Correlated Changes in the Activity, Amount of Protein, and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons

Abstract

Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.

Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis[W]

“…Pchlide and chlorophyllide contents of green plants were determined according to Yoshida et al (1995). The fluorescence emission spectra (600 to 720 nm) of total acetone extracts and hexane-washed extracts were recorded at room temperature with the LS50 luminescence spectrophotometer (Perkin-Elmer, Rotkreuz, Switzerland) set at an excitation wavelength of 433 nm.…”

Chlorophyll a oxygenase ( CAO ) is involved in chlorophyll b formation from chlorophyll a

“…Two micrograms of poly(A) ϩ RNA were electrophoresed on a 1% agarose͞formamide gel and blotted onto Hybond-N nylon membrane. Hybridization with 32 Plabeled probe were performed as described (13).…”

Differential Expression of Two hemA mRNAs Encoding Glutamyl-tRNA Reductase Proteins in Greening Cucumber Seedlings

“…Total RNA was extracted from cucumber cotyledons, hypocotyls, and roots as described by Yoshida et al (1995). Total RNA (4 pg) was electrophoresed in a 1% agarose/ formamide gel and transferred to Biodyne-B membrane (Pall).…”

“…hanced by light (Yoshida, 1991;Yoshida et al, 1995), but that of heme in chloroplasts is only slightly increased upon illumination (Ohashi et al, 1992).…”