher in bearing shoots in October (Fig. 2(B)). This content fluctuated within a small range throughout the measurement in all treatment, and no significant differences could be found except that the content in bearing shoots was higher than in vegetative shoots in October. 4. Discussion From the result of defoliation treatment at different seasons, flower induction of satsuma mandarin initiated from September to March (Osaki and Saso, 1942,

7 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185± Fig. 2. Differences of GA 1/3 (A) and GA 4/7 (B) contents in bearing and vegetative shoot leaves. Data points represent the means of four trees determinations SE. Bars represent the SE and were not shown when smaller than the data symbol. 1943). From microscopic observation, 40% of flower bud are on differentiation stage in middle January (Iwahori and Oohata, 1981). In any season from November up to sprouting, however, exogenous GA inhibited flower formation (Goldschmidt and Monseile, 1972; Guardiola et al., 1982). Moreover, this inhibition occurred even after flower bud differentiation had already been confirmed microscopically (Nir et al., 1972). Therefore, the possibility of transition from floral to vegetative buds by exogenous GA was suggested. These data implied that flower bud formation occurred and was affected by environmental and exogenous conditions from early fall to sprouting. Exogenous GAs have been shown to inhibit flower bud formation not only in citrus (Monselise and Halevy, 1964; Goldschmidt and Monseile, 1972;

8 192 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185±194 Davenport, 1983) but also in apples (Guttridge, 1962; Marcelle and Sironval, 1963), pears (Griggs and Iwakiri, 1961), and cherries and peaches (Hull and Lewis, 1959). This hormone is thus believed to strongly participate in flower bud formation. In the present study, GAs content in October in the leaves of bearing shoots which produced little florescence in the following spring was three fold higher than in the leaves of vegetative shoots which produced florescence in abundance (Table 1, Fig. 2). The higher endogenous GAs levels may be one of the reasons for vegetative growth in the following spring. These data confirmed that there is a relationship between endogenous GAs and flower bud formation. On the contrary, only slight difference was observed in the GAs content between bearing and vegetative shoot leaves in December and in February (Fig. 1). These data also support the result that showed no relationship between flower bud formation and GAs content (Southwick and Davenport, 1987a, b). Thus, knowledge of the fluctuation of plant hormones is essential, since, as mentioned above, the process between bud formation and differentiation in citrus is lengthy. GAs in the satsuma mandarin were identified previously. Goto et al. (1989) identified and quantified GA 1,4,9,19,20,24,25,44 and GA 53 in young fruits of satsuma mandarin. They suggested that the early-13-hydroxylation pathway seemed to be the major pathway in the young fruit. In our data, GA 1/3 were major throughout the measurement (Fig. 2). These data suggested that early-13-hydroxylation pathway may be the major not only in young fruits but also in the leaves of satsuma mandarin. In this work, we quantified the levels of GAs by ELISA. It is possible that some GA 1/3 isomers react with the antibody raised against GA 4. But from the result of Goto et al. (1989), the levels of such GAs must be very low. Although the relationship between flower bud formation and GAs has been widely investigated, there are few reports on the relationship between IAA, ABA and flower bud formation in citrus. In the experiment of in vitro bud culture, sprouting of summer buds was delayed by IAA, while, GA enhanced it and ABA completely inhibited it (Altman and Goren, 1972). Nevertheless, the effect of exogenous hormones varied at different periods during the season. Recently, inhibitory effect of exogenous IAA and ABA, and endogenous IAA were reported in Pharbitis nil in which exogenous GA induce flowering (Wijayanti et al., 1997). In our report, the ringing treatment in October enhanced the leafless inflorescence ratio (Table 1) and total number of flower buds per node (Table 2). Further, IAA and ABA content increased in December (Fig. 1). These data implied that these hormones might also be related to the flower bud development. In summary, flower buds were few in the bearing shoots and GA 1/3 content in the leaves was significantly higher in these shoots than in vegetative shoots in October. These data suggested that not only exogenous but also endogenous GAs reduce flower bud formation. In addition, the increase of leafless inflorescence and enhancement of the ABA content in December and February, and of the IAA

9 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185± content in December by ringing suggested that endogenous IAA and ABA influence the pattern of inflorescence; in other words, these hormones might affect flower bud development. References Altman, A., Goren, R., Growth and dormancy cycles in Citrus bud cultures and their hormonal control. Physiol. Plant. 30, 240±245. Atzorn, R., Weiler, E.W., The immunoassay of gibberellins. II. Quantitation of GA 3,GA 4 and GA 7 by ultra-sensitive solid-phase enzyme immunoassays. Planta 159, 7±11. Davenport, T.L., Daminozide and gibberellin effects on floral induction of Citrus latifolia. HortScience 18, 947±949. Goldschmidt, E.E., Monseile, S.P., Hormonal control of flowering in citrus and other woody perennials. In: Carr, D.J. (Eds.), Plant growth substances. Springer, NY pp. 758±766. Goldschmidt, E.E., Aschkenazi, N., Herzano, Y., Schaffer, A.A., Monselise, S.P., A role for carbohydrate levels in the control of flowering in citrus. Scientia Hort. 26, 159±166. Goto, A., Yamane, H., Takahashi, N., Hirose, K., Identification of nine gibberllins from young fruit of Satsuma mandarin (Citrus unshiu Marc.). Agric. Biol. Chem. 53, 2817±2818. Griggs, W.H., Iwakiri, B.T., Effects of gibberellin and 2,4,5-Trichlorophenoxy-propionic acid sprays on Bartlett pear trees. Proc. Amer. Soc. Hort. Sci. 77, 73±89. Guardiola, J.L., Monerri, C., Agusti, M., The inhibitory effect of gibberellic acid on flowering in Citrus. Physiol. Plant. 55, 136±142. Guttridge, C.G., Inhibition of fruit bud formation in apple with gibberellic acid. Nature 196, 920±921. Hirose, K., Control of citrus flower bud formation. I. The effect of gibberellic acid spraying on flower bud formation in satsuma orange. Bull. Hort. Res. Stn. B8, 1±11. Hull Jr., J., Lewis, L.N., Response of one year old cherry and mature bearing cherry, peach, and apple trees to gibberellin. Proc. Amer. Soc. Hort. Sci. 74, 93±100. Iwahori, S., Oohata, J.T., Control of flowering of satsuma mandarins (Citrus unshiu Marc.) with gibberellin. Proc. Int. Soc. Citriculture pp. 247±249. Iwahori, S., GarcõÂa-Luis, A., Santamarina, P., Monerri, C., Guardiola, J.L., The influence of ringing on bud development and flowering in satsuma mandarin. J. Exp. Bot. 41, 1341±1346. Lenz, F., Karnatz, A., The effect of GA 3, Alar, and CCC on citrus cuttings. Acta Hort. 49, 147±155. Marcelle, R., Sironval, C., Effect of gibberellic acid on flowering of apple trees. Nature 197, 405. Monselise, S.P., Halevy, A.H., Chemical inhibition and promotion of citrus flower bud induction. Proc. Amer. Soc. Hort. Sci. 84, 141±146. Nir, I., Goren, R., Leshem, B., Effects of water stress, gibberellic acid and 2- chloroethyltrimethylammoniumchloride (CCC) on flower differentiation in `Eureka' lemon trees. J. Amer. Soc. Hort. Sci. 97, 774±778. Osaki, M., Saso, H., Studies on seasons of citrus flower bud formation I. J. Japan Soc. Hort. Sco. 13, 24±29 (in Japanese). Osaki M., Saso H., Studies on seasons of citrus flower bud formation II. J. Japan Soc. Hort. Sco. 14, 103±106 (in Japanese). Southwick, S.M., Davenport, T.L., Characterization of water stress and low temperature effects on flower induction in citrus. Plant Physiol. 81, 26±29.

10 194 Y. Koshita et al. / Scientia Horticulturae 79 (1999) 185±194 Southwick, S.M., Davenport, T.L., 1987a. Modification of the water Stress-induced floral response in `Tahiti' lime. J. Amer. Soc. Hort. Sci. 112, pp. 231±236. Southwick, S.M., Davenport, T.L., 1987b. The role of gibberellins and ABA in citrus flowering. In: proc. Plant Growth Regulator Society of America, pp. 487±488. Takagi T., Suzuki, T., GA-like substances in spring flush and preceeding shoot at the blooming time in Satsuma mandarin trees. Bull. Fac. Agr. Shizuoka University 35, pp. 25±28 (in Japanese with English summary). Takahara, T., Hirose, K., Iwagaki, I., Ono, S., Enhancement of suppression effect of flowerbud formation of citrus varieties by addition of machine oil emulsion to gibberllin. Bull. Fruit Tree Res. Stn. 18, pp. 77±89 (in Japanese with English summary). Weiler, E.W., Zenk, M.H., Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 15, 1537±1545. Wijayanti, L., Fujioka, S., Kobayashi, M., Sakurai, A., Involvement of abscisic acid and indole-3-acetic acid in the flowering of Pharbitis nil. J. Plant Growth Regul. 16, 115±119.