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Responses of rice seedlings to UV-B radiation (UV-B) were investigated, aiming to establish rice as a model plant for UV-B signalling studies. The growth of japonica rice coleoptiles, grown under red light, was inhibited by brief irradiation with UV-B, but not with blue light. The effective UV-B fluences (10-1 -103 μmol m-2 ) were much lower than those reported in Arabidopsis. The response was much less in indica rice cultivars and its extent varied among Oryza species. We next identified UV-B-specific anthocyanin accumulation in the first leaf of purple rice and used this visible phenotype to isolate mutants. Some isolated mutants were further characterized, and one was found to have a defect in the growth response. Using microarrays, we identified a number of genes that are regulated by low-fluence-rate UV-B in japonica coleoptiles. Some up-regulated genes were analysed by real-time PCR for UV-B specificity and the difference between japonica and indica. More than 70% of UV-B-regulated rice genes had no homologs in UV-B-regulated Arabidopsis genes. Many UV-B-regulated rice genes are related to plant hormones and especially to jasmonate biosynthetic and responsive genes in apparent agreement with the growth response. Possible involvement of two rice homologs of UVR8, a UV-B photoreceptor, is discussed.

To understand the unique green–blue color of Puya alpestris (Bromeliaceae) flowers, we investigated their constituent anthocyanin and related compounds. An anthocyanin, two undescribed flavonols, and two flavones were isolated and identified as delphinidin 3,3′,5′-tri-O-β-glucopyranoside, myricetin 3-O-[α-rhamnopyranosyl-(1 → 6)-β-glucopyranoside]-3′,5′-di-O-β-glucopyranoside, myricetin 3,3′,5′-tri-O-β-glucopyranoside, luteolin 4′-O-glucoside, and apigenin 4′-O-glucoside. Furthermore, the presence of chlorophyll has also been revealed. P. alpestris petals show a gradient color appearance: Green–blue at the tip and blue at the base. This color difference between the tip and base was used to analyze the pigment components underlying the green–blue color expression. It was found that the petal tip contains the anthocyanin, flavonols, flavones, and chlorophyll in high quantities. Furthermore, the pH of petal juice was 6.2 and 5.6 at the tip and base, respectively. In vitro reconstruction revealed the blue color expression occurred via an intermolecular copigmentation between the anthocyanin and flavones, as well as yellow color expression, which was due to an increase in the absorption at 400–450 nm of the flavonols under the higher pH conditions. Furthermore, we found that the petal extract obtaining using 50% acetone containing chlorophyll showed the same absorption spectrum as that observed for the raw petal. These results indicate that the green–blue color of P. alpestris flowers is developed via an intermolecular co-pigmentation of the anthocyanin (delphinidin 3,3′,5′-tri-O-β-glucopyranoside) with flavones, such as luteolin 4′-O-glucoside, the yellow color expression of flavonols, such as myricetin 3,3′,5′-tri-O-glucoside under relatively high pH conditions in the cell sap, and the presence of chlorophyll.

Jasmonate plays key roles in plant growth and stress responses, as in defense against pathogen attack. Jasmonoyl-isoleucine (JA-Ile), a major active form of jasmonates, is thought to play a pivotal role in plant defense responses, but the involvement of JA-Ile in rice defense responses, including phytoalexin production, remains largely unknown. Here we found that OsJAR1 contributes mainly to stress-induced JA-Ile production by the use of an osjar1 Tos17 mutant. The osjar1 mutant was impaired in JA-induced expression of JA-responsive genes and phytoalexin production, and these defects were restored genetically. Endogenous JA-Ile was indispensable to the production of a flavonoid phytoalexin, sakuranetin, but not to that of diterpenoid phytoalexins in response to heavy metal stress and the rice blast fungus. The osjar1 mutant was also found to be more susceptible to the blast fungus than the parental wild type. These results suggest that JA-Ile production makes a contribution to rice defense responses with a great impact on stress-induced sakuranetin production.

†‡ § ¶ SUMMARY Two 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.

Summary The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T-DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch-angle phenotype and the gene consequently named AtLAZY1. Time-resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch-angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch-angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery.

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl2-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.

We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.

Although circumnutation occurs widely in higher plants, its mechanism is little understood. The idea that circumnutation is based on gravitropism has long been investigated, but the reported results have been controversial. We used dark-grown coleoptiles of rice (Oryza sativa L.) to re-investigate this issue. The following results supported the existence of a close relationship between gravitropism and circumnutation: (1) circumnutation disappears on a horizontal clinostat; (2) circumnutation is interrupted by a gravitropic response and re-initiated at a definable phase after gravitropic curvature; (3) circumnutation can be re-established by submergence and a brief gravitropic stimulation in the coleoptiles that have stopped nutating in response to red light; and (4) lazy mutants show no circumnutation. In spite of these results, however, there were cases in which gravitropism and circumnutation could be separated. Firstly, the non-circumnutating lazy coleoptile showed nearly a wild-type level of gravitropic responsiveness in its upper half, although this part was an active site of both gravitropism and circumnutation in wild-type coleoptiles. Secondly, coleoptiles could nutate without overshooting the vertical when developing phototropic curvature. It is concluded that gravitropism influences, but it is not directly involved in the process of circumnutation. It is further suggested that a gravity signal, shared with gravitropism, contributes to the maintenance of circumnutation.

Tropisms of higher plants have been investigated for well over a century. Only recently, however, we have begun to establish their mechanisms firmly, mainly thanks to the availability of mutants and genome sequence information. For example, the starch-statolith hypothesis is now best supported as the main mechanism by which plants perceive gravity direction. Phototropins have been identified as the photoreceptors for the major blue-light-sensitive phototropism. Investigations have been extended to elucidate the relationships among tropisms and the controlling roles played by environmental factors, such as light. We are now finding examples in which phototropic and hydrotropic responses are modified through the environmental control of counteracting gravitropism. We are also finding that seedlings generally become phototropically competent only after phytochrome is activated. Such results are providing insights into how plants use tropisms to achieve adaptive growth movements.

We identified a gene of maize (Zea mays L.) that is transcriptionally activated in decapitated coleoptiles. The amino acid sequence deduced from its full-length cDNA indicated that the identified gene encodes a novel leucine-rich-repeat receptor-like kinase. The gene is named WOUND-RESPONSIVE AND PHYTOCHROME-REGULATED KINASE1 (WPK1) based on the findings of this study. Database searches revealed two and three homologs of WPK1 for Arabidopsis thaliana and rice, respectively. These homologs occurred along with WPK1 on a phylogenetic branch separated from all reported receptor kinases. We uncovered that the level of WPK1 transcripts is up-regulated rapidly and transiently in response to wounding and red light. The response to red light was reversible by far-red light, indicating that it is mediated by phytochrome. Applied jasmonic acid activated the expression of WPK1, while ethylene, salicylic acid and abscisic acid had no such effect. These results strongly suggested that WPK1 is a component of the jasmonate-mediated signaling that participates in both wound-induced defensive and phytochrome-mediated photomorphogenetic responses. Furthermore, it was found that both wounding and red light up-regulate the transcript level of ZmAOS, a gene for the jasmonate biosynthesis enzyme allene oxide synthase, and that auxin inhibits the expression of WPK1 but not of ZmAOS. We present a model of jasmonate-mediated signaling to explain the results obtained.

It has been found that coleoptiles of dark-grown rice (Oryza sativa L.) seedlings undergo regular circumnutation in circular orbits with periods of about 180 min. Both clockwise and counter-clockwise movements were observed, but individual coleoptiles continued to rotate only in one direction. Light-grown seedlings did not show circumnutation. In fact, dark-grown seedlings were found to cease circumnutating in response to a pulse of red light (R). This light-induced inhibition of circumnutation was demonstrated to involve both a FR-inducible very-low-fluence response, solely mediated by phytochrome A, and a FR-reversible low-fluence response, mediated by phytochrome B and/or C. The R-induced inhibition of circumnutation showed temporal agreement with the R-induced inhibition of coleoptile growth, suggesting that the former results from the latter. However, about 25% of growth activity remained after R treatment, indicating that circumnutation is more specifically regulated by phytochrome. The R-treated coleoptile showed gravitropism. Investigation of the growth differential for gravitropic curvature revealed that gravitropic responsiveness was rather enhanced by R. The results suggested that gravitropism is not a cause of circumnutation. It remained probable, however, that gravity perception is a part of the mechanism of circumnutation. It is speculated that the circumnutation investigated aids the seedling shoot in growing through the soil.

We isolated a mutant, named coleoptile phototropism1 (cpt1), from γ-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.

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.

A mutant showing a long coleoptile phenotype under white light was isolated from gamma-ray-mutagenized rice (cv. Nihonmasari). This mutant, named cpm1 (coleoptile photomorphogenesis 1), has been found to be impaired in phytochrome-mediated inhibition of coleoptile growth. Another outstanding feature of the mutant is impaired anthesis. Under red light (R), cpm1 coleoptiles elongate at a higher rate than wild-type (WT) coleoptiles, owing to substantially reduced responsiveness to R. This phenotype occurs in an age-dependent manner, and cpm1 coleoptiles become responsive to R as they elongate. The impairment was found in both very-low-fluence and low-fluence responses. Mutant coleoptiles also elongate longer than WT coleoptiles in darkness, but in this case the long coleoptile results from an extended elongation period. The cpm1 mutation does not affect the following phytochrome responses: the growth stimulation in submerged coleoptiles (uncovered in this study), potentiation of greening, and down-regulation of PHYA transcription. The cpm1 mutation does not significantly affect the level of spectroscopically detectable phytochrome and the transcription levels of three phytochrome genes (PHYA-C). It is concluded that the CPM1 gene is involved in the phytochrome signal transduction that specifically leads to growth inhibition. Some aspects of rice seedling photomorphogenesis are discussed in relation to the results obtained.

Protoplasts isolated from the laminar pulvinus of Phaseolus vulgaris and bathed in a medium containing KCl as the major salt were found to swell in response to IAA and to shrink in response to ABA. The protoplasts of flexor cells and those of extensor cells responded similarly. The results indicate that the cellular content of osmotic solutes is enhanced by IAA and reduced by ABA. The IAA-induced swelling was abolished when either the K(+) or the Cl(-) of the bathing medium was replaced by an impermeant ion or when the medium was adjusted to neutral pH (instead of pH 6). The response was inhibited by vanadate. It is concluded that the swelling is caused by enhanced influxes of K(+) and Cl(-), which probably occur through K(+) channels and Cl(-)/H(+) symporters, respectively. The ABA-induced shrinking was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid, an anion-channel inhibitor, suggesting that it is caused by Cl(-) efflux through anion channels and charge-balancing K(+) efflux through outward-rectifying K(+) channels. It appears that the two plant hormones act on pulvinar motor cells to regulate their turgor pressure, as they do in stomatal guard cells. The findings are discussed in relation to the pulvinar movements induced by environmental stimuli.

Protoplasts isolated from red-light-grown maize (Zea mays L.) coleoptiles shrank transiently upon brief exposure (e.g. 30 s) to blue light under background irradiation with red light. The maximal volume reduction (about 4% at a saturating fluence) occurred about 5 min after blue-light stimulation. The response was prevented by the anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Red light and far-red light did not induce any comparable response. Protoplasts of different sizes and those isolated from different coleoptile positions showed similar responses. After treatment with a saturating blue-light pulse, the protoplasts became responsive to a second pulse and gained full responsiveness within 5 min, suggesting that the photoreceptor system involves a dark-reversible component. The response to continuous blue light was also found to be transient. The protoplast volume was reduced during about 6 to 9 min of irradiation and returned within the next 30 min to the control level. The response to continuous blue light was saturated at 30 [mu]mol m-2 s-1. However, when the fluence rate was enhanced 10-fold after a period of irradiation at 30 [mu]mol m-2 s-1, the protoplasts showed another shrinking response. These and other kinetic results indicate that the photoreceptor system undergoes a photosensory adaptation. Growth in different zones of the coleoptile was inhibited by blue light transiently after pulse stimulation, as well as during continuous stimulation. It was concluded that the observed protoplast shrinking is related to the blue-light-induced inhibition of coleoptile growth.

A microbeam irradiation technique was used to analyze phytochrome-mediated phototropism of the protonema of the fern Adiantum capillus-veneris. One side of the sub-apical zone of a dark-adapted protonema was irradiated with a red-light (R) microbeam to induce phototropic curvature toward the irradiated side. Except the cases where fluence-response relationships were examined, the protonema was stimulated with the microbeam for about 30 s to provide a fluence (5.3–5.5 mmol m-2) optimal for curvature. When the whole protonema was pretreated with a high fluence of R (about 9 mmol m-2), no significant curvature could be induced by immediately subsequent one-sided microbeam stimulation. It was found, however, that curvature became inducible progressively as the time of microbeam stimulation was delayed after the R pretreatment. The protonema gained nearly full responsiveness within 40 min after the pretreatment. Moreover, the inductive effect of microbeam R escaped rapidly from the reversing effect of far-red light. These results indicated that most of the far-red-absorbing form of phytochrome (Pfr), which mediates the phototropic response, undergoes relatively fast dark reversion to the red-absorbing form. The dark reversion kinetics was analyzed further by taking into account that the phototropic responsiveness after the R pretreatment was measured in the presence of background Pfr over the protonema. This analysis revealed a rate constant of about 0.002 s-1 (t1/2˜ 6 min) for the dark reversion. It is considered that the Pfr dark reversion plays a role in establishing a lateral Pfr gradient in the unilaterally irradiated protonema.

Phototropism of rice (Oryza sativa L.) coleoptiles induced by unilateral blue light was characterized using red-light-grown seedlings. Phototropic fluence-response relationships, investigated mainly with submerged coleoptiles, revealed three response types previously identified in oat and maize coleoptiles: two pulse-induced positive phototropisms and a phototropism that depended on stimulation time. The effective ranges of fluences and fluence rates were comparable to those reported for maize. Compared with oats and maize, however, curvature responses in rice were much smaller and coleoptiles straightened faster after establishing the maximal curvature. When stimulated continuously, submerged coleoptiles developed curvature slowly over a period of 6 h, whereas air-grown coleoptiles, which showed smaller phototropic responsiveness, established a photogravitropic equilibrium from about 4 h of stimulation. The plot of the equilibrium angle against log fluence rates yielded a bell-shaped optimum curve that spanned over a relatively wide fluence-rate range; a maximal curvature of 25 degrees occurred at a fluence rate of 1 micromole m-2 s-1. This optimum curve apparently reflects the light sensitivity of the steady-state phototropic response.

Gravitropism of oat (Avena sativa L.) and wheat (Triticum aestivum L.) coleoptiles was investigated in relation to the displacement angle or to the initially set stimulation angle (SA). We measured curvature rates at the early phase of curvature, before it was affected by the drop in SA resulting from the curvature response itself. The plot of the rates against the sines of initial SAs revealed similar curves for oats and wheat, which approached saturation as the sine increased to unity. The two species and previously analyzed rice [Iino et al. (1996) Plant Cell Environ. 19: 1160] appeared to have similar gravisensitivities. Initial SAs below and over 90 degrees yielded comparable rates when the sine values were the same, indicating that the extent of gravitropism is determined by the gravity component perpendicular to the organ's long axis. Long-term curvature kinetics at different SAs indicated that the net curvature rate dropped sharply before the tip reached the vertical position and then the tip approached the vertical slowly, with clear oscillatory movements in the case of wheat. During this late curvature phase, the coleoptile straightened gradually, although none of its parts had yet reached the vertical. When rotated on horizontal clinostats or displaced upwards to reduce SA in the late curvature phase, coleoptiles bent in the opposite direction. These results demonstrated that autotropism counteracts gravitropism to straighten coleoptiles.

Gravitropism of maize and rice coleoptiles was investigated with respect to its dependence on the angle of displacement or the initial stimulation angle (ISA). Close examination of curvature kinetics and the response to a drop in stimulation angle (SA) indicated that the gravtropic response during an early but substantial part of the curvature development is directly related to the ISA, there being no effect of the reduction of SA resulting from the curvature response itself. On the basis of this finding, the relationship between the steady SA and the curvature rate was determined. In maize, the curvature rate increased linearly with the sines of SAs up to an SA of 90 degrees. Rice coleoptiles, however, showed a saturation curve in the same range of SAs. The saturation profile was nearly identical between coleoptiles grown in air and those submerged in water, although the latter elongated much faster. Rice coleoptiles appeared to be far more sensitive to gravity than maize coleoptiles. It is concluded that the sensitivity to gravity, assessed through dependence on ISA, is a property inherent to a given gravitropic organ. Long-term measurements of curvature indicated that the coleoptiles bend back past the vertical. This overshooting was marked in submerged rice coleoptiles.

— Phytochrome-mediated phototropism of the protonema of the fern Adiantum capillus-veneris was studied in view of the hypothesis that phototropism is controlled by the gradient of Pfr (phytochrome in the far-red-absorbing form) across the short axis of the protonema. Fluence-response relationships were investigated using a microbeam irradiation technique that allowed simultaneous stimulation of the two sides of the subapical portion of the protonema with different fluences of red light. Mathematical models describing the tropic response as a function of fluence were derived from the hypothesis in consideration of the minimal phototransformation kinetics of phytochrome. and the fitness of the functions to the experimental data was examined. The analytical results were then evaluated in view of the photochemical properties of phytochrome known from the literature. It is concluded that the extent of the tropic response is determined by the difference in the Pfr concentrations between the two sides of the protonemal cell. It is further suggested that, even if phytochrome exists as a dimer in vivo, the physiological unit of phytochrome is the monomer.

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.

Plants respond to light to undergo adaptive changes in their growth patterns.The idea that these responses are mediated by plant hormones has long been investigated. In fact, the first-identified plant hormone auxin was discovered and the original plant hormone concept was formulated through the studies of coleoptile phototropism, a light-induced growth movement. The role for auxin has since been a central subject of phototropism research. The elongation growth of seedling organs such as mesocotyls and hypocotyls is subject to marked lightinduced inhibition. Evidence has been provided that auxin and other plant hormones participate in these typical photomorphogenetic responses. This chapter reviews and discusses the mechanisms of phototropism and photomorphogenesis, focusing on the role played by the native auxin indole-3-acetic acid (IAA). Our understanding of the molecular mechanisms by which auxin mediates these physiological processes is far from complete, but recent molecular genetic studies have began to yield useful information.

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.

Blue light was found to induce shrinkage of the protoplasts isolated from first-leaf lamina pulvini of 18-day-old Phaseolus vulgaris. The response was transient following pulse stimulation, while it was sustainable during continuous stimulation. No apparent difference was found between flexor and extensor protoplasts. Protoplasts of the petiolar segment located close to the pulvinus showed no detectable response. In the plants used, the pulvinus was fully matured and the petiole was ceasing its elongation growth. When younger, 12-day-old, plants were used, however, the petiolar protoplasts did respond to blue light. The pulse-induced response was similar to that in pulvinar protoplasts, although the response to continuous stimulation was transient and differed from that in pulvinar protoplasts. No shrinkage was induced in pulvinar protoplasts when the far-red-light-absorbing form of phytochrome was absent for a period before blue-light stimulation, indicating that the blue-light responsiveness is strictly controlled by phytochrome. Inhibitors of anion channels and H(+)-ATPase abolished the shrinking response, supporting the view that protoplasts shrink by extruding ions. The response of pulvinar protoplasts is probably involved in the blue-light-induced, turgor-based movement of pulvini. The blue-light responding system in pulvini is suggested to have evolved from that functioning in other growing organs.

Phototropism refers to the curvature movements of organs whose directionality is determined by the direction or uneven distribution of impinging light. Optimizing the acquisition of light energy in shade, and directing the growth of shoots away from shade, appear to be the most common roles played by phototropism. Seedling organs such as coleoptiles and hypocotyls are typically phototropic and have served as model systems in laboratory studies. Results with these organs have revealed the following basic aspects of the process of phototropism. The light signal is perceived by the photoreceptor which is most sensitive to blue light. The direction of movement is determined by the transverse light gradient generated within the photosensing zone. The phototropic curvature results from an asymmetry of growth, which is not caused by local light-growth response but by growth redistribution. The plant hormone auxin is laterally translocated in response to the transverse light gradient and mediates the growth redistribution. This chapter summarizes most of the results obtained during the last century, including the recent advances made in molecular genetic studies. The field has experienced controversies over some of its basic aspects. It will be noted that many of the apparently complex and conflicting results represent aspects of the overall phototropic system that is undoubtedly highly sophisticated.

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 inhibitorN-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.

Time-dependent phototropism (TDP), sometimes called second positive curvature, occurs when the duration of phototropic stimulation with blue light (B) exceeds a few minutes. TDP was characterized in maize (Zea mays L.) coleoptiles raised under continuous red light (R). Subsequently, coleoptiles adapted to darkness were used to investigate the effect of R on TDP. It was found that TDP, which is induced in R-grown coleoptiles, does not occur in dark-adapted coleoptiles and that dark-adapted coleoptiles begin to show TDP after treatment with R. The TDP responsiveness became maximal 1-2 h after treatment with a R pulse and decreased during the next few hours. At least 10 min was required after a short pulse of R before the coleoptile began to respond to B for the induction of TDP. The effect of R in establishing the TDP responsiveness was totally suppressed by a pulse of far-red light given immediately after an inductive pulse of R. It is concluded that the mechanism of TDP requires for its establishment a R signal perceived by phytochrome. The TDP of R-grown and R-pretreated coleoptiles showed relationships to stimulation times and fluence rates that are similar to those reported for oat coleoptiles, except that TDP of maize showed a sharp increase in its magnitude within a narrow range of stimulation times as short as 5-10 min.

Coleoptile tips (about 2.5 mm in length) were excised from 3-day-old dark-adapted maize {Zea mays L.) seedlings and incubated in darkness in potassium phosphate buffer that contained u C-L-tryptophan (Trp). Subsequent analysis by gas chromatography-mass spectrometry indicated that a significant portion of endogenous indole-3-acetic acid (IAA) had been labeled with 14 C. About 8% of the IAA that diffused from the tissue into the medium during incubation from 0.5 to 2 h was labeled, and 12% of the IAA extracted from the tissue after a 2-h incubation was labeled. On the other hand, 30% of the Trp extracted from the tissue after a 2-h incubation was l4 C-Trp, which was more than those determined for IAA. Since the experiments were carried out under the non-steady-state conditions in which the tissue content of u C-Trp increased with time, and since the extracted Trp included the M C-Trp in the apoplastic space, it seemed that synthesis de novo from Trp was the major means by which IAA was produced in the coleoptile tip. The conversion of Trp to IAA was not detected in sub-apical segments (5-7.5 mm from the top) that were incubated similarly, an indication that synthesis of IAA occurs specifically in the tip region. When intact seedlings were irradiated with a pulse of red light 2 h before excision of tips and the application of u C-Trp, the amounts of extractable and diffusible IAA were reduced by 40-60% without a change in the rate of I4 C incorporation. This result indicated that the production of IAA from

Change of membrane potential is well known to play a key role in signal transduction and turgor movement in excitable plant tissue (Sibaoka 1966). The electrogenic nature of some ion pump in fungal and plant cells has already been demonstrated (Slayman 1965, Higinbotham et al. 1970). This electrogenic ion pump was found to be a proton extruding one fueled by ATP (Kitasato 1968, Shimmen and Tazawa 1977). At the same time, the acid growth hypothesis for the action of auxin-induced promotion of plant elongation growth appeared (Hager et al. 1971, Rayle and Cleland 1970). A close correlation between electrogenic proton transport and elongation growth was indicated in the stem (Katou and Okamoto 1970). Furthermore, fusicoccin-induced promotion of growth with electrogenic H § extrusion (Marre 1979) and IAA-induced hyperpolarization of the plasma membrane have been demonstrated (Cleland et al. 1977). Through these studies, it has been gradually recognized that proton transport plays an important role in plant growth. Much progress has been made in the biophysical, biochemical and cell physiological studies of membrane transport. It has been revealed that proton pump and ion channels play crucial roles in signal transduction, turgor regulation, cell motility, etc. As a consequence, it has become possible to discuss various aspects of the function of membranes in the integrated activities of plants such as growth and stress adaptation (Dainty et al. 1989). During the last fifteen years or so, we have held a series of symposia and workshops on membrane function in plants (Tazawa et al. 1989). The last symposium was held at Nagoya in November 1994 on the occasion of the retirement of Professor Hisashi Okamoto from Yokohama City University. Professor Okamoto did the major portion of his work at Nagoya University. This symposium was focused on "membrane function and growth regulation in plants". The present issue of the JPR-symposium is a selection of papers highlighting the various aspects of opinion presented to this symposium. We hope that this issue will evoke the interest of readers on the subjects and fields discussed. We are very much indebted to Professors Masashi Tazawa, Yoshio

Phototropisms induced by a pulse (1-30 seconds) of blue light in red-light-grown coleoptiles of oats (Avena sativa L.) and maize (Zea mays L.) were investigated in terms of fluence-response relationships and time courses. Phototropic stimulation was made by a laser beam (457.9 nanometers), allowing application of high-fluence pulses. The phototropic fluence-response curves for oats and maize revealed two peaks in the positive range, thus indicating the occurrence of two separable pulse-induced positive responses. The response at low fluences corresponded to the `first positive curvature.' The response at high fluences was very small in oats, but was large in maize. Reciprocity was valid in this high-fluence response (tested only for maize), indicating that it is distinct from the so-called `second positive curvature.' In oats, the trough between the two positive responses fell into the negative range. This negative response, corresponding to the `first negative curvature,' showed time courses distinct from those of `first positive curvature:' the negative response was induced after a longer time lag and developed with a more gradual increase of the rate of bending. The maximal rate of the negative response was as high as one-half of that of first positive curvature. In maize, the trough between the two responses was in the positive range, and the time-course result revealed no apparent response counteracting the positive responses. Physiological and ecological implications of the pulse-induced phototropisms are discussed.

Phototropism is a common property of plants, but it is not known if different species use the same photoreceptor for their response. We have determined fluence-response relations for phototropism in response to brief, broad-band blue irradiation for four plant species grown under red light (Amaranthus paniculatus, Linum usitatissimum, Vigna radiata and Medicago sativa) and compared these to ones previously obtained for Pisum sativum and Zea mays, grown under similar conditions. Curves for all species showed a bell-shaped dependence on fluence, a characteristic of first positive curvature as originally defined for the Avena coleoptile, and had a similar optimal fluence, near 3 H.mol m−2. We have obtained an action spectrum in the blue and UV spectral regions for first positive phototropism of the hypocotyl of alfalfa grown under red light. Fluence-response curves at wavelengths between 300 and 500 nm were nearly identical in shape and magnitude; whereas below 300 nm, their slopes and maximum curvatures were reduced. The action spectrum showed that activity rose sharply at wavelengths below 500 nm, peaked at 450 nm with shoulders on either side of that peak, and had lesser peaks at 380 and, in the far ultraviolet, at 280 nm. This action spectrum was very similar to ones in the literature (obtained between 350 and 500 nm) for first and second positive phototropism of oat coleoptiles. We conclude that the same photoreceptor mediates phototropism in oat and alfalfa.

— Intact leaves of Commelina communis irradiated with high fluence rates of red light, showed discrete increases in stomatal conductance in response to pulses (1-100 s) of blue light (250 μmol m−2 s−1). Red light pulses were ineffective, indicating that the conductance increases were not mediated by photosynthesis and that they constitute a specific stomatal response to blue light. The response peaked 15 min after the pulse and was completed within50–60 min. Conductance increases were proportional to pulse duration up to about 30 s and saturated at longer exposures. The relationship between stomatal responses and pulse duration approximately fitted an exponential function, with a t 9s. Pulse responses at two different fluences indicated that reciprocity held. Responses to two consecutive pulses varied with time between pulses. A saturating pulse applied immediately after a preceding one induced no additional response; two saturating pulses 50 min apart caused two identical, consecutive responses. Total increases in conductance induced by two pulses separated by intermediate time intervals increased with time between pulses with a = 9 min. These results point to a blue light-dependent photoconversion of a molecular form, with the activity of the photoconversion product decaying in a thermal reaction. Under continuous blue light, prevailing fluence rates and rates of the light and thermal reactions are postulated to determine steady-state activities of the photoconversion product and proportional increases in conductance levels. These findings have implications for the environmental and metabolic roles of the stomatal response to blue light.

Green light affects the growth of etiolated cereal seedlings. Green ‘safelights’ are therefore not safe for photomorphogenetic studies. Invisible infrared (IR) radiation, in which objects can be viewed using an IR-scope, is proposed as a safelight. The IR radiation exerts no measurable effect on the growth of cereal seedlings.

The stomatal response to blue light was analyzed with gas-exchange techniques in Commelina communis L. leaves by using high-fluence-rate short pulses. Pulses of blue light were given under a background of high-fluence-rate red light, which maintained photosynthesis at near saturation and stomatal conductance at a steady state. A single blue light pulse of 1-100 sec induced an increase in stomatal conductance, which peaked after 15 min and then returned to the initial steady-state level within 50-60 min after the pulse. The response could be repeatedly induced in the same leaf. Red light pulses on a red background did not induce any comparable response. The stomatal response quantified by integrating the conductance increases after pulse application approached saturation with increasing pulse duration ( t ½ ≈9 sec with 250 μmol·m -2 ·sec -1 of blue light). After a saturating pulse, sensitivity to a second pulse was restored slowly. This recovery response, quantified from the conductance increases caused by the two pulses, approached saturation with a t ½ of ≈9 min. These results were used to test a model in which a molecular component in the sensory transduction process is considered to exist in two interconvertible forms, A and B . If B is the physiologically active form inducing stomatal opening, then A is the inactive form. The A to B conversion is a light-induced reaction and the B to A conversion is a thermal reaction. Rate constants for these reactions were estimated from single- and double-pulse experiments (at a fluence rate of 250 μmol·m -2 ·sec -1 , k 1 = 0.075 sec -1 ; thermal rate constant k d = 0.0014 sec -1 ), allowing the calculation of steady-state concentration of B under continuous irradiation. The calculated values accurately predicted the steady-state stomatal conductances under continuous blue light.

The first cell cycle of Adiantum spores, which is induced by red light (R), was studied with regard to its temporal dependence on Pfr (the active form of phytochrome) and Ca2+. Responses were quantified with increases of the spore content of DNA, thus limiting the investigation to the reactions taking place before the S phase of the cell cycle (i.e. during G0/G1/S transitions). Spores hydrated for more than 2 days in the standard medium (includes 1 millimolar free Ca2+) showed, after stimulation with a saturating R pulse, an increase of DNA beginning at about 25 hours and saturating at about 55 hours. Reversal by far-red light of the inductive effect of R was used to examine the temporal requirement for Pfr. Spores became dependent on the supply of external Ca2+ when incubated in a low Ca medium (32 nanomolar free Ca2+ with 10 millimolar EGTA); this culture condition was used, after observing that the DNA increase occurs similarly if Ca2+ is supplied after the R pulse, to examine the temporal requirement for external Ca2+. It was concluded that the G1 phase of the spore is separated into three subphases: (a) the Pfr-dependent period which immediately follows the R pulse and varies among individual spores from a few to about 25 hours, (b) the Ca2+-dependent period (about 10 hours) which occurs immediately before the S phase, and (c) a gap (15-20 hours) between the two periods. In the Ca2+-dependent period, spores require the presence of extracellular Ca2+. This period occurs only during a determined time after the R pulse, and the competence of spores to enter the S phase is lost sharply if external Ca2+ is not available continuously during this period.

Effects of batatasins I, III and V, phenolic growth inhibitors occuring in dormant bulbils of Dioscorea batatas Decne., on photosynthetic reactions of chloroplasts from spinach (Spinacia oleracea L.) and on respiration of mitochondria from potatoes (Solanum tuberosum L.) were investigated. In chloroplasts, the batatasins effectively inhibited CO2-dependent oxygen evolution and electron flow from water to acceptors such as dichlorophenolindophenol, ferricyanide and methylviologen. Photosystem-I dependent electron transport from ascorbate to oxygen was stimulated. The proton conductivity of thylakoid membranes was increased and phosphorylation was uncoupled from electron transport. Inhibition of electron transport with water as electron donor appeared to precede uncoupling. In mitochondrial, batatasin I did not much inhibit succinate-dependent O2 uptake in the absence of ADP, but caused strong inhibition in the presence of ADP. Batatasins III and V inhibited oxygen uptake irrespective of the presence or absence of ADP. Inhibition of chloroplast and mitochondrial reactions by batatasins was shown to be reversible.

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.

Positive phototropic curvature of Phycomyces sporangiophores (stage I) induced by a short pulse (10 sec) of blue light has been characterized. The curvature response, measured 40 min after irradiation, increases with increasing fluences up to ≈10 -4 J·m -2 ; it then decreases and becomes nearly 0 at a fluence of ≈10 -1 J·m -2 . The threshold fluence is found near 10 -7 J·m -2 , indicating over 1000-fold higher sensitivity than the first positive phototropism of grass coleoptiles. Positive curvature of the sporangiophore becomes detectable 5 min after irradiation and increases with time up to ≈40 min. During the course of this curvature development, overall elongation growth of the sporangiophore is first stimulated and then inhibited. The relationship between the phototropic response and the response in overall elongation growth is discussed, and it is suggested that the primary mechanisms of these responses are distinct.

Growth redistribution which occurs as a result of phototropic stimulation was studied in red light-grown, maize (Zea mays L.) seedlings. The pattern of elongation of small areas (0.1mm2) of coleoptile epidermis on intact plants was analysed from time-lapse, photomicrographic records. Growth following unilateral, pulse irradiation with blue light was depressed on the illuminated side and was stimulated on the shaded side. The time at which the change in growth rate occurred, on both illuminated and shaded sides, was significantly earlier in apical patches than it was in basal patches. Both kinds of change in the growth rate (stimulation and depression) occurred rapidly such that a new, constant growth rate was often established within five minutes. Micrographic, time-lapse records were also obtained of growth changes induced by sub-apical, unilateral application of a spot of an indole-3-acetic acid (IAA) and lanolin mixture. Growth on the side of the coleoptile to which IAA had been applied was similar to the growth on shaded sides of phototropically stimulated plants. The distance between apical and basal patches and the elapsed time between their changes in growth rate gave a velocity at which the growth response moved basipetally. Calculation of this velocity for blue light and auxin treatment gave values that were not significantly different. Thus, basipetal movement of a transverse auxin gradient could mediate growth changes that cause curvature of the coleoptile towards first positive fluences of blue light.

Brief irradiation of 3-d-old maize (Zea mays L.) seedlings with red light (R; 180 J m(-2)) inhibits elongation of the mesocotyl (70-80% inhibition in 8 h) and reduces its indole-3-acetic acid (IAA) content. The reduction in IAA content, apparent within a few hours, is the result of a reduction in the supply of IAA from the coleoptile unit (which includes the shoot apex and primary leaves). The fluence-response relationship for the inhibition of mesocotyl growth by R and far-red light closely resemble those for the reduction of the IAA supply from the coleoptile. The relationship between the concentration of IAA (1-10 μM) supplied to the cut surface of the mesocotyl of seedlings with their coleoptile removed and the growth increment of the mesocotyl, measured after 4 h, is linear. The hypothesis that R inhibits mesocotyl growth mainly by reducing the IAA supply from the coleoptile is supported. However, mesocotyl growth in seedlings from which the coleoptiles have been removed is also inhibited by R (about 25% inhibition in 8 h). This inhibition is not related to changes in the IAA level, and not relieved by applied IAA. In intact seedlings, this effect may also participate in the inhibition of mesocotyl growth by R. Inhibition of cell division by R, whose mechanism is not known, will also result in reduced mesocotyl elongation especially in the long term (e.g. 24 h).

Evidence is presented that more than one blue-light photoreceptor plays a role in morphogenesis, and that there are at least three, distinguishable on the basis both of action spectra and other criteria, which may be found both in green plants and fungi. One of these has been tentatively identified as a flavoprotein-cytochrome complex, most probably located in the plasma membrane. Studies with oat seedlings suggest that it may be involved in photoreception for phototropism, at least for the first positive curvature response. Both photoreduction of the cytochrome, via excitation of the flavin, and phototropic sensitivity in the first positive curvature range are similarly affected by diphenylether herbicides. The second class of photoreceptors can be distinguished from the first inNeurosporaby both genetic and physiological evidence, as well as by the action spectrum. It could be either flavin or carotenoid, although a different moiety is not excluded. The third class, distinguished only by action spectroscopy, shows a single sharp action peak near 475 mm, and seems unlikely to be either a flavin or a carotenoid, though they are not rigorously excluded. The first positive phototropic curvature response in maize shows a redistribution of growth consonant with the Cholodny-Went hypothesis for tropic responses, with an increase in the growth rate of the shaded side over dark controls, a concomitant decrease on the illuminated side, and no net change in overall growth rate.

Blue-light-induced phototropism of maize (Zea mays L.) coleoptiles was studied with a view to kinetic models. Red-light-grown plants were used to eliminate complication arising from the activation by blue light of phytochrome-mediated phototropism. In the first part, mathematical models were developed to explain the phototropic fluence-response data, which were obtained for the responses induced by a single unilateral pulse (30 s) and those induced by a unilateral pulse (30 s) given immediately after a bilateral pulse (30 s, fixed fluences). These data showed bell-shaped fluence-response curves, characteristic of "first positive curvature". Modelling began with the assumptions that the light gradient plays a fundamental role in phototropism and that the magnitude of the response is determined by the gradient, or the concentration difference, in a photoproduct between the irradiated and the shaded sides of the tissue. Minimal mathematical models were then derived, by defining chemical kinetics of the photoreaction and introducing the minimum of parameters needed to correlate the incident fluencerate to the functional fluence-rates within the tissue, the functional fluence-rate to the rate constant of the photoreaction, and the photoproduct concentration difference to the curvature response. The models were tested using a curve-fitting computer program. The model obtained by assigning first-order kinetics to the photoreaction failed to explain the fluence-response data, whereas application of second-order kinetics led to a successful fit of the model to the data. In the second part, temporal aspects of the photosystem were examined. Experimental results showed that a high-fluence bilateral pulse eliminated the bell-shaped fluence-response curve for an immediate unilateral pulse, and that the curve gradually reappeared as the time for unilateral stimulation elapsed after the bilateral pulse. The model based on a second-order photoreaction could be extended to explain the results, with assumed changes in two components: the concentration of the reactant for the photoproduct, and the light-sensitivity of the reaction. The reactant concentration, computed with the curvefitting program, showed a gradual increase from zero to a saturation level. This increase was then modelled in terms of regeneration of the reactant from the photoproduct, with an estimated first-order rate constant of about 0.001·s(-1). The computed value for the constant reflecting the light-sensitivity showed a sharp decline after the high-fluence pulse, followed by a gradual return to the initial level. From these analytical results, the appearance of "second positive curvature" was predicted.

Two new growth inhibitors, R-dihydromaleimide and R-dihydromaleimide β- d -glucoside, were isolated from 2-week-old pea shoots.

Unilateral irradiation with red light (R) or blue light (BL) elicits positive curvature of the mesocotyl of maize (Zea mays L.) seedlings raised under R for 2 d from sowing and kept in the dark for 1 d prior to curvature induction. The fluenceresponse curve for R-induced mesocotyl curvature, obtained by measuring curvature 100 min after phototropic induction, shows peaks in two fluence ranges, designated first positive range (from the threshold to the trough), and second positive range (above the trough). The fluence-response curve for BL is similar to that for R but shifted two orders of magnitude to higher fluences. Blue light elicits the classical first positive curvature of the coleoptile, whereas this response is not found with R. Positive mesocotyl curvature induced by either R or BL is eliminated by R given from above just before the unilateral irradiation, whereas BL-induced coleoptile curvature is not eliminated. The above results collectively offer evidence that phototropic curvature of the mesocotyl is induced by R-sensitive photosystem(s). Mesocotyl curvature in the second positive range is reduced by vertical far-red light (FR) applied after phototropic induction with R, but is not affected by FR applied before R. Unilateral irradiation with FR following vertical irradiation with a high R fluence leads to negative curvature of the mesocotyl. It is concluded that mesocotyl curvature in the second positive range results from a gradient in the amount of the FR-absorbing form of phytochrome (Pfr) established across the plant axis. Mesocotyl curvature in the first positive range is inhibited by vertical FR given either before or after phototropic induction with R. Since the FR used here is likely to produce more Pfr than the very low fluences of R eliciting the mesocotyl curvature in the first positive range, it is assumed that FR reduces the response in this case by adding Pfr at both sides of the plant axis. By rotating seedlings on a clinostat with its axis horizontal, the kinetics of mesocotyl curvature can be studied in the absence of a counteracting gravitropic response. On the clinostat, the R-induced mesocotyl curvature develops after a lag, through two successive phases having different curvature rates, the late phase is slower than the early phase. Negative curvature of the coleoptile can be induced by either R or BL; the BL-induced negative curvature is found at fluences higher than those giving positive curvature. The clinostat experiments show that the negative coleoptile curvature induced by either R or BL is a gravitropic compensation for positive mesocotyl curvature.