Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination

Wang, Xuechen; Outlaw, William H.; Bedout, J. A. De; Du, Zhirong

doi:10.1007/bf00157964

Cited by 13 publications

(8 citation statements)

References 46 publications

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“…Our results are in general agreement with those of Donovan et al (1985) and with other previous reports that assert that L-malate inhibits and Glc-6-P activates GC PEPC; these metabolites are more effective at physiological pH than at more alkaline pH (8.0-8.5), at which GC PEPC ir; most efficient (Denecke et al, 1993;Wang et al, 1993). The present approach complements and extends conclusions based on earlier ones in severa1 significant ways: (a) the isolaiion of protoplasts, which has been reported to affect PEPC kinetics (Petropoulou et al, 1990;Devi and Raghavendra, 1992), was av~ided; (b) the [Glc-6-P] in GC in planta was detennined, and a surrogate in planta value for cytoplasmic malate concentration is now available (Bodson et al, 1991); these values permitted experimental design to cover the physiological concentration ranges of these effectors; (c) the assay protocol provided for completion of the PEPC assay within 2 or 3 min from extraction, which limits postextraction artifacts such as proteolysis and changes in aggregation state or phosphorylation status; (d) the tissue incubation conditions were physiological; i.e.…”

Section: Dlscusslonsupporting

confidence: 93%

“…Small but statistically significant differences (e.g. Glc-6-P effects on the L-malate-inhibited enzyme) are discussed elsewhere (Wang et al, 1993). 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).…”

Section: Resultsmentioning

confidence: 99%

“…Equally problematic is the situation with GC PEPC, which differs kinetically (Outlaw, 1990;Wang et al, 1993)'and physically from the PEPC complement of bulk C3 leaf. Neither here (Figs.…”

Section: Dlscusslonmentioning

confidence: 99%

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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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Section: Dlscusslonsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

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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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“…On a leaf area basis, the rate of 14C02 incorporation was 0.013 pmol CO, cmp2 leaf area h-' (conversion factors in Outlaw, 1983). Whereas we caution that the individual values for 14C incorporation by guard cells were imprecise because of the low levels of 14C in the initial samples, in aggregate the rates are in the range of organic anion synthesis in planta in guard cells (guard-cell malate accumulation rate: 0.33 pmol cell-I h-', Outlaw and Kennedy, 1978; guard-cell PEP carboxylase activity: 3-4.5 pmol ce1l-l h-', Tarczynski and Outlaw, 1990;Wang et al, 1994). Corroboratively, on a cell basis, the total amount of ['4C]Suc in palisade parenchyma was approximately 30-fold that of the guard-cell symplast at 20 min (332 versus 12 mBq cell-', Fig.…”

Section: Dlscusslon In Planta Rates Of 14c Lncorporation By Palisade mentioning

confidence: 97%

A New Mechanism for the Regulation of Stomatal Aperture Size in Intact Leaves (Accumulation of Mesophyll-Derived Sucrose in the Guard-Cell Wall of Vicia faba)

et al. 1997

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At various times after pulse-labeling broad bean (Vicia faba 1.) leaflets with 14C0,, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents, whereas those from rinsed peels contained only symplastic contents. Sucrose (Suc)-specific radioactivity peaked (1 11 CBq mol-') in palisade cells at 20 min. I n contrast, the 14C content and Sucspecific radioactivity were very low in guard cells for 20 min, implying little CO, incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum Suc-specific radioactivity (204 CBq mol-') and a high Suc influx rate (0.05 pmol stoma-' min-'). These and other comparisons implied the presence of (a) multiple Suc pools i n mesophyll cells, (b) a localized mesophyllapoplast region that exchanges with phloem and stomata, and (c) mesophyll-derived SUC in guard-cell walls sufficient t o diminish stomatal opening by approximately 3 pm. Factors expected to enhance Suc accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and (b) high apoplastic SUC concentration, which i s elevated when mesophyll Suc efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal aperture size by this previously unrecognized mechanism.For many years sugars played a prominent role in the explanations of stomatal movements. The classical theory (starch + sugar during stomatal opening, and vice versa) invoked an osmotic role for sugars within guard cells sufficient to create the requisite turgor for stomatal opening. This theory was based on the usual observation of a reciprocal relationship between guard-cell starch content and stomatal aperture size. For the lack of methods at the time, the theory was tested only semiquantitatively for starch and not at a11 for sugars. Upon the discovery that massive K+ accumulation in guard cells accompanies stomatal opening and that K+ loss from guard cells accompanies stomatal closure, the classical theory was discarded (for history, see Hsiao, 1976;Raschke, 1979; Outlaw, 1983 Poffenroth et al., 1992;Talbott and Zeiger, 1993), also working with V. fubu, reported that red light causes an increase in stomatal aperture size on epidermal peels and a decrease in guardcell qs without either an increase in guard-cell K+ concentration or a decrease in guard-cell starch content. Under other conditions, they found that stomata also open without an increase in guard-cell K+ concentration but with a loss of guard-cell starch content. They indicated that their data are not consistent with K' being the universal guardcell osmoticum, and they suggested, as additional osmotica, interna1 sugars arising from the PCRP or starch breakdown. Important to comparisons made with our work, they noted that stomata induced to open in intact leaves have a substantially higher K+ content than those of epidermal peels discussed above. In summary, the work discussed above a...

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“…It consists of an interaction between this allosteric enzyme and its negative (L-malate) and positive (G6P, triose-P) effectors and a complex regulatory phosphorylation cycle that modulates several important kinetic parameters of PEPC, most notably Ki (L-malate) and K, (G6P) (Duff et al, 1995). In contrast, relatively little is known about the possible posttranslational regulation of the nonphotosynthetic C, enzyme, although it too is subject to allosteric control (Outlaw, 1990;Schuller et al, 1990aSchuller et al, , 1990bWang et al, 1994). However, only a single report has appeared dealing directly with the in vivo phosphorylation of the C,-leaf enzyme (Van Quy et al, 1991a).…”

mentioning

confidence: 99%

In Vivo Regulation of Wheat-Leaf Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

Duff¹,

Chollet²

1995

Plant Physiol.

116

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Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination

Cited by 13 publications

References 46 publications

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

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

A New Mechanism for the Regulation of Stomatal Aperture Size in Intact Leaves (Accumulation of Mesophyll-Derived Sucrose in the Guard-Cell Wall of Vicia faba)

In Vivo Regulation of Wheat-Leaf Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

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