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  • Periodic Report Summary 1 - 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)

Periodic Report Summary 1 - 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)

• Summary description of the project objectives

The overall goal of the GRASSWALL project is to gain insights into the molecular mechanisms controlling biosynthesis and metabolism of cell wall components in maize as a grass model species. The first part of the project involves the generation of new mutants affected in cell wall composition and/or properties. These candy-leaf (cal) mutants represent unique tools to study the biology of the specific cell wall of grasses. The second part of the project aims to characterize these cal mutants to address two major questions: 1) what is the molecular basis of the recalcitrance of lignocellulosic materials to enzymatic breakdown into fermentable sugars suitable for biofuel production; and 2) what cell wall components and/or proprieties play roles in the interaction with fungal pathogens, particularly Fusarium graminearum (Fg) and Cochliobolus heterostrophus (Ch), two important cereal pathogens which use the release of cell wall degrading enzymes as infection strategy.

• Description of the work performed since the beginning of the project and description of the main results achieved so far

During the first 2 years of the project we have generated and characterized of a suite of new maize cal mutants with different alterations in lignocellulosic composition and/or properties. We screened an EMS-mutagenized population looking for mutants with differences in 3 different analytical assays: composition of non-cellulosic cell wall monnosaccharides; total wall acetylation and saccharification yield. After the cell wall analysis of 8900 plants, we identified 7 new maize cell wall mutants (cal-1 to 7) with the following alterations in cell wall composition and/or properties:
cal1: high saccharification (30%) and high glucose
cal2: low saccharification (30%)
cal3: low saccharification yield (30%) and high xylose and arabinose (15%)
cal4: low saccharification yield (30%)
cal5: high saccharification yield (25%)
cal6: High saccharification (25%)
cal7: Low saccharification yield (25%)

We developed a marker-based mapping method combined with next generation RNA sequencing to identify the mutations responsible for the cal-1 chemotype. We have identified Mo17 as a good inbred line to generate the mapping populations as the cell wall composition and saccharification yield of both A619 (cal mutant genetic background) and Mo17 are very similar and introduce less variation in the F2 cross than other inbreds tested. The method consists on crossing the different cal mutants with Mo17 inbred line to generate an F2 mapping population. After that, we analyze the cell wall composition of 200-500 individuals of this segregating F2 population. We isolate DNA and RNA from individual plants and roughly define a mapping interval using available markers between A619 and Mo17. Based on the cell wall phenotype information, we pool equal amounts of RNA isolated from individuals showing the mutant phenotype on the one hand (sample A), and individuals showing the wild-type phenotype on the other (sample B). Finally we use next generation RNA sequencing technology to deep sequence samples A and B in order to identify both the differences in gene expression and in the gene coding region sequence between the two samples focusing only on the previously identified mapping interval. This method allowed us to identify a mutation in cal-1 affecting the coding region of a gene encoding 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. Additional alleles of cal-1 were identified in publically available Uniform Mu transposon collection ( showing similar results. In addition, overexpression of a functional copy of the licheninase gene in the cal-1 mutant background was able to complement the chemotype. The corn transformation was performed by our collaborator Prof. Sarah Hake in the US Department of Agriculture (Albany, CA). The gene was expressed in Nicotiana benthamiana and the purified enzyme showed specific activity against (1,3; 1,4)-β-D-glucan. Collectively these results demonstrate that CAL1 encodes a licheninase.
Based on sequence similarity and co-expression analyses we were able to identify the putative CAL1 homolog in Brachypodium distachyon, a model grass specie. BdCAL1 knocked-down plants showed an increased accumulation of MLG consistent with a decreased degradation rate, suggesting that the mechanism of MLG degradation is conserved between grasses. 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 a energy storage polymer in monocots.
The cal-1 mutant is just an example showing that the approach used in this project could be very successful to identify new players regulating the cell wall composition in grasses. The mapping populations of cal-2 to -7 have been also generated and initially the same mapping method could be applied. Interestingly, all the remaining cal mutants are affected in the saccharification yield. The identification of the respective mutations would greatly increase our knowledge on the molecular basis of the maize cell wall recalcitrance to enzymatic breakdown as well as how the different polymers are synthetized and deposited in the plant cell walls.

• Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).

One of the main limitations of modern plant biology is the transference of the acquired knowledge from plant model species such as Arabidopsis thaliana to crops. In the case of cell wall biology, it has been demonstrated that Arabidopsis and grass walls (lignocellulosics) are significantly distinct in terms of composition and properties. The GRASSWALL project has generated a unique tool, 7 new maize mutants, whose study will be able to give novel specific insights into the major question of how grass wall composition, biosynthesis and metabolism are regulated. This aim includes the ultimate goal to develop plants whose cell walls can be efficiently broken down to produce biofuel and yet have enough mechanical strength to grow tall and resist pathogen attacks for use in the European Community and beyond. The identification of maize cal mutants may provide a direct translational value as these new varieties are non-transgenic and can be used directly by appropriate industries. That would be particularly interesting in the case of cal-1, -5 and -6 as they show an increase in the glucose released by enzymatic digestion (saccharification) and could be used as new varieties with improved characteristics as biofuel feedstock.

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Life Sciences
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