Vers l'identification de gènes contrôlant la dégradabilité de la paroi secondaire lignifiée chez le maïs à travers l'élucidation de QTLs à effets forts

La dégradabilité des parois des plantes fourragères est un facteur limitant à la fois pour l'alimentation des ruminants et pour la production de biocarburants de seconde génération. Les recherches conduites ont donc visé à identifier les gènes contrôlant des propriétés des parois lignocellulosiques chez le maïs en prenant comme modèle un cluster de QTLs à effets forts localisé dans le bin 6.06 des lignées recombinantes (RILs) F288 x F271. Après avoir mis en évidence que les positions de ces QTLs se situaient dans une région monomorphe entre les deux lignées parentales, une densification ciblée de la carte génétique a permis de révéler que ces QTLs "fantômes" correspondaient en fait à des QTLs localisés sur deux positions proches (bins 6.05 et 6.07). De nouveaux QTLs majeurs au bin 4.09 ont également été détectés. La détection de QTLs avec de nouvelles mesures phénotypiques a également permis de conforter l'implication des acides p-hydroxycinnamiques et de la composition monomériques des lignines dans la dégradabilité des parois. Afin d'identifier les gènes candidats présents sous ces QTLs, des études d'expression et du séquençage ont été entreprises, en plus de la recherche a priori de gènes potentiellement impliqués dans la formation des parois lignifiées, à partir de la bibliographie. L'étude d'expression entre le parent F271 et quatre RILs porteuses des allèles favorables à la dégradabilité des parois (F288) aux QTLs majeurs du bin 6.06 a permis de mettre en évidence 360 gènes différentiellement exprimés. Le séquençage ciblé de BACs porteurs de la région d'intérêt chez F271 et F288, quant à lui, a souligné le très grand polymorphisme entre ces deux lignées.Discovering the genetic determinants of the lignified cell wall assembly in grasses is a major challenge for both basic research and for plant breeding based on marker-assisted selection. Cell wall degradability is a limiting factor of plant energy value for cattle feeding, as well as for the production of second-generation biofuel. The research conducted thus aimed at identifying genes involved in cell wall related traits, taking as model a cluster of strong effect QTLs located in the bin 6.06 of the maize recombinant inbred line (RIL) progeny F288 x F271. Having shown that these QTL positions were located in a monomorphic area between the two parental lines, targeted densification of the genetic map revealed that these "ghost" QTLs correspond in fact to QTLs located on two close positions (bins 6.05 and 6.07). New major QTLs in bin 4.09 have also been detected. New QTL detection from new field experiments has also allowed to consolidate the involvement of p-hydroxycinnamic acids and of the lignin monomeric composition, in the variation of cell wall degradability. In order to identify the candidate genes underlying these QTLs, expression studies and sequencing were undertaken, besides the a priori search for genes potentially involved in the lignified cell wall formation, from the bibliography. The expression studies between the F271 parental line and four RILs carrying favorable alleles for the cell wall degradability (F288) at the major QTLs of bin 6.06 allowed to highlight 360 differentially expressed genes. The targeted sequencing of BACs carrying the QTL region of interest, for F271 and F288, underlined the great polymorphism between these parental maize lines

Breeding grasses for capacity to biofuel production or silage feeding value: an updated list of genes involved in maize secondary cell wall biosynthesis and assembly

In the near future, maize, sorghum, or switchgrass stovers and cereal straws will be a significant source of carbohydrates for sustainable biofuel production, in addition to the current use of grass silage in cattle feeding. However, cell wall properties, including the enzymatic degradability of structural polysaccharides in industrial fermenters or animal rumen, is greatly influenced by the embedding of cell wall carbohydrates in lignin matrix, and the linkages between lignins, p-hydroxycinnamic acids, and arabinoxylans. Breeding for higher and cheaper biofuel or silage production will thus be based on the discovery of genetic traits involved in each cell wall component biosynthesis and deposition in each lignified tissue. Due to its considerable genetic and genomic backgrounds, maize is the relevant model species for identifying traits underlying cell wall degradability variations in grasses. Maize genes involved or putatively involved in the biosynthesis of cell wall phenolic compounds, cell wall carbohydrates and regulation factors were therefore searched for using data available in grass, Arabidopsis, and woody species (mostly poplar and eucalyptus). All maize ortholog genes were searched for using protein sequences and a “blastp” strategy against data available in the www.maizesequence.org database. Genes were also mapped in silico considering their physical position in the same database. Finally, 409 candidate genes putatively involved in secondary cell wall biosynthesis and assembly were shown in the maize genome, out of which 130 were related to phenolic compound biosynthesis, 81 were related to cell wall carbohydrate biosynthesis, and 198 were involved in more or less known regulation mechanisms. Most probable candidate genes involved in regulation and assembly of secondary cell wall belonged to the MYB (45 genes) and NAC (38 genes) families, but also included zinc finger and HDZipIII encoding genes. While genes involved in ferulic acid cross-linkages with other cell wall components were little known, several families putatively involved in (arabino)-xylan chain biosynthesis and in feruloyl transfer were shown, including especially arabinosyl-CoA-acyltransferases, feruloyl-AX b-1,2-xylosyl transferases, and xylan-O-3-arabinosyl transferases. This candidate gene list, which focused on genes and orthologs known to be involved in cell wall component biosynthesis and regulation, cannot be considered as exhaustive. Other genes, whose role in cell wall lignification and deposition have not yet been defined, should very likely be added to the list of candidates required for secondary cell wall assembly. Genes encoding proteins of still unknown function should also be added to the list, as several of the latter are probably involved in lignified tissue biosynthesis and deposition

Different mutations in the ZmCAD2 gene underlie the maize brown-midrib1 (bm1) phenotype with similar effects on lignin characteristics and have potential interest for bioenergy production

The maize ZmCAD2 gene has been fully sequenced in several normal and bm1 maize lines, highlighting a large diversity of mutations underlying the bm1 phenotype. Mutations in three bm1 lines (F2bm1, A619bm1, and 511Jbm1) were found corresponding to short InDels inducing premature stop codons and truncated proteins. In two lines (511Kbm1 and 5803Cbm1), mutations were limited to an only SNP or to a few SNP, modifying the catalytic sites, and likely inactivating the proteins. Results also established that the 5803Ibm7 mutant was in fact a bm1 mutant, with a sequence fully identical to the 5803Cbm1 sequence. The two new F7803bm1 (natural mutant) and Ev2210bm1 (transposon tagging Mutator investigations) both had a transposon insertion in the ZmCAD2 DNA, resulting in a truncated protein, even if the mRNA was produced. The biochemical characteristics of the Ev2210bm1 lignins corroborated the signature of CAD2 deficiency in plants, with the presence of aldehydes and atypical compounds and linkages. Considering lignin structure and content, CAD2 is likely a good target for the improvement of energy production based on maize and grass lignocellulosic biomass, including a greater susceptibility to environmentally friendly pretreatments, as it was shown in bmr sorghum. The interest in maize bm1 hybrids for cattle feeding also should be considered because there seem to be little or limited negative effects of CAD2 mutations on other agronomical traits

Different mutations in the ZmCAD2 gene underlie the maize brown-midrib1 (bm1) phenotype with similar effects on lignin characteristics and have potential interest for bioenergy production

The maize ZmCAD2 gene has been fully sequenced in several normal and bm1 maize lines, highlighting a large diversity of mutations underlying the bm1 phenotype. Mutations in three bm1 lines (F2bm1, A619bm1, and 511Jbm1) were found corresponding to short InDels inducing premature stop codons and truncated proteins. In two lines (511Kbm1 and 5803Cbm1), mutations were limited to an only SNP or to a few SNP, modifying the catalytic sites, and likely inactivating the proteins. Results also established that the 5803Ibm7 mutant was in fact a bm1 mutant, with a sequence fully identical to the 5803Cbm1 sequence. The two new F7803bm1 (natural mutant) and Ev2210bm1 (transposon tagging Mutator investigations) both had a transposon insertion in the ZmCAD2 DNA, resulting in a truncated protein, even if the mRNA was produced. The biochemical characteristics of the Ev2210bm1 lignins corroborated the signature of CAD2 deficiency in plants, with the presence of aldehydes and atypical compounds and linkages. Considering lignin structure and content, CAD2 is likely a good target for the improvement of energy production based on maize and grass lignocellulosic biomass, including a greater susceptibility to environmentally friendly pretreatments, as it was shown in bmr sorghum. The interest in maize bm1 hybrids for cattle feeding also should be considered because there seem to be little or limited negative effects of CAD2 mutations on other agronomical traits

Comparative expression of cell wall related genes in four maize RILs and one parental line of variable lignin content and cell wall degradability

A comparison of gene expression in maize between the parental line F271 and four RILs derived from the cross F288 x F271 was investigated based on hybridization on the 17,555 probes Affymetrix micro-array, targeting nearly one third of the genes present in maize genomes. The parental line had unfavorable alleles for cell wall degradability traits at the major QTL position in bin 6.06, while the set of RILs had both the favorable allele and high cell wall degradability. 360 genes were differentially expressed in the four RIL in comparison to F271, including nine genes underlying the major QTL position and 36 underlying two other QTL positions. However, their proposed function (whenever is described) do not allow us to firmly consider their involvement in the observed variation of cell wall related traits. Only a few genes involved in monolignol biosynthesis or polymerization located elsewhere in the genome were differentially expressed between the four RILs and F271, corroborating with the fact that these genes are probably not involved in major determinants of cell wall degradability in the studied set of lines. Among the investigated regulation factors, three ZmMYB, one NAC and one C3HC4 zinc finger were differentially expressed between the four RILs and F271, but they were not located in bin 6.06. Notwithstanding, the obtained results especially strengthened the probable involvement of these genes in maize secondary wall assembly and/ or lignification

Comparative expression of cell wall related genes in four maize RILs and one parental line of variable lignin content and cell wall degradability

A comparison of gene expression in maize between the parental line F271 and four RILs derived from the cross F288 x F271 was investigated based on hybridization on the 17,555 probes Affymetrix micro-array, targeting nearly one third of the genes present in maize genomes. The parental line had unfavorable alleles for cell wall degradability traits at the major QTL position in bin 6.06, while the set of RILs had both the favorable allele and high cell wall degradability. 360 genes were differentially expressed in the four RIL in comparison to F271, including nine genes underlying the major QTL position and 36 underlying two other QTL positions. However, their proposed function (whenever is described) do not allow us to firmly consider their involvement in the observed variation of cell wall related traits. Only a few genes involved in monolignol biosynthesis or polymerization located elsewhere in the genome were differentially expressed between the four RILs and F271, corroborating with the fact that these genes are probably not involved in major determinants of cell wall degradability in the studied set of lines. Among the investigated regulation factors, three ZmMYB, one NAC and one C3HC4 zinc finger were differentially expressed between the four RILs and F271, but they were not located in bin 6.06. Notwithstanding, the obtained results especially strengthened the probable involvement of these genes in maize secondary wall assembly and/ or lignification

En route pour ma 1re année de droit : préparez votre rentrée 2021

National audienceUne initiation aux notions juridiques de base et aux exercices de la première année de droit (dissertation, fiche d'arrêt ou de décision, commentaire de texte juridique ou cas pratique). Avec des conseils pour organiser son travail, des encarts pour prendre des notes et des entraînements corrigés

En route pour ma 1re année de droit : préparez votre rentrée 2022

Une initiation aux notions juridiques de base et aux exercices de la première année de droit (dissertation, fiche d'arrêt ou de décision, commentaire de texte juridique ou cas pratique). Avec des conseils pour organiser son travail, des encarts pour prendre des notes et des entraînements corrigés

Colocalizations between several QTLs for cell wall degradability and composition in the F288 x F271 early maize RIL progeny raise the question of the nature of the possible underlying determinants and breeding targets for biofuel capacity

Understanding the genetic basis of the different traits which contribute to a given value of cell wall degradability is a key issue towards the breeding of grasses with higher feeding value or higher capability for bioenergy production. A quantitative trait loci (QTL) investigation for cell wall degradability and several cell wall component traits including lignin content, p-hydroxycinnamic acid content, and lignin monomeric structure was thus done with a simultaneous search for underlying candidate genes in the F288 x F271 recombinant inbred line progeny. Genotype effects were highly significant for all cell wall investigated traits (P < 0.001) and much higher than genotype x environment interaction effects. Out of 42 QTLs mapped, 11 and 23 QTLs explained more than 20 and 15 % of the observed trait phenotypic variation, respectively. Twenty-three QTLs were gathered into four large clusters shown in bins 3.06, 4.09, 6.05, and 6.07. Colocalizations of cell wall degradability QTLs occurred with lignin content QTLs and lignin structure QTLs. Moreover, for two positions, there were also colocalizations with etherified ferulic acid QTLs. Such simultaneous colocalizations between QTLs for cell wall degradability and both lignin- and ferulate-related traits led to questioning the possible underlying genetic determinant(s). A cluster of (linked) genes involved in the different mechanisms of cell wall biosynthesis and/or assembly is likely the simplest situation to consider. However, a single "master" regulation factor located upstream in the pathway of cell wall biosynthesis and assembly cannot be definitely ruled out. Candidate genes putatively involved in cell wall degradability variations highlighted especially the presence of ZmMYB Hv5/EgMYB1-like and COV-like genes in any of the clusters. Moreover, besides potential regulatory candidates, there are a number of candidates of still unknown functions. The question of the nature of the possible QTL underlying determinants is still partly unanswered, even if the results obtained strongly suggested that, in this progeny, genes involved in monolignol biosynthesis and important Arabidopsis NAC are not likely candidates. In addition, the positions of candidate genes suggested that ghost QTL positions should also be considered