African Journal of Agricultural Research Vol. 6(20), pp. 4785-4788, 26 September, 2011 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR11.902 ISSN 1991-637X ©2011 Academic Journals Full Length Research Paper Endogenous gibberellin levels during early fruit development of macadamia Stephen J. Trueman Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC 4558, Australia. E-mail: [email protected]. Tel: +61 7 54565033. Accepted 12 August, 2011 Gibberellins play a key role in flower and fruit development, and fruits often contain high gibberellin concentrations. This study quantified endogenous gibberellins in flowers and immature fruits of the macadamia cultivars, H2 and 246, to assist in developing growth-regulator treatments for improving fruit set. GA 1, GA 3, GA 8, GA 9 and GA 20 was detected in flowers or fruits of both cultivars, and GA 4 were detected in flowers of cultivar 246. The timing of gibberellin accumulation was consistent between cultivars but total concentrations were very low (< 7 pmol g -1 fresh weight). Low gibberellin levels are consistent with the inability of the gibberellin-synthesis inhibitor, paclobutrazol, to influence fruit retention in macadamia. Key words: Abscission, fruit drop, kernel, nut, paclobutrazol, plant growth regulators, Proteaceae. INTRODUCTION Macadamia ( Macadamia integrifolia , Macadamia development (Wilkie et al., 2008; de Jong et al., 2009), tetraphylla and hybrids) is a subtropical evergreen tree and fruits often contain high levels of endogenous that is grown in plantations in South Africa, Australia, gibberellins (Zhang et al., 2007; Ayele et al., 2010). This Hawaii and Brazil. Macadamia trees produce masses of paper describes the identification and quantification of racemes in spring, each bearing 100 to 300 flowers gibberellins in developing macadamia fruits, and (Trueman and Turnbull, 1994a; Olesen et al., 2011). discusses their relationship with the effects of gibberellic More than 98% of flowers and immature fruits abscise acid and paclobutrazol applications on fruit retention during the first 10 weeks after anthesis (Trueman and (Trueman, 2010a). Such information is useful for Turnbull, 1994b). Fruits reach maturity about 24 weeks developing growth regulator treatments to improve post-anthesis, after which they fall to the orchard floor macadamia yield and kernel quality. and are harvested and dehusked to provide the nut-in- shell and edible kernel (Trueman et al., 2000, 2002; Walton and Wallace, 2008, 2009). MATERIALS AND METHODS Auxins, cytokinins, gibberellins and the gibberellin- Fruit samples synthesis inhibitor, paclobutrazol, have been tested on macadamia to reduce flower and fruit drop but none has Fruits were collected from macadamia cultivars ‘H2’ (‘Hinde’) and increased final fruit set (Williams, 1980; Trueman, ‘246’ (‘Keauhou’) at Hidden Valley Plantations, Beerwah, Queensland (26 °50’S 152°56’E). Racemes were selected and 2010a). The cytokinin, benzyladenine, increases fruit tagged on four trees of each cultivar, approximately 3 d pre- retention for up to 8 weeks after anthesis but does not anthesis (28 Sep 1989). All selected racemes were pollinated using increase fruit set beyond the major abscission period at the test-tube method of Trueman and Turnbull (1994a) and Wallace 10 weeks post-anthesis (Trueman, 2010a). Endogenous et al. (1996), with cv. H2 racemes pollinated using cv. 246 pollen, cytokinin levels are very high in macadamia fruits around and cv. 246 racemes pollinated using cv. H2 pollen. Fruit samples 10 weeks post-anthesis, which suggests that fruits may were collected 2, 4, 7, 11, 14, 21, 30, 36, 42, 56 and 70 days post- anthesis. Each sample consisted of all the fruits from four racemes, be, in effect, cytokinin-saturated and insensitive to with one raceme from each tree. Samples at 2 to 7 days post- applied cytokinins at this stage (Trueman, 2010b). anthesis also included sepals that had not yet abscised from some Gibberellins also play a key role in flower and fruit fruits. Fresh weights of all samples were recorded, and samples 4786 Afr. J. Agric. Res. Table 1. Gibberellin concentrations (pmol g -1 fresh weight) in macadamia cv. H2 fruits. Time post-anthesis (d) 2 4 7 11 14 21 30 36 42 56 70 GA 1 0.78 nd nd nd nd 0.40 0.29 1.26 0.52 0.57 nd GA 3 nd nd nd 0.49 0.92 0.75 1.45 nd nd 1.33 1.36 GA 4 nd nd nd nd nd nd nd nd nd nd nd GA 8 4.75 0.55 0.33 nd nd nd 0.91 nd 0.33 nd nd GA 9 nd nd nd nd nd 3.20 2.63 nd nd nd nd GA 20 nd 0.81 nd nd 0.39 1.96 0.42 0.81 0.33 4.10 0.60 Total 5.53 1.36 0.33 0.49 1.31 6.31 5.70 2.07 1.18 6.00 1.96 nd’ = Not detected. were stored in liquid nitrogen. Extractions were performed on entire and GA 9. All samples and standards were assayed in duplicate. samples except for the 56 to 70 days samples, where only two fruits Each assay tube contained 100 µl Tris-HCl buffer (0.1 M pH 7.1) were used per extract due to the higher fresh weights of individual plus 0.1 M NaCl, 200 µg bovine immunoglobulin and 170 Bq tracer 3 fruits. ([ H]GA 1-Me, corresponding to a concentration of 0.7 nM). To this was added 50 µl of standard (GA 1-Me, 5 – 5000 pg) or sample. Water was used for determination of B max . Finally 50 µl of Extraction procedure appropriate diluted antiserum was added (water for non-specific binding blank) and the tubes were mixed and incubated for 1 h. Tissue samples were homogenised with an Ultra-Turrax tissue Next, 200 µl of saturated ammonium sulphate solution was added, disintegrator together with 90% methanol (10 ml g-1 fresh weight) thoroughly mixed and left to precipitate the antibody-antigen 3 12 -1 and 330 Bq of [1,2- H]GA 1 (1.21 x 10 Bq mmol ). Extracts were complexes for 20 min. Tubes were centrifuged (10000 g for 3 min) cleared by centrifugation. Insoluble material was reextracted in 90% and supernatants discarded. Pellets were washed with 150 µl of methanol. The combined extracts were reduced to an aqueous 50% saturated ammonium sulphate, centrifuged as before and residue by rotary evaporation at 40 °C. The extract was frozen to - decanted again. Pellets were dissolved in 225 µl water, mixed 20 °C, then thawed and centrifuged (10000 g for 5 min) to remove thoroughly for 1 h, before 1.2 ml of Beckman ReadyValue precipitated lipids and chlorophyll. The extract was then passed scintillation cocktail was added. Tubes were counted on a Packard through a column of insoluble polyvinylpolypyrrolidone (6 ml g-1 Minaxi 4000 scintillation counter for 2 min. Data was processed fresh weight) to remove phenolic compounds. Three volumes of using a custom-written program. The assays were calibrated using water were then passed through the column to recover the a least-squares linear regression plot of logit [B/B max ] v. log gibberellins. The eluate was rotary evaporated to < 3 ml, adjusted [standard concentration]. Compounds were identified on the basis to pH 2.9 with HCl, and extracted 5 times with 2/3 volume of ethyl of immunoreactivity and HPLC retention times, compared with acetate. The combined ethyl acetate fractions were washed with standard gibberellins. The lowest standard (5 pg) was regarded as water to remove excess acid, rotary evaporated to dryness, the detection limit for all samples. dissolved in 2 ml methanol, and then desiccated. The gibberellins were methylated with ethereal diazomethane, and the sample was again rotary evaporated to dryness. RESULTS Compounds with HPLC and radioimmunoassay behaviour HPLC of GA 1, GA 3, GA 8, GA 9 and GA 20 were detected in extracts of both cultivars, H2 (Table 1) and 246 (Table 2). Gibberellins were fractionated on a Shandon ODS 5 µm Hypersil column (250 × 4.6 mm) using a gradient of methanol in water at a GA 4 was only detected in the 2- and 4-days post- flow rate of 1 ml min -1. Samples were firstly dissolved in 900 µl of pollination extracts of cv. 246 flowers. The timing of 30% methanol and centrifuged at 10000 g for 5 min to remove gibberellin accumulation was consistent between particulate matter prior to injection. Samples were then fractionated cultivars (Figure 1), but the total concentrations of using the following gradient: 35% for 4 min, 35 to 47% over 7 min, extractable gibberellins were very low (max. 6.31 pmol g -1 47 to 87% over 10 min, 87 to 100% over 5 min, and 100% for 5 min. Thirty-five 0.6-min fractions were collected from 6 to 27 min. All fresh weight) throughout the first 70 d after anthesis collected fractions were dried in a Savant SVC100H centrifugal (Tables 1 and 2). sample concentrator at 45 °C, and then dissolved in 200 µl water. 3 Recoveries of [ H]GA 1 were estimated from the appropriate HPLC fractions to be 6 to 73%. DISCUSSION Macadamia fruits contained several gibberellins (viz. GA 1, Radioimmunoassay GA 3, GA 4 and GA 9) that have also been detected at low concentrations (max. 11.48 pmol g -1 fresh weight) in Radioimmunoassay was performed on all HPLC fractions. An apical buds or lateral buds of macadamia cv. 344 antiserum against GA 1 was used. This antiserum had a broad specificity for C -GAs, which allowed accurate simultaneous (Fletcher and Mader, 2007). The gibberellin levels in 19 -1 measurement of GA 1, GA 3, GA 4, GA 20 and, at lower sensitivity, GA 8 macadamia fruits (< 7 pmol g fresh weight) were much Trueman 4787 Table 2.