Sawers Ruairidh J H, Linley Philip J, Gutierrez-Marcos Jose F, Delli-Bovi Teegan, Farmer Phyllis R, Kohchi Takayuki, Terry Matthew J, Brutnell Thomas P
Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA.
Plant Physiol. 2004 Sep;136(1):2771-81. doi: 10.1104/pp.104.046417. Epub 2004 Sep 3.
The light insensitive maize (Zea mays) mutant elongated mesocotyl1 (elm1) has previously been shown to be deficient in the synthesis of the phytochrome chromophore 3E-phytochromobilin (PPhiB). To identify the Elm1 gene, a maize homolog of the Arabidopsis PPhiB synthase gene AtHY2 was isolated and designated ZmHy2. ZmHy2 encodes a 297-amino acid protein of 34 kD that is 50% identical to AtHY2. ZmHY2 was predicted to be plastid localized and was targeted to chloroplasts following transient expression in tobacco (Nicotiana plumbaginifolia) leaves. Molecular mapping indicated that ZmHy2 is a single copy gene in maize that is genetically linked to the Elm1 locus. Sequence analysis revealed that the ZmHy2 gene of elm1 mutants contains a single G to A transition at the 3' splice junction of intron III resulting in missplicing and premature translational termination. However, flexibility in the splicing machinery allowed a small pool of in-frame ZmHy2 transcripts to accumulate in elm1 plants. In addition, multiple ZmHy2 transcript forms accumulated in both wild-type and elm1 mutant plants. ZmHy2 splice variants were expressed in Escherichia coli and products examined for activity using a coupled apophytochrome assembly assay. Only full-length ZmHY2 (as defined by homology to AtHY2) was found to exhibit PPhiB synthase activity. Thus, the elm1 mutant of maize is deficient in phytochrome response due to a lesion in a gene encoding phytochromobilin synthase that severely compromises the PPhiB pool.
先前已表明,光不敏感型玉米(Zea mays)突变体伸长中胚轴1(elm1)在植物色素发色团3E-植物胆色素(PPhiB)的合成方面存在缺陷。为了鉴定Elm1基因,分离出了拟南芥PPhiB合酶基因AtHY2的玉米同源基因,并将其命名为ZmHy2。ZmHy2编码一个由297个氨基酸组成、分子量为34 kD的蛋白质,该蛋白质与AtHY2有50%的同源性。预测ZmHY2定位于质体,在烟草(Nicotiana plumbaginifolia)叶片中瞬时表达后靶向叶绿体。分子定位表明,ZmHy2在玉米中是单拷贝基因,与Elm1基因座存在遗传连锁。序列分析显示,elm1突变体的ZmHy2基因在第III内含子的3'剪接位点处存在一个由G到A的单碱基转变,导致剪接错误和翻译提前终止。然而,剪接机制的灵活性使得一小部分符合读框的ZmHy2转录本能够在elm1植株中积累。此外,野生型和elm1突变体植株中都积累了多种ZmHy2转录本形式。ZmHy2剪接变体在大肠杆菌中表达,并使用偶联的脱辅基植物色素组装测定法检测产物的活性。结果发现,只有全长ZmHY2(根据与AtHY2的同源性定义)表现出PPhiB合酶活性。因此,玉米的elm1突变体由于编码植物胆色素合酶的基因发生损伤,严重损害了PPhiB库,从而导致对植物色素反应缺陷。