Auxin-induced growth of coleoptiles depends on the presence of potassium and is suppressed by K ؉ channel blockers. To evaluate the role of K ؉ channels in auxin-mediated growth, we isolated and functionally expressed ZMK1 and ZMK2 (Zea mays K ؉ channel 1 and 2), two potassium channels from maize coleoptiles. In growth experiments, the time course of auxin-induced expression of ZMK1 coincided with the kinetics of coleoptile elongation. Upon gravistimulation of maize seedlings, ZMK1 expression followed the gravitropic-induced auxin redistribution. K ؉ channel expression increased even before a bending of the coleoptile was observed. The transcript level of ZMK2, expressed in vascular tissue, was not affected by auxin. In patch-clamp studies on coleoptile protoplasts, auxin increased K ؉ channel density while leaving channel properties unaffected. Thus, we conclude that coleoptile growth depends on the transcriptional up-regulation of ZMK1, an inwardly rectifying K ؉ channel expressed in the nonvascular tissue of this organ.
Potassium ions constitute the most important macronutrients taken up by plants. To unravel the mechanisms of K+ uptake and its sensitivity to salt stress in the model plant rice, we isolated and functionally characterized OsAKT1, a potassium channel homologous to the Arabidopsis root inward rectifier AKT1. OsAKT1 transcripts were predominantly found in the coleoptile and in the roots of young rice seedlings. K+ channel mRNA decreases in response to salt stress, both in the shoot and in the root of 4-day-old rice seedlings. Following expression in HEK293 cells, we were able to characterize OsAKT1 as a voltage-dependent, inward-rectifying K+ channel regulated by extracellular Ca2+ and protons. Patch-clamp studies on rice root protoplasts identified a K+ inward rectifier with similar channel properties as heterologously expressed OsAKT1. In line with the transcriptional downregulation of OsAKT1 in response to salt stress, inward K+ currents were significantly reduced in root protoplasts. Thus, OsAKT1 seems to represent the dominant salt-sensitive K+ uptake channel in rice roots.
In search of K؉ channel genes expressed in the leaf of the C 4 plant Zea mays, we isolated the cDNA of KZM1 (for K ؉ channel Zea mays 1). KZM1 showed highest similarity to the Arabidopsis K ؉ channels KAT1 and KAT2, which are localized in guard cells and phloem. When expressed in Xenopus oocytes, KZM1 exhibited the characteristic features of an inward-rectifying, potassiumselective channel. In contrast to KAT1-and KAT2-type K ؉ channels, however, KZM1 currents were insensitive to external pH changes. Northern blot analyses identified the leaf, nodes, and silks as sites of KZM1 expression. Following the separation of maize leaves into epidermal, mesophyll, and vascular fractions, quantitative real-time reverse transcriptase-PCR allowed us to localize KZM1 transcripts predominantly in vascular strands and the epidermis. Cell tissue separation and KZM1 localization were followed with marker genes such as the bundle sheath-specific ribulose-1,5-bisphosphate carboxylase, the phloem K ؉ channel ZMK2, and the putative sucrose transporter ZmSUT1. When expressed in Xenopus oocytes, ZmSUT1 mediated proton-coupled sucrose symport. Coexpression of ZmSUT1 with the phloem K ؉ channels KZM1 and ZMK2 revealed that ZMK2 is able to stabilize the membrane potential during phloem loading/unloading processes and KZM1 to mediate K ؉ uptake. During leaf development, sink-source transitions, and diurnal changes, KZM1 is constitutively expressed, pointing to a housekeeping function of this channel in K
Auxin redistribution along gravistimulated maize coleoptiles causes differential expression of the auxin-induced K ؉ -channel gene ZMK1 (Zea mays K ؉ channel 1) and precedes the curvature response. To evaluate the role of ZMK1 during phototropism, we here investigated blue light-stimulated coleoptiles. Four hours of blue light stimulation resulted in phototropic bending (23°). Rotation on a clinostat, at nominally ''zero'' gravity, and simultaneous stimulation with unidirectional blue light, however, resulted in up to 51°bending toward the light. Differential ZMK1 transcription reached a maximum after 90 min of blue light stimulation under gravity, whereas ZMK1 expression remained asymmetric for at least 180 min in photostimulated coleoptiles on a clinostat. We therefore conclude that the stronger phototropic bending under nominally ''zero'' gravity results from prolonged differential expression of ZMK1. Under both conditions, asymmetric expression of ZMK1 could be superimposed on the lateral auxin gradient across the coleoptile tip, whereas the gene for the blue light receptor phototropin 1 (PHOT1), expressed in the tip only, was not differentially regulated in response to blue light. The activation of the two different receptors eliciting the photo-and gravitropic response of the coleoptile thus feeds into a common signaling pathway, resulting in auxin redistribution in the coleoptile tip and finally in differential transcription of ZMK1. In the process of signal integration, gravity transduction restricts the magnitude of the blue light-inducible ZMK1 gradient. The spatial and temporal distribution of ZMK1 transcripts and thus differential K ؉ uptake in both flanks of the coleoptile seem to limit the stimulus-induced bending of this sensory organ. Studying the tropistic curvature of plant organs, Charles Darwin (1) postulated the existence of a signal molecule being synthesized in the tip of photostimulated grass coleoptiles and enhancing growth in one flank of the organ. Went (2) and Cholodny (3) could show that this substance, called auxin, is laterally translocated in photostimulated coleoptiles, resulting in a curvature of the plant toward the light source. Using radioactively labeled auxin, Parker and Briggs (4) and Iino (5) confirmed the Cholodny-Went hypothesis (6). Whereas lateral auxin translocation in photostimulated maize seedlings is restricted to the tip (7), auxin redistribution in gravistimulated seedlings occurs along the length of the entire coleoptile (8). Tropistic curvature is preceded by the asymmetric distribution of apoplastic protons (9), the drop in extracellular pH being caused by increased indole-3-acetic acid (IAA) levels in the fastergrowing flank of the organ (for the Acid Growth Theory, see ref. Although the incoming signal is transduced with the help of one single phytohormone, signal perception is different in graviand photostimulated plants. The light signal is perceived by photoreceptor proteins (phototropins). In Arabidopsis thaliana, two phototropins, phot1 and -2, act tog...
The objective of this flow cytometric study was to examine plasma membrane integrity, mitochondrial membrane potential (MMP) and the degree of DNA fragmentation of cryopreserved bovine sperm immediately (0 h) and 3 h after thawing and to compare the results with each other and with the fertility of bulls. Cryopreserved spermatozoa from 4 consecutive ejaculates of 20 bulls were examined. Percentages of plasma membrane intact sperm (PMI) and sperm showing a high MMP (HMMP), respectively, were determined by the SYBR14/PI- and the JC-1 assays. DNA fragmentation was analysed by the standard deviation of the DNA fragmentation index (SD-DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSA(TM). The mean non-return rate on day 56 (NRR 56) ranged from 63.7% to 78.0% (mean +/- SD: 71.8% +/- 3.7%). Mean values for PMI and HMMP decreased from 37.4% +/- 6.8% to 31.2% +/- 6.1% and from 38.8% +/- 7.1% to 23.8% +/- 7.7% respectively. SD-DFI increased from 56.9% +/- 8.0% to 69.0% +/- 12.9% and %DFI from 6.4% +/- 2.5% to 12.4% +/- 5.8%. The correlation between PMI 0 h and HMMP 0 h (r = 0.95; p < 0.0001) was higher (p < 0.05) than that between PMI 3 h and HMMP 3 h (r = 0.88; p < 0.0001). %DFI 0 h was neither related to PMI 0 h nor to HMMP 0 h (p > 0.05), nor was there a correlation (p > 0.05) between DFI 3 h and PMI 3 h; but %DFI 3 h and HMMP 3 h were significantly correlated (r = -0.31; p < 0.05). SD-DFI and %DFI 3 h were the only parameters related to NRR 56 (r = -0.58; p < 0.05). In conclusion, plasma membranes and mitochondria are similarly affected by the freezing and thawing process, but not during the incubation period after thawing.
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