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

白纹突变体表现出典型的叶片杂色表型，是大麦（Hordeum vulgare L.）性状最好的突变体之一。白体特异性杂交种表型不能完全传递，因此纯合突变株可能表现为野生型表型。这种植物的后代将分离为野生型绿色（10%），白化（10%）和杂色（80%）植物。对白纹大麦的研究导致了质体向核（逆行）信号传导和核基因编码质体RNA聚合酶NEP的发现。对白纹生物的研究清楚地表明，功能性质体/叶绿体对植物与病原体的相互作用很重要，线粒体的遗传装置对叶绿体的发育/代谢状态作出反应。在之前的核心资助项目阶段，我们分离了大麦潜在的单一核基因。我们可以通过遗传连锁和独立突变等位基因分析来确认该基因的功能。通过GFP标记的白体基因和YFP标记的叶绿体标记的共定位实验，可以短暂地显示推定的蛋白质在叶绿体中的分配。由于白体基因可能是早期胚胎发生过程中叶绿体不可逆成熟的重要因素，我们的目标是对新发现的基因进行功能分析，以便更好地了解基因功能，这对基础和应用植物科学特别感兴趣。我们希望获得证据，证明该基因不是最初提出的质体突变，不影响叶绿体DNA，而是直接或间接参与质体核糖体的生物发生。我们想进一步了解白纹体功能的阈值是否与杂色叶表型有关。我们将进行：通过转基因分析对鉴定的白纹基因进行功能验证；WT基因在WT和MT遗传背景下的过表达；利用rnguided /Cas9对白白WT进行基因组编辑，验证不同截短蛋白中残余基因功能的阈值假设拟南芥cia2/cia2样突变体与大麦白体基因的异源互补。野生型（As/As、As/As）和突变型白体植物超微结构差异的测定ALBOSTRIANS蛋白的胞内定位，以确定在发育的哪个阶段，ALBOSTRIANS功能障碍导致质体核糖体的缺乏和变异？通过ha标记白纹体蛋白支持的CHipSEQ揭示白纹体基因产物的相互作用网络；定量RNAseq;Yeast-two-hybrid分析;大麦白体基因平行体的耕作分析。

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.