Plant Metabolism at UWB focuses on primary metabolism, primarily
during the course of seed germination and development. Ultimately, our aim is
to understand the metabolic forces regulating the transition from heterotrophy
to autotrophy in plants. We also have a number of other interests revolving
around plant primary and secondary metabolism and interactions with the
environment.
Acetate metabolism and signalling: (BBSRC grant 5/P14659)
We have devised a program of genetic screens to identify mutants of Arabidopsis
that are disrupted in acetate metabolism and organic acid signalling. We have
selected for mutants that germinate and establish in the presence of the toxic
acetate analogues monofluoroacetic acid and monofluoroacetamide (Fig. 1). Characterisation of these
mutants led to the identification of the enzymatic steps for the transport of
acetate into peroxisomes and for its activation to
acetyl-CoA. Surprisingly, the transport protein is
COMATOSE, which is the plant homologue of the human adrenoleukodystrophy
protein. Using microarrays and 1H-NMR profiling we
have determined that gene expression and metabolite levels, respectively, are
significantly altered in the acn1 mutant suggesting that acetate and
short-chain fatty acid metabolism is more important during early development
than first thought. In collaboration with Professor George Ratcliffe
at Oxford University
and Steve Smith at the University
of Western Australia, we
are investigating the fates of acetyl-CoA from both
fatty acids and acetate in glyoxylate cycle and other
mutants of Arabidopsis.
Related publications:
Hooks
MA, Turner JE, Murphy EC, Johnston K, Burr S, Jaroslawski
S (2007) The ATP-Binding Cassette Protein COMATOSE is instrumental in glyoxysomal acetate transport. Biochem. J. 406, 399-406
Turner
JE, Greville, K, Murphy EC and Hooks MA (2005)
Characterization of Arabidopsis Fluoroacetate
Resistant Mutants Reveals the Principal Mechanism of Acetate Activation for
Entry into the Glyoxylate Cycle. J. Biol. Chem. 280,
2780-2787
Hooks
MA, Turner JE, Murphy E, and Graham IA (2004) Acetate non-utilizing mutants of
Arabidopsis: Evidence that organic acids influence carbohydrate perception in
germinating seedlings. Mol. Genet. Genomics 271: 249-256
Figure 1.
Growth of Arabidopsis mutant seedlings on FAc. Seeds of wild-type Col-0 and the M3 generation of
mutants acn1, acn2, and acn3 were germinated and grown on standard agar plates
containing ½ Murashige and Skoog
Salts and 500 ?M FAc with and without 20 mM sucrose. The M3 generation of the EMS
mutants are shown. Photos were taken 6 days after germination.
Acyl-Activating Enzyme (AAE): (BBSRC
grant 5/P19408)
ACN1 is a member of an unknown family of proteins, which is a subgroup of the
AMP-binding protein (AMPBP) superfamily that contains
the acyl-CoA synthetases
and other proteins. We are addressing the question if any of the other AAEs have acyl-CoA synthetase activity and are involved in fatty acid
degradation. In collaboration with Prof. Ian Graham at York University,
we are taking are profiling acyl-CoAs and fatty acids
in the various AAE mutants. Other approaches that we are taking are the tissue
expression of the genes by quantitative RT-PCR, subcellular
localisation of the proteins by GFP fusions (Fig 2), and heterologous
expression of the enzymes in E. coli for the analysis of substrate specificity.
We are also investigating the structure/function relationships of ACN1.
Figure 2.
Expression of AAE15 in plastids of
cultured cells of tobacco.
Plant Responses to Cauliflower Mosic Virus (CaMV): BBSRC grant
In collaboration with Dr. Joel Milner at the University of Glasgow and Prof. Deri
Tomos at UWB, we are studying the signaling
pathways involved in host responses to the compatible virus CaMV.
Mutants in salicylic acid, jasmonic acid, and
ethylene signalling pathways show different responses to CaMV
infection. Constitutive SA producers show resistance to CaMV,
but Et signalling mutants are more less sensitive to CaMV. Et appears to be a pathogenicity factor, whereas SA likely interacts with
other signalling pathways to try to inhibit pathogen spread.
Relevant publications:
Roberts
K, Love AJ, Laval V, Laird J, Tomos AD, Hooks MA,
Milner JJ (2007) Long-distance movement of Cauliflower mosaic virus and host
defence responses in Arabidopsis follow a predictable pattern that is
determined by the leaf orthostichy New Phytol. 175, 707-717
Love
AJ, Laval V, Geri C, Laird J, Tomos AD, Hooks MA,
Milner JJ (2007) Components of Arabidopsis defence- and ethylene-signaling pathways regulate susceptibility to Cauliflower
mosaic virus by restricting long-distance movement. Mol. Plant Microbe
Interact. 20, 659-670
Laval
V, Koroleva OA, Murphy E, Lu C, Milner J, Hooks MA, Tomos AD (2002) Distribution of actin
gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells. Planta 215, 287-292
Other projects:
Metabolic regulation of the transition from heterotrophy to autotrophy
in seedlings:
This transition sees seedling go from using carbon reserves in seeds to
producing their own carbon resources by photosynthesis. There are likely to be
metabolic signals that help to govern this transition. We are taking a holistic
approach to try to identify metabolic signals regulation gene expression during
young seedling growth. We have done microarray
expression measurements of imbibed seeds to seedlings 8 days old. Metabolites
have been profiled in the same samples using 1H-NMR by Prof. Dominique Rolin’s team at INRA-Bordeaux. With the help of Dr.
Tim Ebbels at Imperial College London, we are
currently looking for correlations between metabolites and genes to identify
potential metabolic signals. We observe that metabolite profiles from 2-day-old
seeds (radical just emerged) cluster with those from days 3-8, whereas
2-day-old gene expression profiles cluster with those from days 0 -1.
The role of aconitases in metabolism and
metal homeostasis:
In higher plants, aconitases work both in the TCA and
glyoxylate cycles. In animals, aconitase
also functions as an iron responsive element binding protein to promote
translation of enzymes critical for iron uptake and metabolism. There are three
full aconitase isozymes in
Arabidopsis, apparently one cytosolic and the other
two mitochondrial. Expression of the the cytosolic aconitase is upregulated in the zinc accumulating species A. halleri compared to the zinc non-accumulator A. petraea. Which of the aconitases
predominate in organic acid metabolism, and if either functions in metal
nutrition are questions we are currently addressing.
Fluorine metabolism in higher plants:
It is commonly believed that the mechanism of fluoroacetate
(FAc) toxicity in eukaryotic cells is the inhibition
of aconitase upon conversion of FAc
to fluorocitrate. Besides the transport step
mutations in the peroxisomal acetyl-CoA synthetase and peroxisomal citrate synthase 3
prevent FAc toxicity. However, the isocitrate lyase mutant
(enzymatic step after aconitase) is slightly
resistant to FAc and 19F-NMR signals we observe upon
feeding wild-type FAc are not entirely consistent
with aconitase being the only target for inhibition.
In collaboration with Prof. George Ratcliffe, we are
feeding glyxoylate cycle mutants FAc
with the objective of using 19F-NMR resonances to explain our physiological
results.
A second study entails demonstrating if plants can make fluoroacetate.
We have obtained the S. cattleya fluorinase
gene from Prof. David O’Hagan at St. Andrews University
and are expressing it in the fluoroacetate resistant
mutant ACN1.
Metabolite QTLs for aroma in rice:
Aroma in rice is governed by a suite of about 60 different secondary compounds
although some like 2-acetylproline appear to dominate. The Centre for Arid Zone
Studies (CAZS) at UWB has developed recombinant inbred lines of rice that show unusal segregation for aroma. We are looking to profile
fragrance compounds in these recombinant inbred lines to identify metabolite QTLs using the extensive genetic markers developed for this
population of RILs. We will also associate fragrance
compounds with primary metabolites to investigate synthetic pathways and
morphological traits to understand how growth affects aroma. This information
will help guide breeding studies to develop or improve varieties with aroma that
will bring higher premiums for produce from farmers of underdeveloped
countries.