A major component (50-90%) of the nitrogen (Ni in berry juice is in the form of free
1 The term amino acid will be extended to include proline which is an imino acid.
2 Nitrogen (N), reters to nitrogenous compounds such as free amino acids, the ammonium ions, peptides and nucleotides.
87
K.A. Roubelakis-Angelakis (ed.), Molecular Biology & Biotechnology of the Grapevine, 87-108.
© 2001 Kluwer Academic Publishers.
88 R. V AN HEESWIJCK et al.
amino acids with the remainder present as ammonium ions, peptides, proteins and ni- trates as well as trace amounts of vitamins, nucJeotides and amines (Castor, 1953; Nassar and Kliewer, 1966; Ough, 1969; Kliewer, 1968,1969,1970; Huang and Ough, 1991). Of the free amino acids, the most predominant are commonly proline3 and arginine which can be present at concentrations of up to 2 giL.
The actual content and composition of berry amino acids is influenced by a variety of factors, most notably cultivar (cv) type and level of fruit maturity. Early studies reported significant variations in the free amino acid composition of different cultivars, however the influence of berry maturity or vineyard location was not always taken into account (Lafon-Lafourcade and Peynaud, 1952; Gallander et aI., 1969). Later studies, in which the examined grape cultivars were grown under uniform soil, climatic and cultural con- ditions, have provided data such as that presented in Table 4.1, confirming that amino acid profiles differ considerably between cultivars, although proline and/or arginine are almost always predominant (Kliewer 1969, 1970; Huang and Ough, 1991; Stines et al., 2000). Absolute values for the content of each amino acid vary considerably between studies, however the different ratios of proline:arginine remain relatively consistent for each cultivar. Certain cultivars, for example Cabernet Sauvignon and Chardonnay al- ways accumulate high levels of proline, while others, for example Gewurztraminer and Muscat Gordo Blanco, predominantly accumulate arginine. This suggests that the basic pattern of amino acid accumulation is determined by genetic factors with environmental and cultural factors only having a moditying effect. The nature of the genetic differences leading to the differences in proline and arginine accumulation currently remains un- known, however they may be manifested as the presence or absence of particular alleles that contribute to the operation and regulation of certain metabolic pathways.
Whilst the total amino acid content of berries rises significantly during maturation, in parallel with increasing total N (Lafon-Lafourcade and Guimberteau, 1962; Kliewer, 1968, 1969, 1970; Ough, 1969), the relative amounts of different amino acids change significantly between early and late-harvested fruit from the same cultivar (Kliewer, 1968, 1969, 1970; Stines et aI., 2000). The changes which can be observed in glutamate, glutamine, arginine and proline, all members of the so-called glutamate family of amino acids, are presented in Figure 4.1. In particular, the content of proline changes dramati- cally in cv Chardonnay berries, although only during the later stages of fruit ripening, when its accumulation parallels the increasing sugar concentration. In this cultivar the content of arginine increases slightly during preveraison, then remains somewhat stable, while the content of glutamate, and glutamine in particular, both decrease throughout fruit development. In berries of cv Gewurztraminer, proline also accumulates post- veraison, although to a lesser extent. In this cultivar, however, the arginine concentration increases during ripening to levels similar to that of proline. Again, in contrast to the ac- cumulation of proline, this accumulation of arginine appears to begin before veraison.
3 For the remainder of this chapter. the terms proline, arginine etc. will refer to the free amino acid. i.e. that not incorporated into protein, unless otherwise indicated.
PROLINE ACCUMULATION IN GRAPE BERRIES 89 Table 4.1. Free amino acid composition of whole berry homogenates of V.vinifera harvested from the Waite Variety Block (University of Adelaide, Urrbrae, South Australia) at the maturity level indicated. Sangiovese (SA, 21.9 °Brix), Riesling (RI, 20.4 °Brix), Pinot noir (PN, 22.0 °Brix), Cabemet Sauvignon (CS, 20.5 °Brix), Muscat Gordo (MG, 22.4 °Brix) and Grenache (GR, 20.9
°Brix). Results are expressed as J.lg amino acid/ g fresh weight".
Amino acid
SA RI PN CS MG GR
(J.lg/gfw}
Asp 47.70 29.80 40.60 55.60 23.90 33.90
Glu 47.20 79.50 56.40 71.60 49.40 53.60
HydroxyPro 27.90 7.99 15.80 18.60 9.70 2.64
Asn 25.50 1O.l0 17.70 7.34 3.60 20.80
GIn 77.66 104.20 119.70 139.60 47.94 182.60
Ser 47.64 74.01 94.87 75.40 28.93 41.11
His 54.30 51.57 82.24 55.25 46.00 42.02
Gly 21.50 19.20 33.(;0 23.90 10.50 21.30
Thr 96.65 77.51 182.40 86.59 29.18 70.67
Ala 67.59 121.30 125.80 152.50 46.79 93.27
GABA 82.51 152.70 174.30 146.60 79.56 90.53
Pro 1079.90 688.98 1262.70 1794.70 573.67 424.02
Tyr 30.20 31.20 76.70 49.30 31.50 56.50
Arg 847.22 703.80 1056.00 468.93 1035.10 854.31
lie 27.50 38.70 58.20 38.20 16.50 24.50
Leu 9.51 21.90 9.57 14.10 25.00 18.80
Val 21.10 43.60 58.80 30.60 18.60 25.30
Met 21.90 35.30 59.60 31.40 24.50 26.80
Phe 17.20 51.10 46.60 20.60 42.70 38.40
Om 10.20 13.30 41.40 36.80 48.00 34.10
Lys 35.20 65.22 68.63 70.07 67.71 97.50
Total 2696.14 2421.06 3681.76 3387.69 2260.37 2255.34
• Source: Reproduced from Stines et at. (2000) Australian Journal of Grape and Wine Research 6: 150-158, with permission from the Australian Society of Viticulture and Oenology.
In addition to cultivar type and berry maturity, a number of other factors including season (Huang and Ough, 1991; Spayd and Andersen-Bagge, 1996), use of rootstocks (Huang and Ough, 1989; Treeby et a!., 1998), nitrogen fertilisation (Kliewer, 1971;
Goldspink and Gordon, 1991; Spayd et al., 1994), temperature (Buttrose et a!., 1971) and crop level (Kliewer and Ough, 1970) have also been reported to have an influence on the amino acid composition of grape berries. The mechanisms underlying the effects of these factors are unknown.
Analysis of the free amino acid composition of a range of tissues of cv Chardonnay (Table 4.2), confirms the disproportionate accumulation of proline within berry tissues, compared to flowers, leaves and roots. In berry pulp, the percentage of total amino acids present as proline, on a molar basis reached 59.4%. As pulp accounts for approximately 80010 of the berry weight, the whole berry homogenate results presented previously (Fig. 4.1;
Table 4.1), predominantly reflect the amino acid composition of this berry component.
Interestingly, the pattern and degree of arginine accumulation in cv Chardonnay differs
90
~ 1600
~ 1400
.g: 1200
.3 1000 '0 800
'0 <II 0 600 c 400 'E 200
~ 0
i 3000
!;!> 2500
0>
.3 2000 '0 1500
'0 <II .S; 0 1000
E 500
~ 0 4
R. VAN HEESW]JCK et al.
glulam ine glulam ale
4 6 8 10 12 14
Gewurztram iner - weeks poslflowering glulamine
glutamate arginine
roline
6 8 10 12
Chardonnay -weeks poslflowenng 14 16
Figure 4.1. Changes in glutamine, glutamate, arginine and proline content of homogenates of whole berries sampled from V. vinifera cv Chardonnay and cv Gewurtztraminer. Arrows indicate time of veraison, Final maturity levels were 25.0 °Brix (CH) and 24.1 °Brix (GT). Source:
Adapted from Stines et ai, (2000) Australian Journal of Grape and Wine Research 6: 150-158, with permission from the Australian Society of Viticulture and Oenology,
between skin and pulp leading to a higher proline:arginine ratio in the pulp of ripe fruit, compared to that of the skin (Table 4.2). This is consistent with results presented elsewhere for cv Cabemet Sauvignon (Stines et aI., 2000). In that study it was also shown that in ber- ries of cv Riesling, there is only a moderate accumulation of proline, and that the proline:arginine ratio remained essentially constant throughout the later stages of ripening in Table 4.2. Content of proline, arginine, glutamate and glutamine in a range of V. vinifera cv Char- donnay tissues. Source: Stines (1999).
Ti ue Amino acid (J,lmoL!g fwt)
proline arginine gl utamate glutamine %_proline*
Berry pulp 14,06 1.70 0.26 1.60 59.4
Berry kin 11.02 9.29 1.29 2.04 25 .0
Berry eed 6.94 3.41 5.04 4.05 15.0
Flower buds 4.19 0.92 6.42 29.23 5.8
Flowers 3.00 0.48 3.47 10.03 6.2
Young leaves 1.31 1.43 5.20 7. 17 3.5
Mature leaves 0.88 0.31 4.05 0.96 4.6
Roots 0.36 0.64 2.53 1.29 2.2
' Indicates molar percentage OrlOlal free amino acid presenl as proline,
PROLINE ACCUMULATION IN GRAPE BERRIES 91 both the skin and the pulp. It appears therefore that particular physiological events dur- ing berry development lead to accumulation of both proline and arginine in skin and pulp of some cultivars (classified as moderate proline accumulators), whilst in other cultivars (high proline accumulators) they lead to higher levels of proline in the pulp in associa- tion with lower levels of arginine.