800 600 400 200
POL YAMINES IN GRAPEVINE
o ~~----~---~~----~~
HI J K M N
Baggiolini
123
Medium green berries
Figure 5.12. Total po\yamines contents in flowers and berries of normal grapes (Merlot) during their development.
Carranza et aI., 1998). The mechanisms involved in the control of flower and fruitlet abscission are complex. In various fruit plants, this event is largely considered to be as- sociated to metabolic and hormonal disorders and depletion in the nutrient supply to the inflorescence (Kaska, 1989; Osborne, 1989; Gonzalez-Carranza et al., 1998). Abscisic acid and ethylene are considered to be the most important drivers of the abscission proc- ess (Sexton and Roberts, 1982; Ruperti et aI., 1998). In the past 20 years, considerable research has suggested a close connection between PAs and the physiological events leading to flowering (for review see Galston et al., 1997; Martin-Tanguy, 1997). Certain tobacco mutants and transformants with aberrant flowering habits have an unusual me- tabolism and PA content (Malmberg and McIndoo, 1983). Specific inhibitors ofPA syn- thesis have produced parallel changes in the levels of PAs and flowering behavior. In A.
thaliana, a high amount of Spd, probably due to the increased activities of Spd synthase, seems to be related to flowering (Tassoni et al., 2000). As reported in previous studies (Caffaro et al., 1994), the translocation under photoperiodic flowering induction of free Spd from leaves to the axillary and apical buds seems to be part of the complex mecha- nism during the transition of vegetative buds to flowers.
Early work showed that exogenous application of PAs during fruit set increased the nuinber and the size of the fruit that set (Egea-Cortines and Mizrahi, 1991; Evans and Malmberg, 1989). Male sterile mutants of maize lack conjugated P A accumulation in anthers (Flores et al., 1989). Tobacco carrying an oat ADC gene under the control of ABA-inducible promoter showed accelerated growth and development and increased flower and capsule numbers (Limami et aI., 1999). However, in spite of the regulatory role attributed to PAs in the floral development, little is known on their involvement in the control of the flower or young fruit abscission in perennial plant species.
124 K.A. P ASCHALTDIS et af.
5.3. J. Polyamines and abscission potential
Like many fruit trees species, grape exhibits massive fruitlet abscission, which is under hormonal and metabolic regulation (Gillaspy et af. , 1993). Monitoring grafted vines on the same rootstock and fruiting cuttings from various cultivars exhibiting different ab- scission potentials (Merlot, GewUrztraminer, Pinot noir and Meunier) indicated some variability in the corresponding abscission potential and their endogenous PA levels.
Floral organs started to drop before or at anthesis depending to some extent on the meta- bolic activity of each cultivar. At fruit set stage (Fig. 5.13A), more than 65% of flowers and young fruits of Merlot and Gewurztraminer plants had abscised against about 40%
on those of Pinot noir and Meunier. No significant differences were observed in abscis- sion percentage between grafted vines and fruiting cuttings of each cultivar. Merlot and Gewurztraminer were more sensitive to abscission than Pinot noir and Meunier even their inflorescence and fruit number are different.
The most abundant PAs in the grapevine organs were Spd, Spm and Dap (a product of Spd and lor Spm oxidation), while Put and Agm were present at low levels. At full flowering, free PAs were quantitatively lower in grapevine cultivars exhibiting a high abscission level, like Merlot and Gewurztraminer (Fig. 5.13B). This difference was more pronounced in the inflorescences than in the leaves. In the roots, free PA level remained very low. These results raise the possibility that abscission potential or fruit set could depend upon free P A level in the floral organs (Biasi et al., 1999).
The increased percentage of flowers abscised in grapevine was associated with a de- crease of free PA level in the corresponding inflorescences (Aziz et af., 2001). The two
t 80 , . - - - -- - - - ,
.. A I
G 70 ,-l
c: " 60
"
... 50 -;::
.:: 40
~ 30 o
" 20
:e o 10
1l 0 +-'---....,..-'-'---r-'---'-r--'-"~
-<: PN PM ~ IRT GW 1.6 r : : - - - ,
~ I 4 .:: ~ 12
~ 1.0
~ 0.8 ... 0.6
~ t 0.4
PN P~ I MRT GW
Figure 5.13. Fruitlet abscission (A) and total free polyamine levels (B) in different grapevine culti- vars. PN, Pinot noir; PM, Meunier; MRT, Merlot;
GW, Gewiirztraminer. Abscission was determined at fruit set. Grafted vines (0); fruiting cuttings (_); roots (ill); leaves (f,jj); inflorescences (ill) (from Aziz, Brun and Audran, unpublished).
POLY AMINES IN GRAPEVINE 125 processes are strongly correlated starting from total free PA concentration of less than 0.7 Ilmol. gfw-I . In the inflorescences of the low abscising cultivars free Spd, Spm and Dap levels decreased gradually after anthesis and remained important even after full bloom. No major change was observed in the level of Put. However, in inflorescences of the sensitive plants the content of all free PAs decreased before anthesis and remained very low during flowering. This suggests that the amounts of free Spd, Spm or their oxi- dation product (Dap) in the floral organs may contribute to regulatory processes involved in grapevine fruitIet drop.
5.3.2. Polyamines counteract abscission
When considering total PAs in grapevine, changes in free and conjugated PA contents followed opposite trends before anthesis in all organs examined (Fig. 5.14). The conju- gated PAs in grapevine organs were mainly present as acid soluble forms of Put, Spd and Dap, while the insoluble forms were quantitatively very weak during development (data not shown). In contrast to the tolerant-cultivar (Fig. 5.14B, D), the decrease of total free P A content in the sensitive cultivar was more important in the inflorescences than in leaves (Fig. 5.14A, C). However, the level of conjugated PA in tolerant cultivar in inflorescences increased to a maximum value at anthesis and decreased gradually thereafter. Change of these compounds was not highly significant in leaves. At anthesis, the high level of soluble conjugated PAs in the inflorescences was con-elated to a low sensitivity to fruitIet abscission. After anthesis, soluble conjugates were lower in inflo-
3 A 3 B
~
~ ... 2 2
'0 E ~
..51
'"
<
c.
0 0
3 C 3
D
~
J: 2 2
""
'0 ~
~ I <
c.
0 0
-10 0 10 20 30 -10 0 10 20 30 Days after anthesis Days after anthesis
Figure 5.14. Changes in total free and conjugated polyamine contents in the inflorescences (A,B) and leaves (C,D) during development of Merlot (A,C) and Pino! noir (B,D). Free PAs (_); solu- ble-conjugated PAs (0) (from Aziz, Brun and Audran, unpublished).
126 K.A. PASCHALIDIS et al.
rescences of abscising cultivar but higher in their leaves. These adjustments might result from a conversion of free PA to conjugated forms through an activation of PA trans- ferases in both, leaf and floral tissues and their subsequent accumulation in the inflores- cences. It has been reported that soluble conjugated PAs move from leaves to the young floral buds of Sinapis alba (Havelange et aI., 1996). These forms of PAs are assumed to be markers for female reproductive organ fertility (Martin-Tanguy, 1997). PA conjuga- tion to a cinnamoyl moiety would be important in the detoxification of phenolic com- pounds known to inhibit growth (Martin-Tanguy, 1997; Biasi et aI., 1999). In addition, hydroxycinnamic acids are antioxidants and phenylpropanoid accumulation could be involved in preventing abscission. It was proposed that the protective effect of exoge- nous PAs against the damage of the superoxides was dependent on their prior conversion to conjugated forms.
The level of PAs in the inflorescences might also depend on developmental stage of leaves and floral organs. Hence, before anthesis the free PA mobilization appeared more pronounced in inflorescences than in leaves of the abscising cultivar. This suggests that the PA partitioning between source and sink organs might be a critical factor controlling the grapevine plant productivity. For instance, it seems that inflorescences of grapevine must receive at least part of their PAs, under free and/or conjugated forms, from leaves needed for reproductive organ stability and/or fertility as reported for other plants (Bagni and Torrigiani, 1992). In this context, P A could serve either as nitrogenous source, since their endogenous concentration was higher in grapevine, or as signal molecules controlling the abscission processes in target organs.
The ultimate importance of the abscission level in grapevine was influenced by addi- tion of PAs to the nutritive medium of fruiting cuttings or by spraying them on the grafted vines. Thus, application of Spd 7 days prior to anthesis, reduced significantly abscission in all cultivars (Fig. 5.15). However, Put or Dap treatment at the same devel- opmental stage did not cause any significant change in the abscission levels in both cul- tivars. Similar results were obtained when PAs were sprayed on the grafted vines before anthesis. When cuttings were treated with 0.5 mM Spd, the amount of endogenous free
'E 100
~ 80
"
.~ = " 60
j:~ 40
<~
"
~ 7.0
0 Control Put
• Pinotnoir
o Merlol
Spd Dap
Figure 5.15 Eftects of exogenous polyamines on Hower and fruitlet abscission in Pinot noir and Merlot.Putrcscine (Put), spermidine (Spd) and diaminopropane (Dap) were added separately in the nutritive medium at 0.5 mM 7 days before anthesis. Abscission was determined at fruit set (from Aziz, Brun and Audran, unpublished).
POL Y AMINES IN GRAPEVINE 127 Spd and Spm increased to a great extent in different organs. In inflorescences Spd reached a maximum level at anthesis and decresed during flowering (Fig. 6.16). How- ever, Agm and Dap levels decreased in both, leaves and inflorescences. PAs levels re- mained lower in roots. Spd application also led to a doubling or almost so in the level of conjugated Put and Spd (result not shown). However, application of Put and Dap had no significant effect on neither free nor conjugated Spd level. Thus, Spd-treated plants clearly take up this P A from the medium and redistribute' it in the inflorescence organs both in free and conjugated forms. These results are in agreement with recent findings in A. thaliana (Tassoni et aI. , 2000), which also showed that the uptaken Spd was converted to Put suggesting a possible interconcersion of acetylspermidine to Put via a
1.2 :::-- 1.0
~ bfI ' 08
~ 0.6
~ 0.4 E
"0
rE °.2
o ~~~""":'::;:::::ii,;;;;;;a--l
-10 0 10 20 30
Figure 5.16. Spermidine accumulation in the inflorescences of Merlot after tretament of the corre- sponding fruiting cuttings with exogenous Spd. Spd was applied at 0.5 mM 7 days before anthesis (from Aziz, Brun and Audran, unpublished).
putative PAO (De Agazio et aI., 1995). As reported in previous studies (Caffaro et aI., 1994), the translocation under photoperiodic flowering induction of free Spd to the inflo- rescences seems to be part of the complex mechanism, which occurs during the transi- tion of vegetative buds to flowers. Our results strengthen the hypothesis that Spd may be involved in the regulation of abscission processes in grapevine, through their preferential accumulation in the inflorescences under both free and conjugated forms. It is suggested that the fertility and fruit set could be tightly and positively influenced by limitation of free Put accumulation and increases of free and conjugated Spd production in the floral organs.
Other processes including the activity of PA synthesis and degradation could also regulate the fruit set (Tiburcio et aI. , 1990; Slocum and Galston, 1985). The sharp decline observed in Put, Spd and Spm contents during floral development might result from either stimulation of DAO and PAO or inhibition of enzymes responsible for their synthesis from arginine and/or ornithine or from both. The marked decrease in the level ofDap suggests that its synthesis from Spd andlor Spm was blocked or its degradation to p-alanine was stimulated during floral development.
128 K.A. PASCHALlDIS et al.
5.3.3. Polyamine biosynthesis and abscission
The relative contribution of ADC and ODC to the biosynthesis of PAs is dependent upon plant species and growth process (Martin-Tanguy, 1997). It has been reported that, dur- ing vegetative growth and floral bud formation, ADC but not ODC activity was the likely source of the PA synthesis (Tiburcio et aI., 1988). Subsequent development, how- ever, appears to be dependent on ODC activity. Similarly, in the pea plants, gene expres- sion of the ADC is high in young developing tissues, like shoot tips and flowers buds (Perez-Amador et aI., 1995). When the nutritive solution was supplemented with I mM DFMA, a specific and irreversible inhibitor of ADC, the percentage of abscission in grapevine increased by about 40 and 20% in Pinot and Merlot, respectively, compared to the corresponding control (Fig. 5.17). In contrast, abscission level remained relatively unchanged in both cultivars after application of DFMO, an inhibitor of ODC. Addition- ally, DFMA exerted a dramatic inhibitory effect on the levels of free and conjugated PAs at the end of flowering. In contrast, DFMO, added at the same stage of development, promoted slight production of free PAs without significant effect on fruitlet abscission.
This suggests that PA synthesis before anthesis might require ADC rather than ODC activity. The P A pool produced via ADC may counteract floral organ abscission, al- though the effects of DFMA on both PA level and abscission were greater in the no ab- scising cultivar. However, the paradoxical effect ofDFMO on PA content could be rele- vant to its consistently observed stimulatory effect on ADC activity (Burtin et al., 1989), since Agm level was enhanced in response to DFMO treatment. Our results are consis- tent with the hypothesis that fruitlet abscission could be induced at least in part, by depressing PA synthesis via ADC pathway.
Similarly, grapevine fruitlet abscission was extremely sensitive to cyclohexylamine (CHA), a Spd synthase inhibitor. The abscission percentage increased in response to CHA added in the external medium. It reached values of about 60 and 80% in Pinot and Merlot, respectively (Fig. 5.17). Furthermore, the addition of CRA revealed the contri-
120 • Pinotnoir ';;'
.. .. 100 o Merlot
= ~ Q 80 or;; -; QC .~ .... '" = - "" 60
.J:l .-
< :;;
1, 1,
I, .... Q 40
~ 20
0 I I
Control DFMO DFMA CHA AG HEH
Figure 5.17. Effect of inhibitors of polyamine synthesis and catabolism on flower and fruitlet abscission in Pinot noir and Merlot. The target sites of inhibitors are shown in Figure 5.2. Each inhibitor was added to the nutritive medium 7 days before anthesis and abscission was determined at fruit set (from Aziz, Brun and Audran, unpublished).
POLY AMINES IN GRAPEVINE 129 bution of the Spd synthase pathway in modulating PA concentration in both leaves and inflorescences. In cuttings treated with 1 mM CHA, Agm and Put contents increased in both roots and leaves, while Spd, Spm and Dap decreased in all organs (not shown). In the inflorescences, Spd and Spm levels decreased by 80 and 50 % respectively, whereas Agm, Put and Dap remained relatively unchanged. This is in accordance with the fmd- ings of Burtin et at. (1991) showing that inhibition of Spd synthesis in tobacco caused malformation of anthers, including lack of pollen, replacement of anthers by petals and infertility. Other studies (Kaur-Sawhney et at., 1988) reported that inhibition of Spd syn- thesis by CHA lead to a decreased flowering in parallel with the decline in Spd titer.
Recently, Spd synthase activity was detected in pea ovaries and one of the two genes encoding this enzyme was highly expressed in young leaves and flowers at anthesis (Alabadi and Carbonell, 1999).
It should be pointed out the interconnection between P A metabolism and ethylene, since Spd synthase requires an aminopropyl group donor (decarboxylated SAM) as also a precursor of ethylene synthesis. Recent report (Ruperti et al., 1998) indicates that in- creased ethylene production during floral development was accompanied by an increased fruitlet abscission in peach. In contrast, treatment with inhibitors of ethylene production stimulated the accumulation of free and conjugated PA (Martin-Tanguy et at., 1993;
Kumar et aI., 1997). Thus, abscission might be related to the metabolic flux from decar- boxylated SAM, which could be highly in favor of ethylene synthesis in the abscising
cultiVlm. In thi~ context, fA. can be vieWed ~ a bnoce, and balance between r A and
ethylene synthesis may be one of the major determinants in regulating the abscission process.
5.3.4. Polyamine catabolism and abscission
P A catabolism through DAO and PAO appear to be important for plant developmental processes such as floral induction, reproductive processes and root formation (Martin- Tanguy, 1997). In grape plants the addition of aminoguanidine (AG), an inhibitor of DAO, and B-hydroxyethyJhydrazine (HEH), a potent inhibitor of PAO, to nutritive me- dium, strongly increased abscission of floral organs in both cultivars (Fig. 5.17). In this study, PA catabolism seems to be involved in the regulation of PA pools in grapevine organs as suggested for tobacco during sexual differentiation (Martin-Tanguy and Tepfer, 1998). The application of CHA and HEH in the external medium caused a de- crease of Spm and Dap levels in both leaves and inflorescences (not shown). The Spd level remained however unchanged, while Put increased in all organs. These results sug- gest that the effects of Spd on fruitlet abscission might be linked to its synthesis from Put via Spd synthase and its catabolism through increased PAO activity, which also depend on Spd availability.
The results support that both P A synthesis and catabolism are tightly coordinated and could be required for an optimal level of Spd and subsequently for a regular fruitlet ab- scission. PA oxidation ensures recycling of the reduced carbon and nitrogen from PAs to Krebs cycle through the formation of ill-pyrroline and GABA (Aziz et aI., 1998). The
130 K.A. P ASCHALIDIS et al.
Spd effect also might result from an inhibition of the protease activity, which seems to be due at least in part to a repression of the synthesis of the enzyme, and to direct inhibi- tion of its action as reported for PA-mediated inhibition of protease activities (Balestrieri etal.,1987).
5.3.5. Photodependance ofpolyamine levels and abscission
It has been shown that PAs interact in some way with all plant hormones (Smith, 1990;
Altman, 1989; Scaramagli et aI., 1995; Bagni et al., 1998). Recently, most biochemical, genetic and molecular studies on plant hormone action and biosynthesis have been carried out in the model plant Arabidopsis thaliana (McGrath and Ecker, 1998; Phillips, 1998). On the other hand, it has been reported that PAs playa role in the structure and functioning of the photosynthetic apparatus (Kotzabasis et aI., 1993).
It has been reported that competition for photoassimilates is thought to be responsible for fruit abscission (Moss et aI., 1992; Goldschmidt and Koch, 1996; Gomez-Cadenas et aI., 2000). In grape free PA pools were strongly photodependent since darkening the Pinot noir plants resulted in a decrease of free and conjugated P A levels in both leaves and inflorescences. In the later organs, free and conjugated PAs decreased by about 55 and 45%, respectively upon 24 h. Dark had also a detrimental effect on growth, flower- ing and fruit set (more than 85% of floral organs had abscised). Similar trends were ob- served by reducing the photoperiod from 16 h to 12 h. This could be related to the local-
ilation of ADC ~nIYm~ in th~ (hloropla~t (fiormll ~[ at., l~~j). fA~ Me pm~ent in
chloroplasts, thylakoid and PSII membranes and in the light-harvesting complex (Kotza- basis et aI., 1993). Andreadakis and Kotzabasis (1996) suggested that PAs as well as the corresponding enzyme activities of ADC, ODC and DAO are photoregulated and un- dergo considerable change during chloroplast photodevelopment. These results led us to suggest that PAs could have a functional role in the photosynthetic metabolism or in the regulation of photoassimilate biosynthesis or partitioning between organs. To our knowledge, no relationship between these processes has been carried out in higher plants. However, data show that depletion in free PA level in inflorescences was associ- ated to a decrease in total sugar content particularly in that of sucrose (not shown). The concept that initial fruit set might be limited under nOlmal conditions by the availability of carbohydrates was questioned. Some experiments have shown that defoliation in- duced fruitlet abscission in citrus (Mehouachi et at., 1995). The authors suggested that the sucrose status of the fruitlets is a major factor in the regulating mechanism of citrus abscission.
5.3.6. Modulation of carbohydrate and amino acid levels by polyamines
It is generally stated that in grapevines, the inflorescence competes with the shoot apex for photosynthetic assimilates from the onset of its growth to flowering and the early stage of berry development. The question arises how do the grapevine plants determine the balance between productive and aborted fruit set? It is well known that carbon and nitrogen metabolisms are linked because they share organic carbon and energy. Thus,