Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels.

The interactive effects of light and temperature on C(4) phosphoenolpyruvate carboxylase (PEPC) were examined both in vivo and in situ using the leaves of Amaranthus hypochondriacus collected at different times during a day and in each month during the year. The maximum activity of PEPC, least inhibition by malate, and highest activation by glucose-6-phosphate were at 15.00 h during a typical day, in all the months. This peak was preceded by maximum ambient light but coincided with high temperature in the field. The highest magnitude in such responses was in the summer (e.g. May) and least in the winter (e.g. December). Light appeared to dominate in modulating the PEPC catalytic activity, whereas temperature had a strong influence on the regulatory properties, suggesting interesting molecular interactions. The molecular mechanisms involved in such interactive effects were determined by examining the PEPC protein/phosphorylation/mRNA levels. A marked diurnal rhythm could be seen in the PEPC protein levels and phosphorylation status during May (summer month). In contrast, only the phosphorylation status increased during the day in December (winter month). The mRNA peaks were not as strong as those of phosphorylation. Thus, the phosphorylation status and the protein levels of PEPC were crucial in modulating the daily and seasonal patterns in C(4) leaves in situ. This is the first detailed study on the diurnal as well as seasonal patterns in PEPC activity, its regulatory properties, protein levels, phosphorylation status, and mRNA levels, in relation to light and temperature intensities in the field.

Interaction of polyethylene glycol-6000 with C4 phosphoenolpyruvate carboxylase in crude leaf extracts as well as in purified protein form from Amaranthus hypochondriacus L.: evidence for oligomerization of PEPC in vitro and in vivo.

The interaction of phosphoenolpyruvate carboxylase (PEPC) with a compatible solute, PEG-6000, was examined using crude leaf extracts as well as the purified protein from leaves of Amaranthus hypochondriacus, a NAD-malic enzyme type C4 plant. The inclusion in the assay medium of PEG-6000 stimulated the activity of purified PEPC by about 2.5-fold over control. The addition of PEG during both extraction and assay, stimulated PEPC activity by almost 5.0 fold in crude extracts. The stimulation by PEG of the dark-form of PEPC (2.4 fold) was more than that of the light-form (1.7 fold). Gel filtration of PEPC in leaf extracts on Sephadex G-200, showed the existence of three different oligomeric forms: tetramer, dimer and monomer. The exclusion of PEG and glycerol during extraction and elution on Sephadex resulted in a marked shift of the enzyme into dimer and/or monomer, with a very small proportion of tetramer but on the contrary, the inclusion of PEG and glycerol resulted in the enzyme maintaining predominantly a tetrameric shape. Thus, the activity and the structural properties of PEPC can be influenced by the presence or absence of compatible solutes (PEG or glycerol), obviously due to changes in the microenvironment of the enzyme.

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus.

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.