7.1. Ammonium and glutamine metabolism
Proline accumulation in developing grape berries could be regulated through availability of the substrate glutamate, or its precursor glutamine, which in turn is a product of am- monium assimilation through the activities of glutamine synthetase (GS) (EC 6.3.1.2), glutamate synthetase (GOGAT) (EC 1.4.1.14) and possibly glutamate dehydrogenase (GDH) (EC 1.4.1.2) (Ghisi et aI., 1984; Kanellis and Roubelakis-Angelakis, 1993). A recent study in tobacco concluded that in some situations GS plays a major role in regu- lating proline production (Brugiere et al. 1999). This study demonstrated using trans- genic plants that a phloem-specific reduction in GS activity resulted in decreased gluta- mine availability and consequently decreased proline accumulation when plants were forced to assimilate large amounts of ammonium. Thus, in wild-type plants proline was
100 R. V AN HEESWJJCK et al.
proposed to constitute a strong sink for amino-nitrogen derived from ammonia, with glutamine acting as the primary precursor regulating its synthesis. Glutamate was a metabolic intermediary, but its rate of use appeared similar to its rate of synthesis, result- ing in very little change in steady-state levels of this amino acid under the conditions examined.
Both GS and GDH activities have been detected in grape tissues, including fruit, dem- onstrating that berries possess the ability to assimilate ammonium ions via these pathways (Ghisi et aI., 1984; Kanellis and Roubelakis-Angelakis, 1993). The synthesis of proline is, therefore, likely to be influenced by the flow of glutamate, glutamine and ammonium ions into the berry and also by the metabolic conversions that take place between these three substrates. Glutamine is a major constituent of the phloem sap (Gholami, 1996). [t is not surprising therefore, that glutamine is initially the predominant amino acid in berries after which it appears to be converted to other amino acids, such as proline, at a rate which is faster than its import or synthesis (Fig. 4.1). Thus, relatively early in berry development, the concentration of glutamine could become rate-limiting for proline synthesis. Interest- ingly, the content of ammonium ions is relatively high in immature grape berries, but steadily declines during maturation of the fruit (Lafon-Lafourcade and Guimberteau, 1962). The post-veraison assimilation of ammonium ions, via GS or GDH, could provide additional glutamine for proline biosynthesis, possibly enhancing the rate of its synthesis.
Information on GS, GDH and GOGAT gene expression and enzyme activity throughout berry development will be important to better describe the metabolic environment in which proline synthesis and accumulation is taking place. The cloning of cDNAs encoding at least some isoforms of these enzymes from V vinifera (Loulakakis and Roubelakis- Angelakis, 1996; Syntichaki et al., 1996; Loulakakis and Roubelakis-Angelakis, 1997) represents a valuable step towards achieving this.
7.2. Arginine metabolism and regulation of OAT
The cloning of cDNAs encoding P5CS and OAT from grape berries indicates that both the glutamate and ornithine pathways of proline biosynthesis are in operation during development of this fruit. Many studies have demonstrated that the glutamate pathway is the predominant pathway of proline synthesis in osmotically stressed plants, however the role and significance of the ornithine pathway to proline synthesis is still subject to de- bate. Based on work in V aconitifolia and A. thaliana, it has been suggested that OATis primarily involved in the recycling of amino acids such as glutamate, when levels of nitrogenous compounds are high (Delauney et aI., 1993; Roosens et aI., 1998). This is supported by the previous demonstration in other plant species that arginine, which acts as a nitrogen reserve in cotyledons and germinating seeds, is mobilised by hydrolysis to ornithine [by arginase (EC 3.5.3.1)] followed by conversion to P5C (by OAT) and thence to proline or glutamate (Mazelis and Fowden, 1969; Splittstoesser and Fowden, 1973). OAT gene expression is induced by arginine in yeast (Brandriss and Magasanik, 1980) and by excess nitrogen in V aconitifolia (Delauney et aI., 1993). In the bacterium
PROLINE ACCUMULATION IN GRAPE BERRIES 101 Bacillus subtilis, OATis also considered to be part of the arginine catabolism pathway (Harwood and Baumberg, 1977).
The patterns of amino acid accumulation observed in developing grape berries (Fig.
4.1) suggest that proline and arginine metabolism may be linked, with arginine possibly acting as a precursor for at least some of the proline accumulated (Kliewer, 1968). Argi- nase is present in developing grape berries, with a peak of activity at veraison (Roube- lakis-Angelakis and Kliewer, 1981) and ornithine is detected in berry extracts, albeit at low levels (Table 4.1). It is feasible therefore that OAT links proline biosynthesis to ar- ginine catabolism in grape berries.
Vvoat mRNA and protein have been shown to be present throughout berry develop- ment and in a range of other grapevine tissues, although there is little, if any, expression of Vvoat in berry skin (Fig. 4.5) (Stines, 1999). This tissue specific pattern of expression could contribute to the different patterns of proline and arginine accumulation observed in berry skin and pulp of cultivars such as Chardonnay (Table 4.2). Vvoat enzymatic activity can also be detected throughout berry development in cv Chardonnay with only a slight increase in activity late in ripening (data not shown). The final level ofVVOAT enzymatic activity is similar in mature berries of both cv Chardonnay and cv Gewurz- traminer. Together, these results suggest that whilst VVOA T may be involved in proline synthesis in the berry, it is not a key regulatory factor per se.
7.3. Proline degradation
The first step of proline degradation in plants is mediated by the mitochondrial enzyme proline dehydrogenase (PDH) (EC 1.5.99.8) which converts proline to P5C (Huang and Cavalieri, 1979). Under osmotic stress conditions, proline degradation is regulated coor- dinately with proline synthesis in A. thaliana (Kiyosue et a!., 1996; Peng et al., 1996).
Whilst PDH mRNA levels are normally induced by high proline levels, this induction is inhibited during osmotic stress, when levels of P5CS mRNA are high. During subse- quent recovery from osmotic stress, PDH mRNA levels become increased, concomitant with a reduction in P5CS mRNA and levels of free proline. This coordinate, reciprocal regulation of PDH and P5CS mRNA levels indicates that cellular free proline levels are tightly controlled, at least during the osmotic stress response (Kiyosue et aI., 1996; Peng et al., 1996; Nakashima et a!., 1998). Under "normal" non-stressed conditions, however, PDH mRNA levels are highest in those tissues of A. thaliana which also contain the highest concentrations of proline i.e. pollen, pistils and seeds (Nakashima et al., 1998).
The coexistence of high proline concentrations and high levels of PDH mRNA, in the same tissue, suggests that either the location of proline and its degradative pathway are separated into distinct subcellular compartments, or that a high rate of proline turnover is occurring in these tissues.
In order to investigate whether proline accumulation in ripening berries could result from a developmentally programmed decrease in the proline degradation, polyclonal antibodies raised against A. thaliana PDH (N. Verbruggen, University of Ghent, Bel-
102 R. VAN HEESWIJCK et al.
gium, unpublished), were used in western blot analyses of cv Chardonnay grape berries (Fig. 4.7) (Stines et aI. , 1999). A cross-reactive protein of~55kDa representing a puta- tive grapevine PDH homologue was detected. Steady-state levels of this protein in- creased throughout berry development to 13 weeks postflowering, possibly induced by the increases in proline which occur during this period. The levels of the grapevine PDH homologue remain relatively high late in berry development, suggests that the rapid ac- cumulation of proline which occurs at this time, is not due to a decrease in proline deg- radation. It is of course possible that post translational mechanisms may regulate PDH activity in berries. To assess this possibility, direct measurements of PDH enzyme activ- ity will need to be made.
weeks postflowering
4 8 10 12 13 14 16
,---'---=---...;...::=----'----'---'---, kDa
94
PDH~ 68
~ - - ---
43 30 21
Figure 4.7. Steady-state levels of PDH protein throughout development of VVinifera cv Chardonnay berries, detected by Western blot hybridisation of protein extracts from whole berry homogenates (equivalent amounts per lane on a fresh weight basis), using anti AtPDH antibodies.
Source: Adapted from Stines et af. (1999) Plant Physiology 120: 923-931 (with permission from the American Society of Plant Physiologists).
7.4. Protein accumulation
Changes in the rate of berry growth and net accumulation of proteins during development would, at least in part, determine the cellular demand for proline for biosynthetic purposes and could therefore have an impact on the levels of free proline accumulation. Measure- ment of the total protein present in extracts of cv Cabernet Sauvignon berries demonstrates that protein accumulation has ceased by 12 weeks postflowering (Fig. 4.8) (Stines, 1999).
This is consistent with previous reports, which demonstrate that the accumulation of pro- tein occurs primarily during stage I of berry development with a second increase in protein content at veraison, after which it remains relatively constant (Ghisi et aI., 1984; Tattersall et al. , 1997). In cv Cabernet Sauvignon at least, there appears to be a correlation between the cessation of berry growth and net protein accumulation during the late phases of berry development and rapid proline accumulation. The relative contribution of this decreased de-
PROLINE ACCUMULATION IN GRAPE BERRIES 103
A kDa B
94 0.8
sa § 06 06 25 ..,
43 :E 2' C .. C 20 2-S"
~ 04 80.4 15 !.
30 1:
~ 10 &
21 1: 02 ~
-' 5
.. !
0 ~ 0 0
2 4 S a 12 15 16 2 .. 6 8 10 12 14 16
weeks postflowering weeks postftowering
Figure 4.8. The increase in free proline accumulation in V vinifera cv Cabernet Sauvignon berries correlates with cessation of berry growth and protein synthesis. A) SDS-PAGE of total berry pro- tein extracted from samples collected throughout development. B) Relative amounts of total pro- tein and free proline in each sample and berry weight.
mand for proline in protein synthesis to net proline accumulation remains to be deter- mined.