Low Temperature Sweetening in Potato Tubers: the Role of the Amyloplast Membrane

J. Plant Physiol. Vol. 145. pp. 335 - 341 (1995)

Low Temperature Sweetening in Potato Tubers: the Role of the Amyloplast Membrane EILEEN

P.

O'DONOGHUE, RICKEY Y. YADA*,

and ALEJANDRO G.

MARANGONI

Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada Received August 31,1993 . Accepted June 10, 1994

Summary

Properties of the amyloplast membrane were examined for potato cultivars susceptible (Norchip) and resistant (ND860-2) to low temperature sweetening following storage for 4 months at either 12°C or 4°C. Chips from Norchip stored at 4°C were darker and the tissue contained higher levels of glucose, fructose and sucrose as compared to ND860-2 tubers stored at 12°C and 4°C and Norchip tubers at 12°C. Membrane lipid phase transitions of intact amyloplasts assessed using differential scanning calorimetry were higher for Norchip than ND860-2 tubers after storage at both 12°C and 4°C (45 vs. 38°C and 35 vs. 27°C, respectively). Fluorescence depolarization measurements using the probes 1,6-diphenyl1,3,5-hexatriene (DPH) and trans-parinaric acid (t-PA) indicated that the structural order parameters of Norchip amyloplasts membranes at 4°C were higher than at 12°C (0.655 vs. 0.459 with DPH and 0.658 vs. 0.531 with t-PA) while the opposite was true for ND860-2 (0.402 vs. 0.549 with DPH and 0.481 vs. 0.566 with t-PA). Small differences in the SDS-PAGE protein patterns of amyloplasts were observed between Norchip and ND860-2. Degradation of amyloplast unsaturated fatty acids was greater for Norchip membranes (93 % decrease in double bond index) than for ND860-2 (70 % decrease in DBI). The above results suggested that amyloplast membranes from Norchip potatoes senesced to a greater extent than ND860-2 amyloplast membranes upon exposure to low temperature and that this event may be associated with low-temperature sweetening.

Key words: Amyloplast membrane, low-temperature sweetening, Solanum tuberosum tubers. Introduction

Potato tubers, as well as many other plants and plant parts, often undergo a phenomenon known as low-temperature sweetening (L TS), resulting from the conversion of starch to sugars, after exposure to low temperatures (i.e., < 10°C) (Isherwood, 1973; Isherwood, 1976; apRees et aI., 1981; Sowokinos, 1990 a, b; Wismer et aI., 1995). Although this phenomenon has been well documented, the causes and mechanisms by which LTS occurs are still not established (Sowokinos, 1990a,b; Wismer et aI., 1995). Evidence, however, suggests that a fine metabolic control rather than a coarse metabolic control is involved in this process (ap Rees et aI., 1981). While much research has been, and is presently, devoted towards understanding potato tuber metabolism in the pres-

* Corresponding Author. © 1995 by Gustav Fischer Verlag, Stuttgart

ence and absence of stress (Stark et aI., 1992; Sowokinos et aI., 1993), very little work has been directed towards determining the effects of low, but nonfreezing temperatures on the structure of the potato amyloplast membranes and the relationship between amyloplast membrane damage and the phenomenon of low-temperature sweetening. Studies on the structure of amyloplast membranes during low temperature sweetening seem to indicate that this membrane remains intact, albeit at a macroscopic scale, during the stress period (Isherwood, 1976; Wetstein and Sterling, 1978; Sowokinos et aI., 1987; Yada et aI., 1990). A study by Ohad et ai. (1971), however, reported damage to the amyloplast membrane upon exposure to cold temperatures while Isherwood (1976) noted that the amyloplast membranes became more fragile due to the low temperature stress. As Sowokinos et ai. (1987) have suggested, gross observations do not preclude changes that may occur in the molecular com-

336

EILEEN P. O'DONOGHUE, RICKEY Y. YADA, and ALEJANDRO G. MARANGONI

position and organization of the amyloplast membranes, influencing their permeability and!or transport properties. As a matter of fact, very little and inconclusive work has been performed addressing this latter aspect of the low temperature sweetening phenomenon. Biological membranes undergo a series of reactions during senescence that eventually lead to decompartmentation and cellular death (Paliyath and Droillard, 1992). It was therefore our objective to determine whether a stress-induced accelerated rate of senescence, of the amyloplast membranes, was a factor in the development of the stress response. In this light, a study was undertaken to investigate the structure and function of amyloplast membranes from chilling-resistant and chilling-sensitive potato tubers that have been stored at temperatures below (4°C) and above (12°C) the critical temperature required for triggering low temperature sweetening.

Materials and Methods

Plant material The potato tubers Norchip, North Dakota 860-2 (ND860-2) and Monona were grown at the Cambridge Agriculture Research Station, Ontario Ministry of Agriculture and Food (Cambridge, ON) using standard agronomic practices (Coffin et al., 1987). Tubers were cured for 2 weeks at 12°C then stored at 12 °C and 4°C for 4 months at approximately 95 % relative humidity.

Chemicals Sigma (St. Louis, MO): Bovine serum albumin (BSA), cytochrome c, DL-dithiothreitol (DTT), 1,6-diphenyl-l,3,5-hexatriene (DPH), disodium ethylenediamine tetraacetic acid (EDTA), L-glutamine, (3nicotinamide adenine dinucleotide (NAD), N-(1-naphthyl) ethylenediamine hydrochloride, oxoglutarate, Percoll, insoluble polyvinylpyrrolidone (PVP), sorbitol, sulfanilamide, Tricine. Calibiochem (San Diego, CA): trans-parinaric acid (t-PA). Fisher (Toronto, ON): All chemicals were Fisher Certified ACS grade unless otherwise specified. Acetonitrile, L-ascorbic acid, ethanol, 37 % formaldehyde, 50% hydrogen peroxide, isooctane (Optima grade), methanol (HPLC grade), magnesium chloride, nitric acid, potassium dichromate, sodium azide, sodium dithionite, sodium metabisulfite, Triton X-l00. Bio·Rad (Mississauga, ON): {3-mercaptoethanol, acrylamide, Bis (n,n'-methylene-bis) acrylamide, ammonia persulfate, coomassie blue R-250, glycine, Tris, Protein Assay Dye Reagent Concentrate, Protein Assay Standard I (bovine serum albumin), sodium dodecyl sulfate, SDS-PAGE molecular weight standards low and high. Supelco (Bellefonte, PA): Methanolic-base reagent. Extraction and purification ofamyloplasts Amyloplasts were isolated using the method of Emes and England (1986) with some modifications. Approximately 100g of potatoes were peeled, cut into 3-cm cubes and homogenized in a Waring blender for 30s in an equal volume of 50mM Tricine buffer pH 7.9 containing 330 mM sorbitol, 1mM disodium ethylenediaminetetraacetic acid (EDTA), 1 mM magnesium chloride, 28 mM ascorbic acid, 5mM dithiothreitol, 2.5mM sodium metabisulphite, 0.1 % (w/v) defatted bovine serum albumin (BSA) and 0.5 % (w/v) insoluble polyvinylpyrrolidone. The homogenate was then filtered through 4 layers of grade 90 cheesecloth and centrifuged at 200 x g for 1 min at room temperature. Aliquots (10-15 mL) of supernatant were underlaid with 15 % (w/v) Percoll and centrifuged at 4000 x g for 5 min at room temperature. Fifteen percent Percoll was

prepared by adding equal volumes of 30 % Percoll (as purchased from supplier) and 50 mM Tricine buffer pH 7.9 containing 330 mM sorbitol, 1 mM EDTA, 1 mM magnesium chloride, and 0.1 % (w/v) defatted BSA (Buffer A). The resulting pellet was washed three times in Buffer A, containing no BSA, by gently resuspending and pelleting (4000 x g, 5 min).

Enzyme assays The activities of marker enzymes were assayed as described by Emes and England (1986). The glutamate synthase assay for determination of intactness of amyloplasts was performed using the protocol of Emes and Fowler (1979).

Chip colour Chips were processed as described by Coffin et al. (1987). Chip colour was determined based on 3 determinations using an Agtron Model M30A colorimeter (Chism Machinery, Niagara Falls, Canada). Acceptable colour score was defined as those chips that received scores of 50 or greater.

Sugar analysis The fructose, glucose, and sucrose contents of potatoes were determined using the high-performance liquid chromatographic (HPLC) method of Wilson et al. (1983). The HPLC was equipped with a Waters Associates injector (Milford, Massachusetts), a Beckman 1l0B solvent delivery model pump (Mississauga, Ontario), a Jones Chromatography 4.6 mm i.d. x 25 em Apex amino 51l column (Mid Glam, England), and a Waters Associates differential refractometer R401 detector. The mobile phase was acetonnitrile: water (80 : 20). The column was operated at a flow rate of 2.0 mLi min. Three samples of 15 ilL were injected into the column.

Differential scanning calorimetry Amyloplast membrane phase transition properties were assessed using differential scanning calorimetry (DSC) with a Dupont Model 1090 thermal analyzer equipped with a Model 910 thermal unit. The Interactive DSC Data Analysis Program «Heats and Temperatures of Transition Version 3.0" (Dupont Instruments, Wilmington, DE) was used to obtain transition temperatures. Indium was used to calibrate melting temperatures and heats of transition. Aluminum DSC pans containing 10 ilL of amyloplast membranes in extraction buffer A were hermetically sealed and heated from 0 °C to 90°C at a rate of 5 °C/min. An equal volume of G-25 Sephadex in extraction buffer A was used as a reference. Phase transition temperatures correspond to the average peak temperature for 5 determinations.

Lipid analysis Membrane lipids were extracted as described by Higgins (1987). The methylation of free fatty acids was carried out as directed using methanolic base reagent (Supelco, Bellefonte, PA). Gas chromatography was performed with a Shimadzu GC-14A (TekScience, Mississauga, ON) gas chromatograph equipped with a flame ionization detector, using high purity hydrogen as the carrier gas. The split ratio of the column was 1: 100. A O.5-IlL volume of the fatty acid methyl esters in isooctane was injected. The injector temperature was 220°C, while the detector temperature was 250 °C. The column used was a 25 m x 0 22 . mm i.d. fused silica capillary coated with BPX70 (Scientific Glass Engineering PTY. LID. Australia). Column initial temperature was 50°C, held for 3.0 min. This was followed by an increase in temperature to 185°C at 20 °C/min, and

Low temperature sweetening in potato tubers held at this temperature for 3 min, then heated at 20°C/min to 200°C, at which point it ran isothermally. Individual peak heights of fatty acids were determined with a Shimadzu C-R4A chromatopac (TekScience, Mississauga, ON). Fatty acid composition was determined based on three determinations of samples concentrated 20 times under a stream of nitrogen. Fluorescence Polarization

Fluorescence polarization measurements were performed as described by Nealon et al. (1984) on a Shimadzu RF-540 spectrofluorophotometer (TekScience, Mississauga, ON) using 1,6-diphenyl1,3,5-hexatriene and trans-parinaric acid fluorescent probes. Anisotropy values were measured between 5 and 30°C every 5 0c. Structural order parameters (S) were derived as described by Marangoni (1992) by averaging the anisotropy values over the whole temperature range. Three determinations were performed on the membrane preparation. Protein analysis

Protein concentrations were determined using the Bio-Rad dye binding assay (Bradford, 1976). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12 % acrylamide gel, using a BioRad Mini-protean II electrophoresis system (Bio-Rad, Mississauga, ON) as indicated by the manufacturer, according to the method of Laemmli (1970). Twenty microliters of amyloplast membrane protein (51.6I!g/mL) in denaturing buffer were loaded in each lane. Protein samples were prepared by heating the amyloplast extracts in SDS-denaturing buffer (1:4 v/v) at 5560°C for 15 min. The gels were run at 200 V for 45 min. Protein bands were visualized using silver staining as described by Merril (1990). Dried gels were scanned using a Zeineh Soft-Laser Densitometer model SLR-504-XL (Biomed Instruments, Fullerton, CA).

Results and Discussion Both the low-temperature sweetening (L TS)-resistant ND860-2 and sensitive Norchip potato tubers accumulated fructose, glucose, and sucrose when stored at 4 °C relative to 12°C stored tubers (Table 1). The accumulation of fructose, glucose, and sucrose, however, was greater for Norchip cultivar than for ND860-2. Agtron color scores for Norchip and ND860-2 potato chips decreased (darker chips) due to low-temperature sweetening. However, the decrease in the Agtron color scores was greater for Norchip than for ND860-2 (55.4 to 18.9 for Norchip and 49.4 to 33.3 for Table 1: Potato tuber glucose, fructose and sucrose contents (g/ 10kg of wet tissue) determined by high-performance liquid chromatography and chip color scores from ND860-2 and Norchip cultivars stored at 4°C and 12°C for a period of 4 months'. Cultivar

Tem perature

Fructose (g/10 kg)

Glucose (gI10kg)

Norchip

12°C 4°C

ND' 2.5 ± 0.11

ND'

ND860-2

12°C 4°C

NO' 0.8 ± 0.17

Sucrose

Color score b

(g/10kg)

5.5 ± 0.35

2.0 ± 0.13 8.0 ± 0.40

55.4 ±0.22 18.9 ± 0.15

ND' 3.0 ± 0.12

1.9 ± 0.24 3.2 ± 0.25

49.4 ± 0.09 33.3± 0.12

Values represent the average of three determinations ± standard error. b High color scores are associated with lighter potato chips. A color score below 50 is deemed undesirable for a potato chip. , ND = not detectable. a

337

ND860-2), closely mirroring the sugar content trends. Nonenzymatic browning of the chips is caused by the reaction of the anomeric carbon in sugars with amino groups of proteins or free amino acids (Whistler and Daniel, 1985). These results confirmed that ND860-2 was more resistant to low temperature sweetening than Norchip (Coffin et al., 1987). However, ND860-2 tubers were darker and contained more glucose, fructose and sucrose than Norchip tubers after four months of storage at 12°C. This was interpreted as ND8602 tubers having senesced to a greater extent than Norchip tubers. The respiration rate and metabolic activity of ND 860-2 tubers is greater than that of Norchip tubers, hence ND8602 senesced on average faster than Norchip at temperatures above the critical temperature for chill-damage (Barichello et al., 1990). In this study, as well as in any study dealing with time-temperature effects, the non-chilled control tubers are senescing while the cold-stored tubers are both senescing and experiencing stress at the same time. For the L TS-resistant cultivars, cold-temperature slows down the natural senescent processes, while for the L TS-sensitive cultivars, cold-temperature accelerate the senescent and/or stress-induced physiological damage processes. Amyloplasts were isolated according to the method of Emes and England (1986). Latency of glutamate synthase activity is used as a marker for amyloplast intactness (Emes and Fowler, 1979). For ND860-2 amyloplasts derived from tubers stored at 12°C and 4°C, glutamate synthase activity increased 3.9-fold and 26-fold, respectively, in the presence of 0.3 % (v/v) Triton-X-IOO (Table 2). For the Norchip amyloplasts derived from tubers stored at 12°C and 4°C, glutamate synthase activity increased 123-fold and 8.3-fold, respectively (Table 2). The non-ionic detergent Triton-X-100 dissolves the amyloplast bilayers, destroying the compartmentation provided by this organelle. Hence, any enzyme located in the interior of the amyloplast would display increased activity when assayed in the presence of Triton-X100. Depending on the extent of damage to the amyloplast double membrane, different degrees of latency would be observed. From the patterns observed for glutamate synthase activity prior to Triton-X-100 addition, and from the -fold Table 2: Glutamate synthase activity associated with amyloplasts derived from Norchip and ND860-2 potato tuber cultivars in the presence and absence of the non-ionic detergent Triton X-IOO. Higher activities after addition of detergent are interpreted as high levels of amyloplast membrane intactness. Assays were performed as described in the materials and methods section. Variety I Storage temperature

Glutamate synthase activity' (I1moll mini mg) No Triton X-l00

ND860-2 12°C (0.3 mL; 0.85 g/L)b

12.2

± 6.60

ND860-24°C (0.3 mL; 0.26g/L)b

6.38 ± 0.15

Norchip 12 °C (0.3 mL; 1.13 g/L)b

0.189 ± 0.10

Norchip 4°C (0.3 mL; 0.30 g/L)b

16.9

± 6.39

0.3 % Triton X-100 47.2 ± 7.50 166 23.3

± 14.7

±

6.00

140.0 ± 51.9

, Values represents average of 3 determinations ± standard error. b Values in parenthesis represent the volume of potato amyloplast suspensions used in the assay and their protein concentrations.

338

EILEEN P. O'DONOGHUE, RICKEY Y. YADA, and ALEJANDRO G. MARANGONI

increases after detergent addition, it became clear that ND 8602 amyloplast membranes had senesced to a greater extent than Norchip amyloplast membranes at 12°C since the degree of membrane intactness was greater for the Norchip cultivar. For 4°C-stored cultivars, however, the degree of intactness for ND860-2 membranes relative to 12°C-stored tubers was greater than for Norchip's. ND860-2 tubers senesced faster than Norchip tubers, however, this process was slowed down at 4°C for ND860-2 and accelerated for Norchip. These observations were interpreted as a cold temperature induction of amyloplast membrane breakdown for Norchip tubers (LTSsensitive) and a cold temperature-induced decrease in the rate of senescence-induced breakdown of amyloplast membranes for ND860-2 tubers (LTS-resistant). One of the characteristic symptoms of senescence is the loss of membrane lipid (Paliyath and Droillard, 1992). Amyloplast membrane lipid yield decreased approximately 24 % for the Norchip cultivar stored at 4 DC relative to 12 DC (from 21 to 16 mg/kg fresh tissue) while a relative increase in the amyloplast membrane yield was observed for the ND860-2 cultivar stored at 4 DC relative to 12 DC (from 7.0 to 10.0 mg/kg fresh tissue). The above data would suggest that membrane lipid breakdown was occurring in the LTS sensitive Norchip cultivar and not in the LTS resistant ND860-2 cultivar due to cold temperatures. According to amyloplast membrane protein yield, however, both Norchip and ND860-2 displayed lower protein yields due to low-temperature storage (from 29.1 to 5.9 and from 27.0 to 8.1 mg/kg fresh tissue at 12 DC and 4 DC, respectively). Obviously, the ratio of lipid/protein in the amyloplast membrane does not remain constant during low-temperature stress. Fluorescence polarization spectroscopy was used to probe the structure of amyloplast membranes. For this study we chose two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and trans-parinaric acid (t-PA). DPH probes deeper regions of the membrane while t-PA probes regions closer to the surface of the bilayer. Amyloplasts derived from the cultivars stored at chilling and non-chilling temperatures and labelled with either of the two probes, did not display any phase transitions in the temperature range studied (5 - 30 DC). The structural order parameter (Table 3), however, increased for

the Norchip cultivar stored at 4 DC relative to 12 DC for both DPH (from 0.459 to 0.655) and t-PA (from 0.531 to 0.658) labelled membranes. For ND860-2 amyloplast membranes, the structural order parameter of amyloplast membranes for the 4 DC stored potatoes decreased relative to the 12 DC stored tubers (from 0.549 to 0.402 for DPH-labelled membranes and from 0.566 to 0.481 for t-PA-labelled membranes). Increases in the structural order parameter are indicative of changes in the structure of the amyloplast membranes characteristic of senescence-related processes (Legge et al., 1986). As expected, the structural order parameter of t-PA-labelled membranes is, in all cases, higher than that of DPH-labelled membranes since this probe is localized close to the surface of the membrane bilayer, a region of higher structural order, in which the motion of the fluorophore would be restricted to a greater extent. The ND860-2 amyloplast membranes had senesced to a greater extent than the Norchip membranes at 12 DC. At 4 DC, however, ND860-2 membranes were less senesced than Norchip membranes stored at 4 DC and their 12 DC-stored counterparts, suggesting a slowing-down of cold temperature-induced membrane degradation. Norchip membranes stored in the cold, on the other hand, had senesced to a much greater extent than their 12 DC counterparts. Differential scanning calorimetric studies of isolated amyloplasts detected phase transitions that corresponded to characteristic lipid phase transitions of bilayers (Fig. 1). No significant differences were observed for phase transition temperatures (PTT) between Norchip and ND860-2 amyloplast membranes from tubers stored at 12 DC (p >0.05) (Table 3). Higher membrane lipid phase transition temperatures have

Norchlp 12·C

-a '" 0

Cultivar / Storage Temperature

Structural Order Parameter DPHa

ND860-2 12°C ND860-24 °C Norchip 12°C Norchip 4°C

0.549 0.402 0.459 0.655

± 0.022 ± 0.054 ± 0.023 ± 0.017

t-PAb 0.566 ± 0.015 0.481 ± 0.018 0.531 ± 0.025 0.658± 0.019

Tm'

(0C) 37.9 ± 6.95 27.2 ± 1.72 45.1± 2.26 35.3 ;t: 3.04

a Means ± standard error of all temperatures between 5 °C-30 °C every 5 °C for 3 b

determinatio ns. Means ± standard error of all temperatures between 5 °C - 30 °Cevery 5 °C for 2 determinatio ns.

, Means of 4 d eterminations ± standard error.

Norchlp "DC

'-'

~

Table 3: Structural order parameter and phase transition temperatures (Tm) for amyloplasts derived from the chilling sensitive Norchip and the chilling resistant ND860-2 varieties. Structural orders parameters were determined using fluorescence polarization spectroscopy of 1,6-diphenyl-hexatriene (DPH) and trans-parinaric (TPA) acid labelled membranes while membrane phase transition temperatures were determined by differential scanning calorimetry.

100

~

....as

Q)

II:

t

50

0

..

'0

1:1 0

o

H

C>

t o

5

10

15 20

25

30

35

40

45

50

Temperature (oC)

Fig. 1: Differential scanning calorimetric traces of isolated amyloplast derived from low-temperature sweetening sensitive Norchip potato tubers and from resistant ND860-2 potato tubers. Aluminum DSC pans containing 10 ilL of amyloplast membranes in extraction buffer A were hermetically sealed and heated from 0 °C to 90°C at a rate of 5 °CI min. An equal volume of G-2S Sephadex in extraction buffer A was used as a reference. A representative DSC trace for each amyloplast preparation is presented in the figure.

Low temperature sweetening in potato tubers been traditionally associated with a greater sensItivIty to chilling injury (Lyons, 1973) and/or a greater extent of senescence (Paliyath and Droillard, 1992). Phase transition temperatures decreased for both cultivars when potatoes were stored at 4°C, 28 % for the ND860-2 and 22 % for the Norchip cultivar (Table 3). The reason for this decrease may be due to an active retailoring process of the membrane lipids and/or proteins and/or a slowing down of senescent processes, which were more effective in ND860-2 tubers. The PTT for ND860-2 at 4°C was lower than for Norchip, suggesting that the ND860-2 amyloplast membranes had acclimated to cold temperatures to a greater extent than Norchip. However, according to our previous results, we expected the PTT of Norchip membranes at 12°C to be lower than at 4°C. This proved to be opposite to what we expected. We have no explanation for this aberration, other than that phase transition temperatures are complex parameters that do not necessarily faithfully reflect the changes that are occurring in membranes in response to stress or senescence. The fatty-acid profile of the lipids isolated from the amyloplast membranes of the Norchip and ND860-2 cultivars is presented in Table 4. The double bond index (DBI) of Norchip amyloplast fatty acids from tubers stored at 12°C was higher (more unsaturation) than for ND860-2. This again suggested that ND860-2 tubers had senesced to a greater extent than Norchip tubers. The DBI of the Norchip cultivar stored at 4°C decreased dramatically (93 % decrease) upon storage at 4 DC, suggesting a massive loss of unsaturated fatty acids upon exposure of the potato tuber to cold temperatures. The DBI for the ND860-2 cultivar also decreased (70 % decrease), albeit to a lesser extent than for Norchip, upon storage at 4°C relative to 12°C storage. This suggested that the Norchip cultivar was more susceptible to cold-temperature injury than ND860-2, this effect being reflected by the degree of unsaturation of the amyloplast membrane lipids. For the Norchip cultivar, the most dramatic changes were decreases in linoleic acid and linolenic acid contents, 26 to 2.3 and 9.3 to 0.9 mol %, respectively. The loss of these unsaturated fatty acids was accompanied by an increase in the capric acid content, from 17.5 to 50.3 mol % and an increase in the palmitic acid content, from 33.4 to 43 mol %. The 32.1 mol % loss in fatty acids attributed to losses in linoleic and linolenic acid corresponded very closely to the 32.8 mol % increase in capric acid. Both linolenic and linoleic acids are prone to oxidative breakdown due to their high

339

degree of unsaturation. Breakdown, for example, of C-9 hydroperoxide fatty acid of linoleic acid can produce 2,4-decadienal, which could elute from the gas chromatograph at the retention time corresponding to capric acid. Breakdown of linoleic and linolenic acids in the sensitive cultivar could be mediated by lipoxygenase (Kumar and Knowles, 1993). Lipoxygenase has been associated with the development of senescence and corresponding membrane degradation (Paliyath and Droillard, 1992). If lipoxygenase is responsible for the observed lipid oxidation, this would lend strength to the argument that chilling stress induces an accelerated rate of membrane lipid degradation, and therefore, a greater senescence in sensitive cultivars. Changes in the fatty acid profile of ND860-2 amyloplast membranes due to exposure to cold temperatures included losses in the degree of unsaturation, primarily through the loss of linoleic and linolenic acids as well. Accompanying this decrease in unsaturation was a corresponding large increase in the relative content of palmitic acid and a smaller increase in the relative content of oleic acid. The relatively high content (73.4 mol %) of palmitic in ND860-2 amyloplast membrane lipids derived from potato tubers stored at 4 °C for four months was surprising, and we have no suitable explanation for this effect. Kumar and Knowles (1993) presented evidence of extensive lipid peroxidation during prolonged cold-storage of seed tubers (up to 32 months at 4°C). These authors reported that increased levels of malonaldehyde in the lipid extract from seed tubers stored in the cold were positively correlated with reducing sugar levels (r2 = 0.92). Accumulation of malonaldehyde is an index of lipid peroxidation. Kumar and Knowles (1993) went as far as to suggest that the observed increase in reducing sugars may be due to a progressive degeneration of amyloplast membranes, which facilitates the enzymatic hydrolysis of starch. As well, Spychalla and Desborough (1990) reported an inverse relationship between lipid un saturation and sugar content in potato tubers. Cultivars with higher levels of total-lipid fatty-acid unsaturation displayed lower rates of electrolyte leakage and lower sugar contents upon storage for 40 weeks at 3 dc. Knowles and Knowles (1989) also reported that increased saturation of phospholipids during aging of potato seed-tubers correlated well with age-induced loss of membrane integrity. It is possible, therefore, that an age-induced loss in amyloplast membrane integrity, via a gradual peroxidation of amyloplast membrane lipids, leads to senescent sweetening of potato tubers (Kumar and Knowles, 1993) and that this age-related phenomenon

Table 4: Relative fatty acid composition of amyloplast membranes (%mol/mol) derived from the Norchip and ND860-2 cultivars stored at 4°C and 12°C for a period of 4 months. * Cultivar / Temperature Norchip 12°C Norchip 4°C ND860-2 12 °C ND860-24°C

ClO ,o 17.5a 50.3b 26.7e 8.3d

C I2 ,O 2.0a NDf 2.5e LCd

C 14 ,o 3.0' 1.2b 5.6e 1.9d

C 16 ,O 33.4· 43.0 b 30.Oc 73 .3d

C 16,1 LOa NDf 2.6e 0.8d

C1S,o 4.9a l.3 b 5.8e 1.6d

CIS,1 2.9 a l.4 b 4.Oc 7.7d

Cls,z

CIs,)

DBI'

26.0' 2.3 b 17.5e 4.0d

9.3a 0.9b 5.2e l.3b

1.38 0.09 0.81 0.24

* Values r epresent means of three determinations. For each fatty acid column only, values with the same letter are not significantly different by the Duncan's multiple range test (p > 0.05). Statistical analysis was performed using the general linear methods procedure of the SAS sta-

tistical package (SAS, 1985). DBI (double bond index) = (1 x %monoenes + 2 x %dienes + 3 x %trienes)/(%saturates). f ND = not detectable. e

340

EILEEN P. O'DONOGHUE, RICKEY Y. YADA, and ALEJANDRO G. MARANGONI

further evidence of protein degradation. The thick band at 66-kDa corresponds to bovine serum albumin, which was carried over from the initial extraction buffer and was still present after several washes. In summary, low temperature stress affected the structure of the amyloplast membranes in a senescence-like fashion. It would seem that the structure and probably the function of the amyloplast membranes from low-temperature sweetening cultivars are affected to a greater extent than the amyloplast membranes from resistant cultivars. A senesced membrane will affect the compartmentation provided by the organelle, affecting the transport of inorganic phosphate, glucose-6-phosphate and other effectors and intermediaries of starch metabolism. The effects of low temperature storage on the amyloplast double membrane structure may also be a determining factor in the development of low temperature sweetening in potato tubers. Acknowledgements

We acknowledge the technical assistance of Dr. Yukio Kakuda with the fatty acid analysis and helpful comments throughout the study and the technical assistance of Vanessa Locke, S. T. Ali-Khan and Sascha Wijsman. This research was supported by the Ontario Ministry of Agriculture and Food, the Ontario Potato Cultivar Evaluation Association, the Canadian Potato Chip and Snack Food Association, the Ontario Potato Growers' Marketing Board and the Natural Sciences and Engineering Research Council of Canada and Agriculture Canada. 97,4 66,2

45

31

21,5

14,4

Molecular mass (kOa) Fig. 2: Densitometric scan of an electrophoretic separation of isolated amyloplasts derived from low-temperature sweetening sensitive Norchip potato tubers and from resistant ND860-2 potato tubers stored at 4 or 12°C for 4 months, performed on a 12 % (w/v) sodium-dodecyl sulfate polyacrylamide gel (SDS-PAGE). (A) Norchip stored at 12°C, (B) Norchip stored at 4°C, (C) ND860-2 stored at 12°C, (D) corresponds to ND860-2 stored at 4°C. Protein peaks indicated by the arrows correspond to the 50.6 and 48.5-kDa proteins present in Norchip but not in ND860-2. Gels were loaded with 1 /!g of protein per lane and stained with silver after separation.

shares similar biochemical mechanisms with the temperature-induced sweetening observed in LTS-sensitive potato tubers. Amyloplast protein patterns, as determined by SDSPAGE, are presented in Figure 2. The patterns observed were very similar, however, the separation achieved on the 12 % acrylamide gel, as depicted in this densitometric scan (Fig. 2), showed that two protein bands of apparent molecular weight 50.8 and 48.7-kDa were present in the Norchip variety and not in the ND860-2 variety, stored at either 4 or 12°C. As well, amyloplasts derived from the Norchip cultivar and run on a 12 % acrylamide gel showed a large band migrating close to the electrophoretic front, suggesting proteolysis had taken place (not shown). This sample also displayed substantial streaking during the electrophoretic run,

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