Breeding maize for silage and biofuel production, an illustration of a step forward with the genome sequence.

The knowledge of the gene families mostly impacting cell wall digestibility variations would significantly increase the efficiency of marker-assisted selection when breeding maize and grass varieties with improved silage feeding value and/or with better straw fermentability into alcohol or methane. The maize genome sequence of the B73 inbred line was released at the end of 2009, opening up new avenues to identify the genetic determinants of quantitative traits. Colocalizations between a large set of candidate genes putatively involved in secondary cell wall assembly and QTLs for cell wall digestibility (IVNDFD) were then investigated, considering physical positions of both genes and QTLs. Based on available data from six RIL progenies, 59 QTLs corresponding to 38 non-overlapping positions were matched up with a list of 442 genes distributed all over the genome. Altogether, 176 genes colocalized with IVNDFD QTLs and most often, several candidate genes colocalized at each QTL position. Frequent QTL colocalizations were found firstly with genes encoding ZmMYB and ZmNAC transcription factors, and secondly with genes encoding zinc finger, bHLH, and xylogen regulation factors. In contrast, close colocalizations were less frequent with genes involved in monolignol biosynthesis, and found only with the C4H2, CCoAOMT5, and CCR1 genes. Close colocalizations were also infrequent with genes involved in cell wall feruloylation and cross-linkages. Altogether, investigated colocalizations between candidate genes and cell wall digestibility QTLs suggested a prevalent role of regulation factors over constitutive cell wall genes on digestibility variations.

Impact of the brown-midrib bm5 mutation on maize lignins.

We have investigated the impact of the brown-midrib bm5 mutation on lignins and on p-coumaric acid and ferulic acid ester-linked to maize (Zea mays L.) cell walls. Lignified stalks or plant aerial parts (without ears) collected at grain maturity were studied in three genetic backgrounds. Relative to the control, bm5 mutants displayed lower levels of lignins and of p-coumarate esters but increased levels of ferulate esters. Thioacidolysis revealed that bm5 lignins display an increased frequency of free-phenolic guaiacyl units. More importantly, thioacidolysis provided unusual amounts of 1,2,2-trithioethyl ethylguaiacol, a marker compound diagnostic for the incorporation of free ferulic acid into lignins by bis 8-O-4 cross-coupling. As the resulting acetal bonding pattern is a chemically labile branch point introduced in maize lignins by the bm5 mutation, this alteration is prone to facilitate the delignification pretreatments used in the cellulose-to-ethanol process.

Impact of lignin structure and cell wall reticulation on maize cell wall degradability.

In this study, eight maize recombinant inbred lines were selected to assess both the impact of lignin structure and the impact of cell wall reticulation by p-hydroxycinnamic acids on cell wall degradability independently of the main "lignin content" factor. These recombinant lines and their parents were analyzed for in vitro degradability, cell wall residue content, esterified and etherified p-hydroxycinnamic acid content, and lignin content and structure. Lignin structure and esterified p-coumaric acid content showed significantly high correlation with in vitro degradability (r=-0.82 and r=-0.72, respectively). A multiple regression analysis showed that more than 80% of cell wall degradability variations within these 10 lines (eight recombinant inbred lines and their two parents) were explained by a regression model including two main explanatory factors: lignin content and estimated proportion of syringyl lignin units esterified by p-coumaric acid. This study revealed new biochemical parameters of interest to improve cell wall degradability and promote lignocellulose valorization.

Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis.

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.