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.

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.