Study of polyamines during grape ripening indicate an important role of polyamine catabolism

Plant Physiology and Biochemistry 67 (2013) 105e119

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Research article

Study of polyamines during grape ripening indicate an important role of polyamine catabolism Patricia Agudelo-Romero a, Cristina Bortolloti b, Maria Salomé Pais a, Antonio Fernández Tiburcio b, Ana Margarida Fortes a, * a b

Universidade de Lisboa, Faculdade de Ciências de Lisboa, BioFIG, Campo Grande, 1749-016 Lisboa, Portugal University of Barcelona, Pharmacy Faculty, Av. Diagonal 643, 08028 Barcelona, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 November 2012 Accepted 22 February 2013 Available online 14 March 2013

Grapevine (Vitis species) is the most economically important fruit crop worldwide. Ripening of nonclimacteric fruits such as grapes has been the subject of intense research. Despite this interest, little is known on the role played by polyamines in the onset of ripening of non-climacteric fruits. These growth regulators have been involved in plant development and stress responses. Molecular and biochemical studies were developed in three important Portuguese cultivars (Trincadeira, Touriga Nacional and Aragonês) during the year 2008 and in Trincadeira during 2007 in order to gather insights on the role of polyamines in grape ripening. Microarray and real-time qPCR studies revealed up-regulation of a gene coding for arginine decarboxylase (ADC) during grape ripening in all the varieties. This increase was not accompanied by an increase in free and conjugated polyamines that presented a strong decrease. Putrescine and Spermidine levels were higher at earlier stages of development, while Spermine level remained constant. Berries of Trincadeira cultivar presented the highest content in total free and conjugated polyamines at earlier stages of fruit development in particular in the year 2007. The decrease in polyamines content during grape ripening was accompanied by up-regulation of genes coding for diamine oxidase (CuAO) and polyamine oxidase (PAO), together with a significant increase in their enzymatic activity and in the hydrogen peroxide content. These results provide, for the first time, strong evidence of a role of polyamine catabolism in grape ripening possibly through interaction with other growth regulators. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Copper amine oxidase Gene expression Grape ripening Polyamine catabolism Polyamine oxidase Vitis vinifera

1. Introduction Grapevine (Vitis vinifera) is one of the most widely cultivated fruit crops. The current interest in understanding ripening of grape berry is due to the economic relevance of grape quality and of their processed products (wine, juice and dried fruit). Moreover, grapes present nutritional and health benefits for humans due to the presence of polyphenolic and anti-oxidant compounds [1,2]. Grape berry development consists of two successive sigmoidal growth periods separated by a lag phase. From anthesis to ripening, three major phases are considered [3]; the detailed descriptive

Abbreviations: ADC, arginine decarboxylase; CuAO, copper amine oxidase; PAO, polyamine oxidase; PAs, polyamines; PH, bound fraction of PAs; S, free fraction of PAs; SAM, S-adenosylmethionine; SAMDC, SAM decarboxylase; SH, conjugated fraction of PAs; SPDS, spermidine synthase; SPMS, spermine synthase. * Corresponding author. Tel.: þ351 21 7500382; fax: þ351 21 7500048. E-mail addresses: [email protected], [email protected] (A.M. Fortes). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.02.024

designations, known as the modified EeL system, being used to define more precise growth stages over the entire grapevine lifecycle [4]. The first growth period corresponds to the formation of the seed embryos and the pericarp. It is characterized by exponential growth of the berry, biosynthesis of tannins and hydroxycinnamic acids, and accumulation of the organic acids, tartrate and malate (EL 31e34). The onset of ripening, véraison (EL 35), constitutes a transition phase during which growth declines and there is initiation of color development (anthocyanin accumulation in red grapes) and berry softening. Ripening (the last phase) is characterized by pH increase, and additional berry growth mainly due to cell expansion and accumulation of soluble sugars, anthocyanins and flavor-enhancing compounds (EL 36e38). Molecular evidence is lacking for a single master switch controlling ripening initiation, such as the established role for ethylene in climacteric fruit ripening. Abscisic acid, brassinosteroids, and, to a lesser extent, ethylene, have been implicated in control of fruit ripening initiation in grapevine but their modes of action at the

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molecular level are far from understood [5e7]. Moreover, certain growth regulators such as polyamines have been little studied in the context of grape ripening. In fact, the majority of studies in fruit development and ripening have been focused on climacteric fruits such as tomato [8,9]. In addition, to our knowledge few reports have been focused mainly on the study of changes in the endogenous polyamines levels [10e12]. Thus, little is known on PA metabolism (biosynthesis and catabolism) in non-climacteric fruits particularly in V. vinifera. Polyamines (PAs) are growth regulators that have been implicated in a wide range of metabolic processes in plants such as floral initiation and development [13,14], organogenesis and embryogenesis [15,16] leaf senescence [17] and fruit development and ripening [9,18], as well as abiotic and biotic plant stress responses [14,19,20]. The PAs are small aliphatic amines positively charged at physiological pH in both prokaryotic and eukaryotic cells. The PAs, diamine Putrescine, triamine Spermidine and tetraamine Spermine are present in all living organisms [21]. In nature, PAs often occur as free molecular bases (S-), but they can also be associated with small molecules like phenolic acids (conjugated forms, SH) and also with various macromolecules like proteins (bound forms, PH). The most common conjugated PAs are those which are covalently linked to cinnamic acids [22]. Metabolic studies suggest that the intracellular levels of PAs in plants are mostly regulated by anabolic and catabolic processes, as well as by their conjugation with hydroxycinnamic acids [14]. In mammals and fungi, Putrescine is exclusively synthesized from ornithine, via ornithine decarboxylase (ODC; EC 4.1.1.17) activity, whereas in plants and bacteria Arginine can also be used as a metabolic precursor, via Arginine decarboxylase (ADC; EC 4.1.1.19) activity [23]. Spermidine synthase (SPDS, EC 2.5.1.16) catalyzes Spermidine synthesis from Putrescine and Spermine synthase (SPMS, EC 2.5.1.16) catalyzes Spermine synthesis from Spermidine. Decarboxylated S-adenosylmethionine (dcSAM), the donor of aminopropyl groups, is formed by decarboxylation of S-adenosylmethionine (SAM) in a reaction catalyzed by SAM decarboxylase (SAMDC, EC 4.1.1.50) [14]. The polyamine metabolic pathway is also interconnected with other metabolic routes such as ethylene, gaminobutyric (GABA), nitric oxide (NO), Krebs cycle (TCA) and abscisic acid (ABA) [14,24]. Oxidation of Putrescine at the primary amino groups is catalyzed by Copper amine oxidases (CuAO; EC 1.4.3.6) which are copper-containing enzymes. This reaction produces 4-aminobutanal (which spontaneously cyclizes to D1pyrroline), hydrogen peroxide (H2O2) and ammonia [24]. The Arabidopsis genome carries at least twelve DAO-like genes, but only one of them (At4g14940; AtAO1) has been characterized [25]. Polyamine oxidases (PAOs; EC 1.5.3.3) are flavin adenine dinucleotidedependent (FAD) enzymes. PAOs are involved in the terminal catabolism of Spermidine and Spermine, producing pyrroline and 1,5-diazabicyclononane, along with 1,3-diaminopropane and H2O2 [23]. The maize PAO (ZmPAO) belongs to this PAO class and it is the best characterized [24]. A second class of plant PAO catalyzes the back-conversion of Spermine to Spermidine, which resemble the mammalian Spermine oxidases (SMO; EC 1.5.3.3) [14]. The Arabidopsis genome contains at least five genes (AtPAO1-5) coding putative PAOs [26]. Takahashi et al. [27] have reported that AtPAO1 and AtPAO4 catalyze the back-conversion reaction corresponding to SMO, with specificity by the substrate; in which, AtPAO1 prefers Thermospermine and Norspermine, while AtPAO4 seems to be Spermine specific. AtPAO2 and AtPAO3 act in the back-conversion from Spermine to Spermidine and Spermidine to Putrescine; both enzymes show similar preference of Spermidine as substrate but less specificity to Spermine. Catalytic properties of AtPAO5 have not been determined yet.

Previous studies on grape ripening carried out in Trincadeira cultivar using transcriptomic and metabolomic approaches suggested an important role of PAs in ripening process [28]. Moreover, complementary metabolic profiling of Trincadeira, Touriga Nacional and Aragonês cultivars has shown accumulation of GABA and L-arginine during grape ripening [29]. In order to evaluate the role played by PA metabolism in grape ripening, berries of three important Portuguese cultivars (Trincadeira, Touriga Nacional and Aragonês) were analyzed in the year 2008. In the case of Trincadeira cultivar a study was also performed in 2007 because it has been reported as a cultivar with irregular growth between years [28]. Moreover, the fact that the study was carried out for one cultivar for 2 years (2007 and 2008) gave information about climatic influence on PA metabolism. In this work, it was performed a detailed analysis of the PA pathway using microarrays and real-time qPCR studies complemented by HPLC analysis of PA contents, as well as enzymatic activity assays of the PA catabolic enzymes (CuAO and PAO). 2. Results 2.1. Phenotypic characteristics of grape berries revealed differences in berry weight among cultivars and between years Grape berries of Trincadeira, Touriga Nacional and Aragonês grape cultivars were collected at four developmental stages (EL 32, EL 35; EL 36 and EL 38) during 2008 season, and additionally, grape berries of the Trincadeira cultivar were also assayed during 2007 season. The developmental stages identified were: EL 32 characterized by small hard green berries accumulating organic acids; EL 35 corresponding to véraison; EL 36 characterized by sugar and anthocyanins accumulation and active growth due to cell enlargement and EL 38 corresponding to harvesting time. For the three varieties, the stage véraison was set at approximately 9 weeks post-flowering. From EL 32 to EL 38, berry weight increased considerably in all varieties. For Trincadeira cultivar, a significant difference (1.81  0.11 fold; P ¼ 0.000) was observed at EL 38 (Fig. 1), when 2007 and 2008 seasons are compared. This was most probably due to differences in climatic conditions between years. Considering all the cultivars in the season 2008, the differences among them were less evident; Aragonês cv. presents the highest berry weight at EL 38 (1.93 g  0.06) followed by Trincadeira (1.78 g  0.08) and Touriga Nacional (1.46 g  0.001) (Fig. 1). A significant difference was observed between Touriga Nacional and Aragonês cultivars (P ¼ 0.043). The onset of véraison is characterized by the initiation of anthocyanin accumulation in red grapes and berry softening. Fortes et al. [28] reported on a considerable difference between the anthocyanin content in Trincadeira grape berries between 2007 and 2008 seasons mostly due to the fact that berries did not expand during 2008 season as in 2007 and berry weight almost doubled at EL 38 in the later season (Fig. 1). Regarding the differences in anthocyanin content among the cultivars assayed in 2008 at EL 36 and EL 38 stages, Trincadeira and Touriga Nacional presented the highest anthocyanin content compared to Aragonês that presented the lowest [67]. 2.2. Expression of genes involved in polyamines and ethylene metabolisms during grape ripening suggested reprogramming of these metabolisms The mRNA expression profiles of two time points (EL35 vs EL36) were compared using the Affymetrix GrapeGenÒ genome array containing 23,046 probesets corresponding to 18,712 unique

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Fig. 1. Fresh weight of the berries from Trincadeira, Touriga Nacional and Aragonês cultivars during four stages of fruit development: green (EL 32), véraison (EL 35), ripe (EL 36) and harvest (EL 38). Vertical bars indicate the SE.

sequences. It was carried out in Trincadeira, Touriga Nacional and Aragonês cultivars during 2008 season and in addition in Trincadeira during 2007 season. Testing was performed using biological triplicates for each time point. After performing a Bayes t-statistics from the linear models for microarray data (Limma) for differential expression analysis, P-values were corrected for multiple-testing using the BenjaminieHochberg’s method [30]. Table 1 shows that thirty nine probesets annotated as being involved in PA and ethylene metabolisms were differentially expressed (fold change 1.5 and FDR <0.05 or fold change  .1.5 and FDR < 0.05). They were classified into two main functional categories; these were “Metabolism” accounting for 74.4% and it is represented by three subcategories: Primary, Secondary and Cellular metabolisms; and “Signalling” category accounting for 25.6% and it is represented by hormone signaling subcategory. The assignment to functional categories was performed assigning each gene to a category according to its putative molecular function. Among the transcripts modulated, it has been found that several of them code for eight enzymes involved in PA metabolism. Six enzymes are involved in the PA biosynthesis (ADC, ARG, OTC, ASS, SPDS and SPMS) and the remaining two enzymes are implicated in PA catabolism (CuAO and PAO) (Table 1). Concerning ethylene metabolism, it was also found several probesets coding for three enzymes involved in ethylene biosynthesis (SAMS, ACC and ACO) besides SAMDC enzyme, which establishes the link between the biosynthesis of the polyamines and ethylene (Table 1). Fig. 2 summarizes gene expression related to PA and ethylene metabolisms evaluated in Trincadeira, Touriga Nacional and Aragonês cultivars, regarding the comparison EL36 vs EL35 stages of development. In Fig. 2 and Table 1, it can be observed a gene up-regulated coding for ADC only in Touriga Nacional. However, real-time qPCR data showed that the expression of this ADC gene (VVTU12839_at) increased during ripening in all cultivars (Fig. 3). The same holds true for a probeset representing a gene coding for ARG (VVTU5482_at) (Fig. 3). A gene coding for OCT (VVTU866_at) was down-regulated in all cultivars (Table 1). OCT enzyme catalyzes

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the first step in the conversion of ornithine to arginine, suggesting that this part of the pathway may be less active. Two probesets coding for SPDS were either down-regulated in Trincadeira or presented no differential expression in the other cultivars according to microarray results though real-time qPCR data showed down-regulation of one probeset (VVTU33098_s_at) in all cultivars at least when EL 36 is compared with EL 35. Interestingly, this gene is up-regulated when EL 35 is compared with EL 32 suggesting a role of SPDS in the onset of ripening (Fig. 3). As shown in Fig. 2, different patterns of genes coding for SPMS can be observed: they were up-regulated (VVTU24933_at; VVTU10365_at and VVTU5224_at), down-regulated (VVTU6650_at and VVTU22442_at) or presented no differential expression depending on the variety (Table 1). It should be noted that Aragonês presented no differential expression of genes coding for SPDS and SPMS (Fig. 2) which was partially confirmed by real-time qPCR data (Fig. 3). The profiles revealed by real-time qPCR showed that a gene coding for SPDS (VVTU33098_s_at) decreased during ripening in all cultivars. In the case of a transcript encoding for SPMS (VVTU10365_at), its expression increased from véraison until harvest in Trincadeira and Touriga Nacional whereas Aragonês presented no differential expression (Fig. 3). Most genes coding for CuAO and PAO were up-regulated. In addition, real-time qPCR results showed a sharp rise in their expression (VVTU37047_at; CuAO and VVTU851_at; PAO) during ripening in all cultivars (Fig. 3) indicating increased catabolism of PAs following véraison. Opposite profiles were obtained for genes coding for SAMDC with one gene being up-regulated (VVTU12964_s_at) and another being down-regulated (VVTU16123_s_at). To further investigate these profiles real-time qPCR was performed and showed that a gene coding for SAMDC1 (VVTU12964_s_at) presented an increase during ripening mainly in Touriga Nacional and Aragonês cultivars (Fig. 4) in agreement with microarray data (Fig. 2). On the other hand, a gene coding for SAMDC2 (VVTU16123_s_at) exhibited a cultivar dependent profile (Fig. 4). Four probesets coding for SAMS were differentially expressed, two of them (VVTU8738_s_at and VVTU25643_x_at) up-regulated and the other two (VVTU13925_ s_at and VVTU893_x_at) down-regulated in Touriga Nacional and Aragonês whereas Trincadeira presented no differential expression (Table 1). These results were further confirmed by real-time qPCR with a transcript encoding SAMS1 (VVTU8738_s_at) showing a general tendency to increase during ripening in all cultivars and a transcript encoding SAMS2 (VVTU13925_s_at) exhibiting a sharp decrease in expression for the Aragonês cultivar (Fig. 4). Most genes coding for ACS and ACO were down-regulated at EL36 when compared to EL 35 suggesting a decrease in ethylene biosynthesis during ripening as previously reported for other grape varieties [31,32]. Nevertheless, one exception coding for ACO (VVTU2507_ s_at) was found and further confirmed by real-time qPCR. Interestingly, Aragonês presented a huge increase in the expression of this gene contrasting with the above mentioned sharp decrease in expression of a gene coding for SAMS2 (VVTU15167_at) (Fig. 4). In order to discriminate between the behavior of the cultivars concerning the PAs and ethylene pathways, Multivariate Data Analysis was performed with real-time qPCR data using the unsupervised method of Principal component analysis (PCA). The first and second principal components accounted for 68.38% and 18.56% of variance, respectively (Fig. 5). A good discrimination was obtained for the three cultivars in what concerns the data from the 2008 season. Samples of Trincadeira from 2007 and 2008 tend to cluster together suggesting that the season plays a less important role than the type of cultivar (genotype) in discrimination of PA and ethylene metabolisms. Touriga Nacional and Trincadeira are separated from Aragonês by PC1 whereas Trincadeira is separated from Touriga Nacional by PC2 (Fig. 5). Among the genes contributing for

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Table 1 Genes differentially expressed involved in Polyamine and Ethylene metabolisms evaluated in Trincadeira, Touriga Nacional and Aragonês cultivars, regarding the comparison EL36 vs EL35 (considering a fold change  1.5 and FDR < 0.05 or fold change  1.5 and FDR < 0.05). The transcripts shown in bold were chosen for real-time qPCR experiments. Annotation Primary metabolism Arginine decarboxylase (ADC) Arginase (ARG) Ornithine-carbamoyl transferase (OTC) Arginosuccinate synthase (ASS) SAM decarboxylase (SAMDC) SAM synthetase (SAMS)

Secondary metabolism Spermidine synthase (SPDS) Spermine synthase (SPMS)

Grapegen unique ID

a

Grapegen probeset

Trincadeira 2007 EL36/EL35

Trincadeira 2008 EL36/EL35

Touriga N.2008 EL36/EL35

Aragonês 2008 EL36/EL35

VIT_15s0048g00420 VIT_18s0001g12780

VVTU12839_at VVTU8198_at VVTU5482_at VVTU866_at

e e 1.51 1.62

e e 1.54 1.87

2.59 2 e 2.11

e e 1.66 1.8

VIT_17s0000g01690

VVTU2465_at

e

1.96

1.74

e

VIT_01s0010g00990 VIT_11s0037g00950 VIT_08s0007g05000 CN007600 VIT_07s0005g02230

VVTU12964_s_at VVTU16123_s_at VVTU8738_s_at VVTU25643_x_at VVTU13925_s_at VVTU7893_x_at

e e e e e e

e 1.8 e e e e

2.49 e 6.58 1.57 e 1.84

2.77 2.6 3.26 e 1.79 1.61

VIT_01s0026g00240

VVTU33098_s_at VVTU1269_s_at VVTU6650_at VVTU22442_at VVTU24933_at VVTU10365_at VVTU5224_at

1.52 1.55 e e 5.17 13.13 2

1.57 e 1.58 10.9 3.37 5.8 e

e e e 36.1 2.97 5.39 e

e e e e e e e

VVTU17481_s_at VVTU36933_at VVTU6472_at VVTU2557_at VVTU27834_at VVTU2983_s_at VVTU37047_at

1.57 1.77 2.03 1.68 e e e

e e e e e e 2.22

e e 3.04 e e e 3.75

e e e e 1.63 1.52 e

VVTU8008_at

1.61

2.83

3.13

e

VVTU851_at VVTU5226_at VVTU28144_at

e e 1.64

e e 1.63

1.88 1.85 1.55

2.3 e e

VIT_18s0001g03300 VIT_02s0025g00360 VIT_00s1764g00010

VVTU5165_at VVTU6382_at VVTU12042_at

e e e

1.53 5.37 e

2.03 8.17 1.75

1.58 4.9 e

VIT_00s2086g00010

VVTU13344_at VVTU20527_at VVTU15455_at VVTU22021_at VVTU24731_s_at VVTU15167_at VVTU2507_s_at

1.58 3.41 4.94 e e e 2.37

4.19 4.63 4.49 1.82 e 1.93 e

4.11 5.45 4.52 3.27 e 4.28 4.15

2.54 2.3 3.53 2.04 1.65 e 2.2

VIT_03s0038g00760

VIT_05s0029g00720 VIT_04s0008g01740 VIT_05s0020g03200 VIT_16s0050g02670

Cellular metabolism Cooper amine oxidase (CuAO)

GSVIVT00011290001 VIT_00s0225g00090

VvCuAO1 VvCuAO2

VIT_00s1682g00010 VIT_02s0025g04560

VvCuAO3 VvCuaO4

VIT_17s0000g09100

VvCuAO5

VvCuAO5 Polyamine oxidase (PAO)

Hormone signalling ACC synthase (ACS)

ACC oxidase (ACO)

VIT_04s0043g00220 VIT_12s0028g01120 VIT_13s0019g04820

VIT_02s0012g00360 VIT_12s0028g02420 VIT_15s0021g00950 VIT_02s0012g00450 VIT_12s0059g01380 a

VvPAO4 VvPAO5 VvPAO7

Nomenclature used in the present study for Copper amine oxidases (CuAO) and polyamine oxidases (PAOs) from V. vinifera (Table S2).

separation are genes coding for PAO and SAMDC1 that enables the discrimination of the two seasons of Trincadeira (2007 and 2008), and a gene coding for SPMS that discriminates Touriga Nacional and SAMS1, SAMS2 and ACO1 that discriminates Aragonês. Altogether the data indicate increased expression of genes involved in polyamine biosynthesis and catabolism whereas expression of genes involved in ethylene biosynthesis seems to be reduced. 2.3. Quantification of endogenous levels of polyamines during grape ripening showed decrease in these growth regulators Free (S), conjugated (SH) and bound (PH) polyamine contents at EL 32, EL 35; EL 36 and EL 38 developmental stages were studied in the pericarp extracts from the 2007 season for the Trincadeira cultivar and from the 2008 season for all cultivars. During grape

ripening, the three main PAs Putrescine, Spermidine and Spermine were present both in free and conjugated forms. In all cultivars the levels of Putrescine and Spermidine presented a strong decrease during ripening whereas Spermine had a more or less constant content during ripening (Fig. 6). A general profile was detected in all cultivars with the highest values of the free total PAs found at earlier stages of development. In particular, at EL32 stage of Trincadeira samples from 2007 season showed a difference of 1.44  0.12 fold more than samples from 2008 season in the content of free total PAs though at EL38 no significant differences were found. It is clear that 2007 and 2008 seasons of Trincadeira tend to present higher contents of free total PAs (mainly Putrescine and Spermidine) especially in the earliest stages of the berry grape development. Although there was a decrease in their contents at EL 35 stage in comparison with EL 32 stage, Trincadeira remains with

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Fig. 2. Gene expression related to polyamine and ethylene metabolic pathways. The grids represent the gene expression from microarray data comparison (EL36 vs EL35) where green represent down-regulated genes, purple up-regulated genes and black genes not differentially expressed. Each column represent from left to right: Trincadeira 2007, Trincadeira 2008, Touriga Nacional 2008 and Aragonês 2008 and each row represent different Grapegen Unique IDs. The following enzymes involved in the pathway are shown: ADC: Arginine decarboxylase; ODC: Ornitine decarboxylase; SPDS: Spermidine Synthase: SPMS: Spermine Synthase; CuAO: Copper Amine Oxidase; PAO: Polyamine Oxidase; SAMDC: SAM decarboxylase (SAMDC); SAMS: SAM synthetase; ACS: ACC synthase and ACO: ACC oxidase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

higher contents with respect to the other cultivars from 2008, this decrease was around 1 (0.17) fold less for 2007 season and only 0.2 (0.04) fold less for 2008 season (Fig. 6). On the other hand, Aragonês tends to present lower levels at EL 36 and EL 38 stages particularly for Putrescine (Fig. 6). The same tendency was observed for conjugated forms while SH fraction is present in all cultivars in lower levels (Fig. 7). In Trincadeira samples from the 2007 season an accumulation of SH content was only detected at EL32 stage, whereas in Trincadeira samples from 2008 values remained low. Conjugated PAs levels of Touriga Nacional and Aragonês decreased following EL35 (Fig. 7). On the other hand, endogenous level of PH polyamines was not found throughout ripening in any cultivar. In conclusion, the contents in free and conjugated polyamines decreased in all varieties during grape ripening with Trincadeira exhibiting higher content in these growth regulators at pre-véraison stage. 2.4. Activity of amine oxidases increased during grape ripening An activation of the PA pathway during grape ripening was suggested by the enhanced expression of ADC gene in all the varieties. Previously, an increase in GABA and arginine were detected during grape ripening [29] suggesting that the oxidation of PAs produced a decreased in the PAs titers. This flux through the PA pathway may be related with the rise in the expression of catabolism-related genes (DAO and PAO). Therefore, the enzymatic activity of PAO and DAO enzymes was studied. In Fig. 8, it is observed in all samples from 2008 season the increase during ripening (EL 32, EL 35, EL 36 and EL 38 stages) of DAO and PAO enzymatic activities, mainly from EL 35 to EL 36 stage followed by a small rise until EL 38 stage. In the case of DAO activity at EL 38 stage, Trincadeira presented the highest values (2.03  0.00) and Aragonês presented the lowest (1.58  0.09). In case of PAO activity at EL 38 stage, Aragonês showed the highest

values (0.82  0.04) whereas Touriga Nacional presented the lowest (0.44  0.00). The values of DAO activity were higher than the ones obtained for PAO activity. Comparing the increment of DAO with respect to PAO, the difference at EL38 stage was around 3.08 (0.01) fold more for Trincadeira; 4.03 (0.25) fold more for Touriga Nacional and only 1.92 (0.11) fold more for Aragonês. This is in agreement with the less Putrescine content in comparison with the high Spermidine content detected throughout developmental grape stages. The increase in the activity of DAO and PAO in all varieties indicated increased catabolism of polyamines following véraison. 2.5. In situ staining of hydrogen peroxide showed increase in this reactive oxygen species in skin of ripe berries In order to detect hydrogen peroxide content in skin of grapes berries DAB staining was performed in samples from Trincadeira cultivar (2007 season) at three developmental stages (EL 32, EL 35, and EL 38). The in situ detection of hydrogen peroxide in the skin of green berries showed no dark spots (Fig. 9A). At véraison DAB staining is already recognized by the appearance of dark spots (Fig. 9B and C). In the skin of ripe berries a strong DAB staining is observed (Fig. 9D and F) indicating higher hydrogen peroxide content than in the previous developmental stages. Similar results were obtained for the other cultivars (not shown). 2.6. Phylogenetic analysis suggested activity during grape ripening of CuAO and PAO isoforms putatively located in the peroxisome There are five genes of V. vinifera coding for CuAO isoforms in Genoscope database, and all of them are differentially expressed in the comparison EL36 vs EL35 stages of development. Three of them were down-regulated, while the other two were up-regulated. On the other hand, of the seven genes coding for PAO that are

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Fig. 3. Changes in the relative expression of genes involved in polyamine metabolism during grape ripening. Vertical bars indicate the SE.

annotated in the Genoscope database, just three of them were up-regulated. Phylogenetic analysis of the five CuAO isoforms of V. vinifera (VvCuAO1 to 5) and other known plant CuAOs was carried out. The resulting tree revealed that CuAOs are divided into three major branches (Fig. 10A). VvCuAO1, 3 and 4 belong to the branch in which atCuAO4, 6 to 8 and SlCuAO (Solanum lycopersicum) are included; a second branch is formed by AtCuAO1 to 3, together with

GmCuaO (Glycine max), LcCuAO (Lens culinaris) and PsCuAO (Pisum Sativum). The third branch is composed by VvCuAO2, 5 and AtCuAO5. On the contrary, AtCuAO5 is placed alone between the second and third branches. Alignment of 18 copper amine oxidase sequences revealed 35 highly conserved residues at the C-terminal tails (Supplementary material, Fig. S1). VvCuAO2 and VvCuAO5 showed high identity (83%) and AtCuAO5 (Reumann et al., 2009) shared 81% and 79%

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Fig. 4. Changes in the relative expression of genes involved in ethylene metabolism during grape ripening. Vertical bars indicate the SE.

identity with them, respectively. VvCuAO 1 and VvCuAO4 presented high identity (61%). On the other hand, VvCuAO1 showed 61%, 60% and 58% identity with AtCuAO4, AtCuAO6 and AtCuAO8, respectively. VvCuAO4 also presented high levels of identity with these AtCuAOs (57%, 55% and 65%, respectively). SlCuAO (S. lycopersicum) showed an identity of 51% with VvCuAO1, and 61% with VvCuAO4. VvCuAO3 presented low levels of identity with other plant CuAOs.

Fig. 11A shows that when CuAOs 2 and 5 sequences from grape were aligned, their carboxyl-distal sequences displayed putative peroxisomal target signals type 1 (PTS1); such is also the case of AtCuAO5 (At2g42490). Moreover, the correspondent protein has been reported in Arabidopsis to be located in peroxisomes [33]. Phylogenetic analysis was performed using the seven amino acid sequences of V. vinifera obtained from Genoscope database together with five Arabidopsis PAOs, two Hordeum vulgare PAOs

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three Arabidopsis members (AtPAO3, AtPAO4 and AtPAO5; ranged between 60% and 79%) and three rice members (OsPAO3, OsPAO4 and OsPAO5; ranged between 58% and 71%). For all of them, a PTS1 domain has been predicted (Fig. 11B). In the case of Arabidopsis [39] and rice [35] these isoforms have been reported to be localized in peroxisomes. 3. Discussion

Fig. 5. Score plot of PCA showing discrimination of developmental stages (EL EL 32, EL 35, EL 36 and EL 38) corresponding to Trincadeira, Touriga Nacional and Aragonês cultivars. It was performed from real-time qPCR data of expression of genes involved in polyamine and ethylene pathways. PC1 accounts for 68.38% of the variance whereas PC2 accounts for 18.56% of the variance.

and seven Oriza sativa PAOs, and with PAOs belonging to other plant species. Plant PAOs were divided into four major branches (Fig. 10B). In the first branch are grouped AtPAO1, NtPAO (Nicotiana tabacum [34]) and MdPAO (Malus domestica). The second branch comprises two grape members (VvPAO1 and 2), three from rice (OsPAO 2, 6 and 7 [35], other two from barley (HvPAO1 and 2 [36]) and one from maize (ZmPAO [37]). The third branch is formed by two grape members (VvPAO3 and 7) with AtPAO5, OsPAO1 and BjPAO (Brassica juncea [35]). In the fourth branch, two grape PAOs (VvPAO4 and 5) were grouped together with three Arabidopsis members (AtPAO2 to 4 [26,38]) and three PAOs from rice (OsPAO3 to 5). VvPAO6 is placed alone in this un-rooted tree. Alignment of 25 polyamine oxidase sequences was performed (Supplementary material, Fig. S2). VvPAO1 and VvPAO2 showed 66% identity. In addition, VvPAO1 and VvPAO2 showed high identity with the other members of the second branch which ranged between 48% and 62%. On the other hand, VvPAO4 and VvPAO5 showed 63% of identity; they also presented a high identity with

The PAs are natural growth substances known to be implicated in fruit ripening helping to control the quality of climacteric fruits [40,41]. The idea that PAs may play a regulatory role in these processes is supported by the fact that when PAs are exogenously supplied they improve quality features such as fruit set, fruit size and post-harvest decay [40]. In this study, we noticed increased levels of PAs at earlier stages of grape development. Elevated concentrations of free PAs in the pericarp of the early phase of fruit development were reported in climacteric fruits, such as apple [42], avocado [43] and peach [44], together with a gradual decrease shortly afterwards. Moreover, in the pericarp of non-climacteric fruits such as grape, it has also been mentioned that high levels of free PAs during early development may be associated with cell proliferation [10,12]. On the other hand, it has also been suggested that amine oxidases have a role in the regulation of different physiological stages through other reactions products or through modulation of PA cellular content [45]. It should be noticed that the higher levels of these PAs were detected at EL 32 (fruit set stage) from Trincadeira samples collected in 2007 which were considerably heavier than the berries from 2008 season. The same was observed in two cultivars of olives (Olea europaea L.), in which the differences from the fruit size could be related with cell division to the developmental acquisition of cell size, depending on PA concentrations [46]. However, little is known about the role of the PAs on the maturity of non-climacteric fruits [47]. In particular the role of PAs in grape ripening has been little explored [10,12]. Recent transcriptomic studies in Trincadeira cultivar showed that a gene coding for ADC was up-regulated during grape ripening [28] in agreement with previous reports for Cabernet Sauvignon cultivar [31]. This aspect was confirmed for Trincadeira as well as

Fig. 6. Changes in the endogenous levels of the free PAs (S) in pericarp of berries from Trincadeira, Touriga Nacional and Aragonês cultivars during grape ripening. To obtain total free polyamines content were summed Putrescine, Spermidine and Spermine contents. Vertical bars indicate the SE.

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Fig. 7. Changes in conjugated total Polyamines (SH) in pericarp of berries from Trincadeira, Touriga Nacional and Aragonês cultivars during grape ripening. To obtain the conjugated total polyamines was subtracted to soluble total polyamines content the free total polyamine content. Vertical bars indicate the SE.

for Touriga Nacional and Aragonês by the real-time qPCR results shown here. This prompted us to deepen the study of the role played by PAs in grape ripening and the putative interaction with ethylene. On the other hand, ODC gene was not differentially expressed in all the cultivars, in agreement with what was reported

Fig. 8. Changes in the enzymatic activity of CuAO and PAO from grape berries pericarp of Trincadeira, Touriga Nacional and Aragonês cultivars during grape ripening of the 2008 season. Vertical bars indicate the SE.

by Delùc et al. [31] for Cabernet Sauvignon. Although ODC activity has been reported in the tomato ovary, it decreases rapidly after fruit set coinciding with the cessation of cell division [48]. Furthermore, ODC activity has also been detected in the early stages of fruit development of avocado [43] and peach [44], suggesting that the pathway for PA synthesis during the early stages of these climacteric fruits is at least partially through ornithine. However, ODC pathway does not appear to contribute to the biosynthesis of PAs in non-climacteric fruits such as grapes [12]. It is known that ethylene biosynthesis decreases during ripening of non-climacteric fruits such as grape [49]. Our results support this since most of the genes coding for enzymes involved in their biosynthesis such as ACS and ACO are down-regulated during ripening in all the cultivars. Nevertheless, one specific isoenzyme of ACO may be active at later stages of ripening to account for some ethylene synthesis. In fact, the ethylene biosynthetic enzymes ACS and ACO belong to multigene families. Different patterns of transcript accumulation were observed that would suggest different roles for the above-cited isoenzymes during fruit development. On the other hand, SAMS is involved in the biosynthesis of SAM and its activity is related as precursor in the biosynthesis of the PAs Spermidine and Spermine as well as ethylene. Previously, Negri et al. [50] have reported a sharp decline of the expression of SAMS protein in mature grape skins of cv. Pinot Noir. Nevertheless, in this work and by using real-time qPCR we have detected an increase in expression of a gene coding for SAMS (SAMS1; Fig. 4); whereas another one is down-regulated (SAMS2; Fig. 4), mainly in Aragonês. Although this may be due to the fact that these experiments were performed in whole berries, it is also possible that some specific isoforms are activated during ripening. The fact that ethylene decreases and the genes coding for enzymes involved in biosynthesis of PAs increase during ripening suggested that the levels of these growth regulators would increase too since they compete for the same precursor, SAM. However, we observed decreased levels of PAs. Previously, maximum ADC expression was associated with non-dividing expanding cells, including fruit tissues [41,51], and with the response to various stresses [52]. In peach [41], a climacteric fruit, changes in ADC transcript levels were not accompanied by analogous changes in

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Fig. 9. Tissue distribution of H2O2 following staining with DAB in skin of grapes of Trincadeira cultivar from 2007 season. Green berries (EL 32) showed no dark spots (A) whereas berries at véraison (B, C), and in particular ripe berries (D, E) showed intense DAB staining.

enzyme activity supporting the view that ADC is not mainly regulated at the transcriptional level. As it has already been reported in climacteric fruits such as peach [41], free PA levels generally change from a maximum in early developmental stages, in which cell division may still be present, to minimum levels at ripening when the growth of fruit ceases and the climacteric ethylene emission starts [40]. Similar decreases on the PA contents were noticed by Bauza et al. [12]. These authors analyzed two cultivars of grape (Granache Noir and Syrah) and detected that Putrescine, Spermidine, Spermine, and Agmatine decrease during ripening. These results suggest that the major pathway of PAs biosynthesis in grape is via Arginine and Agmatine [12]. Furthermore, the PA contents of the grape pericarp of cv. Muscat Bailey A (Vitis labrusca L.  Vitis vinifera L.) have also been analyzed, in which the levels of free Putrescine and Spermidine were higher during earlier development [10]. This decrease in PA levels are in agreement with our results for all grape cultivars. The levels of free PAs are regulated by conjugation either to small molecules, especially hydroxycinnamic acids named as conjugated PAs (SH) [53], or with high molecular mass substances, such as hemicelluloses and lignin and, to a lesser extent, proteins also called insoluble conjugated PAs (PH) [19]. Cinnamic acids are higher at earlier stages in grapes [29]. Metabolic studies indicate that the intracellular levels of PAs in plants are mostly regulated by anabolic and catabolic processes, as well as by their conjugation to hydroxycinnamic acids [14]. It should be noted that although PH polyamines were not detected in our study, it has been reported its presence in the earlier stages of grape berry development together with a decrease of its content during ripening in cv. Muscat Bailey A [10]. Recent studies indicate that PAs may act as cellular signals in intricate crosstalk with hormonal pathways, including ABA regulation of abiotic stress responses [14]. Alcázar et al. [14] showed that ABA is responsible for the induction of genes encoding for enzymes in the biosynthetic pathway of PAs in Arabidopsis. Besides, Moschou et al. [26] reported that the PA catabolic genes are also ABA- inducible in tobacco. This was further tested in the perennial plant grapevine which highlights the interplay between PA anabolism and ABA signaling pathway [54]. Since ABA is known to increase in grape at véraison [28,31,32], it can hypothesized that ABA is triggering increased PA synthesis which is subjected to a rapid turnover by amine oxidases as shown here by increasing their

activity during ripening. This PA catabolism may constitute a source of reactive oxygen species as H2O2 to signal downstream stress defense events toward abiotic and biotic stress factors [24,55] that are enhanced following véraison along with sugar accumulation. In fact, we have observed increased content in H2O2 in the skin of grapes from véraison until ripening stages; green berries presented few spots indicative of H2O2 presence (Fig. 9). The ABA-induced increase of PAs and the simultaneous DAO and PAO induction and PA exodus to the apoplast could promote a PA-dependent H2O2 generation process. To test this, Toumi et al. [55] supplied ABA exogenously, which resulted in significant H2O2 amounts even at low concentrations, further confirming the view of PA-derived ROS production. The PAO-generated apoplastic H2O2 resulting from osmotic stress may act as a secondary messenger in the signaling pathway leading to stoma closure a phenomena known to occur at véraison. Indeed, ABA has been reported to activate Putrescine catabolism and H2O2 production through DAO activities during the induction of stomatal closure in Vicia faba guard cells [56]. Additionally, hydrogen peroxide resulting from Copper amine oxidase has been shown to contribute to cell reinforcement during plantepathogen interaction [57]. This may be a response in grape cells known to be subjected to pathogen attack following véraison concomitant with sugar accumulation. It is also possible that the genes coding for VvCuAO5 and VvPAO 4 and 5 shown here to be up-regulated during ripening, and whose protein products present a putative peroxisomal target may constitute a source of reactive oxygen species for signaling events. In fact, plant peroxisomes play a role in development as well as abiotic and biotic stress responses [58]. In this work we have observed increased H2O2 content in the skin of mature grapes (Fig. 9) corroborating a previously detected increase of the anti-oxidant glutathione during ripening [28]. GABA, a product of DAO catabolism was previously shown to increase also during grape ripening for all cultivars described in this work [29]. This is in agreement with hereby reported increases in DAO and PAO gene expression in addition to their enzymatic activity. In the present study it has been observed that as grapes reach maturity there is a general reduction in the concentration of Putrescine what may imply an increase in the concentration of the precursor Arginine. In fact, Ali et al. [29] showed increased arginine synthesis during ripening in agreement with decreased PA levels.

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Furthermore, the levels of PAs and Proline could be interrelated because they share certain common intermediates in their biosynthetic and catabolic pathways [59]. Oxidative deamination of Putrescine generates a pyrroline that can act as a substrate for Proline synthesis after further metabolic processing [53]. Proline also increases in ripe berries [29] as it is known to increase in response to various abiotic stresses [60]. In addition, PA catabolism is closely related to Proline accumulation in response to salt accumulation [59]. Metabolite profiling by using NMR from transgenic tomato lines transformed with yeast SAM decarboxylase showed an accumulation in Spermidine and Spermine and revealed that these higher PAs influence multiple cell pathways in tomato fruit ripening [61]. Levels of Choline, Glu, Gln, Asn, Citrate, Malate and Fumarate increased and those of Asp, Thr, Val, glucose and sucrose decreased in the transgenics compared to the wild type and azygous control lines [61]. In our case the decrease in PAs observed during ripening may contribute to the previously detected decrease in organic acids and glutamate and increased sucrose and glucose, Valine and Threonine [28,29]. This fact suggests further relevance of higher PAs in the regulation of TCA cycle and nitrogen metabolism. In conclusion the up-regulation of a gene coding for ADC during grape ripening found in this study suggests that the PA production is led by the Arginine and Agmatine pathway. This increase in ADC expression was not accompanied by increased PA levels which suggested diminished synthesis or increased levels of catabolism, since we also observed decreased levels of conjugated PAs. Therefore, the decrease in PA contents may be due to the action of amine oxidases that fastly metabolize those growth regulators. Indeed, this seems to be the case in grape ripening since significant increases in the activity of DAO and PAO were observed. It remains to be elucidated whether intensification of polyamine catabolism during ripening is a feature common to both non-climacteric and climacteric fruits. In fact, up to our knowledge this is the first report mentioning an important role of polyamines’ catabolism in fruit ripening. 4. Methods 4.1. Plant material Berries of Trincadeira, Touriga Nacional and Aragonês cultivars of grapevine (Vitis vinifera L.), were collected between July and September. Three-four biological replicates of each cultivar of 80e 100 berries from 8 to 10 plants were collected from vineyard at Montemor (Alentejo, Portugal) during 2007 season for Trincadeira cultivar and during 2008 season for all cultivars. The sampling of berries was performed based on EeL system developed by Coombe [4] considering EL32 (green grapes), EL35 (berries at véraison), EL36

Fig. 10. (A) Phylogenetic tree representing the relationship of grapevine CuAO sequences with other known plant CuAOs. Genes and accessions numbers used were: VvCuAO1 (V. vinifera CuAO1, GSVIVT00011290001); VvCuAO2 (V. vinifera CuAO2, VIT_00s0225g00090); VvCuAO3 (V. vinifera CuAO3, VIT_00s1682g00010); VvCuAO4 (V. vinifera CuAO4, VIT_02s0025g04560); VvCuAO5 (V. vinifera PAO5, VIT_17s0000g09100); AtCUAO1 (At1g31670, NM_102902); AtCUAO2 (At1g31690, NM_102904); AtCUAO3 (At1g31710, NM_102906); AtCUAO4 (at1g62810, NM_104959); AtCUAO5 (At2g42490, BT000029); AtCUAO6 (At3g43670, NM_114235); AtCUAO7 (At4g12280, NM_117298); AtCUAO8 (At4g12290, NM_117299); AtCUAO9 (At4g14940, NM_117580); PsCuAO (Pisum sativum CuAO, AAA62490); GmCuAO1 (Glycine max CuAO, CAE47488); LcCuAO (Lens culinaris CuAO, AAB34918) and SlCuAO (Solanum lycopersicum CuAO, CAI39243). (B) Phylogenetic tree representing the relationship of grapevine PAO sequences with other known plant PAOs. Genes and accessions

numbers used were: VvPAO1 (V. vinifera PAO1, VIT_01s0127g00750); VvPAO2 (V. vinifera PAO2, VIT_01s0127g00800); VvPAO3 (V. vinifera PAO3, VIT_03s0017g01000); VvPAO4 (V. vinifera PAO4, VIT_04s0043g00220); VvPAO5 (V. vinifera PAO5, VIT_12s0028g01120); VvPAO6 (V. vinifera PAO6, VIT_12s0055g00480); VvPAO7 (V. vinifera PAO7, VIT_13s0019g04820); AtCuAO1 (At5g13700, NM_121373); AtCuAO2 (At2g43020, NM_129863); AtCuAO3 (At3g59050, AY143905); AtCuAO4 (At1g65840, AF364953); AtCuAO5 (At4g29720, AK118203); OsPAO1 (Os01g0710200, NM_001050573); OsPAO2 (Os03g0193400, NM_001055782) OsPAO3 (Os04g0623300, NM_001060458); OsPAO4 (Os04g0671200, NM_001060753); OsPAO5 (Os04g0671300, NM_001060754); OsPAO6 (Os09g0368200, NM_001069545); OsPAO7 (Os09g0368500, NM_001069546); BjPAO (Brassica juncea PAO, AY188087); HvPAO1 (Hordeum vulgare PAO1, AJ298131); HvPAO2 (Hordeum vulgare PAO2, AJ298132); MdPAO (Malus domestica PAO, AB250234); NtPAO (Nicotiana tabacum PAO, AB200262); ZmPAO1 (Zea mays PAO, NM_001111636). The Neighbor-joining analysis and the consensus tree were performed with 1000 bootstrap replicates. Bootstrap values are indicated.

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Fig. 11. Alignment of amino acids sequences of CuAO (A) and PAO (B). Multialignment was accomplished by using the program ClustalW sequence alignment. Numbering of amino acid residues is shown on the right. Putative peroxisomal targeting signals type 1 (PTS1) is located at the C-terminal, and PTS1s are highlighted in green with yellow letters. CRT motif is also highlighted in clear green with black letters; this motif has been suggested as a possible functional motif of PTS1 [35]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(ripe berries) and EL38 (harvest stage berries). Berries sampled at each time point were immediately frozen in liquid nitrogen, and stored at 80  C until further analysis. It should be emphasized that the experiments were conducted on healthy grapes presenting no visible signs of fungal attack or other microbiological alteration.

4.2. RNA extraction Grapes were grinded in liquid nitrogen, seeds removed, and then RNA extraction was carried out according to Fortes et al. [28]. A DNAse treatment was performed according to suppliers’ instructions (Invitrogen, San Diego, CA, USA). RNA was further purified using RNeasy Plant Mini kit according to the manufacturer’s instructions (Quiagen, Valencia, CA, USA).

4.3. Target preparation and hybridization of oligo arrays RNA quality was checked using the Agilent 2100 Bioanalyzer (Agilent technologies, Palo Alto, CA). cDNA was synthesized from 4 mg of total RNA using One-cycle target labeling and control reagents (Affymetrix, Santa Clara, CA) to produce biotin labeled cRNA which was then fragmented at 94  C for 35 min into 35e200 bases in length. Three biological replicates corresponding to each stage of growth were independently hybridized to the GrapeGen 520510F array (Affymetrix, Santa Clara, CA). Each sample was added to a hybridization solution containing 100 mM 2-(N-morpholino) ethanesulfonic acid, 1 M NaCl, and 20 mM of EDTA in the presence of 0.01% of Tween-20 to a final cRNA concentration of 0.05 mg mL1. Hybridization was performed for 16 h at 45  C. Each microarray was washed and stained with streptavidin-phycoerythrin in a Fluidics station 450 (Affymetrix) and scanned at 1.56 mm resolution in a GeneChipÒ Scanner 3000 7G System (Affymetrix).

4.4. Data and sequences analysis and gene annotation Robust Muti-array Analysis (RMA) algorithm was used for background correction, normalization and expression levels summarization [62]. Next, differential expression analysis was performed with the Bayes t-statistics from the linear models for Microarray data (Limma), included in the affylmGUI package. P-values were corrected for multiple-testing using the Benjaminie Hochberg’s method (Benjamini and Hochberg, 1995). Data obtained from hybridization of GrapeGen chips was filtered considering a fold change 1.5 and corrected P-value <0.05 or fold change  1.5 and a corrected P-value <0.05. The microarray data were submitted to Gene Expression Omnibus (NCBI) and are accessible through GEO accessions numbers GSE28779 and GSE35172. The probesets sequences were blasted against the genes predicted from the genome (blastn, e-value < e20, minimum of 100 pb alignment) available at the NCBI website. Gene annotation was performed by updating the annotation performed in Grimplet et al. [63] following the same protocol as described by the authors to the new genes from the 12X coverage release of the genome assembly. 4.5. Real-time semi quantitative PCR (RT-qPCR) Complementary DNA was synthesized using an RevertAidTM H Minus M-MuLV Reverse Transcriptase (Fermentas, Burlington, Canada) according to the manufacturer’s instructions were using 1.5 mg RNA per reaction. Primers were selected using Primer express software 3.0 (Applied Biosystems, Forster City, CA). Real-time qPCR reactions were prepared using MaximaTM SYBR Green qPCR Master Mix (2X) (Fermentas, Burlington, Canada) and performed using the StepOneTM Real-Time PCR System (Applied Biosystems, Foster City, CA). Expression was determined for duplicate biological replicates and triplicate technical replicates using a serial dilution

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cDNA standard curve per gene. The standard curve was drawn from four points corresponding to serial dilutions of mixture of all RNAs analyzed. Real-time PCR was performed with StepOneTM RealTime PCR System (Applied Biosystems, Foster City, CA) under conditions of 95  C for 10 min, then 40 cycles of 95  C for 15 s and 60  C for 45 s and analyzed with StepOne software 2.1. Data were calculated from the calibration curve and normalized using the expression curve of Actin gene (VVTU17999_s_at). All primers used for this assays are shown in supplemental data, Supplementary Table S1.

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(0.3 mL total volume) was incubated at 37  C for 1 h and was stopped by adding 0.2 mL of saturated sodium carbonate. Labeled D1-pyrroline was extracted immediately in 1 mL toluene. The 0.5 mL aliquots were placed in scintillation liquid (0.5% [w/v] 2,5-diphenyloxazole and 0.05% [w/v] 1,4-bis[5-phenyloxazoyl] benzene in toluene) and counted in an Tri-carb 2100TR Liquid Scintillation Analyzer (Packard BioScience, Boston, USA). Protein content of the supernatant was determined according to Bradford’s method. 4.9. ROS staining

4.6. Polyamine extraction Samples were grinded in liquid nitrogen, seeds removed and lyophilized at 40  C. One hundred mg of dried tissue were extracted in 1 mL of 5% perchloric acid and the extracts were centrifuged at 13,000 rpm for 30 min at 4  C. Supernatant fraction was used for the determination of free (S) and soluble conjugated (SH) PAs. The pellet was used for the determination of insoluble bound PAs (PH). It was washed in 1 mL of perchloric acid, centrifuged for 1 min at 13,000 rpm and resuspended in the original volume with NaOH 1 M and left overnight at 4  C. The pellet suspension and the original supernatant (0.1 mL each) were hydrolyzed over night at room temperature with 0.1 mL of 37% HCl. The hydrolysate was centrifuged and was dried under vacuum at 60  C, then dissolved in 0.1 mL perchloric acid. 4.7. Dansylation of polyamines and HPLC analysis The extracts were dansylated as described by Marcé et al. [64]. An aliquot (0.1 mL) of the extract was added to 0.1 mL saturated sodium carbonate and 0.2 mL dansyl chloride in acetone (0.5 g mL1). The mixture was incubated overnight at room temperature in the dark. Dansylated PAs were extracted with 0.25 mL toluene by vortexing for 30 s, and a 0.2 mL aliquot of toluene was dried in speed vac. The derivatives were analyzed by HPLC (PU-980 Jasco, Tokyo, Japan) on a reverse phase C18 column (Spherisorb ODS2, 5-mm particle size, 4.6  250 mm) using a programmed acetonitrile: water step gradient that allowed PA separation in 22 min. Eluted peaks were detected by a spectrofluorometer (821FP Jasco, excitation 345 nm, emission 455 nm), and their areas were recorded and integrated relative to those of internal standard PAs using the ChromNAV software package (Jasco-Borwin). Polyamine content was expressed in units per fresh weight. In order to estimate the free total PAs, Putrescine, Spermidine and Spermine contents were summed whereas the estimation of conjugated PAs was carried out by subtracting to soluble PA content the free polyamines content. 4.8. Enzyme assays CuAO (EC 1.4.3.6) and PAO (EC 1.4.3.4) activities were assayed by a radiometric method measuring the [14C]pyrroline formed from [14C]Putrescine and [14C]Spermine, respectively; for Trincadeira, Touriga Nacional and Aragonês cultivars from 2008 season during ripening (EL 32, EL 35, EL 36 and EL 38 stages). Grapes were grinded in liquid nitrogen and 0.3 g of fresh weight were homogenized in 3 volumes of 100 mM potassium phosphate (CuAO: pH 7 and PAO: pH 8), 2% (w/v) polyvinylpyrrolidone, 5 mM 1,4-dithiothreitol buffer. The homogenate was centrifuged at 10,000 rpm for 30 min at 4  C and was collected 0.15 mL of supernatant to which was added 0.15 mL of 100 mM potassium phosphate, 2 mL [1-414C] Putrescine or [14C]Spermine (specific activity 3.7 GBq mmole1, American Radiolabeled Chemicals Inc, St Louis, USA), 10 mM unlabeled Putrescine or Spermine, and 30 mg catalase. The reaction

Hydrogen peroxide was detected by incubation in DAB-HCl solution (1 mg/mL) (pH 3.8) (Sigma, MO; # D-8001) for 30 min in the dark, after which the material was transferred to ethanol 96% (v/v) and maintained at 4  C until observation. 4.10. Statistical analysis The statistical analysis of data was performed with one-way ANOVA and using SPSS 15.0 software to determine whether statistically significant differences occurred among groups. Principal component analysis (PCA) was performed using MultiExperiment Viewer (version 4.6) software. 4.11. In silico analysis The sequences of five putative CuAO isoforms and seven putative PAO isoforms annotated in Genoscope database (Vitis genome browser) of Vitaceae family have been analyzed [http://www. genoscope.cns.fr/spip/Vitis-vinifera-e.html]. The PAOs and CuAOs of grape were termed as VvPAO (1e7) and VvCuAO (1e5) (Supplementary Table S2). Isoforms were numbered consecutively depending on the chromosome number on which the gene is located. Genes on the same chromosome were number in descending order by their assigned accession number. For the other plant sequences of CuAOs and PAOs analyzed, it was used the nomenclature already reported. Up to our knowledge, there is no uniform short nomenclature to CuAO genes from Arabidopsis; therefore, isoforms were numbered as it was already explained above (AtCuAO) (Table S2). Nine of twelve AtCuAO genes annotated in the TAIR (www.arabidopsis.org/) have been analyzed. Multiple sequence alignment of the amino acid sequences was performed with ClustalW using default parameters with the aid of molecular evolutionary genetic analysis (MEGA) software version 5.0 [65]. Based on the alignment of amino acid sequences it was performed the Neighbor-joining analysis and the consensus tree with 1000 bootstrap replicates. Trees were represented with the help of TreeView [66]. To seek for candidate sequences of signal peptide, domains and motifs were used the following bioinformatic tools: eukaryotic linear motif (ELM) resource for functional sites in proteins (http:// elm.eu.org/) and simple modular architecture research tool (SMART) (http://smart.embl-heidelberg.de/). Signal peptide was confirmed using SignalP 4.0 Server (http://www.cbs.dtu.dk/ services/SignalP/) and for prediction of the subcellular localization TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/) was used. Acknowledgments Funding was provided by the Portuguese Foundation for Science and Technology (FCT) through a Post-doctoral fellowship to Patricia Agudelo-Romero (SFRH/BPD/72070/2010) and Auxiliar Researcher contract Ciência 2008 to Ana M. Fortes (C2008-UL-BioFIG-5).

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Additional funding was provided by the FCT project PTDC/ AGR-GPL/100919/2008 and COST Action FA0605 (STSM-FA0605131210-003763). AFT acknowledges funding from MEC-BIO201129683. This work was developed within the BioFig Center with reference PEst-OE/BIA/UI4046/2011. We would like to thank Dr Rachel Webster (University of Manchester, UK) for revising the English.

[27]

[28]

[29]

Appendix A. Supplementary material [30]

Supplementary material related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2013.02.024.

[31]

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