naringin and neohesperidin levels development of leaves ...naringin and neohesperidin levels in...

Plant Physiol. (1992) 99, 67-730032-0889/92/99/0067/07/$01 .00/0

Received for publication July 30, 1991Accepted December 2, 1991

Naringin and Neohesperidin Levels during Development ofLeaves, Flower Buds, and Fruits of Citrus aurantium

Julian Castillo, Obdulio Benavente, and Jose A. del Rio*

Department of Plant Biology (Plant Physiology), University of Murcia, Campus de Espinardo,E-30071 Murcia, Spain

ABSTRACT

The distribution of the flavanones naringin and neohesperidinhas been analyzed during the development of the leaves, flowerbuds, and fruits of Citrus aurantium. These flavonoids are atmaximum concentration in the organs studied during the logarith-mic phase of growth, gradually decreasing until the organs reachmaximum development. However, this decrease in the naringinand neohesperidin concentration in leaves, flower buds, and fruitsis due to a dilution of the flavonoids caused by cell growth,because total content per organ continues to increase. The levelsof neohesperidin are always greater than those of naringin, al-though the ratio between the relative concentrations is differentin the three organs studied. Leaves have the highest ratios,varying between 8.83 and 5.18, followed by flowers (3.15-1.85),and fruits (2.23-1.02). These observations suggest different re-lationships between the respective enzymic activities in theirbiosynthetic pathway.

The flavonoids are an important class of plant secondarymetabolites and have been the subject of intense biochemicalstudies in recent years (7). Citrus species are of much interestbecause they accumulate large amounts of flavanone glyco-sides, whose aglycons are early intermediates in the flavonoidbiosynthetic pathway (9). Naringin, the 7-,3-neohesperidosideof naringenin (4',5,7-trihydroxy flavanone) and neohesperi-din, the 7-,3-neohesperidoside of hesperetin (3',5,7-trihy-droxy-4'-methoxy flavanone) are the most abundant flavo-noids in Seville orange (Citrus aurantium) (12, 16, 24).The interest in flavonoids in Citrus is due to their potential

physiological and biochemical activity (10), as well as to theirorganoleptic properties. Flavanone-7-neohesperidosides, par-ticulary naringin (1 1), are extremely bitter, whereas the iso-meric flavanone-7-rutinosides, narirutin and hesperidin, aretasteless (27). In addition, flavanone distribution within dif-ferent Citrus species can be quite distinctive (1). Therefore,naringin and neohesperidin concentrations can be used todifferentiate grapefruit, sour orange, and K-early (tangerinex grapefruit hybrid) juice (28). Neohesperidin is a naturalsource of an intense sweetening agent, neohesperidindihydrochalcone (3).

Little information is available conceming the individualreactions of the biosynthesis of these compounds in Citrus.The enzymes that take part in the biosynthesis of naringenin,as well as its subsequent glycosylation to naringin, have beencharacterized (4, 8, 19, 20, 22, 23, 26). However, the biosyn-

thetic pathways of 4'-methoxylated glycosyl flavanones havenot been fully described.As part of a continuous study of the biosynthesis of glyco-

side flavonoids in Citrus, we have studied the distribution ofnaringin and neohesperidin during the development of leaves,flower buds, and fruits of Seville orange. This study of narin-gin and neohesperidin distribution will permit us to know inwhich stages of the development their levels are highest, toisolate and characterize in the future the key enzymes of thebiosynthetic pathways of both flavonoids.

MATERIALS AND METHODS

Plant Materials

Leaves, flower buds, and fruits were obtained from 5-year-old Citrus aurantium cv Sevillano trees, grown in greenhousesof the University of Murcia. The phloematic fluid from thereceptacles of decapitated flower buds were collected with acapillary inserted in the ovary hole. The capillaries (1.5 mmi.d.) were left in the plant for 48 h, and their content wasweighed and immediately dissolved in DMSO for analysis.

Chemicals

Naringin, neohesperidin, naringenin, and hesperetin wereobtained from Zoster S.A., Murcia, Spain.

Extraction, Chromatographic Analysis, Isolation, andHydrolysis Conditions of Flavonoids

Ten leaves and five flower buds and fruits were collected,immediately dried at 50°C (13), and ground; the flavonoidswere extracted with DMSO in the ratio of 2 mg/mL foranalytical chromatography. These measurements were re-peated for four trees, and the mean values obtained at eachage were used to express the distribution of flavonoids inleaves, buds, and fruits. For the isolation ofcompounds 4 and7 (Fig. 1), 15 g of leaves (1 1-20 d old) and 15 g of fruits (3-20 mm diameter) were extracted with DMSO in the ratio 200mg/mL. The solutions were filtered through a 0.45-,um nylonmembrane.For the location of naringin and neohesperidin in the young

leaf, flower bud, and fruit extracts of Citrus aurantium, weused a gBondapak C18 (250 x 4 mm i.d.) analytical columnwith an average particle size of 5 ,um and several solvents: (a)isocratic H20-Me'OH-acetonitrile-HO acetyl (15:2:2:1); (b)

' Abbreviation: Me, methyl.67

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isocratic MeOH-0.0l M H3PO4 (1:2); and (c) gradient solventA (MeOH) and B (0.01 M H3PO4), the linear gradient profilebeing 30 to 70% of A in 20 min. In all solvent systems, theflow rate was 1 mL/min at room temperature. The HPLCanalysis was performed using a Beckman liquid chromato-graph with a model llOB solvent delivery module and aSystem Gold Module 168 diode array detector (BeckmanInstruments, San Ramon, CA). The absorbance change wasmonitored at 280 nm. Naringin and compound 4 showed, inthe different solvent systems, the following retention times:20.33 min (solvent 1), 24.72 min (solvent 2), and 15.34 min(solvent 3). Neohesperidin and compound 7 showed the fol-lowing retention times: 30.34 min (solvent 1), 37.11 (solvent2), and 16.61 min (solvent 3).For the isolation ofcompounds 4 (naringin) and 7 (neohes-

peridin), the following semipreparative column was used:Nucleosil C,8, 5 ,um (250 x 10 mm i.d.), eluted with H20-MeOH-acetonitrile-HO acetyl (15:2:2:1) and a flow rate of 3mL/min. For the identification and isolation of the sugars ofboth compounds (rhamnose and glucose), an IR detector(Shimadzu Co., Kyoto, Japan) and the following semiprepar-ative column was used: Nucleosil-NH2 7 um (250 x 10 mmi.d.) eluted with acetonitrile-H20 (85:15) and a flow rate of 3mL/min at 30°C (14). Products 4 and 7 and the respectivesugars of the hydrolysis were isolated by several chromato-graphs in their respective semipreparative columns, and thefractions were collected with a Pharmacia FRAC 100 (Phar-macia LKB Biotechnology, Uppsala, Sweden).

Product 4 (1 g), from several semipreparative chromato-graphs ofplant material extracts, was hydrolyzed in 20 mL of2N sulfuric acid under reflux for 2 h, and a white precipitatewas obtained. The cooled suspension was neutralized withNaOH and the precipitate filtered and washed with distilledwater. A white precipitate (0.4 g) was obtained (compoundIV). Product 7 (1.5 g), from several semipreparative chromat-ographs of plant material extracts, was hydrolyzed in 250 mLof 2N sulfuric acid under reflux for 6 h, and a white-beigeprecipitate was obtained. The cooled suspension was neutral-ized with NaOH and the precipitate filtered and washed withdistilled water. A white-beige precipitate (0.6 g) was obtained(compound VII). Compounds 4, 7, IV, and VII were identifiedby their melting points (Gallenkamp, Loughborough, Eng-land), mass spectra (electron impact MS) (Hewlett-PackardCo., Palo Alto, CA), and the 'H NMR (200 MHz) and '3CNMR (50 MHz) spectra (Bruker, Bremen, Germany) in hex-adeutero-DMSO, which was capable of dissolving the prod-ucts. The sugars were identified by their mass spectra (electronimpact MS) (Hewlett-Packard).

RESULTS

Identification of Naringin and Neohesperidin in Leaves,Flower Buds, and Fruits of C. aurantium

The analysis by HPLC ofDMSO extracts of leaves, flowerbuds, and fruits during their development shows the presenceof many flavonoid structures. Figure 1 shows a characteristicchromatogram of an extract from C. aurantium fruits (5-6mm diameter). The chromatograms of leaf and bud extractsshowed the same peaks, although with different relative pro-

z 0.20

0.0

N 4

0.1

0.

0 10 20 30 40

TIME (MAIN)Figure 1. Characteristic HPLC of an extract with DMSO from

immature fruits (5-6 mm diameter) of C. aurantium, using a MBonda-pak C18 (250 x 4 mm i.d.) analytical column with an average particlesize of 5 um. It was eluted with water-methanol-acetonitrile-aceticacid (15:2:2:1) with a flow rate of 1 mL/min at room temperature.Eluent monitored at A28o. 1, Neoeriocitrin (25); 2, 3, 6, unknownflavones/flavonols; 4, naringin; 5, unknown flavanone; 7, neohesper-idin; 8, rhoifolin; 9, neodiosmin (6).

portions. Peaks whose spectra are similar to or identical withflavanone-, flavone-, or flavonol-type structures are numbered(1-9). In this chromatogram, compounds 4 and 7 are of notebecause they present a retention time identical with that ofnaringin and neohesperidin and coincide with those shownby the other chromatographic systems (see "Materials andMethods"). Despite this similarity, these compounds wereisolated to confirm their identity.The absorption spectra ofthese compounds show two max-

ima (in the elution solvent of Fig. 1), at 283 and 326 nm forcompound 4 and 284 and 327 for compound 7. These dataare consistent with the compounds having flavanone skeletonsidentical with those of naringin and neohesperidin. Thesecompounds were isolated with a semipreparative column (see"Materials and Methods"), and two white products wereobtained, with melting points of 171 and 244°C, respectively.

Hydrolysis of compounds 4 and 7 in 2N sulfuric acid gavea white (compound IV) and white-beige (compound VII)precipitate, respectively. Compounds IV and VII had UVspectra, melting points, mass, 'H NMR and "3C NMR spectraidentical with those of the flavanones, naringenin and hesper-etin, respectively. The sugars resulting from hydrolysis wereisolated by semipreparative chromatography (see "Materialsand Methods") and identified as rhamnose and glucose bycomparing their mass spectra with those of authentic sugars.Both 'H and 13C NMR spectra for compounds 4 and 7corresponded to the flavanone structure (21) with glycosyla-tion at position 7, which is characteristic ofa neohesperidosidestructure (18). This led us to believe that the compounds inquestion referred to naringin and neohesperidin, respectively(18, 21).

Changes in the Levels of Naringin and Neohesperidinduring Development of C. aurantium LeavesBoth flavanones are at their highest concentration in the

logarithmic phase of development (Fig. 2). Neohesperidin is

68 CASTILLO ET AL.



NARINGIN AND NEOHESPERIDIN LEVELS IN CITRUS AURANTIUM

150

30 125w

100 ow20l

25~

0 *o00 10 20 30 40 50

LEAF AGE (DAYS)

Figure 2. Accumulation of naringin (A) and neohesperdin (A) con-

centration and changes in the mean total content per leaf of naringin(0) and neohesperidin (0) according to age in C. aurantium. Verticalbars, ± SE when larger than symbols.

always more concentrated than naringin and is 8.83 timesgreater in 1 l-d-old leaves. However, these concentrations are

not constant and gradually decrease as leaves grow. Thesedecreases in concentration take place at different rates, andwhen the leaf is fully developed, the neohesperidin concentra-tion is only 5.18 times greater than that of naringin.Both naringin and neohesperidin increase on a per leaf

basis as the leaf grows (Fig. 2). Furthermore, the rate ofneohesperidin synthesis and/or accumulation is greater thanthat for naringin. These results confirm that the decreases inconcentration ofboth flavonoids in Figure 2 are possibly dueto a dilution effect (15). However, the decrease in the relativeproportions ofthe concentrations ofneohesperidin and narin-gin seems to indicate the presence of a different turnover foreach.

Changes in the Levels of Naringin and Neohesperidinduring Development of C. aurantlum Flower Buds

Buds reach their maximum development after 27 to 30 d,and the fresh weight decreases drastically as the petals drop(Fig. 3). Both flavonoids show an initial decrease in concen-

tration as the bud swells. No differentiated structures appearat this stage. Old buds (9 to 11 d) show an increase in naringinconcentration from 6.49 to 11.37 ,umol/g fresh weight by 19d. The neohesperidin concentration increases after 16 d andreaches 25.52 gmol/g fresh weight at 18 d. These increasescoincide with the formation of the different organs of theflower (ovary, petals, stigma, filaments). Subsequently, duringbud enlargement, there is a second decline in concentrationbefore it increases again, coinciding with pollination and thefall of petals and stamens.The observed decrease in the concentration of both flavo-

noids seem to be linked to dilution resulting from cell growthin the buds, similarly to that described in the leaves, ratherthan to a degradative process. The first increases detected innaringin and neohesperidin are observed during the divisionand differentiation stages of the cells. However, the subse-quent increase after pollination (3 l-d-old buds) is principally

%0c:i 30400

o 0

w

0 5 10 1 $2 5 0 3590.-O ;gj:C Xt6

0 == -"~ i i

15 20 25 30 35 40

TIME (DAYS)

Figure 3. Accumulation of the mean fresh weight per flower bud (A)and of naringin (0) and neohespendin (0) concentration according toage in C. aurantium. Vertical bars, ± SE when larger than symbols.

due to the fall ofpetals. The petals represent a large proportionof total flower weight and dilute the total concentration ofthe flavonoids. This can be confirmed by analyzing naringinand neohesperidin levels in the different parts of the flower.Table I shows the concentration (gmol/g fresh weight) andpercentage of each of these flavonoids contributed by thedifferent parts ofthe flower and the neohesperidin to naringinratio in each one of these parts. The ovary and stigma showthe highest concentrations of both flavonoids, followed byfilaments, receptacles, petals, and anthers, in decreasing orderof concentration. When the contribution of each of the struc-tures to the total content per flower of these flavonoids isanalyzed, we find that the petals are responsible for 25.09 and15.61% of neohesperidin and naringin, respectively, because,although their concentrations are low (4.57 and 5.41 imol/gfresh weight, respectively), they represent a high percentageof the total weight of the flower (42.34%). The unequaldistribution of both flavonoids within the flower is shown inTable I. In the receptacle and petals, they are present inalmost equal proportions, whereas in the stigma and anthersneohesperidin is, respectively, 3.83 and 3.41 times more con-

centrated than naringin and is more than double in the ovary

(2.42). These different ratios between the different bud struc-

Table I. Levels of Neohesperidin and Naringin in Different Parts ofthe 30-d-Old Flower Buds of Citrus aurantium

The concentration and contribution of each flower structure to thetotal content in the flower and the ratio between the concentrationsof both flavanones in the different structures are represented.

Concentration % of TotalStructure Ratio

Nanngin Neohespendin Nanngin Neohesperidin

pnmoI/g fresh wtReceptacle 8.10 7.93 18.17 9.34 0.98Ovary 59.82 144.81 26.28 33.39 2.42Stigma 16.91 64.86 11.37 22.88 3.83Petals 4.57 5.41 25.09 15.61 1.18Filaments 6.79 12.42 18.50 17.75 1.83Anthers 1.22 4.16 0.59 1.02 3.41

69




tures suggest the existence of different turnovers for eachflavonoid.The overall levels of naringin and neohesperidin in buds

show a similar pattern to those in leaves, and their develop-ment is accompanied by an increase of both flavonoids (Fig.4). Maximum values are reached at full development of thebud, and the final decrease in both values is due to petal lossbecause these are responsible for approximately 25% of over-all flavonoid content, as mentioned above. Neohesperidinlevels are greater than those of naringin throughout develop-ment, as in leaves.

Changes in the Levels of Naringin and Neohesperidinduring Development of C. aurantium Fruits

Growth of the fruit of bitter orange, as measured by fruitdiameter, is sigmoidal (Fig. 5), reaching its maximum size (61mm diameter) at approximately 200 d. From this time, theprocesses of maturation begin, with no appreciable change indiameter. Neohesperidin levels are higher in the first stages ofdevelopment, coinciding with the "lag," and reach their high-est concentration in fruit of 5 to 6 mm diameter. When thelinear phase begins, a marked decrease in neohesperidin isobserved. Naringin concentration remains constant in fruitsup to 15 mm diameter, at which stage it decreases to reachthe levels of neohesperidin in the senescence phase. Withregard to the overall content ofboth flavanones per fruit (Fig.6), the increase in synthesis and/or accumulation rates in theday following anthesis is similar to the pattern found in leavesand buds. The neohesperidin synthesis and/or accumulationrate exceeds that of naringin (Fig. 6, inset), although afterfruits reach 10 mm diameter these rates even out. Thiscontrasts with that which occurs in leaves, in which neohes-peridin rates remain higher than those for naringin through-out development. This suggests that during fruit developmentboth flavonoids experience a change in their metabolism andreach maximum levels in fruit of 30 to 35 mm diameter.

8.0

6.0

4.0 5 T

0

~2.0

0.0~ ~0 15 20 25 30 35 40

TIME (DAYS)

Figure 4. Changes in the mean total content per flower bud ofnaringin (0) and neohesperidin (@) according to age in C. aurantium.Vertical bars, ± SE when larger than symbols.

Figure 5. Accumulation of mean fruit diameter (0) and naringin (0)and neohesperidin (A) concentration according to age in C. aurantium.Vertical bars, ± SE when larger than symbols.

Relationships between Neohesperidin and Nanngin in theDevelopment of Leaves, Flower Buds, and Fruit ofC. aurantium

The ratio between neohesperidin and naringin concentra-tions during the development of the leaves decreases from8.83 for those 11 d old to 5.18 when they reach their maxi-mum size (43 d) (Fig. 2). In buds and fruits, the values of thisratio are always smaller than those described for leaves. Inbuds, they are higher than 3 during the first phase of thelogarithmic stage of growth (1-10 d) and decrease sharply tovalues slightly greater than 2 during the stages in which thedifferent parts of the buds are differentiated. There is thenanother slight decrease to 1.61 while the bud finishes devel-oping. The bud then opens, is fecundated, and loses petalsand stamens; this process increases again slightly to almost 2(Fig. 3).

In fruits, this ratio varies between 3.15 and 1.02, and threedistinct zones are discernible where these values oscillate. Itincreases from 2.23 to its maximum of 3.15 as fruits grow

from 3 to 7 mm in diameter. The ratio then decreases sharplyto 1.13 in fruits of 35 mm diameter. At this size, the fruit ofC. aurantium accumulates maximum overall levels of both

1.0 T TT

0 1 0 20 30 4050.100O.6- .0

0.10

0.4-/.0

0E26 00.0046

~0 -4 6 -8 10

0.0-oe

0 10o 20 30 40 50 60

DIAMETER FRUIT (MM)

Figure 6. Changes in mean total content per fruit of naringin (0) andneohesperidin (0) according to age in C. aurantium. Vertical bars, +SE when larger than symbols.

(AL&

0

Lfl0I

2

E

* t=

TIME (DAYS)

70 CASTILLO ET AL.




flavonoids. From this stage onward and until maximum sizeis reached, the ratio decreases gradually to reach a value of1.02. At this stage, the fruit completes its development, andthe period of maturation begins.

Relationships of Neohesperidin and Naringin Levelsamong Leaves, Flower Buds, and Fruits during the FirstStages of the Reproductive Cycle in C. aurantium

Table II shows the distribution of these flavanones in thedifferent organs of C. aurantium during the initial stages ofthe reproductive cycle. The respective concentrations of neo-hesperidin and naringin and the relationship between themare shown in leaves before anthesis, during the final stage offlowering, and during the logarithmic and linear phase of fruitgrowth; in 30-d-old buds; in the stems that attach these budsto the tree; in the phloematic fluids of the receptacles of thesebuds; and finally, in fruits of 10 and 25 mm diameter.Neohesperidin and naringin concentrations are higher inleaves than in the buds and the stems ofthese buds. However,even higher concentrations are found in the phloematic fluids,especially in the case of naringin. The highest ratio betweenthe two is found in leaves, followed by stems and fluids; thelowest ratio is in buds at 30 d.As indicated before, the synthesis and/or accumulation rate

of neohesperidin in fruits up to 10 mm diameter is greaterthan that of naringin, and the values tend to even out whenthe fruits reach this size (Fig. 6, inset). When the fruits reach25 mm in diameter, the flavanones begin to reach their highestlevels with regard to quantity per fruit (Fig. 6). Fruits of 10mm diameter show high quantities of neohesperidin andnaringin, which is much higher than those of leaves, and theratio between both concentrations is almost three timesgreater.

This tendency remains the same when the fruits reach 25mm diameter, although the concentration of both flavonoidsdecreases in leaves and fruits. The greatest decrease in con-centration is shown by neohesperidin, which is approximately

Table II. Concentration of Neohesperidin and Naringin and theNeohesperidin to Naringin Ratio in Different Organs of C. aurantiumat the Outset of the Reproductive Cycle

Nanngin Neohespendin Ratioa

pmol/g fresh weightLeaf (130 mm in length)

Before anthesis 3.83 19.84 5.18In flowerb 4.56 19.86 4.35In logarithmic phase of fruit 2.56 2.74 1.07growth

In the linear phase of fruit 1.64 1.56 0.95growth

Fluids in flower receptaclec 9.74 20.74 2.13Stemsd 1.55 3.73 2.40Fruit10 mm diameter 56.07 158.99 2.8325 mm diameter 49.39 76.91 1.56a Ratio between the concentration of both flavanones. b Leaves

near 30-d-old buds. c In receptacles of decapitated 30-d-oldbuds. d Stems that attach the 30-d-old buds to the tree.

50% in fruits and 40% in leaves. Naringin decreases muchless drastically in both organs. This means that in leaves theneohesperidin to naringin ratio remains practically constantat approximately 1, whereas in fruits it decreases from 2.83to 1.56.

DISCUSSION

In this paper, we have used a profile protocol to examinethe distribution of the flavanones, naringin and neohesperi-din, in the leaves, buds, and fruits of C. aurantium duringtheir development. By means of semipreparative chromatog-raphy and structural studies ofthe aglycons and sugars presentin the molecules, we confirmed the presence of neohesperidinand naringin in the above-mentioned organs. The main aimof this paper is to describe naringin and neohesperidin levelsduring the development of the three organs. In this way, wehope to obtain the necessary information to improve isolationand characterization of the specific enzymes involved in thebiosynthetic pathways of these flavonoids in C. aurantium inthe future.The quantitative studies described here concentrate on

three particular aspects in the three organs analyzed: themeasurement of the concentrations and overall quantities ofboth flavanones in each organ and the calculation of theneohesperidin to naringin ratio. From these data it is possibleto reach a series of conclusions regarding the metabolism ofthese flavonoids in C. aurantium. Some have already beennoted for each particular organ; an overall summary is givenbelow.The relative levels of these molecules are highly character-

istic of a given organ and are markedly affected by the age ofthe developing organ. Our results confirm previous findingsconcerning flavanone-glycoside accumulation in Citrus par-adisi and Citrus limonia (1, 15, 29), which demonstrated thatthe concentration is greater in young tissues and that there isa decrease in all tissues during maturation (2). The high levelsof these flavonoids during the lag of development might beconnected with protection against both insects and pathogens.However, it must be pointed out that in both leaves and

fruits the decrease in concentration observed, as stated in"Results," is due to dilution of enlarging cells, because duringthe development of these organs, before they enter theirrespective maturation periods, the overall quantities of narin-gin and neohesperidin increase. It is evident that the slopesobserved for the decrease in concentration and increases inoverall flavonoid quantities are different for both organs,suggesting the presence of different metabolisms for bothflavonoids in leaves and fruits of C. aurantium. On the otherhand, different synthesis and/or accumulation profiles existfor naringin and neohesperidin in the same organs, but thisdoes not imply that the flavonoids are derived from differentprecursors. It is more likely that the variations observed are aresult ofthe change in activity ofcertain late enzymes involvedspecifically in the formation of the two related aglycons.

In C. aurantium buds, as has been described in the "Re-sults," no constant pattern is observed for the changes in theconcentration of either flavonoid, because the cellular differ-entiation stages and subsequent growth of the bud organs,which produced respective increases and decreases in these

71




concentrations, do not occur simultaneously. Therefore, thedelay in neohesperidin concentration increase, when com-pared with that of naringin, might indicate the precursor roleof naringenin in neohesperidin synthesis (20).The study of the neohesperidin to naringin ratio during

leaf, bud, and fruit development casts some light on thebiosynthetic pathways ofthese flavanones. This ratio generallydecreases during the development ofthe organs studied. How-ever, the rates of the decline and the absolute values of theneohesperidin to naringin ratio are not the same in thedifferent organs. The high ratios detected in leaves suggestthat there is high activity of the enzymes involved in thesynthesis of neohesperidin from naringenin (20). This is par-ticularly true of methyltransferase activity (5, 17) in the lag ofleaf development, although there is a subsequent diminutionof this activity as the leaf grows. Our previous experimentsshowed the presence of a high 4'-O-methyltransferase activityin C. aurantium leaves of<20 mm in length (data not shown),which seems to confirm this suggestion. The decrease inactivity might be partly due to a diminution in S-adenosyl-methionine levels, given its active participation as a methyl-ating agent in different components of the cell wall, which areactively synthesized during plant cell growth.Buds and fruits present lower neohesperidin to naringin

ratios. During bud development, the ratio between both fla-vonoids oscillates and is related with the stages of cell divisionand the subsequent development of different parts of theflower. These results are in accordance with those of otherstudies that suggest that these flavonoids are rapidly synthe-sized in tissues during the cell division phase and that synthesisslows down in the cell enlargement phase (1).The data in Table I confirm that the flower organs that

present the highest mitotic activity (ovary and stigma) havethe highest concentrations of these flavonoids and the highestneohesperidin to naringin ratios, similarly to that observed inleaves during their cellular division stage (20 mm in length).A study of all the values described in Table II concerning

the first stages of the reproductive cycle of the tree (concen-trations of neohesperidin and naringin and their relationship)provides valuable information regarding the presence or ab-sence of synthesis of these flavanones in the different organs.An analysis ofthe phloematic fluids in the receptacle (Table

II) of these buds suggests that both flavonoids may be trans-ported to the different organs in the tree. Given that theneohesperidin to naringin ratio in leaves during this period(4.35) is much higher than that in the fluids, it can be deducedthat naringin is more readily transported through the phloem.It can also be seen that naringin concentration is higher inbuds than in leaves, the neohesperidin to naringin ratio beingnoticeably lower (1.67). The fact that this ratio is also lowerthan that in the stems and fluids examined seems to indicatethat in this period the buds have a greater capacity forsynthesizing naringin than neohesperidin. These data seem toconfirm that this capacity for naringin synthesis is the causeof the decrease in the ratio between both flavonoids in budsat the conclusion of their swelling stage (1-10 d) and at themoment that the differentiation of the flower organs begins(10-19 d). Thus, the variations observed in neohesperidin tonaringin ratios in the different organs of the buds may be dueto a differential exogenous contribution of both flavonoids to

the tissue and/or to changes in the activity of enzymes in-volved in the synthesis of these compounds.The higher concentrations of neohesperidin and naringin

in fruits of 10 mm diameter than in leaves (Table II) seem toindicate that the fruits are important sites of synthesis and/oraccumulation of these flavanones. Although in fruit of 25mm diameter the concentration of neohesperidin and narin-gin and their ratio remain higher than the respective valuesin leaves (Table II), the decrease in these concentrations andratios points to a diminution in the activity of flavanonesynthase enzyme and of the enzymes involved in the subse-quent synthesis of neohesperidin during the development ofthe fruits (20).The final conclusion that we can reach is that all the organs

analyzed of the C. aurantium tree are capable of synthesizingand/or accumulating these flavanones. It is clear that there isa process of flavonoid transport as is shown by the levels ofneohesperidin and naringin in phloematic fluids and by ourpreliminary results in the study of this phenomenon (data notshown). It can also be concluded that in all organs studies thebiosynthesis of these flavonoids is more intensive during thestages of cell differentiation and less intensive in periods ofgrowth and subsequent maturation and that their biosyntheticpathway is regulated differently in each organ.

LITERATURE CITED

1. Albach RF, Juarez AT, Lime BJ (1969) Time of naringin pro-duction in grapefruit. J Am Soc Hortic Sci 94: 605-609

2. Albach RF, Redman GH, Cruse RR (1981) Annual and seasonalchanges in naringin concentration ofRubi Red grapefruitjuice.J Agric Food Chem 29: 808-81 1

3. Bar A, Borrego F, Benavente 0, Castillo J, Del Rio JA (1990)Neohesperidin dihydrochalcone: properties and applications.Lebensm Wiss U Technol 23: 371-376

4. Berhow MA, Vandercook CE (1989) Biosynthesis of naringinand prunin in detached grapefruit. Phytochemistry 28:1627-1630

5. Brunet G, Ibrahim RK (1980) O-Methylation of flavonoids bycell-free extracts of calamondin orange. Phytochemistry 19:742-746

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