Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding ß-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Epicuticular wax on leaf cuticles does not establish the transpiration barrier, which is essentially formed by intracuticular wax.

It is well established that waxes built up the barrier properties of cuticles, since their extraction in organic solvent e.g. chloroform increases diffusion of water and organic compounds by 1-2 orders of magnitude. Leaf surface waxes can be divided in epicuticular (on the surface of the cuticular membrane) and intracuticular (embedded in the cutin polymer) waxes. Until today there are only limited investigations dealing with the question to what extent epi- or intracuticular waxes contribute to the formation of the transpiration barrier. For Prunus laurocerasus previous studies have shown that epicuticular waxes do not contribute to the formation of the transpiration barrier. This approach successfully established for P. laurocerasus was applied to further species in order to check whether this finding also applies to a broader spectrum of species. Epicuticular wax was mechanically removed using collodion from the surface of either isolated cuticular membranes or intact leaf discs of ten further plant species differing in total wax amounts, wax compositions and transport properties. Scanning electron microscopy, which was performed to independently verify the successful removal of the surface waxes, indicated that two consecutive treatments with collodion were sufficient for a complete removal of epicuticular wax. The treated surfaces appeared smooth after removal. The total wax amounts removed with the two collodion treatments and the residual amount of waxes after collodion treatment were quantified by gas chromatography and mass spectrometry. This showed that epicuticular waxes essentially consisted of long-chain aliphatic molecules (e.g. alkanes, primary alcohols, fatty acids), whereas intracuticular wax was composed of both, triterpenoids and long-chain aliphatic molecules. Cuticular transpiration using combined replicates was measured before and after removal of surface wax. Results clearly indicated that two consecutive collodion treatments, or the corresponding solvent treatments (diethyl ether:ethanol) serving as control, did not increase cuticular transpiration of the ten further leaf species investigated. Our results lead to the conclusion that epicuticular wax does not contribute to the formation of the transpiration barrier of leaves.

Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion.

One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 µm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity.