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Publications (7)16.69 Total impact

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    ABSTRACT: Puroindolines are endosperm lipid binding proteins, which are separated by reversed phase-high-performance liquid chromatography or cation exchange chromatography into two isoforms, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Being very basic and close in molecular weight, PIN-a and PIN-b have never been separated using conventional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A two-dimensional electrophoresis method, linear immobiline pH gradient (IPGxSDS-PAGE), was developed, using 6-11 linear immobiline Dry Strips in the first dimension, which allowed the puroindolines to be focused between isoelectric point 10.5 and 11. Immunoblotting revealed that both PIN-a and PIN-b were each composed of several spots. Two-dimensional patterns from unrelated wheat varieties revealed that several spots can be highlighted among varieties. Matrix-assisted laser desorption/ionization-time of flight spectrometry allowed the majority of the spots revealed in the puroindoline zone to be identified. The two-dimensional IPGxSDS-PAGE of these very basic wheat endosperm proteins, puroindolines and related grain softness proteins should facilitate the identification of the proteins associated with wheat endosperm texture that have a strong effect on milling, dough properties and end-uses of wheats.
    PROTEOMICS 03/2003; 3(2):168-74. · 4.13 Impact Factor
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    ABSTRACT: Seeds from near-isogenic hard and soft wheat lines were harvested at regular intervals from 5 days post-anthesis to maturity and examined for hardness using the single kernel characterisation system (SKCS). SKCS analysis revealed that hard and soft lines could be distinguished from 15 days post-anthesis (dpa). This trend continued until maturity where the difference between the hard and soft lines was most marked. SKCS could not be applied to the small 5- and 10-dpa wheat kernels. Fresh developing endosperm material was examined using light microscopy and no visible differences between the cultivars were detected. When air-dried material was examined using scanning electron microscopy (SEM) differences between soft and hard lines were visible from as early as 5 dpa. Accumulation of puroindoline a and puroindoline b was investigated in developing seeds using both Western blotting and ELISA. Low levels of puroindoline a could be detected in the soft cultivar from 10 dpa, reaching a maximum at 32 dpa. In the hard cultivar, puroindoline a levels were negligible throughout grain development. Puroindoline b accumulates in both the soft and hard cultivars from 15 dpa, but overall contents were higher in the soft cultivar. These findings indicate that endosperm hardness is expressed very early in developing grain when few starch granules and storage proteins were deposited in the endosperm cells. Further, the near-isogenic soft and hard Heron lines could be differentiated by SEM at a stage in development when the accumulation of puroindolines could not be detected by the methods used in this study.
    Planta 03/2003; 216(4):699-706. · 3.38 Impact Factor
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    ABSTRACT: Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.
    Theoretical and Applied Genetics 01/2002; 106(1):19-27. · 3.66 Impact Factor
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    ABSTRACT: Grain hardness (GHa) in hexaploid wheat (Triticum aestivum L.) is a major factor of end use quality. The variation of texture has been related to the Hardness locus, closely linked with the puroindoline-a (PIN-a) and puroindoline-b (PIN-b) genes. In order to study the role of puroindoline content in texture variation, the quantity of puroindolines was determined. Puroindoline-a (PIN-a) and puroindoline-b (PIN-b) content was determined and a total of 11 bread making parameters were obtained from 40 bread wheat cultivars grown in four experimental locations. The 11 parameters were significantly influenced by the genotype whereas location did not significantly affect PIN-a or PIN-b content and loaf volume. PIN-b and grain hardness displayed the highest heritability coefficients (both 0.88). PIN-a and PIN-b content were not correlated with grain protein content (Prot) and grain hardness in hard and soft wheat types. In soft samples PIN-(a+b) content was negatively correlated, with loaf volume in two locations. Multiple regression analyses, carried separately for soft and hard types, revealed that PIN-b content explained variation of dough strength (W) and loaf volume, however their influence was mostly significant in soft types. For each location, from 22 to 91% of the phenotypic variation of strength and loaf volume was explained by combining up to three flour traits. Protein content, PIN-b and the average score of high molecular weight glutenin subunits (HMW-GS) were frequently introduced by multiple regression (without GHa) as explanatory variables of strength and loaf volume. These results strengthened the significant role of PIN-b in breadmaking (loaf volume), and indicated that biochemical factors other than puroindolines are involved in the grain hardness variation.
    Journal of Cereal Science. 01/2001;
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    ABSTRACT: The sequence of the puroindoline-b gene from 15 Australian wheat cultivars was determined. Sequence variation was observed in the WPTKWWKGGCE motif of the deduced puroindoline-b protein sequence. Previously, it has been suggested that this sequence is crucial in determining grain hardness. In this study, no correlation was found between the variation in this sequence and the hardness or softness of the cultivar. The amounts of puroindoline-a and puroindoline-b protein in a selection of hard and soft Australian wheat cultivars were also determined using ELISA techniques. Both soft and hard cultivars had variable amounts of puroindoline-a and puroindoline-b. In particular, it is notable that the hard cultivars Cook and Diaz contained high amounts of puroindoline-a and puroindoline-b and also contained the puroindoline-b sequence previously reported to be associated with grain softness. These results suggest that if the puroindoline proteins are involved in determining grain softness or hardness they do so as part of a multi-component mechanism.
    Functional Plant Biology 01/2000; 27:153-158. · 2.47 Impact Factor
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    ABSTRACT: In wheat endosperm, the main isoforms of puroindolines (PIN-a and PIN-b) and nonspecific lipid transfer protein (ns-LTP1e1), structurally related lipid binding proteins, were asynchronously synthesized during maturation and are partially degraded during germination. These proteins are not detected in roots and hypocotyls of seedlings, while ns-LTP1e1, but not PINs, was synthesized during germination in the scutellum and/or mesocotyl. In mature wheat seeds, ns-LTP1-e1 was specifically localised within aleurone cells but not in cell walls in marked contrast with most other plant ns-LTP1s. PINs are both located in the starchy endosperm and in the aleurone layer. In the latter cells, PINs and ns-LTP1-e1 were both localised in small inclusions within protein-rich aleurone grains. In the mature starchy endosperm, PINs were localised in the protein matrix and at the interface between starch granules and protein matrix. It was shown that both PIN-a and PIN-b, have antifungal properties in vitro and a synergistic enhancement of the antifungal properties of α-purothionins (α-PTH) was observed in the presence of PINs. This synergism could have biological significance since α-PTH and PINs are both located in the protein matrix of starchy endosperm. ns-LTP1e1 is not capable to inhibit growth of fungi and a synergy rather weak in comparison with PINs was also observed between ns-LTP1e1 and α-PTH.
    Plant Science. 01/1998; 138(2):121-135.
  • L. Quillien, T. Gaborit, J. Gueguen
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    ABSTRACT: Antibodies can be used as probes to investigate the structure of 11S storage proteins, their subunit composition and structural modifications induced by technological treatments. Monoclonal antibodies have been raised against Pisum sativum legumin (11S storage protein). Their binding characteristics were examined by direct, sandwich and competitive ELISA and by immunoblotting against legumin and 11S type storage proteins from other species. One of the MAbs, reacting with all 11S proteins tested, recognizes a discontinuous epitope accessible on the surface of the native hexameric protein but destroyed in dissociated legumin. Two antibodies recognize sequential epitopes belonging to a region of the acidic polypeptides present on the surface of the native legumin. These two MAbs cross-react only with pea and bean 11S proteins. Two other MAbs are specific for sequential epitopes buried in the native protein, localized, respectively, on acidic and basic polypeptides. The MAb reacting with the acidic polypeptide exhibits very specific binding for pea legumin. By contrast, the MAb specific to the basic polypeptide cross reacts with 11S proteins studied. This work shows the potential of this approach for the characterization of the conformation of the 11S proteins and the investigation of structural modifications.
    Phytochemistry 07/1995; · 3.05 Impact Factor