Amanda P. De Souza

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.

Composition and structure of sugarcane cell wall polysaccharides: implications for second-generation bioethanol production.

The structure and fine structure of leaf and culm cell walls of sugarcane plants were analyzed using a combination of microscopic, chemical, biochemical, and immunological approaches. Fluorescence microscopy revealed that leaves and culm display autofluorescence and lignin distributed differently through different cell types, the former resulting from phenylpropanoids associated with vascular bundles and the latter distributed throughout all cell walls in the tissue sections. Polysaccharides in leaf and culm walls are quite similar, but differ in the proportions of xyloglucan and arabinoxylan in some fractions. In both cases, xyloglucan (XG) and arabinoxylan (AX) are closely associated with cellulose, whereas pectins, mixed-linkage-β-glucan (BG), and less branched xylans are strongly bound to cellulose. Accessibility to hydrolases of cell wall fraction increased after fractionation, suggesting that acetyl and phenolic linkages, as well as polysaccharide–polysaccharide interactions, prevented enzyme action when cell walls are assembled in its native architecture. Differently from other hemicelluloses, BG was shown to be readily accessible to lichenase when in intact walls. These results indicate that wall architecture has important implications for the development of more efficient industrial processes for second-generation bioethanol production. Considering that pretreatments such as steam explosion and alkali may lead to loss of more soluble fractions of the cell walls (BG and pectins), second-generation bioethanol, as currently proposed for sugarcane feedstock, might lead to loss of a substantial proportion of the cell wall polysaccharides, therefore decreasing the potential of sugarcane for bioethanol production in the future.

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass.

Background Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the worlds second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant. Results Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source. Conclusion Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.

The Biotechnology Roadmap for Sugarcane Improvement

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main São 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 São 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.

Sugarcane as a Bioenergy Source: History, Performance, and Perspectives for Second-Generation Bioethanol

For hundreds of years, sugarcane has been a main source of sugar, used as a sweetener, and alcohol, fermented from the plant juice. The high cost of petroleum towards the end of the twentieth century stimulated the development of new fermentation technologies for producing economically viable bioethanol from sugarcane as an alternative to importing petroleum. More recently, awareness of the effects of greenhouse gas emissions due to the global climate changes propelled bioethanol as a viable renewable fuel. Consequently, sugarcane gained importance as a bioenergy feedstock. However, the lack of knowledge about sugarcane physiology, notably on aspects of photosynthesis and source–sink relationship, has slowed the advance of this expanding bioenergy-producing system. Besides the changes in source–sink relationship, another option to increase bioethanol production even more would be to use a greater fraction of the total biomass of plants, i.e., not only the soluble sugars but also the sugars present in the cell wall fractions. Here, we review the history of sugarcane as a bioenergy crop and discuss some of the relevant routes that could be adopted in the near future to make sugarcane an even better feedstock for producing biofuels.

Ethanol from sugarcane in Brazil: a ‘midway’ strategy for increasing ethanol production while maximizing environmental benefits

This article reviews the history and current state of ethanol production from sugarcane in Brazil and presents a strategy for improving ecosystem services and production. We propose that it is possible to produce ethanol from sugarcane while maintaining or even recovering some of Brazils unique neotropical biodiversity and ecosystem climate services. This approach to the future of sustainable and responsible ethanol production is termed the ‘midway’ strategy. The ‘midway’ strategy involves producing the necessary biotechnology to increase productivity while synergistically protecting and regenerating rainforest. Three main areas of scientific and technological advance that are key to realizing the ‘midway’ strategy are: (i) improving the quality of scientific data on sugarcane biology as pertains to its use as a bioenergy crop; (ii) developing technologies for the use of bagasse for cellulosic ethanol; and (iii) developing policies to improve the ecosystem services associated with sugarcane landscapes. This article discusses these three issues in the general context of biofuels production and highlights examples of scientific achievements that are already leading towards the ‘midway’ strategy.

How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

Highlight The manner in which lignin is linked to polysaccharides and the polysaccharide–polysaccharide interactions within cell walls of Miscanthus sinensis are associated with recalcitrance to hydrolysis.

Breaking the “Glycomic Code” of Cell Wall Polysaccharides May Improve Second-Generation Bioenergy Production from Biomass

Plant cell walls display a highly complex organization that confers resistance (recalcitrance) to enzymatic hydrolysis. This poses a barrier to the development of technologies for second-generation bioenergy production due to the difficulty of enzymes in accessing wall polymers. Here, we examine the fine structure of some of the main cell wall hemicelluloses and present some evidences that lend support to the idea of a glycomic code, which can be defined as the diversity of encrypted results of the biosynthetic mechanisms of plant cell wall polysaccharides that give rise to fine-structural domains containing information in polysaccharides. These are responsible for the formation of polymer composites with different levels of polymer-polymer interactions and recalcitrance to hydrolysis. Polysaccharide motifs that are recalcitrant to hydrolysis are here called pointrons, and the ones that are available to enzyme attack are named pexons. From the biotechnological viewpoint, the understanding of the glycomic code will require further identification of pointrons and possibly the transformation of them into pexons so that walls would become suitable to hydrolysis. This is thought to be key for tackling the cell wall recalcitrance, therefore opening the way for efficient biomass disassembly and efficient bioenergy production.

How endogenous plant cell-wall degradation mechanisms can help achieve higher efficiency in saccharification of biomass

Cell-wall recalcitrance to hydrolysis still represents one of the major bottlenecks for second-generation bioethanol production. This occurs despite the development of pre-treatments, the prospect of new enzymes, and the production of transgenic plants with less-recalcitrant cell walls. Recalcitrance, which is the intrinsic resistance to breakdown imposed by polymer assembly, is the result of inherent limitations in its three domains. These consist of: (i) porosity, associated with a pectin matrix impairing trafficking through the wall; (ii) the glycomic code, which refers to the fine-structural emergent complexity of cell-wall polymers that are unique to cells, tissues, and species; and (iii) cellulose crystallinity, which refers to the organization in micro- and/or macrofibrils. One way to circumvent recalcitrance could be by following cell-wall hydrolysis strategies underlying plant endogenous mechanisms that are optimized to precisely modify cell walls in planta. Thus, the cell-wall degradation that occurs during fruit ripening, abscission, storage cell-wall mobilization, and aerenchyma formation are reviewed in order to highlight how plants deal with recalcitrance and which are the routes to couple prospective enzymes and cocktail designs with cell-wall features. The manipulation of key enzyme levels in planta can help achieving biologically pre-treated walls (i.e. less recalcitrant) before plants are harvested for bioethanol production. This may be helpful in decreasing the costs associated with producing bioethanol from biomass.

Galacturonosyltransferase 4 silencing alters pectin composition and carbon partitioning in tomato

Pectin is a main component of the plant cell wall and is the most complex family of polysaccharides in nature. Its composition is essential for the normal growth and morphology pattern, as demonstrated by pectin-defective mutant phenotypes. Besides this basic role in plant physiology, in tomato, pectin structure contributes to very important quality traits such as fruit firmness. Sixty-seven different enzymatic activities have been suggested to be required for pectin biosynthesis, but only a few genes have been identified and studied so far. This study characterized the tomato galacturonosyltransferase (GAUT) family and performed a detailed functional study of the GAUT4 gene. The tomato genome harbours all genes orthologous to those described previously in Arabidopsis thaliana, and a transcriptional profile revealed that the GAUT4 gene was expressed at higher levels in developing organs. GAUT4-silenced tomato plants exhibited an increment in vegetative biomass associated with palisade parenchyma enlargement. Silenced fruits showed an altered pectin composition and accumulated less starch along with a reduced amount of pectin, which coincided with an increase in firmness. Moreover, the harvest index was dramatically reduced as a consequence of the reduction in the fruit weight and number. Altogether, these results suggest that, beyond its role in pectin biosynthesis, GAUT4 interferes with carbon metabolism, partitioning, and allocation. Hence, this cell-wall-related gene seems to be key in determining plant growth and fruit production in tomato.