The Penn State Research Foundation

Advanced Technologies (cambridge) Limited

The Advanced Technologies Corp.

Simon McQueen-Mason

Daniel J Cosgrove

Mark J Guiltinan

Tatyana Shcherban

Jun Shi

Michael Meyrick Burrell

Martin Maunders

Amanda Pat Cambridge

Martin Jack Maunders

This invention relates to a new class of proteins called expansins, and methods related thereto are presented. This class of proteins can be characterized as catalysts of the extension of plant cell walls and the weakening of the hydrogen bonds in pure cellulose. Two proteins have been isolated from washed wall fragments of cucumber hypocotyls, referred to as 'cucumber expansin-29' and 'cucumber expansin-30'. Moreover, three peptide fragments from the purified cEx-29 protein were sequenced, then oligonucleotide primers were designed to amplify a portion of the expansin cDNA using polymerase chain reaction with a cDNA template derived from cucumber seedlings, and then the PCR fragment was used to screen a cDNA library to identify full length clones. Another expansin protein has been isolated from oat coleoptiles (oat expansin oEx-29), while three additional expansin sequences have been identified inand an additional two in rice.

Purified plant expansin proteins and DNA encoding same

The invention relates to the isolation and characterisation of novel expansin gene sequences from heterologous and homologous tree species and re-introducing such novel genes into trees so as to alter expansin levels. Six novel genes have been identified.has also been transformed using the cucumber EX29 sequence (GenBank, Accession No. U30382-known as Cs-EXP1) (SEQ. ID. NO: 9). A change in the plant height and internode length was observed compared with control plants.

Modification of plant fibers

The sequence is disclosed both as the nucleic acid sequence SEQ ID NO: 1 and as the corresponding amino acid sequence SEQ ID NO: 7. Five other amino acid sequences are also disclosed, two from rice and three from Arabidopsis, SEQ ID NO: 2 through SEQ ID NO:6, respectively.

Purified plant expansion proteins and DNA encoding same

Plant cell expansion is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive 'wall loosening' enzymes. By employing a reconstitution approach, we initially found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicots and monocots, but was less effective on grass coleoptile walls. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD, as measured by SDS-PAGE, associated with such activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall We proposed the name 'expansins' for this class of proteins. Expansins have been isolated from various plant sources including oat, cucumber, broccoli, celery, tomato, cotton, cabbage, and corn, and also from snail and its feces. These proteins weaken the intermolecular bonds between plant wall polysaccharides. They decrease the mechanical strength of commercial products made from polysaccharides, such as paper, and therefore present a novel approach in developing new technologies in industries which make use of such polysaccharides, such as in the paper industry, in the applications of polysaccharide gums and related products. These proteins moreover present a novel approach in the control of plant growth.

Proteins catalyzing the extension of plant cell walls

A new class of proteins and methods related thereto are presented. The proteins, which can be characterized as catalysts of the extension of plant cell walls and the weakening of the hydrogen bonds in pure cellulose, are referred to as expansins. Two proteins have been isolated by fractionation techniques from washed wall fragments of cucumber hypocotyls, referred to as 'cucumber expansin-29' and 'cucumber expansin-30' (abbreviated cEx-29 and cEx-30, with respect to their apparent relative masses as determined by SDS-PAGE). Moreover, three peptide fragments from the purified cEx-29 protein were sequenced, then oligonucleotide primers were designed to amplify a portion of the expansin cDNA using polymerase chain reaction with a cDNA template derived from cucumber seedlings, and then the PCR fragment was used to screen a cDNA library to identify full length clones. Another expansin protein has been isolated from oat coleoptiles (oat expansin oEx-29), while three additional expansin sequences have been identified in Arabidopsis and an additional two in rice. Expansins appear to be broadly distributed throughout the plant kingdom and can be identified in stem and leaf vegetables (i.e., broccoli, cabbage), fruit and seed vegetables (i.e., tomato), fiber crops and cereals (i.e., corn), and forest and ornamental crops (i.e., cotton). An expansin, generally, is a protein which has at least about 60% sequence similarity with the amino acid sequence shown in SEQ. ID. NO:1, and preferably has at least about 70% sequence similarity with SEQ. ID. NO:1.

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York, United Kingdom

Simon McQueen-Mason