Patent Application: US-201314399901-A

Abstract:
the disclosure is based on the discovery of genes which influence lignin biosynthesis . in particular , the inventors have observed that if the expression , function and / or activity of these gene is / are modulated , the lignin content of plants can be altered . as such , this disclosure provides plants , which exhibit modulated expression of one or more lipase / esterase / thioesterase family gene and which may find application in methods for producing biofuels .

Description:
the disclosure will now be described in detail with reference to the following figures which show : fig1 a and 1b : arabidopsis mutants in at1g52760 ( encoding a lysophospholipase / thiolesterase lysopl2 ) have reduced ( mutant mx12 — 7 ) or virtually abolished ( mutant gb9 ) expression of at1g52760 rna compared to wild - type col - 0 . the gene at1 g52760 was identified as being tightly co - expressed with lignin biosynthesis genes using methods similar to [ 6 - 8 ]. levels of at1g52760 mrna were estimated by qrt - pcr . ** 0 . 01 & gt ; p & gt ; 0 . 001 . fig2 a : stem cross sections stained with maule reagent reveal that mutant gb9 with defective at1g52760 expression ( bottom ) shows less red staining in fibres and more irregular shaped xylem vessels compared to wild - type plants ( top ). fig2 b : stem cross sections autofluorescence ( top ) and stained with maule reagent ( bottom ) reveal that mutant gb9 ( right - hand side ) with defective at1g52760 expression shows less lignin autofluorescence , less maule red staining in fibres , and more irregular shaped xylem vessels compared to wild - type plants ( left - hand side ). fig3 a - c : arabidopsis mutant gb9 with defective at1g52760 expression has reduced lignin , with levels significantly lower than wild - type plants ( wt ). lignin was determined by the acetyl bromide method similar to [ 9 ]. ( c ) shows the altered monomer structure ; lignin was determined by the acetyl bromide method similar to [ 9 ] and by thioacidolysis to determine h , g and s units . fig4 a : arabidopsis mutant gb9 with defective at1g52760 expression has increased sugar yield on cell wall saccharification , with levels comparable to known lignin mutants ccr1 and ref3 - 3 , and significantly higher than col - 0 wild - type control plants . saccharification was determined in a relatively mild assay using methods similar to [ 10 ]. greater improvements in saccharification might be expected under conditions with higher enzyme loading left for longer time . fig4 b : arabidopsis mutants gb9 and mx12 — 7 with defective at1g52760 expression have increased cellulose - to - glucose conversion on cell wall saccharification compared to wild - type plants . error bars represent the standard error . * 0 . 05 & gt ; p & gt ; 0 . 01 , ** 0 . 01 & gt ; p & gt ; 0 . 001 , *** 0 . 001 & gt ; p . fig5 a - c : phenolic profiling reveals that there are differences in metabolite accumulation between wild - type ( wt ) and the lysophospholipase / thioesterase mutants mx12 7 and gb9 . pca plots show that the wild - type profiles cluster differently from those of the mutants while an s - plot analysis also confirms that some metabolites accumulate differentially between mutants and wild - type ( revealed by outlying dots in the tails of the s - plot ). fig6 : differential accumulation of three compounds that are present in different levels in the lysophospholipase / thioesterase mutant and the wild - type is illustrated in the right - hand side column of graphs while the mass spectra of these compounds is shown in the left - hand side column . fig7 : arabidopsis mutant with defective at1g52760 expression ( green line ) has more ferulate esters and glucosides on phenolic profiling than wild - type plants ( red line ). two peaks that accumulate in the thioesterase mutant correspond to ferulate glucose ester and one to ferulic acid glucoside . the peak that accumulates 70 - fold is feruloyl malate . fig8 : arabidopsis mutant with defective at1g52760 expression ( green line ) has less lignin oligomers than wild - type plants ( red line ). fig9 : multiple alignment of arabidopsis at1g52760 amino acid sequence and similar sequences from other plant species . at1g52760 has previously been described as a lysophospholipase 2 ( lysopl2 ) involved in tolerance to cadmium - induced oxidative stress [ 11 ]. no basis for a role in lignin biosynthesis has previously been proposed . ( at1g52760 ( seq id no : 2 ); populus ( seq id no : 4 ); pt ( seq id no : 6 ); vitis ( seq id no : 8 ); glycine ( seq id no : 12 ); medicago ( seq id no : 10 ); os ( seq id no : 14 )) a variety of tools [ 6 - 8 ] including act and cressexpress were used to search for genes that have similar expression patterns to individual lignin biosynthesis genes . in total , 255 genes were retrieved , with some of them shared between different analyses ; 102 of them were chosen for further investigation . to investigate the potential biological function of these genes , we searched the nottingham arabidopsis stock centre ( nasc ) for available t - dna insertion mutants in these genes and obtained 66 homozygous mutants , including two , renamed gb9 and mx12 - 7 , that are mutated in at1g52760 , a gene annotated as encoding a lipase / thioesterase enzyme and later described as a lysophospholipase [ 11 ] with no known role in lignin biosynthesis . fig1 shows that mx12 - 7 retains a very small level of the at1g52760 lysophospholipase / thioesterase expression while in gb9 , no expression was detected . expression was quantified by standard qrt - pcr analysis . histochemical staining with maule reagent ( which stains s lignin ) of transverse stem sections from the gb9 mutant showed reduced lignin staining and collapsed xylem indicative of a cell wall defect ( fig2 ). acetyl bromide lignin determinations [ 9 ] carried out on arabidopsis mutant gb9 showed that it has reduced lignin , with levels significantly lower than wild - type plants ( fig3 ). this suggests that the at1g52760 lysophospholipase plays some unknown role in determining the amount of lignin deposited in arabidopsis and possibly other plants . we subsequently demonstrated a significant improvement in the release of sugar from plant cell walls of the gb9 mutant ( fig4 ), which releases levels comparable to that of known lignin mutants ( ccr1 ), and significantly higher than the levels released by col - 0 wild - type plants . this saccharification assay is a very mild treatment and does not indicate the maximum possible sugar release from these genotypes but merely reveals differences between them under mild conditions . saccharification was evaluated by grinding stem material to a fine powder , pretreating it with mild acid ( typically 1 % h2so4 ), washing the residue and subjecting it to enzymic hydrolysis with novozymes 188 plus celluclast . levels of simple reducing sugars released were determined by mbth detection using methods similar to [ 10 ]. these data illustrating improved saccharification of gb9 suggest that it may be a novel point at which to manipulate lignin biosynthesis to improve sugar release for biofuel production . phenolic profiling by ultrahigh pressure liquid chromatography ( uplc ) of methanol - soluble phenolic compounds revealed that some metabolites accumulate differentially between the wild - type and the lysophospholipase / thioesterase mutant . pca plots ( fig5 , top ) confirmed that the wild - type profiles differ from those of the mutant , as did an s - plot analysis ( fig5 , bottom ). dots in the tails of the s - plot designate metabolites that accumulate differentially between mutant and wild - type . the mass spectra of three compounds that accumulate differentially between the lysophospholipase / thioesterase mutant and the wild - type are shown in fig6 . two peaks that accumulate in the mutant correspond to ferulate glucose ester and one to ferulic acid glucoside . a peak that accumulates 70 fold is feruloyl malate ( fig7 ). arabidopsis mutants with defective ati g52760 lysophospholipase / thioesterase expression ( esterase ; green line ) also have less lignin oligomers than wild - type plants ( red line ) ( fig8 ). the figure shows regions in the chromatogram that are rich in small lignin oligomers . chromatograms of the thioesterase mutant have lower peak heights compared to the wild - type . structures of some oligolignols that are reduced in the thioesterase mutant are shown . these data are being studied further to try to deduce the exact role of at1g52760 mutants in lignin biosynthesis and new lines of investigation are being pursued to the same end . in order to determine whether the at1g52760 lysophospholipase interacted directly with lignin biosynthesis genes , the lysophospholipase was used as a bait to trap any interacting protein complexes using a tandem affinity purification system . evaluation of the proteins co - purifying with the lysophospholipase by gc - ms revealed several potential lignin biosynthesis enzymes ( data not shown ). this suggests that the lysophosholipase influences lignin by some direct mechanism modulating lignin biosynthesis . evaluation of at1g52760 orthologues using blast searches of gene sequence data revealed several highly homologous sequences from populus trichocarpa , vitis vinifera , glycine max , medicago truncatula and oryza sativa , suggesting that the role of at52760 is widely conserved in the plant kingdom ( fig7 ). 4 . j . ralph , h . kim , f . lu , j . h . grabber , w . boerjan , j .- c . leplé , j . berrio sierra , m . mir derikvand , l . jouanin and c . lapierre . ( 2008 ) identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins ( and an indicator for cinnamoyl - coa reductase deficiency ). the plant journal , 53 ( 2 ), 368 - 379 . 5 . h . kim , j . ralph , f . lu , s . a . ralph , a .- m . boudet , j . j . mackay , r . r . sederoff , t . ito , s . kawai , h . ohashi and t . higuchi . ( 2003 ) nmr analysis of lignins in cad - deficient plants . part 1 . incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins . organic and biomolecular chemistry , 1 , 268 - 281 . 6 . brown , d . m ., zeef , l . a ., ellis , j ., goodacre , r ., & amp ; turner s . r . identification of novel genes in arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics . plant cell 17 : 8 , 2281 - 2295 ( 2005 ). 7 . manfield , i . w ., jen , c . h ., pinney , j . w ., michalopoulos , i ., bradford , j . r ., gilmartin , p . m ., & amp ; westhead , d . r . arabidopsis co - expression tool ( act ): web server tools for microarray - based gene expression analysis . nucleic acids res . 34 : suppl 2 , w504 - w509 ( 2006 ). 8 . srinivasasainagendra , v ., page , g . p ., mehta , t ., coulibaly , i ., & amp ; loraine , a . e . cressexpress : a tool for large - scale mining of expression data from arabidopsis . plant physiol . 147 : 3 , 1004 - 1016 ( 2008 ). 9 . hatfield , r . d ., grabber , j ., ralph , j ., & amp ; brei , k . using the acetyl bromide assay to determine lignin concentrations in herbaceous plants : some cautionary notes . j . agric . food chem . 47 : 2 , 628 - 632 ( 1999 ). 10 . gomez , l . d ., whitehead , c ., barakate , a ., halpin , c ., & amp ; mcqueen - mason , s . j . 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