Professor Paul Bolwell

Research interests

Overview of current research

The PI has been involved in plant cell wall research for many years and published widely in this area. During his research active time he has developed methodologies for purification of enzymes involved in the biosynthesis of phenolic and polysaccharide components of the wall leading to their immunolocalisation and subsequent gene identification. Particular areas of study are vascular secondary wall formation and papilla formation as part of the resistance response to bacterial and fungal pathogens.

Molecular Model of Lignification-specific Peroxidase from Tobacco
(Upper panel shows a single glycosylation site;
lower panel distribution of surface charge, blue = positive; red = negative)

1. Understanding regulation of secondary wall formation as biomass for second generation biofuels

We have worked for many years on developing model systems starting with French bean and more lately a transformed tobacco culture system to obtain a convenient source of biological material for gene discovery in secondary cell wall biosynthesis. A tobacco culture transformed with the ipt gene has proved to be useful in that it makes reasonable levels of tracheids in a continuous culture system.

Tracheid formation in a tobacco cell culture transformed with ipt gene from Agrobacterium

We have characterized this culture for tracheid formation, biosynthesis of xylan and lignin and a characteristic proteomic profile for cell wall proteins (Blee et al., 2001a). This system was used to identify and clone genes that were important to the success of the EC framework V programme (2000-2004).

"INTEGRATED CONTROL OF POLYSACCHARIDE AND LIGNIN BIOSYNTHESIS TO IMPROVE CELLULOSE CONTENT AND AVAILABILITY AND FIBRE QUALITY (COPOL)"

This programme complied with the Common Agricultural Policy in the diversification of Agriculture in the area of nonfood and forage crops. It underpinned wealth creating-opportunities that are environmentally desirable in a number of developing industrial fields. It is designed as a core discovery programme using proteomics and leading to gene isolation involved in the biosynthesis of cell wall lignins and hemicelluloses in the model species tobacco. The influence of these components on fibre properties and cellulose availability will be determined in modified plants to define the limits to which they can be manipulated beneficially. These deliverables were used to direct molecular breeding programmes and genetic modification to improve fibre in the commercial species, poplar, for biomass energy and improved pulping and paper making, maize, for improved forage digestibility and flax for improved production of industrial fibre.
More information is available on COPOL

The model tobacco system was therefore used to aid novel gene discovery in vascular tissue formation and 44 wall proteins and 110 membrane protein involved in secretion and wall modification have been subjected to proteomic analysis by MALDI and ESI-MSMS to provide sequence data to enable this. Also an EST (EH663598 – E666265) of relatively vascular-specific genes expressed in these cultures was deposited in GENBANK. The EST has been used to design qRT-PCR primers to profile the expression of genes involved in cellulose, xylan and lignin expression in transgenic tobacco plants with interesting insights into co-regulation and consequences for the desired outcomes of engineering secondary cell walls. These plants have been modified as follows:-

  • Flux into lignin by heterologous down regulation of cinnamate 4-hydroxylase (C4H) (Blee et al., 2001a)
  • Lignin monomers by down regulation of cinnamoyl CoA reductase (CCR) (Piquemal et al., 1998. Plant J. 13, 71-83)
  • Lignin polymerization by down regulation of lignin-specific cationic peroxidase (Blee et al., 2003)
  • Flux into xylan by down-regulation of UDP-glucuronate decarboxylase (Bindschedler et al., 2005; Bindschedler et al., 2007)

These lines are currently being examined for effects on the efficiency of delignification and saccharification in the conversion of biomass into biofuel. A number of microorganisms are being used to test the utility of such modifications together with Dr Alessandra Devoto (RHUL).

2. Molecular plant pathology: The Oxidative Burst

We have established a three-component model for the generation of apolplastic hydrogen peroxide in the basal resistance response in both French bean and Arabidopsis. This involves a cell wall peroxidase, the delivery of reductant(s) / substrate(s) and a relative increase in the apoplast pH.  Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a French bean type III peroxidase (FBP1) (Blee et al. 2001) exhibited an impaired oxidative burst in response to avirulent strains of P. syringae as shown by a lack of diaminobenzidine detectable ROS at the cellular level in the leaf and cerium hydroperoxide staining in cell wall appositions at the subcellular level. Moreover FBP1 antisense plants were more susceptible than wild-type plants to both fungal and bacterial pathogens. Work is ongoing with the lab of Prof Fred Ausubel, Harvard University, to establish the role of At3g49110 (AtPrx33) and At3g49120 (AtPrx34) with a high degree of homology to FBP1 and identified as down regulated by transcriptome profiling of these antisense lines. This will utilise a systems biology approach studying effects of knock-outs on gene expression, metabolite profiling and papilla formation in resistance responses. These data indicate that peroxidases play a significant role in generating H2O2 during the Arabidopsis defence response and in conferring resistance to a wide range of pathogens. Dr Dewi Davies is a colleague also involved in this work (see website).

Susceptibility of Arabidopsis transformed with antisense French bean oxidative burst peroxidases to two strains of Pseudomonas syringae

3. Optimising the dietary antioxidant content of fruit and vegetables.

Initial observations of the Bramley group that tomato fruit lines with lowered carotenoid content show elevated levels of flavonoids, has prompted the investigation of the extent of crosstalk between the two pathways. Tomato lines were generated down-regulated for two cytochrome P450s involved in phenolic biosynthesis, CYP73 and CYP84.  Lines were metabolite profiled together with other mutant lines altered for carotenoid content with limited evidence of cross talk between the two pathways revealed. Such studies give fundamental insights into the mechanisms that regulate dietary antioxidant content in order to improve such content in fruit and vegetables.

Long, M., Millar, D.J., Kimura, Y., Donnovan, G., Rees, J., Fraser, P., Bramley, P.M., Bolwell, G.P. (2006) Metabolite profiling of carotenoid and phenolic pathways in mutant and transgenic lines of tomato: identification of a high antioxidant fruit line. Phytochemistry 67, 1750-1757

Millar, D.J., Long, M., Donovan, G., Fraser, P., Boudet A-M., Danoun, S., Bramley, P.M., Bolwell, G.P. (2007)  Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids. Phytochemistry 68, 1497-1509

4. Targets for CDPKs in metabolic regulation

We identified a CDPK responsible for the phosphorylation of Phenylalanine Ammonia-Lyase. This was subsequently shown to be AtCPK1. Several lines of evidence suggest that this kinase could play crucial roles in early events during the responses to elicitor action.

In-gel assay of an Arabidopsis CDPK expressed in maize protoplasts using recombinant PAL as substrate

Either vector alone (tracks 1 and 3) or vector containing a CDPK engineered for constitutive expression of the active kinase (Tracks 2 and 3) were subjected to SDS/PAGE using histone III as substrate (Tracks 1 and 2) or recombinant PAL (tracks 3 and 4). Note the CDPK phosphorylates both PAL and histone III under incubation conditions for CDPK activity.

Research interests (continued)

Research group (1988-date)
Technicians and AssistantsResearch StudentsPostdoctoral FellowsVisiting Fellows
Chris GerrishDennis LintonDave MillarVernon Butt (Emeritus Oxford University)
Andrew BlackJane JohnsonMatthew RodgersKristin Bozak (Cal State Pomona)
Rebekka BeeversJohn TrethowanIwona Beech (now at University of Postsmouth)Przemyslaw Wojtaszek (Poznan University)
Yukiko KimuraJo PartingtonDuncan RobertsonFarida Minibayeva (University of Kazan)
Marianne LongJames HamiltonFred Zimmerlin (now at Syngenta Basel)Mario De Tullio (University of Bari)
Manuela SchuwerackVictoria BonhamSteve JupeMike Burrell (Advanced Technologies, Cambridge – now Professor Sheffield University]
Rebecca OrchardCaroline HagueBarbara McCormackPaul Finch* (RHUL)
Stefanie GruenRia KerryAbigail Gregory 
 Tony StricklandGeoff MitchellGaelle Richard 
 Sian CoxJon ReesAnn O'Connell 
Safina Khan*Adriana FaniguiloKris Blee (now at Cal State Chico) 
 Ed WheatleyEllen Rowntree 
 Stephen KiddSarah Gardner 
  Jon SisonGina Donovan 
  Jose O’Brien*Laurence Bindschedler (now at Reading University) 
  Charis Howard*Elaine Gay 
 Rupesh Paudyal*Dave Millar (now at Royal Free Hospital) 
 Natalie Sykes*Arsalan Daudi* 

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