Photosynthetic carbon reduction pathway is absent in chloroplasts of Vicia faba guard cells

Abstract

Four cell types from Vicia faba Linnaeus 'Long Pod' leaflets were assayed for three enzymes unique to the photosynthetic carbon reduction pathway. The enzymes were ribulosebisphosphate carboxylase [3-phospho-p- Gas exchange between a leaf and the atmosphere is almost exclusively through stomata in the epidermis. Stomatal aperture is varied to minimize H20 loss while admitting CO2. The physical basis for opening is swelling of the surrounding guard cell pair in response to the large negative osmotic potential resulting from K+ influx (1). Potassium uptake is electrically balanced by Cl-uptake (2) and the synthesis of organic anions from starch (3-6).An (9) or as by Outlaw and Manchester (6). Analytical enzymes were from Boehringer except RuP2 carboxylase and ribose-P isomerase, which were from Sigma. Agarose was from Miles; most other chemicals were from Sigma. Microscopy Guard cells in epidermal peels were examined by brightfield and fluorescence microscopy. Filters for fluorescence studies were Leitz BG12 and K580. For transmission electron microscopy, tissue was fixed at room temperature for 2 hr under partial vacuum in 100 mM Na cacodylate (pH 7) containing 3% (wt/wt) glutaraldehyde. The tissue was postfixed for 2 hr with 1% OS04 in the above buffer and then dehydrated in a graded ethanol series. The alcohol was replaced by propylene oxide before the tissue was embedded in Spurr's resin (10). Thin sections were stained with uranyl acetate and lead citrate (11) and then were examined in a Hitachi HU-11C electron microscope operated at 75 kV.Enzyme assays Leaflets were illuminated (200 microeinsteins m-2 s-1 of 400-to 700-nm radiation) for at least 15 min before quenching in liquid N2 that had been reduced to its freezing point by evacuation. [Illumination was to activate the enzymes (12)(13)(14).] Leaflets were then freeze-dried at -35°C. Freeze-drying did not change the activities of any of the enzymes reported here.Samples were dissected and weighed (5-15 ng) on a quartz fiber balance in a room with controlled temperature and humidity. The early analytical steps were conducted in a small droplet under oil to prevent evaporation. All enzyme activities were measured at 230C, with the reaction initiated by addition of the tissue sample through the oil. Acid was used to destroy NAD(P)H before enzymatic amplification of the oxidized pyridine nucleotide (NAD, ref. 15; NADP, ref. 16). Tissue blanks and standards were carried through all steps. Except as noted, enzyme specific activities reported are based on dry weight. The concentration of most substrates was determined Abbreviation: RuP2, ribulose bisphosphate.

Rubisco Activity in Guard Cells Compared with the Solute Requirement for Stomatal Opening

“…This Supported by grants from the Deutsche Forschungsgemeinschaft to K. R. (Ra 122/11) and R. S. (Sche 217/3). conclusion was confirmed by metabolite determinations in mechanically isolated pairs of guard cells (13) as well as in suspensions of guard-cell protoplasts (21).…”

Effect of Light Quality on Stomatal Opening in Leaves of Xanthium strumarium L.

“…The action spectra for the accumulation ofssRb (3) as well as those for the accumulation of malate (5) prove that the metabolism of guard cells can be stimulated by blue and by red light; Zeiger and Hepler (16) showed that even isolated guard cell protoplasts begin to swell when they are exposed to light. We know that photosynthetic reduction of CO2 does not occur in guard cells (6,10,12 5 13 19 This paper, Fig. 5 a Data for leaf temperature 22 C; vapor pressure deficit 8 ml 1-1.…”

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

“…Although a portion of the controversy can be dismissed as semantic-being centered on the words "presence" and "absence"-there are substantive conflicts (e.g. between the immunological results of Outlaw et al [ 14] and those of Zemel and Gepstein [29] 2. Homogeneous tissue samples were dissected from freezedried leaflet (5,10).…”