Roles of the haustorium and endosperm during the development of seedlings of Acrocomia aculeata (Arecaceae): dynamics of reserve mobilization and accumulation.

The mobilization of palm seed reserves is a complex process because of the abundance and diversity of stored compounds and results from the development of a highly specialized haustorium. This work focused on the important Neotropical oleaginous palm Acrocomia aculeata, with the aim of defining phases of seedling development associated with mobilization of reserves and elucidating the role of haustorium and endosperm in this process. Standard methods were performed, including biometric, anatomical, and histochemical analyses, as well as the evaluation of the activities of the enzymes endo-ß-mannanase and lipase, throughout the reserve mobilization in seeds during germination and in seedlings. Seeds of A. aculeata stored large quantities of proteins, lipids, and polysaccharides in the embryo and endosperm. The mobilization of reserves initiated in the haustorium during germination and subsequently occurred in the endosperm adjacent to the haustorium, forming a gradually increasing zone of digestion. Proteins and polysaccharides were the first to be mobilized, followed by lipids and cell wall constituents. The haustorium activates and controls the mobilization, forming transitory reserves and translocating them to the vegetative axis, while the endosperm, which also has an active role, serves as a site of intense enzymatic activity associated with protein bodies. Seedling development can be described as occurring in six phases over a long period (approximately 150 days) due to the large amount of seed reserves. This process exhibits an alternation between stages of accumulation and translocation of protein, lipid, and carbohydrate reserves in the haustorium, which favors the seedling establishment and the reproductive success of the species.

Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa.

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage.

Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa.

Plant peroxidases are presently used extensively in a wide range of biotechnological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å.

Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species.

Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid.

Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase.

Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005), Biomacromolecules, 6, 1360-1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 A. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 116.83, c = 92.24 A, and contain one protein molecule per asymmetric unit. The V(M) value and solvent content are 4.07 A3 Da(-1) and 69.8%, respectively.

Epinephrine quantification in pharmaceutical formulations utilizing plant tissue biosensors.

A plant tissue biosensor associated with flow injection analysis is proposed to determine epinephrine in pharmaceutical samples. The polyphenol oxidase enzymes present in the fibers of a palm tree fruits (Livistona chinensis), catalyses the oxidation of epinephrine to epinephrinequinone as a primary product. This product is then electrochemically reduced (at -0.10 V versus Ag/AgClsat) on the biosensor surface and the resulting current is used for the quantification of epinephrine. The biosensor provides a linear response for epinephrine in the concentration range from 5.0 x 10(-5) to 3.5 x 10(-4) mol l(-1). The limit of detection estimated for this interval was 1.5 x 10(-5) mol l(-1) and the correlation coefficient of 0.998, working under a flow rate of 2.0 ml min(-1) and using a sample loop of 100 microl. The repeatability (R.S.D. for 10 consecutive determinations of a 3.0 x 10(-4) mol l(-1) epinephrine solution) was 3.1%. The results obtained by the method here proposed were compared with the official UV spectrophotometric procedure and also using a plant tissue reactor. The responses obtained with the proposed strategies were in good agreement with both ways of analyses, whereas the values obtained by the official spectrophotometric method was strongly affected by benzoic acid, present in the formulation of pharmaceutical product utilized for inhalation. Such favorable results obtained with the carbon paste biosensor or utilizing the bioreactor, joined with the simplicity of its preparation turns these procedures very attractive for epinephrine quantification in pharmaceutical products.

Spatial genetic structure of two sympatric neotropical palms with contrasting life histories.

The spatial genetic structure within sympatric populations of two neotropical dioecious palm species with contrasting life histories was characterized to evaluate the influence of life history traits on the extent of genetic isolation by distance. Chamaedorea tepejilote is a common wind-pollinated arboreal understory palm. Chamaedorea elatior is an uncommon climbing subcanopy palm with entomophilous pollination syndrome. A total of 59 allozyme alleles for C. tepejilote and 53 alleles for C. elatior was analyzed using both unweighted (Iu) and weighted (Iw) Moran's I spatial autocorrelation statistics. The spatial genetic structure detected within these populations is consistent with those reported for highly dispersed plants. A significance test for differences between mean Moran's I-coefficients revealed less spatial genetic structure within the C. tepejilote population than that in the C. elatior population. Adjacent individuals of C. elatior exhibited significant spatial genetic autocorrelation (Iu=0.039, Iw=0.034), indicating a Wright's neighborhood size of about 100 individuals. For C. tepejilote, nonrandom genetic distribution among nearest neighbors was detected, even from small spatial autocorrelation values (Iu=0.008, Iw=0.009), consistent with a neighborhood size of about 300 individuals. For both species, seed dispersal, mortality among life cycle stages, overlapping generations, and contrasting traits of mating and reproduction influence the standing spatial genetic structure within populations.

Genetic diversity and recruitment of the tropical palm, Euterpe edulis Mart., in a natural population from the Brazilian Atlantic Forest.

The genetic diversity and recruitment of plants of heart-of-palm tree (Euterpe edulis Mart.) were investigated in a natural population located in Southern Brazil. Five categories of plants, from seedlings to adults, were analysed using 16 allozymic loci. The results showed an average population level of genetic diversity (He=0.278) greater than the average of plant species already studied. The recruitment process of E. edulis is related to its genetic characteristics. A significant increase in the heterozygote frequency towards the adult stages was observed at three loci (Pgdh-2, G6pdh-1 and Mdh-1). This suggests the possible action of natural selection in promoting such heterozygote increase. In the same way, a linear increase in allele frequencies was observed at four loci (Prx-3, Prx-4, Pgdh-2 and G6pdh-1), indicating that recruitment is also related to a greater survival of individuals that are carriers of certain alleles. The maintenance of high diversity levels, as well as the increase in the heterozygote frequency, are positive aspects for in situ conservation. Furthermore, an increase in heterozygote frequency is favourable to the management of the species, since the maintenance of a stock of reproductive individuals with high heterozygosity levels favours the maintenance of the population dynamic and structure.