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Amino acids in kiwifruit 1. Distributionwithin the fruitduring fruit maturationE.A. MacRae a b & R.J. Redgwell a ba DSIR Fruit and Trees , Private Bag, Auckland , New Zealandb The Horticulture and Food Research, Institute of New ZealandLtd , Private Bag 92169, Auckland , New ZealandPublished online: 05 Jan 2012.

To cite this article: E.A. MacRae & R.J. Redgwell (1992) Amino acids in kiwifruit 1. Distributionwithin the fruitduring fruit maturation, New Zealand Journal of Crop and Horticultural Science, 20:3,329-336, DOI: 10.1080/01140671.1992.10421775

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NewZealand Journal ojCrop andHorticultural Science, 1992, Vol. 20:329-3360114-067119212003-0329 $2.50/0 The Royal Society of New Zealand 1992

Amino acids in kiwifruit1. Distribution within the fruitduring fruit maturation

329

E. A. MacRAE*R.J.~VVE[JL*

DSIR Fruit and TreesPrivate BagAuckland, New Zealand

*Present address: The Horticulture and Food ResearchInstitute of New Zealand Ltd, Private Bag 92169,Auckland, New Zealand.

Abstract The distribution of free amino acids indifferent tissues ofkiwifrnit (Actinidia deliciosa) andchanges in their concentration during maturation ofthe fruit were investigated. Amino acid concentrationsin the fruit (and each fruit tissue except the skin)decreased as the fruit matured. Asparagine andglutamine were the main amino acids to decrease,allowing arginine and y-aminobutyric acid to becomethe predominant amino acids in fruit harvested at theend ofMay. There were differences in individual andtotal amino acid concentrations between the core,inner and outer cortex fruit tissues. and with the fruitstalk. The core contained the highest concentration ofamino acids and the outer cortex the least InFebruary,asparagine was the major amino acid in the inner andouter cortex, whereas arginine and y-aminobutyricacid were predominant in the core. By May, arginineand y-aminobutyric acid were predominant in alltissues. Arginine and/or y-aminobutyric acid alwaysformed a longitudinal gradient within the fruit,regardless of tissue type. It was highest at the stemend and lowest at the blossom (distal) end.Concentrations of amino acids in the fruit stalkincreased as the fruit matured. Arginine and/or y-aminobutyric acid and asparagine were the majoramino acids to increase, followed by glutamine.

H91093Received11 November 1991; accepted 26 May1992

Keywords Actinidia deliciosa; kiwifruit; cv.Hayward; arginine; asparagine; glutamine; maturation

INTRODUCTIONPrevious investigations of the chemical constituentsofkiwifrnit (Aetinidia deliciosa (A. Chev.) CF. Lianget A.R. Ferguson) have indicated that the differenttissues vary in chemical composition, e.g., organicacid and starch (MacRae et al. 1989a). Calciumconcentrations have also been found to vary withinthe fruit longitudinally (Ferguson 1980), and werehighest in the inner cortex and at the stem end of thefruit. As well, some events in fruit ripening such ascell wall and starch degradation and softening do notoccur synchronously in all tissues (MacRae et al.1989b; Redgwell et al. 1990; Hallett et al. 1992). Inthese investigations no measurement was made offree amino acids in fruit. Fuke & Matsuoka (1982)found little change in total free amino acids duringkiwifruit growth and in fruit held for 1 month afterharvest. In their experiments arginine was thepredominant amino acid in fruit and decreased asfruit matured and ripened. Alanine content ina-easedwith ripening. Clark et aI. (1992) found that theconcentration of glutamine and asparagine ina-easedmarkedly early in fruit development, peaking at 9weeks after fruit set. After this time both amino acidsdeclined 2-3-fold as fruit continued to develop andmature, and the level ofseveral amino acids increasedduring low temperature storage. Arginine is regardedas a key amino acid in terms of nitrogen (N) storage,and Clark et al. (1992) found a strong correlationbetween arginine concentrations in the fruit and Nfertiliser regimes. Increased N resulted in increasedarginine concentrations.

In experiments on the fate of l4C-leaf photo-synthate in fruit during fruit maturation (MacRae &Redgwell 1990), decreased proportions of 14C_photosynthate were allocated to the amino acids asfruit matured. Except for fruit in February, theproportion of label found in amino acids also decreasedwith time after application of radioactivity. However,

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330 New ZealandJournalof Crop and Horticultural Science,1992,Vol.20

increased allocation of 14C-photosynthate to aminoacids in the fruit stalk occurred as the fruit matured.This may well be reflected in amino acid concen-trations,and indicatea major shift in requirementforaminoacids,a factornot found inconcurrentanalysisof leaf petioles.

We describe in this study the composition anddistribution of amino acids in the fruit and fruit stalkat three stagesof fruitmaturation.

MATERIALS AND METHODSPlant materialKiwifruitwere harvestedin 1986from a l();oyear-oldvinegrowingina blockat theDSIRResearch Orchardat Kumeu, New Zealand as previously outlined(MacRae & Redgwell 1990). On each samplingoccassion, fruit were harvested from two lateralsonopposingsides of the vine; four fruit per lateral;with4-6 terminal leaves on each lateral.The experimenttookplaceat threestagesof fruitdevelopment: in lateFebruary wben fruit were 55% of ftnal fresh weight,bad unripe seeds, and a soluble solids concentration00.9%; late Aprilwhenfruitwere82%offmal freshweight, seeds were mature, and fruit bad a solublesolids concentration of 5.4%; and late May wbenfruithada solublesolidsconcentration of9.2%. Fruitset occurredin early Decemberand furtherdetailsoffruitdevelopment arereported inMacRae& Redgwell(1990). Nitrogen as urea. totalling 140 kglha, wasappliedin earlyspring(August)andbeforeflowering(late October)in a ratio of 2:1.

The water solublefractionfromdifferentpartsofeachfruit(fruitstalkandskin; blossom(distal), middle

and stem ends of the outer cortex, inner cortex, andcore)wasextractedaspreviouslydescribed(MacRae& Redgwell 1990), and the amino acid fractionisolated.

Quantitation of free amino acidsAmino acids were analysed as their phenylisothiocyanate derivatives by highperformance liquidchromatography (HPLC) (Heinrikson & Meredith1984). A set of standards were co-derivatised withrepresentative kiwifruit amino acids to checkderivatisation efftciencies. Recoveryof eacb alwaysexceeded 95%. y-aminobutyric acid (GABA) wasnot quantifted by this technique as it co-chroma-tographed witharginine; and arginineconcentrationsreported will be an overestimate and are writtenarginine/GABA. Estimates from thin layerchromatograms (Bieleski& Turner 1966) indicatedthat arginine was present in greater quantities incomponentfruit tissues than GABA.This was in theorder of 100:1 in the core, 10:1 in the inner cortex,and2:1for theoutercortex.Whenadjustedforwholefruit (using 7.9% for core, 35.8% for inner cortex,and 53.6% for outer cortex as relative tissuecontributions) a value of 3:1 is obtained. This issimilar to the value reported by Clark et al. (1992):c. 2.5:1 for wholefruit.

Methionine was measured but has not beenreported because of variability in quantitation,probably because of its known instability and rapidoxidation during most extraction procedures.Methionine concentrations ranged from 0.01 to0.2 ~g1g fwt which representedfrom 0.1 to 1.6%ofthe total amino acids.

Table 1 Concentration of the total amino acids in kiwifruit fruit and fruit stalk during the later stagesof fruit maturation. Whole fruit values were calculated using the formula of core = 7.9 %fwt, innercortex =35.8 %fwt, and outercortex =53.6% fwt (see MacRae et aI. 1989a). The skin was not included.A number ofharvest parameters are included in the Table heading for reference. These are date or meanfruit measurements from eight replicates per harvest.

Total amino acid (Ilg!g fwt)Harvest date:Stalk (g fwt):Fruit (g fwt):

Tissue Soluble solids(%):

Feb0.59633.9

Apr0.67945.4

May0.731159.2

6915P

MacRae & Redgwell-Amino acids in kiwifruit 1. Distributionduring fruit maturation

4000I II Feb

3500 -f~ Apr

c:

3000 ~ ~ May0-co...

-- 2500c:-Q) ~o _

c:20000 C)o .......

C)"'0 ::L- - 15000co0c: 1000E

500

0

331

ASP GLU ASN SER GLN HIS ARG/GABA THR

Amino acidFig.l Concentration of amino acid in the fruit stalk in February, April, and May. Values are means SEM of eightreplicates, and annotated with the standard amino acid abbreviations.

StatisticsData fromtheeightsinglefruitreplicates weremeanedand eitherpresented withSEM,ocwithlevelsof signi-ficance calculated using the standard Students r-test,

RESULTS AND DISCUSSION

Amino acids of the whole fruit and changesduring fruit maturationThe total concentrationof amino acids in each tissueof the fruit decreased as the fruit matured (fable 1).Although there were changes in concentrations ofseveralamino acids these weremostly tissuespecific.The relative contribution of each tissue remainedsimilar at each stage of maturation; with the coreshowing the most variation, from 5% in February to7.9% in May.

In the whole fruit, asparagine and glutamineconcentrationsdecreased as fruit matured (fable 2).

Table 2 Changes in concentration of asparagine,glutamine, and arginine in the fruit during fruit maturation.Whole fruit values were calculated as for Table 1.

Feb Apr May

Amino acid concentration (Jlglgfwt)asparagine 116.7 P < 0.001 6.7 1.7glutamine 113.8 P < 0.001 29.7 12.7arginineiGABA 140.7 P < 0.05 80.6 76.9Amino acid proportion (%)asparagine 25 4 1glutamine 18 16 9arginineiGABA 20 29 36histidine 0.8 1.4 2.1

The decrease in asparagine was found in each tissue.Histidine, isoleucine, and phenylalanine remainedsimilar (data not shown). The proportionalcontribution of histidine and arginine/GABA to the

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50

c:o- 40co....

-c: _

Q) -o ~c: - 30o 0)0 ......

"'C 0)- :::L0_co 20oc:

E 10

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New ZealandJournalof Crop and Horticultural Science,1992,Vol. 20

ASP GLU ASN GLN ARG/GABA-

StemfZI Middleo Blossom

-

-

- ,

1

-

~ ... h nFeb Apr May Feb Apr May Feb Apr May Feb Apr May

Month

Feb Apr May

Fig.2 Amino acid concentrations in the outer cortex of the fruit in February, April, and May. Values are means SEMof eight replicates, and annotated with the standard amino acid abbreviations.

total amino acids increased, and that of asparagineand glutaminedecreased(fable 2).

The asparagine changes appear to be a genuineloss from the fruit and not a dilutioneffect. Ifvaluesare estimated on a per fruit basis, as outlinedaboveand using the fruit weights in Table I, there was7350 Jlg asparagine per fruit in February. In Aprilthis reducedto 630 ug and in May to 195 Jlg.

These results conflict with those of Fuke &Matsuoka(1982). Theyfoundlowlevelsofasparagine(-8 Jlg/gfwt) throughoutfruitmaturationand ripen-ing, implyingan increasein totalasparagineper fruitas fruit matured. Their value is similar to our Aprilsample and perhaps reflects a poor supply of N,differentfertiliserregimes,or a non-reducing sourceof N. Their total amino acid concentrations were,however, slightly higher than ours(~ Jlg/gfwt). The valuesof Clark et al. (1992) for individualamino acids such as asparagine and glutamine are

similar to or higher than ours. The trends with fruitdevelopment are similar, but are more dramatic inour study. For example, in Clark et al. (1992)asparagine decreased from c. 3 to 1.25 umol/g fwt,whereas in the current study asparagine decreasedfrom c. 0.8 to 0.01 umol/g fwt. A key differencebetween the two studies is the N fertiliser regime,and the way in which the amino acids wereextracted and analysede.g., fresh tissue versus drytissue.

ValuesforN inkiwifruitin AprilandMay canbefound in the literature. Ferguson & Eiseman (1983)reporteda valueof 0.53mg N/g fwt for fruit in April!May. Clark & Smith found values of 0.51-0.59 mgN/g fwt at a similar time of the season (1988) and1.42-3.3 mg N/g fwt depending on the stage of fruitgrowth(1991),andPrasadet al. (1984)0.24-0.71 mgN/g fwt. In our experiment, total amino acid inFebruarywas 0.48 mg/g fwt and in April 0.21 mglg

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MacRae & Redgwell-Amino acids in kiwifruit 1. Distribution during fruit maturation 333

200

c:0,-....

IV'-....c: _ 150Q) ....0 ~c:-0

0 C).......

"0 C) 1000 ::L.IV

0c:

E

50

o

ASP GLU ASN GLN ARG/GABA

-

StemIZI Middleo Blossom

-

-

-

In_ I m mFeb Apr May Feb Apr May Feb Apr May Feb Apr May

Month

Feb Apr May

Fig.3 Amino acid concentrations in the inner cortex of the fruit in February, April, and May. Values are means SEMof eight replicates, and annotated with the standard amino acid abbreviations.

fwt and May 0.15 mglg fwt When amino acid iscalculated in terms of N, in February a value of102 ug N/g fwt and in Maya value of 36 ~g1g fwt isobtained for fruit N in the form of free amino acids.When these are compared to reported values 20% offruit N occurred as free amino acids in February and7% in May. These values are lower than those reportedby Clark et al. (1992), and may reflect differences inN fertiliser regimes, all of which appear to be higherthan that in our study. An earlier study by Clark &Smith (1991) found that free amino acids contri-buted to between 7 and 43% of total fruit N, moresimilar to our experiment In their study vines hadone application ofNas urea (480 kg/ha) in September,but at a higher application rate than in ourinvestigation. ,

In our study atginine/GABA could haveaccounted for c. 5% of fruit N in May. In that ofClark & Smith (1991) arginine/GAB A was 50% of

fruit amino acid concentration at commercial maturityin May and hence probably contributed c. 3.5-4% oftotal fruit N.

Amino acids of the fruit stalk and changesduring fruit maturationTotal amino acid concentrations of the fruit stalkincreased as fruit matured (Table 1).The majoraminoacids in the stalk were arginine!GABA, glutamicacid, glutamine, and asparagine (Fig. 1). Arginine!GABA increased from 19% of the total amino acidsin February to 50% in May, and asparagine from 7%in February to 29% in May. Glutamic acid decreasedfrom 25% in February to 4% in May and glutaminefrom 15% in February to 8% in May.

All other amino acids increased in concentrationon a fresh weight basis during fruit maturation;although their proportional contribution to the totalamino acid pool decreased. The exceptions were

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334 New ZealandJournalof Crop and Horticultural Science,1992,Vol. 20

2000 ASP GLU ASN GLN ARG/GABA

1750c::::o

Blossom

IZI Middleo Stem

750

1500

1250

1000en.......

"'0 en

MacRae & Redgwell-Amino acids in kiwifruit 1.Distributionduring fruit maturation 335

inMay; in the innercortextheywere 20%inFebruaryand 38% in May.

Concentrations of isoleucine, leucine, andphenylalanine remained similar in all tissues at allstages of fruit maturation (data not shown). Valine,alanine, and threonine decreased slightly as fruitmatured. Concentrations of all these amino acidswere least in the outer cortex and greatest in the core.

The inner cortexdifferedfrom theother tissuesinthat high concentrations of aspartic and glutamicacids were found at the blossom (distal) end of thefruit Glutamine tended to be similar. This tissuecontainedseeds and the vascular tissuefeeding them.

In the skin of the fruit asparagine was the onlyamino acid to show significant loss during fruitmaturation (data not shown), and together withglutamic acid (30%), arginine!GABA (25%), andasparticacid (20%) formedc. 75% of the total aminoacid pool.

Tadeo et al. (1988) found that asparaginedecreased in the rind of navel orange duringripening,but not the juice sacs. In the rind it was the majoramino acid. Arginine increasedin both rind andjuicesacs, although it was not the predominant form ofamino acid in ripe fruit. Their findings implied thatasparagine in the fruit was continuallybeing utilised,becauseasparaginewas lost at a time when therewasexpected to be reduced synthesis in the roots andtransport to the fruit. The same may well be true forkiwifruit, in that both fruits are autumn/winterharvestedand both exhibita major lossof asparagine.

The increase found in asparagine in the fruit stalkin kiwifruit in April and May also implies however,that there may be some impediment to transfer ofasparagine to the fruit, or that the stalk has a strongerrequirementfor asparagine than the fruit.

Theunusualgradientfoundin thefruitforarginine!GABA isdifficultto interpretwithcurrentknowledge.It is the reverseof that found for sugar concentrationswhere % soluble solids is highest at the blossom(distal) end of the fruit in April and May (HopkirketaI. 1986; MacRae et aI. 1989a). A possibility is thatthere is limited movement of arginine once in thefruit. Because there are such large quantities ofarginine in the fruit stalk in April and May it ispossible that the high concentrationsin the stem endof the fruit are a "spill over" from the stalk.

Both the explanations for the patterns found forasparagine and arginine presume that they aretranslocated intact into the fruit and not synthesisedinsitu.Examinationofthexylemsap showsglutamineto be predominant early in fruit growth (Clark &Smith 1991),but arginine,asparticacid,andglutamic

acid may be found in similar quantities in April orMay.As arginineisnotbelievedtobea goodcandidatefor translocation, examination of phloem exudateandlabelling studiesshouldhelp toclarify thequestionof synthesis in situ versus translocation.

ACKNOWLEDGMENTS

We thank Noel Turner, Ross Ferguson, and Chris Clarkfor helpful discussions. The New Zealand KiwifruitMarketing Board provided partial funding for this work.

REFERENCES

Bieleski, R. L.; Turner, N. A 1966: Separation andestimation of amino acids in crude plant extractsby thin-layer electrophoresis and chromatography.Analytical biochemistry 17: 278-293.

Clark, C. J.; Smith, G. S. 1988: Seasonal accumulation ofmineral nutrients by kiwifruit 2. Fruit. Newphytologist 108: 399-409.

----1991: Seasonal variation of nitrogenouscompounds in components of the kiwifruit vine.Annals ofbotany 68: 441-450.

Clark, C. J.; Smith, G. S.; Boldingh, H. L. 1992: Effect ofnitrogen on the free amino acid composition ofkiwifruit during development and postharveststorage. Scientia horticulturae (in press).

Ferguson, A R; Eiseman, J. A. 1983: Estimated annualremoval of macronutrients in fruit and pruningsfrom a kiwifruit orchard. New Zealandjournal ofagricultural research 26: 115-117.

Ferguson, I. B. 1980: Movement of mineral nutrients intothe developing fruit of the kiwifruit (Actinidiachinensis Planch.). New Zealand journal ofagricultural research 23: 349-353.

Fuke Y.; Matsuoka, H. 1982: Changes in contents ofsugars, starch, organic acids and free amino acidsin kiwifruit during growth and after ripening.Nippon Shokuhin Kogyo Gakkaishi 29: 642-648.

Hallett, I. C.; MacRae, E. A; Wegrzyn, T. F. 1992:Changesin cell wall ultrastructure and cell packing duringpost-harvest ripening. Internationaljournalofplantsciences 153: 49-60.

Heinrikson, R. L.; Meredith, S. C. 1984: Amino acidanalysis by reverse-phase high-performance liquidchromatography: Precolumn derivatisation withphenylisothiocyanate. Analytical biochemistry 136:65-74.

Hopkirk, G.; Beever, D. J.; Triggs, C. M. 1986: Variationin soluble solids concentration in kiwifruit atharvest. New Zealand journal of agriculturalresearch 29: 475-484.

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MacRae, E. A.; Bowen, 1. H.; Stec, M. G. H. 1989a:Maturation of kiwifruit from two orchards:differences in the composition of the tissue zones.Journal ofthe science offood and agriculture 47:401-416.

MacRae, E. A.; Lallu, N.; Searle, A N.; Bowen, 1. H.1989b: Changes in the softening and compositionof kiwifruit (Actinidia deliciosa) affected bymaturity at harvest and postharvest treatments.Journal ofthe science offood and agriculture 49:413-430.

MacRae, E. A; Redgwell, R. J. 1990: Partitioning of 1"C_photosynthate in developing kiwifruit: distributionof radioactivity among metabolite fractions anddifferent fruit tissues. Scientia horticulturae 44:83-95.

Prasad, M.; King, G.; Spiers, M. 1988: Nutrient influenceon storage quality. New Zealand kiwifruit specialpublication no. 2: 22-24.

Redgwell, R. J.; MacRae, E. A. 1992: Amino acids inkiwifruit. 2. Distribution of 14C-Ieaf photosynthateamong free amino acids during fruit maturation.New Zealand journal of crop and horticulturalscience 20: in press.

Redgwell, R. J.; Melton, L. D.; Brasch, D. 1. 1990: Cellwall changes in kiwifruit following postharvestethylene treatment. Phytochemistry 29: 399-407.

Tadeo, J. L.; Ortiz, J. M.; Martin, B.; Estelles, A 1988:Changes in nitrogen content and amino acidcomposition of navel oranges during ripening.Journal ofthe science offood and agriculture 43:201-209.

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