Kolossov Vladimir L, Bohnert Hans J, Rebeiz Constantin A
Rebeiz Foundation for Basic Research, 2209 Edgewater, Champaign, IL 61822, USA.
Anal Biochem. 2006 Jan 15;348(2):192-7. doi: 10.1016/j.ab.2005.07.031. Epub 2005 Aug 10.
Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction. In this undertaking, Arabidopsis mutants that accumulate DV chlorophyllide a and/or DV chlorophyll [Chl(ide)] a are likely to provide an appropriate resource. Because the Arabidopsis genome has been completely sequenced, the best strategy for identifying 4VR and/or putative regulatory 4VR genes is to screen Arabidopsis Chl mutants for DV Chl(ide) a accumulation. In wild-type Arabidopsis, a DV plant species, only MV chlorophyllide (Chlide) a is detectable. However in Chl mutants lacking 4VR activity, DV Chl(ide) a may accumulate in addition to MV Chl(ide) a. In the current work, an in situ assay of DV Chl(ide) a accumulation, suitable for screening a large number of mutants lacking 4-vinyl Chlide a reductase activity with minimal experimental handling, is described. The assay involves homogenization of the tissues in Tris-HCl:glycerol buffer and the recording of Soret excitation spectra at 77K. DV Chlide a formation is detected by a Soret excitation shoulder at 459 nm over a wide range of DV Chlide a/MV Chl a ratios. The DV Chlide a shoulder became undetectable at DV Chlide a/MV Chl a ratios less than 0.049, that is, at a DV Chlide a content of less than 5%.
叶绿素生物合成的异质性主要源于平行的二乙烯基（DV）和单乙烯基（MV）生物合成途径，这两条途径由4-乙烯基还原酶（4VRs）相互连接，4VRs通过将卟啉大环第4位的乙烯基转化为乙基，从而将DV四吡咯转化为MV四吡咯。目前尚不清楚的是，各种4VR活性是由一种具有广泛特异性的酶催化，还是由一个基因或多个基因编码的一系列酶催化，其中每种酶具有狭窄的特异性。需要进一步的研究来确定4-乙烯基还原的各种调控成分。在这项工作中，积累DV叶绿素酸a和/或DV叶绿素[Chl(ide)] a的拟南芥突变体可能会提供合适的资源。由于拟南芥基因组已被完全测序，鉴定4VR和/或假定的4VR调控基因的最佳策略是筛选拟南芥Chl突变体中DV Chl(ide) a的积累情况。在野生型拟南芥（一种DV植物物种）中，只能检测到MV叶绿素酸（Chlide）a。然而，在缺乏4VR活性的Chl突变体中，除了MV Chl(ide) a之外，DV Chl(ide) a也可能积累。在当前的工作中，描述了一种原位检测DV Chl(ide) a积累的方法，该方法适用于筛选大量缺乏4-乙烯基叶绿素酸a还原酶活性的突变体，且实验操作最少。该检测方法包括在Tris-HCl:甘油缓冲液中匀浆组织，并记录77K下的Soret激发光谱。在广泛的DV Chlide a/MV Chl a比例范围内，通过459 nm处的Soret激发肩检测DV Chlide a的形成。当DV Chlide a/MV Chl a比例小于0.049时，即DV Chlide a含量小于5%时，DV Chlide a肩无法检测到。
