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We have cloned a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) cDNA (AtP5K1) from Arabidopsis thaliana. By the application of cell permeabilization and short-term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus-infected insect (Spodoptera frugiperda) cells directs synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. The same phosphoinositides were produced by isolated whole-cell membrane fractions of AtP5K1-expressing insect cells. Their synthesis was not affected by adding defined precursor lipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, in excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1-directed synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 proceeds via 5-phosphorylation of precursors. Analysis of promoter-reporter gene (beta-glucuronidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namely in cells of the lateral meristem, that is the procambium. Single-cell sampling of sap from flower stem meristem tissue and neighbouring phloem cells, when coupled to reverse transcriptase--polymerase chain reaction, confirmed preferential expression of AtP5K1 in procambial tissue. We hypothesize that AtP5K1, like animal and yeast PIP5K, may be involved in the control of cell proliferation.

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor- beta-glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T-DNA insertion in AtMRP5 (mrp5-1). mrp5-1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5-1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5-1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

A cDNA encoding a novel inwardly rectifying potassium (K+(in)) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K+(in) channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For, this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K(+)-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than -50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channel's pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+(in) channels. The pharmacological inhibitor Cs+, applied externally, inhibited K+(in) currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC50) of 21 microM and 17 microM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K+ into root hair cells. Comparison of homologous K+(in) rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships.

It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA (AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35-41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg2+ ions for full activity.