Mesophyll photosynthesis and guard cell metabolism impacts on stomatal behaviour

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere. Guard cells determine stomatal aperture and must operate to ensure an appropriate balance between CO2 uptake for photosynthesis (A) and water loss, and ultimately plant water use efficiency (WUE). A strong correlation between A and stomatal conductance (gs) is well documented and often observed, but the underlying mechanisms, possible signals and metabolites that promote this relationship are currently unknown. In this review we evaluate the current literature on mesophyll-driven signals that may coordinate stomatal behaviour with mesophyll carbon assimilation. We explore a possible role of various metabolites including sucrose and malate (from several potential sources; including guard cell photosynthesis) and new evidence that improvements in WUE have been made by manipulating sucrose metabolism within the guard cells. Finally we discuss the new tools and techniques available for potentially manipulating cell-specific metabolism, including guard and mesophyll cells, in order to elucidate mesophyll-derived signals that coordinate mesophyll CO2 demands with stomatal behaviour, in order to provide a mechanistic understanding of these processes as this may identify potential targets for manipulations in order to improve plant WUE and crop yield. © 2014 New Phytologist Trust

Are Small GTPases Signal Hubs in Sugar-Mediated Induction of Fructan Biosynthesis?

External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling

Osmotic effects on vacuolar ion release in guard cells

Tracer flux experiments in isolated guard cells of Commelina communis L. suggest that the vacuolar ion content is regulated and is reset to a reduced fixed point by abscisic acid (ABA) with no significant change in cytoplasmic content. The effects of changes in external osmotic pressure were investigated by adding and removing mannitol from the bathing solution. Two effects were distinguished. In the new steady state of volume and turgor, the vacuolar ion efflux was sensitive to turgor: efflux increased at high turgor and reduced at lower turgor after the addition of mannitol. These changes were inhibited by phenylarsine oxide and are likely to involve the same channel that is involved in the response to ABA. After a hypoosmotic transfer, there was an additional effect: a fast transient stimulation of vacuolar efflux during the period of water flow into the cell; the size of this hypopeak increased with the size of the hypoosmotic shock, with increased water flow. No corresponding transient in reduced vacuolar efflux was observed upon hyperosmotic transfer. The fast hypopeak was not inhibited by phenylarsine oxide and appears to involve a different ion channel from that involved in the resting efflux, the response to ABA, or the turgor sensitivity. Thus, the tonoplast can sense an osmotic gradient and respond to water flow into the vacuole by increased vacuolar ion efflux, thereby minimizing cytoplasmic dilution. An aquaporin is the most likely sensor and may also be involved in the signal transduction chain

Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae.

Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+

Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae.

Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+

Membrane transport in stomatal guard cells: the importance of voltage control.

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mM external K+ (with 5 mM Ca2(+)-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage (VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM = -143 +/- 4 mV), the other (mean VM = -237 +/- 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+ equilibrium potential (EK+), in 3 and 10 mM external K+. In these cases VM was also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VM was situated positive of EK+ and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 microM ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H(+)-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization.(ABSTRACT TRUNCATED AT 400 WORDS