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.     

Effects of red light on the growth of intact wheat and barley coleoptiles

The final lengths of intact dark-grown coleoptiles vary with species and cultivar. The growth distribution pattern in the apical 25-mm growing zone and the absolute amount of growth in each zone depend on the age and species of the coleoptile. A comparative study of several cultivars of wheat, Triticum vulgare, and barley, Hordeum vulgare, indicates that the growth distribution pattern in 30- to 38-mm coleoptiles varies with the species and cultivar. In barley, there are two patterns of growth distribution among the several cultivars, whereas in wheat, all cultivars exhibit a common zonal growth pattern. The total growth of coleoptiles, initially 30 to 38 mm in length, during a 24-hour dark incubation period is the same in dark-grown coleoptiles as in those irradiated with 3 minutes of red (660 nm) light prior to the incubation period. The growth distribution pattern in the growing zone of this 30- to 38-mm coleoptile is, however, altered by red light. Growth of the apical 5-mm zone is stimulated by red light and the zonal growth 5 to 10 mm below the apex is only slightly affected, whereas growth in the zones 10 to 15 to 20, and 20 to 25 mm below the apex is inhibited. This growth distribution pattern in irradiated coleoptiles changes as the coleoptile increases in length. The response of a zone following exposure to red light is dependent upon the age of the seedlings irradiated. The over-all effect of red light on growth of the intact coleoptile varies with the length of the coleoptile. In young seedling 20 to 29 mm in length, the cells of the coleoptile can compensate for the effects of red light, with the over-all growth of the dark-grown and irradiated coleoptile about the same. As the seedling grows older, the cells of the coleoptile can no longer make up for the effects of red light, and the over-all effect changes from compensation to pronounced inhibition.     

Rapid Growth Responses of Dark-Grown Wheat Seedlings to Red Light-Irradiation: I. Kinetic Studies on the Short-Term Growth Responses of Intact Coleoptiles at Different Seedling Age

Continuous recordings of the effect of red light on intact darkgrown wheat seedlings (Triticum aestivum L. cv. Hatri) weremade at different times after sowing. When the coleoptile tipregion was irradiated 50, 70 or 90 h after sowing with red lightfrom two opposite fibre bundles a decrease in extension ratewas detectable after a latent period of 10 to 15 min. Growthrate reached a fluence dependent minimum at about 60 min, afterwhich growth acceleration towards the dark control rate wasobserved. When continuous red irradiation was started 50 or70 h after sowing the dark control rate was reached 2.5 h afteronset of irradiation and growth rate was little above this levelduring the next 2 h. With older coleoptiles (90 h after sowing)the growth rate recovery was only up to 50% of the dark controlrate and a second phase in growth inhibition became detectableabout 2.5 h after onset of red exposure, characterized by acontinuous decrease in extension rate. Under R/FR pulse irradiationboth the red-light-induced transient growth inhibition and thesecond phase of growth inhibition exhibit far-red reversibilityup to the level of far-red induced growth rate changes. (Received September 19, 1986; Accepted December 8, 1986)     

PHYTOCHROME ACTION IN ORYZA SATIVA L. III. THE SEPARATION OF PHOTOPERCEPTIVE SITE AND GROWING ZONE IN COLEOPTILES, AND AUXIN TRANSPORT AS EFFECTOR SYSTEM

• 1 In 4-day-old etiolated rice seedlings, 3 mm of the coleoptile tip did mainly perceive the photostimulus to cause the phytochrome-dependent inhibition of coleoptile elongation. At this age, cell elongation occurred most in the middle portion of coleoptiles in the dark, and was reversibly controlled by a brief exposure of the tip to red and far-red light. Thus, the photoperceptive site was evidently separated from the growing zone in intact rice coleoptiles.
• 2 The red-light-induced inhibition of coleoptile elongation was nullified by the removal of tip followed by the exogenous application of IAA. The sensitivity of thus treated coleoptiles to IAA was gradually lost during intervening darkness between the irradiation and the decapitation, and a 50% loss was obtained at ca. 6th hour at 26°C.
• 3 Polar auxin transport from coleoptile tips was remarkably prevented at the period between, at least, 2nd and 4th hour after red irradiation, and it recovered to the level of dark control by the 6th hour. Far-red light given immediately after red irradiation reversed the yield of diffusible auxin up to that of far-red control.

     

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.     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome     

Biphasic effect of red light on the growth of coleoptiles in etiolated barley seedlings

InHordeum vulgare cultivar “Kirin-choku No. 1”, the final length of intact coleoptiles of totally etiolated seedlings was approximately twice as long as that of those grown under continuous red light. The fluence response curve of the latter was biphasic; the low-energy effect was saturated by red light of ca. 50 J m⁻² which gave rise to about 40% of the maximum inhibition by continuous irradiation with red light of 1.2 W m⁻², whereas the high-energy effect was induced by irradiation for 1 hr or longer. Coleoptiles of 3-day-old seedlings were most sensitive to light causing the low-energy effect, which was repeatedly red/far-red reversible. The growth inhibition was correlated to the photometrically measured percentage of Pfr so that the maximum effect was induced by red light of 50 J m⁻² which transformed 70% of phytochrome to Pfr in the coleoptile tip. Wavelength dependence of the high-energy effect showed that monochromatic light of 400, 600 and 650 nm greatly inhibited the coleoptile growth, whereas light of 700 and 750 nm promoted it instead. The effect was also induced by intermittent irradiation with red light, and the more frequently the intermittent treatment was given, the more the growth was inhibited.     

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; )     

Restoration of phototropic responsiveness in decapitated maize coleoptiles

The literature indicates that the tip of maize (Zea mays L.) coleoptiles has the localized functions of producing auxin for growth and perceiving unilateral light stimuli and translocating auxin laterally for phototropism. There is evidence that the auxinproducing function of the tip is restored in decapitated coleoptiles. We examined whether the functions for phototropism are also restored by using blue-light conditions that induced a first pulse-induced positive phototropism (fPIPP) and a time-dependent phototropism (TDP). When the apical 5 mm, in which photosensing predominantly takes place, was removed, no detectable fPIPP occurred even if indole-3-acetic acid (lanolin mixture) was applied to the cut end. However, when the blue-light stimulation was delayed after decapitation, fPIPP became inducible in the coleoptile stumps supplied with indole-3-acetic-acid/lanolin (0.01 mg g-1), indicating that phototropic responsiveness was restored. This restoration progressed 1 to 2 h after decapitation, and the curvature response became comparable to that of intact coleoptiles. The results for TDP were qualitatively similar, but some quantitative differences were observed. It appeared that the overall TDP was based on a major photosensing mechanism specific to the tip and on at least one additional mechanism not specific to the tip, and that the tip-specific TDP was restored in decapitated coleoptiles with kinetics similar to that for fPIPP. It is suggested that the photoreceptor system, which accounts for fPIPP and a substantial part of TDP, is regenerated in decapitated coleoptiles, perhaps together with the mechanism for lateral auxin translocation.     

Growth distribution during first positive phototropic curvature of maize coleoptiles*

Abastract Measurements of growth increments on the shaded and the irradiated sides of phototropically stimulated maize (Zea mays L.) coleoptiles, obtained over the entire fluence range of the first positive curvature, indicate that the curvature is induced by growth stimulation on the shaded side and compensating inhibition on the irradiated side (length increments on the coleoptile flanks were determined 100 min after 30 s phototropic induction with blue light). At high fluences of blue light, overall stimulation of growth takes place, but this tendency is largely eliminated when only the tip of the coleoptile is irradiated. Time courses for growth increments obtained for the maximum first positive response show that the growth stimulation on the shaded side and the growth inhibition on the irradiated side commence almost simultaneously 20-30 min after the phototropic induction. The growth on the irradiated side almost ceases, but the growth rate on the shaded side is doubled, relative to the control rate. The onset of differential growth migrates basipetally from the tip at a velocity similar to that for polar auxin transport. The first positive phototropic response of the coleoptile is concluded to be the consequence of lateral redistribution of growth, which is not necessarily accompanied by changes in the net growth. The results are consonant with the Cholodny-Went theory of tropisms, in which lateral redistribution of auxin is considered to be the cause of tropic responses.     

Differential expression of two barley XET-related genes during coleoptile growth

Plant cell elongation depends on the physical properties of the primary cell wall. Because xyloglucan endotransglycosylases (XETs) are enzymes that mediate cleavage and rejoining of the beta(1-4)-XG backbone of primary cell wall, they are potentially involved in cell elongation. In this paper, the growth of the barley coleoptile was related to the expression patterns of two genes from this family (hvEXT, hvXEB) in experiments where coleoptile elongation varied according to light/dark treatments in order to assess the potential role of these genes in cell elongation. In dark-grown and light-grown coleoptiles, growth rate variations were associated with altered levels of expression of hvEXT and hvXEB: they were higher in dark-grown than in light-grown seedlings, and decreased after 5 d in darkness, and after 4 d in continuous light. In 4-d-old seedlings, coleoptile elongation decreased significantly 4 h after the onset of a continuous white- light irradiation, and hvXEB and hvEXT mRNA levels decreased, respectively, 2 h and 4 h after the onset of white-light irradiation. Moreover, the distribution of hvXEB and hvEXT along the coleoptiles of 4-d-old dark-grown seedlings were different. Altogether, these results suggest a complex pattern of temporal and positional expression for the different genes of the XET-related family.     

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     

Response of actin microfilaments during phytochrome-controlled growth of maize seedlings

Summary In seedlings of maize (Zea mays L. cv. Percival), growth is controlled by the plant photoreceptor phytochrome. Whereas coleoptile growth is promoted by continuous far-red light, a dramatic block of mesocotyl elongation is observed. The response of the coleoptile is based entirely upon light-induced stimulation of cell elongation, whereas the response of the mesocotyl involves light-induced inhibition of cell elongation. The light response of actin microfilaments was followed over time in the epidermis by staining with fluorescence-labelled phalloidin. In contrast to the underlying tissue, epidermal cells are characterized by dense longitudinal bundles of microfilaments. These bundles become loosened during phases of rapid elongation (between 2–3 days in irradiated coleoptiles, between 5–6 days in dark-grown coleoptiles). The condensed bundles re-form when growth gradually ceases. The response of actin to light is fast. If etiolated mesocotyls are transferred to far-red light, condensation of microfilaments can be clearly seen 1 h after the onset of stimulation together with an almost complete block of mesocotyl elongation. The observations are discussed in relation to a possible role of actin microfilaments in the signal-dependent control of cell elongation.     

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.     

The biophysical basis of cell elongation and organ maturation in coleoptiles of rye seedlings: implications for shoot development

The relationships between changes in irreversible and reversible organ length, turgor (P), osmotic pressure (pi), and metabolic activity of the cells were investigated in intact coleoptiles of rye seedlings ( Secale cereale L.) that were either grown in darkness or irradiated with continuous white light. Cessation of growth at day 4 after sowing was associated with an apparent mechanical stiffening of the cell walls. Turgor pressure was measured in epidermal and mesophyll cells with a miniaturized pressure probe. No gradient of turgor was found between the peripheral and internal cells. In juvenile (growing) coleoptiles, average turgor was 0.60 MPa and a negative water potential (P - pi) was established in these cells. Upon emergence of the primary leaf, turgor declined, but P was maintained at values of 0.43 and 0.52 MPa in 7-day-old light- and dark-grown coleoptiles, respectively. Water potential in non-growing cells approached zero. The rate of dark respiration and elongation growth were not correlated. Surgical removal of the mature coleoptile revealed that the erect position of the 7-day-old shoot was dependent on the presence of this sturdy, turgid organ sheath. It is concluded that, during the first week of seedling development, the pierced, metabolically active coleoptile fulfills an essential function as an elastic basal tube for the juvenile shoot.     

Rapid suppression of extension growth in dark-grown wheat seedlings by red light

Continuous recordings were made using a linear displacement transducer to investigate short-term growth responses of intact dark-grown wheat (Triticum aestivum L. cv Maris Huntsman) seedlings to red light. To eliminate any effect of light prior to the experimental treatments, the seedlings were grown and mounted on the transducer apparatus in total darkness. The growth kinetics after irradiation were complex and appeared to consist of three successive phases of growth deceleration. When the tip of the intact coleoptile was irradiated with red light from two opposite fiber bundles (fluence rate: 2 × 64 micromoles per square meter per second) for varying periods of time (10 seconds, 1 minute, 5 minutes, continuous), a decrease in extension rate was detectable after a latent period of 8 to 10 minutes. Up to 30 minutes after the start of the irradiation treatment, there was no difference in the kinetics of inhibition (about 20 to 25% inhibition) between the different lengths of irradiation. Extension rate reached a minimum (65% inhibition) at about 85 minutes, after which growth acceleration toward the dark control rate was observed. Far-red reversibility of the rapid effect of red light on growth was not observed, even when far-red light was given only 4 seconds after the end of 10 seconds red light. Short (15 seconds) far-red light did not induce a response.     

Effect of red light on coleoptile growth

The effects of red light in reducing the growth of the oat (Avena sativa L.) coleoptile and the synthesis of auxin in the coleoptile tip are detectable 2 hours after treatment and become more pronounced with time. When the coleoptile tip is supplied with additional tryptophan the synthesis of auxin is doubled both in darkness and when exposed to red light. Treatment of the tip with gibberellic acid or pyridoxal phosphate overcomes the reduction of auxin synthesis caused by red light. The uptake of exogenous indoleacetic acid, at pH 6.5, by coleoptile tissue is doubled by exposure to red light. The effect of red light on coleoptile growth appears to be mediated by phytochrome in the cell membrane which delocalizes the tryptophan utilized for auxin synthesis.     

Gravitropic microtubule reorientation can be uncoupled from growth

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90°), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation. Received: 23 November 1999 / Accepted: 10 May 2000     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.