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Instituto de Biologia Unicamp






ABSTRACTA IB (2) - 1999


P080-99 Sodium chloride-induced leaf senescence in Hydrocotyle bonariensis Lam. and Foeniculum vulgare L.

Haddad CRB* , Mazzafera P

Leaf senescence induced by sodium chloride was studied in Hydrocotyle bonariensis Lam. and Foeniculum vulgare L. Both species belong to Umbelliferae family, however only H. bonariensis grows spontaneously in sandy soils of coastal regions (saline soils). Leaves of plants receiving nutrient solutions containing different concentrations of NaCl were evaluated for fresh and dry weights and chlorophyll content. The denaturating electrophoretic profiles of leaf proteins were also studied. Sodium chloride changed the protein profile of F. vulgare and hastened the leaf senescence of both the species. However, plants of H. bonariensis receiving 599 mM NaCl lasted longer than F. vulgare. Therefore, the occurrence of H. bonariensis in saline soils might be related with mechanisms of salinity tolerance.
Brazilian Archives of Biology and Technology 42: 161-168, 1999

P081-99 Catabolic pathway of caffeine and purification of a xanthine oxidase responsible for methyluric acid production in Pseudomonas L.

Yamaoka-Yano DM, Mazzafera P*

Caffeine catabolism and a xanthine oxidase involved in the alkaloid breakdown were studied in Pseudomonas putida L, a strain displaying high ability to grow on this substrate. Cells cultured with unlabelled caffeine and 14C labeled caffeine and xanthine showed that this alkaloid was broken-down via theobromine/paraxanthine -> 7-methylxanthine -> xanthine -> uric acid -> allantoin -> allantoic acid. Methyluric acids were formed from the oxidation of theobromine, paraxanthine and 7-methylxanthine, although no bacterial growth was observed on these compounds, indicating that this might be due to a wide substrate specificity of xanthine oxidase. This was confirmed by activity staining in PAGE where activity was observed with theophylline and 3-methylxanthine, which are not involved in the alkaloid breakdown. A single band of activity was detected without addition of NAD+, showing an oxidase form of the enzyme. The enzyme optimum temperature and pH were 30oC and 7.0, respectively. The determined Km was 169 (M, and the pI 3.1 - 4.0. The molecular weight determined by side by side comparison of activity staining of the enzyme in PAGE and PAGE of BSA was 192 kDa, which was coincident with the sum (198.4 kDa) of three subunits (71, 65.6 and 61.8 kDa) of the purified protein.Revista de Microbiologia 30: 70-78, 1999. IF= 0.074

P082-99 Extraction de alcaloides de los granos de café canephora usando fluidos supercríticos

Saldaña MA, Mazzafera P*, Mohamed RS

Se ha obtenido datos experimentares de solubilidad para la extracción de dos alcaloides, cafeína y trigonelina, de los granos de café con CO2 supercrítico. Las solubilidades de cafeína pura en CO2 supercrítico fueron determinadas a 313, 323 y 343 K y presiones de 9,5 a 23,5 MPa. Se determino también las cantidades de cafeína y trigonelina extraídas de los granos de café enteros y partidos utilizando CO2 supercrítico a 313 K y 15 MPa. Los resultados obtenidos revelan mayor selectividad del CO2 por la cafeína en comparación con la trigonelina. Mayores cantidades de cafeína y trigonelina fueron extraídos de granos partidos que de granos enteros, indicando una mejor transferencia de masa. Un modelo termodinámico que usa una ecuación de estado de cuarto orden correlacionó eficazmente los datos experimentales de cafeína pura/CO2.
Informacion Tecnologica (Chile) 10: 87-94, 1999

P083-99 Nitrogen compounds in the xylem sap of coffee

Mazzafera P*, Gonçalves KV

Some N containing compounds were analyzed in the xylem sap collected from coffee seedlings. In order to facilitate sap bleeding, the seedlings were kept in a humidified chamber. Asparagine was the major amino acid present, representing 30.5% (257 ?g N ml-1 sap) of the total nitrogen detected, while glutamine represented 6.9% (58 ?g N ml-1 sap). Overall, amino acids accounted for 41.2% (347 ?g N ml-1 sap) of the total sap nitrogen whereas NO3 was the most abundant N compound (51.9% - 437 ?g N ml-1 sap). Caffeine (1.2% - 10.5 ?g N ml-1 sap), and the ureides alantoin (4.2% - 35.6 ?g N ml-1 sap) and allantoic acid (1.4% - 11.9 ?g N ml-1 sap) were also detected in the xylem exudate.Phytochemistry 50: 383-386, 1999. IF= l.l65

P084-99 Xanthine degradation and related enzymes activities in leaves and fruits of two Coffea species differing in caffeine catabolism

Vitória AP, Mazzafera P*

Xanthine is an intermediate during caffeine degradation in fruits and leaves of coffee. Radioisotope feeding experiments have suggested that uric acid, allantoin, allantoic acid, glyoxylic acid and urea are xanthine catabolites, with significant accumulation of radioactivity in the ureides. We have investigated xanthine degradation in young and aged leaves, and in immature and mature fruits of Coffea arabica and C. dewevrei, which differs in respect to caffeine catabolism. Radioisotope feeding experiments showed that leaves degraded xanthine more readily than fruits, however, mature fruits and aged leaves were less efficient than younger tissues. In all cases, significant part of the recovered radioactivity was in ureides. Xanthine dehydrogenase was characterized as the enzyme responsible for xanthine degradation and together with uricase, their activities were consistent with the results obtained in the radioisotope feeding experiments. Activities of allantoinase and allantoate amidohydrolase were not detected even with several changes in the extraction and assay conditions. Considerable levels of endogenous allantoin and allantoic acid were found in fruits and leaves. Although data in the literature show that 14CO2 is released from coffee tissues from labeled xanthine catabolism, ureide accumulation might be a consequence of low enzyme activity. There was no positive correlation between urease activity and the data from the radioisotope feeding experiments. Journal of Agriculture and Food Chemistry 47: 1851-1855, 1999. IF= 1.502

P085-99 Carbon partitioning in soybean infected with Meloidogyne incognita and M. javanica

Carneiro RG, Ferraz LCCB, Mazzafera, P*

Seven days old seedlings of two cultivars (Cristalina and UFV ITM1) of Glycine max were inoculated with 0, 3,000, 9,000 or 27,000 eggs of Meloidogyne incognita race 3 or Meloidogyne javanica and maintained in a greenhouse. Thirty days later, plants were exposed to 14CO2 for 4 hours. Twenty hours after 14CO2 exposure, the root fresh weight, leaf dry weight, nematode eggs per gram of root, total and specific radioactivity of carbohydrates in roots and root carbohydrates content were evaluated. M. javanica produced more eggs than M. incognita on both varieties. A general increase of root weight and a decrease of leaf weight with increased inoculum levels were observed. Gall tissue appeared to account for most of the root mass increase in seedlings infected with M. javanica. For both nematodes there was an increase of total radioactivity in the root system with increased levels of nematodes, and this was positively related with number of eggs per gram fresh weight and root fresh mass, but negatively with leaf dry weight. For most of the cases specific radioactivity of sucrose and reducing sugars were also increased with increased inoculum levels. Highest specific radioactivities were observed with reducing sugars, however although significant changes were not observed in the carbohydrates endogenous levels, sucrose content was higher than reducing sugars. The data show that nematodes are strong metabolic sinks as well as they significantly change the carbon distribution pattern in infected soybean plants. It is discussed that carbon partitioning in plants infected with nematodes may vary with the nematode genotype.
Journal of Nematology 31: 348-355, 1999. IF= 0.618

P086-99 Extraction of purine alkaloids from mate (Ilex paraguariensis) using supercritical CO2

Saldaña MA, Mohamed RS, Baer G, Mazzafera, P*

Experimental data for the supercritical CO2 extraction of purine alkaloids (caffeine, theobromine, and theophylline) from ground herbal mate tea (Ilex paraguaryensis) using a high-pressure apparatus are presented. Caffeine, theophylline, and theobromine were identified in the extracted fractions using HPLC. Results indicated a much higher CO2 selectivity for caffeine in comparison with those for theophylline and theobromine. Solubilities of pure compounds in carbon dioxide were also determined at 313.2, 323.2, 338.2, and 343.2 K, and pressures ranging from 14 to 24 MPa. Caffeine solubility exhibited a retrograde behavior with temperature while theophylline and theobromine manifested a normal behavior at conditions explored in this study. Solubilities in binary CO2/purine alkaloid model systems were much higher than those obtained during extraction of mate tea, demonstrating the difficulty of using binary data in predicting complex multicomponent behavior.
Journal of Agricultural and Food Chemistry 47: 3804-3808, 1999. IF= 1.502

P087-99 Xylem sap nitrogen composition of some Crotalaria species

Vitória AP, Sodek L*

Thirteen species of Crotalaria were analysed for nitrogen compounds in the xylem root bleeding sap. Amino acids were the main form of organic nitrogen found, but only traces of ureides were present. Of the four species analysed for amino acid composition, asparagine was found to predominated the amino acid fraction, accounting for over 68% of the nitrogen transported. No striking deviations from this general pattern were found between species, between vegetative and floral stages of development, nor between nodulated and non-nodulated plants. It is concluded that the Crotalaria species studied here have an asparagine-based nitrogen metabolism, consistent with many other non-ureide-producing legume species. Sci. Agric. 56: 733-737, 1999

P088-99 Waterlogging affects nitrogen transport in the xylem of soybean

Puiatti M, Sodek L*

Transfer of nodulated and non-nodulated plants grown in vermiculite to hydroponic culture without soil was used to study waterlogging and nitrogen transport in the xylem of soybean. Ureides were considerably reduced in waterlogged plants (nodulated) or after transfer to water-culture, especially when not aerated or aerated with nitrogen gas. Although smaller changes in the total amino acid fraction were observed, marked changes occurred in the composition. It is suggested that an alteration in asparagine metabolism may underlie the changes in amino acid transport in the xylem associated with waterlogging. Plant Physiol. Biochemistry 37: 767-773, 1999. IF= 1.613

P089-99 Purification of leghemoglobin from nodules of Crotalaria infected with Rhizobium

Mendonça EHM, Mazzafera P, Schiavinato MA*

The leghemoglobin from nodules of Crotalaria juncea infected with Rhizobium spp. was purified to homogeneity after precipitation with 40-80% (NH4)2SO4. Chromatography was conducted on two anionic exchange and one gel filtration columns. The leghemoglobin has a single component and showed an apparent Mr of ca 17,300 and 23,700 determined by SDS-PAGE and gel filtration, respectively. Amino acid composition showed that asparagine/aspartic acid, glutamine/glutamic acid, alanine, lysine, serine and leucine were the main amino acids. Iron was detected only in the band corresponding to the purified protein. The N-terminal amino acid sequence for the first 19 residues showed high similarities with leghemoglobin from other plants. Phytochemistry 50: 313-316, 1999. IF= 1.165


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