Light Induction of Phosphoenolpyruvate Carboxylase in Etiolated Maize Leaf Tissue

Abstract

An antibody for phosphoenolpyruvate carboxylase was used to isolate and to quantitate the enzyme from greening maize (cv. KOU 6) leaves. The increase in enzyme activity during greening was due to de novo synthesis, which was paralleled by increases in enzyme protein and incorporation of leucine. The light-induced activity was due to one specific isoenzyme. The action spectrum for enzyme synthesis had red and blue peaks.Phosphoenolpyruvate carboxylase [orthophosphate:oxaloacetate carboxylase (phosphorylating), EC 4.1.1.311 activity increases during greening of C4 plants (4,6,10,20). This increase of activity in maize can be prevented by inhibitors of protein synthesis (6,10). This effect may be indirect e.g. due to modification of the enzyme. It has been suggested by the use of density labeling techniques that, shortly after the initiation of greening, the properties of the carboxylase from sugar cane are subjected to modification in the absence of protein synthesis (4). Whether the carboxylase is synthesized or modified during greening is important in cellular control of the enzyme and development of the C4 pathway.We report here that the increase of PEP4 carboxylase activity during the greening of maize leaf tissue is due to de novo synthesis. We also report the effects of light quality on the induction of activity and of illumination on PEP carboxylase isoenzymes. MATERIALS AND METHODSPlant Materials. Zea mays L. cv. Kou 6 was germinated and grown for 6 or 7 days in darkness at 25 C in a sand bed supplemented with the standard medium described previously (19).Greening Experiments. Greening experiments were conducted at 25 to 27 C. The etiolated seedlings or the detached leaves were irradiated with white lamps (300 w National RF and 150 w Toki RF). To study the wavelength effects on light induction of the enzyme, the seedlings were irradiated first by a white fluorescence lamp (1.0 ,uw For larger scale preparations, extraction of PEP carboxylase was carried out using a chilled Omni Mixer. Ten or 16 g primary leaves were blended four times for 30 s each time in 4 volumes 0.2 M Tris-HCl (pH 7.5) containing 10 mm 2-mercaptoethanol, 0.7% sodium ascorbate, and 1.5 g insoluble PVP. The homogenate was filtered through two layers of muslin and centrifuged at 35,000g for 15 min. The 30 to 60% saturated ammonium sulfate fraction was taken, dissolved in a few ml 50 mm Tris-HCl (pH 7.0) containing 100 ,LM EDTA and 10 mm 2-mercaptoethanol, and desalted by passing through a column of Sephadex G-25 equilibrated with the same buffer. All procedures were conducted at 4 C.Preparation of Antibody and Its Purification. Two rabbits (New Zealand White) were immunized over 1 month by injecting PEP carboxylase from mature green maize leaves and purified according to the method reported previously (22). Injections were made subcutaneously using incomplete adjuvant of Freund. During the course of immunization, a total of 3.5 mg PEP carboxylase was administered to each rabbit. IgG was purified by ammonium sulfate fractionation ...

Photocontrol of Sorghum Leaf Phosphoenolpyruvate Carboxylase

“…activity was paralleled by the accumulation of large amounts of PEPC in the leaf (3,9,11,17,28). These results were further extended by Sims and Hague (8,21) who observed a photoinduced increase in translatable mRNA upon greening of maize leaf, and then by Harpster and Taylor (10) who quantitatively characterized the specific mRNA in maize leaf extracts.…”

Comparative Studies of Phosphoenolpyruvate Carboxylase from C3 and C4 Plants

“…5). These results show that there are two types of PEPC expression in rice, one resembling that of PEPC in C4 plants, which is induced by light (9,14). Greening wheat seedlings also had one band induced by light (Fig.…”

“…17). The enzyme in C4 and CAM plants have been well characterized as to their biochemical and kinetic properties (2,19), regulation of gene expression (3,9,15,18), and molecular structure (7,11,12). However, few reports have appeared on the molecular structure and gene expression of PEPC in C3 plants (1).…”

Transcriptional and Posttranscriptional Regulation of Nitrogen-Responding Expression of Phosphoenolpyruvate Carboxylase Gene in Maize

“…In the leaves of maize two distinct isoforms of PEPC have been described (Ting and Osmond, 1973;Hayakawa et al, 1981). The major form (C 4 type) is the most abundant protein in mesophyll cells and is responsible for the primary carboxylation in the C 4 photosynthetic pathway.…”