Lebensm.-Wiss_ u -Techno!., JO, 379-389 (1997)
Actinidin Levels in Fruit of Actinidia Species and Some Actinidia arguta Rootstock-Scion Combinations Stewart Boyes, Peter Striibi and Hinga Marsh
S. Boyes, P. Striibi: Food Science and Technology, The Horticulture and Food Research Institute of New Zealand Ltd., Mt. Albert Research Centre, Private Bag 92169, Auckland (New Zealand) H. Marsh: Te Puke Research Centre, The Horticulture and Food Research Institute of New Zealand Ltd., No. 1 Road, RD2, Te Puke (New Zealand) (Received May 20, 1996; accepted August I, 1996}
Actinidin, the kiwifruit protease, was measured in the fruit of a number ofActinidia species during their growth, at harvest and during subsequent storage. Actlr::ldla deliciosa cv. Hayward (kiwifruit) fruit actinidin levels were compared to fmit levels measured in cutting-grown plants of species to be used as rootstocks, A arguta selections and some rootstock-scion combinations. The fruit showed a wide range of actin/din activities, which were influenced by the scion and rootstock genotype. Some trends were seen in rootstock-sdon combinations; rootstocks whose fruit had low actinfdin levels conferred low levels in the fruit of grafted scion species. Simllariy, high levels were seen in sdon fruit whose rootstock had high levels. There was a significant sdon influence in som1! combinations. The correlation between actinidin and protein levels suggested control at transcription. An SDS-PAGE gel showed that the density of the actin/din band correlated w/th the measured actin/din levels. The data suggested that variation in actfnidin activity was not due to endogenous inhibitor contTo/ or the presence of an inactive zymogen.
©1997 Academic Press Limited
Keywords: Actinfdia; A arguta; actlnidi.n; rootstock; scion
Introduction effect of rootstocks has been simplified to three main effects: water and nutrient absorption and metabolism; Actinidia fruit sold on the wor.:d market are dominated transport of water, nutrient and metabolised com by those of a single cultlvar, Acttnidia delidosa cv. pounds to the scion; and auxin formation (10). Two Hayward (kiwifruit). Other species or cultivars of recent reviews explain the mechanism by which the Actinidia may have commerci2l potential, with changes rootstock affects the scion (13, 14). As the scion and the in characteristics such as taste and harvest time, and/or rootstock retain their own genetic characteristics, the be considered as a separate :'ruit. Actinidia arguta is resuH.:s of a scion-rootstock combination will depend on widely grown, occupying areas in the Northern Hemi the compatibility of transport tissues as well as their sphere where kiwifruit does not survive, as far north as independent metabolism, for example, some organic Eastern Siberia. For this reason it is sometimes known compounds contained in the rootstock may or may not as the 'hardy kiwifruit'. The geographical distribution of pass through the graft union. A. arguta results in a wide variation in physiological One of the trial rootstock-scion combinations is repli characteristics such as time of flowering, yield, size, cated but includes an interstock. Interstock influence colour of fruit and harvest time (1). The edible green on the scion fruit may be similar to the same plant used skin and a distinctively sweete: taste than kiwifruit are as a rootstock, suggesting that control is through the desirable characteristics for the commercialisation of A. stem of the rootstock. In other cases the interstock and arguta. rootstock effects of the same plant on an identical scion The biochemical properties of kiwifruit actinidin have may be different, suggesting influence is through the been studied (2-5), including the development of root system (13). actinidin activity during ripening (6.1') and storage (8). Kiwifruit rootstock trials in New Zealand showed that The possible use of actinidin in the food processing each rootstock produced marked effects on flower industry has been evaluated (S). production, bud burst, starch mobilisation and the Rootstocks are commonly used in many fruit crops and formation of crystalline calcium oxalate in idioblast have been demonstrated to affect characteristics such cells (11, 12). This research was used in the seleclion of as flower number, fruit yield, o::ganic acid and carbohy rootstocks for A. arguta. Grafting trials on rwo kiwifruit drate levels, size. taste and colour of fruit (10-12). The rootstocks in Italy showed that rootstock influenced ------0023-6438/97/040379 + 11 $25.00/0/fs960193 ©1997 Academic Press Limited 379 !;vtlvol. 30 (1997) No. 4
fruit yield. firmness, soluble solids, flesh colour and Table i Experimental rootstock-scion combinations storage quality of the fruit (15). Scion Rootstock The contribution of actinidin to the taste of kiwifruit BILl A. arguta has not been investigated. Ta5.te panels in New Zealand var. arguta (arguta) A. arguta var. purpurea (purpurea) have identified some kiwifruit as causing 'mouth burn' A. arguta x A. deliciosa hybrid (arguta x deliciosa) which varied from an 'unpleasant sensitivity of the A. deliciosa (deliclosa) mouth'. to a 'burning sensation to the tongue, inside of A. polygama (polygama) the mouth and lips in the most extreme cases' (16). This BlFZ A. delidosa P.ffect was different from 'catch' caused by crystalline A.polygama oxalic acid (17). Actinidin could exacerbate the irrita AAME A. delidosa tion and c:oughing response tl1at accompanies catch. A.polygama The aim of the study was to c.etermine the variation A. polygama with A. deliciosa interstock in {polygama-dellciosa) actinidin activity in various Actinidia species, and the influence of scions and rootstocks on the chemical AA A. deliciosa composition of A. arguta fru.t. Factors A.polygama were included A. hemsleyana (hemsleyana) which could affect act!nidin levels, some noted from previous work on kiwifruit. such as soluble solids The botanical rootstock name is followed 0 by an abbreviated content ( Brix), titratable acidity, fruit size and yield version (in parenthesis) to be used in the text to identify rootstocks. per vine and fruit colour (red anthocyanin pigments).
Materials and Methods per channel. Actinidin was identified by co-chromato graphing purified actinidin (19). Actinidin preparation and ass<~ Six fruit samples from each vine, each consisting of 10 to 15 fnJit, were prepared in the following manner. Other methods except for kiwifruit. Whole fresh fruit were weighed. The Coomassie Blue assay (20) cut into quarters was used to determine and put into an equal weight of ice soluble protein levels. cold phosphate buffer (50 mmol!L) containing sodium Soluble solids concentration (SS). quoted as "Brlx, was metabisulphite (Na ~0 ; 80 mmol!L) at pH 6. 2 3 The measured by refractometer (Atago). Titratable acidity tissue was homogenised by Ullra Turrax at low velocity (TA) was measured by IFU method No.3 (21). Results tor 1 min. An aliquot was centrifuged in a microfuge at were expressed as g citric acid per 100 g fw. 12.000rpm (12.718 X g) for 2 min. Titesupernatantwas The green intensity of the outer peri carp was measured pi petted off and enzyme activity measured immediately using a Mino1ta Chroma Meter Model II. The following preparation (9). first 2 to 3 mm of the skin of the fruit was removed with a scalpel Kiwifruit were peeled and longitudinal sections taken. to expose the fruit tissue. Results were interpreted as The outer and inner pericarp, the skin, and core of average green intensity (approximately equal kiwifruit were individually am:lysed. to the colour of fresh kiwifruit) and significantly above The actinidin substrate, N-a-CBS-Iysine-p-nitrophenyl and below the average. Red colour intensity ester. was used of a mid cross at a reaction concentration of 76 ~mol/L section was visually in assessed in a large number of 50 mmol/L phosphate buffer, pH 6 and held at 25 oc fruit. (18) Three millilitres of substrate were added to the sample (l to 100 !-'L) directly in a cuvette to initiate the reaction. Actinidin activity was measured at 348 nm in a Rootstock-scion sejections Varian Cary 1 spectrophotometer. One unit of activity Fruit selection. All fruit were grown at Te Puke was defined as the linear change in absorbance of Research Centre (East Coast, N. Island), characterised 1.0/min. Results were expressed as units per gram of by deep loam-sandy soils. Four scions fresh weight (U/g were used in the fw) or as specific activity quoted as study: two scions were interspecific units per mg protein. F1 hybrids of A arguta and A melanandra and were half-sisters, denom Final preparations retained their activity when stored inaled BILl and B1F2. An open pollinated overnight at 4 A. argu oc. A maximum of 5'7':) activity was lost ta X A melanandraseedlingofB1L1 over 24 h. was denominated AAME. The temaining scion was A arguta var. arguta (AA). Duplicate vines were studied for the BILl and BlfZ scion-rootstocks except B1Ll-arguta and Electrophoresis gel BlLl purpurea which had one each, and six vines for each SDS-PAGE thin layer (0.5 mm) horizontal gels were AA and AAME rootstock-scion combination. Actini run on a linear polyacrylan1ide gradient. Protein was dia polygama and A. deliciosa were used as rootstock stained with silver nitrate. Standards were the low for the four scions in order to show scion influence. molecular weight calibration kit from Pharmacia. Pro Table 1 shows the rootstock-scion combinations tein was measured by the Coomassie blue assay and studied. diluted accordingly to give sim!lar amounts of protein Analysis of some rootstock-scion combinations and of 380 JwtJvol. 30 (1997) No. 4 all independently grown plants was repeated in succes pendent purpurea flower later. in the last week of sive years to show continuity of actlnidin levels. The December to the first week of January. graft was made about 300 mm above ground level. Actinidia arguta, A. polygama and rootstock-scion fruit For clarity, the plants used as rootstocks and scions and were harvested when, by gently shaking a shoot, 10% grown on their own roots wi.ll be denominated 'inde drop occurred. The fruit of the rootstock-scion combi pendent'. Actinidia deliciosa cv. Hayward will be nations and independent arguta were harvested on 1 to denominated kiwifruit when .analysed as an independ 19 March over the 3-year study, with a maximum range ent plant and deliciosa when used as a rootstock. Arguta when grown on its own roots wlll be denominated of 14 days in 1 year. about 6 to 8 weeks before the 'independent arguta', as a rootstock denominated commercial harvest of kiwifruit. Independent polygama 'anzuta', and as a sclon denominated 'AA'. and purpurea were harvested at the end of April to the beginning of May. A brief description of the fruit in this study is given. Actinidia melanandra is closely related to A. arguta and From the time of flowering to harvest of independent the hybrid produced fruit with a green skin and a pink arguta, independent purpurea and rootstock-scion to red star-shaped colour to the inner peric.arp, sur combinations was approximately 4 months, and 5 rounding the core. The intensity of colour varied widely months for independent polygama. In contrast, kiwi within and between vines (22). The arguta X melanan fruit are harvested after 6 months following flowering, dra hybrid produced fruit weighing 8 to 10 g which were but reach eating ripeness after 8 months on the vine. very sweet and were the first red pigmented Actinidia The period to achieve kiwifruit eating ripeness on the grown in New Zealand. Actinidia arguta (AA) was from vine varies widely from 20 to 44 weeks following a selection introduced into N.Z. from Japan in 1977. anthesis (24). Independent arguta are round to ovoid, have green skin Independent arguta and poiygama. and all rootstock and flesh and weigh 5 g at harvest maturity. Independ scion combination fruit. when harvested at 10% drop. ent arguta var. purpurea have a purple skin and a were about 1 week prior to eating ripeness when held at purple-green flesh with an elongated shape. Independ ambient temperature, or 3 to 4 weeks when held at 4 ·c. ent polygama are hairless. like arguta, 5 to 10 g in It was not possible to define eating ripeness of arguta weight. ovoid. and have a light green skin which physio-chemically as in kiwifruit by pressure (kgf) and changes to a yellow-orange on ripening. The fruit have SS (•Brix). Arguta ripeness was defined as fruit where a strong peppery taste wh:.ch diminishes towards the first signs of water logging began, when the skin ripeness (23). visibly darkened. All independent and rootstock-scion Polygama has been shown to be an extremely vigorous fruit for actinidin comparative studies were analysed at vine when used as a rootstock with other Actinidia. this puint. Same species were analysed before and after Arguta and arguta X melammdra have a vigorous the standard harvest date to show the development of growth rate but in comparison to polygama have a act!nidin during growth. lower flower number. AAME was grafted onto polyg ama rootstock and onto polygama rootstock with a deliciosa interstock to give a greater chance of scion rootstock compatibility. Interstocks also influence the 0 16r=- fruit characteristics and development, and in this case. served to moderate the effect of very high fruit number c 0.14~ induced by polygama rootstoc.~. Hemsleyana has been ~ 0.12~ used successfully to increase flower number, lower -g ~ .:9-0.!0f. flower abortion rate, and improve synchrony of bud i:' L burst with kiwifruit scions (11) and has been registered ~ o.osl as a rootstock under the name 'Kaimai'. Fruit of each ~ 0.06 scion and rootstock genotype grown on their own roots .:a '2 0.04 were studied independently with the exception of :.:::: hemsleyana fruit for which only male vines were grown :;: 0.02 in New Zealand at that time.
c------~pP===~---- T----- pH Harvest assessment and grmvth period All kiwifruit were harvested at a minimum of 6.2 oBrix Fig. 1 pH profile of three Actinidia species. (D) = pwpurea (!IlL); (L\.) = kiwifruit (SilL); (O) = polygama (100 IlL). when it is known that upon rip2ning t.l,ey wm have the Lines at the base represent the pH range of citric, phosphate distinctive kiwifruit flavour. Fmit harvested below this and Tris buffers; combination buffers were used at over physiological maturity do not develop the 'full flavour' lapping points to Jessen the effect of buf~er change . on (24). actinidin activity. pH maxima of the three speCies were Similar Independent arguta. polygama and the rootstock-scion at 5.8 to 5.4. Actinldin activity was measured as!:;, abs/min for each sample size due to the large differences in actinidin vines flower in the last 2 weeks of November. Inde- activity 381 lwtJvoL 30 (1997) No. 4
Table 2 Physico-chemical characteristics of rootstock-scion combinations Titratable Fruit Green ·Brix Red acidity weight (g) intemity" !ntensitya,b BILl scion arguta 13.7 1.33 9.28 ++ ++ purpurea 14.7 1.28 9 25 argutaxdel ++ ++ 16.9 1.45 8.73 + deliciosa 15.:8 + 1.41 10.53 +++ ++ polygam a 16.0 n.d. 10.04 + + B1F2 scion deliciosa 14.0 1.72 !0.78 +++ +++ polygama 14.8 n.d. 7.95 ++ + AAMEscion delidosa 14.B 1.10 9.00 n.d. polygama 13J ++ L22 5.96 n.d. +++ pofygama-del 15.'1 115 10.15 n.d. + AAscion deliciosa 14.9 0.96 4.85 polygama n.d. IS.S 0.82 4.22 n.d. hemsleyana 14.?, 0.91 5.48 n.d. 8 Green and red intensity of the fruit perlcarp are defined as: ++ average of total rootstock-scion population (for green thls is approxlmately equal to fresh kiwifruit colour); + significantly lighter; and +++ significantly darker. b(-) denotes absence of red. n.d.=not determined. TA is expressed as g citric acld/100 g fw. Fruit weight (g) at harvest was assessed on 40 to 240 fruit.
Table 3 Actinidin levels at pre-harvest and harvest for the independent scions and rootstocks Average fruit Harvest rlate weight (g) Weight range Actinidin8 SD (n-1) BILl 20 Mar* 10.70 8.96-16.03 29.7 10.1 B1F2 20 Mar* 7.95 4.95-13.28 18.3 4.9 arguta 22 Feb 4.25 3.23-5.55 88.2 12.8 8 Mar* 5.04 4.06-6.66 124.2 18.5 20Mar 5.12 4.14-6.56 120.7 19.2 polygama 22 Feb 6.46 5.19-7.90 0.96 0.27 20 Apr* 9.22 8.20-9.70 1.04 0.68 I May 10.22 9.20-13.14 1.20 0.32 8May 9.85 9.25-14.04 1.64 0.54 purpurea 20 Apr 5.37 4.78-6.55 316.0 76 25 Apr (Gpi) 2.10 1.50-3.24 25 Apr (Gpii) 3~4.5 58 5.54 4.12-5.85 508.0 25 Apr (Gpiii) 59 6.03 5.51-6.43 634.5 25 Apr (GpiV) 56 6.18 5.84-6.79 687 5 25Apr (GpV) 82 5.70 5.38-6.08 637.5 105 1 May* 6.21 5.80-6.84 8May 704.7 76 6.18 5.85-6.76 726.2 14 *Standard harvest time (6.2 ·Brix for kiwifruit and tO% drop for arguta, polygama, purpurea, groups harvested on BILl and B1F2). Purpurea 25 April (range of fruit weight and maturity) are defined in text. •Actinidin units are Ll. abs/(min. g fresh weight).
Results and Discussion 7.3 to 7.6 was found for the proteases found in asparagus, kiwifruit and miut with casein substrate pH profile (5). Two kiwifmit actinidin isoenzy:nes with pH maxima at To ensure the standard assay at pH 6.0 was suitable for 3.9 and 6.5 were reported (6). In other studies when the the measurement of actinidin in all Actinidia species. a substrate was gelatin in citrate-phosphate buffer, kiwi pH profile was done on kiwifruit, independent pur fruit actinidin maximal activity was at pH 4.0 to 4.3 (2). purea and independent polygama fruit (Fig. 1). The and a maximum at pH 5 to 7 wa.s seen with N-benzoyl· profile of the three fruit species showed similar L-arginine ethyl ester as substrate (4). A pH optima of maximal actinidin activity at pH 5.8 to 6.4. 382 lwtlvo/. 30 (1997) No. 4
Table 4 Actinidin levels per g fruit and specific activities of rootstock-sciun combinations Protein Act!nldin Range SD (n-1) (mg/g fruit) SA BIFlscion arguta 80.88 45.3-104 22.65 i.206 66.55 purpurea H0.78 70.0-103 10.45 l.l05 82.12 argutax de/icios.~ 3.70 0.98-11.2 2.88 Ll59 3.55 deliciosa 86.59 54.2-135 21.51 pofygama 1.395 62.01 ·~2.00 [3.4-32.2 6.24 0.931 23.33 BlFZscion deliciosa 14.17 7.39-22.1 5.04 0.837 16.79 polygama 4.45 1.38-8.19 2.52 1.100 4.11 AAMEsclon deliciosa 0.74 0.34-121 0.34 0.407 1.82 polygama 0.41 0.09-0.74 0.29 0.661 0.62 polygama-delido.~a 1.67 0.39-2.92 1.02 0.517 3.23 AAscion delidnsa 36.82 22.2-53.0 12.20 n.d. polygama 27.72 14.1-39.9 11.05 hemslyana 1.033 26.83 38.26 22.2-54.1 11.06 n.d. n.d.=not determined. SA=specific activity (t> abs/(min. mgprotein).
Soluble solids (SS) and titrata/Jle acidity (TA) 58; broccoli 74; peas 95 and silver beet 1148 (26) In the Table 2 lists SS (cBrix) and TA results measured at the same manner, the nature of some arguta species· time of analysis. The differen:es between the various pericarp cells results in a pink to deep red intensity of scion-rootstock combinations indicate that there is at colour at very low anthocyanin concentration in com most. a minor influence by rootstock or scion even parison to other red-coloured fruits (27). though fruit size, fruit number and the resultant yield Anthocyanin inhibition of enzymes has been well per vine, factors which may influence "Brix levels, documented (28,29). In order to test for inhibition by varied widely. In comparison, kiwifruit had cBrix anthocyanins or possibly other endogenous com readings of 12.5 to 13.7 at eating ripeness and a pounds, three preparations were made: one of arguta, titratable acidity of l.O to 1.2. was no correlation ~mere one of arguta x melanandra hybrid and one containing between actinidin levels with TA and •Bnx levels of equal weights of the two fruits. Fruit were halved: one rootstock-scion samples. half was used for a single fruit species preparation and Fruit size of all rootstock-scion combinations agreed the other half for the combination preparation. The with the Independent scion weights: about 10 g for expected result of the combination preparation was the arguta x melanandra combinations and 5 g for arguta average of the actinidin levels measured in the two combinations (Tables Z and 3). single fruit species preparations. However. the combi nation preparation resulted in a 15 to 30% inhibition of actinidin activity. The arguta and the arguta >< me/a Actinidln inhibition by antlwcyanins or a nandra samples prepared individually, when mixed in melanandra -induced compound equal volumes, showed no inhibition of actinidin Arguta X melanandra (BILl, B1F2 and AAME) sci activity. Therefore the inhibitor was not present or ons and their rootstock-scion combinations showed a nonfunctional once the samples had been prepared. variation of flesh colours, from light green with tight Results of one typical experiment were: AAME prepa pink centres to deep green with deep red centres (Table ration 0.54 U/g fw; AA preparation 33.18 U/g fw: 2). In general the intensity of the two colours coincided. expected resull of combination 16.86 U/g fw; The red colour was restricted to the inner pericarp and AA + AAME combination 12.36 U/g fw (25% inhibi was influenced by the scion and rootstock. There was a tion); equal volume of AA and AAME samples, mixed close correlation of the averaged values of lightness, following preparation 16.58 U/g fw. concentration a.'1d saturation of the green colour Measured actinidin levels did not always support between identical vines of rootstock-scions actinidln inhibition by anthocyanins. Arguta X deli (? = 0.98). ciosa hybrid and polygama, when used as rootstocks, The red anthocyanin colour in the seed area of A. produced fruit with the least red intensity of the inner arguta x A. melanandra and i.TJdependent purpurea has pericarp and 1o\V actinidin activity in all their root been identified as three cyanidin glycosides (25). In stock-scion fruit (Tables Z and 4). Arguta, purpurea and kiwifruit. the translucent nature of the pericarp cells deliciosa scion-rootstock combinations with B lLl had results in an intense emerald green although the level medium to high red intensity to the fruit, but the fruit of chlorophyll is low in comparison to green vegetables: had the highest actinidin activities of the rootstock kiwifruit pericarp 25 mg chlorophyWkg; brussel sprouts scion combinations. Independent purpurea fruit with 383 Jwt/voL 30 (1907) No. 4
the deepest red intensity of a.ll Actinidia analysed had 80 · ll:GO the highest measured actinldln levels (Table 3). However, the AA sdon ~ LJr -·0. ~ population (without red c anthocyanins) had higher actinid!n levels than the AAME population. Even with the anecdotal evidence ~-" that anthocyanins, or some other organic compound, s" 0 40 ~ ~ could partially inhibit actinidin activity, the differences ' J 80 ~· between the AA and AAME scion populations were ~ ! g ~ ~ large enough to state that Inhibition was not significant / -\ € to the final result. ~ 20 ~/// 160 An AA and an AAME preparation was made by ~u ---i crushing the fruit to a fine powder in liquid N < 0 2 and 40 w using phenolic scavengers, polyvlnylpolypyrrolidone 80 120 160 200 240 280 Feb Mar Apr (PVPP) and Dowex-L in order to produce a phenolic May Jun Jul Aug free preparation. Only 20% of the activity was recov Days following 90% petal fall ered compared to the standard assay. The actinidin did not bind to the phenolic scavengers Fig. 2 Development of kiwifruit actinidin activity during but the rea~on for growth the poor recovery is unknown. and storage. Fruit were harvested from early February to the nonnal harvest time at the beginning of May ((•) = actinldin levels at harvest date). The da~hed line represents a quadratic best fit of actinidin development Actinidin Following harvest, distribution fruit were stored for various times at 4 ·c and actinidin levels The kiwifruit outer pericarp. representing 70% of the measured (solid lines to .6)_ Fruit harvested in May were stored for 2 months; total fruit weight, had the highest level of actin!din at this stage the fruit were still hard. and were then ripened by ethylene. activity. No activity was found in the The skin and very little actinidin levels were measured over the following month in the core (3.5U/g (dotted core). The inner pericarp level was ltne to 0). Error bars on the fruit weight (0) are 75% of the actinidln level of the outer pericarp. l'irguta SEM had a similar distribution of c!Ctinidin activity. From a selection of fruit that recorded levels of 38.3 U/g fw for Lewis and Luh (8) who found a 3-fold increase in whole fruit, the outer pericarp contained 46.7 U/g fw. actinidin activity in the 3 weeks following harvest. It is unlikely that an actinidin inhibitor was present in immature fruit as an equal weight of fruit harvested Development of actinidin during growth in and storage February and in May in a mixed preparation A study was made of actinidin resulted in development in kiwifruit an expected average activity during fruit development, of the two individually at commercial harvest and prepared samples. during storage (Fig. 2). A less detailed study was done on independent arguta, independent purpurea and independent polygama (Table 3). Independent rootstock and scion actinidin levels Kiwifruit harvested early in February. at an average Table 3 lists the actinidin levels of fruit from vines used fruit weight of 65 g. showed 5 U/g fw actinid!n activity as scions (AA BlLl and B1F2), and rootstocks (8% of maximum); 1 month later at a fruit weight of (poiygama and purpurea), all grown as independent 94 g the actinidin level had increased to 8 U/g fw. At the plants. Arguta were tested 2 weeks before standard beginning of April (125 d following 90% petal fall) fruit harvest date (22 Feb) when the fruit were hard and weighing 106 g had 27 U/g fw. By the end of April the inedible. Polygama were analysed 4 weeks before actinidin levels increased sharply to 50 U/g fw. Fruit standard harvest on 22 Feb when the fruit were rock harvested at the beginning to mid May, the commercial hard. On 20 April the polygama fruit began harvest time (155 to 165 d following to show a 90% petal fall), characteristic yellow tinge to the had 60 to 65 U/g fw. skin. Polygama. which had the lowest actiniilln levels of the Actinidia species, Kiwifruit harvested during February and March mar were tested for !ate development of actinidin on the 1 ginally increased their actinidin levels following pro and 8 May when the fruit had developed a deep yellow longed storage at 4 oc. Stored fruit harvested in April orange colour of the skin, peri carp and core. Purpurea and May increased their actinidin levels at the same were analysed on 20 April when the fruit had green rate as fruit on the vine. Actinldin reached a maximal skins with a star-like pink centre. A second a;1.alysis was level of 70 to 75 U/g fw following 2 months storage and done on 25 April with a larger fruit number which ripening by ethylene (Fig. 2), then gradually declined to varied In weight from 1.04 g to 7.52 g. Purpurea fruit 55 U/g fw at which stage the fruit were 'mushy'. were divided into five groups by size and maturity. Up to 2 months storage after 1:he commercia! harvest Group 1 were underdeveloped fruit (average 2.1 g). had period, the kiwifruit remained hard, and the change in light green skins, a light pink centre and were very hard; actinidin levels was small. The actinidin development group 2 were larger (5.54 g) but very hard with a light results were similar to another study (7); the first pink centre; group 3 were equal in weight to mature appearance of activity at 50% fruit weight and maximal fruit (6.03 g) but were still extremely hard; group 4 were levels at maximal fruit weight. Our results differ from softer, had developed a purple tinge to the skin and a 384 lwtlvol. 30 (1997) No. 4 red centre; and group 5 were soft, and had dark red from less than l to 144 U/g fw. The rootstocks had at flesh and a purple skin. Somedmes examples of the five most a marginal effect on actinidin levels of groups were found growing in the fruit in the same cluster. TI1e the AAME and AA scion populations. actinidin levels agree with the development of actinidln The four scions influenced the level of actinidin activity in kiwifruit; at maximal weight, actlnidin levels were shown by the two rootstocks in common with all scions; highest (Table 3). Purpurea analysed on 1 and 8 May polygama and deliciosa (Table 4). The AA and had a deep purple skin and pericarp. AAME scions which were unrelated had the greater influence The specific activity of the purpurea groups 1 to 4 did on actinidin levels than the rootstocks. The not significantly change; 142, 149, upper levels 145, and 126 U/mg measured in some BILl combinations protein, respectively, were not found demonstrating the high percent in any of the AA age and AAME rootstock-scion combina of ar.tinidin protein to total soluble protein in tions; all six combinations were below the level found in purpurea fruit. Group 5 protein reading was invalid due kiwifruit pericarp. There were no significant differences to the high pectin levels which interfere in the between the three AA -rootstock combinations, averag Coomassie assay. The purpurea specific activity was ing 34.2 U/g fruit (range 14.1 to 54.1 U/g fw). An greater than the specific activity of 55 to 65 U/mg explanation for the lack of rootstock influence with the protein for freshly harvested kiwifruit. Polygama had AAME and AA scions may be that the rootstock did extremely low levels of measured acUnidin but medium not contain high levels of actinidin activity as measured protein levels in comparison to other Actinidia. A in independently grown arguta and purpurea. preparation made in a similar manner was used to test The actinidin levels of rootstock-scion combination for anthocyanin inhibition of actinidin. Ripe yellow fruit were not an average of the two independent skinned polygama and purpurea fruit were halved, one plants. The 13 comhlnations showed the following half for an individual preparation, the other for a trends: (!) rootstock fruit with low actihidin levels combined preparation of the two fruits. The results resulted in rootstock-scion fruit with low to medium showed that there was no actinidin inhibition by activity (polygama and arguta X deliciosa rootstock); endogenous polygama compounds. Although polygama (ii) rootstock with high levels of actinidin were the only matured 2 to 3 weeks before kiwifruit there was no rootstocks to result in high actinidin activity of root significant late development of actinidin up to the stock-scion combinations (arguta, purpurea rootstocks normal kiwifruit harvest date ::n early May {Table 3). and BlLl-deliciosa); (iii) scion influence was significant with the low to medlum actinidin activities found with scion-polygama combinations and particularly Actinidin levels of rootstock-scion noted combination fruit with scion-deliciosa combinations. The seven rootstock-scion combinations with BILl Kiwifruit had consistent actinidin levels from season to and BIF2 as the scions resulted in three rootstock season (kiwifruit levels at harvest over three seasons scions with high actinidln levels, arbitrarily defined as were 66.8, 64.0 and 70.4 U/g fw). Independent arguta > 40 U/g fw, and the remaining four combinations with and rootstock-scion combinations had consistent levels < 25 U/g fw. There was up tc a 2.5-fold variation of over two seasons. Although the intravine range actinidin activity within some of rootstock-scion combina actinidin activity of arguta and tions (Fig. 3). rootstock-scion combi nations was greater than kiwifruit, the coefficient of Rootstocks strongly influenced actinidin levels in the variability of averaged values between identical arguta BILl and BIF2 scion populations; the BILl population rootstock-scion vines was 4.1% (calculated from SED held the extreme low and high sample levels varying on a non-log scale). An explanation for the consistency of actinidin levels of kiwifruit at commercial harvest time was suggested as the smaller variation hemsieyana in maturity polygama and size compared to arguta fruit. dcliciosa AA
poly- del. AAME polygama Protein levels in Actinidia species and rootstock--scion deliciosa combinations B1F2 The actinidin activity and protein concentration for BILl and BlFZ rootstock-scion combinations showed a significant positive correlation (l- = 0.558, Fig. 4a). In general the samples with low actinidin activity also had low protein concentration, with the exception of BlLl arguta x deliciosa. Despite having low actinidin activ ity the protein level of BlLl-arguta X deliciosa Acttnldin activity (LI abs/{min.g fruit)) was above the average of most combinations. The non actinidin soluble protein Fig. 3 Activity range of actinidin within could vary between rootstock and between scion combinations rootstock-scion combinations. The range of actinldin levels and a close fit of protein to actinidin within a vine was as great as that between vines of identical activity for all scion-rootstock combinations was not rootstock-scion combinations. Rootstocks are listed on the expected. Kiwifruit values for actinidin and protein y-axis and the four scions printed inside the box levels at normal harvest (6.2 °Brix) and at eating 385 fP'IqF> 8 ':If 11)
t-...vt/voL 30 (1997) No 4
ripeness, measured over 3 years. agreed closely to the actinidin with a molecular weight of 39 kDa has been best fit line of the rootstock-scion tombinations. identified (31). An inactive zymogen of actinidin is Purpurea fruit showed extremely high levels of actini unlikely to be present in fruit with low actinidin activity din and protein compared to other Actinidia (Fig. 4b); as no significant bands above 30 kDa were seen on even group 1 purpurea fruit (Table 3) had higher electrophoresis gels except for independent polygama actinidin activity and soluble protein than all other which could have a small amount of an inactive Actinidia analysed. Extrapolation of the best fit line of actinidin precursor (line 11; Fig. 5). all combinations, independent scions and rootstocks (f = 0.895) to zero actinldin activity would give a soluble protein concentration of 0.84 mg/g fw. At an Interstock influence average kiwifruit level of 65 U/g fw, actinidin would One scion-rootstock combination (AAME-polygama) represent 25% of the soluble protein and arguta (86 U/g was replicated, but with deliciosa as an interstock. The fw) 31%. Actinidin in purpurea containing 688 U/g fw interstock did not affect SS(.Brix), titratable acidity or activity would represent 83% of the soluble protein. actinidln levels, but did affect the intensity of the red The measured protein level may be an underestimate of the percentage of total protein of actinidin. The Coomassie blue assay is dependent almost totally on arginine content for the measurement of protein (30). 2.l (a) The larger of the actinidin genes encodes a protein of 1.8 302 amino acids of which nine are arginine. The ~ ~I actinidin precursor loses 57 amino acids from the amino !.6 I end and 25 from the carboxy end to become fully 0 functional (31.32). Five of the nine arginines are found in the N-terminal segment le.:.ving only four arginines I in the active 220 amino ac:.d protein. An average protein has 3 to 5% arginine content and thus the I protein assay would be expected to underestimate the i actinidin level by 50%. ~ The results of the measured protein and actinidin levels g --'--' were supported by a SDS-PAGE gel (Fig. 5). The N" 20 40 60 80 100 120 140 !60 0 protein band that represents actinidin is approximately ...c. at the level of the 30 kDa marker protein. Varieties .$ (!J) ~ 5.0 .!l which had extremely low actinidin levels (AAME 0 (I) poiygama (line 3, 1.6 U/g fW) and independent polyg ~ ama (line 11. 1.0 U/g fW)) had little actinidin protein. 4.0 Moderate amounts of actinidln protein were seen in /i0 AAME-deliciosa (line 1, 24.3 U/g fw), AAME-polyg 3.0 ama with deliciosa interstock Cine 2, 16.6 U/g fW), AA <> polygama (line 4, 23.6 U/g fw). and ,~-delidosa (line 5. 40.8 U/g fW). TI1e density of the actinidin band was highest with independent BILl (line 10. 51.2U/g fw). kiwifruit (line 9, 66 U/g fw), independent arguta (line 12. 118 U/g fW). independent unripe purpurea (line 6, 406 U/g fw). and independent ripe purpurea (line 7, 656 U/g fw). Polygama showed a different protein Actinidin activity (A absl(min.g fw)) profile to other Actinidia wi':h two high molecular weight proteins at 35 and 58 kDa which were not seen in other Actinidia species. Some fruit samples of Fig. 4 (a) Relationship between actinidin and protein levels of AAME-rootstock combinations had surprisingly low the BILl and B1F2 populations. Each scion-rootstock soluble protein levels, V
extremely low levels of actin.idin activity may abate 6 HASHINAGA, F., FuKUDOME. T. AND hoo, S. Activity and these effects. Nine staff were each asked to taste one distribution of protease in kiwifruit during ripening. ripe Purpurea fruit (purple skin and flesh, equivalent to Bulll!tin of the Faculty of Agriculture, Kagoshiwa Uni· the group five purpurea; Table 3) and in the following versity. 36, 65-69 (1986) days to eat an arguta fruit. All staff, following eating 7 LrN, E., BuRNs, D. AND GARDNER, R. Fruit developmental regulation of the kiwifruit actinidin promoter is conserved purpurea fruit, complained cf tenderness to the inner in transgenic petunia plants. Plant Molecular Biology. 23, mouth and tongue. and burning of the lips. Four of the 489-499 (1993) nine staff experienced mild to extreme 'catch'. Arguta 8 LEwrs, D. AND LuH, B. Development and distribution of fruit gave either mild tenderness ('tingling') to the actinidin in kiwifruit (Actfnidia chinensis) and Its partial mouth or no effect was noted, although tenderness to characterization. Journal of Food Biochemistry. 12, 109~ 116 (1988) the inside of the mouth increased with the number of 9 BoLAND, M. AND BuRNs, D. Production of attinidin; a Arguta fruit eaten. No staff experienced 'catch' after proteolytic enzyme from kiwifruit. Internal report 1. eating arguta fn1it. Generally. independent arguta have Auckland, N.Z.: DSIR. Mt. Albert Research Centre slightly elevated levels of crystalline oxalic acid com (1980) pared to kiwifruit (results not published) but this 10 ROGERS, W. AND BEAKBANE, B. Stock and scion relations. Annual Review of Plant Physiology, 8, 217~236 (1957) difference would not be sufficient to induce the 11 WA"'G, Z., PATTERSON, K., GouLD, K. AND LoWE, R. extreme irritation caused from eating purpurea fruit. Rootstock effects on budburst and flowering in kiwifruit. Sdenda Horticulturae, 57, 187~199 (1994) 12 LOWE, R. G. Rootstocks - the underground workers in the orchard. New Zealand Kiwifruit, 31, 20~21 (1987) Conclusion 13 WEBSTER, A. Rootstock and lnterstock effects on decid uous fruit tree vigour, precocity, and yield productivity. New Zealand Joumal of Crop and Horticultural Science, While actinidin is a major protein in most Actinidia 23, 373-382 (1995) fruit, there was a wide variation in levels within the 14 CAs·tu:, W. Rootstock as a quality factor in citrus and genus, from 1 to 1000% of the level found in kiwifruit. deciduous tree crops. New Ze<1land Joumal of Crop and Actinidin was developmentally regulated in all fruit. Horticultural Science, 23, 383~394 (1995) and did not depend on other physiological differences 15 MoNASTRA, F. AND TESTON!, A. Horticultural performance such as the harvest date. Fruit with low actinidin and quality characteristics of fruit from kiwifruit plants (cv. Hayward) obtained by in-vitro propagation. by activity did not have an inactive actinidin precursor. cuttings and by grafting. Acta Horticulturae. 297, 197~200 Rootstocks and scions influenced the levels of actinidin (1991) activity with control suggE~ted at the level of 16 HoPKIRK, G. Personal communication (1994) transcription. 17 PERERA, c.. HALLETT, I., NGYYEN, T. Al'-r::l CHARLES, J. Some characteristics of the rootstock fruit were not Calcium oxalate crystals: the Irritant factor in kiwifruit. Journal of Food Science, 55, 1066~1069 (1990) conferred on the scion fruit. such as the 'peppery' 18 BoL'.ND, M.]. AND HARDMAN. M.]. The actinidin-cata flavour of polygama. Some characteristics remain under lysed hydrolysis of N-a-benzyloxycarbonyi-L-tysine p·ni the genetic control of the scion. such as the fruit weight. trophenyl ester, pH dependence and mechanism. Euro The wide variation in actinidin levels and other organic pean Journal of Biochemistry. 36, 575~582 (1973) 19 DAWES, H., STREUBI, P., BoYES, S. AND HEATHERBELL, D. compounds such as sugars and organic acids offers the Kiwifruit juice proteins: characterization and removal opportunity of a wide search for the best commercially during processing of clarified juice. Acta Horticulturae, viable arguta fruit. 297(2). 567~671 (1991) Taste trials will be required to demonstrate the influ 20 BRADFORD. M. A rapid and sensitive method for the ence of actinidin on mild mouth irritation to 'mouth quantitation of microgram quantities of protein utilising burn' and 'catch' in fresh fruit and processed products. the principles of protein-dye binding. Analytical Bio chemistry. 72, 248~254 (1976) Analyses of organic acids and sugars is being com 21 International Federation of Fruit Juice Produr:er~ (IFU). pleted. and will serve to associate positive character Methods of analysis. Switzerland: Zug (1985) istics to taste. 22 SEAL. A. AND McNEILAGE, M. New red and green flushed ActirliJia. New Zealand Kiwifruit. 42, 9 {1988) 23 FERGUSON, A. R. Ecstatic cats among the vines. New Zealand Kiwifruit. 82, 24 (1991) References 24 WARRINGTON, I. AND WEsroN. G. Kiwifruit; science and management. Auckland. NZ: Ray Richards Pub!, pp. i07-!21 (1990) A. R. l FERGUSON, Actinidia arguta - the hardy kiwi fruit. 25 WEBBY, R. F. Determination of the chemical nature of the New Zealand Kiwifruit. 81, 23-24 (1991) red pigmentation in the fruJt of Actinidia. Industrial 2 ARcus, A. C. Proteolytic enzyme of Actinidia chinensis. Research Institute (N.Z.) Internal Report, No. 210 Biochimica et Biophysica Acta. 33, 242~244 (1959) (1994) 3 BoLAND. M.J Properties of actinidln. Chemistry in New 26 KHACHIK. E. BEECHER, G. AND WHIITAKER, N. Separa Zealand, 37 (3). 83~88 (1973) tion, identification and quantification of the major carate· 4 MrDoWJ\U , M. Anionic prott~inase from Actinidia chi noid a.TJd chlorophyll constituents in eA"tracts of several nensis. European Journal of Biochemistry. 14, 214~221 (1970) green vegetables by liquid chromatography. Journaf of Agricultural and Food Chemistry. 54, 603~610 (1986} 5 YAMAGUCHI, Y. YAMASHITA, Y. TAKEDA, I. AND KISO, H 27 WEBBY, R. Personal communication (1995) Proteolytic enzymes in green asparagus, kiwifruit and 28 ANDERSON, ]. Extraction of enzymes and subcellular mlut: occurrence and partial characterisation. Agricultural organelles from plant tissues. Phytochemistry, 7, and Biological Chemistry. 46, 1983-1986 (1982) 1973~ 1988 (1968) 388 lwtlvol. 30 (1997) Nu 4
29 LooMIS, W Overcoming j:roblems of phenolics and 33 PooiVINSKY, E., SNOWDEN, K., KEELING, J.. LJN, E. AND qulnones In the Isolation of plant enzymes and organelles. Methods in Enzymology. 31, 528-544 (1974) GARDNER, R. Expression of actinidin, a kiwifruit cysteine protease. Acta Horticulturae, 297, 140 (1991) 30 CoMPTON, S AND ]ONES, C. Mechanism of dye response and interference in the Bradford protein assay. Analytical 34 GREAVES, A. Root distribution of kiwifruit (Actlnidia Biochemistry, 15, 369-374 (1985) deliciosa) in a deep sandy loam soli of the Te Puke district, New Zealand. New Zealand Journal of Agricultural 31 PRAEKELT, U., McKEE. A. AND SMITH, H. Molecular Research, Z8, 433-436 (1985) analysis of actinidin, the cysteine protease of Actinidia 35 McANENEY, AND JuDD, M. Observations on kiwifruit chinensis. Plant Molecular Biology, 10, 193-202 (1988) K. (Actinidia chinensls Planch.) root exploration, root pres 32 PoDIVINSKY, E., FoRsTER, R. AND GARDNER, R Nucleo sure, hydraulic conductivity and water uptake. New tide sequence of actinidin, a kiwifruit protease. Nucleic Zealand Journal of Agricultural Research. 26, 507-510 Acids Research. 17(20), 8363 (1989) (1983)
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