Purpose

1. The Effects of Chilling on Maize Photosynthesis after Interrupting the C4 Cycle with an Inhibitor of Phosphoenolpyruvate CarboxylaseMelissa L. Sharps and George T. ByrdFerrum College, Ferrum, VA 24088 • Research Plan • Attempts will be made to separate the C4 and Calvin-Benson cycles in the leaf by chemically inhibiting phosphoenolpyruvate (PEP) carboxylase, the enzyme that initially binds CO2 from the atmosphere in C4 plants. Activities of PEP carboxylase and rubisco activities and leaf photosynthetic efficiency (monitored using chlorophyll fluorescence techniques) were monitored throughout the chilling treatments and in the absence of an operational C4 cycle (that is in the presence of the PEP carboxylase inhibitor) • PEPCarboxylaseInhibition • PEP carboxylase in detached maize leaves was inhibited by placing the leaf bases in 4mM 3,3-dihydroxyphosphinoylmethylo-2-propenate (DCDP) for 30 minutes at 25°c under high light. DCDP is a competitive inhibitor of the enzyme. • Chilling Protocol • After the 30 minutes of photosynthetic equilibration of maize leaves (+ or – DCDP) • Leaves were placed in petri dishes (+ or – DCDP) and floated on ice water (cold treatment) or ambient water (control) • Leaves were exposed to high light (1000 µmol m-2 s-1) for 8 hours • After the 8 hours all treatments were maintained at 20°c and low light (100 µmol m-2 s-1) and the leaves in DCDP petri dishes placed in water dishes Photosynthetic Efficiency The effect of chilling on photosynthetic performance in maize leaves (+ or – DCDP) was monitored throughout the experiment using chlorophyll fluorescents techniques (See Table 1) • Assays for Enzyme Activity After extraction of rubisco and PEP carboxylase the oxidation of NADH can be followed spectrophotometrically at 340 nm. • Purpose • To determine how short-term exposure to chilling temperatures in the light affects the activities of the photosynthetic carboxylating enzymes within leaves of the C4 plant maize. Background C4 plants occur rarely in cooler climates in which average temperatures during the growing season are less than about 16°c, presumably because photosynthesis is inhibited by low temperatures (Sage et al., 1999). The mechanism for this inhibition is unclear; however, a breakdown of photosynthesis may result because cool temperatures impair the ability of C4 plants to concentrate CO2 via the C4 cycle into bundle sheath cells where the Calvin-Benson cycle operates. Kubien et al. (2003) proposed that C4 plants in cool climates, compared to their C3 competitors, are constrained because they have less ribulose bisphosphate carboxylase/oxygenase (rubisco), the enzyme that binds CO2 in the Calvin-Benson cycle. Thus, if the C4 cycle is impaired by cool temperatures, rubisco would not operate at maximum efficiency. In addition, because bundle sheath cells resist CO2 diffusion (Brown and Byrd, 1993), CO2 within bundle sheath cells likely would be very low without an operational C4 cycle. Another possible explanation for inhibition of photosynthesis is direct impairment of C3 photosynthesis in chilling sensitive species. Following chilling in the light, Calvin-Benson cycle enzymes including rubisco are reduced in tomato and other species of tropical origin (Powles et al., 1983; Byrd et al, 1995). Specifically, rubisco activation in these species is impaired after short term exposure to low temperatures and high light, which also includes an inability to regenerate rubulose bisphosphate. Values of Fv/Fm reflect changes in photosynthetic efficiency and Indicate the health of a plant leaf. It shows information ultimately related to the assimilation of CO2 giving insight into the stress the plant is undergoing. Results Table 1. Fvm values of maize leaf treatments for chilling in the light and DCDP inhibition • Summary • Found no correlation between Fvm and CO2 levels • PEP assays seem to show that the DCDP did not act as an inhibitor; however, the Chlorophyll Fluorescence showed that it did have some effect • The DCDP appears to have increased the ratio of Chlorophyll Fluorescence in the controlled environment, but decreased the plants ability to recover after chilling. • Ultimately the untreated control recovered a cut above the rest. Fluorescence Light Less than 3 percent of the absorbed light energy is ever lost as chlorophyll fluorescence, this pathway is not considered a major pathway for the safe dissipation of excess light energy. However, chlorophyll fluorescence is a sensitive, intrinsic probe for the function of the photosynthetic apparatus and overall physiological status of the plant. NADP+ KF Kp PSII PQ PSI KD ADP + Pi ATP NADPH Heat Co2 Sugars -Hopkins, William and Huner, Norman. Introduction to Plant Physiology. 3rd Edition. United States: John Wiley & Sons, Inc, 2004