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Aspects of amino acid metabolism in stored potato tubers (cv. Pentland Dell)
Plant Science 127 (1997) 17 – 24
Aspects of amino acid metabolism in stored potato tubers (cv. Pentland Dell) E.R. Brierley, P.L.R. Bonner, A.H. Cobb * Department of Life Sciences, The Nottingham Trent Uni6ersity, Clifton Lane, Nottingham NG11 8NS, UK Received 19 February 1997; received in revised form 10 April 1997; accepted 21 April 1997
Abstract Tubers of the cv. Pentland Dell were stored at 5 and 10°C for up to 33 weeks and analysed for soluble protein and free amino acids. In addition, glutamine synthetase, NADH-GOGAT and acid proteinase activities were measured over the 33-week storage period. An accumulation of asparagine and glutamine occurred during late storage which coincided with an upturn in proteinase activity. The amide content of the major tuber storage proteins suggested that amidation of free amino acids must account for the high proportion of free amides. The activities of glutamine synthetase and NADH-GOGAT further indicated amidation of free amino acids throughout storage, independent of storage temperature. An increase of total soluble protein during storage suggested that much of the observed increase in amides resulted from translocation into the tuber core tissue, perhaps as a result of the chemical suppression of sprout growth. © 1997 Elsevier Science Ireland Ltd. Keywords: Potato tuber storage; Proteinase activity; Free amide content; amidation of free amino acids
1. Introduction A detailed understanding of fundamental nitrogen flux in potato tubers during storage is lacking. There is a paucity of data in the literature regarding nitrogen metabolism in tubers and extrapolation from leaves and roots to tubers should be avoided because of the physiological differences
Abbre6iations: CIPC; chlorpropham. * Corresponding author.
potato tubers exhibit compared to root and green tissues. Such a study of nitrogen flux is commercially important because of the involvement of amino acids in the Maillard browning reaction, responsible for the characteristic fry colours of processed potato products [1]. The tuber reducing sugar content alone is generally used as a marker for processing quality [2]. However, anomalies in the relationship of reducing sugars to fry colour do occur, and studies have suggested that free amino acids may also play a role in determining fry colour [3–5].
0168-9452/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 1 0 9 - X
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E.R. Brierley et al. / Plant Science 127 (1997) 17–24
Previous studies have shown varying trends in free amino acid content during the storage of potato tubers [6,7]. However, in all studies, the amides asparagine and glutamine formed a high proportion of the free amino acid pool [8,9,5]. Hart and Cobb [10] suggested that the conversion of glutamine to asparagine by asparagine synthetase during storage was critical in the determination of fry colour. The high concentration of free amides may result from either amidation of glutamate by glutamine synthetase or the breakdown of storage proteins, such as patatin and the 22-kDa protein complex, both of which contain asparagine and glutamine residues [11]. The amide content may also be influenced by NADHGOGAT activity, the enzyme of glutamine deamidation in non-green plant tissues [12]. In this investigation, nitrogen flux during prolonged tuber storage was examined in the cv. Pentland Dell, a commercially important cultivar to the UK processing industry. The amino acid, amide, patatin and total soluble protein contents were routinely analysed for 33 weeks in order to assess the influence of storage duration and temperature regime on pool size. Glutamine synthetase, NADH-GOGAT and acid proteinase activities [5] were also measured to ascertain their relative roles in controlling amide and amino acid content.
2. Materials and methods
2.1. Storage and sampling of potato tubers Tubers of the cv. Pentland Dell were harvested in October 1992, transported to the Potato Marketing Board Experimental Station (Sutton Bridge, Lincolnshire, UK), cured at 15°C and 95% RH for 2 weeks, before division into boxes for storage at 5 and 10°C (95% RH) for up to 33 weeks. Sprout formation during storage was suppressed by chlorpropham (CIPC) treatments as granules. Tubers were sampled for analysis at 4-week intervals. Fifteen tubers were randomly selected, packed in insulated cold boxes containing vermiculite equilibrated at the storage temperature and transported to The Nottingham Trent University for analysis.
2.2. Extraction of amino acids and soluble proteins Five tubers were used for the extraction and analysis of amino acids and soluble proteins. Longitudinal cores of 10 mm diameter were taken from the centre of each tuber and divided equally into basal, median and apical sections. Basal sections were used for the extraction of amino acids as the greatest changes in concentration occur in this section during storage [10]. Each section was weighed in 5 ml absolute ethanol, homogenised in 5 ml cold water (MilliQ reagent grade, Millipore) and centrifuged at 800 × g for 5 min using a Denley model BR401 refrigerated centrifuge. The supernatant was stored on ice and the pellet resuspended in 3 ml cold Millipore (MilliQ reagent grade) water prior to re-centrifugation (800 × g) for a further 5 min. The supernatants from both centrifugations were pooled and stored on ice. The pellet was resuspended in a further 2 ml of Millipore (MilliQ reagent grade) water and a final centrifugation step (800× g) performed. The pooled supernatants from each tuber were frozen at −70°C prior to lyophilisation and analysis.
2.3. Measurement of free amino acids and soluble protein content Tuber amino acid contents were measured throughout storage by reversed phase HPLC using the method of Brierley et al. [5]. The total free amino acid pool size was determined by ninhydrin assay [13]. Absorbance readings were converted to mg amino acid g − 1 tuber fresh weight using a glycine standard curve. Total soluble protein content was determined by the Coomassie Blue dye-binding assay of Bradford [14]. Tuber patatin content was measured using lyophilised tuber core tissue (4 mg) suspended in 2 ml 200 mM Tris–HCl, pH 7.0, containing 0.1 mM dithiothreitol. The suspension was filtered through a 0.2-mm membrane filter (Millipore) and the total soluble protein content determined by Coomassie Blue dye-binding [14]. The patatin proportion of the soluble protein pool was analysed by gel-filtration as described by Brierley et al. [5], and the patatin content calculated using the total soluble protein content.
E.R. Brierley et al. / Plant Science 127 (1997) 17–24
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2.4. Measurement of acid proteinase acti6ity
2.6. Data analysis
Acid proteinase activity was extracted as described by Brierley et al. [5] and assayed using fluorescein isothiocyanate-labelled casein as substrate [15]. One unit of activity was defined as a change in fluorescence of 1 relative fluorescence unit (i.e. the fluorescence of a 48-mM quinine sulphate solution) min − 1.
Key differences in pool sizes and enzyme activities were analysed for significance using Student t-tests. All results below are presented as means (n=5) with standard errors.
2.5. Measurement of glutamine synthetase and NADH-GOGAT acti6ities
3.1. Tuber free amino acid content
Longitudinal cores were removed from three tubers and the basal sections pooled on an equal weight basis. Tissue was homogenised on ice in 100 mM Tris–HCl, pH 8.5, containing 1 mM MgCl2, 1 mM EDTA and 0.1% (v/v) 2-mercaptoethanol. The homogenate was filtered through two layers of muslin, centrifuged in the cold for 5 min at 10 000× g using an MSE Micro-Centaur centrifuge and the crude extract was desalted using a Pharmacia PD10 column. Glutamine synthetase activity was measured by the method of Lea et al. [16] modified to pH 8.5. Non-enzymic colour production was accounted for by zero-time additions of acidified ferric chloride. The change in absorbance was related to a standard curve for g-glutamylhydroxamate and a unit of activity defined as 1 nmol g-glutamylhydroxamate formed min − 1. NADH-GOGAT (NADH-glutamine:2-oxoglutarate aminotransferase) activity was measured throughout storage. Basal core tissue was pooled from three tubers and homogenised on ice in 100 mM KH2PO4 –KOH, pH 8.5, containing 500 mM EDTA, 100 mM KCl, 0.1% (v/v) 2-mercaptoethanol and 0.1% (v/v) Triton X-100. The homogenate was filtered through two layers of muslin, clarified and desalted as above. A 500-ml aliquot of enzyme was added to a reaction mixture at 30°C which contained 1.4 ml 100 mM KH2PO4 –KOH, pH 8.5, 200 ml 100 mM glutamine, 200 ml 2-oxoglutarate and 1 mM NADH. The change in absorbance was monitored at 340 nm and related to the oxidation of NADH using a molar extinction coefficient of 6220 for a 1-cm path length. One unit of activity was defined as the oxidation of 1 nmol (NADH) min − 1.
3. Results
In tubers of cv. Pentland Dell the amides asparagine and glutamine predominated and comprised between 34 and 90% (w/w) of the total free amino acid pool (Table 1). The total free amino acid content was stable for the first 17 weeks of storage, after which it increased (Fig. 1D) significantly (PB0.05). This trend is comparable to that observed by previous workers [17] during prolonged storage of tubers of cv. Katadhin. The rise of free amino acid pool size consisted primarily of asparagine and glutamine, which comprised over 90% (w/w) of the free amino acid pool after 25 weeks storage at 10°C (Table 1). This significant
Fig. 1. The influence of prolonged storage at 10°C on the: (A) patatin; (B) total soluble protein; (C) total free amide; and (D) total free amino acid content (mg g − 1 fresh weight basis) of cv. Pentland Dell tubers. Values are means (n =5) 9 SE.
a
mean 9 SE (mg g−1 fresh wt.)
% of total poola 18.63 15.82 0.27 1.35
mean 9SE (mg g−1 fresh wt.) 2.07 9 0.24 1.7690.36 0.03 9 0.01 0.159 0.03 11.109 0.95
7.27 91.10 4.60 9 0.99 0.13 90.01 0.329 0.07 18.299 2.72
25 wks storage at 5°C
Harvest
Treatment
Percentages of the total free amino acid pool calculated on a mg g−1 fresh weight basis.
Asn Gln Asp Glu Total free amino acid pool
Amino acid
Table 1 The free amino acid content of cv. Pentland Dell tubers at harvest and after prolonged storage
39.72 25.13 0.73 1.73
% of total poola
9.319 1.36 7.669 1.36 0.229 0.13 0.609 0.32 18.789 2.94
mean9 SE (mg g−1 fresh wt.)
25 wks storage at 10°C
49.57 40.80 1.19 3.21
% of total poola
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E.R. Brierley et al. / Plant Science 127 (1997) 17–24
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and 29 was 1.18:1, 1.19:1, 1.58:1 and 1.14:1, respectively.
3.2. Tuber soluble protein content The total soluble protein content increased (PB0.05) throughout storage (Fig. 1B). In contrast, the content of the storage protein patatin declined (PB 0.05) during late storage, as was clearly illustrated in tubers after 17 weeks storage at 10°C (Fig. 1A). The proportion of the total soluble protein pool consisting of patatin declined with storage duration (PB 0.05). At weeks 0, 13 and 29 at 10°C, patatin comprised 28, 28 and 9% (w/w), respectively, of the total soluble protein pool.
3.3. Acid proteinase acti6ity Fig. 2. The total free amide content of cv. Pentland Dell tubers stored at 5 ( ) and 10°C ( ), correlated to acid proteinase activity at 5 () and 10°C (). Amide values are means of five tubers 9SE and acid proteinase activities are means of five assays9 SE.
accumulation of free amides (P B0.05) was 40% greater at 10 than 5°C after 25 weeks in storage (Fig. 2). The amides thus comprised only 65% (w/w) of the free amino acid pool after 25 weeks at 5°C (Table 1), although this value increased to 85% by 29 weeks. The aspartate and glutamate contents also increased during storage (P B 0.05) reaching a maximum after 33 weeks, this accumulation was 77% greater at 10 than 5°C. Aspartate and glutamate together comprised less than 2% (w/w) of the total free amino acid pool at harvest, this proportion rising to 12% (w/w) after 33 weeks at both 5 and 10°C. As a consequence of the late season increase in the amides, aspartate and glutamate, the proportion of the total free amino acid pool consisting of other amino acids decreased at both 5 and 10°C, with the exception of a peak after 29 weeks at 10°C. Asparagine and glutamine accumulated equally during late storage such that the asparagine:glutamine ratio showed little variation throughout storage. This was clearly illustrated in tubers stored at 5°C, with the exception of a slight peak after 25 weeks, where the free asparagine:glutamine ratio at harvest, weeks 17, 25
Acid proteinase activity declined after 5 weeks, and remained at a minimal activity until 21 weeks of storage (Fig. 2). Activity then increased (PB 0.05) with an accumulation of free amides. This increase of acid proteinase activity was temperature dependent (PB 0.05) with twice the activity at 10 than at 5°C (Fig. 2).
3.4. Glutamine synthetase and NADH-GOGAT acti6ities Activity of NADH-GOGAT rose to a maximum at between 13 and 21 weeks depending on storage temperature (Fig. 3A). Activity of glutamine synthetase was observed to fluctuate throughout storage with no significant temperature dependence (Fig. 3B). Activity of NADHGOGAT was generally lower than that of glutamine synthetase throughout storage (Fig. 3), with the exception of a period at around 29 weeks.
4. Discussion The minimal level of acid proteinase activity and low free amino acid pool size prior to 21 weeks in storage, suggested that the tubers were in a state of dormancy. After 21 weeks, the upturn
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E.R. Brierley et al. / Plant Science 127 (1997) 17–24
Fig. 3. Activities of (A) NADH-GOGAT and (B) glutamine synthetase during the storage of cv. Pentland Dell tubers at 5 () and 10°C (). Activities are means of five assays9 SE. Error bars are included where they exceed the symbol size.
of acid proteinase activity and the resultant increase of free amides, indicated the emergence of tubers from dormancy and the mobilisation of nitrogen reserves to supply sprout initiation and growth [18]. Bailey et al. [19] suggested that reserve mobilisation may be regulated by gibberellic acid, through effects on cell compartmentation. The temperature dependence of the increase of acid proteinase activity and free amide content in cv. Pentland Dell suggested that nitrogen mobilisation may increase with temperature. This contradicts the findings of Nowak and Sckwiercz [20] who suggested low temperature enhancement of protein degradation. The increases in aspartate and glutamate contents, and total free amino acid content with exception of 25 weeks in storage, also demonstrated temperature dependence which could be related to the effect of temperature on acid proteinase activity. Nucleic acid sequencing of the gene encoding patatin, the major tuber protein, showed asparagine and glutamine to comprise only 4.40 and 3.63% respectively, of the amino acid residues in patatin [21]. In contrast, the proportion of the free amino acid pool consisting of the amides was far higher than that present in patatin and in-
creased during storage to a maximum after 25 weeks at 10°C (Table 1) and 29 weeks at 5°C. This, coupled to the relatively minor scale of the decline in patatin content, suggested that patatin does not act directly as the major source of free amide accumulation during storage. It is likely that the free amino acids produced from degradation of storage proteins were subjected to amidation. This was supported by the study of Pereira et al. [22] which showed high levels of cytosolic glutamine synthetase present in tubers at the onset of sprouting. This form of glutamine synthetase was primarily located around the vascular ring and in the inner phloem, suggesting a role for the enzyme in the synthesis of glutamine for export to the sprouts [22]. In this study, the higher specific activity of glutamine synthetase, relative to that of NADH-GOGAT until after 25 weeks in storage, suggests there was a higher rate of free amino acid amidation compared to deamidation. This may have accounted for much of the increase in free amides during prolonged storage. The increasing proportion of the total free amino acid pool consisting of aspartate and glutamate after 29 weeks storage at 5 and 10°C may indicate a switch of amino acid metabolism towards a net de-amida-
E.R. Brierley et al. / Plant Science 127 (1997) 17–24
tion. This is supported by the higher specific activity of NADH-GOGAT relative to that of glutamine synthetase after 29 weeks. The accumulation of free asparagine in conjunction with free glutamine after 25 weeks storage suggested that asparagine synthetase activity was present as postulated by Hart and Cobb [10]. However, as the concentration of free asparagine did not increase relative to glutamine, it is likely that amide metabolism was tightly controlled. It is also possible that asparaginase activity may have negated any net conversion of free glutamine to asparagine through the deamidation of asparagine to aspartate. Tuber nitrogen metabolism may hence have fundamental differences to that observed upon seed germination, where a clear conversion of glutamine into asparagine generally occurs [23]. It is therefore likely that both asparagine and glutamine play an equally important role in nitrogen storage and transport after the break of tuber dormancy. The increase of these amides upon emergence from dormancy, may account for the decline in potato tuber processing quality often observed after prolonged storage [4,5]. The high free amide content of stored tubers probably results from a combination of factors including protein amide residue content, acid proteinase activity and the net rate of free amino acid amidation. However, the control of tuber free amide content was further complicated by the increase of total soluble protein content during storage, possibly due to the formation of low molecular weight proteins, as observed in a previous study [18]. This increase of total soluble protein in conjunction with the accumulation of free amino acids, suggested that nitrogen was supplied from the degradation of insoluble proteins, or that the total nitrogen content of tuber core tissue increased with storage. As previous workers had shown no decrease of insoluble proteins during storage [24], a possible explanation is the inward translocation of free amides into tuber pith tissue, as postulated by Cotrufo and Levitt [25]. A translocation of free amides may be a feature of tubers treated with sprout suppressants, as in this study where CIPC was used. By inhibiting
23
sprout growth, CIPC may have removed the source–sink relationship of nitrogen flux from tubers to sprouts and thus prevented the mobilisation of nitrogen reserves from reaching maximum potential [26]. In the absence of the sink strength provided by growing sprouts, inward translocation of amides may have occurred through the internal phloem in a similar fashion to that which occurs in tuber development [22], providing nitrogen for protein synthesis within the tuber core. This would suggest that nitrogen flux during the prolonged storage of tubers is far more complex than a straightforward mobilisation of nitrogen to support sprout growth. However, further work is needed to confirm this hypothesis.
Acknowledgements The authors wish to acknowledge the Potato Marketing Board, Ministry of Agriculture Fisheries and Food and the Potato Processors Association for financial support, and in particular Dr R.M.J. Storey and staff at the Sutton Bridge Experimental Station for their continued support.
References [1] R.S. Schallenberger, O. Smith, R.H. Treadaway, Role of sugars in the browning reaction in potato chips, J. Agric. Food Chem. 7 (1959) 274. [2] G. Marquez, M.C. An˜on, Influence of reducing sugars and amino acids in the color development of fried potatoes, J. Food Sci. 51 (1986) 157 – 160. [3] M.A. Roe, R.M. Faulks, J.L. Belsten, Role of reducing sugars and amino acids in fry colour of chips from potatoes grown under different nitrogen regimes, J. Sci. Food Agric. 52 (1990) 207 – 214. [4] O.S. Khanbari, A.K. Thompson, Effects of amino acids and glucose on the fry colour of potato crisps, Potato Res. 36 (1993) 359 – 364. [5] E.R. Brierley, P.L.R. Bonner, A.H. Cobb, Factors influencing the free amino acid content of potato (Solanum tuberosum L) tubers during prolonged storage, J. Sci. Food Agric. 70 (1996) 515 – 525. [6] E.A. Talley, R.B. Toma, P.H. Orr, Amino acid composition of freshly harvested and stored potatoes, Am. Potato J. 61 (1984) 267 – 279. [7] P.C.M. Hart, K.E. Pallett, A.H. Cobb, Metabolic changes in Pentland Dell tubers during storage at 5°C and 10°C,
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in: S.F.L. Ball, C. Coll, P. Gans, M.C. Heath, M. Lainsbury, C.S. Speller, R.M.J. Storey, J.B. Sweet, K.G. Sutherland, G.P. Whytock (Eds.), Aspects of Applied Biology, 13, Crop Protection of Sugar Beet and Crop Protection and Quality of Potatoes, Part II, Association of Applied iologists, 1986, pp. 457–464. A.M.C. Davies, The free amino acids of potato varieties grown in England and Ireland, Potato Res. 20 (1977) 9– 21. P. Millard, The nitrogen content of potato (Solanum tubersosum) tubers in relation to nitrogen application- the effect on amino acid composition and yields, J. Sci. Food Agric. 37 (1986) 107–114. P.C.M. Hart, A.H. Cobb, Aspects of carbohydrate and amino acid metabolism in Pentland Dell tubers stored at 5°C and 10°C, in: Proceedings of the European Association for Potato Research Conference on Storage and Utilisation of Potatoes, European Association for Potato Research, 1988, pp. 293–304. S.G. Suh, J.E. Peterson, W.J. Stiekema, D.J. Hannapel, Purification and characterisation of the 22–kilodalton potato tuber proteins, Plant Physiol. 94 (1990) 40–45. G.O. Osuji, R.G. Cuero, A.C. Washington, Effects of a-ketoglutarate on the activities of the glutamine synthetase, glutamate dehydrogenase, and aspartate transaminase of sweet potato, yam tuber and cream pea, J. Agric. Food Chem. 39 (1991) 1590–1596. E.W. Yemm, E.C. Cocking, Determination of amino acids with ninhydrin, Analyst 80 (1955) 209–213. M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248 – 254. S.S. Twining, Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes, Anal. Biochem. 143 (1984) 40 – 45.
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[16] P.J. Lea, R.D. Blackwell, F.-L. Chen, U. Hecht, Enzymes of ammonia assimilation, in: P.J. Lea (Ed), Methods in Plant Biochemistry, vol. 3, Academic Press, New York, 1990, pp. 257 – 276. [17] E.A. Talley, W.L. Porter, Chemical composition of potatoes VII, Relationship of the free amino acid concentrations to specific gravity and storage time, Am. Potato J. 47 (1970) 214 – 224. [18] J. Nowak, Biochemical changes in stored potato tubers with different rest periods, 1. Influence of the storage temperature and isopropyl phenylcarbamates (IPC and CIPC) on protein changes, Z. Pflanzenphysiol. Bodenkunde 8 (1977) 113 – 124. [19] K.M. Bailey, I.D.J. Phillips, D. Pitt, The role of buds and gibberellin in dormancy and the mobilization of reserve materials in potato tubers, Ann. Bot. 42 (1978) 649 – 657. [20] J. Nowak, A. Sckwiercz, Proteolytic enzyme activity in stored tubers with different rest periods, Bull. Acad. Polon. Sci. Ser. Sci. Biol. 23 (1975) 129 – 133. [21] S. Rosahl, R. Schmidt, J. Schell, L. Willmitzer, Isolation and characterization of a gene from S.tuberosum encoding patatin, the major storage protein of potato tubers, Mol. Gen. Genet. 203 (1986) (1986) 214 – 220. [22] S. Pereira, J. Pissarra, C. Sunkel, R. Salema, Tissue-specific distribution of glutamine synthetase in potato tubers, Ann. Bot. 77 (1996) 429 – 432. [23] M.F. Dilworth, L. Dure III, Developmental biochemistry of cotton seed embryogenesis and germination, Plant Physiol. 61 (1978) 698 – 702. [24] D. Racusen, Occurrence of patatin during growth and storage of potato tubers, Can. J. Bot. 61 (1983) 370 – 373. [25] C. Cotrufo, J. Levitt, Investigations of the cytoplasmic particulates and proteins of potato tubers, VI. Nitrogen changes associated with emergence of potato tubers from the rest period, Physiol. Plant. 11 (1958) 240 – 248. [26] H.V. Davies, H.A. Ross, The pattern of starch and protein degradation in tubers, Potato Res. 27 (1984) 373 – 381.
Aspects of amino acid metabolism in stored potato tubers (cv. Pentland Dell) E.R. Brierley, P.L.R. Bonner, A.H. Cobb * Department of Life Sciences, The Nottingham Trent Uni6ersity, Clifton Lane, Nottingham NG11 8NS, UK Received 19 February 1997; received in revised form 10 April 1997; accepted 21 April 1997
Abstract Tubers of the cv. Pentland Dell were stored at 5 and 10°C for up to 33 weeks and analysed for soluble protein and free amino acids. In addition, glutamine synthetase, NADH-GOGAT and acid proteinase activities were measured over the 33-week storage period. An accumulation of asparagine and glutamine occurred during late storage which coincided with an upturn in proteinase activity. The amide content of the major tuber storage proteins suggested that amidation of free amino acids must account for the high proportion of free amides. The activities of glutamine synthetase and NADH-GOGAT further indicated amidation of free amino acids throughout storage, independent of storage temperature. An increase of total soluble protein during storage suggested that much of the observed increase in amides resulted from translocation into the tuber core tissue, perhaps as a result of the chemical suppression of sprout growth. © 1997 Elsevier Science Ireland Ltd. Keywords: Potato tuber storage; Proteinase activity; Free amide content; amidation of free amino acids
1. Introduction A detailed understanding of fundamental nitrogen flux in potato tubers during storage is lacking. There is a paucity of data in the literature regarding nitrogen metabolism in tubers and extrapolation from leaves and roots to tubers should be avoided because of the physiological differences
Abbre6iations: CIPC; chlorpropham. * Corresponding author.
potato tubers exhibit compared to root and green tissues. Such a study of nitrogen flux is commercially important because of the involvement of amino acids in the Maillard browning reaction, responsible for the characteristic fry colours of processed potato products [1]. The tuber reducing sugar content alone is generally used as a marker for processing quality [2]. However, anomalies in the relationship of reducing sugars to fry colour do occur, and studies have suggested that free amino acids may also play a role in determining fry colour [3–5].
0168-9452/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 1 0 9 - X
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E.R. Brierley et al. / Plant Science 127 (1997) 17–24
Previous studies have shown varying trends in free amino acid content during the storage of potato tubers [6,7]. However, in all studies, the amides asparagine and glutamine formed a high proportion of the free amino acid pool [8,9,5]. Hart and Cobb [10] suggested that the conversion of glutamine to asparagine by asparagine synthetase during storage was critical in the determination of fry colour. The high concentration of free amides may result from either amidation of glutamate by glutamine synthetase or the breakdown of storage proteins, such as patatin and the 22-kDa protein complex, both of which contain asparagine and glutamine residues [11]. The amide content may also be influenced by NADHGOGAT activity, the enzyme of glutamine deamidation in non-green plant tissues [12]. In this investigation, nitrogen flux during prolonged tuber storage was examined in the cv. Pentland Dell, a commercially important cultivar to the UK processing industry. The amino acid, amide, patatin and total soluble protein contents were routinely analysed for 33 weeks in order to assess the influence of storage duration and temperature regime on pool size. Glutamine synthetase, NADH-GOGAT and acid proteinase activities [5] were also measured to ascertain their relative roles in controlling amide and amino acid content.
2. Materials and methods
2.1. Storage and sampling of potato tubers Tubers of the cv. Pentland Dell were harvested in October 1992, transported to the Potato Marketing Board Experimental Station (Sutton Bridge, Lincolnshire, UK), cured at 15°C and 95% RH for 2 weeks, before division into boxes for storage at 5 and 10°C (95% RH) for up to 33 weeks. Sprout formation during storage was suppressed by chlorpropham (CIPC) treatments as granules. Tubers were sampled for analysis at 4-week intervals. Fifteen tubers were randomly selected, packed in insulated cold boxes containing vermiculite equilibrated at the storage temperature and transported to The Nottingham Trent University for analysis.
2.2. Extraction of amino acids and soluble proteins Five tubers were used for the extraction and analysis of amino acids and soluble proteins. Longitudinal cores of 10 mm diameter were taken from the centre of each tuber and divided equally into basal, median and apical sections. Basal sections were used for the extraction of amino acids as the greatest changes in concentration occur in this section during storage [10]. Each section was weighed in 5 ml absolute ethanol, homogenised in 5 ml cold water (MilliQ reagent grade, Millipore) and centrifuged at 800 × g for 5 min using a Denley model BR401 refrigerated centrifuge. The supernatant was stored on ice and the pellet resuspended in 3 ml cold Millipore (MilliQ reagent grade) water prior to re-centrifugation (800 × g) for a further 5 min. The supernatants from both centrifugations were pooled and stored on ice. The pellet was resuspended in a further 2 ml of Millipore (MilliQ reagent grade) water and a final centrifugation step (800× g) performed. The pooled supernatants from each tuber were frozen at −70°C prior to lyophilisation and analysis.
2.3. Measurement of free amino acids and soluble protein content Tuber amino acid contents were measured throughout storage by reversed phase HPLC using the method of Brierley et al. [5]. The total free amino acid pool size was determined by ninhydrin assay [13]. Absorbance readings were converted to mg amino acid g − 1 tuber fresh weight using a glycine standard curve. Total soluble protein content was determined by the Coomassie Blue dye-binding assay of Bradford [14]. Tuber patatin content was measured using lyophilised tuber core tissue (4 mg) suspended in 2 ml 200 mM Tris–HCl, pH 7.0, containing 0.1 mM dithiothreitol. The suspension was filtered through a 0.2-mm membrane filter (Millipore) and the total soluble protein content determined by Coomassie Blue dye-binding [14]. The patatin proportion of the soluble protein pool was analysed by gel-filtration as described by Brierley et al. [5], and the patatin content calculated using the total soluble protein content.
E.R. Brierley et al. / Plant Science 127 (1997) 17–24
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2.4. Measurement of acid proteinase acti6ity
2.6. Data analysis
Acid proteinase activity was extracted as described by Brierley et al. [5] and assayed using fluorescein isothiocyanate-labelled casein as substrate [15]. One unit of activity was defined as a change in fluorescence of 1 relative fluorescence unit (i.e. the fluorescence of a 48-mM quinine sulphate solution) min − 1.
Key differences in pool sizes and enzyme activities were analysed for significance using Student t-tests. All results below are presented as means (n=5) with standard errors.
2.5. Measurement of glutamine synthetase and NADH-GOGAT acti6ities
3.1. Tuber free amino acid content
Longitudinal cores were removed from three tubers and the basal sections pooled on an equal weight basis. Tissue was homogenised on ice in 100 mM Tris–HCl, pH 8.5, containing 1 mM MgCl2, 1 mM EDTA and 0.1% (v/v) 2-mercaptoethanol. The homogenate was filtered through two layers of muslin, centrifuged in the cold for 5 min at 10 000× g using an MSE Micro-Centaur centrifuge and the crude extract was desalted using a Pharmacia PD10 column. Glutamine synthetase activity was measured by the method of Lea et al. [16] modified to pH 8.5. Non-enzymic colour production was accounted for by zero-time additions of acidified ferric chloride. The change in absorbance was related to a standard curve for g-glutamylhydroxamate and a unit of activity defined as 1 nmol g-glutamylhydroxamate formed min − 1. NADH-GOGAT (NADH-glutamine:2-oxoglutarate aminotransferase) activity was measured throughout storage. Basal core tissue was pooled from three tubers and homogenised on ice in 100 mM KH2PO4 –KOH, pH 8.5, containing 500 mM EDTA, 100 mM KCl, 0.1% (v/v) 2-mercaptoethanol and 0.1% (v/v) Triton X-100. The homogenate was filtered through two layers of muslin, clarified and desalted as above. A 500-ml aliquot of enzyme was added to a reaction mixture at 30°C which contained 1.4 ml 100 mM KH2PO4 –KOH, pH 8.5, 200 ml 100 mM glutamine, 200 ml 2-oxoglutarate and 1 mM NADH. The change in absorbance was monitored at 340 nm and related to the oxidation of NADH using a molar extinction coefficient of 6220 for a 1-cm path length. One unit of activity was defined as the oxidation of 1 nmol (NADH) min − 1.
3. Results
In tubers of cv. Pentland Dell the amides asparagine and glutamine predominated and comprised between 34 and 90% (w/w) of the total free amino acid pool (Table 1). The total free amino acid content was stable for the first 17 weeks of storage, after which it increased (Fig. 1D) significantly (PB0.05). This trend is comparable to that observed by previous workers [17] during prolonged storage of tubers of cv. Katadhin. The rise of free amino acid pool size consisted primarily of asparagine and glutamine, which comprised over 90% (w/w) of the free amino acid pool after 25 weeks storage at 10°C (Table 1). This significant
Fig. 1. The influence of prolonged storage at 10°C on the: (A) patatin; (B) total soluble protein; (C) total free amide; and (D) total free amino acid content (mg g − 1 fresh weight basis) of cv. Pentland Dell tubers. Values are means (n =5) 9 SE.
a
mean 9 SE (mg g−1 fresh wt.)
% of total poola 18.63 15.82 0.27 1.35
mean 9SE (mg g−1 fresh wt.) 2.07 9 0.24 1.7690.36 0.03 9 0.01 0.159 0.03 11.109 0.95
7.27 91.10 4.60 9 0.99 0.13 90.01 0.329 0.07 18.299 2.72
25 wks storage at 5°C
Harvest
Treatment
Percentages of the total free amino acid pool calculated on a mg g−1 fresh weight basis.
Asn Gln Asp Glu Total free amino acid pool
Amino acid
Table 1 The free amino acid content of cv. Pentland Dell tubers at harvest and after prolonged storage
39.72 25.13 0.73 1.73
% of total poola
9.319 1.36 7.669 1.36 0.229 0.13 0.609 0.32 18.789 2.94
mean9 SE (mg g−1 fresh wt.)
25 wks storage at 10°C
49.57 40.80 1.19 3.21
% of total poola
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E.R. Brierley et al. / Plant Science 127 (1997) 17–24
21
and 29 was 1.18:1, 1.19:1, 1.58:1 and 1.14:1, respectively.
3.2. Tuber soluble protein content The total soluble protein content increased (PB0.05) throughout storage (Fig. 1B). In contrast, the content of the storage protein patatin declined (PB 0.05) during late storage, as was clearly illustrated in tubers after 17 weeks storage at 10°C (Fig. 1A). The proportion of the total soluble protein pool consisting of patatin declined with storage duration (PB 0.05). At weeks 0, 13 and 29 at 10°C, patatin comprised 28, 28 and 9% (w/w), respectively, of the total soluble protein pool.
3.3. Acid proteinase acti6ity Fig. 2. The total free amide content of cv. Pentland Dell tubers stored at 5 ( ) and 10°C ( ), correlated to acid proteinase activity at 5 () and 10°C (). Amide values are means of five tubers 9SE and acid proteinase activities are means of five assays9 SE.
accumulation of free amides (P B0.05) was 40% greater at 10 than 5°C after 25 weeks in storage (Fig. 2). The amides thus comprised only 65% (w/w) of the free amino acid pool after 25 weeks at 5°C (Table 1), although this value increased to 85% by 29 weeks. The aspartate and glutamate contents also increased during storage (P B 0.05) reaching a maximum after 33 weeks, this accumulation was 77% greater at 10 than 5°C. Aspartate and glutamate together comprised less than 2% (w/w) of the total free amino acid pool at harvest, this proportion rising to 12% (w/w) after 33 weeks at both 5 and 10°C. As a consequence of the late season increase in the amides, aspartate and glutamate, the proportion of the total free amino acid pool consisting of other amino acids decreased at both 5 and 10°C, with the exception of a peak after 29 weeks at 10°C. Asparagine and glutamine accumulated equally during late storage such that the asparagine:glutamine ratio showed little variation throughout storage. This was clearly illustrated in tubers stored at 5°C, with the exception of a slight peak after 25 weeks, where the free asparagine:glutamine ratio at harvest, weeks 17, 25
Acid proteinase activity declined after 5 weeks, and remained at a minimal activity until 21 weeks of storage (Fig. 2). Activity then increased (PB 0.05) with an accumulation of free amides. This increase of acid proteinase activity was temperature dependent (PB 0.05) with twice the activity at 10 than at 5°C (Fig. 2).
3.4. Glutamine synthetase and NADH-GOGAT acti6ities Activity of NADH-GOGAT rose to a maximum at between 13 and 21 weeks depending on storage temperature (Fig. 3A). Activity of glutamine synthetase was observed to fluctuate throughout storage with no significant temperature dependence (Fig. 3B). Activity of NADHGOGAT was generally lower than that of glutamine synthetase throughout storage (Fig. 3), with the exception of a period at around 29 weeks.
4. Discussion The minimal level of acid proteinase activity and low free amino acid pool size prior to 21 weeks in storage, suggested that the tubers were in a state of dormancy. After 21 weeks, the upturn
22
E.R. Brierley et al. / Plant Science 127 (1997) 17–24
Fig. 3. Activities of (A) NADH-GOGAT and (B) glutamine synthetase during the storage of cv. Pentland Dell tubers at 5 () and 10°C (). Activities are means of five assays9 SE. Error bars are included where they exceed the symbol size.
of acid proteinase activity and the resultant increase of free amides, indicated the emergence of tubers from dormancy and the mobilisation of nitrogen reserves to supply sprout initiation and growth [18]. Bailey et al. [19] suggested that reserve mobilisation may be regulated by gibberellic acid, through effects on cell compartmentation. The temperature dependence of the increase of acid proteinase activity and free amide content in cv. Pentland Dell suggested that nitrogen mobilisation may increase with temperature. This contradicts the findings of Nowak and Sckwiercz [20] who suggested low temperature enhancement of protein degradation. The increases in aspartate and glutamate contents, and total free amino acid content with exception of 25 weeks in storage, also demonstrated temperature dependence which could be related to the effect of temperature on acid proteinase activity. Nucleic acid sequencing of the gene encoding patatin, the major tuber protein, showed asparagine and glutamine to comprise only 4.40 and 3.63% respectively, of the amino acid residues in patatin [21]. In contrast, the proportion of the free amino acid pool consisting of the amides was far higher than that present in patatin and in-
creased during storage to a maximum after 25 weeks at 10°C (Table 1) and 29 weeks at 5°C. This, coupled to the relatively minor scale of the decline in patatin content, suggested that patatin does not act directly as the major source of free amide accumulation during storage. It is likely that the free amino acids produced from degradation of storage proteins were subjected to amidation. This was supported by the study of Pereira et al. [22] which showed high levels of cytosolic glutamine synthetase present in tubers at the onset of sprouting. This form of glutamine synthetase was primarily located around the vascular ring and in the inner phloem, suggesting a role for the enzyme in the synthesis of glutamine for export to the sprouts [22]. In this study, the higher specific activity of glutamine synthetase, relative to that of NADH-GOGAT until after 25 weeks in storage, suggests there was a higher rate of free amino acid amidation compared to deamidation. This may have accounted for much of the increase in free amides during prolonged storage. The increasing proportion of the total free amino acid pool consisting of aspartate and glutamate after 29 weeks storage at 5 and 10°C may indicate a switch of amino acid metabolism towards a net de-amida-
E.R. Brierley et al. / Plant Science 127 (1997) 17–24
tion. This is supported by the higher specific activity of NADH-GOGAT relative to that of glutamine synthetase after 29 weeks. The accumulation of free asparagine in conjunction with free glutamine after 25 weeks storage suggested that asparagine synthetase activity was present as postulated by Hart and Cobb [10]. However, as the concentration of free asparagine did not increase relative to glutamine, it is likely that amide metabolism was tightly controlled. It is also possible that asparaginase activity may have negated any net conversion of free glutamine to asparagine through the deamidation of asparagine to aspartate. Tuber nitrogen metabolism may hence have fundamental differences to that observed upon seed germination, where a clear conversion of glutamine into asparagine generally occurs [23]. It is therefore likely that both asparagine and glutamine play an equally important role in nitrogen storage and transport after the break of tuber dormancy. The increase of these amides upon emergence from dormancy, may account for the decline in potato tuber processing quality often observed after prolonged storage [4,5]. The high free amide content of stored tubers probably results from a combination of factors including protein amide residue content, acid proteinase activity and the net rate of free amino acid amidation. However, the control of tuber free amide content was further complicated by the increase of total soluble protein content during storage, possibly due to the formation of low molecular weight proteins, as observed in a previous study [18]. This increase of total soluble protein in conjunction with the accumulation of free amino acids, suggested that nitrogen was supplied from the degradation of insoluble proteins, or that the total nitrogen content of tuber core tissue increased with storage. As previous workers had shown no decrease of insoluble proteins during storage [24], a possible explanation is the inward translocation of free amides into tuber pith tissue, as postulated by Cotrufo and Levitt [25]. A translocation of free amides may be a feature of tubers treated with sprout suppressants, as in this study where CIPC was used. By inhibiting
23
sprout growth, CIPC may have removed the source–sink relationship of nitrogen flux from tubers to sprouts and thus prevented the mobilisation of nitrogen reserves from reaching maximum potential [26]. In the absence of the sink strength provided by growing sprouts, inward translocation of amides may have occurred through the internal phloem in a similar fashion to that which occurs in tuber development [22], providing nitrogen for protein synthesis within the tuber core. This would suggest that nitrogen flux during the prolonged storage of tubers is far more complex than a straightforward mobilisation of nitrogen to support sprout growth. However, further work is needed to confirm this hypothesis.
Acknowledgements The authors wish to acknowledge the Potato Marketing Board, Ministry of Agriculture Fisheries and Food and the Potato Processors Association for financial support, and in particular Dr R.M.J. Storey and staff at the Sutton Bridge Experimental Station for their continued support.
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