Cesar Valmor Rombaldi

Characteristics of the tomato chromoplast revealed by proteomic analysis

Chromoplasts are non-photosynthetic specialized plastids that are important in ripening tomato fruit (Solanum lycopersicum) since, among other functions, they are the site of accumulation of coloured compounds. Analysis of the proteome of red fruit chromoplasts revealed the presence of 988 proteins corresponding to 802 Arabidopsis unigenes, among which 209 had not been listed so far in plastidial databanks. These data revealed several features of the chromoplast. Proteins of lipid metabolism and trafficking were well represented, including all the proteins of the lipoxygenase pathway required for the synthesis of lipid-derived aroma volatiles. Proteins involved in starch synthesis co-existed with several starch-degrading proteins and starch excess proteins. Chromoplasts lacked proteins of the chlorophyll biosynthesis branch and contained proteins involved in chlorophyll degradation. None of the proteins involved in the thylakoid transport machinery were discovered. Surprisingly, chromoplasts contain the entire set of Calvin cycle proteins including Rubisco, as well as the oxidative pentose phosphate pathway (OxPPP). The present proteomic analysis, combined with available physiological data, provides new insights into the metabolic characteristics of the tomato chromoplast and enriches our knowledge of non-photosynthetic plastids.

Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits

The enzyme which converts 1-aminocyclo-propane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses☆

The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.

Carotenoid Biosynthetic and Catabolic Pathways: Gene Expression and Carotenoid Content in Grains of Maize Landraces

Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 μg·g−1, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase) expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase) expression levels were positively correlated with β-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase) expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase) was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which would thus better enable us to select promising varieties for biofortification efforts.

Produtividade e qualidade de uva, cv. Isabel, em dois sistemas de produção

Estudou-se a produtividade e a qualidade da uva, cv. Isabel, produzida em dois sistemas de producao, convencional e alternativo. O sistema convencional corresponde aquele empregado pela maioria dos produtores dessa cultivar, que consiste no controle de plantas concorrentes com o emprego de capinas e/ou herbicidas e no controle de doencas com fungicidas sinteticos orgânicos e nao orgânicos. O sistema alternativo corresponde aquele onde se manteve a cobertura do solo e se inseriu a aveia como complemento, e o controle de doencas foi feito apenas com o emprego de calda bordalesa. Essas praticas foram realizadas desde 1998 e, nas safras 2001-2002 e 2002-2003, avaliaram-se a produtividade e a qualidade da uva produzida nos dois sistemas. Pelos resultados, observou-se que a produtividade e a qualidade da uva sao mais afetadas pelas condicoes climaticas de safra do que pelo sistema de producao, indicando que o sistema de producao alternativo, onde se empregou menor numero de pulverizacoes, nao se usou herbicida, nem fungicida orgânico sintetico, tem elevado potencial de adocao para essa cultivar.

Ácido giberélico no retardamento da maturação de caquis (Diospyrus kaki, L.), cultivar Fuyu

Objetivou-se estudar o efeito da aplicacao de Acido Giberelico (AG3) no retardamento da maturacao de caquis da cultivar Fuyu. Adotou-se o delineamento inteiramente casualizado, com quatro repeticoes. O tratamento foi realizado por pulverizacao das plantas com 30ppm de AG3. Como tratamento controle pulverizou-se agua. A partir da instalacao do experimento, na colheita das frutas e a periodos de 7 dias coletaram-se amostras para a avaliacao da firmeza de polpa, acidez total titulavel, do conteudo de solidos soluveis, carotenoides, clorofilas e fenois, da coloracao e da producao de etileno. Os resultados mostraram que o AG3 agiu retardando a maturacao em aproximadamente 20 dias, retardando a diminuicao da firmeza de polpa, da acidez, do conteudo de clorofilas e fenois, e da producao de etileno. Tambem, agiu retardando o acumulo de carotenoides e a evolucao da coloracao externa.

CONSERVATION OF PERSIMMONS FRUITS (Diospyros kaki, L.), cv. FUYU WITH APLICATION OF 1- METHYLYCLOPROPENE

The present work evaluated the effects of the 1-MCP (1-methylcyclopropene) on persimmons fruits (Diospyrus kaki L.) of cv. Fuyu stored at 0oC for up to 90 days. Fruits were picked from a commercial orchard in Farroupilha-RS, when skin color was yellow-orange. Three concentrations of 1-MCP (312, 625 and 1250 nL.L-1) were applied for 24 hours in an ambient temperature (±25oC). Control fruit were kept under identical conditions without 1-MCP treatment. Immediately after treatment application, the fruits were transferred to a cold storage at 0oC and approximately 90% of relative humidity. Persimmons were analysed on the day of harvest, after 30, 60 and 90 days of refrigerated storage plus 3 more days at ambient temperature to simulate the commercialization period. Total soluble solids, pH and titratable acidity were not influenced by 1-MCP treatments. Ethylene production rates did not reach detectable levels. Skin color showed a higher development of red color in fruits treated with 1-MCP. Flesh firmness was significantly higher in treated fruits in comparison to control fruits, indicating a positive effect of 1-MCP in the postharvest life of persimmons fruits. There was no significant difference among the different concentrations of 1-MCP.

Micronutrient and Functional Compounds Biofortification of Maize Grains

Maize, in addition to being the main staple food in many countries, is used in the production of hundreds of products. It is rich in compounds with potential benefits to health, such as carotenoids, phenolic compounds, vitamin E, and minerals that act as cofactors for antioxidant enzymes. Many of these compounds have been neglected thus far in the scientific literature. Nevertheless, deficiencies in the precursors of vitamin A and some minerals, such as iron and zinc, in maize, in association with the great genetic variability in its cultivars and our genomic, transcriptomic, and metabolomic knowledge of this species make targeted biofortification strategies for maize promising. This review discusses the potential of the main microconstituents found in maize with a focus on studies aimed at biofortification.

Importance of heat shock proteins in maize

Abiotic and biotic stress conditions cause extensive losses to maize production, mainly due to protein dysfunction in these conditions. In higher plants, the occurrence of heat-shock proteins (HSPs) in response to different environmental stresses is a universal phenomenon and has been well documented. Many studies have demonstrated that most HSPs are involved in many regulatory pathways, act as molecular chaperones for other cell proteins, and have strong cytoprotective effects. Although many functional roles for HSPs are known, the mechanisms for these multiple functions are not entirely understood. Here we reviewed the correlation among HSP genes/proteins and plant tolerance, especially maize, in different environmental stresses. Due to the low availability of information regarding the expression of HSP genes in response to different stresses in maize, we decided to mine databases in order to generate new insights related to this topic.