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Guadalupe Cumplido-Laso,
Laura Medina-Puche, Enriqueta Moyano,
Thomas Hoffmann,
Quirin Sinz,
Ludwig Ring,
Claudia Studart-Wittkowski,
José Luis Caballero,
Wilfried Schwab,
Juan Muñoz-Blanco,
Rosario Blanco-Portales
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ABSTRACT: Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.
Journal of Experimental Botany 05/2012; 63(11):4275-90. · 5.36 Impact Factor
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ABSTRACT: The flavor of strawberry (Fragaria x ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of approximately 37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria x ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.
The Plant Cell 05/2006; 18(4):1023-37. · 8.99 Impact Factor
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ABSTRACT: A strawberry (Fragaria x ananassa cv. Chandler) fruit cDNA (Fahyprp -cDNA) and its corresponding gene (Fahyprp) showing sequence homology to higher plant hyprp genes have been isolated. The cDNA contains an open reading frame encoding a 16 kDa protein with 156 amino acids. The peptide has an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxy-terminal region homologous to other HyPRP proteins. Northern blot and QRT-PCR analysis have shown that the strawberry transcript is specifically expressed in fruit, not being detected in other plant tissues. " In situ " hybridization and immunolocalization studies have indicated that the Fahyprp gene is strongly expressed in achene sclerenchyma cells, in the vascular and receptacle cells of immature green fruit and in the vascular cells of mature red fruits. The achenes removal from unripe green fruits induced the expression of this Fahyprp gene. This induction was reverted by treatment of deachened fruit with the auxin NAA, supporting the idea that Fahyprp gene expression is regulated by auxins. Furthermore, the HyPRP protein has been localized in parenchymatic cells of immature fruits associated to structures containing condensed tannins. The results are discussed supporting a putative role of this protein in the anchoring of polymeric polyphenols in the strawberry fruit during growth and ripening.
Plant Molecular Biology 09/2004; 55(6):763-80. · 4.15 Impact Factor
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ABSTRACT: cDNA and genomic clones encoding a strawberry (Fragariaxananassa cv. Chandler) non-specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio-temporal expression pattern and structural features of this gene were studied for the first time in strawberry, a non-climacteric fruit of agricultural importance. The architecture and the encoded amino acid sequence of this non-climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an N-terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using the PLACE database predicted the presence of several putative cis-regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5'-flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.
Journal of Experimental Botany 09/2003; 54(389):1865-77. · 5.36 Impact Factor
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ABSTRACT: Two genomic clones corresponding to putative pectate lyase genes (plA and plB) were isolated and characterized in strawberry (Fragaria x ananassa cv. Chandler). The corresponding ORFs for the plA and plB genes revealed deduced proteins of 451 and 439 amino acids, respectively, that differ from that of the previously isolated strawberry plC gene. Southern blot analysis has shown that while the plB gene is a single copy gene, the plA gene is probably encoded by a small multigene family. By using specific probes corresponding to the untranslated 3' terminal region of the pl genes, and QRT-PCR methodology, the spatio-temporal expression pattern of both strawberry pl genes have been compared with that of the plC gene. The three transcripts were specifically expressed only in fruit and mainly during the ripening stages. Moreover, the expression of the plA and plB genes was induced in green de-achened fruit, but this increase was reduced by the external application of auxins as was the expression of plC. The expression of both pl genes was also strongly reduced in harvested fruit kept in controlled atmosphere (CA) containing high CO(2) levels. Immunolocalization studies using antibodies raised against the strawberry PL proteins placed the proteins in the cell wall of parenchymatic cells of the fruit receptacle. The role of pl genes in cell-wall disassembly and fruit ripening softening is discussed.
Journal of Experimental Botany 03/2003; 54(383):633-45. · 5.36 Impact Factor
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ABSTRACT: The intracellular localization of the activity and synthesis of three isozymes of NAD(P)(+)-glutamate dehydrogenase from the unicellular green alga Chlamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.
Plant physiology 12/1992; 100(3):1575-9. · 6.53 Impact Factor
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ABSTRACT: Two strawberry cDNAs showing sequence similarity to pyruvate decarboxylase (PDC) genes (Fapdc1 and Fapdc3 genes) from higher plants have been isolated by a differential display-PCR approach (DDRT-PCR). Sequence comparisons, northern and RT-PCR analysis showed that these strawberry genes are different and are expressed in a different expression pattern in vegetative tissues and in fruits during fruit development and ripening processes. RT-PCR studies indicated that only Fapdc1 gene is induced during fruit ripening whereas Fapdc3 gene is constitutively present in all tissues studied. The removal of the achenes from unripe green fruits induced the expression of Fapdc1 gene and this induction was inhibited by treatment of de-achened fruits with naphthaleneacetic acid (NAA) and indoleacetic acid (IAA), thus indicating that only the expression of Fapdc1 gene is under hormonal control. Also, Fapdc1 gene expression was increased in anoxia conditions in cultured cells. However, Fapdc3 is accumulated to high levels in strawberry cultured cells, irrespective of oxygen availability and probably controlled by sugar supply. The expression pattern of both genes in response to stress and post-harvest treatments is evaluated. All these results suggest that the strawberry Fapdc1 gene could play an important role in fruit ripening and aroma biogenesis, and also under stress conditions, whereas Fapdc3 would be involved in general metabolism to support energy production and biosynthesis of higher molecular weight compounds. The Fapdc1 genomic region has been cloned and characterised to isolate an inducible fruit ripening-related promoter for biotechnological purposes in strawberry.
Plant Science.
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ABSTRACT: Two genomic clones corresponding to putative pectate lyase genes ( plA and plB ) were isolated and characterized in strawberry ( Fragaria × ananassa cv. Chandler). The corresponding ORFs for the plA and plB genes revealed deduced proteins of 451 and 439 amino acids, respectively, that differ from that of the previously isolated strawberry plC gene. Southern blot analysis has shown that while the plB gene is a single copy gene, the plA gene is probably encoded by a small multigene family. By using specific probes corresponding to the untranslated 3′ terminal region of the pl genes, and QRT‐PCR methodology, the spatio‐temporal expression pattern of both strawberry pl genes have been compared with that of the plC gene. The three transcripts were specifically expressed only in fruit and mainly during the ripening stages. Moreover, the expression of the plA and plB genes was induced in green de‐achened fruit, but this increase was reduced by the external application of auxins as was the expression of plC . The expression of both pl genes was also strongly reduced in harvested fruit kept in controlled atmosphere (CA) containing high CO 2 levels. Immunolocalization studies using antibodies raised against the strawberry PL proteins placed the proteins in the cell wall of parenchymatic cells of the fruit receptacle. The role of pl genes in cell‐wall disassembly and fruit ripening softening is discussed.
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I Congreso de la Cultura del Olivo, 2007-01-01, ISBN 978-84-96047-57-0, pags. 663-672.
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ABSTRACT: Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800–1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266 000–269 000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60–62°C) and isoelectric points (7.9–8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5′-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentra tion-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13–53 mM; 0.36–1.85 mM for 2-oxoglutarate and 0.07–0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64–3.52 mM for l-glutamate and 0.20–0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperatively (n = 0.8).
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology.
