NEW FINDINGS ON DEVELOPMENTAL, BIOCHEMICAL AND MOLECULAR RESPONSES TO ENVIRONMENT
3. RECENT DEVELOPMENTS OF MOLECULAR BIOLOGY APPLIED TO GRAPEVINE PHYSIOLOGY
3.1. Genes involved in general berry development and maturation
Grape berries are non-climacteric fruits, and their growth pattern follows a double-sigmoid
PLANT ORGANIZATION BASED ON SOURCE-SINK RELATIONSHIPS 271 curve, from anthesis to maturity (Harris et aI., 1968). Development prior to the color change "veraison" is referred to as the "first growth period" (I), while subsequent growth to the point of maturity is referred to as the "second growth period" (IJI). A lag phase (II) is observed between these two growth periods. Yeraison occurs at the beginning of the sec- ond growth period, and corresponds to the inception of ripening. Mechanisms of veraison and the triggers of ripening in grape, as non-climacteric fruit, are poorly understood. The objective is therefore to improve knowledge of the molecular physiology of grapevine berry development and its response in relation to biotic or abiotic stress.
The expression of one alcohol dehydrogenase gene (GY-Adh 1) was shown during ripening of grapevine berry (Tesniere et aI., 1993; Sami-Manchado et aI., 1997). Filion et al. (1999) studied the expression of gene hexose transporter (VvhtJ) during berry rip- ening. Vvht is strictly conserved between two grape varieties, Pinot noir and Ugni-blanc.
Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase suggests a possible co-regulation of the expression of these genes. During berry ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, two cDNAs fragments (gin1 and gin2) were cloned (Davies and Robinson, 1996). Invertase activity increased from flowering, was maximal 8 weeks post flowering, and remained constant throughout ripening. Expression of gin1 and gin2 in berries, which was very marked early in berry development, decreased significantly at the beginning of hexose accumula- tion. The expression of the genes and the synthesis of the enzymes seems to precede the onset of hexose accumulation.
Yeraison is the key stage of grape berry development, and few studies have been carried out to identify the nature of the signal that initiates ripening. Hormones such as ethylene is not considered to have a major role in controlling ripening in non-climacteric fruit such as grapes (Coombe and Hale, 1973). Other hormones such as abscisic acid and auxin have been studied in relation to berry ripening. ABA increases in concentration during berry ripening, and treatments that delay this increase seem to delay ripening (Coombe and Hale 1973; DUring et aI., 1978). Davies et al. (1997) have shown on grapevine that treatment with a synthetic auxin BTOA (benzothiazole-2-oxyacetic acid), delays the onset of berry ripening by two weeks. Nevertheless, the nature of the signal that initiates ripening in non- climacteric fruits remains unidentified. Salicylic Acid (SA) is known as an important molecule implicated as one of the key components in the signal transduction pathways leading to plant resistance to various pathogens (Ryals et aI., 1996), but also in some de- velopmental events as plant flowering (Raskin, 1992). We have demonstrated that exoge- nous SA is able to delay, or even inhibit berry ripening (Fig. 11.3). Ripening was delayed in berries treated by dipping in a solution of SA or when SA was injected into berries using a syringe (Kraeva et ai., 1998). A possible link between SA and the onset of grape berry ripening is suggested by this experimentation.
3.2. Pathogenesis related proteins
Certain proteins - Pathogenesis-Related proteins (PRs) - are known to be involved in plant
272 A. CARBONNEAU and A. DELOlRE
Figure 11.3. The lower half part of a Syrah bunch was dipped in a solution of Salicylic Acid (SA) at the "berry touch complete" phenological stage. Ripening of treated berries was retarded for more or less than 2 weeks.
defence and are also expressed during some developmental events (Van Loon, 1997).
Robinson et al. (1997) cloned two closely related chitinases cDNAs (VvChi4A and VvChi4B) from grapes. These two clones represent alleles of the same gene. The VvChi4 genes are highly constitutively expressed in ripening berries of Syrah. In the same vari- ety, Kraeva et al. (1998) demonstrated transcription of a P-1,3-glucanase genes (VvGLull7 and VvGlu26) at the beginning of veraison and during ripening (Fig. 11.4). In mature berries, the translation of p-I ,3-glucanase mRNA to protein was shown by im- munodetection with Tobacco antibodies (Kraeva, 1999). Induction of the synthesis of p-
1,3-glucanase mRNA could be obtained artificially from green berries by wounding or wounding in conjunction with the injection of salicylic acid (Fig. 11 .5). These results questioned the role of chitinase and P-I ,3-glucanase during normal berry development or in response to elicitation. A thaumatine-Iike protein (VVTLl) has been identified in V vinifera cv Muscat of Alexandria. VVTLl was found only in the berry and is encoded by a single gene that is expressed in conjunction with the onset of sugar accumulation and softening (Tattersall et al., 1997).
PLANT ORGANIZATION BASED ON SOURCE-SINK RELATIONSHIPS 273
- 1.4 kb
I 2 3
Figure 11.4. Kinetics ofVvGlu 117 ~-1 ,3-glucanase mRNA synthesis during berry development: I - stage of "berry touch complete"; 2 -onset of ripening; 3 - stage of "harvest berries ripe". Each lane was loaded with 10 fig of total RNA from berries without seed. Blot corresponds to exposition of 4d. RNA size (in kb) is indicated at the right side (Kraeva et at., 1998).
3.3. Phenolic compounds
Anthocyanins and stilbenes are considered to play important roles in determining the quality of wine and in crop protection, respectively. Genes involved in flavonoid and stilbene biosynthesis were isolated from different grape cultivars by Sparvoli et af.
(1994). These authors showed that the expression of genes for flavonoid biosynthesis - but not for pal and Stilbene synthases - is enhanced by light.
Expression of the genes for anthocyanin biosynthesis was also studied in Syrah grape berries (Boss et af., 1996). Expression of seven genes of the anthocyanin biosynthetic pathway was determined: phenylalanine amonia lyase (pal), chalcone synthase (chs), chalcone isomerase (chi), flavanone-3-hydroxylase (j3h), dihydroflavonol 4-reductase (dfr), leucoanthocyanidin dioxygenase (ldax) and UDP glucose-flavonoid 3-o-glucosyl transferase (ufgt). In the berry skin, most genes in the pathway were expressed briefly during the early stages of berry development, and again after veraison, when the color change occurred. ufgt was not expressed in flowers and during the first 4 weeks post- flowering. No pal nor ufgt were detected in grape berry flesh, at any developmental stage.
Thus these results have shown that the major control point of anthocyanin biosynthesis in grape berry skins is ufgt.
-1.4 kb
1 2 3
Figure 11.5. Induction ofVvGlu 117 ~-1 , 3-glucanase mRNA synthesis in young berries (stage of
"berry touch complete" ) by elicitation and wounding. I - wounded berries; 2 -berries wounded and treated with salicylic acid; 3 -control berries. Each lane was loaded with IO fig of total RNA from berries without seed. Time after the treatment: 7 d. (Kraeva et aI., 1998).
274 A. CARBONNEAU and A. DELOIRE
Wiese et al. (1994) isolated a 13 kb DNA fragment from the grapevine (Vitis vinifera cv Optima) genomic library. Two full-size stilbene synthase genes (Vstl and Vst2) were located within the 13 kb fragment. The expression of those genes was tested using a fun- gal cell wall elicitor on cell suspension cultures of Optima. The expression of VstI and Vst2 differed by a factor of 10 (after 5 hours' induction) or by a factor of 100 (after 12 hours' induction), respectively.
3.4. Biochemical and molecular responses to biotic stress
Regarding other grapevine organs, mainly the leaves, studies were carried out at the mo- lecular level which involved the plant-pathogen relationship. A basic class I (vchitlb) and a class III (vch3) chitinase cDNAs were cloned from cultured Pinot noir cells and used by the authors to probe the induction response of grapevine cells to salicylic acid or a yeast elicitor (Busam et aI., 1997). Single leaves of Vitis vinifera cvPinot noir and of Vitis rupestris were inoculated with Plasmopora viticola spore suspensions. Selective expression of vch3 was observed in leaves of both infected genotypes. mRNA also in- creased transiently in the healthy tissue of the younger next stage leaf of V. vinifera, but not in next stage leaf of V. rupestris. That result suggests that grapevine is capable of generating a systemic acquired resistance response. The kinetics of grapevine 13-1,3- glucanase (VvGlul/7) expression pattern of V. vinifera cv Chardonnay, in response to Botrytis cinerea leaf inoculation, was studied at the transcription stage by Renault et ai.
(1997, 2000). Northern blot hybridization showed that 13-1,3-glucanase mRNAs strongly accumulated in the leaves from day 3 to day 7 post-infection, with a maximum rate ob- served on day 5 (Fig. 11.6). At the translation stage, proteins synthesized in response to B. cinerea infection were nevertheless unable to inhibit the fungal growth development on leaves.
In relation to the biochemical response, Gianakis et al. (1998) collected leaves of 21 different grapevine genotypes with varying resistance to powdery mildew disease caused by Uncinuia necator. A correlation between the resistance rating and the sum of the chitinase and 13-1,3-glucanase activities was discussed.
3.5. Biochemical and molecular responses to abiotic stress
In response to abiotic stress such as drought, no significant work have been carried out on the grapevine at the molecular level. Nevertheless, in order to permit evaluation of the in- fluence of different levels of hydric stress on berry cell multiplication and enlargement, Ojeda et al. (1999) have developed a technique using DNA extraction to determine indi- rectly the rate of cell division and enlargement in the grape pericarp (Fig. 11.7). They stud- ied on growing Syrah berries, the biochemical effects of various degrees of water deficit on The phenolic compounds that were studied were analyzed according to protocols devel- oped by Andary (Andary et ai., 1996) and by Ojeda (1999). The flavan-3-0Is (catechins and proanthocyanins) and anthocyanins, have the same precursor (flavan-3,4-diol) in their biosynthesis, although their biosyntheses do not occur in the same time. The biosynthesis
PLANT ORGANIZATION BASED ON SOURCE-SINK RELATIONSHIPS 275
A
PR2a
B
~_-JII 0 3
( H.llryl/\ ftlocul:llIon (I)
Figure 11.6. Time chart of glucanase synthesis in Chardonnay leave, after Botrytis cinerea infec- tion (Renault et al., 2000). A) No ~-l ,3-glucanase protein was detected in non-inoculated leaves at day 1 and 7 (lanes C 1 and C7). The ~-l ,3-glucanase proteins were first detected at day 3 after B.
cinerea inoculation and steadily accumulated through day 7 (lanes 13 to 17). Western blot analysis of acido-soluble proteins (15 ~g/lane) harvested at different times (0 to 7 days). Proteins were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the anti-PR2a (acidic gluca- nase). B) No ~-1,3-glucanase mRNAs were detected before inoculation (lanes CI and C7). Infec- tion with B. cinerea induced an accumulation of ~-1,3-glucanase mRNAs from 3 to 7 days after inoculation. At day 7, the signal seems to have slightly decreased. Northern blotting of total RNA (15 ~gllane) harvested at various times (0 to 7 days). The blot was probed with the 32P labelled~-
1,3-glucanase cDNA. Equal loading of RNA populations was checked by staining the membrane with methylene blue.
of Total Tannins (TT) is intense during the flowering-veraison period; it then decreases and stabilizes a few days after veraison - the starting point of anthocyanin biosynthesis which continues until maturation. The early water deficit treatments, between flowering and veraison, have a negative effect on the biosynthesis of TT and could have a positive effect on the flavonol biosynthesis, but if the intensity of water deficit is very severe dur- ing this period, it can also reduce the synthesis of anthocyanins. On the other hand, wa- ter deficit level applied during the maturation stage was found to stimulate both flavonol and anthocyanin biosynthetic pathways (Fig. 11.8 and 11.9). Finally, in all cases, the water deficit increased the polymerization of tannins.
276 A. CARBONNEAU and A. DELOIRE
Average daily air temperature sum (basis 10°C)
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25 42 60 75 100% of DNA accumulated
Figure ]].7, Changes in the total amount of peri carp DNA (fig) and of the relative DNA increase (fig day'l figãl) of Syrah berries related to the average daily air temperatures sum (basis 10°C) and the number of days after anthesis. Total DNA of the peri carp increased from anthesis until the average daily temperature sum (basis J00e.) reached about 340°e., approximately 35d. after an- thesis. The changes of the relative DNA content indicate that the highest mitotic activity occured before day 5. The arrow indicates the start of the veraison (softening of 10% of berries). The dot- ted vertical lines delimit phases I, II and 1Il. (Ojeda et al., 1999).
Various studies in the field have implicated polyphenols (phenol acids and flavon- oids) in numerous defence mechanisms against attack by pathogens. Relations between abiotic stress as drought and grapevine defence mechanisms require more in depth inves- tigations, considering the plant development phases. However, it would appear that this is currently the most advanced example of techniques at the moment that allow the study of "gene x environment" interactions and the test ofthe basic triptych organization.
Molecular aspects of berry development will be detailed by Davis, Robinson, Romieu and Tesniere in the other chapters of th is book.
PLANT ORGANIZATION BASED ON SOURCE-SINK RELATIONSHIPS 277
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Figure 11.8. Flavonols content expressed in mg rutin equivalent, of Syrah berries of control plants and of plants subjected to water deficit treatment. C = control (100% ETP). S = water deficit be- tween veraison and maturity (30% ETP). The arrows indicate the onset of veraison (softening of 10% of the berries).
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Figure J 1.9. Anthocyanins content, expressed in mg malvidin equivalent, of Syrah berries of con- trol plants and of plants subjected to water deficit treatment. C = control (I 00% Erp). S = water deficit between veraison and maturity (30% ETP). The arrows indicate the onset of veraison (sof- tening of 10% of the berries).
278 A. CARBONNEAU and A. DELOIRE
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