Daniel R. C. Hite

Elevated levels of both sucrose-phosphate synthase and sucrose synthase in vicia guard cells indicate cell-specific carbohydrate interconversions

A long series of reports correlate larger stomatal aperture size with elevated concentration of sucrose (Suc) in guard cells. To assess the role and autonomy of guard cells with respect to these changes, we have determined quantitatively the cellular distribution of the synthetic enzyme, Suc-phosphate synthase (SPS) and the degradative enzyme Suc synthase (SS) in Vicia leaflet. As expected for Suc-exporting cells, the photosynthetic parenchyma had a high SPS:SS ratio of approximately 45. Also as expected, in epidermal cells, which had only few and rudimentary plastids, the SPS:SS ratio was low (0.4). Of all cells and tissues measured, those that had the highest specific activity of SPS (about 4.8 [mu]mol mg-1 of protein h-1) were guard cells. Guard cells also had a very high relative specific activity of SS.

Stomata: Biophysical and Biochemical Aspects

Regulation of gas exchange by stomata of adjustable aperture size in the leaf epidermis is crucial to a plant’s physiology: sufficient CO2 must be admitted into the leaves for growth, but loss of water, usually the limiting resource for terrestrial plants, must be minimized. In essence, the study of stomatal movements is an inquiry into accumulation and dissipation of K + salts, which account for the bulk of the osmotic change associated with stomatal movements.

Enzymic potential for fructose 6-phosphate phosphorylation by guard cells and by palisade cells in leaves of the broad beanVicia faba L

SummaryGuard cells and palisade cells were dissected from freeze-dried leaflets of the broad bean,Vicia faba L. Individual cell samples (6–12 ng) were assayed for ATP-dependent and pyrophosphate-dependent phosphofructokinases. The assay indicator, NADH loss, was monitored in real time in oil droplets with a computer-driven microfluorometer. On a protein basis, both activities were 10-fold higher in guard cells than in palisade cells, indicating (i) elevated carbon metabolism in guard cells to meet demands for energy and carbon skeletons required during stomatal opening, and (ii) parallel glycolytic pathways in guard cells, one responsive to the potent regulatory metabolite fructose 2,6-bisphosphate and the other not. Future work will be devoted to clarifying the roles of the cytosolic and chloroplastic compartments in guard cells.

Molecular, cellular, and plant mechanisms of ABA control of stomatal aperture size

The physical basis for stomatal opening is simple: the flanking pair of guard cells accumulate solutes, notably K+ salts (Outlaw, 1983; Zeiger, 1983). In response, the cells take up water osmotically and swell, which enlarges the pore between them. Closing is the reverse: cellular solutes dissipate. The motor that drives solute fluctuations is the H+-extruding ATPase, an electrogenic pump (stomatal reviews: MacRobbie, 1988; Raschke et al.1988; Mansfield et al. 1990; general reviews: Serrano,1989; Briskin, 1990). Understanding how this pump and the associated secondary processes are regulated is of paramount importance to the plant’s physiology, as water is usually the most limiting resource for terrestrial plants. It will become apparent that this regulation is multifaceted; some signals are processed by the guard cells themselves, whereas others are involved in the integration, overall, of the plant’s responses to the environment.

Regulation of ion transport in guard cells

Stomatal aperture size is controlled by volume changes in the subtending guard-cell pair (Fig. 1). Basically, stomatal aperture size increases as guard cells swell because of asymmetric distension of their cell walls. Guard-cell swelling occurs in response to K+ uptake (Imamura, 1943), balanced by Cl- uptake and anion (malate2-) synthesis from starch. These salts accumulate in vacuoles. Accumulation lowers guard-cell solute potential; as a result, water flows into the cell. Water influx increases the pressure potential, which causes cell swelling. Stomata close by dissipating these salts, including malate2- (Van Kirk & Raschke, 1978).

Removal of contaminating nucleoside diphosphates from commercial preparations of uridine diphosphoglucose

Nucleoside diphosphate contamination in commercial preparations of UDP-Glc poses potential problems in activity assays for enzymes that use this substrate. For removing these contaminants, we report a simple, inexpensive, and rapid method that obviates the need for uncertain corrections in assay calculations.

Evaluation of two approaches to the quantitative histochemical localization of sucrose-P synthase in leaves

SummaryTwo methods for determining the quantitative localization of sucrose-P synthase in plant tissues were evaluated. The single-cell method (rapid freezing, freeze-drying, microdissection, micro-analysis) was validated in several ways, including comparative biochemistry, comparative histochemistry, and kinetics. In contrast, bulk isolation of cells by protoplast-forming methods resulted in loss of sucrose-P synthase activity. This latter approach is widely used and, as far as we are aware, can be successfully used for measurement of other enzymes. Thus, our observations form the basis for a specific caution against the use of protoplast-forming methods in an assay protocol for sucrose-P synthase.