Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L.

Abstract

The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vkia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Tissue Preparation and Protein ExtractionTwo different procedures, each utilizing plants from three different growth lots, were used:1. Guard-cell protoplast preparations were made by differential digestion essentially as described as method 3 by Gotow et al. (3), except pepstatin A was not included in the digestion mixture, and epidermal peels were brushed to lower contamination. The protoplasts that were released after 180 min were further purified by differential sieving through a cascade of nylon meshes (210, 70, and 20 ,um)

Rethinking Guard Cell Metabolism

“…Light-stimulated increases in PEPC activity have been demonstrated with enhanced malate accumulation and increased NADPor NAD-dependent MDH activity, which facilitates the reduction of OAA to malate (Rao and Anderson, 1983;Scheibe et al, 1990). A recent study using stable isotope labeling to explore guard cell metabolism revealed that tobacco guard cells fix CO 2 via both Rubisco and PEPC (Daloso et al, 2015), which agrees with earlier studies showing that guard cells have all the necessary machinery to operate the two CO 2 fixation pathways Tarczynski et al, 1989;Parvathi and Raghavendra, 1997;Zeiger et al, 2002;Outlaw, 2003;Suetsugu et al, 2014;Wang et al, 2014). The importance of PEPC in stomatal behavior has been shown by Cousins et al (2007) in PEPC-deficient mutants of the C 4 plant Amaranthus edulis, which exhibited reduced stomatal opening and low final conductance compared with wild-type plants.…”

“…Evidence exists for all three of these pathways. However, it seems that starch degradation and guard cell photosynthesis can provide only limited amounts of Suc, implying that guard cell apoplastic Suc is the most important source (Tarczynski et al, 1989;Reckmann et al, 1990;Vavasseur and Raghavendra, 2005). Outlaw and colleagues (Lu et al, 1995;Ritte et al, 1999;Outlaw and De Vlieghere-He, 2001;Kang et al, 2007) suggested that Suc produced by mesophyll photosynthesis is transported to the guard cells via the apoplast and is taken up into the guard cell symplast.…”

Tobacco guard cells fix CO2 by both Rubisco and PEPcase while sucrose acts as a substrate during light‐induced stomatal opening

“…; Tarczynski et al . ). Probably not only PEPcase but also the Calvin–Benson cycle plays a positive role during stomatal opening particularly in circumstances wherein the alternate pathway is restricted (Parvathi & Raghavendra ).…”

Alterations in light‐induced stomatal opening in a starch‐deficient mutant of Arabidopsis thaliana L. deficient in chloroplast phosphoglucomutase activity

“…Several reports lead to the conclusion that the activity of the PCRP is considerably reduced in guard cell chloroplasts Vaughn 1987;Tarczynski et al 1989;Reckmann, Scheibe & Raschke 1990). Using mass spectrometric measurements, Gautier et al (1991) observed that in a guard cell protoplast suspension of C. communis the net CO2 influx did not balance the net O2 efflux under red light, as with mesophyll cell protoplasts, suggesting that light energy could be used for other processes than for PCRP only.…”