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

Albostrians突变体表现出特有的叶片杂色表型，是大麦(Hordeum uggare L.)最具特征的突变体之一。白纹菌特有的杂色表型不是完全传递的，因此纯合突变植株可能表现出野生型表型。这样的植物的后代将被分离为野生型绿色(10%)、白化(10%)和杂色(80%)植物。对白纹菌大麦的研究导致了叶绿体到核(逆行)信号的发现和核基因编码的叶绿体RNA聚合酶NEP的发现。对白纹藻的研究清楚地表明，功能质体/叶绿体对于植物与病原菌的相互作用是重要的，线粒体的遗传机构对叶绿体的发育/代谢状态做出反应。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标记的白纹菌基因和YFP标记的叶绿体标记的共定位实验，可以瞬时地显示推测的蛋白质对叶绿体的分配。由于基因albostrians可能是参与早期胚胎发育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，以便更好地了解该基因的功能，这对基础和应用植物科学具有特别重要的意义。我们想要获得的证据是，该基因不是最初提出的一个叶绿体突变体，不影响叶绿体DNA，但直接或间接参与了叶绿体核糖体的生物发生。我们将进行以下工作：通过转基因分析对已鉴定的白粉菌基因进行功能验证：WT基因在WT和MT遗传背景中的过表达；用RNAGuided/Cas9编辑WT白粉菌的基因组，以检验不同截短蛋白中残留基因功能的阈值假说；拟南芥cia2/cia2类突变体与大麦白粉菌基因的异源互补。野生型(AS/AS，AS/AS)和突变型Albostrians植株之间超微结构差异的测定；ALBOSTRIANS蛋白在细胞器内的定位，以确定ALBOSTRIANS的功能障碍在什么发育点导致缺乏叶绿体核糖体和多样性？通过以下方式解开涉及白纹菌基因产物的相互作用网络：由HA标记的白纹菌蛋白支持的CHipSEQ；定量RNASEQ；酵母双杂交分析；白纹菌基因大麦平行序列的TILLING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.