Cloning, bioinformatics and the enzyme activity analyses of a phenylalanine ammonia-lyase gene involved in dragon's blood biosynthesis in Dracaena cambodiana.

Phenylalanine ammonia-lyase (PAL) is the key enzyme of the phenylpropanoid pathway, playing an important role in plant development and defence. We cloned a partial cDNA of PAL gene, DcPAL1, from Dracaena cambodiana seedlings using RT-PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. DcPAL1 shows highly homologous to other known PAL genes registered in GenBank, being closest to that of Musa acuminata. DcPAL1 has a relatively high GC content and most of the GC is in the third codon position. It has 768 bp in size with a maximum open reading frame (ORF) of 765 bp, encoding a 255 amino acid-polypeptide. The deduced PAL protein is a stable protein, having classical PAL domains and consisting of three major hydrophobic domains. Analysis of effective number of codons (ENC) shows that DcPAL1 codons are used at equal frequency. Relatively higher usage frequency appears randomly in codons ended with any of the four bases; six codons have no usage bias. There are 45 codons showing distinct usage preference between DcPAL1 and E. coli, 20 between DcPAL1 and yeast. Therefore, the yeast system may be more suitable for the expression of DcPAL1. Upon the elicitation of Fusarium proliferatum, a potent elicitor of dragon's blood, the PAL enzyme activity in the leaves and stems of D. cambodiana and other two Dracaena spp. significantly increased, accompanying with the formation of dragon's blood, indicating the involvement of PAL in the biosynthesis of dragon's blood, a precious traditional medicine.

The potential of SiK® fertilization in the resilience of chestnut plants to drought - a biochemical study.

Silicon is an essential mineral nutrient, that plays a crucial role in the metabolic, biochemical, and functional mechanisms of many crops under environmental stress. In the current study, we evaluated the effect of SiK® fertilization on the biochemical defense response in plants exposed to water stress. Castanea sativa plants were fertilized with different concentrations of potassium silicate (0, 5, 7.5, and 10 mM of SiK®) and exposed to a non-irrigation phase and an irrigation phase. The results indicate that silicon promoted the synthesis of soluble proteins and decreased the proline content and the oxidative stress (reduced electrolyte leakage, lipid peroxidation, and hydrogen peroxide accumulation) in tissues, due to an increase in ascorbate peroxidase, catalase, and peroxidase activity, which was accompanied by the rise in total phenol compounds and the number of thiols under drought conditions. This study suggests that exogenous Si applications have a protective role in chestnut plants under water deficit by increasing their resilience to this abiotic stress.

Chestnut Brown Rot and Gnomoniopsis smithogilvyi: Characterization of the Causal Agent in Portugal.

Sweet chestnut (Castanea sativa Miller) is a nutritious food with high social and economic impacts in Portugal. The fungus Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) is the causal agent of chestnut brown rot, and is currently considered one of the major threats to the chestnut production chain worldwide. Considering the lack of knowledge on both the disease and the causal agent in Portugal, studies were conducted in an attempt to develop the necessary control strategies towards the mitigation of the disease in a timely way. Isolates of G. smithogilvyi were selected from three varieties of chestnut from the northeast of Portugal, and were characterized at the morphological, ecophysiological and molecular levels. Tests of pathogenicity and virulence were also developed. Gnomoniopsis smithogilvyi was confirmed as the causal agent of brown rot disease in Portuguese chestnut varieties, which showed high susceptibility. The fungus showed high adaptability to chestnut substrates. The Portuguese isolates of G. smithogilvyi are morphologically and genetically similar to those from other countries, even though some physiological variability was observed among them.

The Dynamics of Flower Development in Castanea sativa Mill.

The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein-protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.

Bentazon triggers the promotion of oxidative damage in the Portuguese ricefield cyanobacterium Anabaena cylindrica: response of the antioxidant system.

Rice fields are frequently exposed to environmental contamination by herbicides and cyanobacteria, as primary producers of these aquatic ecosystems, are adversely affected. Anabaena cylindrica is a cyanobacterium with a significantly widespread occurrence in Portuguese rice fields. This strain was studied throughout 72 h in laboratory conditions for its stress responses to sublethal concentrations (0.75-2 mM) of bentazon, a selective postemergence herbicide recommended for integrated weed management in rice, with special reference to oxidative stress, role of proline and intracellular antioxidant enzymes in herbicide-induced free radicals detoxification. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione S-transferase (GST) increased in a time- and herbicide dose-response manner and were higher than those in the control samples after 72 h. A time- and concentration-dependent increase of malondialdehyde (MDA) levels and the enhanced cell membrane leakage following bentazon exposure are indicative of lipid peroxidation, free radicals formation, and oxidative damage, while increased amounts of SOD, CAT, APX, GST, and proline indicated their involvement in free radical scavenging mechanisms. The appreciable decline in the reduced glutathione (GSH) pool after 72 h at higher bentazon concentrations could be explained by the reduction of the NADPH-dependent glutathione reductase (GR) activity. The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure. As the toxic mechanism of bentazon is a complex phenomenon, this study also adds relevant findings to explain the oxidative stress pathways of bentazon promoting oxidative stress in cyanobacteria.

Comparative toxicity of bentazon and molinate on growth, photosynthetic pigments, photosynthesis, and respiration of the Portuguese ricefield cyanobacterium Nostoc muscorum.

Bentazon and molinate are selective herbicides recommended for integrated weed management in rice. Their toxicity on growth and some biochemical and physiological parameters of Nostoc muscorum, an abundant cyanobacterium in Portuguese rice fields, was evaluated under laboratory conditions during time- and concentration-dependent exposure for 72 h. Results showed that toxic concentrations (0.75-2 mM) of both herbicides have pleiotropic effects on the cyanobacterium. Molinate was more toxic than bentazon to growth, respiration, chlorophyll-a, carotenoids, and phycobiliproteins contents. Protein content was increased by both herbicides although the effect was particularly evident with higher concentrations of molinate (1.5-2 mM). The herbicides had contrasting effects on carbohydrates content: molinate increased this organic fraction whereas bentazon decreased it. Photosynthesis and respiration were inhibited by both herbicides.

First interspecific genetic linkage map for Castanea sativa x Castanea crenata revealed QTLs for resistance to Phytophthora cinnamomi.

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenata allowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 498,9 cM and contains 217 markers mapped with an average interval of 2.3 cM. For QTL analyses, the progression rate of P. cinnamomi lesions in excised shoots inoculated was used as the phenotypic metric. Using non-parametric and composite interval mapping approaches, two QTLs were identified for ink disease resistance, distributed in two linkage groups: E and K. The presence of QTLs located in linkage group E regarding P. cinnamomi resistance is consistent with a previous preliminary study developed in American x Chinese chestnut populations, suggesting the presence of common P. cinnamomi defense mechanisms across species. Results presented here extend the genomic resources of Castanea genus providing potential tools to assist the ongoing and future chestnut breeding programs.

Study of the temperature effect in three chestnut (Castanea sativa Mill.) cultivars' behaviour.

The aim of this work was to analyse the effect of temperature in three chestnut cultivars, Aveleira, Judia and Longal. For this purpose, gas exchange, thylakoid membrane potential, photosynthetic pigment and lipid content data in July, September and October under different temperatures (31, 26 and 18 degrees C) were determined. With respect to gas exchanges, significant changes in photosynthesis rate of Aveleira were observed between July and September (7mumol CO2m(-2)s(-1)). In contrast, Judia and Longal showed a strong increase in this period, 6.1-8.5 and 4.9-6.7 micromol CO2m(-2)s(-1), for Judia and Longal, which represent an increase of about 15% and 43%, respectively. Similar patterns were detected in daylight photosynthesis measurements for Judia and Longal, in which an almost 60% decrease was observed, in contrast to 40% for Aveleira, from morning to midday, when temperatures increased from 27 to 34 degrees C. In addition to high photosynthetic rates in the hottest month, Aveleira was also the sunniest cultivar according its highest value on chlorophyll a/b ratio (3.65). Cultivars also presented maximal thylakoid membrane potential at different temperatures, with their values being 20.8, 17.8 and 17.2 degrees C for Aveleira, Longal and Judia, respectively. These results were also supported by thylakoid fatty acid composition which indicated that the unsaturation index of Aveleira (158) was the lowest in comparison with other two cultivars, 168 and 175, for Longal and Judia, respectively.

Detoxification of Jatropha curcas kernel cake by a novel Streptomyces fimicarius strain.

A huge amount of kernel cake, which contains a variety of toxins including phorbol esters (tumor promoters), is projected to be generated yearly in the near future by the Jatropha biodiesel industry. We showed that the kernel cake strongly inhibited plant seed germination and root growth and was highly toxic to carp fingerlings, even though phorbol esters were undetectable by HPLC. Therefore it must be detoxified before disposal to the environment. A mathematic model was established to estimate the general toxicity of the kernel cake by determining the survival time of carp fingerling. A new strain (Streptomyces fimicarius YUCM 310038) capable of degrading the total toxicity by more than 97% in a 9-day solid state fermentation was screened out from 578 strains including 198 known strains and 380 strains isolated from air and soil. The kernel cake fermented by YUCM 310038 was nontoxic to plants and carp fingerlings and significantly promoted tobacco plant growth, indicating its potential to transform the toxic kernel cake to bio-safe animal feed or organic fertilizer to remove the environmental concern and to reduce the cost of the Jatropha biodiesel industry. Microbial strain profile essential for the kernel cake detoxification was discussed.

Antioxidant activities of chestnut nut of Castanea sativa Mill. (cultivar 'Judia') as function of origin ecosystem.

The antioxidant properties of different ecotypes of chestnut nut (cv. Judia) were studied. Total phenolics and flavonoids were also determinated. Total phenolics amount ranged from 9.6mg/g of GAE (hottest ecotype, Murça) to 19.4mg/g of GAE (coldest ecotype, Valpaços). Gallic and ellagic acid were the predominant compounds and Valpaços had the highest values while, Murça had the lowest ones. The antioxidant capacity of ethanolic extracts were evaluated through several biochemical essays: ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity, FRAP (ferric reducing/antioxidant power) and inhibition of oxidative haemolysis in erythrocytes. In order to evaluate the antioxidant efficiency of each ecotype, the EC50 values were calculated. Once again Valpaços revealed the best antioxidant properties, presenting much lower EC50 values. Climatic conditions influence seems to be a limiting factor for production of phenolic compounds and consequently for the antioxidant properties of chestnut nuts.

Exposure of the cyanobacterium Nostoc muscorum from Portuguese rice fields to Molinate (Ordram(®)): Effects on the antioxidant system and fatty acid profile.

Herbicide contamination of aquatic ecosystems is a serious global environmental concern. Several herbicides enhance the intracellular formation of reactive oxygen species, and can lead to the damage of macromolecules and to a decrease of oxidant defenses in a wide range of non-target microorganisms including cyanobacteria. The effects of molinate (a thiocarbamate herbicide used for controlling grassy weeds in rice fields) on the activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione S-transferase were evaluated in Nostoc muscorum, a freshwater cyanobacterium with a significant spread in Portuguese rice fields. These were determined in N. muscorum cultures acutely (72h) exposed to concentrations ranging from 0.75 to 2mM of molinate. This study also analyzed the effects of molinate on: (1) the nonenzymatic antioxidant contents (reduced and oxidized glutathione, carotenoids, and proline), (2) the oxidative cell damage measured in terms of lipid peroxidation (MDA level) and electrolyte leakage (intactness of plasma membrane), and (3) the total fatty acid profile. The results showed that the activities of all antioxidant enzymes decreased dramatically with the rising concentration of molinate after 72h. Time-dependent and concentration-dependent increase in MDA and enhanced cell membrane leakage were indicative of lipid peroxidation, formation of free radicals and oxidative damage. Compared to control, 72-h herbicide exposure increased lipid peroxidation by 5.4%, 19% and 28% with 0.75, 1.5 and 2mM of molinate, respectively. Similarly, herbicide stress induced an increase in electrolyte leakage (5.8%, 29.5% and 30.2% above control, with 0.75, 1.5 and 2mM of molinate, respectively). The increased production of proline at higher molinate concentrations (the values rose above control by 45%, 95% and 156% with 0.75, 1.5 and 2mM, respectively) indicated the involvement of this osmoprotectant in a free radical scavenging mechanism. Moreover, a radical decline in both glutathione pool, carotenoids and saturated fatty acids were also observed. The results of the present study lead us to conclude that: (1) both enzymatic and nonenzymatic antioxidative defense system of N. muscorum are dramatically affected by molinate, (2) the herbicide induces peroxidation, (3) it contributes to an increase of the unsaturation level of cell membrane fatty acids. These evidences should be taken in account when using N. muscorum as an environmental indicator species in studies of herbicide biotransformation and biomarker response as well as in environmental monitoring programmes.

Production of dragon's blood in Dracaena cochinchinensis plants by inoculation of Fusarium proliferatum.

Dragon's blood is a traditional medicine widely used in the world from ancient times. However, little is known about its formation mechanism. This work aimed to gain some insights into its formation mechanism and to control its production. The results demonstrate that wounding plus causal fungal infection and keeping the wound moist are essential for efficient dragon's blood formation in Dracaena cochinchinensis. Two fungal isolates YM-266 and YM-71213 of Fusarium proliferatum increased the yield of dragon's blood in D. cochinchinensis trees by 2.7- and 3.3-times compared to that of the control (wounding alone and keeping the wound moist), respectively. The fungal induced dragon's blood had almost identical chemical constituents to that of the natural dragon's blood with a higher loureirins a and b content as analyzed by TLC and HPLC. In addition, the induced dragon's blood had similar antimicrobial activity and similar or higher antioxidant activity than that of the natural dragon's blood. The novel biological technology developed here for the production of dragon's blood is safe, repeatable, practical, and feasible for the farmers, enabling the production of dragon's blood in a sustainable way without destroying the endangered trees and environment.

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli.

The effects of the herbicides 1,1'-dimethyl-4,4'-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1 microM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.

Structural analysis of Castanea sativa Mill. leaves from different regions in the tree top

The aim of this work was to perform the histological characterization of the C. sativa leaves of three Portuguese cultivars to establish comparison among the leaves of the different quadrants in accord and with the cardinal points of the tree top and among different cultivars of this species, using light microscopy (OM) and scanning electron microscopy (SEM). Measurements were also carried out for the leaf tissue thickness, stomatal density, leaf area in the four tree top quadrants. The leaves turned to the North had lesser thickness of mesophyll mainly due to lower amount of palisade parenchyma. The stomatal density was significantly lower in these leaves, unlike the leaf area that has the highest expression.