Abstract:
The present invention relates to expression in plants of pectinolytic enzymes and of pectin methylesterase inhibitors for increasing the degradability of plant tissues through enzymatic digestion, thus improving saccharification efficiency.

Description:
This application claims priority to and the benefit of Italian Application No. RM2008A000696 filed on Dec. 30, 2008, the content of which is incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to a process for improving conversion of lignocellulosic biomasses into fermentable sugars, a process called saccharification, which then allows the production, on an industrial scale, of any product obtained by fermentation, including ethanol (bioethanol). 
     The present invention relates to the steady expression in plants of pectinolytic enzymes and of pectin methylesterase inhibitors for increasing the degradability of plant tissues through enzymatic digestion and thus the saccharification efficiency. 
     BACKGROUND OF THE INVENTION 
     The increasing demand for fuels alternative to oil derivatives is directing towards processes for production of bio fuels. The production of ethanol and biogas through the digestion process starting from complex organic matrices of a biological nature (biomasses) coming from agricultural production lines (for example residual biomasses of food or industrial crops and agro-industrial or urban waste) represents a promising way to produce renewable energy and is one of the preferred ways of disposing of organic residues and waste. A considerable portion of biomasses (about 75%) is represented by the cell walls of plants. These consist of a heterogeneous matrix composed of polymeric carbohydrates associated to other components, such as lignin and proteins. Wall polysaccharides produce simple sugars by degradation (saccharification) which can then be used by the micro organisms present in aerobic and anaerobic fermentation for the production of bioethanol and biogas, respectively. Enzymatic hydrolysis is currently regarded as the most promising technology and with the lowest environmental impact for “saccharification”, that is, the conversion of raw plant material into fermentable sugars. The limiting factor in this process is the natural resistance of cell walls to enzymatic degradation. Pre-treatment technologies currently available for making biomasses sensitive to hydrolytic enzymes that degrade the single components of the plant cell wall are expensive and in some cases require, along with mechanical break-up processes, the use of toxic and/or polluting substances, such as acids, peroxides and ammonia. 
     The availability of more easily degradable plant material would considerably improve the use of biomasses and would reduce the need for expensive pre-treatments with a strong environmental impact. Since the accessibility of the cellulose component to the degrading enzymes is prevented by the presence of pectins, hemicelluloses and lignin, qualitative and quantitative modifications of these components in the plant walls may improve the efficiency of saccharification. 
     The technical problem the invention intends to solve is to decrease the recalcitrance of cell walls found in lignocellulosic biomasses to the enzymatic hydrolysis by cellulases and other degrading enzymes. 
     The authors have set up a process that reduces, in the cell wall of a plant, the presence of de-esterified homogalacturonan, a component of plant pectins. The reduction is achieved by expression of a hexogen polygalacturonase, for example fungal polygalacturonase (PG), and/or overexpression of a pectin methylesterases inhibitor (PMEI). The process lead to an increase in the efficacy of cellulose enzymatic hydrolysis. 
     The biomasses containing the pectin thus modified do not need expensive and polluting pre-treatments and allow improving the saccharification process. 
     PRIOR ART 
     Plant biomasses, that is, plants and/or parts thereof at any vegetative and desiccative state, are one of the alternative source of energy, and of renewable materials and industrial products. The plant cell walls comprise about 75% by weight of lignocellulosic biomass (1) and they can be an abundant potential source of ethanol (2-4). They consist of a heterogeneous matrix of carbohydrates polymers associated to other components, such as lignin and some proteins. The degradation of the cell wall into fermentable sugars, called saccharification, is the key process for making ethanol from such biomasses. Enzymatic hydrolysis is the most promising technology that is also compatible with the protection of the environment for saccharification (5, 6). However, such process exhibits several limits, especially for an application at industrial level, due to the recalcitrance of the cell walls to hydrolysis (7). Among the causes of such recalcitrance are: the heterogeneity and complexity of the structural components of the cell walls themselves, and the inter-polymeric interactions thereof (8); the lower efficacy of the enzymes for insoluble substrates, components of the walls (9); the presence of microbial enzyme inhibitors (10); the degree of lignification (11). 
     In order to make the plant biomasses accessible to cell wall degrading enzymes (CWDEs), expensive pre-treatments are currently required, which use toxic chemical products such as acids, peroxides or ammonia, often accompanied by passages of mechanical rupture. An example of pre-treatment is represented by the hydrolysis with sulphuric acid 1.3% (w/w) and autoclaving at 130° C. for 40 min (14). Such pre-treatments are in some cases inefficient, as they cause the degradation of potentially useful products that may be obtained during the biomass degradation. 
     It is therefore clear that the reduction or elimination of such pre-treatments would represent a significant improvement of the saccharification process (12). 
     To this end, the modification of the cell wall structure is a non-polluting bio-technological alternative (13). 
     It has been proven, for example, that the reduction in the lignin contents in the cell walls of alfalfa improves the saccharification process but reduces the plant growth (14). 
     In dicotyledons, particularly critical polysaccharide component for tissue integrity and accessibility to CWDEs enzymes is the cohesive pectin network that surrounds the cellulose-hemicellulose matrix, which in turn contains the main elements that impart resistance. It is also known that intermolecular pectin bonds affect wall plasticity (15-17) and that the acid form of homogalacturonan (HGA) forms cross-links with Calcium ions (Ca ++ ) to form “junction zones” that stiffen the cell wall (18-20). The amount of HGA, its methylation level and the distribution of methyl groups affects the formation of such “junction zones”. HGA is synthesised and secreted in a highly methyl esterified form, not capable of forming the network with ions Ca ++  (21). Afterwards, it is de-esterified by pectin methylesterases (PMEs), enzymes located in the walls, which produce long chains of free carboxylic residues, capable of interacting with ions Ca ++ , and thus forming the “junction zones” (22). 
     De-esterification of galacturonans in lignified tissues may also increase the formation of lignin-carbohydrates complex forming benzyl-ester links which could cause a further resistance of the cell wall (29). 
     The generation of PMEI plants and tobacco plants expressing pgallm (PG plants) has been described (23,24), however these documents do not mention their use in reducing resistance to saccharification of lignocellulosic biomasses. 
     DESCRIPTION OF THE INVENTION 
     The authors of the present invention have surprisingly found that the saccharification process of plant biomasses may be improved by reducing the regions of de-esterified HGA in pectin, decreasing the “glue” feature of the middle lamellas, rich in pectins, which join the cell walls of two adjacent cells. 
     It is therefore the object of the present invention the use of plants having a reduced content of de-esterified homogalacturonan (HGA) in the pectins of said plants cell walls and a reduced resistance to saccharification with respect to control plants in a saccharification process of plant biomasses. 
     Preferably, the plants are obtained by genetic transformation with a gene encoding an agent able to reduce the content of de-estherified HGA in the pectins. 
     Preferably, the agent is a polygalacturonase. 
     Still preferably the gene encoding a polygalacturonase is from  Aspergillus niger.    
     Yet preferably the gene encoding a polygalacturonase is modified to produce an enzyme having a reduced specific activity. 
     In a preferred embodiment the gene encodes a polygalacturonase having a reduced specific activity and having the amino acid sequence of SEQ ID No. 2. 
     In a still preferred embodiment the gene encoding a polygalacturonase having a reduced specific activity has the nucleotide sequence of SEQ ID No. 1. 
     Preferably, the plants are obtained by transformation with a gene encoding an inhibitor of pectin methylesterases or with a gene encoding encoding a pectate lyase. 
     Still preferably, the gene encoding an inhibitor of pectin methyl esterases is of plant origin. 
     Yet preferably, the gene encodes an inhibitor of pectin methyl esterases having the amino acid sequence of SEQ ID No. 4. 
     Still preferably, the gene encoding an inhibitor of pectin methyl esterases has the nucleotide sequence of SEQ ID No. 3. 
     Preferably, the gene encoding encoding a pectate lyase is of bacterial origin. 
     Still preferably, the gene encodes a pectate lyase having the amino acid sequence of SEQ ID No. 6. 
     Yet preferably, the gene encoding a pectate lyase has the nucleotide sequence of SEQ ID No. 5. 
     In a preferred embodiment the plants are obtained by selecting natural or mutagenesis induced variants. 
     Plants with reduced contents of de-esterified HGA in pectins are obtainable with different methods, all comprised within the protection scope of the invention. 
     They are produced transgenically, transforming wild plants with a gene coding for an agent capable of reducing the contents of de-esterified HGA in pectins. 
     As particular embodiment, the plants are transformed with a nucleic acid of a nucleotidic sequence coding for a polygalacturonase, preferably from  Aspergillus niger , more preferably a mutant (variant) thereof, such as to code for an enzyme with reduced specific activity; even more preferably, a mutant (variant) of the sequence of gene pgaII (GenBank ID N. XM 001397030, NT 166530), preferably deleted of the sequence from nt. 1 to nt. 81 coding for the signal peptide of 21 aa. and for the propeptide from aa. 22 to aa. 27), and with such modifications as to have a deletion of the treonin amino acid in position 34 and a replacement of the amino acidic residue asparagin 178 with an aspartate (N178D). Such positions refer to the non-mature native protein. The nucleotidic sequence coding for the mature mutated protein (pgaIIm) is as follows (SEQ ID No. 1): 
     
       
         
               
             
           
               
                 GACAGCTGCACGTTCACCGCTGCCGCTGCTAAAGCGGGCAAGGCGAAATG 
               
               
                   
               
               
                 CTCTACTATCACCCTTAACAACATCGAAGTTCCAGCTGGAACCACCCTCG 
               
               
                   
               
               
                 ACCTGACCGGTCTCACCAGCGGTACCAAGGTCATCTTCGAGGGCACCACG 
               
               
                   
               
               
                 ACCTTCCAGTACGAAGAATGGGCAGGCCCCTTGATCTCCATGAGTGGCGA 
               
               
                   
               
               
                 ACATATCACCGTCACTGGTGCCTCCGGCCACCTCATCAATTGCGATGGTG 
               
               
                   
               
               
                 CGCGCTGGTGGGATGGCAAGGGAACCAGCGGAAAGAAGAAGCCCAAGTTC 
               
               
                   
               
               
                 TTTTACGCCCATGGCCTTGACTCCTCGTCTATTACTGGATTAAACATCAA 
               
               
                   
               
               
                 AAACACCCCCCTTATGGCGTTTAGTGTCCAGGCGAATGACATTACGTTTA 
               
               
                   
               
               
                 CCGATGTTACCATCAATAATGCGGATGGCGACACCCAGGGTGGACACGAC 
               
               
                   
               
               
                 ACTGATGCGTTCGATGTTGGCAACTCGGTCGGGGTGAATATCATTAAGCC 
               
               
                   
               
               
                 TTGGGTCCATAACCAGGATGACTGTCTTGCGGTTAACTCTGGCGAGAACA 
               
               
                   
               
               
                 TCTGGTTCACCGGCGGCACCTGCATTGGCGGCCACGGTCTCTCCATCGGC 
               
               
                   
               
               
                 TCTGTCGGCGACCGCTCCAACAACGTCGTCAAGAACGTCACCATCGAACA 
               
               
                   
               
               
                 CTCCACCGTGAGCAATTCCGAAAACGCCGTCCGAATTAAGACCATCTCTG 
               
               
                   
               
               
                 GCGCCACTGGCTCCGTGTCCGAGATTACGTACTCCAACATCGTCATGTCT 
               
               
                   
               
               
                 GGCATCTCCGATTACGGCCTGGTCATTCAGCAGGATTACGAAGACGGCAA 
               
               
                   
               
               
                 GCCTACGGGTAAGCCGACGAACGGTGTCACTATTCAGGATGTTAAGCTGG 
               
               
                   
               
               
                 AGAGCGTGACTGGTAGCGTGGATAGTGGGGCTACTGAGATCTATCTTCTT 
               
               
                   
               
               
                 TGCGGGTCTGGTAGCTGCTCGGACTGGACCTGGGACGATGTGAAAGTTAC 
               
               
                   
               
               
                 CGGGGGGAAGAAGTCCACCGCTTGCAAGAACTTCCCTTCGGTGGCCTCTT 
               
               
                   
               
               
                 GTTAG. 
               
             
          
         
       
     
     The amino acidic sequence coding the mature mutated protein (pgaIIm) is as follows (SEQ ID No. 2): 
     
       
         
               
               
             
           
               
                   
                 DSCTFTAAAA KAGKAKCSTI TLNNIEVPAG TTLDLTGLTS 
               
               
                   
                   
               
               
                   
                 GTKVIFEGTT TFQYEEWAGP LISMSGEHIT VTGASGHLIN 
               
               
                   
                   
               
               
                   
                 CDGARWWDGK GTSGKKKPKF FYAHGLDSSS ITGLNIKNTP 
               
               
                   
                   
               
               
                   
                 LMAFSVQAND ITFTDVTINN ADGDTQGGHD TDAFDVGNSV 
               
               
                   
                   
               
               
                   
                 GVNIIKPWVH NQDDCLAVNS GENIWFTGGT CIGGHGLSIG 
               
               
                   
                   
               
               
                   
                 SVGDRSNNVV KNVTIEHSTV SNSENAVRIK TISGATGSVS 
               
               
                   
                   
               
               
                   
                 EITYSNIVMS GISDYGVVIQ QDYEDGKPTG KPTNGVTIQD 
               
               
                   
                   
               
               
                   
                 VKLESVTGSV DSGATEIYLL CGSGSCSDWT WDDVKVTGGK 
               
               
                   
                   
               
               
                   
                 KSTACKNFPS VASC. 
               
             
          
         
       
     
     As an alternative example, the plants are transformed with a gene coding a pectin methylesterase inhibitor, preferably from a plant source, more preferably of sequence (Locus tag: At3g17220; NCBI n. NM — 112599 and NP — 188348). 
     The full length nucleotide sequence is as follows (SEQ ID No. 3): 
     
       
         
               
             
           
               
                 ATGGCAGCAT ACCTGACGAA CAGAGTTTTA ATGTCTTCTC 
               
               
                   
               
               
                 TGATGTTTTT TGTAATGACT GGTTCTTTGA ACGCACAAGT 
               
               
                   
               
               
                 GGCAGACATA AAAGCGATAT GTGGAAAAGC GAAAAACCAA 
               
               
                   
               
               
                 TCCTTCTGTA CGAGCTACAT GAAATCCAAC CCAAAGACCT 
               
               
                   
               
               
                 CAGGTGCTGA TCTTCAAACG CTTGCAAATA TCACATTTGG 
               
               
                   
               
               
                 TTCTGCACAA ACAAGTGCAT CAGAAGGTTT CAGGAAAATT 
               
               
                   
               
               
                 CAATCTCTAG TCAAGACAGC AACCAACCCC ACTATGAAGA 
               
               
                   
               
               
                 AAGCATACAC CTCATGTGTA CAACATTATA AGAGTGCAAT 
               
               
                   
               
               
                 AAGCAGTCTC AATGATGCTA AGCAGAGCCT GGCGTCAGGC 
               
               
                   
               
               
                 GATGGCAAAG GGTTGAACAT TAAGGTTTCA GCAGCTATGG 
               
               
                   
               
               
                 AAGGACCTTC AACATGTGAA CAAGACATGG CGGATTTCAA 
               
               
                   
               
               
                 AGTTGATCCT TCAGCTGTGA AGAACAGTGG TGATTTTCAG 
               
               
                   
               
               
                 AATATTTGTG GCATTGTACT TGTCATCTCA AACATGATGT GA 
               
             
          
         
       
     
     The amino acid sequence coding the protein is as follows (SEQ ID No. 4): 
     
       
         
               
             
           
               
                 MAAYLTNRVLMSSLMFFVMTGSLNAQVADIKAICGKAKNQSFCTSYMKSN 
               
               
                   
               
               
                 PKTSGADLQTLANITFGSAQTSASEGFRKIQSLVKTATNPTMKKAYTSCV 
               
               
                   
               
               
                 QHYKSAISSLNDAKQSLASGDGKGLNIKVSAAMEGPSTCEQDMADFKVDP 
               
               
                   
               
               
                 SAVKNSGDFQNICGIVLVISNMM 
               
             
          
         
       
     
     As a further and alternative example,  Arabidopsis  plants are transformed with a gene coding a bacterial pectate lyase from a bacterial source (pel1 pectate lyase 1 of  Pectobacterium carotovorum ) (Bartling, 1995) (NCBI n. X81847 and CAA57439) here named PL1 plants. 
     The full length nucleotide sequence is as follows (SEQ ID No. 5): 
     
       
         
               
               
             
           
               
                   
                 cccgggggat ctcaaagcaa tcgggtagcg atgctgaatc 
               
               
                   
                   
               
               
                   
                 aataatgagc gaatgatagc gggtgaccgt caaaggctga 
               
               
                   
                   
               
               
                   
                 gctagccctg cgaaaacccc cgtttcgctg tgcgcaatct 
               
               
                   
                   
               
               
                   
                 cagaggtttt cccgtgcatg acctgccgtg ctcgcaccac 
               
               
                   
                   
               
               
                   
                 acgcgcgccg aacgcctgtc ccatcgcctg atggccaaga 
               
               
                   
                   
               
               
                   
                 cacacgccca gaataggcag tttatcggca aagtgacgga 
               
               
                   
                   
               
               
                   
                 tagcagccag tgaaatgccc gcctcatccg gcgtacaagg 
               
               
                   
                   
               
               
                   
                 gccaggtgaa ttaaccaatc gctcaggggc aagccgttca 
               
               
                   
                   
               
               
                   
                 atctcgcgca gcgtcagttc atcattacgc ttcaccacga 
               
               
                   
                   
               
               
                   
                 cctgtgcgcc aagctcgcaa aagtattggt aaaggttgta 
               
               
                   
                   
               
               
                   
                 ggtaaaggag tcgtagttat cgataattag cagcatagtc 
               
               
                   
                   
               
               
                   
                 attgcactgt tagtcggaaa agccgtacta acatacatga 
               
               
                   
                   
               
               
                   
                 aatccgcgtc aggtacccac ccctcgtcac catcaaagac 
               
               
                   
                   
               
               
                   
                 aaaggtggcg atctcgctgt ttaagaaatt agcatggtaa 
               
               
                   
                   
               
               
                   
                 taattttatc gatcataaat catttatttc atcagtaaac 
               
               
                   
                   
               
               
                   
                 atctttatta ataggcctta tttattatcc caattcacag 
               
               
                   
                   
               
               
                   
                 taaacgatta ccttgaaatt atttttaaca aaaaaaataa 
               
               
                   
                   
               
               
                   
                 taagaaaaaa ccgcctatga attaattcat tttttttaaa 
               
               
                   
                   
               
               
                   
                 aggaagaaaa actaaaggga tcatttctta cgtgatattt 
               
               
                   
                   
               
               
                   
                 tttggtggcg atcacaatcg ttcaacaagc gaataaccac 
               
               
                   
                   
               
               
                   
                 gcatataaac gggataaaaa ataaagaacc cttaaaaaca 
               
               
                   
                   
               
               
                   
                 taaagacatg aatttaaatg atttaaatag aaattggttt 
               
               
                   
                   
               
               
                   
                 ctatttgaaa tagatagaca caaatcctct caactgtcct 
               
               
                   
                   
               
               
                   
                 ctgttattta attaatatat ttaacgcccc atcctgtatt 
               
               
                   
                   
               
               
                   
                 gtttgttttt ctggtaacac ttatctcggc tttatagaca 
               
               
                   
                   
               
               
                   
                 aaccttattt tcatttttgt tgaaacagcc tttttaaatt 
               
               
                   
                   
               
               
                   
                 acacagggtg tgtagtgata aatacccaaa aaattctatg 
               
               
                   
                   
               
               
                   
                 tcaaggagag tacataatga aatacctact gccttctgca 
               
               
                   
                   
               
               
                   
                 gcagctgggc tggtgctgct cgctgcccaa ccgacaatgg 
               
               
                   
                   
               
               
                   
                 cggcaaatac ggggggttat gccaccactg acggcggcga 
               
               
                   
                   
               
               
                   
                 cgtttctggt gcggtgaaaa aaacggcgcg ttctctgcaa 
               
               
                   
                   
               
               
                   
                 gagatcgtcg atatcattga agccgcgaaa aaggactcaa 
               
               
                   
                   
               
               
                   
                 gcggtaaagc ggtcaaaggc ggagcctacc cgctcgttat 
               
               
                   
                   
               
               
                   
                 tacctacaac ggtaatgaag atgcgctaat caaagccgcc 
               
               
                   
                   
               
               
                   
                 gaagctaaca tctgcggcca gtggagcaaa gatccgcgcg 
               
               
                   
                   
               
               
                   
                 gtgtggaaat caaagagttc accaaaggga tcaccattct 
               
               
                   
                   
               
               
                   
                 cggaaccaac ggttcttccg ctaacttcgg tatctggatg 
               
               
                   
                   
               
               
                   
                 gttaactctt ccaacgttat cgtacgtaac atgcgcttcg 
               
               
                   
                   
               
               
                   
                 gctatatgcc gggcggtgca aaagatggtg atgccatccg 
               
               
                   
                   
               
               
                   
                 tatcgataat tcaccgaacg tctggatcga ccacaacgag 
               
               
                   
                   
               
               
                   
                 atcttcgcca agaacttcga atgtgccggt acgccagaca 
               
               
                   
                   
               
               
                   
                 acgacaccac ctttgaatcc gcggttgata tcaagaaagc 
               
               
                   
                   
               
               
                   
                 ctcaaccaac gtcaccgtgt cgtacaacta tatccatggc 
               
               
                   
                   
               
               
                   
                 gtgaaaaagg tagggttgag cggttcaagc aacacggata 
               
               
                   
                   
               
               
                   
                 ccggtcgtaa cctgacttac catcacaata tttacagcga 
               
               
                   
                   
               
               
                   
                 tgttaactca cgtctgccgc tgcaacgtgg tggccaagtc 
               
               
                   
                   
               
               
                   
                 catgcttata acaacctgta tggcggcatc aaaagttcag 
               
               
                   
                   
               
               
                   
                 gctttaacgt ccgtcagaaa gggatcgcac tgatcgaaag 
               
               
                   
                   
               
               
                   
                 taactggttc gaaaatgcgc tcaacccagt gaccgcacgt 
               
               
                   
                   
               
               
                   
                 aatgacgatt ccaacttcgg tacttgggaa ctccgtaaca 
               
               
                   
                   
               
               
                   
                 acaacattac cagcccatct gattttgcta aatacaacat 
               
               
                   
                   
               
               
                   
                 cacctggggt aaaccatcca caccgcacat caatgcggat 
               
               
                   
                   
               
               
                   
                 gactggaaga gcaccggtaa attcccagcc gtcccatata 
               
               
                   
                   
               
               
                   
                 gctactctcc agtttctgca cagtgcgtga aggataaact 
               
               
                   
                   
               
               
                   
                 ggcaagctac gctggcgtag gtaaaaacca ggcagtactg 
               
               
                   
                   
               
               
                   
                 acagcagcca actgtaaata aacgcgttaa gcccgacgca 
               
               
                   
                   
               
               
                   
                 ttcgttctgc gtcgggttct tcctgagcga gtgcttcggc 
               
               
                   
                   
               
               
                   
                 ctcgctcatt tttgaaaggc cggagcaaga ttgtcagcgt 
               
               
                   
                   
               
               
                   
                 gaccgccatg aaaatagacg tcgtactcat gacaatttca 
               
               
                   
                   
               
               
                   
                 ccaataagcc gaggtctatt tttcttttat cacgtaaaga 
               
               
                   
                   
               
               
                   
                 aacgccatat agcgattaat aatcgttatt tcttaaaaaa 
               
               
                   
                   
               
               
                   
                 actaaaattc gcgataaaac tgactcatca cgcgaacaat 
               
               
                   
                   
               
               
                   
                 attgaatacg tctgtgatag tatattttga cttaaggtag 
               
               
                   
                   
               
               
                   
                 tgttttacgt tgacggcagc gatcgccaca agaaaaataa 
               
               
                   
                   
               
               
                   
                 caatttaaaa accagccaat aaagaaactc gagtgcattt 
               
               
                   
                   
               
               
                   
                 attatctact aaaaaagtaa ccttatgatt taccgttact 
               
               
                   
                   
               
               
                   
                 ttaaagggaa attattttct ataaaaaata aaaccatcca 
               
               
                   
                   
               
               
                   
                 atcatcagta ttaccaaatg tttcatccgc aatacattta 
               
               
                   
                   
               
               
                   
                 acatttcacc cttggactga tcttattttt tgaccacact 
               
               
                   
                   
               
               
                   
                 ccccttggtt tttcaccaaa attgaaattc atttttgttg 
               
               
                   
                   
               
               
                   
                 aaaaatttac acttgttaca tcgggcatag ggatcgataa 
               
               
                   
                   
               
               
                   
                 atgcccatga aaattctatt ccaaggagac agtgataatg 
               
               
                   
                   
               
               
                   
                 aaatacctat tgcctacggc agccgctgga ttgctattac 
               
               
                   
                   
               
               
                   
                 tcgcggctca acccgcaatg gccgcaaata cgggcggcta 
               
               
                   
                   
               
               
                   
                 tgccactacg gatggtggag aagtgtccgg tgccgtgaaa 
               
               
                   
                   
               
               
                   
                 aaaacggcac gttccatgaa agaaattgtg gatattattg 
               
               
                   
                   
               
               
                   
                 aagccgcgca agtggattca aaaggcaaga aagtcaaagg 
               
               
                   
                   
               
               
                   
                 cggtgcttac ccgctcatca tcacctatag cggtaatgaa 
               
               
                   
                   
               
               
                   
                 gactcattaa tcaaagcggc tgaaaagaat atctgcggcc 
               
               
                   
                   
               
               
                   
                 agtggagtaa agacgcacgc ggcgtacaaa tcaaagagtt 
               
               
                   
                   
               
               
                   
                 caccaaaggc attactatcc agggcaccaa tggctcatcc 
               
               
                   
                   
               
               
                   
                 gccaacttcg gtgtctggat tgtgaactct tctaatgtcg 
               
               
                   
                   
               
               
                   
                 tgctacgtaa tatccgcttt ggctatatcc cgggcggcgc 
               
               
                   
                   
               
               
                   
                 gcaacacggc gatgccattc gtatcgataa ctccccgaac 
               
               
                   
                   
               
               
                   
                 gtctggatcg accacaacga aatctttgcc aagaactttg 
               
               
                   
                   
               
               
                   
                 agtgtaaagg cacgccagac aatgacacca cctttgaatc 
               
               
                   
                   
               
               
                   
                 ggctgtcgat atcaaaaaag ggtcaactaa cgtcacggta 
               
               
                   
                   
               
               
                   
                 tcctacaact atattcatgg tatcaagaaa gtcggcctga 
               
               
                   
                   
               
               
                   
                 gcggcgcaag caatacggat acgggccgta acctgactta 
               
               
                   
                   
               
               
                   
                 ccatcacaat atttatagcg atgttaactc acgcctgccg 
               
               
                   
                   
               
               
                   
                 ctgcaacgtg gtggtctggt tcacgcgtac aacaacctgt 
               
               
                   
                   
               
               
                   
                 atgacggcat caccggttca ggctttaatg tgcgtcagaa 
               
               
                   
                   
               
               
                   
                 agggatcgca ctgattgaaa gcaactggtt cgagaatgcg 
               
               
                   
                   
               
               
                   
                 ctcaacccag tgacagcacg taacgacagc tcaaactttg 
               
               
                   
                   
               
               
                   
                 gtacctggga gctgcgtaac aacaacatca cgaaaccggc 
               
               
                   
                   
               
               
                   
                 agacttctcc aaatacaaaa tcacctgggg caagccttcc 
               
               
                   
                   
               
               
                   
                 tctcctcaca tcaatgcgga tgactggaag agcaccggta 
               
               
                   
                   
               
               
                   
                 agttccctgc cgtctcctat aagtacactc cagtttctgc 
               
               
                   
                   
               
               
                   
                 acagtgcgtg aaggataaac tggcaaacta tgctggcgtc 
               
               
                   
                   
               
               
                   
                 ggtaaaaacc tggcagtact gacagcagct aactgcaaat 
               
               
                   
                   
               
               
                   
                 aaacgcggtc aggctttctc cgtcgtcgca agacaggaag 
               
               
                   
                   
               
               
                   
                 caataagtct gaatatcccg cgccgtgact cttcatacag 
               
               
                   
                   
               
               
                   
                 aagcatggtt ctacccaagc gagcctctta gagctcgctc 
               
               
                   
                   
               
               
                   
                 atttttttat ggaaactcag gcgcgaacat ggatatgcat 
               
               
                   
                   
               
               
                   
                 tcctaaagag caggcataaa ttaaatatca ggctaattat 
               
               
                   
                   
               
               
                   
                 taatataatg aattaatagc ttgatttatt aatggagaaa 
               
               
                   
                   
               
               
                   
                 accatccttt aattaatgat taatcaatag aaataacaac 
               
               
                   
                   
               
               
                   
                 agcccatcaa cacgtgatta attaataatt tgatcggcat 
               
               
                   
                   
               
               
                   
                 caactatttc tgacgccatc atctcagcga gtaataaaac 
               
               
                   
                   
               
               
                   
                 gaaatataat tttatttttc ataaaactct caatggccta 
               
               
                   
                   
               
               
                   
                 attttagaaa aaatgaaaaa aatattacca tttaaaatag 
               
               
                   
                   
               
               
                   
                 gcacttatta gtctttgtga tttccaccgt tactcactct 
               
               
                   
                   
               
               
                   
                 atatatttaa tatcgaaata ttgaattctt ttaattttat 
               
               
                   
                   
               
               
                   
                 ggtaagaatt aatcgggatt tcaataaacc ttgatttcat 
               
               
                   
                   
               
               
                   
                 ttttattgaa acaccaaaat aatataatct gggttatgtg 
               
               
                   
                   
               
               
                   
                 gatcataaat gcccaaacaa aaattctatt ccaaggagag 
               
               
                   
                   
               
               
                   
                 taccctaatg aaatacctac tgccttctgc agccgctggg 
               
               
                   
                   
               
               
                   
                 ctgttattgc ttgcggccca accaacgatg gtggcaaata 
               
               
                   
                   
               
               
                   
                 cggggggtta tgccaccact gacggtggtg acgttgccgg 
               
               
                   
                   
               
               
                   
                 tgccgtgaaa aaaacagcgc gctccatgca agatattatt 
               
               
                   
                   
               
               
                   
                 gatatcatcg aagccgcaaa gctggattcc aatggcaaga 
               
               
                   
                   
               
               
                   
                 aagtcaaagg tggcgcttta ccgcttgtca tcacctttat 
               
               
                   
                   
               
               
                   
                 cggtaatgaa gacgcgctga tcaaagccgc tgaggccaac 
               
               
                   
                   
               
               
                   
                 atctgcggcc agtggagtaa ggatgcccga ggtgtggaaa 
               
               
                   
                   
               
               
                   
                 tcaaagagtt caccaaaggg attaccatcc ttggaactaa 
               
               
                   
                   
               
               
                   
                 cggatcgtcc gccaacttcg ggatctggct gacaaagtca 
               
               
                   
                   
               
               
                   
                 tccgatatcg tcatacgtaa catgcgtttt ggttacatgc 
               
               
                   
                   
               
               
                   
                 cgggcggcgc gcaggatggc gatgccattc gtatcgataa 
               
               
                   
                   
               
               
                   
                 cacgccgaac gtctggattg accacaacga gatcttcgcg 
               
               
                   
                   
               
               
                   
                 aaaaactttg aatgcgcagg tacaaaagac ggtgacacga 
               
               
                   
                   
               
               
                   
                 cattcgagtc cgcgattgat attaagaaag cttcgaccaa 
               
               
                   
                   
               
               
                   
                 cgtgaccatt tcgtacaact acattcatgg catcaaaaaa 
               
               
                   
                   
               
               
                   
                 gtggggctga gcggcttcag cagcagcgat acgggccgtg 
               
               
                   
                   
               
               
                   
                 acctgactta tcatcacaat atttacgacg acgttaacgc 
               
               
                   
                   
               
               
                   
                 tcgcctccca ctgcaacgtg gtggtcaggt tcatgcctac 
               
               
                   
                   
               
               
                   
                 aacaacttgt atactggcat caccagctct ggcctgaacg 
               
               
                   
                   
               
               
                   
                 tgcgtcagaa agggattgcg ctgatcgaac gtaactggtt 
               
               
                   
                   
               
               
                   
                 cgagaatgcg aaaaacccag tgacctcacg ttatgacggt 
               
               
                   
                   
               
               
                   
                 tccaacttcg gtacgtggga actgcgtaat aacaacgtca 
               
               
                   
                   
               
               
                   
                 tgagcccagc cgacttcgct aaatacaaca tcacttggga 
               
               
                   
                   
               
               
                   
                 taaagatacc aaaccctacg tgaattccga agactggaaa 
               
               
                   
                   
               
               
                   
                 aacaccggta cgtttgcttc tgttccttac agctactctc 
               
               
                   
                   
               
               
                   
                 cagtttctgc acagtgtgtg aaggacaaac tggcgaacta 
               
               
                   
                   
               
               
                   
                 tgctggcgtg aaccaaaacc tttccgtgct gtcagcagca 
               
               
                   
                   
               
               
                   
                 aattgcaact agttgcaaag cgtgaaaggt aaataagcga 
               
               
                   
                   
               
               
                   
                 atagcaggcc ccgcccctca ttgtcctgtg ctaactggag 
               
               
                   
                   
               
               
                   
                 cctgcaaaca agcttaacgc ccgtcggttt atcgttcggg 
               
               
                   
                   
               
               
                   
                 ccgcacttga gcgagcccta agggtcgctc atttttatcg 
               
               
                   
                   
               
               
                   
                 ttaatattca aggaaatgaa atgaaacgtt ctcttctgtt 
               
               
                   
                   
               
               
                   
                 cgccgcactg ttcagcaccg gcttagtgta tagtgttggt 
               
               
                   
                   
               
               
                   
                 ataccgatgg cgggcgccga cacaccggca gcaccagagt 
               
               
                   
                   
               
               
                   
                 tgaaaggatt cggaacggat accgtagcag gcagcggtgg 
               
               
                   
                   
               
               
                   
                 acgcattatt cgcgtcacca cgctagcctc ttctggagca 
               
               
                   
                   
               
               
                   
                 ggttcactca gggaagcgct ggccgccaaa ggaccgcgca 
               
               
                   
                   
               
               
                   
                 ttatcgtttt tgaggttggc ggcattatcg acctaaataa 
               
               
                   
                   
               
               
                   
                 aagtgacctt cggttatcag aaccgtttgt caccattgcc 
               
               
                   
                   
               
               
                   
                 ggccagaccg ctccctctcc cggtatcacc atcattcgcg 
               
               
                   
                   
               
               
                   
                 gcggaatggg gatcagcaca catgatgtgc tgatgcagca 
               
               
                   
                   
               
               
                   
                 tattcgtttc cgcatcggcg atgccggtac gggtaaaaaa 
               
               
                   
                   
               
               
                   
                 agcggctttg aacgcgatgt ttccatcaac ggcaaagatg 
               
               
                   
                   
               
               
                   
                 cctacaacgt cgtgatcgac cactctagtt ttcgcctggg 
               
               
                   
                   
               
               
                   
                 gtacggacga aaacctgtct atttccggcc cacgctatga 
               
               
                   
                   
               
               
                   
                 cggaccgcag ggcaccgcac accgcattac gctttcgaat 
               
               
                   
                   
               
               
                   
                 aatatcgttg ctgaaggcct atacgattcg gctcacacca 
               
               
                   
                   
               
               
                   
                 aaggtattca ctcgatgggg acgctggttc acgataacgt 
               
               
                   
                   
               
               
                   
                 gaccgacgta tcgatcgtgg gcagcctgta cgcgcacaac 
               
               
                   
                   
               
               
                   
                 aacgagcgta atgcctggtt taaagcaggt tctacaggtg 
               
               
                   
                   
               
               
                   
                 tcatggtcaa taacctgatc tacaaccccg gcatttgggg 
               
               
                   
                   
               
               
                   
                 cgttcgcgtt ggtggcgtaa aaggagagtg ggaagggaaa 
               
               
                   
                   
               
               
                   
                 accatgcccg ccagcccacg cgtctccgtt gcgggtaacg 
               
               
                   
                   
               
               
                   
                 tgatgcacta cggcacgaat accaaagcag gtttaggact 
               
               
                   
                   
               
               
                   
                 ggtaggaagc aacagctccg gcgatgtctg gatgtcagat 
               
               
                   
                   
               
               
                   
                 aacctcgcgt acgatgctca gggaaaagcg gcaccgcaaa 
               
               
                   
                   
               
               
                   
                 cgtcaggcag tgggattaac ttactcaaag tgtcccctat 
               
               
                   
                   
               
               
                   
                 ttggcctgcg ggcttaacgg catcaccggc cagcgccgtc 
               
               
                   
                   
               
               
                   
                 accaatcagg tactgcaaag cgcaggcgcg cgtcctaaag 
               
               
                   
                   
               
               
                   
                 gtcgtgatgc cgttgataaa cgtatcgtgg ataatttcaa 
               
               
                   
                   
               
               
                   
                 acagcggaaa ggtggtttcg tgaacagcca ggaagacgtc 
               
               
                   
                   
               
               
                   
                 ggcggctatc ccgttgcaac agcgacctac cgtcagctga 
               
               
                   
                   
               
               
                   
                 acgtaccgag cactggcgta gatgcctggc tacagcagat 
               
               
                   
                   
               
               
                   
                 ggccaacgcg ctggaataaa acggcgctca tggaatagca 
               
               
                   
                   
               
               
                   
                 gagtaaaatg ctaaaaggcc acatcagtgg cctttgtcgt 
               
               
                   
                   
               
               
                   
                 attctcggtt ctttgcttat ctattttgcc aatccgggca 
               
               
                   
                   
               
               
                   
                 gcactttcgc ggactgaatc acgactggcg tggtcggcac 
               
               
                   
                   
               
               
                   
                 attctggtaa ggcccgacat tcttcgttgg cacctgagaa 
               
               
                   
                   
               
               
                   
                 atcttatcca cgacgtccat gcccttcact actttaccga 
               
               
                   
                   
               
               
                   
                 acacggcgta gcccaaatca cgctggccat gatccccggg 
               
             
          
         
       
     
     The amino acid sequence coding the protein is as follows (SEQ ID No. 6): 
     
       
         
               
               
             
           
               
                   
                 mkyllpsaaa glvllaaqpt maantggyat tdggdvsgav 
               
               
                   
                   
               
               
                   
                 kktarslqei vdiieaakkd ssgkavkgga yplvityngn 
               
               
                   
                   
               
               
                   
                 edalikaaea nicgqwskdp rgveikeftk gitilgtngs 
               
               
                   
                   
               
               
                   
                 sanfgiwmvn ssnvivrnmr fgympggakd gdairidnsp 
               
               
                   
                   
               
               
                   
                 nvwidhneif aknfecagtp dndttfesav dikkastnvt 
               
               
                   
                   
               
               
                   
                 vsynyihgvk kvglsgssnt dtgrnltyhh niysdvnsrl 
               
               
                   
                   
               
               
                   
                 plqrggqvha ynnlyggiks sqfnvrqkgi aliesnwfen 
               
               
                   
                   
               
               
                   
                 alnpvtarnd dsnfgtwelr nnnitspsdf akynitwgkp 
               
               
                   
                   
               
               
                   
                 stphinaddw kstgkfpavp ysyspvsaqc vkdklasyag 
               
               
                   
                   
               
               
                   
                 vgknqavlta anck 
               
             
          
         
       
     
     The use of plants already with reduced contents of de-esterified HGA made transgenic as described above, or double transgenic plants, is also within the scope of the invention. 
    
    
     
       The present invention shall now be described by the following figures with reference to explanatory examples non-limiting the scope of protection. 
         FIG. 1 . Saccharification of PG (fungal polygalacturonase) and PMEI (pectin methylesterase inhibitor) plants. The efficiency of enzymatic hydrolysis, expressed as percentage of total sugars released in the medium, at the indicated times was measured in leaf tissues of non transformed (WT) and transgenic plants with cellulases (Celluclast 1.5 L) and (A), WT tobacco plants and AnPGII ( Aspergillus niger  endopolygalacturonase II, PG)) showing an enzymatic hydrolysis efficiency for PG of at least 55% at 24 hours, compared to an enzymatic hydrolysis efficiency of about 20% for WT at 24 hours; (B)  Arabidopsis  WT and expressing pgallm (PG), (C)  Arabidopsis  WT and expressing AtPMEI2 ( A. thaliana -specific pectin methyl esterase inhibitors, PMEI); (D) Saccharification of leaf tissues of WT, PG and PMEI plants pre-treated with diluted acids before enzymatic hydrolysis. The bars represent the average ±s.e.m (n&gt;6). The asterisks indicate statistically significant differences between WT and transgenic plants, according to the t test of Student (*, P&lt;0.05; ***, P&lt;0.01). The panels in the inserts demonstrate the maceration of the WT and transgenic representative samples after 48 hours of digestion. 
         FIG. 2 . Immunodot analysis of fractions of cell wall. Fractions of cell wall enriched in pectin (ChASS Chelating Agents Soluble Solids) extracted from cell wall material from leaves of WT, PMEI or AnPGII (PG) plants were applied on nitrocellulose to the dilutions indicated and tested with JIM5 and PAM1 monoclonal antibodies. 
         FIG. 3 . Efficiency of saccharification of  Arabidopsis  WT plantules in the presence of beta-estradiol and PL1 plants with and without beta-estradiol inductor. Saccharification carried out on seedlings of non transformed (WT) and transgenic (PL1) lines with cellulose (Cellulast 1.5 L 0.1% v/v) previously treated and not treated with beta-estradiol. The efficiency of the enzymatic hydrolysis is expressed as percentage of reducing sugars released in the medium compared to the total sugars of the tissue at the times indicated. The panel in the inserts shows the maceration after 24 hours of digestion of WT seedlings incubated with 50 μM beta-estradiol, PL1 seedlings non induced and induced with beta-estradiol. 
     
    
    
     MATERIALS AND METHODS 
     Transgenic Plants 
     Plants of  Arabidopsis thaliana , ecotype Columbia (Col.-0) were obtained from G. Redei and A. R. Kranz (Arabidopsis Information Service, Frankfurt, Germany). The generation PMEI plants and tobacco plants expressing pgallm has been described (23,24). For the transgenic expression of pgallm in  Arabidopsis , an expression cassette was used which included the promoter of the 35S of the Cauliflower Mosaic Virus (CaMV) of the binary vector pBI121 (Stratagene: GenBank ID AF485783), the coding gene pgallm fused with peptide signal of PGIP1 from  Phaseolus vulgaris  (corresponding to the first 87 nt. of the sequence X64769 (3) and the terminator of the gene nopaline synthase (NOS) of the vector pBI121, excised from the construction described in (2) by double digestion of plasmid with Pstl and EcoRI. The fragment of DNA excised was cloned in the binary vector Pcambia3300 (cambia, Can berra, Australia) and the recombinant vector used to transform the strain GV3101 (pMP90RK) of  Agrobacterium tumefaciens  through electroporation (31).  Arabidopsis  (ecotype Col-0) plants of 4 week were stably transformed with the floral-dip method (32). The transgenic plants PG or PMEI T2 plants were selected on the soil after irroration with BASTA (300 uM Phosphinothricin, PPT). The resistant lines were transferred to soil without herbicide and the seeds collected. The T2 progeny was selected on solid sterile Murashige-Skoog soil with 8 mg L −1  of PPT, and the lines with a ratio of segregation of 3:1 by resistance to PPT were selected for analysis. The homozygous lines were analyzed for protein expression and activity by Western blot analysis and test of diffusion in agar as described in (24). The growth of WT and transformed plants was carried out in controlled environment rooms, at 22° C., 70% of relative humidity with a photoperiod of 16-h light and 8-h dark (100 μmol m −2 s −1  of fluorescent light). 
     For the quantification of the fresh weight (FW) and dry weight (DW) of the rosette after 15 days of growth, the plants were transferred to a photoperiod of 12-h (100 m −2 s −1  of fluorescent light). 
     The Tobacco plants were grown in a greenhouse at 23° C. and 60% relative humidity with a photoperiod of 16-h light and 8-h darkness (130 μmol m −2 s −1  of fluorescent light). 
     For the transgenic expression of pel1 in  Arabidopsis , the gene was fused to the peptide signal of PGIP1 from  Phaseolus vulgaris  (corresponding to the first 87 nt of the sequence X64769) and to the HA of the hemagglutinin of the virus of human influenza (corresponding to the 27 nt from 10 to 37 of the sequence EF014106) and was cloned in the vector of expression in plant inducible by estrogen pMDC7 (Zuo, 2000). Such vector was used to transform strain GV3101 of  Agrobacterium tumefaciens  (31) by electroporation.  Arabidopsis  (Col-0) plants of 4 week were transformed with floral-dip method (32). The T1 PL1 transgenic plants were selected after a growth of 4 days in darkness on solid soil Murashige-Skoog (MS), 0.5% sterile sucrose containing Hygromycin 20 mg/l. The plants resulting positive after selection were transferred to soil and the seeds collected. The PL1 lines selected with antibiotic were induced with 50 μM beta-estradiol and analyzed for protein expression enzymatic activity by western blot analysis using commercial antibodies against HA and enzymatic activity was detected by agar diffusion assay as described in (24) with the following modifications: the substrate was prepared by dissolving polygalacturonic acid (PGA) in 0.1M Tris-HCI pH 8 and 0.3 mM CaCl 2 . 
     Enzymatic Hydrolysis 
     PL1 seedlings selected in solid soil containing Hygromycin were grown in light in rooms with controlled atmosphere (22° C. photoperiod 16 h light/8 h darkness 100 μmol m −2  s −1  of fluorescent light) for three days and afterwards 10 plants (fresh weight of approximately 200 mg) were transferred to liquid sterile culture medium MS, 0.5% sucrose. After 7 days following the transfer, the plants were transferred to sterile water containing 50 iM beta-estradiol to induce the expression of the pel1. After 24 hours of induction with beta-estradiol, the plants were treated with 0.1% cellulose (Cellulast 1.5 L) at various times as indicated in the figure at 37° C. in a solution of 50 mM buffer sodium acetate at pH5.5, sterilized through filtration. Leaf explants from WT, PG and PMEI plants (100 mg of fresh weight sterilized in a solution of 1% sodium hypochlorite for 1 minute and washed two times with sterile water to avoid microbial contamination) were incubated for 20 hours at 37° C. in a solution containing 50 mM sodium acetate buffer pH 5.5, and 0.5% Celluclast 1.5 L (cellulase from  Trichoderma reesei ; Sigma, St Louis, Mo.), already sterilized through filtration. The reducing sugars released in solution were quantified with the test PAHBAH assay (4) after centrifugation. The total sugars before enzymatic hydrolysis are determined with the Dubois (33) method. 
     Chemical Pre-Treatment 
     The leaf material was mixed with dilute sulfuric acid (final concentration 1.3%) and pre-treated at 110° C. for 20 minutes. After the pre-treatment, the hydrolyzates were separated and collected through filtration and the residual biomass washed with water. 
     Immunodot Assay 
     AIS (Insoluble solids in alcohol) were extracted as described in (23). After washing with chloroform:methanol, the material was washed twice with 80% acetone and air dried. To obtain fractions of soluble solids in chelating agents (ChASS), the AIS (approximately 10 mg) were homogenized twice in a buffer containing 50 mM TRIS-HCI and 50 mM trans-1,2-Cyclohexanediaminetetraacetic acid (CDTA) pH 7.2, at 80° C. After centrifugation at 10,000 rpm for 10 minutes, the two supernatants were united and lyophilized. Squares of 6×6 mm were marked on membranes of nitrocellulose (Amersham, UK) with a pencil and equal quantities of ChASS fractions from each line were dissolved in water and applied in the squares drawn on the nitrocellulose, respectively in dilutions of about 3×. Specific peptic epitopes were revealed with the monoclonal antibodies PAM1 (25, 26) and JIM5 (26) (provided by Prof. P. Knox University of Leeds). 
     The membranes were blocked in MPBS (1×PBS with 3% “Membrane blocking reagent powder”, Amersham, UK) for 1 hour before the incubation with the primary ab (supernatants of hybridomas of JIM5 and LM7 diluted 1/10 or of PAM1 diluted 1/20 in 3% MPBS) for 1.5 h. After washing in 1×PBS, the membranes were incubated with the secondary ab (anti-rat conjugate with peroxidase from radish, Amersham, UK) diluted 1/1000 for JIM5 and with an anti-histidine antibody conjugate with peroxidase from radish (Sigma A-7058) diluted 1/1000 for PAM1. The membranes were washed as described and subsequently treated with the ECL reagent (Amersham, UK) for the measurement of the peroxidase activity. 
     Results 
     The authors analyzed the efficiency of saccharification from leaf tissues of transgenic plants expressing a polygalacturonase from  Aspergillus niger  (PG plants) and of plants overexpressing an inhibitor of the PME (PMEI plants) (24). 
     The PG plants show reduced levels of HGA, while the PMEI plants have a reduced activity of PME and an increased methylation of HGA. 
     In PG plants of  Arabidopsis  or tobacco ( FIGS. 1A  and B) and PMEI plants of  Arabidopsis  ( FIG. 1C ), the treatment of leaf tissues with commercial cellulase (Celluclast 1.5 L) for 24 hours causes a release of higher amount of sugars than in control WT plants. 
     The saccharification of PG and PMEI plants is accompanied by an increased maceration of the tissues (see inserts in the  FIG. 1A-C ). After an incubation of 24 hours without cellulase no release or sugars or maceration of the tissues was noted in both transformed and WT plants. This proves that the reduction in the HGA content or in its methylation does not cause by itself the disassembling of the tissues and the saccharification, but rather promotes the capacity of the cellulases to degrade the cellulose in the intact tissue. Furthermore, the efficiency of enzymatic hydrolysis on acid pre-treated leaves from WT transgenic (PG and PMEI) plants does not differ significantly ( FIG. 1D ). It must be noted that, after 24 hours of saccharification, the efficiency of enzymatic hydrolysis obtained with acid pre-treated leaves of all the plants is the same as that observed with the transgenic plants not pre-treated. No significant release of sugars is obtained after acid pre-treatment alone, regardless of the plants used. The results indicated that the degradability of the cellulose is improved in tissues from PG and PMEI plants and that it is not necessary to pre-treat such plants with acid to obtain a good saccharification. 
     A possible explanation is the reduced content of “junction zones” due to the particular HGA characteristics in these plants. 
     PG plants from tobacco have been described to show a reduced content of galacturonic acid (GalA) (23), which reduces the possibility that long chains of HGA are formed, which are necessary for the formation of “junction zones”. On the other hand, even if the PMEI plants have the same content of GalA as the WT, they show an increased level of pectin methylation (24) which also prevents the formation of “junction zones”. 
     To verify the presence of de-esterified regions of HGA in PG and PMEI plants compared to the WT ones, the following are used: a monoclonal antibody PAM 1, which specifically recognizes large de-esterified blocks of HGA (at least 30 continuous units of GalA) (25, 26) and a monoclonal antibody JIM5, which binds to pectin of low methylation level (level of methylesterification up to 40%) (26). Serial dilutions of pectic polysaccharides enriched in polyuronides (chelating agents solid solubles, ChASS), extracted from leaves of WT or  Arabidopsis  transgenic plants as in Lionetti et al. (Lionetti, V. et al. 2007). The PAM1 antibody binds epitopes both in PG and PMEI plants, but to a lesser extent than in WT plants, indicating that both the transformed plants show a reduced quantity of de-methylated HGA. 
     The JIM5 also binds epitopes in PG and PMEI plants, but to a lesser extent than in WT plants ( FIG. 2 ), indicating the presence of HGA with a higher degree of methylesterification. 
     In conclusion, the authors have demonstrated that the reduction of de-esterified HGA in cell walls increase the efficiency of enzymatic hydrolysis in the plant tissues. This change is advantageously used to improve the process of saccharification used in the production of bio fuels and other bio-products. 
     The reduction of de-esterified HGA in the cell walls of plant tissues can be obtained in different ways, such as genetic transformation for the obtention of PG and/or PMEI transgenic plants; selection of natural or mutagenesis-induced variants having elevated levels of endogenous PMEI or lower level of PME. 
     The PMEI plants have a better saccharification and also an increased yield of biomass production (approximately 80% increase) (Table I) (17, 24, 28). 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Fresh weight (FW) and dry weight (DW) of the rosette 
               
               
                 of WT, PG and PMEI  Arabidopsis  plants. 
               
             
          
           
               
                   
                 Line 
                 Rosette FW (mg) 
                 DW/FW ratio (%) 
               
               
                   
                   
               
               
                   
                 WT 
                 137.9 ± 10    
                 11.4 ± 1.8 
               
               
                   
                 PG 
                 81.6 ± 2.9 *** 
                  9.9 ± 1.9 
               
               
                   
                 AtPMEI 
                 253.4 ± 18.3 *** 
                 11.2 ± 0.2 
               
               
                   
                   
               
               
                   
                 The data represents the average ± s.e.m. of at least 6 independent samples. The asterisks indicate significant differences compared to WT according to the t test of Student (P &lt; 0.001). 
               
             
          
         
       
     
     Moreover, the plants of the invention, in particular PMEI plants display an increased resistance to microbial pathogens (24) and are therefore an ideal source of biofuels and of other commercial products. 
     BIBLIOGRAPHIC REFERENCES 
     
         
         1. Poorter, H. &amp; Villar, R. in Plant resource allocation. eds. Bazzaz F A &amp; Grace J. 39-72 (Academic Press, San Diego, USA; 1997). 
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         7. Himmel, M. E. et al Science 315, 804-807 (2007). 
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         9. Himmel, M. E. et al Science 315, 804-807 (2007). 
         10. Klinke, H. B., et al., Appl. Microbiol. Biotechnol. 66, 10-26 (2004). 
         11. Cosgrove, D. J. Nat. Rev. Mol. Cell Biol. 6, 850-861 (2005). 
         12. Lynd, L. R. et al Nat. Biotechnol. 26, 169-172 (2008). 
         13. Carpita, N. C. &amp; McCann, M. C. in Biochemistry and Molecular Biology of Plants. eds. Buchanan, B. B., Gruissem, W., &amp; Jones, R. 52-109 (American Society Plant Physiologists, Rockville, Md.; 2000). 
         14. Chen, F. &amp; Dixon, R. A. Nat. Biotechnol. 25, 759-761 (2007). 
         15. Ezaki, N., et al., Plant and Cell Physiology 46, 1831-1838 (2005). 
         16. Proseus, T. E. &amp; Boyer, J. S. Ann. Bot. 98, 93-105 (2006). 
         17. Derbyshire, P., McCann, M. C., &amp; Roberts, K. Bmc Plant Biology 7, (2007). 
         18. Ridley, B. L., O&#39;Neill, M. A., &amp; Mohnen, D. Phytochemistry 57, 929-967 (2001). 
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