Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca(2+)-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca(2+)-binding and -release, respectively (rate constants >> 10(3) sec-1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10(3) sec-1). With 10 mM K+ and 1 mM Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of -105 +/- 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mM Ca2+) and for conductance (1.2 mM Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.

Protoplasts are frequently isolated from maize coleoptiles with cell-wall-degrading enzymes such as pectolyase (PEC), mazerozyme, and cellulase. Incubation of coleoptiles with these enzymes caused rapid depolarizations of the membrane voltage (VM). The depolarizing effect of 0.5% (w/v) mazerozyme or 1.5% (w/v) cellulase was unaffected by denaturation of the enzymes. In the case of pectolyase (0.1%, w/v), however, the active enzyme was significantly more potent than the denaturated enzyme in depolarizing coleoptile cells. Exposure to 0.1% active PEC but not to inactive PEC also caused an oxidative burst in coleoptiles and enhanced K + efflux. Together this suggests that pectic breakdown products of the cell wall act as signal for wounding. Typically addition of 10 gM 1-naphthylene acetic acid (NAA) to coleoptiles causes a transient depolarization followed by a slow hyperpolarization of VM. However, in the presence of PEC, VM only depolarized in NAA. After PEC-treated coleoptiles were washed free of the enzyme, NAA caused only small fluctuations of VM-A similarly small VM response to NAA appeared in eoleoptiles pretreated with heatdenaturated supernatant (SUP) from a protoplast isolation buffer, the latter suspected to contain the PEC-generated wounding signal. Comparable pretreatment of coleoptiles with PEC or SUP had no significant effect on the spontaneous and NAA-evoked acidification of the incubation medium. Pretreatment with SUP also had no significant effect on the NAA-stimulated elongation of coleoptile segment. Hence, PEC treatment of coleoptile tissue affects the membrane transport properties of the cells. This effect is partly maintained after removal of the enzyme from the incubation medium, an effect not significant for NAA-generated acidification and cell elongation.

Two genes (lhca5 and lhcb1) from the unicellular, green alga Pyrobotrys (formerly Chlamydobotrys) stellata were isolated, coding for Chlorophyll a/b-binding proteins that putatively represent constituents of the light-harvesting complexes connected with Photosystem I and Photosystem II, respectively. Expression of both genes on the mRNA-level is markedly inhibited by CO2-depletion. The lhca5 transcript-level was reduced to about 25%, and the lhcb1-expression was completely blocked 9 h after removal of CO2 from the growth medium. Simultaneous addition of acetate, which can substitute for CO2 as a carbon source during photoheterotrophic growth of P. stellata, did not compensate for the diminishing effect of CO2-depletion on lhcb1. However, the amount of lhca5 transcript was comparable to that during photoautotrophic growth. These results are interpreted in terms of the specific metabolic demands of photoheterotrophic growth in P. stellata. Cyclic electron-transfer along Photosystem I must be sustained for ATP-production. Linear electron transport fed by Photosystem II and concomitant production of NADPH for CO2-reduction is no longer required.The sequences reported in this article have been deposited at the EMBL data library under the accession numbers X69434 (CSCAB1) and X71965 (CSCAB2MR).

Summary. The contribution of protein synthesis and secretion to indol acetic acid (IAA) induced polarisation of the plasma membrane voltage (VM) was investigated. The VM of coleoptiles from Zea mays was measured in the presence of known inhibitors of protein-and RNA synthesis, as well as those of Golgi-mediated vesicle secretion. Inhibitors were applied under conditions at which they are known to abolish IAA stimulated H + secretion and cell elongation effectively. Cycloheximide (CHI), an inhibitor of protein synthesis, caused depolarisation of I.q,~i with a half maximal concentration of approximately 20 vtM. At 100 viM CHI, k~ depolarised to a new stable voltage with a half time of 9.8 + 0.6 min. The temporal similarity of CHI-induced depolarisation and cessation of coleoptile elongation suggests that the induced change in VM underlies inhibition of elongation. CHI evoked membrane depolarisation to a final voltage of about -100 mV irrespective of the presence or absence of auxin in the external medium. Thus, CHI probably affected constitutive membrane transport properties independently of IAA-induced modulation of transport proteins. Cordycepin (COR), an inhibitor of RNA synthesis, had no significant effect at 400 ~tM on VM of IAA-treated cells, suggesting that gene transcription for transport-or regulatory protein synthesis was not essential for IAA-generated polarisation of VM. Brefeldin-A (BFA), an inhibitor of Golgi-mediated vesicle secretion in maize coleoptiles, had no perceivable effect at 20 rag/1 on VM of IAA-treated coleoptile cells, demonstrating that constitutive or IAA-stimulated protein secretion was not essential for the mechanism underlying IAA-evoked V• polarisation. Hence, IAAstimulated and COR/BFA-depressed H + extrusion in elongating coleoptiles may not be entirely mediated by auxin-enhanced ATPase activity.