3. ENDOGENOUS POLY AMINES IN GRAPEVINE ORGANS 1. Polyamines in various grapevine organs
3.2. Spatial and temporal free and conjugated polyamine distribution in grapevine leaves
Commercial practices, such as plant micropropagation, and also various biotechnological
POLY AMINES IN GRAPEVINE
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113
Figure 5.3. Free, conjugated and wall-bound polyamines in various grapevine organs at different stages of development (anthesis, fruit set, veraison; from Geny et al., 1997a).
applications, such as genetic transformation, somatic hybridization, somatic embryo- genesis, etc, are based on plant tissue culture techniques. However, grapevine, one of the most widely cultivated perennial plant species, is recalcitrant with regard to plant regen- eration from mesophyll protoplasts (Theodoropoulos and Roubelakis-Angelakis, 1991;
Katsirdakis and Roubelakis-Angelakis, 1992; Siminis el aI., 1993 ; 1994; Reustle et aI., 1995; de Marco and Roubelakis-Angelakis, 1996a, 1996b, 1999; Papadakis and Roube- lakis-Angelakis, 1999) and some other morphogenic programs, such as direct organoge- nesis from callus. PAs have been proposed to affect morphogenic potential. Tissues with lowest PA levels, e.g. internode explants, were superior for vegetative or floral bud for- mation (Tiburcio et al., 1988), whereas the opposite has been found for induction of so- matic embryogenesis (Yadav and Rajam, 1997; Pedroso el al. , 1997). Also, correlations between P A biosynthetic gradient and morphogenic expression of stem pith explants in tobacco (Altamura el aI., 1993)and of hypocotyl explants in eggplant (Sharma and Ra- jam, 1995) have been reported. Furthermore, the use of mutants or antisense transgenic
114 K.A. PASCHALIDIS et al.
plants and of inhibitors of PA biosynthesis has led to the conclusion that adequate en- dogenous PA levels are indispensable for cell growth and differentiation, and also for expression of organogenesis and embryogenesis (Malmberg and Mclndoo, 1983 ; 8agni et aI. , 1993; Kumar et aI. , 1996). Within this context, a comparative biochemical study of the S, SH, and PH contents of Put, Spd, and Spm in the leaves of in vitro grown grapevine plants along the shoot axis was attempted, and also any differences among different areas of leaf lamina and the petiole were dissected (Paschalidis and Roubelakis- Angelakis, unpublished).
Although the content of all fractions of PAs was of the same order of magnitude (nmol per gfw), higher content of almost all PA fractions was found in the marginal leaf region, compared to the central region. The petiolar stub exhibited much lower endoge- nous PA contents than the lamina and these values were much closer to the petiole's contents (Figs SA, 5.5 and 5.6). Leaf size is known to be attained by early cell division on the marginal lamina regions and is mainly integrated by subsequent cell elongation. If in fact PAs are linked with cell division, the slightly greater PA contents in the marginal leaf regimes indicate that cell division was about to be terminated in the first apical leaf.
In the youngest leaf, the S/SHIPH-Put, S/SH/PH-Spd and S/SH/PH-Spm ratios were 1I0AIO.I , 111.6/0.1 and III AIO.2 , respectively. The highest contents were those of SH- Spd and SH-Spm. Increased Spd and Spm biosynthesis during the transition from G I to the S phase of the cell cycle preceding the onset of DNA synthesis is a universal phenome-
300 200
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o 1 234 5 6 0 1 234 5 6 Leaf age
200 150 100 -;' ~
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Figure 5.4. Soluble (S), soluble hydrolyzed (SH), pellet hydrolyzed (PH) and total (S+SH+PH) putrescine (Put) content in different leaf regions during development of grapevine leaves. Leaf numbering started from the apex leaf being designated as leaf I. Margin (0); center (0); petiole (6); petiolar stub (x); entire leaf (dotted line). Error bars represent ± SE (from Paschalidis and Roubelakis-Angelakis, unpublished).
120
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POLY AMINES IN GRAPEVINE
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lIS
200 150 -:'
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Figure 5.5. Soluble (S), soluble hydrolyzed (SH), pellet hydrolyzed (PH) and total (S+SH+PH) spermidine (Spd) content in different leaf regions during development of grapevine leaves. Leaf numbering started from the apex leaf being designated as leaf l. Margin (0); center (0); petiole (t.); petiolar stub (x); entire leaf (dotted line). Error bars represent ± SE (from Paschalidis and Roubelakis-Angelakis, unpublished).
non in animals and plants (Fuller et at., 1997). The ratio of Spd+SpmlPut is positively correlated with the activity of macromolecular biosynthesis (Lin et aI. , 1984). Spd and especially Spm interact ionically with the negative charges of pectic substances, which are known to act on morphogenesis in plants. Tn grapevine leaves, the (Spd + Spm) IPut ratios decreased with increasing leaf age. Tn the oldest leaves, the S/SHfPH- Put, S/SHfPH-Spd and S/SH/PH-Spm ratios were 1/0.7/0.3 , 1/4.9/0.4 and 1/1.8/0.7, respec- tively. In leaves of all developmental stages, the SH-fraction of the higher PAs (Spd and Spm) predominated and the S- and the PH-fractions followed, whereas in Put, the S- fraction predominated. PA conjugation to a hydroxycinnamoyl moiety would possibly be important in the regulation of the size of the S-PA pool, and/or the detoxification of compounds known to inhibit growth such as phenolics; grapevine tissues contain high levels ofphe nolic compounds. Conjugated PAs have also been proposed to act as means for PA translocation (Havelange et aI., 1996), or they could be the preferred substrates for amine oxidases (Aribaud et aI., 1994), whereas the protective antioxidant effect of exogenous PAs was dependent on their prior conversion to conjugated forms (Lange- bartels et aI., 1991).
All Put fractions increased with increasing leaf age and the increase was more pro- nounced in the lamina than in the petiolar stub and the petiole (Fig. 5.4); on the contrary, the Spd and Spm fractions decreased (Fig. 5.5 and 5.6). The decline in Spd and Spm with increasing leaf age is consistent with the facts that these higher PAs and ethylene, the aging phytohormone, have a common precursor, S-adenosyl-methionine (SAM), and
116
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~ 0
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K.A. PASCHALIDIS et al.
~ ~ ~ SH
PH Total
~ ~
o 1 234 5 601 2 3 456
Leaf a!1e
60 40 -; G ..
20 ~
"0 0 E c:
120 E c-
oo 80 40 0
Figure 5.6. Soluble (S), soluble hydrolyzed (SH), pellet hydrolyzed (PH) and total (S+SH+PH) spermine (Spm) content in different leaf regions during development of grapevine leaves. Leaf numbering started from the apex leaf being designated as leaf I. Margin (0); center (D); petiole (~); petiolar stub ( x); entire leaf (dotted line). Error bars represent ± SE (from Paschalidis and Roubelakis-Angelakis, unpublished).
ethylene inhibits the biosynthesis of PAs and vice versa (Apelbaum, 1990; Saftner and Menta, 1990).
On the other hand, high Put has been implicated in stress-responses by plant cells. Di Tomaso et al. (1989) have suggested that toxicity observed by exogenous Put could be due to the formation of active oxygen species (i.e. hydrogen peroxide and free radicals) by apoplastic DAO, which most probably damage membranes. Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts but also, only protoplasts that are able to supply extracellularly H202 can actually divide (Siminis et al. 1993, 1994; de Marco and Roubelakis-Angelakis 1996a, 1996b, 1999).
In summary, our data indicate that leaf laminas contain significantly higher endogenous PA content than the petiolar stubs and the petioles, which in some cases exhibit higher morphogenic potential. With increasing leaf age an increase in Put and a decrease in higher PAs (Spd and Spm) fractions was found (S, SH, PH, Figs 5.4, 5.5, 5.6).
3.3. Polyamines and berry development
3.3. J. Polyamine oxidase activities and diaminopropane contents during floral development in grapevine
Colin et al. (1999) reported for the first time the occurrence and the possible implication of polyamine oxidase activities and diaminopropane during flowering and fruit set. In flowers and young berries, there was an inverse correlation between polyamine oxidase
POLY AMINES IN GRAPEVINE 117 activities and diaminopropane content. At the beginning of anthesis, polyamine oxidase activities were maximum in flowers and diaminopropane levels were low. In contrast, after fruit set polyamine oxidase activities were low in young berries and diaminopro- pane levels increased. After, during fruit set and fruit ripening, polyamine oxidase activi- ties and diaminopropane content decreased (Fig. 5.7). These correlations were the same for both varieties (Merlot and Cabemet Sauvignon) and were independent of the date but dependent on the stage of development. The maximum polyamine oxidase activities were exhibited later than the accumulation of diaminopropane: flowering was character- ised by the highest polyamine oxidase activities and fruit set by the highest diaminopro- pane content; these values could be considered as good indicators of these stages of development.
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Figure 5.7. DAP contents (A) and PAO activities (B) in floral buds and in young berries of grape- vine (from Colin et al., 1999).
These data raised the question of the function of conjugated [soluble and insoluble (cell wall bound)] polyamines and especially the function of diaminopropane. Diamino- propane was the major polyamine found in young berries during the onset of fruit devel- opment (Geny et al., 1997). In other species, putrescine and spermidine predominated (Egea-Cortines and Mizrahi, 1991). In plants, amine oxidases are involved in the regula- tion of polyamine concentrations and transport at the subcellular level, depending on the
118 K.A. P ASCHALIDIS et al.
physiological stage of tissues (Arribaud et aI. , 1994). The data presented by Shih et al.
(1982) demonstrated that diaminopropane is a potent inhibitor of dark-induced senes- cence by inhibiting ethylene production. Diaminopropane may bind to polyribosomes and affect protein synthesis, which may account for the decrease in protease activity.
Antagonism by Ca2+, however, suggests that the initial step in the retardation of senes- cence by diaminopropane probably involves its attachment to membranes (Shih el aI., 1982).
3.3.2. Hydroxycinnamic acid amines in flowers and berries of grapevine
Geny el al. (1999) reported the presence of phenol amines in grapes of Vilis vinifera L.
cv Merlot and Cabernet Sauvignon during their development. Hydroxycinnamic acid amines are found in large amounts in the reproductive organs. The ovaries contain neu- tral phenolamines (spermine and spermidine) and the anthers the basic compound para- coumaryldiaminopropane. After flowering (Stage 19 of Eichorn and Lorenz), insoluble in water and conjugated with p-coumaric acid polyamines rapidly and dramatically de- creased. In young berries, the levels of polyamines increased; they were found to be soluble in water and conjugated with ferulic acid (Fig. 5.8 and 5.9).
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Figure 5.S. Total water soluble and insoluble PAs in berries of Cabernet Sauvignon (A) and Mer- lot (B) during development (from Geny et al., 1999).
The function of conjugated polyamines during cell division and cellular differentia- tion is still unclear. Are they a storage form of polyamines, or are conjugates physiologi- cally active? The observation of a temporal correlation between changes in the levels of
POLY AMINES IN GRAPEVINE
Cabcrnct auvignon Merlot
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119
Figure 5.9. Soluble (A) and insoluble PAs (B) in berries of Cabernet Sauvignon and Merlot dur- ing development (from Geny et aI. , 1999).
conjugated amines in reproductive organs during maturation could suggest, but in no way prove, that they have a causal role in the corresponding processes.