Light-induced modification of plant plasma membrane ion transport.

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

VFK1, a Vicia faba K(+) channel involved in phloem unloading.

In search of a K(+) channel involved in phloem transport we screened a Vicia faba cotyledon cDNA library taking advantage of a set of degenerated primers, flanking regions conserved among K(+) uptake channels. We cloned VFK1 (for Vicia faba K(+) channel 1) characterised by a structure known from the Shaker family of plant K(+) channels. When co-expressed with a KAT1 mutant in Xenopus oocytes, heteromers revealed the biophysical properties of a K(+) selective, proton-blocked channel. Northern blot analyses showed high levels of expression in cotyledons, flowers, stem and leaves. Using in situ PCR techniques we could localise the K(+) channel mRNA in the phloem. In the stem VFK1 expression levels were higher in the lower internodes. There channel transcripts increased in the light and thus under conditions of increased photosynthate allocation. VFK1 transcripts are elevated in sink leaves, and rise in source leaves during the experimental transition into sinks. Fructose- rather than sucrose- or glucose-feeding via the petiole induced VFK1 gene activity. We therefore monitored the fructose sensitivity of the sieve tube potential through cut aphid stylets. In response to an 1 h fructose treatment the sieve tube potential shift increased from 19 mV to 53 mV per 10-fold change in K(+) concentration. Under these conditions K(+) channels dominated the electrical properties of the plasma membrane. Based on the phloem localisation and expression patterns of VFK1 we conclude that this K(+) channel is involved in sugar unloading and K(+) retrieval.

KAT1 is not essential for stomatal opening.

It is generally accepted that K(+) uptake into guard cells via inward-rectifying K(+) channels is required for stomatal opening. To test whether the guard cell K(+) channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K(+) uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K(+) channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K(+) currents across the plasma membrane of both wild-type and KAT1En-1 plants. This study demonstrates that multiple K(+) channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk.

Distribution of K, Ca, Cl, S, and P in freeze-dried sections of Arabidopsis flower stalk was analyzed by energy dispersive x-ray imaging. Concentrations of these elements in different cell types were quantified by microanalysis of single-cell samples and phloem exudates. Results showed a differential pattern of distribution for all five elements. K concentration was found to be highest in the parenchymatous tissue around vascular bundles. Ca and Cl were present mainly in the central part of the flower stalk. P was largely located in the bundles and in the parenchyma surrounding them. S signal was extraordinary high in groups of cells (S-cells) situated between the phloem of every vascular bundle and the endodermis. Enzymatic hydrolysis by thioglucosidase of cell sap collected from S-cells using a glass microcapillary resulted in the release of glucose, indicating that these cells contain glucosinolates at high (> 100 mM) concentration, which is consistent with the concentration of S (> 200 mM) estimated by x-ray analysis of cell sap samples. Since their position outside of the phloem is ideally suited for protecting the long-distance transport system from feeding insects, the possible roles of these cells as components of a plant defense system are discussed.

Developmental and light-dependent regulation of a phloem-localised K+ channel of Arabidopsis thaliana.

K+ channels in plants can currently be classified into six families with individual members being involved in nutrient uptake, loading of the xylem and the physiology of stomatal movement. In this study we have focused on akt2/3. This K+ channel, as shown by GUS-expression analysis, is expressed in the phloem and xylem of the aerial parts of Arabidopsis thaliana. Northern blot analyses revealed the highest akt2/3-concentrations in the flower stalk, followed by the leaf, flower and stem. During the light period (8 am to 4 pm), transcripts reached a peak around noon (11 am), decayed to almost 50% in the afternoon and reached a low background level in the following dark period. In continuous darkness, however, the K+ channel mRNA content had already decreased beyond the background level by noon. In leaves and flower stalk, the light-induced transcription of akt2/3 was suppressed by CO2-free air, indicating that gene activity is under the control of photosynthates. Additionally, when rosette leaves were illuminated and flower stalks shaded, akt2/3-mRNA transcription was still inhibited in the shaded region. This indicates that channel gene activation is sensitive to photosynthesis-derived factors from neighboring cells rather than factors mobile in the phloem. We propose that the coupling between sugar production and allocation involves the photosynthate- and light-dependent phloem K+ channel AKT2/3.

AKT3, a phloem-localized K+ channel, is blocked by protons.

The potassium-channel gene, AKT3, has recently been isolated from an Arabidopsis thaliana cDNA library. By using the whole-mount and in situ hybridization techniques, we found AKT3 predominantly expressed in the phloem. To study the physiological role of this channel type, AKT3 was heterologously expressed in Xenopus oocytes, and the electrical properties were examined with voltage-clamp techniques. Unlike the plant inward-rectifying guard cell K+ channels KAT1 and KST1, the AKT3 channels were only weakly regulated by the membrane potential. Furthermore, AKT3 was blocked by physiological concentrations of external Ca2+ and showed an inverted pH regulation. Extracellular acidification decreased the macroscopic AKT3 currents by reducing the single-channel conductance. Because assimilate transport in the vascular tissue coincides with both H+ and K+ fluxes, AKT3 K+ channels may be involved in K+ transport accompanying phloem loading and unloading processes.

Light-repressible receptor protein kinase: a novel photo-regulated gene from Arabidopsis thaliana.

To identify light-regulated genes in Arabidopsis thaliana (L.) Heynh. a clone was isolated which contains a cDNA fragment with sequence similarity to receptor-like protein kinases (RLKs). Sequence analysis of the corresponding genomic DNA as well as determination of transcribed regions revealed that the gene comprises 12 exons. Sections of the deduced polypeptide exhibit homologies with kinase domains and the entire protein possesses structural features indicating that it is a novel member of the RLK family. The protein consists of a signal peptide, a putative receptor site including a leucine zipper region with a new motif, a transmembrane helix and 11 subdomains characteristic of serine/threonine kinases. The gene is designated light-repressible receptor protein kinase (lrrpk), as the specific mRNA is predominantly expressed in the absence of light. The lrrpk mRNA steady-state levels were assessed by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and found to be very low after light pulses, irrespective of the wavelength applied. Blue light was least effective in this respect, and the repression was not reversible by far-red light. Employment of in-situ RT-PCR revealed elevated lrrpk mRNA levels in the cotyledons of etiolated seedlings. The mRNA was also increased in the outer regions of the roots of greenhouse-grown A. thaliana, but was not detectable in any other part of the plants. An explanation of the relatively low lrrpk mRNA levels and the photophobic expression of the gene could be the finding that in the 5' upstream region of the lrrpk gene sequence elements are present that are similar to those identified in promoters of phytochrome A genes.

Performance of diltiazem tablet and multiparticulate osmotic formulations in the dog.

The in vivo performance of two extended-release (ER) osmotic formulations of diltiazem were evaluated in the beagle dog. Both ER formulations had similar bioavailabilities (F) as the diltiazem solution. Although F was somewhat variable following ER administration, this variability may be related to the drug entity since intra- and interanimal variability of orally administered diltiazem solutions was substantial. Deconvolution of the ER plasma diltiazem data with absorption data from the orally administered diltiazem solutions provided an estimate of the in vivo drug release from the ER formulations. The two ER formulations, designed with different in vitro release profiles, reflected these differences in vivo, with nearly identical respective in vivo and in vitro release profiles.