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 var. arguta (arguta) has not been investigated. Ta5.te panels in New Zealand 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 A. polygama with A. deliciosa interstock The aim of the study was to c.etermine the variation in {polygama-dellciosa) actinidin activity in various Actinidia species, and the AA A. deliciosa influence of scions and rootstocks on the chemical A.polygama composition of A. arguta fru.t. Factors 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 by an abbreviated content (0 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) was used to determine cut into quarters 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 (Na2 ~03 ; 80 mmol!L) at pH 6. 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 first 2 to following preparation (9). 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 to the kiwifruit were individually am:lysed. colour of fresh kiwifruit) and significantly above and The actinidin substrate, N-a-CBS-Iysine-p-nitrophenyl below the average. Red colour intensity of a mid cross ester. was used at a reaction concentration of 76 ~mol/L section was visually assessed in a large number of in 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 were used in the fresh weight (U/g fw) or as specific activity quoted as study: two scions were interspecific F 1 hybrids of A units per mg protein. arguta and A melanandra and were half-sisters, denom­ Final preparations retained their activity when stored inaled BILl and B1F2. An open pollinated A. argu­ overnight at 4 oc. A maximum of 5'7':) activity was lost ta X A melanandraseedlingofB1L1 was denominated over 24 h. 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 BlLl­ Electrophoresis gel 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.