Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes.
BACKGROUND
Hydroperoxide lyase (HPL) is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX) to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively.


RESULTS
To study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two M. truncatula HPL cDNAs and the localisation of the corresponding chimeras were verified by confocal microscopy in tobacco protoplasts and leaves. Results reported here indicated a distribution of M.truncatula 9/13-HPL (HPLF) between cytosol and lipid droplets (LD) whereas, as expected, M.truncatula 13-HPL (HPLE) was targeted to plastids. Notably, such endocellular localisation has not yet been reported previously for any 9/13-HPL. To verify a possible physiological significance of such association, purified recombinant HPLF was used in activation experiments with purified seed lipid bodies. Our results showed that lipid bodies can fully activate HPLF.


CONCLUSIONS
We provide evidence for the first CYP74C enzyme, to be targeted to cytosol and LD. We also showed by sedimentation and kinetic analyses that the association with LD or lipid bodies can result in the protein conformational changes required for full activation of the enzyme. This activation mechanism, which supports previous in vitro work with synthetic detergent micelle, fits well with a mechanism for regulating the rate of release of volatile aldehydes that is observed soon after wounding or tissue disruption.
De Domenico
Stefania
S
Institute of Sciences of Food Production C.N.R. Section of Lecce, via Monteroni, 73100, Lecce, Italy. stefaniadedomenico@yahoo.it
Tsesmetzis
Nicolas
N
Di Sansebastiano
Gian Pietro
GP
Hughes
Richard K
RK
Casey
Rod
R
Santino
Angelo
A
D000446:Aldehyde-Lyases / Q000378:metabolism*; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D005453:Fluorescence; D050356:Lipid Metabolism; D046913:Medicago truncatula / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology*
D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase