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NAC transcription factors play critical roles in xylem secondary development and in regulation of stress response in plants. NAC proteins related to secondary cell wall development were recently identified and characterized in

Teak is known as one of the world’s premier hardwood tree species. However, despite the commercial interest and the large number of scientific investigations, comprehensive understanding on the genetic, molecular, and biochemical processes of this species has only recently begun to be explored. Functional genomics is the field of molecular biology that utilizes widespread combination of genetic information (e.g., enhancer DNA and non-coding RNA) and genome modification technologies to understand interactions between genes and functional regulatory elements. These technologies have emerged as a novel way to understand plant gene functions. Importance of teak has propelled various studies on transcriptomics and molecular genetics. In the last decade, transcriptomics of T. grandis of secondary wood, vegetative to flowering transition stage, and seedlings under water stress were documented. These transcriptomic studies have led to identification of genes involved in xylogenesis, production of secondary metabolites, flower formation, and drought stress. Teak transformation has not been fully achieved, but model plants are currently being used for studying several teak genes. Thus, future of functional genomics of teak is promising, particularly when the existing transcriptome data are used for wide range of analysis.

NAC transcription factors play critical roles in xylem secondary development and in regulation of stress response in plants. NAC proteins related to secondary cell wall development were recently identified and characterized inTectona grandis(teak), one of the hardwood trees of highest economic importance in the world. In this work, we characterized the novelTgNAC01gene, which is involved in signaling pathways that mediate teak response to stress. Abscisic acid (ABA) increasesTgNAC01expression in teak plants. Therefore, this gene may have a role in signaling events that mediate ABA-dependent osmotic stress responsive in this plant species. Stable expression in tobacco plants showed that the TgNAC01 protein is localized in the cell nucleus. Overexpression ofTgNAC01in two out three independent transgenic tobacco lines resulted in increased growth, leaf senescence and salt tolerance compared to wild type (WT) plants. Moreover, the stress tolerance of transgenic plants was affected by levels ofTgNAC01gene expression. Water potential, gas exchange and chlorophyll fluorescence were used to determine salt stress tolerance. The 35S:TgNAC01-6 line under 300 mM NaCl stress responded with a significant increase in photosynthesis rate, stomatal conductance, transpiration and carboxylation efficiency, but lower water potential compared to WT plants. The data indicate that theTgNAC01transcription factor acts as a transcriptional activator of the ABA-mediated regulation and induces leaf senescence.

NAC proteins are one of the largest families of plant-specific transcription factors (TFs). They regulate diverse complex biological processes, including secondary xylem differentiation and wood formation. Recent genomic and transcriptomic studies of Tectona grandis L.f. (teak), one of the most valuable hardwood trees in the world, have allowed identification and analysis of developmental genes. In the present work, T. grandis NAC genes were identified and analyzed regarding to their evolution and expression profile during wood formation. We analyzed the recently published T. grandis genome, and identified 130 NAC proteins that are coded by 107 gene loci. These proteins were classified into 23 clades of the NAC family, together with Populus, Eucalyptus, and Arabidopsis. Data on transcript expression revealed specific temporal and spatial expression patterns for the majority of teak NAC genes. RT-PCR indicated expression of VND genes (Tg11g04450-VND2 and Tg15g08390-VND4) related to secondary cell wall formation in xylem vessels of 16-year-old juvenile trees. Our findings open a way to further understanding of NAC transcription factor genes in T. grandis wood biosynthesis, while they are potentially useful for future studies aiming to improve biomass and wood quality using biotechnological approaches.

Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1–4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

Drought stress is an increasingly common and worrying phenomenon because it causes a loss of production in both agriculture and forestry. Teak is a tropical tree which needs alternating rainy and dry seasons to produce high-quality wood. However, a robust understanding about the physiological characteristics and genes related to drought stress in this species is lacking. Consequently, after applying moderate and severe drought stress to teak seedlings, an infrared gas analyzer (IRGA) was used to measure different parameters in the leaves. Additionally, using the root transcriptome allowed finding and analyzing the expression of several drought-related genes. As a result, in both water deficit treatments a reduction in photosynthesis, transpiration, stomatal conductance and leaf relative water content was found. As well, an increase in free proline levels and intrinsic water use efficiency was found when compared to the control treatment. Furthermore, 977 transcripts from the root contigs showed functional annotation related to drought stress, and of these, TgTPS1, TgDREB1, TgAREB1 and TgPIP1 were selected. The expression analysis of those genes along with TgHSP1, TgHSP2, TgHSP3 and TgBI (other stress-related genes) showed that with moderate treatment, TgTPS1, TgDREB1, TgAREB1, TgPIP1, TgHSP3 and TgBI genes had higher expression than the control treatment, but with severe treatment only TgTPS1 and TgDREB1 showed higher expression than the control treatment. At the end, a schematic model for the physiological and molecular strategies under drought stress in teak from this study is provided. In conclusion, these physiological and biochemical adjustments in leaves and genetic changes in roots under severe and prolonged water shortage situations can be a limiting factor for teak plantlets’ growth. Further studies of those genes under different biotic and abiotic stress treatments are needed.

NAC proteins are one of the largest families of plant-specific transcription factors (TFs). They regulate diverse complex biological processes, including secondary xylem differentiation and wood formation. Recent genomic and transcriptomic studies of Tectona grandis L.f. (teak), one of the most valuable hardwood trees in the world, have allowed identification and analysis of developmental genes. In the present work, T. grandis NAC genes were identified and analyzed regarding to their evolution and expression profile during wood formation. We analyzed the recently published T. grandis genome, and identified 130 NAC proteins that are coded by 107 gene loci. These proteins were classified into 23 clades of the NAC family, together with Populus, Eucalyptus, and Arabidopsis. Data on transcript expression revealed specific temporal and spatial expression patterns for the majority of teak NAC genes. RT-PCR indicated expression of VND genes (Tg11g04450-VND2 and Tg15g08390-VND4) related to secondary cell wall formation in xylem vessels of 16-year-old juvenile trees. Our findings open a way to further understanding of NAC transcription factor genes in T. grandis wood biosynthesis, while they are potentially useful for future studies aiming to improve biomass and wood quality using biotechnological approaches.

The cinnamyl alcohol dehydrogenase (CAD) enzyme catalyzes the last step of monolignols synthesis in the lignin pathway. Tectona grandis (teak) is a tropical tree with high valuable tim­ber. As there is few genetic information about lignin formation in teak, the purpose of this study is to characterize members of CAD family in this species. As methodology, PCR amplification using cDNA samples, vector cloning, sequencing, bioinforma­tics analyses and gene expression studies using real time RT-qPCR were performed. As results, four members (TgCAD1- TgCAD4) were obtained. Comparative analyses showed that all of them have conserved residues for catalytic zinc action, structural zinc ligation, NADPH binding and substrate specifici­ty, consistent with the mechanism of alcohol dehydrogenases. Phylogenetic analysis showed that TgCADs are present in three main classes and seven groups. Expression analyses revealed that TgCAD1 was highly expressed in leaves and could be rela­ted with pathogen defense. TgCAD2 was more expressed in branches and roots. Differently, TgCAD3 and TgCAD4 were highly expressed in juvenile and mature sapwood, suggesting a crucial role in wood development and lignin biosynthesis, with tissue-specialized expression profiles. Furthermore, TgCAD4 could be related with teak maturation for being more expressed in sapwood of mature teak trees. As conclusion, this work is the first to characterize genes of CAD family in Tectona grandis. These genes could be interesting to develop transge­nic plants for basic research and field applications.

A transformação genética possibilita a introdução de genes de interesse nos genomas, podendo assim ser empregada na tentativa de melhorar características agronômicas e florestais. No entanto, para a obtenção de plantas transgênicas são necessários protocolos eficientes de regeneração de plantas in vitro. Em teca, dados sobre cultura de tecidos são escassos, havendo a necessidade de determinar condições ótimas para a mesma. Com isso, o trabalho teve por objetivos estudar a organogênese in vitro de teca visando desenvolver um método de regeneração eficiente, avaliar condições para o processo de transformação e testar a susceptibilidade da lagarta Hyblaea puera a toxinas produzidas pelo Bacillus thuringiensis. Foram avaliadas a influência de TDZ e BAP na indução da competência organogenética em hipocótilos, nó cotiledonar e cotilédones de teca. Os biorreguladores AIB, BAP, NAA e GA3 foram utilizados na regeneração de segmentos de hipocótilo, nó cotiledonar, raiz, epicótilo e cotilédone. Antibióticos supressores de Agrobacterium tumefaciens e a higromicina (seleção de células transgênicas), foram também avaliados. Finalmente, testes com o inseticida biológico DipelTM e esporos de B. thuringiensis crescidos em laboratório foram realizados com as lagartas de Hyblaea puera. Na aquisição de competência organogenética o TDZ proporcionou um aumento de 46% na regeneração e o BAP 26% quando comparados ao controle. Para a organogênese in vitro foi avaliado um máximo de 70% de regeneração em nó cotiledonares em meio MS adicionado de 1 mg.L-1 de BAP + 0,5 mg.L-1 de GA3. Entretanto, em outras concentrações dos meios de regeneração hipocótilos, raiz, cotilédones e epicótilos tiveram máximas frequências de regeneração em torno de 60%, 60%, 30% e 10%, respectivamente. Os antibióticos supressores da Agrobacterium tumefaciens tiveram efeitos diferentes para cada explante. Timentin e cefotaxima na concentração de 300 mg.L-1 aumentaram o número de brotos em hipocótilos e nó cotiledonar em 1,6 e 2,0 vezes, respectivamente. Em cotilédone esses antibióticos tiveram efeitos negativos no número de brotos. Carbenicilina em todas as doses influenciou negativamente a regeneração em todos os explantes utilizados. A higromicina a 2,5 mg.L-1 inibe em 100% a regeneração de cotilédones, nó cotiledonar e hipocótilo. Os ínstares mais novos de H. puera são susceptíveis tanto ao produto comercial DipelTM quanto aos esporos crescidos em laboratório, apresentando 100% de mortalidade a concentrações de 2x105 UFC após 24 horas de ingestão. Mostrando assim seu potencial na transgenia visando à expressão de genes de Bt para a resistência a insetos. Os resultados apresentados nesse trabalho contribuem para o ganho de informação sobre os fatores que influenciam a organogênese desta espécie, bem como, definir parâmetros que possam ser utilizados em experimentos futuros visando à transformação genética da espécie. Genetic transformation allows the introduction of genes in host genomes and can therefore be used to improve forestry and agronomic traits like insect resistence. However, efficient plant regeneration protocols are necessary to obtain transgenic plants. Thus far, information about in vitro teak (Tectona grandis L. f) organogenesis is scarce. Therefore, the aims of this study were: develop an efficient protocol for in vitro organogenesis of teak, assess conditions for its genetic transformation and test the susceptibility of the caterpillar Hyblaea puera to toxins produced by Bacillus thuringiensis. We evaluated the influence of TDZ and BAP on the induction of organogenic competence in hypocotyl, cotyledonary nodes and cotyledons. Growth regulators IBA, BAP, NAA and GA3 were used in the regeneration of the hypocotyl, cotyledonary node, root, epicotyl and cotyledon. Antibiotics for suppression of Agrobacterium tumefacien (timentin, cefotaxime and carbenicillin) and for selection of transgenic cells (hygromycin) were also evaluated. Finally, tests with the biological insecticide DipelTM and spores of B. thuringiensis grown in laboratory were performed with the caterpillar of Hyblaea puera. TDZ increases 46% the regeneration frequency and BAP 26% when compared to controls. Cotyledonary nodes showed the best regeneration frequency (70%) growing on MS medium added of 1 mg.L-1 BAP + 0.5 mg.L-1 GA3. Hypocotyls, roots, cotyledons and epicotyls presented variable frequency of regeneration (60%, 60%, 30%, and 10% respectively) growing on distinct concentrations of grown regulators. We tested three antibiotics (timentin, cefotaxime, and carbenicillin) to suppress A. tumefaciens in vitro growth and they presented different effects on the organogenesis of each explants used in this study. Timentin and cefotaxime at concentration of 300 mg.L-1 increased the number of buds on hypocotyls and cotyledonary nodes. Conversely, these antibiotics had negative effects on the number of shoots of cotyledonary explants. Carbenicillin at all doses presented a negative influence on regeneration of all explants. Hygromycin at concentration of 2.5 mg.L-1 inhibits 100% of regeneration of cotyledons, cotyledonary nodes, and hypocotyls. The young instars of H. puera are susceptible to likely both commercial product DipelTM and spores grown in the laboratory, presented 100% mortality at concentrations of 2x105 CFU after 24 hours of ingestion. These findings suggest its potential to be used in teak transgenic approaches for insect resistance. Our results contribute to information about factors that influence the organogenesis of this specie, as well as define parameters that can be used in future experiments aimed at the genetic transformation of the specie.

The aim of this study was to develop a shoot regeneration protocol for Tectona grandis by examining the effects of pretreatments, regeneration culture medium, adventitious rooting, and acclimatization. The best organogenic potential was achieved when hypocotyls and cotyledonary segments from seed - lings germinated on a medium containing TDZ, were cultured for four weeks on MS medium supplemented with BAP/GA 3 . With TDZ pretreatment during seed germination, up to 70% of the cotyledonary segments (0.5 TDZ pretreatment and 1.0 BAP + 0.5 GA 3 during regeneration, mg L -1 ) and 60% of the hypocotyl explants (0.1 TDZ pretreatment and 1.0 BAP + 0.5 GA 3 during regeneration, mg L -1 ) resulted in shoot regeneration. Finally, 65% of the regenerated shoots rooted, of which 60% were successfully acclimatized ex vitro. Our protocol increases regeneration efficiency for teak and it can serve as a platform for genetic transformation to improve this important tree species. Key words : Adventitious rooting, plant growth regulator, plant tissue culture, woody plant.

Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.

Summary The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C4 grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.

The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main Sao Paulo state research funding agency, launched in 2008 the FAPESP Bioenergy Research Program (BIOEN, http://bioenfapesp.org). BIOEN aims to generate new knowledge and human resources for the improvement of the sugarcane and ethanol industry. As part of the BIOEN program, a Workshop on Sugarcane Improvement was held on March 18th and 19th 2009 in Sao Paulo, Brazil. The aim of the workshop was to explore present and future challenges for sugarcane improvement and its use as a sustainable bioenergy and biomaterial feedstock. The workshop was divided in four sections that represent important challenges for sugarcane improvement: a) gene discovery and sugarcane genomics, b) transgenics and controlled transgene expression, c) sugarcane physiology (photosynthesis, sucrose metabolism, and drought) and d) breeding and statistical genetics. This report summarizes the roadmap for the improvement of sugarcane.

A expectativa de o crescimento populacional atingir 9 bilhões de habitantes em 2050 em adição às questões da sustentabilidade e do aquecimento global nos desafiam a aumentar a oferta de alimentos. Uma metodologia alternativa que contribua para a redução do impacto desse cenário envolve a biotecnologia, que, nas últimas décadas, trouxe marcantes oportunidades tecnológicas na agricultura, resultando em relevante desenvolvimento na obtenção de novas variedades de plantas, na melhoria da qualidade de diversos alimentos e atualmente também na bioenergia. As técnicas biotecnológicas envolvendo os marcadores moleculares, a genômica e a transformação genética estão transformando a agricultura e são discutidas neste artigo.The expected population growth to reach 9 billion by 2050 in addition to issues of sustainability and global warming challenges us to increase the supply of food. An alternative approach to help reducing the impact of this scenario involves biotechnology which in recent decades has brought remarkable technological opportunities in the agriculture that resulted in relevant development in obtaining new plant varieties, improved quality of different foods, and now also in bioenergy. The biotechnology techniques involving molecular markers, genomics and genetic transformation are transforming agriculture and will be discussed in this article.

Sugarcane is a proven biofuel feedstock and accounts for about 40% of the biofuel production worldwide. It has a more favorable energy input/output ratio than that of corn, the other major biofuel feedstock. The rich resource of genetic diversity and the plasticity of autopolyploid genomes offer a wealth of opportunities for the application of genomics and technologies to address fundamental questions in sugarcane towards maximizing biomass production. In a workshop on sugarcane engineering held at Rutgers University, we identified research areas and emerging technologies that could have significant impact on sugarcane improvement. Traditional plant physiological studies and standardized phenotypic characterization of sugarcane are essential for dissecting the developmental processes and patterns of gene expression in this complex polyploid species. Breeder friendly DNA markers associated with target traits will enhance selection efficiency and shorten the long breeding cycles. Integration of cold tolerance from Saccharum spontaneum and Miscanthus has the potential to expand the geographical range of sugarcane production from tropical and subtropical regions to temperate zones. The Flex-stock and mix-stock concepts could be solutions for sustaining local biorefineries where no single biofuel feedstock could provide consistent year-round supplies. The ever increasing capacities of genomics and biotechnologies pave the way for fully exploring these potentials to optimize sugarcane for biofuel production.

The availability of chloroplast genome (cpDNA) sequences of Atropa belladonna, Nicotiana sylvestris, N. tabacum, N. tomentosiformis, Solanum bulbocastanum, S. lycopersicum and S. tuberosum, which are Solanaceae species, allowed us to analyze the organization of cpSSRs in their genic and intergenic regions. In general, the number of cpSSRs in cpDNA ranged from 161 in S. tuberosum to 226 in N. tabacum, and the number of intergenic cpSSRs was higher than genic cpSSRs. The mononucleotide repeats were the most frequent in studied species, but we also identified di-, tri-, tetra-, penta- and hexanucleotide repeats. Multiple alignments of all cpSSRs sequences from Solanaceae species made the identification of nucleotide variability possible and the phylogeny was estimated by maximum parsimony. Our study showed that the plastome database can be exploited for phylogenetic analysis and biotechnological approaches.

An important topic in genomic sequence analysis is the identification of protein coding regions. In this context, several coding DNA model-independent methods, based on the occurrence of specific patterns of nucleotides at coding regions, have been proposed. Nonetheless, these methods have not been completely suitable due to their dependence on an empirically pre-defined window length required for a local analysis of a DNA region. We introduce a method, based on a modified Gabor-wavelet transform (MGWT), for the identification of protein coding regions. This novel transform is tuned to analyze periodic signal components and presents the advantage of being independent of the window length. We compared the performance of the MGWT with other methods using eukaryote datasets. The results show that the MGWT outperforms all assessed model-independent methods with respect to identification accuracy. These results indicate that the source of at least part of the identification errors produced by the previous methods is the fixed working scale. The new method not only avoids this source of errors, but also makes available a tool for detailed exploration of the nucleotide occurrence.

The physiological responses of sugarcane (Saccharum officinarum L.) to oxidative stress induced by methyl viologen (paraquat) were examined with respect to photochemical activity, chlorophyll content, lipid peroxidation and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. Thirty-day-old sugarcane plants were sprayed with 0, 2, 4, 6 and 8 mM methyl viologen (MV). Chlorophyll fluorescence was measured after 18 h and biochemical analyses were performed after 24 and 48 h. Concentrations of MV above 2 mM caused significant damage to photosystem II (PSII) activity. Potential and effective quantum efficiency of PSII and apparent electron transport rate were greatly reduced or practically abolished. Both chlorophyll and soluble protein contents steadily decreased with MV concentrations above 2 mM after 24 h of exposure, which became more pronounced after 48 h, achieving a 3-fold decrease. Insoluble protein contents were little affected by MV. Oxidative stress induced by MV was evidenced by increases in lipid peroxidation. Specific activity of SOD increased, even after 48 h of exposure to the highest concentrations of MV, but total activity on a fresh weight basis did not change significantly. Nondenaturing PAGE assayed with H2O2 and KCN showed that treatment with MV did not change Cu/Zn-SOD and Mn-SOD isoform activities. In contrast, APX specific activity increased at 2 mM MV but then dropped at higher doses. Oxidative damage induced by MV was inversely related to APX activity. It is suggested that the major MV-induced oxidative damages in sugarcane leaves were related to excess H2O2, probably in chloroplasts, caused by an imbalance between SOD and APX activities, in which APX was a limiting step. Reduced photochemical activity allowed the early detection of the ensuing oxidative stress.

1 Laboratorio de Biotecnologia Agricola, Departamento de Ciencias Biologicas, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ), Universidade de Sao Paulo (USP), Avenida Padua Dias, 11, 13.418-900, Piracicaba, SP, Brasil. *E-mail: hecarrer@esalq.usp.br 2 Companhia de Cigarros Souza Cruz, Av. Gal Pinto Tourinho, 3200, 83.880-000, Bom Jesus, Rio Negro, PR, Brasil 3 Departamento de Genetica, ESALQ, USP 4 Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, D-14476, Potsdam, Golm, Germany ABSTRACT Intergenic spacers of chloroplast DNA (cpDNA) are very useful in phylogenetic and population genetic studies of plant species, to study their potential integration in phylogenetic analysis. The non-coding trnE-trnT intergenic spacer of cpDNA was analyzed to assess the nucleotide sequence polymorphism of 16 Solanaceae species and to estimate its ability to contribute to the resolution of phylogenetic studies of this group. Multiple alignments of DNA sequences of trnE-trnT intergenic spacer made the identification of nucleotide variability in this region possible and the phylogeny was estimated by maximum parsimony and rooted with Convolvulaceae Ipomoea batatas, the most closely related family. Besides, this intergenic spacer was tested for the phylogenetic ability to differentiate taxonomic levels. For this purpose, species from four other families were analyzed and compared with Solanaceae species. Results confirmed polymorphism in the trnE-trnT region at different taxonomic levels.

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.br sebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.

Length and number of sequences for branch differentially expressed genes. (PDF 125Â kb)

Melting curves and efficiencies of primers for quantitative real- time PCR. (PDF 192Â kb)