Determination of endogenous hormones, sugars and mineral nutrition levels

#Determination of endogenous hormones,sugars and mineral nutrition levels during the induction,initiation and differentiation stage and their effects on flower formation in olive

Salih Ulger 1,*,Sahriye Sonmez 2,Mustafa Karkacier 3,Nisa Ertoy 1,Ozgur Akdesir 1and Mehmet Aksu 3

1

Department of Horticulture,2Department of Soil Science,and 3Department of Food Engineering,Faculty of Agriculture,Akdeniz University,Antalya,Turkey;*Author for correspondence (e-mail:ulger@https://www.360docs.net/doc/5a19255118.html,.tr)

Received 27November 2002;accepted in revised form 11September 2003

Key words:ABA,Ca,Cu,Fe,Fructose,GA 3,GA 4,Glucose,IAA,K,Mg,Mn,N,Olea europaea ,Olive,P,Sucrose,Zeatin,Zn

Abstract

Olive (Olea europaea L.)is one of the most important crop plants grown in the Mediterranean region.Varying levels of hormones,sugars and mineral nutrient are thought to influence flower bud formation.The objective of this study was to evaluate the changes in endogenous sugar,mineral nutrition and hormone levels in leaf,node and fruit samples of ‘Memecik’olive during the induction,initiation and differentiation periods in on (bearing)and off (non-bearing)years.Leaf,node and fruit samples of mature 15-year-old Memecik olive were used.The samples were taken during the induction,initiation and differentiation periods of olive in on (2000)and off (2001)years.Sugar (glucose,fructose and sucrose),mineral nutrient (N,P,K,Ca,Mg,Fe,Zn,Mn and Cu)and hormone [abscisic acid (ABA),indole acetic acid (IAA),gibberellic acid (GA 3,GA 4)and zeatin (Z)]levels were determined in on and off years.Hormone and sugar levels were measured by gas chromatography and high-performance liquid chromatography.The K,Ca,Mg,Fe,Mn,Zn and Cu levels were quantified by an atomic absorption spectrophotometry.Nitrogen was determined by the Kjeldahl procedure,and P by a spectrophotometric method.The differences in any of the sugar concentrations,with the exception of fructose,were not significant in on and off years.Hormone levels,however,were significantly different in on and off years.Glucose had the highest concentrations in both years,followed by sucrose and fructose,respectively.The highest macro and micro element concentra-tions were found to be Ca and Fe,respectively.Thus,the results suggest that carbohydrates and mineral nutrients may not have a direct effect to induce flower initiation.However,high GA 3level exhibited an inhibitory effect on floral formation during the induction and initiation periods.On the other hand,the high concentrations of GA 4,ABA and certain cytokinin levels may have a positive effect on flower formation in olive during the induction and initiation periods.

Abbreviations:AAS –atomic absorption spectrophotometry;ABA –abscisic acid;Ca –calcium;Cu –copper;Fe –iron;FID –flame ionization detector;GAs (GA 3,GA 4)–gibberellic acid;GC –gas chromatography;HPLC –high performance liquid chromatography;IAA –indole-3-acetic acid;K –potassium;Mg –magnesium;Mn –manganese;N –nitrogen;P –phosphorus;RI –refractive index detector;TLC –thin layer chromatography;TS –total sugars;Z –zeatin;Zn –zinc

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2004Kluwer Academic Publishers.Printed in the Netherlands.

Plant Growth Regulation 89–95,2004.

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Introduction

Olive(Olea europaea L.)is native to the Mediterranean basin where about97%of the world’s olive fruit are produced.Unlike deciduous fruits with a short induction-to-initiation cycle, induction in olive may take up to8months,start-ing as early as July until about6weeks after full bloom,later in February.Although floral initiation occurs in November,the process of developing of visible flower parts does not start until March. Differentiation takes place in late February and bloom in May when the formation of each flower part occurs in the inflorescence(Martin et al.1994). In apple(Malus communis L.),flower induction occurs early in the season,5–10weeks after full bloom,as in olive,and is controlled by complex physiological process.External factors such as tem-perature,light,water supply and mineral nutrients, as well as internal factors such as carbohydrate and hormone levels are involved in growth and devel-opment(Gur1985;Forshey and Elfying1989; Stephan et al.1999).

Researchers have investigated the relationships among carbohydrates(Fahmi1958;Sarmiento et al.1976;Monselise and Goldschmidt1981; Stutte and Martin1986),hormones(Ramirez and Hoad1981;Mullins and Rajasekaran1981;Lavee et al.1983;Chen1987;Stephan et al.1999),mineral nutrients(Hartmann et al.1966;Priestly1977; Golomp and Goldschmidt1981)and flower bud formation in different fruit types.

The objective of this study was to determine the changes in endogenous plant hormones,carbohy-drates and mineral elements in leaf,node and fruit samples of‘Memecik’olive and assess the effects of these concentrations on flower bud formation dur-ing flower and fruit induction,initiation and differ-entiation cycles in on and off years.

Materials and methods

Plant material

Fifteen-year-old trees of the‘Memecik’olive culti-var growing at the Research Station of the Faculty of Agricultural,University of Akdeniz,Antalya, Turkey,were used in this experiment.The Research Station is located3km from the Mediterranean Sea at an elevation of50m.The soil type is clay loam(lithic xerorthent)with low organic matter(2.69%)and a pH of8.23. Supplemental irrigation was provided and stan-dard fertilisation practices were followed. Hormone,sugar and mineral nutrient analyses were conducted during the current season growth on leaf,node and fruit samples.Samples were taken three times with a one-week interval during the induction,initiation and differentiation periods of olive in2000(on year)and in2001(off year). The sampling dates were5,12and19July for induction,2,9and16November for initiation, and23February,2and9March for differentia-tion.The collected samples were stored atà20 C until analysis.Samples consisted of1g fresh weight of each plant part selected from the periphery of the tree at a height of1.5m.Data were analysed using the randomised complete block design with three replications.Each block has three trees. Hormone analysis

Analyses of abscisic acid(ABA),indole acetic acid (IAA),gibberellic acid(GA3,GA4)and Zeatin(Z) were determined according to Topc?uo g lu and Unyayar(1995).This method is a combination of the methods used by Ames et al.(1979),Prakash and Prathapasenan(1990),Staden and Nicholson (1989),Weiler et al.(1981)and Zieslin and Geller (1983).Leaf,node and fruit samples(1g)were homogenised in a cold methanol:chloroform mixture(14:6,v/v)at room temperature,and then were stored atà20 C for1week.The extracts were filtered through a Whatman No.5filter paper,the supernatants were re-homogenised with the same solution mixture and the extracts were combined.The aqueous residue was adjusted to pH8.5with1N NaOH,and transferred to a separa-ting funnel to separate chloroform from the metha-nol after which the chloroform phase was discarded.The methanol phase was reduced to an aqueous phase under reduced pressure on a rotary evaporator at40 C.It was then adjusted to pH2.5 with1N HCl and extracted with ethyl acetate (3?v).The aqueous phase was adjusted to pH7.0 with1N NaOH,extracted with ethyl acetate(3?v) and then the acidic and neutral ethyl acetate phases including free hormones were combined.In order to extract conjugate a hormones,the aqueous

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phase was adjusted to pH11.0with1N NaOH and incubated in a water bath at70 C for1h.It was adjusted to pH7.0with1N HCl and extracted with ethyl acetate(3?v).The aqueous phase was then adjusted to pH2.5with1N HCl and extracted with ethyl acetate(3?v).Conjugated acidic and neutral ethyl acetate phases were combined,and then these combined conjugated extracts were combined with the previously combined free hormone extracts, and dried under vacuum at40 C.The residue was dissolved in1ml methanol,and transferred to an Eppendorf tube.The methanol was reduced to100 l under vacuum,and then line-loaded onto a20?20cm2,0.25mm thick silica gel60F254TLC plate(Merck Plc,Darmstadt,Germany).Standard ABA,IAA,GA3,GA4and Z were also spot-loaded in scored strips at both edges of the plates.The plate was allowed to develop for15cm in the vertical direction using isopropyl alcohol:ammonia:water (84:8:8,v/v/v)as the solvent system.After develop-ment,the positions of ABA,IAA,GA3,GA4and Z were detected under UV light(254nm wave length) and marked.A band of silica corresponding to the Rf values of the standards was scraped off,dis-solved in0.5ml methanol in an Eppendorf tube and then dried under vacuum.The purified samples were methylated with diazomethane(Schlenk and Gellerman1960)dissolved in ethyl ether and metha-nol(9:1,v/v).The derivatives were dried under vacuum and re-dissolved in100 l ethyl acetate for GC analysis.The ABA,IAA,GA3and Z contents were determined with a Fisons8560HRGC Mega 2series equipped withFID,andusingaSPB-1(30m?0.32mm I.D.)capillary column.Injection and detector temperatures were200and300 C,respec-tively.Samples(1 l)were injected into the column at80 C.The temperature was programmed to5 C minà1until the column was at280 C The helium flow rate was1ml minà1,and inlet pressure was 22psi.ABA,IAA,GA3,GA4and Z contents were determined using peak areas.The ratio of response of detector to putative ABA,IAA,GA3,GA4and Z peaks in the plant samples were compared with the response ratio of the detector for authentic ABA,IAA,GA3,GA4and Z standards(Sigma). Sugar analysis

Sugar concentrations were determined according to Camara et al.(1996)with some modifications.Ten grams of homogenised sample were mixed with 40ml water.The mixture was homogenised by using an ultratorax at24000rpm and centrifuged at6000rpm for30min at ambient temperature. The supernatant was filtered through Whatman42 filter paper,and then filtered through a Sep-Pack C18(Waters)cartridge.A2.5ml aliquot of filtrate was blended with7.5ml of acetonitrile;subse-quently,the mixture was filtered through a0.45 m membrane filter before20- l injections.

High-pressure liquid chromatography(HPLC) analysis of the sugars was performed by reversed-phase HPLC on a Varian Model9050equipped with a Varian Model9010solvent delivery system and a Varian Model9040Refractive Index(RI) detector.Separation and determination were con-ducted at25 C using an Alltech Amino Bonded Carbohydrate Column(300mm?4.1mm I.D.). The column was eluted with a30%methanol aceto-nitrile:water(75:25)mobile phase at a flow rate of 1.4ml minà1.The concentrations of glucose,fruc-tose and sucrose were quantified using a Varian Star Chromatography Vorkstation(Ver. 4.51), from the peak area ratios on the basis of the corre-sponding calibration parameter values(Sigma Car-11sugar Kids).

Mineral nutrition analysis

Leaf,node and fruit samples of olive were col-lected as described by Bouat(1960),and trans-ported to the laboratory in closed polyethylene bags.In order to eliminate surface contamina-tion,the samples were carefully washed with liquid soap(Teepol,0.1%),then rinsed in tap and deionised water.The sample surfaces were dried with clean blotting papers,placed in paper bags and dried in a forced-air oven at70 C for 72h.The dried samples were ground in a stain-less steel Wiley mill.The oven-dried-ground sam-ples were wet digested in a mixture of nitric–perchloric acids[(HNO3:HClO4(4:1)]and K, Ca,Mg,Fe,Mn,Zn and Cu concentrations in the digest were quantified by atomic absorption spectrophotometry(Varian Model Spectra-400 Plus)(Kacar1972).Nitrogen was determined by the Kjeldahl procedure(Kacar1972),and P was measured by spectrophotometry using the Olsen and Sommers(1982)method.

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Statistical analysis

Statistical analysis of the data was carried out using a SAS(SAS Inc.,Raleigh,NC,USA)package program,and data were subjected to analysis of variance with mean separation by Duncan’s multi-ple range test.

Results

Sugar contents

The highest sugar level was that of glucose in both on and off years in the‘Memecik’olive cultivar, followed by sucrose and fructose,respectively. Fructose,sucrose and TS levels were significantly different during the differentiation,induction and initiation periods(P0.05).The glucose level, however,was not significantly different during the same periods.The highest fructose,sucrose and TS levels occurred in the induction period,followed by the initiation and differentiation periods,respec-

tively(Table1).Fructose,glucose and TS levels, but not sucrose,were different in leaves,nodes and fruit samples(P0.05).Glucose and TS levels were higher in leaves although the fructose level was higher in fruits(Table2).Glucose,sucrose and TS levels were not different in on and off years (P0.05),but the fructose level was higher in the on year than in the off year(Table3).

Table1.Sugar,hormone and mineral nutrient concentrations of‘Memecik’olive at the three floral stages.

Differentiation Induction Initiation Fructose(%)0.18c*0.34a0.26b Glucose(%) 3.08a 3.26a 3.12a Sucrose(%)0.23b0.36a0.37a Total sugar(%) 3.48b 3.99a 3.81ab IAA( g gà1)0.72b0.70b 1.75a

Z( g gà1)18.09a 5.42c8.63b ABA( g gà1)0.47a0.21b0.15b GA3( g gà1) 4.12a 1.53b 3.88a GA4( g gà1)40.62a23.70b41.36a

N(%) 1.04a0.97b0.61c

P(%)0.11c0.12b0.14a

K(%)0.59c0.91a0.73b Ca(%) 2.06a 1.30b 1.98a Mg(%)0.17a0.13a0.17a Fe( g gà1)93.77a63.98b60.09b Mn( g gà1)33.82a22.73b19.20b Zn( g gà1)21.61a16.95b11.21c Cu( g gà1)29.13a17.77b16.04b *Mean separation within columns and main effects by Duncan’s multiple range test,P0.05.Table2.Sugar,hormone and mineral nutrient concentrations of‘Memecik’olive as affected by plant organ.

Leaf Node Fruit Fructose(%)0.19b*0.21b0.48a Glucose(%) 3.66a 2.86b 2.87b Sucrose(%)0.29a0.37a0.33a Total sugar(%) 4.18a 3.46b 3.73b IAA( g gà1) 1.29a0.72b 1.37a Z( g gà1)11.76a9.90b 6.68c ABA( g gà1)0.31a0.17b0.28a GA3( g gà1) 3.27a 2.61b 3.41c GA4( g gà1)33.38b40.91a26.78c N(%) 1.04a0.97b0.61c P(%)0.11c0.12b0.14a K(%)0.59c0.91a0.73b Ca(%) 2.06a 1.30b 1.98a Mg(%)0.17a0.13a0.17a Fe( g gà1)93.77a63.98b60.09b Mn( g gà1)33.82a22.73b19.19b Zn( g gà1)21.61a16.95b12.21c Cu( g gà1)29.13a17.77b16.04b *Mean separation within columns and main effects by Duncan’s multiple range test,P0.05.

Table3.Main effects of on and off years on sugar,hormone and mineral nutrient concentrations of‘Memecik’olive.

20002001 Fructose(%)0.31a*0.23b Glucose(%) 3.05a 3.28a Sucrose(%)0.33a0.34a Total sugar(%) 3.69a 3.90a IAA( g gà1)0.28b 1.92a Z( g gà1)8.71b10.88a ABA( g gà1)0.15b0.35a GA3( g gà1) 3.62a 2.50b GA4( g gà1)29.16b39.95a N(%)0.99a0.77b P(%)0.12a0.12a K(%)0.71b0.82a Ca(%) 1.81a 1.61a Mg(%)0.16a0.15a Fe( g gà1)82.91a59.85b Mn( g gà1)30.55a19.23b Zn( g gà1)21.52a11.77b Cu( g gà1)29.80a11.24b *Mean separation within columns and main effects by Duncan’s multiple range test,P0.05.

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Endogenous plant hormones

The IAA,Z,ABA,GA 3and GA 4levels were dif-ferent during all developmental stages (differentia-tion,induction,differentiation)and organs (leaves,nodes,fruits)in on and off years (P 0.05).IAA concentration was highest during the initiation per-iod,but ABA,Z,GA 3,GA 4concentrations were highest during the differentiation period.The IAA,Z,GA 3and GA 4levels were at their lowest during the induction period (Table 1).The ABA and Z concentrations were higher in leaves while GA 3and IAA levels were higher in fruits,and only the GA 4level was higher in nodes (Table 2).The IAA,Z,ABA and GA 4levels were higher in the off year,whereas GA 3was higher in the on year (Table 3).Mineral nutrients

The N,P,K,Ca,Fe,Mn,Zn and Cu concentra-tions were different during the differentiation,induction,differentiation periods and between leaf,node and fruit organs (P 0.05),but the Mg concentration was not different.The N,Ca,Fe,Mn,Zn and Cu concentrations were highest during the differentiation period while K and P were higher in induction and initiation periods,respectively.

Except for K and Zn,macro and micro element levels were approximately the same during both the induction and initiation periods (Table 1).The N,Ca,Fe,Mn,Zn and Cu levels were highest in leaves,but P and K levels were higher in fruits and nodes,respectively.Macro and micro element levels were generally lower in the node samples (Table 2).The P,Ca and Mg levels were not differ-ent in on and off years (P 0.05),but,N,K,Fe,Mn,Zn and Cu levels were different.N,Fe,Mn,Zn and Cu levels were highest in the on year and only the K level was higher in the off year (Table 3).Discussion

Hartmann et al.(1966)indicated that carbohy-drates might limit the formation of flowers on the tree during or following a heavy cropping season.Priestly (1977)suggested that a high carbohydrate level alone would not promote flowering if there was an adequate level of N in the leaves.However,our results showed that sugar levels did not appear to limit flower induction of olive,similar to the observations of Stutte and Martin (1986)and Sarmiento et al.(1976).

The results presented in the Table 4show that TS/N ratios during the differentiation,induction

Table 4.Total sugars (TS)and nitrogen (N)concentrations during the induction,initiation and differentiation periods in ‘on’and ‘off’year in Memecik olive tree.

TS**

TS #N**N #TS/N**on year TS/N #off year Off/on Differentiation 3.99* 2.99 1.01 1.07 3.95 2.790.71Induction 4.07 3.970.980.96 4.15 4.14 1.00Initiation

3.14

4.33

1.02

0.87

3.13

4.98

1.59

*Mean (%)of leaf,node and fruit content.**On year.#Off

year.

Figure 1.ABA,IAA,GA 3,GA 4and Z levels during the induction,initiation and differentiation periods of ‘Memecik’olive cultivar in on (*)and off (#)year.

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and initiation periods were different in the on and off years.They indicate that N levels during the differentiation period may play a role in bud differ-entiation in the on year,and since the off/on years ratio is1during the induction period,the C/N ratio seems to have no effect on induction.On the other hand,a higher C/N ratio(1.5times more in off than on year)during the initiation period,and an increased yield(data not shown)in the following year,suggest that sugar levels are associated with floral initiation in olives.This is consistent with the observations of Shimuzi et al.(1978)and Monselise and Goldschmidt(1981).However,Stutte and Martin(1986)suggested that starch and sugar increases during the year had no effect on flower initiation.In accordance with these results,we also suggest that carbohydrates may not have a direct effect on flower initiation.

The ABA,IAA and GA4levels in leaves,nodes and fruits during the induction,initiation and dif-ferentiation periods in the on year were lower than those in the off year(Figure1).However,GA3 levels during these periods were higher in the on year.Similarly,Pal and Ram(1978)in mango (Mangifera indica L.),Ulger et al.(1999)in olive and Chen(1990)in litchi(Litchi chinensis Sonn.) demonstrated higher GA3level in the on year.The fact that GA3decreased and GA4levels increased during the induction and initiation periods in the off year suggest that they affect flower bud forma-tion.Looney et al.(1985)determined that exogen-ous GA4application to apple(Malus domestica Borkh.)trees promoted flowering and yield,and Stephan et al.(1999)also noted that lack of GA4 induced a biennial bearing habit in apples.

Zeatin increase during the induction period in the off year suggests that an increase in cytokinins during the induction period possibly has a positive effect on floral formation.Chen(1987,1990), Mullins and Rajasekaran(1981)and Ramirez and Hoad(1981)mentioned similar results.

Many experiments show the direct involvement of different growth regulators in promoting or inhi-biting flower bud induction and differentiation. However,all these studies relate to the effect of a single regulator or its quantitative change before, during or after flower bud induction(Lavee1989). However,certain results contribute to the hypoth-esis that flowering is controlled by endogenous GA s(Lavee1985).Since TS and mineral nutrient levels were not different in on and off years in our experiment,we suggest that sugars and mineral nutrients are not directly involved in the control of flower formation in olive. Acknowledgements

This study was supported by The Scientific and Technical Research Council of Turkey,and Research Fund of the University of Akdeniz. The authors thank to Dr Levell Moser and Dr M.Z.Firat for critical reading and statistical analysis.

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