High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.Abbreviations FR far-red light - FRc continuous far-red light - FRp pulses of far-red light - HIR high-irradiance responses - LFR low-fluence responses - OPHYA transgenic rice overexpressing oat phytochrome A - Pfr far-red light-absorbing form of phytochrome - phyA phytochrome A - R red light - Rc continuous red light - VLFR very low-fluence responses - WT wildtype We thank Marcelo J. Yanovsky for his help with the photographs and Professor Rodolfo A. Sanchez for providing a reprint of the paper by P.J.A.L. de Lint. This work was supported by grants from UBA (AG041) and Fundacion Antorchas (A-13218/1-15) to J.J.C.     

Time course of phytochrome-mediated changes in growth gradients on coleoptiles of Triticum aestivum L.

The length of parenchyma cells along the axis of dark-grown coleoptiles of Triticum aestivum L. and the pattern of competence for red-light-(R-) induced stimulation or inhibition of cell elongation in the course of coleoptile development were determined by microscopic measurements in a file of 240 cells from the tip to the base. On the basis of these measurements distinct zones (responding in different ways to R) were selected for studying the early time course of phytochrome-mediated growth-rate changes in intact coleoptiles by use of a sensitive transducer system. Between 2 d and 4 d after sowing dark-grown coleoptiles showed a graded incline in cell growth activity from the apex to the base (growth gradient). Whereas cell elongation in the coleoptile base ceased 4 d after sowing, cell elongation speeded up in the tip and middle region at that time. Those cells that grew slowly in darkness (tip and middle region between 2d and 3 d after sowing) were stimulated in growth by R-pulse irradiation (1 min R, 660 nm, 1000 J · m^–2). In contrast, the growth of fast-growing cells (base between 2 d and 4 d after sowing, tip and middle region between 4 d and 5 d after sowing) was inhibited by R. However, the starting time for R-induced growth changes was different for different coleoptile zones. The respective data point to the storage of a phytochrome-mediated signal in the cells of the middle region, until these cells become competent to respond to it; alternatively, Pfr, the far-red-light-absorbing form of phytochrome, may be stored in a stable form. Continuous recordings on the effect of R, far-red (FR) and R/FR on the zonal growth responses were made on intact coleoptiles, selected 3 d after sowing. During a 5-h investigation period the R-induced changes in growth rate could be divided into two phases: (i) A transient growth inhibition which started approx. 15 min after R. This response was qualitatively the same in all coleoptile zones investigated (tip, middle region, base). (ii) Zonal-specific growth responses which became measurable approx. 2.5 h after R, i.e. growth promotion in the tip, growth inhibition in the base and an adaptation of growth rate to the dark control level in the middle region. The R-induced growth rate changes were reversible by FR for both phases. Additional growth experiments on excised coleoptile segments under R and auxin application indicated that the zonal-specific growth promotion or inhibition may be not mediated by an influence of R on the auxin level.Abbreviations FR far-red light - Pfr far-red-light-absorbing form of phytochrome - R red light The technical assistance of Mrs. B. Liebe is gratefully acknowledged.     

The Rice COLEOPTILE PHOTOTROPISM1 gene encoding an ortholog of Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin

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.     

Circumnutation of rice coleoptiles: its occurrence, regulation by phytochrome, and relationship with gravitropism

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.     

Comparative analysis of anoxic coleoptile elongation in rice varieties: relationship between coleoptile length and carbohydrate levels, fermentative metabolism and anaerobic gene expression

Rice ( Oryza sativa L.) seeds can germinate under anoxia and can show coleoptile elongation. The anoxic coleoptile is usually longer than aerobic coleoptiles. Although several hypotheses have been proposed to explain the ability of rice to elongate coleoptiles under anoxia, conclusive experimental evidence explaining this physiological trait is lacking. In order to investigate whether metabolic and molecular markers correlate with anoxic coleoptile length, we screened 141 Italian and 23 Sri Lankan rice cultivars for their ability to elongate coleoptiles under anoxia. Differences in anoxic coleoptile length were used to evaluate whether a correlation exists between coleoptile length and biochemical and molecular parameters. The expression of genes coding for glycolytic and fermentative enzymes showed a very low correlation with anoxic coleoptile length. Although differences were found in carbohydrate content between the varieties tested, this parameter also does not appear to be critical in terms of coleoptile elongation. Efficient ethanol fermentation does, however, correlate well with the elongation of coleoptiles under anoxic conditions.     

Inhibitory action of red light on the growth of the maize mesocotyl: evaluation of the auxin hypothesis

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 M) 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).Abbreviations FR far-red light - IAA indole-3-acetic acid - P_fr phytochrome in the far-red-absorbing form - P_r phytochrome in the red-absorbing form - R red light     

A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable,B-Like Phytochrome

We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.     

PHYTOCHROME ACTION IN ORYZA SATIVA L.: I. GROWTH RESPONSES OF ETIOLATED COLEOPTILES TO RED, FAR-RED AND BLUE LIGHT

1. Under continuous irradiation, the growth of intact rice coleoptilewas strongly inhibited by red light, and somewhat preventedby blue and far-red light. The inhibitory effect of red lighton coleoptile elongation was caused by a low-energy brief irradiation,and a single exposure of 1.5 kiloergs cm^–2 incidentenergy of red light brought about the 50% inhibition. This photoinhibitionof growth was observed only after the coleoptile had elongatedto about 10 mm or longer. The red light-induced effect was reversedby an immediately following brief exposure to far-red light,and the photoresponses to red and far-red light were repeatedlyreversible. The escape reaction of red lightinduced effect tookplace at a rate so that 50% of the initial reversibility waslost within 9 hr in darkness at 27. The inhibition by bluelight and reversal by far-red irradiation was also achievedrepeatedly with successive treatments of the coleoptiles. Theevidence for a low intensity red far-red reversible controlof coleoptile growth, indicative of control by phytochrome,seems clearly established in etiolated intact seedlings.
2. Incontrast, the elongation of apically excised rice coleoptilesegments was promoted by a brief exposure to red light in 0.02M phosphate buffer, pH 7, and the effect was almost completelynullified by an immediately subsequent exposure to far-red light.It becomes evident that the growth of intact coleoptiles wasinhibited by a exposure to red light, while that of excisedsegments in a buffer was rather promoted by red irradiation.The direction of red light induced responses, either promotiveor inhibitory, depends upon the method of bioassay using intactcoleoptiles or their excised segments.

(Received July 24, 1967; )     

Impaired induction of the jasmonate pathway in the rice mutant hebiba

The elongation of rice (Oryza sativa) coleoptiles is inhibited by light, and this photoinhibition was used to screen for mutants with impaired light response. In one of the isolated mutants, hebiba, coleoptile elongation was stimulated in the presence of red light, but inhibited in the dark. Light responses of endogenous indolyl-3-acetic acid and abscisic acid were identical between the wild type and the mutant. In contrast, the wild type showed a dramatic increase of jasmonate heralded by corresponding increases in the content of its precursor o-phytodienoic acid, whereas both compounds were not detectable in the mutant. The jasmonate response to wounding was also blocked in the mutant. The mutant phenotype was rescued by addition of exogenous methyl jasmonate and o-phytodienoic acid. Moreover, the expression of O. sativa 12-oxophytodienoic acid reductase, an early gene of jasmonic acid-synthesis, is induced by red light in the wild type, but not in the mutant. This evidence suggests a novel role for jasmonates in the light response of growth, and we discuss a cross-talk between jasmonate and auxin signaling. In addition, hebiba represents the first rice mutant in which the induction of the jasmonate pathway is impaired providing a valuable tool to study the role of jasmonates in Graminean development.     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.     

Functional and signaling mechanism analysis of rice CRYPTOCHROME 1

Cryptochromes (CRY) are blue-light photoreceptors that mediate various light responses, such as inhibition of hypocotyl elongation, enhancement of cotyledon expansion, anthocyanin accumulation and stomatal opening in Arabidopsis. The signaling mechanism of Arabidopsis CRY is mediated through direct interaction with COP1, a negative regulator of photomorphogenesis. CRY has now been characterized in tomato, pea, moss and fern, but its function in monocots is largely unknown. Here we report the function and basic signaling mechanism of rice cryptochrome 1 (OsCRY1). Overexpresion of OsCRY1b resulted in a blue light-dependent short hypcotyl phenotype in Arabidopsis, and a short coleoptile, leaf sheath and leaf blade phenotype in rice (Oryza sativa). On fusion with beta-glucuronidase (GUS), the C-terminal domain of either OsCRY1a (OsCCT1a) or OsCRY1b (OsCCT1b) mediated a constitutive photomorphogenic (COP) phenotype in both Arabidopsis and rice, whereas OsCCT1b mutants corresponding to missense mutations in previously described Arabidopsis cry1 alleles failed to confer a COP phenotype. Yeast two-hybrid and subcellular co-localization studies demonstrated that OsCRY1b interacted physically with rice COP1 (OsCOP1). From these results, we conclude that OsCRY1 is implicated in blue-light inhibition of coleoptile and leaf elongation during early seedling development in rice, and that the signaling mechanism of OsCRY1 involves direct interaction with OsCOP1.     

Ethylene and the Responses to Light of Rice Seedlings

The growth of rice seedlings (Oryza satira L.) in the presence of ethylene caused a change in the response to light of coleoptile elongation. In plants grown in air without added ethylene coleoptile elongation was promoted by red, far-red and yellow-green light only in very young seedlings; in older plants irradiation inhibited the growth of the coleoptile. The effect of growing plants in the presence of ethylene was to prolong the period during which light promoted coleoptile growth. Elongation of the first internode was inhibited by light whether or not the seedlings were grown in the presence of ethylene. A correlation existed between the growth effect of an irradiation and the initial decay rate of phytochrome which was established by the treatment. Regardless of wave length, light sources whose intensities were adjusted to produce a decay rate of about 10% per hour or less induced a moderate rate of coleoptile elongation which persisted for a relatively long period. Irradiation with red or yellow-green light of higher intensity which produced a higher rate of phytochrome decay induced a higher rate of coleoptile elongation, but growth stopped after several hours. Other observations, however, showed that one cannot establish a general simple correlation between the rate of elongation of rice coleoptiles under light and the status of measurable phytochrome in the plant.     

Light induces jasmonate‐isoleucine conjugation via OsJAR1‐dependent and ‐independent pathways in rice

The bioactive form of jasmonate is the conjugate of the amino acid isoleucine (Ile) with jasmonic acid (JA), which is biosynthesized in a reaction catalysed by the GH3 enzyme JASMONATE RESISTANT 1 (JAR1). We examined the biochemical properties of OsJAR1 and its involvement in photomorphogenesis of rice (Oryza sativa). OsJAR1 has a similar substrate specificities as its orthologue in Arabidopsis. However, osjar1 loss‐of‐function mutants did not show as severe coleoptile phenotypes as the JA‐deficient mutants coleoptile photomorphogenesis 2 (cpm2) and hebiba, which develop long coleoptiles in all light qualities we examined. Analysis of hormonal contents in the young seedling stage revealed that osjar1 mutants are still able to synthesize JA‐Ile conjugate in response to blue light, suggesting that a redundantly active enzyme can conjugate JA and Ile in rice seedlings. A good candidate for this enzyme is OsJAR2, which was found to be able to catalyse the conjugation of JA with Ile as well as with some additional amino acids. In contrast, if plants in the vegetative stage were mechanically wounded, the content of JA‐Ile was severely reduced in osjar1, demonstrating that OsJAR1 is the most important JA‐Ile conjugating enzyme in the wounding response during the vegetative stage.     

Phytochrome action inOryza sativa L. VI. Red far-red reversible effect on early development of coleoptiles

When 3–4 mm long coleoptiles of etiolated rice seedlings (cv. Koshijiwase) were irradiated with continuous red light their growth was seriously inhibited. If a brief exposure of red light (4×10³ ergs cm⁻²) was given to the short coleoptiles, the growth rate dropped immediately after the irradiation, but the growth did not stop till the coleoptile reached some calculated length. If another brief red irradaition of the same order was given 24 hr after the first, the growth rate and the final length dropped further. The effect of red light was reversed by successively given far-red light, and this response was repeatedly red and far-red reversible. The escape reaction was rather slow so that photoreversibility was not lost at all by 8th hr, and 50% of the initial reversibility was lost within ca. 16 hr at 25±0.5 C. Blue light also induced the inhibition of coleoptile elongation, the effect was reversed by subsequent far-red irradiation, and this could be obtained repeatedly. Thus, the photoinhibition of the young coleoptile can be concluded to be under the control of phytochrome, and the mode of action appeared quite different from the previously reported results with longer coleoptiles.     

Coupling of phytochrome B to the control of hypocotyl growth in Arabidopsis

Etiolated seedlings of the wild-type (WT) and of the phyB-1 mutant of Arabidopsis thaliana (L.) Heynh. were exposed to red-light (R) and far-red light (FR) treatments to characterize the action of phytochrome B on hypocotyl extension growth. A single R or FR pulse had no detectable effects on hypocotyl growth. After 24-h pre-treatment with continuous FR (FRc) a single R, compared to FR pulse inhibited (more than 70%) subsequent hypocotyl growth in the WT but not in the phyB-1 mutant. This effect of FRc was fluence-rate dependent and more efficient than continuous R (Rc) or hourly FR pulses of equal total fluence. Hypocotyl growth inhibition by Rc was larger in WT than phyB-1 seedlings when chlorophyll screening was reduced either by using broadband Rc (maximum emission 610 nm) or by using narrow-band Rc (658 nm) over short periods (24 h) or with seedlings bleached with Norflurazon. Hourly R or R + FR pulses had similar effects in WT and phyB-1 mutant etiolated seedlings. It is concluded that phytochrome B is not the only photoreceptor of Rc and that the action of phytochrome B is enhanced by a FRc high-irradiance reaction. Complementary experiments with the phyA-201 mutant indicate that this promotion of a phytochrome B-mediated response occurs via co-action with phytochrome A.Abbreviations D darkness - FR far-red light - FRc continuous FR - Pfr FR-absorbing form of phytochrome - HIR high-irradiance reaction - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - Rc continuous R - WT wild-type I thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands) and Professor J. Chory (Salk Institute, Calif., USA) for their kind provision of the original WT and phyB-1 and phyA-201 seed, respectively. This work was financially supported by grants PID and PID-BID from CONICET, AG 040 from Universidad de Buenos Aires and A 12830/1-000019 from Fundación Antorchas.