The role of tetrapyrrole intermediates in the interorganellar communication in plants.

(1)新的质体信号突变体分析。我分离了600多个拟南芥突变系，在强光下，在去氟唑松（NF）的存在下，Lhcb表达水平升高。叶绿素积累量低于野生型的23个品系与gun5等位。GUN5编码mg螯合酶h亚基，在植物叶绿素合成途径中将原卟啉IX转化为mg -原卟啉IX至关重要。这些gun5等位基因在gun5 （CHLH）编码区存在错义突变。我比较了这些新的gun5等位基因中GUNS基因的突变位置和MgProtoIX水平以及lhcb -抑制表型（gun表型）。很明显，在一些突变系中，MgProtoIX水平和枪表型没有显著的相关性。这一结果与MgProtoIX的积累抑制Lhcb表达的假设不一致（Strand et al. 2003）。对于不具有叶绿素相关表型的突变体，…更多的是至少有两个不同的新位点。进一步详细的基因图谱正在进行中。(2) MgProtoXI和MgProtoIXme水平升高的突变体分析。s -腺苷- l-蛋氨酸：mg -原卟啉IX甲基转移酶（CHLM）和mg -原卟啉IX单甲基酯氧化环化酶（CRD1）是叶绿素合成所必需的酶。我测试了chlM和crd1突变体中MgProtoIX和MgProtoIXme的积累。在正常生长条件下，chIM突变体积累的MgProtoLX是野生型的10倍。crd1突变体的MgProtoIXme积累量是野生型的2-8倍。这些突变体的Lhcb mRNA水平与野生型相当。然后，我测试了MgProtoIX和MgProtoIXme在chIM gun5和crd1 gun5双突变体中的水平。它们分别显示了MgProtoIX和MgProtoIXme的显著积累。尽管在这些双突变体中MgProtoIX和MgProtoIXme的表达水平较高，但chIM gun5和crd1 gun5的gun表型并未受到抑制。与我们的结果相反，有一些强有力的证据支持MgProtoIX是一个强有力的信号因子的观点。首先，在拟南芥细胞培养中外源应用MgProtoIX抑制Lhcb的表达。其次，2,2 '-dipyridyl （CRD1和铁螯合酶抑制剂）的应用导致MgProtoIX和MgProtoIXme积累水平极高，可以抑制gun5表型。详细分析细胞内MgProtoIX水平及其识别机制对于理解质体到细胞核的信号通路是必要的。我目前的工作假设如下：(a) MgProtoIX是一种有效的信号因子。(b) MgProtoIX的亚细胞浓度（定位）对质体信号传导很重要。少

(1) Analysis of new plastid-signaling mutants.I have isolated over 600 Arabidopsis mutant lines that showed an elevated level of Lhcb expression in the presence of Norflurazon (NF) under the strong light. 23 lines that accumulate less chlorophyll than wild type were allelic to gun5. GUN5 encodes H-subunit of Mg-chelatase that is essential for the conversion of Protoporphyrin IX to Mg-Protoporphyrin IX in plant chlorophyll biosynthesis pathway. These gun5 alleles have missense mutations in the GUN5(CHLH) coding region. I compared the locations of the mutation in GUNS gene and the MgProtoIX levels as well as Lhcb-derepression phenotypes (gun phenotype) in these new gun5 alleles. It became clear that the MgProtoIX levels and gun phenotypes have no significant correlation in some mutant lines. This result is inconsistent with the hypothesis that the accumulation of MgProtoIX inhibit Lhcb expression (Strand et al. 2003).With regard to the mutants that have no chlorophyll-related phenotypes, … More there are at least two different new loci. Further detailed genetic mapping is underway.(2) Analysis of mutants that have elevated level of MgProtoXI and MgProtoIXme.S-adenosyl-L-methionine : Mg-protoporphyrin IX methyltransferase (CHLM) and Mg-protoporphyrin IX monomethyl ester oxidative cyclase (CRD1) are the enzymes essential for the chlorophyll synthesis. I tested chlM and crd1 mutant for the accumulation of MgProtoIX and MgProtoIXme. The chIM mutant accumulated 10 times more MgProtoLX than wild type under the normal growth condition. The crd1 mutant had 2-8 fold accumulation of MgProtoIXme than wild type. The Lhcb mRNA level in these mutants are equivalent to that in the wild type. I then tested the MgProtoIX and MgProtoIXme levels in chIM gun5 and crd1 gun5 double mutants. They showed a significant accumulation of MgProtoIX and MgProtoIXme, respectively. In spite of the high level of MgProtoIX and MgProtoIXme in these double mutants, the gun phenotype of chIM gun5 and crd1 gun5 were not suppressed. Contrary to our results, there are some strong evidences that support the idea that MgProtoIX is a potent signaling factor. Firstly, exogenously applied MgProtoIX to the Arabidopsis cell culture repress the Lhcb expression. Secondly, the extremely high level of MgProtoIX and MgProtoIXme accumulation caused by the application of 2, 2'-dipyridyl (inhibitor of CRD1 and Fe-chelatase) can suppress the gun5 phenotype. Detailed analysis of the intracellular MgProtoIX level and its recognition mechanisms is necessary for the understanding plastid-to-nucleus signaling pathways.My present working hypothesis is as follows; (a) MgProtoIX is a potent signaling factor. (b) Subcellular concentration (localization) of MgProtoIX is important for the plastid signaling. Less