Rates of sugar uptake by guard cell protoplasts of Pisum sativum L. related to the solute requirement for stomatal opening.
by Ritte G., Rosenfeld J., Rohrig K., Raschke K. (1999)
Gerhard Ritte, Johanna Rosenfeld, Kerstin Rohrig, Klaus Raschke
in Plant Physiology 1999;121: 647-656. – DOI: https://doi.org/10.1104/pp.121.2.647 –
http://www.plantphysiol.org/content/121/2/647
Abstract
We wished to determine whether the capacity of the sugar uptake mechanisms of guard cells of the Argenteum mutant of pea (Pisum sativumL.) sufficed to support a concurrent stomatal opening movement. Sugar uptake by guard cell protoplasts was determined by silicone-oil-filtering centrifugation. The protoplasts took up [14C]glucose, [14C]fructose, and [14C]sucrose (Suc), apparently in symport with protons. Mannose, galactose, and fructose competed with Glc for transport by a presumed hexose carrier. The uptake of Glc saturated with aK m of 0.12 mm and aV max of 19 fmol cell−1h−1. At external concentrations <1 mm, the uptake of Suc was slower than that of Glc. It exhibited a saturating component with a K m varying between 0.25 and 0.8 mm and a V max between 1 and 10 fmol cell−1 h−1, and at external concentrations >1 mm, a non-saturating component. At apoplastic sugar concentrations below 4 mm, sugar import was estimated to be mainly in the form of hexoses and too slow to support a simultaneous stomatal opening movement. If, however, during times of high photosynthesis and transpiration, the apoplastic Suc concentration rose and entered the range of non-saturating import, absorbed Suc could replace potassium malate as the osmoticum for the maintenance of stomatal opening.
During stomatal opening, the solute content of guard cells increases dramatically. For example, in the Argenteum mutant of pea (Pisum sativumL.), the subject of this study, the content of osmotica increases by 1,600 fosmol per guard cell, with an initial rate of 900 fosmol per guard cell per hour (Reckmann et al., 1990). There are several possible mechanisms for this increase. The predominant osmotically active species are K+, malate, Cl−, Suc, and possibly other sugars. More than a century ago, Kohl (1886)suggested that carbohydrates formed by the assimilatory activity in the “chlorophyll grains” of guard cells caused osmotic water uptake and turgescence. Lloyd (1908)doubted “that the substances which effect the opening are produced (in the guard cells) in great enough quantities by photosynthesis alone”; he proposed that starch “is quickly changed under appropriate stimuli into powerfully osmotic substances.” Recently, Suc accumulation in guard cells of fava bean was determined by Tallman and Zeiger (1988), Poffenroth et al. (1992), and Talbott and Zeiger (1993), and in guard cells of Commelina benghalensisby Reddy and Rama Das (1986). The controversy over the importance of salts of K+ versus that of sugars as solutes of osmotic consequence in guard cells was resolved by the discovery by Talbott and Zeiger (1996) that which osmoticum dominated depended on the phase of the photoperiod, normally correlated with the time of day; Suc gained in significance about the time when stomatal aperture culminated.
Because differentiated guard cells are not connected to neighboring cells by plasmodesmata (Palevitz and Hepler, 1985), increases in solute concentration during stomatal opening can occur via uptake across the plasmalemma; organic solutes can also be produced within. Guard cells have low levels of Rubisco activity (Outlaw et al., 1979; Gotow et al., 1988; Outlaw, 1989; Reckmann et al., 1990). In the Argenteum mutant of pea, for example, photosynthetic carbon reduction can only account for 10% of the reduced carbon required for stomatal opening if potassium malate is the osmoticum (Reckmann et al., 1990). If hexoses are produced, photosynthesis accounts for only 2% of the requirement. If Suc turns out to be the major osmoticum for stomatal opening in pea, then uptake of Suc (or other sugars) and starch breakdown must account for Suc production. Guard cells of epidermal strips floating on solutions of [14C]Glc or [14C]Suc were able to incorporate radioactivity (Dittrich and Raschke, 1977), and Lu et al. (1995) determined on whole leaves of Vicia faba that a close positive correlation existed between the Suc content of the stomatal apoplast and that of the protoplasts of the guard cells embedded in it.
In this study, isolated guard cell protoplasts from the Argenteum mutant of pea were used because the osmotic requirement for stomatal opening and Rubisco activity had already been determined in this material (Reckmann et al., 1990). Our objective was to analyze the capacity of the sugar uptake mechanisms in the plasmalemma of guard cells and to determine whether it sufficed to provide reduced carbon at a rate that would support a stomatal opening movement.
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