In an Arabidopsis thaliana forward genetic screen aimed at identifying mutants with altered structures of their hemicellulose xyloglucan (axy mutants) using oligosaccharide mass profiling, two nonallelic mutants (axy4-1 and axy4-2) that have a 20 to 35% reduction in xyloglucan O-acetylation were identified. Mapping of the mutation in axy4-1 identified AXY4, a type II transmembrane protein with a Trichome Birefringence-Like domain and a domain of unknown function (DUF231). Loss of AXY4 transcript results in a complete lack of O-acetyl substituents on xyloglucan in several tissues, except seeds. Seed xyloglucan is instead O-acetylated by the paralog AXY4like, as demonstrated by the analysis of the corresponding T-DNA insertional lines. Wall fractionation analysis of axy4 knockout mutants indicated that only a fraction containing xyloglucan is non-O-acetylated. Hence, AXY4/AXY4L is required for the O-acetylation of xyloglucan, and we propose that these proteins represent xyloglucan-specific O-acetyltransferases, although their donor and acceptor substrates have yet to be identified. An Arabidopsis ecotype, Ty-0, has reduced xyloglucan O-acetylation due to mutations in AXY4, demonstrating that O-acetylation of xyloglucan does not impact the plant's fitness in its natural environment. The relationship of AXY4 with another previously identified group of Arabidopsis proteins involved in general wall O-acetylation, reduced wall acetylation, is discussed.

All members of the YidC/Oxa1/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that Alb4 is required in assembly and/or stability of the CF1CF0-ATP synthase (ATPase). alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not Alb3 physically interacts with the subunits CF1beta and CF0II. Summarizing, the data indicate that Alb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.

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Members of the Alb3/Oxa1/YidC protein family function as insertases in chloroplasts, mitochondria, and bacteria. Due to independent gene duplications, all organisms possess two isoforms, Oxa1 and Oxa2 except gram-negative bacteria, which encode only for one YidC-like protein. The genome of Arabidopsis thaliana however, encodes for eight different isoforms. The localization of three of these isoforms has been identified earlier: Alb3 and Alb4 located in thylakoid membranes of chloroplasts while AtOxa1 was found in the inner membrane of mitochondria. Here, we show that the second Oxa1 protein, Oxa1b as well as two Oxa2 proteins are also localized in mitochondria. The last two isoforms most likely encode truncated versions of Oxa-like proteins, which might be inoperable pseudogenes. Homozygous mutant lines were only obtained for Oxa1b, which did not reveal any significant phenotypes, while T-DNA insertion lines of Oxa1a, Oxa2a and Oxa2b resulted only in heterozygous plants indicating that these genes are indispensable for plant development. Phenotyping heterozygous lines showed that embryos are either retarded in growth, display an albino phenotype or embryo formation was entirely abolished suggesting that Oxa1a and both Oxa2 proteins function in embryo formation although at different developmental stages as indicated by the various phenotypes observed.