Chloroplast biogenesis 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry.
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%.
Kolossov
Vladimir L
VL
Rebeiz Foundation for Basic Research, 2209 Edgewater, Champaign, IL 61822, USA.
Bohnert
Hans J
HJ
Rebeiz
Constantin A
CA
D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics*; D018553:Cucumis sativus / Q000201:enzymology / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D010944:Plants / Q000201:enzymology / Q000235:genetics; D013053:Spectrophotometry
D010088:Oxidoreductases; C097964:(4-vinyl)chlorophyllide a reductase