Functional analysis of the gene albostrians in barley and its impact on variegation and chloroplast biogenesis (Acronym: ALBOSTRIANS)

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The albostrians mutant shows a characteristically variegated leaf phenotype and it is one of the best characterized mutants of barley (Hordeum vulgare L.). The albostrians specific variegation phenotype is not fully transmissive, hence homozygous mutant plants may exhibit a wild-type phenotype. Progeny of such a plant will segregate for wildtype green (10%), albino (10%) and variegated (80%) plants. Studies on albostrians barley led to the discovery of plastid-to-nucleus (retrograde) signaling and of the nuclear-gene encoded plastid RNA polymerase NEP. Studies on albostrians clearly demonstrated that functional plastids/chloroplasts are important for plant-pathogen interactions and that the genetic apparatus of mitochondria responds to the developmental / metabolic state of chloroplasts. In a previous, core funded, project phase we isolated the underlying single nuclear gene of barley. We could confirm the gene function by genetic linkage and independent mutant allele analysis. Allocation of the putative protein to chloroplasts could be shown transiently by co-localization experiments of a GFP tagged albostrians gene and an YFP labeled chloroplast marker. Since the gene albostrians is potentially an important factor involved in non-reversible chloroplast maturation during early embryogenesis we aim to perform functional analysis of the newly identified gene in order to reach a better understanding of the gene function, which is of particular interest for basic and applied plant sciences. We want to obtain evidence for the hypothesis that this gene is not a plastome mutator as originally suggested and does not affect the chloroplast DNA but is involved directly or indirectly in the biogenesis of plastid ribosomes. We want to find out further whether a threshold of albostrians function is involved in the variegated leaf phenotype.We will perform:Functional validation of the identified albostrians gene by transgene analysis: Overexpression of the WT gene in WT and MT genetic background; Genome editing of WT albostrians by RNAguided/Cas9 to test the threshold hypothesis of residual gene function in differently truncated proteins; Heterologous complementation of Arabidopsis cia2/cia2-like mutants with the barley albostrians gene.Determination of ultrastructural differences between wild-type (As/As, As/as) and mutant albostrians plants; intraorganellar localization of the ALBOSTRIANS protein to determine at what point of development the dysfunction of ALBOSTRIANS leads to the lack of plastid ribosomes and to variegation?Unravelling the interactory network that involves the albostrians gene product by: CHipSEQ supported by HA-tagged albostrians protein; Quantitative RNAseq; Yeast-two-hybrid analysis; TILLING analysis of the barley paralog of the albostrians gene.