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.

     

Photomodulation of mesocotyl elongation in maize seedlings: Is there a correlative relationship between phytochrome,auxin and cell wall extensibility?

Three h white light irradiation of etiolated maize seedlings ( Zea mays L. cv. Jubilee) inhibited mesocotyl elongation and caused a sharp decrease in cell wall plastic extensibility as measured by the Instron technique. The plastic extensibility following white light irradiation (3 h) was photomodulated by phytochrome. Although the photomodulation of the plastic extensibility was correlated with growth during 20 h, no such correlation was observed at shorter times. The addition of indole-3-acetic acid to light-inhibited intact seedlings, or seedlings from which the coleoptile and inner leaves were excised, resulted in a stimulation of growth. However, none of the IAA concentrations could reverse light inhibition. The possibility of a correlative relationship between phytochrome, auxin and cell wall extensibility is discussed.     

Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported thatthe cell walls of growing rye coleoptiles exhibit irreversible(plastic) extensibility in a rheological extension test. Basicallysimilar measurements with cell walls of maize coleoptiles hadpreviously shown that the apparent plastic extensibility determinedin this material is in reality due to the slowly reversible(viscoelastic) extensibility of the walls. A recent reinvestigationof this discrepancy showed that rye coleoptile walls also behaveas a perfectly viscoelastic material if precautions are takento prevent measuring artefacts. Similar results were obtainedwith cell walls from the growing zone of various other seedlingorgans (maize mesocotyl, maize root, cucumber hypocotyl). Itis concluded that plastic extensibility has not yet been convincinglydemonstrated by rheological tests that determine the intrinsicmaterial properties of cell walls. Reported changes in mechanicalmaterial properties of cell walls produced by growth-controllingfactors such as auxin or light may generally be attributed tochanges in viscoelasticity which are not directly related tothe chemo-rheological processes controlling wall extension ofgrowing cells. Key words: Cell wall extensibility, extension growth, plastic cell wall extensibility, viscoelastic cell wall extensibility     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

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.     

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m^-2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine-and tyrosine-ammonia-lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize ( Zea mays L. cv. Cross Bantam T51). Growth rate of dark-grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p -coumaric acid ( p -CA) increased by severalfold in cell walls of light-grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p -CA and the decrease in cell wall extensibility. Light decreased the wall-bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p -CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.     

Auxin increases elastic wall-properties in rye coleoptiles: implications for the mechanism of wall loosening

Auxin-induced changes of wall-rheological properties during different growth rates of rye coleoptile segments (Secale cereale L.) were investigated. In addition, changes of osmotic concentration and turgor pressure were measured. Decrease of turgor and of osmotic concentration followed a synchronous time course. Auxin-incubated segments exhibited a faster decrease and eventually lower values of both parameters. Creep test extensibility measurements demonstrate that apparent plastic as well as elastic extensibility of distilled-water-incubated segments strongly decreased during 24 h. In auxin-incubated segments apparent plastic as well as elastic extensibilities were strongly increased, even in the absence of growth due to insufficient turgor pressure. The increasing effect of auxin on elastic wall properties is also reflected by an increase in relative reversible length (part of segment length by which segments shrink after freezing/thawing as referred to total length) and a complementary decrease of relative irreversible length (remaining length after turgor elimination as referred to turgid length); again the effects were independent of growth rate and turgor pressure. Cellulose synthesis inhibition of approx. 80% by dichlorobenzonitrile (DCB) had no significant effect either on growth or on wall-rheological properties. Independent of whether the changed rheological wall behaviour of auxin-incubated segments is causally related to the mechanism of auxin-induced wall loosening, it indicates changes of wall polymer properties and/or interactions which are conserved when no actual length increase occurs due to insufficient turgor pressure. The results suggest that IAA-induced wall loosening may be primarily mediated by cell wall changes other than cleavage of covalent, load-bearing bonds as hypothesized in various wall loosening models.     

Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space

We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.     

Influence of Auxin on Young's Modulus in Stems and Roots of Pisum and the Theory of Changing the Modulus in Tissues

The effect of auxin on elastic extensibility has been investigated by means of the resonance frequency melhod in Pisum, sativum. The time lag for the decrease in Young's modulus E, caused by IAA, was between 2 and 3 minutes in etiolated stem internodes. The time lag for growth was about 7 minutes. The measurements of E in root segments were only qualitative owing to the structural characteristics; IAA decreases E in roots as it does in stems, but only in the region where IAA is assumed to enhance elongation. The connexion between elastic modulus and growth is discussed with reference to other investigations. The assumption has been made that a decrease in elastic modulus indicates a change in the cell wall which in some way is conducive to growth (induction of elongation). The theoretical possibilities of changing E have been discussed with reference to the formula for water fluxes. Both a change in a cell wall properly and a change in the cytoplasmic permeability are able to cause a change in E in the same way as auxin does. An early action of auxin must be located in the cell-wall-plasmalemma region.     

Changes in cellular osmotic potential and mechanical properties of cell walls during light-induced inhibition of cell elongation in maize coleoptiles

The growth rate of maize ( Zea mays L. cv. Cross Bantam T51) coleoptiles in the dark was highest at the basal zone and decreased towards the tip. Growth was strongly inhibited by white fluorescent light (5 W m⁻²), especially in the basal zone of coleoptiles. Light irradiation caused an increase in the values of stress-relaxation parameters, the minimum stress-relaxation time and the relaxation rate and a decrease in the extensibility (strain/stress) of the cell walls at all zones. In addition, during growth, the accumulation of osmotic solutes was strongly inhibited by white light irradiation, resulting in an increased osmotic potential. The influences of white light on the mechanical properties of the cell wall and the osmotic potential of the tissue sap were most prominent in the basal zone. Significant correlations were observed between the increment of coleoptile length and the mechanical properties of the cell walls or the osmotic potential of the tissue sap and osmotic solutes content. Furthermore, light inhibited the outward bending of split coleoptile segments. These facts suggest that white light inhibits elongation of maize coleoptiles by modifying both the mechanical properties of the cell walls and cellular osmotic potential, which control the rate of water uptake.     

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.     

The Role of the Epidermis in the Control of Elongation Growth in Stems and Coleoptiles

The peripheral cell wall(s) of stems and coleoptiles are 6 to 20 times thicker than the walls of the inner tissues. In coleoptiles, the outer wall of the outer epidermis shows a multilayered, helicoidal cellulose architecture, whereas the walls of the parenchyma and the outer wall of the inner epidermis are unilayered. In hypocotyls and epicotyls both the epidermal and some subepidermal walls are multilayered, helicoidal structures. The walls of the internal tissues (inner cortex, pith) are unilayered, with cellulose microfibrils oriented primarily transversely. Peeled inner tissues rapidly extend in water, whereas the outer cell layer(s) contract on isolation. This indicates that the peripheral walls limit elongation of the intact organ. Experiments with the pressure microprobe indicate that the entire organ can be viewed as a giant, turgid cell: the extensible inner tissues exert a pressure (turgor) on the peripheral wall(s), which bear the longitudinal wall stress of the epidermal and internal cells. Numerous studies have shown that auxin induces elongation of isolated, intact sections by loosening of the growth-limiting peripheral cell wall(s). Likewise, the effect of light on reduction of stem elongation and cell wall extensibility in etiolated seedlings is restricted to the peripheral cell layers of the organ. The extensible inner tissues provide the driving force (turgor pressure), whereas the rigid peripheral wall(s) limit, and hence control, the rate of organ elongation.     

Auxin-induced cell expansion in relation to cell wall extensibility

Decapitation of 30 mm oat coleoptiles, which are commonly usedfor growth tests, resulted in a decrease in their elastic extensibility(DE) but not in their plastic extensibility (DP). By auxin treatmentunder osmotic stress, old coleoptile (45 mm) cells showed noincrease in subsequent expansion in water, whereas RNA synthesisin these cells was stimulated just as in young ones. Auxin increasedthe DE of young coleoptile cell walls but not that of old ones.Significant increase of DE occurred in only 10 min, and themaximum level of DE was reached in 15 min of the auxin treatment.An antiauxin (2,4,6-trichlorophenoxyacetic acid), mitomycinC and cycloheximide inhibited auxin-induced increases in expansionand DE (or Rex, reversible extensibility) of young coleoptilecells. (Received July 23, 1968; )     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Phytochrome-regulated growth of excised coleoptile tips of Triticum aestivum induced by blue, red and far-red light

The elongation growth of 5-mm tip sections of 3-day-old etiolatedwheat coleoptiles was promoted by light flashes of 436, 650and 750 nm in comparison with the dark control. As short as7/1,000 sec red light (R) of 14,420 W/m² led to saturation ofthe R-induced growth response. At quantum-identical far-redlight (FR) irradiation, a plateau developed at about 9/1,000sec which, however, was interrupted at longer irradiation periods.Varying dark periods between R or FR activation of growth anda second FR flash, a light-independent period with a half-lifeof 45 to 60 sec was found. Only after this light-independentperiod could R or FR activation of growth be reversed by FR.A second light-independent period was found by the followingirradiation schedule: 1 sec R/100 sec darknees(D)/l sec FR/varied D/l sec R. Our experimental conditions enabled us (a) to distinguish betweenactivation and inactivation by FR, (b) to clarify the reactionchain of different phytochrome forms and (c) to determine thehalf-life of light-independent phytochrome reactions.     

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice ( Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10–50°C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30–40°C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40°C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30–40°C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40°C were substantially higher than those grown at 10, 20 and 50°C. Furthermore, the activities of (1→3),(1→4)- β -glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1→3),(1→4)- β -glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30–40°C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of β -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of β -glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.     

植物光敏素在玉米胚芽鞘不同类型向光性反应中的作用模式

先前的研究考察了红光对玉米（Zea mays L.cv.Royaldent Hit85)胚芽鞘向光性反应的影响,本研究进一步分析了红光对蓝光照射时间依赖型向光性（TDP）影响的光具－反应曲线,发现该反应不像红光对脉冲蓝光诱导的向光性的影响那样属于超低光量反应,而是一种低光量反应,且之后的脉冲远红光可以逆转红光对TDP影响的效果。但远红光预处理延长后,逆转了的TDP反应性可以得到恢复。不仅如此,暗适应胚芽鞘接受不同时间的单独远红光预处理后可同样获得与预处理光量成比例的TDP反应性,表明暗适应胚芽鞘在接受远红光顶处理后亦可建立起长时间向光性蓝光照射的反应能力。远红光对TDP反应性影响的光量－反应曲线分析揭示,该远红光影响依赖于照射时间并要求高光量。鉴于高光量范围内上述远红影响不符合所谓反比定律,远红光对TDP反应性的影响很可能属一高辐照反应,根据上述研究发现探讨了植物光敏素作用模式与不同光性反应信号转导途径之间可能存在的相互关系。     

Modes of Action of Phytochromes in Establishing DifferentPhototropic Responsiveness of Maize Coleoptiles

Previous studies have examined the effects of red light (R) on phototropism of maize ( Zea mays L. cv. Royaldent Hit 85) coleoptiles. The R effect on time-dependent phototropism (TDP) was further studied by characterizing its fluence-response relationship. The results showed the R effect was a low-fluence-response, unlike those on pulse-induced phototropisms that show a very-low-fluence-response mode. A subsequent pulse of far-red light (FR) could reverse the R effect. TDP responsiveness, however, recovered as the following FR was extended. The FR-dependent increase in TDP responsiveness was obtained even coleoptiles were pretreated only with FR. It suggested that TDP responsiveness could also be established in response to a FR signal. The fluence-response relationship for the effect of FR was then investigated. The effect of FR depended on the time of irradiation and required high photon fluences. Because reciprocity was invalid at the higher fluence range, the effect of FR would be a high-irradiance-response mode. Relation between phytochrome action modes and possible multiple pathways for phototropic signal transduction was analyzed based on the experiment results.     

Auxin-dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC

Mutants in rice (Oryza sativa L. cv. japonica) were used to study the role of the cytoskeleton in signal-dependent morphogenesis. Mutants obtained by gamma ray irradiation were selected that failed to show inhibition of coleoptile elongation by the antimicrotubular drug ethyl-N-phenylcarbamate (EPC). The mutation EPC-Resistant 31 (ER31), isolated from such a screen, caused lethality in putatively homozygous embryos. Heterozygotes exhibited drug resistance, impaired development of crown roots, and characteristic changes in the pattern of cell elongation: cell elongation was enhanced in mesocotyls and leaf sheaths, but inhibited in coleoptiles. The orientation of cortical microtubules changed correspondingly: for etiolated seedlings, compared with the wild-type, they were more transverse with respect to the long cell axis in mesocotyls and leaf sheaths, but more longitudinal in coleoptiles. In mutant coleoptiles, in contrast to wild-type, microtubules did not reorient in response to auxin, and their response to microtubule-eliminating and microtubule-stabilizing drugs was conspicuously reduced. In contrast, they responded normally to other stimuli such as gibberellins or red light. Auxin sensitivity as assayed by the dose-response for callus induction did not show any significant differences between wild-type and mutant. The mutant phenotype is interpreted in terms of an interrupted link between auxin-triggered signal transduction and microtubule reorientation.     

Gravitropism and Phototropism of Maize Coleoptiles: Evaluation of the Cholodny-Went Theory Through Effects of Auxin Application and Decapitation

It was investigated whether or not gravitropism and phototropismof maize (Zea mays L.) coleoptiles behave as predicted by theCholodny-Went theory in response to auxin application, decapitationand combinations of these treatments. Gravitropism was inducedat an angle of 30° from the vertical, and phototropism,by a pulse of unilateral blue light. Either tropism of the coleoptilewas inhibited by IAA, applied as a ring of IAA-lanolin pasteto its sub-apical part, and by decapitation. The dose-responsecurves for the effects of applied IAA on tropisms and growthof intact coleoptiles as well as the time courses of tropismsinduced in decapitated coleoptiles could be explained by thethree conclusions in the literature: (1) the tip of the coleoptileis the site of auxin production, (2) lateral translocation ofauxin in gravitropism occurs along the length of the coleoptile,and (3) lateral translocation of auxin in phototropism occursin the coleoptile tip. By examining the effects of decapitationmade at different distances from the top and of IAA appliedto the cut surface of decapitated coleoptiles, it was indicatedthat auxin is produced in the apical 1 mm zone of an intactcoleoptile and that lateral auxin translocation for phototropismtakes place in an apical part that somewhat exceeds the zoneof auxin production. (Received October 14, 1994; Accepted December 26, 1994)