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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 butwere insensitive to lowfluence 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 andmediates 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.

Several members of the AGCVIII kinase subfamily, which includes PINOID (PID), PID2, and WAVY ROOT GROWTH (WAG) proteins, have previously been shown to phosphorylate PIN-FORMED (PIN) auxin transporters and control the auxin flow in plants. PID has been proposed as a key component of the phototropin signaling pathway that induces phototropic responses, although the responses were not significantly impaired in the pid single and pid wag1 wag2 triple mutants. This raises questions about the functional roles of the PID family in phototropic responses. Here, we investigated hypocotyl phototropism in the pid pid1 wag1 wag2 quadruple mutant in detail to clarify the roles of the PID family in Arabidopsis (Arabidopsis thaliana). The pid quadruple mutants exhibited moderate responses in continuous light-induced phototropism with a decrease in growth rates of hypocotyls and normal responses in pulse-induced phototropism. However, they showed serious defects in enhancements of pulse-induced phototropic curvatures and lateral fluorescent auxin transport by red light pretreatment. Red light pretreatment significantly reduced the expression level of PID, and the constitutive expression of PID prevented pulse-induced phototropism, irrespective of red light pretreatment. This suggests that the PID family plays a significant role in phytochrome-mediated phototropic enhancement but not the phototropin signaling pathway. Red light treatment enhanced the intracellular accumulation of PIN proteins in response to the vesicle-trafficking inhibitor brefeldin A in addition to increasing their expression levels. Taken together, these results suggest that red light preirradiation enhances phototropic curvatures by upregulation of PIN proteins, which are not being phosphorylated by the PID family.

Development of the mutualistic arbuscular mycorrhiza (AM) symbiosis between most land plants and fungi of the Glomeromycota is regulated by phytohormones. The role of jasmonate (JA) in AM colonization has been investigated in the dicotyledons Medicago truncatula, tomato and Nicotiana attenuata and contradicting results have been obtained with respect to a neutral, promotive or inhibitory effect of JA on AM colonization. Furthermore, it is currently unknown whether JA plays a role in AM colonization of monocotyledonous roots. Therefore we examined whether JA biosynthesis is required for AM colonization of the monocot rice. To this end we employed the rice mutant constitutive photomorphogenesis 2 (cpm2), which is deficient in JA biosynthesis. Through a time course experiment the amount and morphology of fungal colonization did not differ between wild-type and cpm2 roots. Furthermore, no significant difference in the expression of AM marker genes was detected between wild type and cpm2. However, treatment of wild-type roots with 50 μM JA lead to a decrease of AM colonization and this was correlated with induction of the defense gene PR4. These results indicate that JA is not required for AM colonization of rice but high levels of JA in the roots suppress AM development likely through the induction of defense.

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.

We isolated a mutant, named coleoptile phototropism1 (cpt1), from [gamma]-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 ³H-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-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.

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 Ca2+. 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 Ca2+. The swelling by either pathway required extracellular K+ and Cl-. 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 Ca2+ 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.

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied 3H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants

ABSTRACT Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light photoreceptor phototropin1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We identified seven phosphorylation sites of NPH3 proteins in the etiolated seedlings of Arabidopsis and generated unphosphorable and phosphomimetic NPH3 proteins on those sites. Unphosphorable NPH3 showed a shortening of its subcellular localization in the cytosol and caused an inability to adapt to very low fluence rates of blue light (∼10−5 µmol m−2 s−1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened subcellular localization in the cytosol and could not lead to the adaptation for blue light at fluence rates of 10−3 µmol m−2 s−1 or more. Our results suggest that the activation levels of phot1 and the corresponding phosphorylation levels of NPH3 determine the rate of plasma membrane-cytosol shuttling of NPH3, which moderately maintains the active state of phot1 signaling across a broad range of blue-light intensities and contributes to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls. One sentence summary The phosphorylation status of NON-PHOTOTROPIC HYPOCOTYL3 proteins affects their subcellular localization and the photosensory adaptation of phot1 signaling. Footnotes * Funding information: This work was supported by the Japan Society for the Promotion of Science (JSPS: KAKENHI 25120710, 16H01231, 17H03694 to T.S., 16J01942 to T. K, 17K07451 to K.H., 16H06280, 17K19380, 18H05492 to T.H.), the Max-Planck-Gesellschaft to H.N., the Sasaki Environment Technology Foundation (to T.S.), Takeda Science Foundation (to T.S.), Ohsumi Frontier Science Foundation (to T.S.), and the NAGAI NS Promotion Foundation for Science of Perception (to T.S.).

The relationships between the distribution of the native auxin indole-3-acetic acid (IAA) and tropisms in the epicotyl of red light-grown pea (Pisum sativum L.) seedlings have been investigated. The distribution measurement was made in a defined zone of the third internode, using 3H-IAA applied from the plumule as a tracer. The tropisms investigated were gravitropism, pulse-induced phototropism, and time-dependent phototropism. The investigation was extended to the phase of autostraightening (autotropism) that followed gravitropic curvature. It was found that IAA is asymmetrically distributed between the two halves of the zone, with a greater IAA level occurring on the convex side, at early stages of gravitropic and phototropic curvatures. This asymmetry was found in epidermal peels and, except for one case (pulse-induced phototropism), no asymmetry was detected in whole tissues. It was concluded, in support of earlier results, that auxin asymmetry mediates gravitropism and phototropism and that the epidermis or peripheral cell layers play an important role in the establishment of auxin asymmetry in pea epicotyls. During autostraightening, which results from a reversal of growth asymmetry, the extent of IAA asymmetry was reduced, but its direction was not reversed. This result demonstrated that autostraightening is not regulated through auxin distribution. In this study, the growth on either side of the investigated zone was also measured. In some cases, the measured IAA distribution could not adequately explain the local growth rate, necessitating further detailed investigation.