A role for LKP2 in the circadian clock of Arabidopsis.

A third member of the ZTL gene family was identified in the Arabidopsis genome and was named LKP2 (for LOV kelch protein2). A cDNA was isolated corresponding to this gene, and plants overexpressing LKP2 were generated. The overexpression of LKP2 resulted in arrhythmic phenotypes for a number of circadian clock outputs in both constant light and constant darkness, long hypocotyls under multiple fluences of both red and blue light, and a loss of photoperiodic control of flowering time. The LKP2 mRNA is not regulated by the circadian clock and was detected in all tissues examined. These results suggest that LKP2 functions either within or very close to the circadian oscillator in Arabidopsis. A model is presented for its mode of action.

LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in arabidopsis.

In plants, light is not only an energy source but also a very important signal that modulates development and differentiation. Here, we report a putative photo-regulatory factor sequence in LKP1 (LOV kelch protein 1). LKP1 cDNA encodes a protein of 610 amino acids and with a molecular weight of 65 905 with an LOV domain and kelch repeats. LOV domains are present in a number of sensor proteins involved in the detection of light, oxygen or voltage. The LKP1 LOV is very similar to the LOV domains in NPH1, a plasma membrane-associated blue light receptor kinase that regulates phototropism (Huala, E., Oeller, P.W., Liscum, E., Han, I-S., Larsen, E. & Briggs, W.R. (1997) Science, 278, 2120-2123). LKP1 mRNA accumulates in roots, stems, flowers and siliques. It is most abundant in leaves, and least abundant in seeds. Transgenic plants with a beta-glucuronidase (GUS) reporter gene driven by a 1.5 kb LKP1 promoter display strong GUS activity in leaves. Transgenic plants with a 35S:LKP1 cDNA gene overexpress LKP1 mRNA. These plants have elongated hypocotyls and petioles with elongated cells, and exhibit distinct cotyledon movement during the day. Expression of 35S:LKP1 in transgenic Arabidopsis promotes late flowering in plants grown under long-day, but not under short-day conditions. Vernalization does not affect the late flowering phenotype of the 35S:LKP1 plants. Transgenic plants possessing the 35S:GFP-LKP1 construct also have long hypocotyles and petioles, and a late flowering phenotype, suggesting that the GFP-LKP1 fusion protein is active. The GFP-associated fluorescence in 35S:GFP-LKP1 plants is observed in nuclei and cytosol, indicating that LKP1 is a new nucleo-cytoplasmic factor that influences flowering time in the long day pathway of Arabidopsis.

Effects of JTV-519, a novel anti-ischaemic drug, on the delayed rectifier K+ current in guinea-pig ventricular myocytes.

We studied the effects of a newly synthesized anti-ischaemic agent, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2, 3, 4, 5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) on the delayed rectifier potassium current (IK), using guinea-pig ventricular myocytes and whole-cell voltage-clamp techniques, under blockade of the L-type calcium current (ICa,L) by D600 (1 microM) or nitrendipine (5 microM). The IK in guinea-pig ventricular cells consists of two different components; the rapidly activating, E4031-sensitive component (IKr) and the slowly activating E4031-resistant component (IKs). Under steady-state conditions, JTV-519 (1 and 5 microM) did not change the amplitude of IKs remaining after blockade of IKr with 5 microM E4031. The effect of JTV-519 on IKr was assessed using short (50 ms) pulses which evoked a tail current that was sensitive to E4031 but not to chromanol 293B, a specific blocker of IKs. JTV-519 suppressed the IKr with a half-maximal inhibitory concentration of 1.2 microM. Selective inhibition of IKr by this agent was confirmed by using the "envelope of tails" test. These results suggest that the blockade of IKr may underlie the prolongation of action potential duration in ventricular muscle and QT-intervals alleged to occur in animal as well as human hearts.

Bepridil differentially inhibits two delayed rectifier K(+) currents, I(Kr) and I(Ks), in guinea-pig ventricular myocytes.

1. We investigated the effects of bepridil on the two components of the delayed rectifier K(+) current, i.e., the rapidly activating (I(Kr)) and the slowly activating (I(Ks)) currents using tight-seal whole-cell patch-clamp techniques in guinea-pig ventricular myocytes, under blockade of L-type Ca(2+) current with nitrendipine (5 microM) or D600 (1 microM). 2. Bepridil decreased I(Ks) under blockade of I(Kr) with E4031 (5 microM), in a concentration-dependent manner. The concentration-dependent inhibition of I(Ks) by bepridil was fitted by a curve, assuming one-to-one interactions between the channel and the drug molecule. The concentration of half-maximal inhibition (IC(50)) was found to be 6.2 microM. 3. The effect of bepridil on I(Kr) was assessed using an envelope-of-tails test. In the control condition, a ratio of the tail current to the time-dependent current measured during depolarization was large (>1) at shorter pulses (<200 ms), and it decreased to a steady state value of approximately 0.4 with increases in the pulse duration. Bepridil at a concentration of 2 microM did not decrease this ratio at shorter pulses. 4. In a short-pulse (duration=50 ms) experiment that largely activates I(Kr), the drug was found to block I(Kr) in a cooperative manner (Hill coefficient=3.03) and the IC(50) was 13.2 microM. 5. These results suggest that bepridil at a clinical therapeutic concentration ( approximately 2 microM) selectively blocks I(Ks) but does not inhibit I(Kr). This may relate to the characteristic frequency-dependent effects of bepridil on the action potential duration (APD), e.g., the non-reverse use-dependent prolongation of APD.

Identification of clp genes expressed in senescing Arabidopsis leaves.

Clp protease is a highly selective protease in E. coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP. In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier [Kiyosue et al. (1993) Biochem. Biophys. Res. Commun. 196: 1214]. We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6). We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence. The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier [Nakashima et al. (1997) Plant J. 12: 851], while that of AtclpC was decreased. Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed. Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma. These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts.

Effects of pimobendan on the L-type Ca2+ current and developed tension in guinea-pig ventricular myocytes and papillary muscle: comparison with IBMX, milrinone, and cilostazol.

In this study, we compared the effects of pimobendan (PIM), a putative Ca(2+)-sensitizer and phosphodiesterase (PDE) inhibitor, on the L-type Ca2+ current (ICa) of guinea-pig ventricular myocytes and contractile tension of ventricular papillary muscles with those of a nonselective PDE inhibitor, isobutylmethylxanthine (IBMX), and selective PDE-III inhibitors, that is, milrinone (MIL) and cilostazol (CIL). The efficacy (maximum attainable effect) of these drugs for increasing ICa or developed tension (DT) ranged in the order of IBMX >> MIL > PIM > CIL. This finding suggests that the positive inotropic effect of each drug is roughly proportional to its increasing effect on ICa. The additional effect of PIM (a Ca(2+)-sensitizing effect) was not identified in "intact" preparations, and the potentiating effects of PIM on the DT and ICa were virtually the same as those observed for MIL. To isolate the Ca(2+)-sensitizing effect of PIM on the DT, we studied the effects of PIM in the presence of H89, an isoquinoline derivative possessing a selective inhibitory effect on cAMP-dependent protein kinase. In the absence of H89, 50 microM PIM increased the DT by 68 +/- 11% (mean +/- SE, n = 6). However, in the presence of 20 microM H89, which completely blocked the PIM-induced increase in ICa, PIM (50 microM) significantly increased the DT by 19 +/- 6% (n = 6), thereby indicating the presence of a positive inotropic effect attributable to a mechanism other than increased intracellular cAMP, that is, a Ca(2+)-sensitizing effect. The latter notion was supported by the finding that in the presence of H89 (20 microM), the PIM-induced augmentation of DT was accompanied by a prolongation of the time to 50% relaxation of contractile tension. In contrast, MIL (50 microM) and forskolin, a direct activator of adenylate cyclase (1-10 nM), did not increase DT in the presence of 20 microM H89. These results suggest that the fraction of positive inotropic effect of PIM attributable to its Ca(2+)-sensitizing effect is masked by its potent PDE-III inhibitory effect in "intact" ventricular preparations.

Inhibition of Na(+)-K+ pump alleviates the shortening of action potential duration caused by metabolic inhibition via blockade of KATP channels in coronary perfused ventricular muscles of guinea-pigs.

The Na(+)-K+ pump is a consumer of intracellular ATP. We therefore examined whether blockade of the Na(+)-K+ pump by cardiac glycosides could inhibit ATP-sensitive K+ (KATP) channels and prolong the action potential duration (APD) of the guinea-pig ventricular muscles perfused with Tyrode's solution via the coronary artery and stimulated at 3 Hz. The metabolic inhibition (MI) achieved by application of 0.1 microM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (a mitochondrial uncoupler) shortened the APD in a time-dependent manner. When dihydroouabain (DHO, 5 microM) was introduced 5 min but not 10 min after introduction of MI, the APD shortening was significantly attenuated. Application of glibenclamide (1 microM), a blocker of KATP channels, introduced both 5 and 10 min after MI also alleviated the APD shortening: DHO did not alleviate the APD shortening effect produced by cromakalim (5 microM), a KATP-channel opener. In separate experiments using whole-cell patch-clamp techniques, we found that this concentration of DHO (5 microM) depressed the Na(+)-K+ pump current of the guinea-pig ventricular myocytes from 210 to 100 pA (at 0 mV) or by 49.5%. We conclude that, during early phase (approximately 5 min) of MI, the APD shortening mostly results from the activation of KATP channels, and that even a approximately 50% inhibition of the Na(+)-K+ pump by DHO leads to the blockade of KATP channels and eventual lengthening of the APD.

Control of fertilization-independent endosperm development by the MEDEA polycomb gene in Arabidopsis.

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis, f644, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA (MEA), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446-450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

Effect of bepridil on intracellular calcium concentration and contraction in cultured rat ventricular myocytes.

We studied the effects of a new antiarrhythmic and antianginal agent, bepridil, on the intracellular calcium transient and contraction of cultured neonatal rat ventricular cells, and compared the effects with those caused by an authentic Ca2+ -entry blocker, D600 (methoxyverapamil). The Ca2+ transient was measured by using dual-wavelength microfluorometry of fura-2. The contraction was measured as a shortening of cell aggregates with the use of a video image-analyzing system. Both bepridil (1-30 microM) and D600 (1-30 microM) decreased the peak systolic amplitude of the Ca2+ transient in a concentration- and frequency-dependent manner. Bepridil, but not D600, significantly shortened the half-decay time of the Ca2+ transient and prolonged the time course of the contraction. D600 decreased the contraction in parallel with the decrease in the peak Ca2+ transient, whereas bepridil exerted no significant effect on the contraction. Bepridil (10 microM) induced a leftward shift (to lower amplitude of peak systolic Ca2+ transient) of the relation between the magnitude of contraction and the peak systolic Ca2+ transient, which was obtained by changing external Ca2+ concentration. In contrast, D600 (10 microM) did not affect the relation. The results suggest that the negative inotropic effect of bepridil (caused by its Ca2+ channel-blocking effect) is offset by its simultaneous increase in the sensitivity of contractile protein(s) to intracellular Ca2+, which may be a unique characteristic of this antiarrhythmic agent in a clinical setting.

A gene encoding proline dehydrogenase is not only induced by proline and hypoosmolarity, but is also developmentally regulated in the reproductive organs of Arabidopsis.

The cDNA clone ERD5 (early responsive to dehydration), isolated from 1-h-dehydrated Arabidopsis, encodes a precursor of proline (Pro) dehydrogenase (ProDH), which is a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The transcript of the erd5 (ProDH) gene was undetectable when plants were dehydrated, but large amounts of transcript accumulated when plants were subsequently rehydrated. Accumulation of the transcript was also observed in plants that had been incubated under hypoosmotic conditions in media that contained L- or D-Pro. We isolated a 1.4-kb DNA fragment of the putative promoter region of the ProDH gene. The beta-glucuronidase (GUS) reporter gene driven by the 1.4-kb ProDH promoter was induced not only by rehydration but also by hypoosmolarity and L- and D-Pro at significant levels in transgenic Arabidopsis plants. The promoter of the ProDH gene directs strong GUS activity in reproductive organs such as pollen and pistils and in the seeds of the transgenic plants. GUS activity was detected in vegetative tissues such as veins of leaves and root tips when the transgenic plants were exposed to hypoosmolarity and Pro solutions. GUS activity increased during germination of the transgenic plants under hypoosmolarity. The relationship between Pro metabolism and the physiological aspects of stress response and development are discussed.

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes.

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.

A chromo box gene from carrot (Daucus carota l.): its cDNA structure and expression during somatic and zygotic embryogenesis.

A cDNA clone, designated DcDB1, was isolated from a cDNA library prepared from embryogenic cell clusters of carrot (Daucus carota L.) and characterized. The cDNA (1416 bp) encoded for a protein of 392 amino acid residues that contained a conserved chromo domain. The chromo domain is a 37 aa region found in both the Polycomo gene product, which is a repressor of homeotic genes, and a heterochromatin protein 1 of Drosophila. This domain is postulated to function in the binding of proteins to chromatin. Genomic blot hybridization experiments suggested that the number of DcCB1 genes in the carrot genome is low. The level of DcCB1 mRNAs was high in somatic embryos at globular and heart-shaped stages but low in torpedo-shaped somatic embryos. The level of DcCB1 transcripts decreased during the formation of seeds. The existence of both homeo and chromo box genes in plants suggests that regulatory mechanisms of developmental genes in plants may resemble those in Drosophila.

ERD6, a cDNA clone for an early dehydration-induced gene of Arabidopsis, encodes a putative sugar transporter.

Previously, we constructed a cDNA library from Arabidopsis plants that were exposed to dehydration stress for 1 h and obtained the ERD6 clone. Here we report that the ERD6 cDNA consists of 1741 bp and encodes a polypeptide of 496 amino acids having a predicted molecular weight of 54,354. The putative polypeptide of ERD6 is related to those of sugar transporters of bacteria, yeasts, plants and mammals. Hydropathy analysis revealed that ERD6 protein has 12 putative transmembrane domains and a central hydrophilic region. Sequences that are conserved at the ends of the 6th and 12th membrane-spanning domains of sugar transporters are also present in ERD6. These data suggest that ERD6 encodes a sugar transporter. Genomic Southern blots indicate that the ERD6 gene is a member of a multigene family in the Arabidopsis genome. The expression of the ERD6 gene was induced not only by dehydration but also by cold treatment.

Regulation of levels of proline as an osmolyte in plants under water stress.

Compatible osmolytes are potent osmoprotectants that play a role in counteracting the effects of osmotic stress. Proline (Pro) is one of the most common compatible osmolytes in water-stressed plants. The accumulation of Pro in dehydrated plants is caused both by activation of the biosynthesis of Pro and by inactivation of the degradation of Pro. In plants, L-Pro is synthesized from L-glutamic acid (L-Glu) via delta(1)-pyrroline-5-carboxylate (P5C) by two enzymes, P5C synthetase (P5CS) and P5C reductase (P5CR). L-Pro is metabolized to L-Glu via P5C by two enzymes, proline dehydrogenase (oxidase) (ProDH; EC 1.5.99.8) and P5C dehydrogenase (P5CDH; EC 1.5.1.12). Such metabolism of Pro is inhibited when Pro accumulates during dehydration and it is activated when rehydration occurs. Under dehydration conditions, when expression of the gene for P5CS is strongly induced, expression of the gene for ProDH is inhibited. By contrast, under rehydration conditions, when the expression of the gene for ProDH is strongly induced, the expression of the gene for P5CS is inhibited. Thus, P5CS, which acts during the biosynthesis of Pro, and ProDH, which acts during the metabolism of Pro, appear to be the rate-limiting factors under water stress. Therefore, it is suggested that levels of Pro are regulated at the level of transcriptional the genes of these two enzymes during dehydration and rehydration. Moreover, it has been demonstrated that Pro acts as an osmoprotectant and that overproduction of Pro results in increased tolerance to osmotic stress of transgenic tobacco plants. Genetically engineered crop plants that overproduce Pro might, thus, acquire osmotolerance, namely, the ability to tolerate environmental stresses such as drought and high salinity.

Rate-dependent prolongation of action potential duration in isolated rat ventricular myocytes.

Rate-dependent alterations of action potential duration (APD) in rat ventricular myocytes were investigated. Action potentials of the isolated myocytes were recorded with patch electrodes containing EGTA (11 mM), and showed a marked rate-dependent prolongation in the APD (0.2-5 Hz). This prolongation was significantly inhibited in the presence of 4-aminopyridine (4-AP), a blocker of the transient outward K+ current (Ito). Thus, the rate-dependent decrease in Ito may underlie the change in APD. In contrast, the action potentials recorded from rat ventricular papillary muscles with conventional microelectrodes did not show rate-dependent alterations in the APD, i.e., the APD remained practically unaltered at the frequency range of 0.2-5 Hz. These results suggest that the rate-dependent prolongation of APD (due to rate-dependent blockade of Ito) becomes evident when the intracellular Ca2+ was chelated by the internal application of EGTA via patch pipette. We speculate that the rate-dependent prolongation of APD (via decreases in Ito) is masked in the ventricular papillary muscles, probably due to rate-dependent decreases in the inward current (e.g., electrogenic Na(+)-Ca2+ exchange current) that is regulated by the intracellular calcium.

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats.

We investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca2+]i) of cultured neonatal rat ventricular cells using microfluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 11.5% of control length), moderately so (to 115%-125% of control length), or extensively (to over 125% of the control length). "Quick stretches" (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca2+]i in the early phase of stretch (30 s-2 min), followed by a small but sustained increase during the late phase of stretch (5-10 min). The initial transient increase in [Ca2+]i after the "quick stretch" was preserved in the presence of gallopamil (10(-7) M) or ryanodine (10(-5) M), but was absent in Ca(2+)-free medium or in the presence of gadolinium (10(-7) M). The late or steady state [Ca2+]i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca(2+)-free medium. A steady-state increase in [Ca2+]i was also evoked by "slow stretch" in which cells were slowly pulled to the final length within 1-2 min. As the presence of external Ca2+ was indispensable, increased trans-sarcolemmal Ca2+ influx appears to be involved in both initial and steady-state increases in [Ca2+]i. The initial increase in [Ca2+]i after the "quick stretch" can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.

A novel gene of tomato preferentially expressed in fruit encodes a protein with a Ca2+-dependent lipid-binding domain.

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54,633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin 1 of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.

A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana.

A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the beta-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.