Eugênio César Ulian

RNA Expression Profiles and Data Mining of Sugarcane Response to Low Temperature

Tropical and subtropical plants are generally sensitive to cold and can show appreciable variation in their response to cold stress when exposed to low positive temperatures. Using nylon filter arrays, we analyzed the expression profile of 1,536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp. cv SP80-3280) exposed to cold for 3 to 48 h. Thirty-four cold-inducible ESTs were identified, of which 20 were novel cold-responsive genes that had not previously been reported as being cold inducible, including cellulose synthase, ABI3-interacting protein 2, a negative transcription regulator, phosphate transporter, and others, as well as several unknown genes. In addition, 25 ESTs were identified as being down-regulated during cold exposure. Using a database of cold-regulated proteins reported for other plants, we searched for homologs in the sugarcane EST project database (SUCEST), which contains 263,000 ESTs. Thirty-three homologous putative cold-regulated proteins were identified in the SUCEST database. On the basis of the expression profiles of the cold-inducible genes and the data-mining results, we propose a molecular model for the sugarcane response to low temperature.

Elevated CO2 increases photosynthesis, biomass and productivity, and modifies gene expression in sugarcane

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (approximately 370 ppm) and elevated (approximately 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

Signal transduction-related responses to phytohormones and environmental challenges in sugarcane

BackgroundSugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins.ResultsAdopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases.ConclusionAn extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties.

Sugarcane genes associated with sucrose content

Background -Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcanes sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants.Results -We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways.Conclusion -Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

Sugarcane: A Major Source of Sweetness, Alcohol, and Bio-energy

Sugarcane is an important tropical crop having C4 carbohydrate metabolism which, allied with its perennial nature, makes it one of the most productive cultivated plants. It is mostly used to produce sugar, accounting for almost two thirds of world production. Recently it has gained increased attention because of its important potential for bio-fuel production. However, sugarcane has one of the more complex crop genomes, which has long hampered the development of sugarcane genetics to support breeding for crop improvement programs. Sugarcane belongs to the genus Saccharum L, part of the Poaceae familly (Grasses) and the Andropogonae tribe, which encompasses only polyploid species.With the advent of molecular genomics, the sugarcane genome has become less mysterious, although its complexity has been confirmed in many aspects. Shortcuts to genomic analyses have been identified thanks to synteny conservation with other grasses, in particular sorghum and rice. Over time, new tools have become available for understanding the molecular bases behind sugarcane productivity and a renewed interest has surfaced in its genetics and physiology.

Analysis of genomic and functional RFLP derived markers associated with sucrose content, fiber and yield QTLs in a sugarcane (Saccharum spp.) commercial cross

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.

Genetically engineered trees for plantation forests: key considerations for environmental risk assessment

Forests are vital to the worlds ecological, social, cultural and economic well-being yet sustainable provision of goods and services from forests is increasingly challenged by pressures such as growing demand for wood and other forest products, land conversion and degradation, and climate change. Intensively managed, highly productive forestry incorporating the most advanced methods for tree breeding, including the application of genetic engineering (GE), has tremendous potential for producing more wood on less land. However, the deployment of GE trees in plantation forests is a controversial topic and concerns have been particularly expressed about potential harms to the environment. This paper, prepared by an international group of experts in silviculture, forest tree breeding, forest biotechnology and environmental risk assessment (ERA) that met in April 2012, examines how the ERA paradigm used for GE crop plants may be applied to GE trees for use in plantation forests. It emphasizes the importance of differentiating between ERA for confined field trials of GE trees, and ERA for unconfined or commercial-scale releases. In the case of the latter, particular attention is paid to characteristics of forest trees that distinguish them from shorter-lived plant species, the temporal and spatial scale of forests, and the biodiversity of the plantation forest as a receiving environment.

Identification of new ABA- and MEJA-activated sugarcane bZIP genes by data mining in the SUCEST database.

Sugarcane is generally propagated by cuttings of the stalk containing one or more lateral buds, which will develop into a new plant. The transition from the dormant into the active stage constitutes a complex phenomenon characterized by changes in accumulation of phytohormones and several other physiological aspects. Abscisic acid (ABA) and methyl-jasmonate (MeJA) are major signaling molecules, which influence plant development and stress responses. These plant regulators modulate gene expression with the participation of many transcriptional factors. Basic leucine zipper proteins (bZIPs) form a large family of transcriptional factors involved in a variety of plant physiological processes, such as development and responses to stress. Query sequences consisting of full-length protein sequence of each of the Arabidopsis bZIP families were utilized to screen the sugarcane EST database (SUCEST) and 86 sugarcane assembled sequences (SAS) coding for bZIPs were identified. cDNA arrays and RNA-gel blots were used to study the expression of these sugarcane bZIP genes during early plantlet development and in response to ABA and MeJA. Six bZIP genes were found to be differentially expressed during development. ABA and MeJA modulated the expression of eight sugarcane bZIP genes. Our findings provide novel insights into the expression of this large protein family of transcriptional factors in sugarcane.

Identification of genes responsive to the application of ethanol on sugarcane leaves

The control of gene expression in precise time and space is a desirable attribute of chemically inducible systems. Ethanol is a chemical inducer with favourable features, such as being inexpensive and easy to apply. The aim of this study was to identify ethanol-responsive genes in sugarcane. The cDNA macroarray technique was adopted to identify transcript changes in sugarcane leaves (Saccharum spp. cv SP80-3280) exposed to ethanol. The expression profiles of sugarcane genes were analysed using nylon filters containing 3,575 cDNA clones from the leaf roll library of the SUCEST project. Seventy expressed sequence tags (ESTs) presented altered expression patterns, including ESTs corresponding to genes related to transcriptional and translational processes, abiotic stress and others. Several genes of unknown function were also identified. Among the 48 ESTs up-regulated by ethanol, an abiotic stress-responsive protein and an unknown function gene presented rapid induction by ethanol. The macroarray data of selected ethanol-responsive EST were confirmed by RNA-blot hybridisation. The expression profile of the 48 up-regulated genes was compared in two other cultivars: SP89-1115 and SP90-3414. Surprisingly, no gene showed a similar expression profile in the three cultivars. This result suggests that sugarcane plants have a high diversity in their responses to ethanol.

Identification of methyl jasmonate-responsive genes in sugarcane using cDNA arrays

O acido jasmonico (JA) e seu ester metil-jasmonato (MeJA) sao moleculas sinalizadoras derivadas do acido linolenico e estao envolvidas no desenvolvimento da planta e na resposta aos estresses. MeJA regula a expressao genica ao nivel transcricional, do processamento do RNA e da traducao. Investigamos as mudancas na expressao genica em folhas de cana-de-acucar expostas ao MeJA usando arranjos de cDNA. O RNA total isolado a 0, 0,5, 1, 3, 6 e 12 horas apos o tratamento com MeJA foi utilizado para a sintese de sondas contendo a-33P-dCTP, as quais foram, posteriormente, hibridizadas em membranas de nailon contendo 1.536 clones de cDNA. Um aumento significativo na expressao genica em resposta ao MeJA foi detectado em genes que respondem a estresses e tambem em genes com funcao desconhecida, enquanto os genes que participam da fotossintese e da assimilacao de carboidrato foram reprimidos. A busca por dominios conservados em proteinas desconhecidas e a analise digital do perfil de expressao de mRNA revelaram possiveis proteinas novas relacionadas a estresses induzidas por MeJA e os tecidos onde os genes regulados por MeJA sao preferivelmente expressos.