William H. Outlaw

Integration of cellular and physiological functions of guard cells

A plants aerial surfaces are covered by a water-impermeable barrier that limits water loss to stomata, which also permit the uptake of carbon dioxide. Water loss through stomata is required for the ascent of nutrient-laden sap, and evaporation of water from the leaf can be an important cooling mechanism. In most terrestrial environments, however, water is a limiting resource, and its loss must be regulated lest the plant desiccate. Balanced against the essential demand for regulated water loss is the less urgent requirement to acquire carbon dioxide. Regulation of water loss and carbon dioxide uptake is achieved through turgor fluctuations of the pair of guard cells that surround each stoma. When a guard-cell pair accumulates solutes, the resultant turgor and volume changes cause the guard cells to bow outward because of cell-wall architecture, enlarging the pore between them. This simple explanation belies the underlying complexity of guard-cell turgor regulation and whole-plant responses, aspects of wh...A plants aerial surfaces are covered by a water-impermeable barrier that limits water loss to stomata, which also permit the uptake of carbon dioxide. Water loss through stomata is required for the ascent of nutrient-laden sap, and evaporation of water from the leaf can be an important cooling mechanism. In most terrestrial environments, however, water is a limiting resource, and its loss must be regulated lest the plant desiccate. Balanced against the essential demand for regulated water loss is the less urgent requirement to acquire carbon dioxide. Regulation of water loss and carbon dioxide uptake is achieved through turgor fluctuations of the pair of guard cells that surround each stoma. When a guard-cell pair accumulates solutes, the resultant turgor and volume changes cause the guard cells to bow outward because of cell-wall architecture, enlarging the pore between them. This simple explanation belies the underlying complexity of guard-cell turgor regulation and whole-plant responses, aspects of which have been the topics of many focused reviews. This review is general and provides an overview of cellular mechanisms that are involved in turgor regulation. It emphasizes regulatory substances that can be altered externally and therefore includes a description of integrative environmental and whole-plant physiological signals that coordinate guard-cell responses.

Sucrose: a solute that accumulates in the guard-cell apoplast and guard-cell symplast of open stomata

Stomatal conductances of Vicia faba leaves were recorded over a day. Coordinately, (a) guard cells dissected from leaflets were assayed, providing total sucrose content, and (b) guard cells dissected from rinsed epidermes were also assayed, providing symplastic sucrose content. Compared with that of pre‐dawn samples, apoplastic sucrose content increased 4.8 – 5.2 × (2 experiments), reaching 1,130 – 1,300 fmol·guard‐cell‐pair−1 at midday, when conductance was highest (ca. 0.13 mol·m−2·s−1); symplastic sucrose content increased 2.5 – 3.5 ×, reaching 350 – 390 fmol·guard‐cell‐pair−1. Thus, there is a correlation between transpiration and guard‐cell sucrose content, particularly that portion localized to the apoplast. Moreover, apoplastic sucrose is apparently a source of guard‐cell nutrition and, possibly, osmoticum.

Carbon Metabolism in Guard Cells

In the past decade stomatal physiology has been the subject of review articles,1–17 two published symposia,18, 19 a multiauthored book,20 and a forthcoming book, 21 all in addition to the older but excellent book of Meidner and Mansfield.22 Two reviews23,24 have been restricted to the topic of carbon metabolism alone. Thus, there are adequate general summaries, and it will not be my purpose to update these articles. Instead, I will presently “inventory” our current knowledge about carbon metabolism in guard cells. I will rely less on the authors’ interpretations than on direct examination of the data and the methods used to obtain the data. With few exceptions (e.g. ref. 25), there were no quantitative studies on guard cell biochemistry prior to 1973. Since then, numerous reports have appeared. It is time to “take stock,” so, to an extent, this is an internal report to stomatal biologists. As a result, I anticipate that readers will spend more time evaluating the tabular data and less time with my narrative. I will retrace the steps leading to our understanding the basic outline of guard cell biochemistry involved in stomatal

Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phosphoenolpyruvate carboxylase during fusicoccin-stimulated stomatal opening

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K+ or other osmolytes. K+ accumulation is stoichiometric with H+ extrusion, which is compensated for by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [32P]orthophosphate (32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H+-ATPase. [32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In -FC controls, stomatal size, guard-cell malate, and [32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by approximately 80% when K+ uptake was associated with Cl- uptake and was essentially abolished when stomatal opening was sucrose--rather than K+--dependent. Finally, alkalinization by NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H+-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H+-ATPase influence the phosphorylation state of this PEPC isoform.

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.

Microdissection and Biochemical Analysis of Plant Tissues

Biological tissues are not homogenous; instead they consist of cells having specific functions. A typical bifacial leaf, for example, contains not only photosynthetic mesophyll cells (palisade parenchyma plus spongy parenchyma) but also epidermal, guard, and bundle-sheath cells, as well as myriad minor cell types. Their specific functions indicate that profound biochemical differences exist among adjacent cells. These differences are obliterated by tissue homogenation, which precedes most analytical biochemistry. Biologists have thus been challenged to develop methods that allow for selective sampling of specific cell types and analysis of the resultant small amounts of material.

Kinetic Properties of Guard-Cell Phosphoenolpyruvate Carboxylase

Summary The pivotal role of guard-cell phospho enol pyruvate carboxylase (PEPC) in stomatal function is discussed. Appropriate tissue sources for this enzyme are suggested. The few kinetic data for guard-cell PEPC are compared with each other and to those of other PEPCs. Finally, an attempt is made to place these findings in the context of guard-cell cytoplasm. Directions for future research are indicated.

Guard cell apoplastic photosynthate accumulation corresponds to a phloem-loading mechanism

Apoplastic phloem loaders have an apoplastic step in the movement of the translocated sugar, prototypically sucrose, from the mesophyll to the companion cell-sieve tube element complex. In these plants, leaf apoplastic sucrose becomes concentrated in the guard cell wall to nominally 150 mM by transpiration during the photoperiod. This concentration of external sucrose is sufficient to diminish stomatal aperture size in an isolated system and to regulate expression of certain genes. In contrast to apoplastic phloem loaders and at the other extreme, strict symplastic phloem loaders lack an apoplastic step in phloem loading and mostly transport raffinose family oligosaccharides (RFOs), which are at low concentrations in the leaf apoplast. Here, the effects of the phloem-loading mechanism and associated phenomena on the immediate environment of guard cells are reported. As a first step, carbohydrate analyses of phloem exudates confirmed basil (Ocimum basilicum L. cv. Minimum) as a symplastic phloem-loading species. Then, aspects of stomatal physiology of basil were characterized to establish this plant as a symplastic phloem-loading model species for guard cell research. [(14)C]Mannitol fed via the cut petiole accumulated around guard cells, indicating a continuous leaf apoplast. The (RFO+sucrose+hexoses) concentrations in the leaf apoplast were low, <0.3 mM. Neither RFOs (<10 mM), sucrose, nor hexoses (all, P >0.2) were detectable in the guard cell wall. Thus, differences in phloem-loading mechanisms predict differences in the in planta regulatory environment of guard cells.

Lessened malate inhibition of guard-cell phosphoenolpyruvate carboxylase velocity during stomatal opening

Leaflets of Vicia faba with closed stomata or with opening stomata were freeze‐dried. Excised guard‐cell pairs were assayed individually under suboptimal conditions (pH 7.1 and subsaturating substrate) for phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) using quantitative histochemical procedures. l‐Malate, 400 μM, significantly inhibited guard‐cell PEPC activity of closed stomata but not that of opening stomata. We postulate that the lessened sensitivity of guard‐cell PEPC activity to malate inhibition is an important regulatory feature of stomatal opening, which is associated with malate accumulation.

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 (Glc-6-P) on the Vmax and Km of guard-cell (GC) phosphoenolpyruvate (PEP) carboxylase (PEPC) of Vicia faba L. were determined. Leaves of three different physiological states (closed stomata, opening stomata, open stomata) were rapidly frozen and freeze dried. GC pairs dissected from the leaves were individually extracted and individually assayed for the kinetic properties of PEPC. Vmax was 6 to 9 pmol GC pair-1 h-1 and was apparently unaffected to a biologically significant extent by the investigated physiological states of the leaf, pH (7.0 or 8.5), L-malate (0, 5, or 15 mM), and Glc-6-P (0, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the Km(PEP.Mg) was about 0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a GC [PEP] of 0.27 mM. In the study reported here, we determined that the in situ GC [Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Glc-6-P was included in the GC PEPC assay mixture, the Km(PEP.Mg) decreased to about 0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Glc-6-P at endogenous concentrations would seem both to activate the enzyme and to diminish the dramatic effect of pH on Km(PEP.Mg). Under assay conditions, L-malate is an inhibitor of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8 mM. Inclusion of 5 mM L-malate increased the Km(PEP.Mg) to about 3.6 mM (pH 7.0) or 0.4 mM (pH 8.5). Glc-6-P (0.5 mM) was sufficient to relieve L-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM Glc-6-P was required to relieve L-malate inhibition at pH 7.0. No biologically significant effect of physiological state of the tissue on GC PEPC Km(PEP.Mg) (regardless of the presence of effectors) was observed. Together, these results are consistent with a model that GC 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 GC PEPC directly or indirectly (by comparison of the concentration of L-malate that causes a 50% inhibition of GC PEPC).