Sophie Laurie

Multiple inward channels provide flexibility in Na+/K+ discrimination at the plasma membrane of barley suspension culture cells

Ion transport across the plasma membrane of suspension-culture cells derived from immature barley embryos has been studied in low (15 mM KCl) and high (additional 150 mM NaCl) salt conditions to understand how plants discriminate between K(+) and Na(+) during ion uptake. In both media about 50% of the cells exhibited resting potentials more negative than any of the passive diffusion potentials. In whole-cell patch clamp experiments membrane hyperpolarization activated large inward currents. Whilst the instantaneous current components did not discriminate between K(+) and Na(+), the time-dependent current, I(in), was selective for K(+) over Na(+). Further analysis of I(in) revealed the following properties: double exponential current activation (time-constants 0.03 s and 0.3 s, half activation potential - 171 mV); no inactivation; complete block by Ba(2+) (30 mM in 100 mM KCl) and part block by TEA(+) (maximum 50% with 20 mM); dependence on millimolar concentrations of cytoplasmic ATP; no block by external or cytoplasmic Na(+). The selectivity sequences K(+) ≫ Rb(+) > NH(+)(4) > Na(+) ≫ Cl(-) and K(+) ≫ NH(+)(4) > Na(+) > Rb(+) were determined from measurements of reversal potentials and relative steady-state currents respectively. P(Na):P(K) was 0.07 ± 0.02 (from reversal potentials) and I(Na):I(K) was 0.17 + 0.05 (from relative currents). A high variance among the observed permeability ratios suggested that several channels with different ion-selectivities contributed to the time-dependent whole-cell currents. In single channel experiments, several inward channels with distinct properties were found. The major channels were (i) a voltage-gated, K(+)-selective channel (12 pS), (ii) an ATP-activated non-selective cation channel (7 pS) and (iii) an inward-rectifying anion-channel (150 pS, all unitary conductances given for 100 mM KCI). No significant differences were found in whole-cell currents or single-channel characteristics between cells that had been adapted to a high-salt growth-medium (150 mM NaCl) and non-adapted cells. The idea that differential regulation of plasma membrane ion channels gives rise to a physiological flexibility, allowing the cells to control Na(+) uptake under varying external conditions, is discussed.

Identification of SnIP1, a novel protein that interacts with SNF1-related protein kinase (SnRK1)

Plant SNF1-related protein kinase (SnRK1) phosphorylates 3-hydroxy-3-methylglutaryl-Coenzyme A, nitrate reductase and sucrose phosphate synthase in vitro, and is required for expression of sucrose synthase in potato tubers and excised leaves. In this study, a barley (Hordeum vulgare) endosperm cDNA, SnIP1, was isolated by two-hybrid screening with barley SnRK1b, a seed-specific form of SnRK1. The protein encoded by the SnIP1 cDNA was found to interact with barley SnRK1b protein in vitro. Southern analysis suggested that barley contains a single SnIP1 gene or small gene family. SnIP1 transcripts were detected in RNA isolated from leaf, root and mid-maturation seed. Sequence similarity searches against protein, nucleotide and expressed sequence tag databases identified hitherto uncharacterized sequences related to SnIP1 from maize (Zea mays, accession number AI691404), arabidopsis (Arabidopsis thaliana, AC079673 and AB016886) and poplar (Populus balsamifera, AI166543). No homologous sequences were identified from outside the plant kingdom, but weak sequence similarity was found between the SnIP1 peptide and yeast (Saccharomyces cerevisiae) SNF4 and its mammalian homologue AMPKγ. Nevertheless, SnIP1 failed to complement a yeast snf4 mutant. SnIP1 was found to have little overall sequence similarity with the PV42 family of SNF4-like plant proteins, but proteins of both the SnIP1 and PV42 families contain a conserved hydrophobic sequence we named the SnIP motif.

The role of protein kinases in the regulation of plant growth and development

We review the role of protein kinases in plant hormone-mediatedsignalling, nutrient signalling and cell cycle control and in the crosstalkbetween these different contributors to plant growth regulation. The areas ofhormone-mediated signalling covered include ABA-mediated responses to osmoticstress, wounding and pathogen attack, as well as ethylene and cytokininsignalling pathways. These areas involve members of several major protein kinasefamilies, including the SNFl-related protein kinase-2 (SnRK2) subfamily, thecalcium-dependent protein kinase (CDPK) family, the mitogen activated protein(MAP) kinase family, the glycogen synthase kinase (GSK)- 3/shaggy family and thereceptor-like protein kinase (RPK) family. In the section on nutrient signallingwe review the role of SnRK1 protein kinases in the global regulation of carbonmetabolism, including aspects of sugar sensing and assimilate partitioning, andwhat is known about nitrogen and sulphur nutrient signalling. In the cell cyclesection, we summarise progress in the elucidation of cell cycle control systemsin plants and discuss the interaction between cell cycle control anddevelopment. We expand further on the hypothesis of crosstalk between differentsignalling pathways in a separate section in which we discuss evidence forinteraction between plant growth regulators and the cell cycle, betweendifferent nutrient signalling pathways, between nutrient and cell cyclesignalling and between nutrient and ABA signalling.

Accelerated production and identification of fertile, homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T0 tissue culture-derived plants and their T1 seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T0 tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.