Histochemical Approach to Properties of <i>Vicia faba</i> Guard Cell Phosphoenolpyruvate Carboxylase

Outlaw, William H.; Manchester, Jill K.; Dicamelli, Cynthia A.

doi:10.1104/pp.64.2.269

Cited by 29 publications

(5 citation statements)

References 8 publications

(9 reference statements)

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“…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)(4)(5)(6).…”

mentioning

confidence: 99%

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

Outlaw¹,

Manchester²,

Dicamelli³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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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.

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mentioning

confidence: 99%

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

Outlaw¹,

Manchester²,

Dicamelli³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…GC PEPC V, , ranged from 6 to 9 pmol GC pair-' h-' (7.23 f 0.9, mean f SD), which is equal to about 1200 mmol kg(* mass)-' h-' or 22 pmol mg-' of protein h-' (for compiled conversion factors, see Outlaw et al, 1985). For comparison, GC PEPC V, , , exceeds by nominally 7-to 10-fold the required in vivo pathway flux (Outlaw and Kennedy, 1978;Outlaw et al, 1979;Tarczynski and Outlaw, 1990); on a protein basis, GC are enriched 8-to 12-fold in PEPC activity compared with palisade cells (Outlaw, 1980).…”

Section: Resultsmentioning

confidence: 98%

“…Neither here (Figs. 1-3) nor before (Outlaw et al, 1979;Tarczynski and Outlaw, 1990) have we detected physiological isoforms of GC PEPC that correspond to Lmalate accumulation (for the sake of clarity, the relatively long activation times used for other PEPCs are irrelevant for stomatal movements). It is possible that our assay methods would not permit detection of the modest light activation (2-fold decrease in K, ) of PEPC observed with Pisum sativum GC protoplast extracts (Raschke et al, 1988) at short times (10 min) or K+-induced oscillations (Michalke and Schnabl, 1990), which were also measured over a short time course.…”

Section: Dlscusslonmentioning

confidence: 99%

The Interactive Effects of pH, L-Malate, and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase

Tarczynski

Outlaw

1993

Plant Physiol.

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The interactive effeds of pH, i-malate, and glucose-6-phosphate (Clc-6-P) on the V , , and K, of guard-cell (CC) phosphoenolpyruvate (PEP) carboxylase (PEPC) of Vicia f a h 1. were determined. Leaves of three different physiological states (closed stomata, opening stomata, open stomata) were rapidly frozen and freeze dried. CC pairs dissected from the leaves were individually extraded and individually assayed for the kinetic properties of PEPC. VmaX was 6 to 9 pmol CC pair-' h-' and was apparently unaffected to a biologically significant extent by the investigated physiological states of the leaf, pH (7.0 or 8.5), i-malate (O, 5, or 15 mM), and Glc-6-P (O, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the K , ( W~. M S ) was about 0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a CC [PEP] of 0.27 mM. In the study reported here, we determined that the in situ CC [Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Clc-6-P was included in the GC PEPC assay mixture, the Km(PEr.Mg) decreased to about 0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Clc-6-P at endogenous concentrations would seem both to adivate the enzyme and to diminish the dramatic effect of pH on K , ( p t p .~~) . Under assay conditions, imalate is an inhibitor of CC PEPC. In planta, cytoplasmic [i-malate] is approximately 8 mM. lnclusion of 5 mM i-malate increased the Km(pEp.Ms) to about 3.6 mM (pH 7.0) or 0.4 mM (pH 8.5). Clc-6-P (0.5 mM) was sufficient to relieve i-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM Ck-6-P was required to relieve i-malate inhibition at pH 7.0. No biologically significant effect of physiological state of the tissue on CC PEPC K,(wP.M~) (regardless of the presence of effectors) was observed. Together, these results are consistent with a model that CC PEPC is regulated by its cytosolic chemical environment and not by posttranslational modification that is detectable at physiological levels of effectors. It is important to note, however, that we did not determine the phosphorylation status of CC PEPC directly or indirectly (by comparison of the concentration of i-malate that causes a 50% inhibition of CC PEPC).Stomatal function is inextricably linked to GC proton extrusion (Raschke et al., 1988;Tallman, 1992), which causes K+-salt accumulation (Outlaw, 1983 Boulder, CO 80303.

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“…21,24). The remainder of K+ is balanced by organic anion synthesis (1,13) via PEP-C4 (12,16,32). The flux of PEP through this pool can be accounted for by guard cell starch hydrolysis (14).…”

Section: Methodsmentioning

confidence: 99%

High Levels of Malic Enzyme Activities in Vicia faba L. Epidermal Tissue

Outlaw¹,

Manchester²,

Brown³

1981

Plant Physiol.

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Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase

Cited by 29 publications

References 8 publications

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

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

The Interactive Effects of pH, L-Malate, and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase

High Levels of Malic Enzyme Activities in Vicia faba L. Epidermal Tissue

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