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.

Living organisms adapt to changing light environments via mechanisms that enhance photosensitivity under darkness and attenuate photosensitivity under bright light conditions. In hypocotyl phototropism, phototropin1 (phot1) blue light photoreceptors mediate both the pulse light-induced, first positive phototropism and the continuous light-induced, second positive phototropism, suggesting the existence of a mechanism that alters their photosensitivity. Here, we show that light induction of ROOT PHOTOTROPISM2 (RPT2) underlies photosensory adaptation in hypocotyl phototropism of Arabidopsis thaliana. rpt2 loss-offunction mutants exhibited increased photosensitivity to very low fluence blue light but were insensitive to low fluence blue light. Expression of RPT2 prior to phototropic stimulation in etiolated seedlings reduced photosensitivity during first positive phototropism and accelerated second positive phototropism. Our microscopy and biochemical analyses indicated that blue light irradiation causes dephosphorylation of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) proteins and mediates their release from the plasma membrane. These phenomena correlate closely with the desensitization of phot1 signaling during the transition period from first positive phototropism to second positive phototropism. RPT2 modulated the phosphorylation of NPH3 and promoted reconstruction of the phot1-NPH3 complex on the plasma membrane. We conclude that photosensitivity is increased in the absence of RPT2 and that this results in the desensitization of phot1. Light-mediated induction of RPT2 then reduces the photosensitivity of phot1, which is required for second positive phototropism under bright light conditions.

SUMMARYTwo photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map-based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST-OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower-related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild-type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blastinduced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.

We isolated a mutant, named coleoptile phototropism1 (cpt1), from g-ray-mutagenized japonica-type rice (Oryza sativa). This mutant showed no coleoptile phototropism and severely reduced root phototropism after continuous stimulation. A map-based cloning strategy and transgenic complementation test were applied to demonstrate that a NPH3-like gene deleted in the mutant corresponds to CPT1. Phylogenetic analysis of putative CPT1 homologs of rice and related proteins indicated that CPT1 has an orthologous relationship with Arabidopsis thaliana NPH3. These results, along with those for Arabidopsis, demonstrate that NPH3/CPT1 is a key signal transduction component of higher plant phototropism. In an extended study with the cpt1 mutant, it was found that phototropic differential growth is accompanied by a CPT1-independent inhibition of net growth. Kinetic investigation further indicated that a small phototropism occurs in cpt1 coleoptiles. This response, induced only transiently, was thought to be caused by the CPT1-independent growth inhibition. The 3 H-indole-3-acetic acid applied to the coleoptile tip was asymmetrically distributed between the two sides of phototropically responding coleoptiles. However, no asymmetry was induced in cpt1 coleoptiles, indicating that lateral translocation of auxin occurs downstream of CPT1. It is concluded that the CPT1-dependent major phototropism of coleoptiles is achieved by lateral auxin translocation and subsequent growth redistribution.

Auxin efflux carrier PIN-FORMED (PIN) proteins are thought to have central roles in regulating asymmetrical auxin translocation during tropic responses, including gravitropism and phototropism, in plants. Although PIN3 is known to be involved in phototropism in Arabidopsis (Arabidopsis thaliana), no severe defects of phototropism in any of the pin mutants have been reported. We show here that the pulse-induced, first positive phototropism is impaired partially in pin1, pin3, and pin7 single mutants, and severely in triple mutants. In contrast, such impairment was not observed in continuous-light-induced second positive phototropism. Analysis with an auxin-reporter gene demonstrated that PIN3-mediated auxin gradients participate in pulse-induced phototropism but not in continuous-light-induced phototropism. Similar functional separation was also applicable to PINOID, a regulator of PIN localization. Our results strongly suggest the existence of functionally distinct mechanisms i.e. a PIN-dependent mechanism in which transient stimulation is sufficient to induce phototropism, and a PIN-independent mechanism that requires continuous stimulation and does not operate in the former phototropism process. Although a previous study has proposed that blue-light photoreceptors, the phototropins, control PIN localization through the transcriptional down-regulation of PINOID, we could not detect this blue-light-dependent down-regulation event, suggesting that other as yet unknown mechanisms are involved in phototropin-mediated phototropic responses.

SummaryRecently, two K + channel genes, ZMK1 and ZMK2, were isolated from maize coleoptiles. They are expressed in the cortex and vasculature, respectively. Expression in Xenopus oocytes characterized ZMK1 as an inwardly rectifying K + channel activated by external acidi®cation, while ZMK2 mediates voltage-independent and proton-inhibited K + currents. In search of the related gene products in planta, we applied the patch±clamp technique to protoplasts isolated from the cortex and vasculature of Zea mays coleoptiles and mesocotyls. In the cortex, a 6±8 pS K + channel gave rise to inwardly rectifying K + currents. Like ZMK1, this channel was activated by apoplastic acidi®cation. In contrast, protoplasts from vascular tissue expressing the sucrose transporter ZmSUT1 were dominated by largely voltageindependent K + currents with a single-channel conductance of 22 pS. The pronounced sensitivity to the extracellular protons Ca 2+ , Cs + and Ba 2+ is reminiscent of ZMK2 properties in oocytes. Thus, the dominant K + channels in cortex and vasculature most likely represent the gene products of ZMK1 and ZMK2. Our studies on the ZMK2-like channels represent the ®rst in planta analysis of a K + channel that shares properties with the AKT3 K + channel family.

Allene oxide synthase (AOS) is a key enzyme for the biosynthesis of jasmonic acid (JA). We identified four AOS gene homologs, named OsAOS1-4, in the database of a japonica rice genome and cloned a full-length cDNA of OsAOS1. The analysis of deduced amino acid sequences indicated that only OsAOS1 has a chloroplast transit peptide among all the identified monocot AOSs including OsAOSs. We found that the transcripts of OsAOS1 and OsAOS4 are up-regulated by red and far-red light in seedling shoots. The response in OsAOS1 transcripts occurred rapidly and transiently, while the response in OsAOS4 transcripts was slower and more sustainable; the maximal enhancement was greater in OsAOS1 transcripts than in OsAOS4 transcripts. The transcript of OsAOS1 was also up-regulated transiently in response to wounding, as reported for dicot AOSs. No wound-induced enhancement occurred, however, in OsAOS4 transcripts. Our results also indicated that OsAOS1, responding to both light and wounding, is the most highly expressed of all the OsAOSs in seedling shoots. By using phyA mutants of rice, it was demonstrated that the photoregulation of the AOS transcript level is mediated by phytochrome. It is suggested that this transcriptional photoregulation participates in the phytochrome-mediated inhibition of rice coleoptile growth.

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca 2ϩ . In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca 2ϩ . The swelling by either pathway required extracellular K ϩ and Cl Ϫ . The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca 2ϩ requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.