Cândido Pinto Ricardo

Invertase activity during the development of carrot roots

Abstract Evidence was obtained that roots of carrot ( Daucus carota L.) contain an acid invertase (optimum pH 4·5) and an alkaline invertase (optimum pH 7·4). The acid and alkaline invertases were partially purified and K mm for sucrose found to be 5·6 × 10 −2 M and 2·4 × 10 −2 M respectively. The acid invertase was distributed between the cell wall and supernatant fractions of root homogenates. This distribution was readily and reversibly altered by varying the pH of the extraction medium. Almost all of the alkaline invertase activity was found in the supernatant fraction of the homogenates. Sugar content and the activities of both invertases were shown to vary widely during the development of carrot roots. High acid invertase activity was found in rapidly growing tissue in which the sucrose content was low or was declining rapidly. With the exception of young seedlings, alkaline invertase activity was most marked in tissues that were storing sucrose. Possible roles for the two invertases in the control of sucrose metabolism are proposed.

Lupinus albus L. Pathogenesis-Related Proteins That Show Similarity to PR-10 Proteins

We describe a group of three acidic proteins, pathogenesis-related (PR)-p16.5a, PR-p16.5b, and PR-p16.5c, that accumulate in the leaves of Lupinus albus L. cv Rio Maior plants when infected with the fungus Colletotrichum gloeosporioides Penz. These proteins co-migrate in sodium dodecyl sulfate-polyacrylamide gels as a single band of 16.5 kD, behaving as charge isomers, and are related to several members of the defense-related PR-10 protein family. Localization of the proteins was investigated by techniques of tissue printing and immunogold electron microscopy; they are predominantly associated with the vascular system and are localized extra-cellularly. The accumulation of PR-p16.5a, PR-p16.5b, and PR-p16.5c also seems to be induced by cucumber mosaic virus and by two forms of abiotic stress, salicylic acid and ultraviolet, suggesting a general defense role for these proteins.

Reference Gene Selection for Quantitative Real-time PCR Normalization in Quercus suber

The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

Development of Tuberous Roots and Sugar Accumulation as Related to Invertase Activity and Mineral Nutrition

SummarySucrose storage in tuberous roots was not observed when the tissues had very high activities of acid invertase. High activities of the enzyme were always present in the roots at early stages of their development. In species where the activity of the enzyme decreased during root development, sucrose was stored. Thus, acid invertase was undetectable in mature roots of carrots (Daucus carota L.) where sucrose formed almost 80% of the dry matter. Conversely, radish (Raphanus sativus L.) and turnip (Brassica rapa L.) roots, in which the activity of the enzyme remained high until maturity, did not store appreciable amounts of sucrose (2% and 9%, respectively, of the dry matter in the mature roots), reducing sugars being the main reserve (more than 80% of the dry matter in mature turnips). The correlation between sucrose content and acid invertase activity was furthermore evident in both sucrose- and hexose-storing roots when the activity of this enzyme was affected by changes in the mineral nutrition. Deficiencies of nitrogen and sulphur reduced the activity of acid and alkaline invertases and led to increase in sucrose content and decrease in reducing sugars. However, the decline of alkaline invertase activity in tissues low in acid invertase had no clear effect on sugar content. Sodium chloride (10−1M) affected acid invertase and sugars in a manner similar to that of the two deficiencies, but had practically no effect on alkaline invertase. The changes in sugar content produced by the variations in mineral nutrition were small in hexose-storing roots in relation to those of sucrose-storing roots. It is possible that this result is related to the different levels of acid invertase in the two types of roots.

Analysis of carbohydrates in Lupinus albus stems on imposition of water deficit, using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and application of a negative ion mode online LC-ESI-MS method for studying the effect of water deficit on the carbohydrate content of Lupinus albus stems, using a porous graphitic carbon (PGC) stationary phase and an ion trap mass spectrometer. Using this method, separation and detection of several water soluble carbohydrates, ranging from mono-, di-, and oligosaccharides (raffinose, stachyose, and verbascose) to sugar alcohols was achieved in approximately 10 min. This on-line PGC-LC-ESI-MS method shows good linearity with correlation coefficients R(2)>0.99, selectivity, short analysis time, and limits of detection (LOD) ranging from 0.4 to 9 pmol for sugars and 4-20 pmol for sugar alcohols. This PGC-LC-ESI-MS method is sensitive and allowed us to detect even small alterations in carbohydrate levels in L. albus stems that resulted from a mild/early water deficit (nmol g(-1)DW). This paper describes details of our method and its application to the quantitative analysis of water soluble underivatised carbohydrates extracted from L. albus stem tissues that have been subjected to early and severe water deficit conditions, followed by a rewatering period.

A proteomic study to identify soya allergens--the human response to transgenic versus non-transgenic soya samples.

Background: In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya® versus its non-transgenic control. Methods: We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples. Results: We identified 2 new potential soybean allergens – one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study. Conclusion: Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment.

Study of the intercellular fluid of healthy Lupinus albus organs. Presence of a chitinase and a thaumatin-like protein.

Proteins in the intercellular fluid (IF) of healthy Lupinus albus leaves were characterized. Silver staining of the proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed more than 30 polypeptides, with the major ones having a molecular mass lower than 36 kD. After amino-terminal amino acid sequence analysis, one of the major polypeptides, IF4, was shown to have no identity with any of the proteins present in the data bases. Two others, IF1 and IF3, showed identity with previously reported pathogenesis-related proteins, IF1 with an antifungal protein from Hordeum vulgare that belongs to the thaumatin family (PR-5 family), and IF3 with class III chitinase-lysozymes. IF3 was also present in the IF of stem and root and it represents the major polypeptide in the medium of L. albus cell-suspension cultures. The ubiquitous presence of this enzyme in healthy, nonstressed tissues of L. albus cannot be explained.

A Biochemical and Molecular Characterization of LEP1, an Extensin Peroxidase from Lupin

An analysis of apoplastic extensin cross-linking activity in vegetative organs of Lupinus albus indicated that leaves contained the highest specific activity. Assays of peroxidases fractionated from this material demonstrated that this activity could be largely attributed to a soluble and apoplastic 51-kDa peroxidase, denoted LEP1. Relative to other purified peroxidases, LEP1 demonstrates high extensin cross-linking activity and can be classified as an extensin peroxidase (EP). Optimal conditions for the in vitro oxidation of other phenolic substrates included 1.5–3.0 mm peroxide at pH 5.0. EP activity of LEP1 was low under these conditions but optimal and substantially higher with 100 μm peroxide and neutral pH, suggesting that physiological changes in pH and peroxide in muro could heavily influence the extensin cross-linking activity of LEP1 in vivo. Analysis of LEP1 glycans indicated 11–12 N-linked glycans, predominantly the heptasaccharide Man3XylFucGlcNAc2, but also larger structures showing substantial heterogeneity. Comparative assays with horseradish peroxidase isoform C and peanut peroxidases suggested the high level of glycosylation in LEP1 may be responsible for the high solubility of this EP in the apoplastic space. A full-length cDNA corresponding to LEP1 was cloned. Quantitative reverse transcriptase-PCR demonstrated LEP1 induction in apical portions of etiolated hypocotyls 30–60 min after exposure to white light, prior to the onset of growth inhibition. Comparative modeling of the translated sequence indicated an unusually unobstructed equatorial cleft across the substrate access channel, which might facilitate interaction with extensin and confer higher EP activity.

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

Abstract. A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein.

Analysis of Lupinus albus leaf apoplastic proteins in response to boron deficiency

Boron is essential for plant development and although its precise functions are not fully understood there is evidence for its importance in the extracellular matrix. So we have analysed by two-dimensional gel electrophoresis the effect of B deficiency in the soluble apoplast proteins of Lupinus albus leaf. Twenty-three polypeptide spots varied significantly between the control and the B deficiency patterns. Of these polypeptides only 9 could be identified by mass spectrometry techniques: PR-1 like protein, β-1,3-glucanases, class III chitinases, thaumatin like proteins and an expansin-like protein, all of them being involved in plant defence mechanisms. Only PR-1 like protein was denovo expressed under B deficiency, while the remaining proteins also responded to water stress. Although general response mechanisms seem to be triggered by both B and water stress, the pattern of protein expression was distinct, suggesting that under B deficiency specific regulatory mechanisms may be induced.