The Woody-preferential Gene Egmyb88 Regulates The Biosynthesis Of Phenylpropanoid-derived Compounds In Wood

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Comparative phylogenetic analyses of the R2R3-MYB transcription factor family revealed that five subgroups were preferentially found in woody species and were totally absent from Brassicaceae and monocots (Soler et al., 2015). Here, we analyzed one of these subgroups (WPS-I) for which no gene had been yet characterized. Most Eucalyptus members of WPS-I are preferentially expressed in the vascular cambium, the secondary meristem responsible for tree radial growth. We focused on EgMYB88, which is the most specifically and highly expressed in vascular tissues, and showed that it behaves as a transcriptional activator in yeast. Then, we functionally characterized EgMYB88 in both transgenic Arabidopsis and poplar plants overexpressing either the native or the dominant repression form (fused to the Ethylene-responsive element binding factor-associated Amphiphilic Repression motif, EAR). The transgenic Arabidopsis lines had no phenotype whereas the poplar lines overexpressing EgMYB88 exhibited a substantial increase in the levels of the flavonoid catechin and of some salicinoid phenolic glycosides (salicortin, salireposide, and tremulacin), in agreement with the increase of the transcript levels of landmark biosynthetic genes. A change in the lignin structure (increase in the syringyl vs. guaiacyl, S/G ratio) was also observed. Poplar lines overexpressing the EgMYB88 dominant repression form did not show a strict opposite phenotype. The level of catechin was reduced, but the levels of the salicinoid phenolic glycosides and the S/G ratio remained unchanged. In addition, they showed a reduction in soluble oligolignols containing sinapyl p-hydroxybenzoate accompanied by a mild reduction of the insoluble lignin content. Altogether, these results suggest that EgMYB88, and more largely members of the WPS-I group, could control in cambium and in the first layers of differentiating xylem the biosynthesis of some phenylpropanoid-derived secondary metabolites including lignin.7Centre National pour la Recherche Scientifique (CNRS)University Paul Sabatier Toulouse III (UPS)French Laboratory of Excellence project "TULIP" [ANR-10-LABX-41, ANR-11-IDEX-0002-02]TREEFORJOULES project [ANR-2010-KBBE-007-01]"Beatriu de Pinos" from Departament d'Universitats, Recerca i Societat de la Informacio de la Generalitat de Catalunya [2009 BP-A 00185]TREEFORJOULESTULIPMinistere de l'Education Nationale, de l'Enseignement Superieur et de la RechercheSao Paulo Research Foundation (FAPESP) [2013/17846-0]CNPQ-Brazil [202228/2015-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale

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

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale

Transcriptional regulation of the lignin biosynthetic pathway revisited: new players and insights

The discovery that AC elements coordinated the regulation of genes belonging to the entire lignin biosynthetic pathway was the first breakthrough in understanding how lignin biosynthesis is regulated. Since then, tremendous progress has been made in the identification and characterization of many transcription factors (TFs) that regulate the genes of the phenylpropanoid branch pathway leading to lignin. A major breakthrough consisted in the discovery of a hierarchical transcriptional network regulating the biosynthesis of lignified secondary walls (SWs) in Arabidopsis. The NAC TFs (VND/NST/SND) work as the first layer of master switches activating the whole SW biosynthetic network through the regulation of a cascade of downstream TFs. Among these, MYB46/83 act as a second layer of master switches. Recent findings, however, reveal that the regulation of SW formation is far more complex than initially thought, involving both positive and negative regulators, dual function regulators, feedback loops, combinatorial complexes and cross talk between pathways. Finally, because of the great potential that lignocellulosic biomass represents for the production of bioenergy, there is a great interest in further elucidating the molecular mechanisms underlying the regulation of lignified SW and subsequently applying this knowledge to improve their saccharification potential for the generation of biofuels61173218sem informaçã

Transcriptional Regulation of the Lignin Biosynthetic Pathway Revisited: New Players and Insights

The discovery that AC elements coordinated the regulation of genes belonging to the entire lignin biosynthetic pathway was the first breakthrough in understanding how lignin biosynthesis is regulated. Since then, tremendous progress has been made in the identification and characterization of many transcription factors (TFs) that regulate the genes of the phenylpropanoid branch pathway leading to lignin. A major breakthrough consisted in the discovery of a hierarchical transcriptional network regulating the biosynthesis of lignified secondary walls (SWs) in Arabidopsis. The NAC TFs (VND/NST/SND) work as the first layer of master switches activating the whole SW biosynthetic network through the regulation of a cascade of downstream TFs. Among these, MYB46/83 act as a second layer of master switches. Recent findings, however, reveal that the regulation of SW formation is far more complex than initially thought, involving both positive and negative regulators, dual function regulators, feedback loops, combinatorial complexes and cross talk between pathways. Finally, because of the great potential that lignocellulosic biomass represents for the production of bioenergy, there is a great interest in further elucidating the molecular mechanisms underlying the regulation of lignified SW and subsequently applying this knowledge to improve their saccharification potential for the generation of biofuels.6117321

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale

Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses