GRASSWALL · Maize Candy-leaf mutants: a tool for the study of grass-specific cell wall biology with potential applications in renewable energy production and cereal crop pathogen protection

Maize Candy-leaf mutants: a tool for the study of grass-specific cell wall biology with potential applications in renewable energy production and cereal crop pathogen protection

A modern bioeconomy needs to valorize the potential of sustainable carbon neutral resources to replace greenhouse gas emitting, finite fossil fuels. One resource with such attributes is highly abundant agricultural residue from crops. The majority of agricultural residue consists of so called lignocellulosics, a complex non-food plant composite material, whose sugars, once released, can be used for microbial fermentation to commodity chemicals or biofuels. Lignocellulosics also provide structural strength for the plant and are the first line of defence against invading pathogens, so the biggest challenge in converting lignocellulosics to chemicals is overcoming its recalcitrance to degradation without affecting the mechanical strength of the plants to grow tall and resist pathogen attacks. Since these properties are determined by the structure and composition of the different polysaccharides composing the lignocellulosic material, it is essential to understand how plants regulate the biosynthesis of these structural features. While the focus of past research has been on model species (e.g. Arabidopsis), very little is known about how the lignocellulosic polysaccharides are formed and integrated into the cereal cell walls. The GRASSWALL project has developed a platform to identify new non-transgenic corn varieties allowing us to gain insights into the cereal-specific molecular mechanisms controlling biosynthesis and metabolism of lignocellulosic polysaccharides. As an example, the cal-1 corn variant accumulates high amounts of glucan resulting in a 30% increase in sugar release after saccharification without affecting the disease resistance to some important fungal pathogens. We identified a mutation in cal-1 affecting the coding region of a putative licheninase. These enzymes catalyze the hydrolysis of (1,4)-β-D-glucosidic linkages in β-D glucans containing (1,3)- and (1,4)-bonds. The cal-1 mutant shows an increased accumulation of mixed-linkage glucan (MLG) consistent with a reduction on the MLG degradation rate and explaining the associated increase in the saccharification yield. Based on sequence similarity and co-expression analyses we were able to identify the putative CAL1 homolog in other cereal crops such as barley, sorghum, rice or the grass model species Brachypodium distachyon. Similar to the cal-1 corn variant, Bdcal-1 knocked-down plants showed an increased accumulation of MLG consistent with a decreased degradation rate, accompanied by an increase in saccharification yield. In addition, CAL1 gene expression analyses revealed a circadian clock-regulation of the abundance of MLG in both maize and Brachypodium favoring the hypothesis that MLG acts as an energy storage polymer in monocots. These results suggest that the mechanism of MLG degradation is conserved between grasses, and serves as an example of the potential of the GRASSWALL project to translate the knowledge obtained in corn to other grass species. Six other corn variants have been also identified with altered polysaccharide composition and/or properties affecting the saccharification yield. The GRASSWALL project established the foundation to develop algorithms predicting saccharification yields based on structural lignocellulosic parameters and to breed crops with enhanced, advantageous lignocellulosic attributes to alleviate e.g. processing costs and energy investment. In addition, the response of all these new corn variants to various fungal pathogens has been characterized. The results obtained in the GRASSWALL project provide an important step towards understanding how diverse alterations in lignocellulosic composition and/or structure affects the ability of corn plants to efficiently respond to the attack of important crop pathogens, opening new avenues to engineer crops with enhanced characteristics as biofuel feedstock and improved tolerance to pests. During the Outgoing phase at University of California Berkeley, I acquired complementary technical skills to my genetics and molecular biology background ranging from cutting-edge carbohydrate analytical methods (to determine composition and structure of lignocellulosic polysaccharides from plant material), to bioinformatics and genetics of complex genomes (in order to identify the mutations responsible for the cal chemotype). Assisting to the weekly seminar series at the Energy Biosciences Institute exposed me to a broad spectrum of exciting talks covering primary areas of bioenergy development such as cellulosic fuels (derived from non-food plants) and fossil fuel microbiology, but also other applications of biological knowledge to energy solutions, such as biolubricants, biosequestration and to other topics such as economic and policy factors influencing biofuel development. During the GRASSWALL project I have been invited to several international conferences to present the results obtained, expanding my network in the cell wall biology field and helping me to establish new international collaborations. As a result of the collaborations with the group of Prof. Sarah Hake from the Plant Genome and Expression Center in Albany, CA, USA, an article was published in The Plant Cell, a leading scientific journal in the field and a second article is in preparation. Another article is in preparation as a result of the collaboration with the group of Prof. Henrik V. Sheller from the Joint BioEnegy Institute, Emeriville, CA, USA. A first author article published in Plant Direct resulted from the GRASSWALL project, including results obtained in collaboration with Prof. Shinjiro Yamaguchi, from the Tohoku University in Sendai, Japan. The GRASSWALL project has created a fertile ground for making rapid progress in our understanding of cell wall biology in grasses addressing fundamental and questions of cell wall biology and with a direct translational value for the world's cereal crops. The execution of this project has represented a fundamental step in my carrier. The experience gained during the Marie Curie IOF Fellowship in crop cell wall biology has allowed me to reach a position of professional maturity helping me to obtaining a permanent position at Heinrich Heine University where I will start my own independent research group.

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