Molecular characterization of the fatty alcohol oxidation pathway for wax-ester mobilization in germinated jojoba seeds.

Jojoba (Simmondsia chinensis) is the only plant species known to use liquid wax esters (WEs) as a primary seed storage reserve. Upon germination, WE hydrolysis releases very-long-chain fatty alcohols, which must be oxidized to fatty acids by the sequential action of a fatty alcohol oxidase (FAO) and a fatty aldehyde dehydrogenase (FADH) before they can be ß-oxidized. Here, we describe the cloning and characterization of genes for each of these two activities. Jojoba FAO and FADH are 52% and 68% identical to Arabidopsis (Arabidopsis thaliana) FAO3 and ALDH3H1, respectively. The genes are expressed most strongly in the cotyledons of jojoba seedlings following germination, but transcripts can also be detected in vegetative tissues. Proteomic analysis indicated that the FAO and FADH proteins can be detected on wax bodies, but they localized to the endoplasmic reticulum when they were expressed as amino-terminal green fluorescent protein fusions in tobacco (Nicotiana tabacum) leaves. Recombinant jojoba FAO and FADH proteins are active on very-long-chain fatty alcohol and fatty aldehyde substrates, respectively, and have biochemical properties consistent with those previously reported in jojoba cotyledons. Coexpression of jojoba FAO and FADH in Arabidopsis enhanced the in vivo rate of fatty alcohol oxidation more than 4-fold. Taken together, our data suggest that jojoba FAO and FADH constitute the very-long-chain fatty alcohol oxidation pathway that is likely to be necessary for efficient WE mobilization following seed germination.

MAP20, a microtubule-associated protein in the secondary cell walls of hybrid aspen, is a target of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile.

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

Evolution of a domain conserved in microtubule-associated proteins of eukaryotes.

The microtubule network, the major organelle of the eukaryotic cytoskeleton, is involved in cell division and differentiation but also with many other cellular functions. In plants, microtubules seem to be involved in the ordered deposition of cellulose microfibrils by a so far unknown mechanism. Microtubule-associated proteins (MAP) typically contain various domains targeting or binding proteins with different functions to microtubules. Here we have investigated a proposed microtubule-targeting domain, TPX2, first identified in the Kinesin-like protein 2 in Xenopus. A TPX2 containing microtubule binding protein, PttMAP20, has been recently identified in poplar tissues undergoing xylogenesis. Furthermore, the herbicide 2,6-dichlorobenzonitrile (DCB), which is a known inhibitor of cellulose synthesis, was shown to bind specifically to PttMAP20. It is thus possible that PttMAP20 may have a role in coupling cellulose biosynthesis and the microtubular networks in poplar secondary cell walls. In order to get more insight into the occurrence, evolution and potential functions of TPX2-containing proteins we have carried out bioinformatic analysis for all genes so far found to encode TPX2 domains with special reference to poplar PttMAP20 and its putative orthologs in other plants.

The Phanerochaete chrysosporium secretome: database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium.

The white rot basidiomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose and lignin. Towards the identification of participating enzymes, 268 likely secreted proteins were predicted using SignalP and TargetP algorithms. To assess the reliability of secretome predictions and to evaluate the usefulness of the current database, we performed shotgun LC-MS/MS on cultures grown on standard cellulose-containing medium. A total of 182 unique peptide sequences were matched to 50 specific genes, of which 24 were among the secretome subset. Underscoring the rich genetic diversity of P. chrysosporium, identifications included 32 glycosyl hydrolases. Functionally interconnected enzyme groups were recognized. For example, the multiple endoglucanases and processive exocellobiohydrolases observed quite probably attack cellulose in a synergistic manner. In addition, a hemicellulolytic system included endoxylanases, alpha-galactosidase, acetyl xylan esterase, and alpha-l-arabinofuranosidase. Glucose and cellobiose metabolism likely involves cellobiose dehydrogenase, glucose oxidase, and various inverting glycoside hydrolases, all perhaps enhanced by an epimerase. To evaluate the completeness of the current database, mass spectroscopy analysis was performed on a larger and more inclusive dataset containing all possible ORFs. This allowed identification of a previously undetected hypothetical protein and a putative acid phosphatase. The expression of several genes was supported by RT-PCR amplification of their cDNAs.