Professor Nick Smirnoff in the plant room

Professor Nick Smirnoff in the plant room

Professor Nicholas Smirnoff
Professor of Plant Biochemistry

Research

Research interests

The main focus is on the synthesis and function of vitamin C (ascorbic acid) and other antioxidants in plants, and the functions of reactive oxygen species (ROS). The lab also has wider interests in plant metabolism, metal tolerance, and responses to environmental stresses, particularly the involvement of ROS.

Research projects

The synthesis and function of vitamin C (ascorbate).

Ascorbate is a key vitamin in the human diet and plants are the main dietary source. Recent work has completed identification of all the genes and enzymes involved in the GDP-mannose (“Smirnoff-Wheeler”) pathway of ascorbate synthesis. This shows that the pathway is the major source of ascorbate and that it is essential for plant growth, as well as for stress resistance. Current research is aimed at understanding the functions of ascorbate and how its biosynthesis is controlled.

  • The control of ascorbate synthesis by light and its role in photoprotection.
  • The role of ascorbate and ascorbate oxidase in plant cell growth and development.
  • The role of ascorbate, along with other antioxidants and reactive oxygen species, in plant-pathogen interactions.

Physiological, biochemical, molecular and -omics (transcriptomics and metabolomics) approaches are being used to investigate these topics. The main experimental material is the ascorbate-deficient (vtc) mutants of the model plant Arabidopsis thaliana.

Plants and mammals employ different ascorbate biosynthesis pathways while fungi have an analogue (erythroascorbate). Biosynthetic pathways in protists have been little explored. Biochemical approaches and genome sequence information are being used to define biosynthetic pathways in a wide range of organisms with the aim of understanding the evolution and function(s) of ascorbate.

Imaging hydrogen peroxide production in plants using HyPer, a genetically-encoded YFP sensor (PDRA- Marino Exposito Rodriguez).

Understanding the role of hydrogen peroxide as a signalling molecule is hampered by inadequate methods to measure its production and concentration in specific subcellular compartments. In collaboration with Phil Mullineaux (Essex University) we are expressing the ratiometric YFP-based hydrogen peroxide sensor HyPer in plants. Along with a new pH sensor (pHRed, to correct for the pH sensitivity of HyPer)) and evaluating their use in measuring H2O2. By the end of the project, HyPer constructs targeted to multiple subcellular compartments will be provided to the research community.

Decreasing the oxygenase activity of Rubisco: a synthetic biology approach (PDRA- Chloe Singleton)

The oxygenase activity of the primary CO2 fixing enzyme Rubisco introduces inefficiency into photosynthesis giving rise to "wasteful" photorespiration (PR). Some plants and algae have evolved CO2 concentrating mechanisms (e.g. C4 photosynthesis, HCO3- transport) to increase [CO2]/[O2] at the Rubisco active site thus reducing PR. Because of decreased PR, C4 plants can have high productivity and require less water (stomata can be more closed) and nitrogen (less Rubisco is required). Cyanobacteria concentrate CO2 by packaging Rubisco and carbonic nanhydrase (CA) in protein microcompartments (carboxysomes). CA catalyses CO2 release from bicarbonate in the vicinity of Rubisco, thereby out-competing oxygenase. Although there has been speculation about transferring carboxysomes to plants their structural complexity makes this a formidable task. Also, introduction of C4 photosynthesis or production of Rubisco are possible approaches. We propose an alternative approach in which a protein scaffold tethers Rubisco and CA in close proximity. The scaffold is a synthetic polypeptide which contains concatenated protein-peptide interaction domains that will bind Rubisco and CA that have been tagged with the appropriate interaction peptides. This method has been used to produce a three enzyme metabolon that greatly improves local substrate concentration, resulting in greater flux through the mevalonate pathway in E. coli. Although the ultimate aim is to engineer plants, it is more convenient to carry out proof of concept in a cyanobacterium. The carboxysome-based CO2 concentrating mechanism of Synechocystis sp. PCC 6803 will be disrupted and the synthetic Rubisco/CA complex introduced. The effectiveness of the Rubisco/CA complex in reducing the oxygenase reaction will be assessed by measuring the photosynthetic characteristics of the cells. In parallel, the kinetic properties of the complex will be investigated in vitro.

Frequency-modulated stimulated Raman scattering microscopy for label-free functional imaging in planta (with Julian Moger, Physics Exeter).

Stimulated Raman Scattering (SRS) microscopy is an optical imaging technique that provides label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade it has been shown to have clear advantages for a vast range of biological applications. It provides quantitative functional analysis; does not suffer from photobleaching; allows near real time imaging; and has good depth penetration (several hundreds of microns) into scattering tissues. However, due to the susceptibility of current detection schemes to optical absorption from highly pigmented samples its application in plant science has remained virtually unexplored. This project aims to overcome the limitations of SRS for plant imaging by develop

The objectives of the project are:

 

i) To develop a new detection scheme for SRS microscopy, based on Frequency Modulation (FM-SRS), which is impervious to interference from pigments.

ii) To perform a short series of proof-of-principle investigations that demonstrate how label-free imaging with FM-SRS can be used to answer fundamental biological questions that are not possible with current analytical techniques.

Metabolite profiling and metabolism of planktonic microalgae (Debbie Salmon, PhD student).

We are assessing the use of NMR and LC-MS metabolite profiling to distinguish between genotypes of Emiliania huxleyi, a globally important phytoplankton species. The aim is to assess the genetic diversity of this species both geographically and within blooms and to understand the role of calcification.

Research networks

  • Patricia Conklin (State University of New York, Cortland)
  • Takahiro Ishikawa (Shimane University, Japan)
  • Prof. Phil Mullineaux (Essex University)
  • Jim Beynon and Vicky Buchanan-Wollaston (Warwick-HRI)
  • Glen Wheeler (Plymouth Marine Laboratories)

Research grants

  • 2013 BBSRC
    Frequency-Modulated Stimulated Raman Scattering Microscopy for Label-Free Functional Imaging In-Planta
  • 2013 BBSRC
    Harnessing synthetic biology to improve photosynthesis: preparation of joint grant proposal for BBSRC-NSF photosynthesis initiative
  • 2011 BBSRC
    Decreasing the oxygenase activity of Rubisco: a synthetic biology approach
  • 2010 BBSRC
    Development of plant-based hydrogen peroxide YFP nanosensors targeted to multiple sub-cellular locations
  • 2010 EPSRC
    The Exeter Science Exchange:trading ideas to promote multi-disciplinary collaboration
  • 2009 BBSRC
    The control of ascorbate biosynthesis: the role of light and GDP-L-galactose phosphorylase
  • 2008 BBSRC
    Elucidating Signalling Networks in Plant Stress Responses. (Part of SABR consortium with Warwick and Essex Universities)
  • 2008 BBSRC
    Liquid chromatography-mass spectrometry equipment for plant metabolism and host-pathogen interactions (REI).
  • 2006 NERC
    GFP based biosensors for ascorbate and hormones
  • 2006 Leverhulme Trust
    GFP based biosensors for ascorbate and hormones
  • 2003 NERC
    Genomics of zinc hyperaccumulation in Arabidopsis halleri. (Environmental Genomics programme)
  • 2003 NERC
    Evolution of zinc hyperaccumulation.
  • 2001 BBSRC
    The role of RACs, ROS and antioxidants in cell growth: Arabidopsis root hairs as a model system.
  • 2001 BBSRC
    Metabolic profiling of sugars, sugar phosphates, nucleotide sugars and antioxidants in plants and fungi. (JREI equipment grant)

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