Phototropism in Hypocotyls of Radish : I. Isolation and Identification of Growth Inhibitors, cis- and trans-Raphanusanins and Raphanusamide, Involved in Phototropism of Radish Hypocotyls

Three growth inhibitors which might be involved in phototropism of Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls, were isolated as crystalline forms from light-exposed radish seedlings and identified as cis- and trans-raphanusanins and 6-methoxy-2,3,4,5-tetrahydro-1,3-oxazepin-2-one (designated raphanusamide). The cis- and trans-raphanusanins inhibited growth of etiolated radish hypocotyls at concentrations higher than 1.5 micromolar, raphanusamide at concentrations higher than 20 micromolar.     

Phototropism in Hypocotyls of Radish : III. Influence of Unilateral or Bilateral Illumination of Various Light Intensities on Phototropism and Distribution of cis- and trans-Raphanusanins and Raphanusamide

When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light at 0.05, 0.1, or 1 watt per square meter, the suppression of the growth rate on the lighted side depended on the light intensity. The growth rate at the shaded side was only a little affected by the illumination at 0.05 and 0.1 watt per square meter but considerably suppressed by that at 1 watt per square meter. Upon a continuous unequal bilateral illumination, the growth rate was more strongly suppressed on the side of the higher intensity than on the side of the lower one, resulting in phototropic curvature toward the light source of the higher intensity. It was calculated from correlation analysis of light intensity and growth rate that, on an average, 6.9% of the irradiation applied to one side reached the opposite side. The amounts of cis- and trans-raphanusanins and raphanusamide in hypocotyls subjected to unilateral or unequal bilateral illumination increased much more at the side of the lighted or the higher intensity than at the opposite side. The present study demonstrates that phototropism in radish hypocotyl is correlated with and we conclude caused by a gradient of growth inhibition in the hypocotyl, depending on irradiation-induced amounts of cis- and trans-raphanusanins and raphanusamide.     

Phototropism in Hypocotyls of Radish: IV. Flank Growth and Lateral Distribution of cis- and trans-Raphanusanins in the First Positive Phototropic Curvature

The first positive phototropic curvature induced by a pulse of unilateral white irradiation (0.1 watt per square meter, 30 seconds) of etiolated and de-etiolated Sakurajima radish (Raphanus sativus var hortensis f. gigantissimus Makino) hypocotyls was analyzed in terms of differential growth and growth inhibitor contents of the hypocotyls. In both etiolated and de-etiolated hypocotyls, the growth rates at the lighted sides were suppressed whereas those at the shaded ones showed no change. De-etiolation treatment induced a larger difference between the growth rates at the lighted and shaded sides of the hypocotyls, resulting in a larger curvature of de-etiolated seedlings than of etiolated ones. The contents of growth inhibitors, cis- and trans-raphanusanins, increased in the lighted but not in the shaded halves of the hypocotyls of etiolated seedlings. In de-etiolated seedlings, the two inhibitors increased due to the de-etiolation treatment. When de-etiolated seedlings were exposed to a pulse of unilateral irradiation the level of the two inhibitors remained high along the lighted side for 1 h following the light pulse, whereas at the shaded side the contents of the inhibitors abruptly decreased upon transfer to the dark, the difference between their amounts in the lighted and shaded sides being larger than in etiolated seedlings. Another growth inhibitor, raphanusamide, did not respond to the phototropic stimulus, although its amounts increased by the de-etiolation treatment. These data suggest that cis- and trans-raphanusanins are involved in the first positive phototropic response of radish hypocotyls, and that de-etiolation magnifies the phototropic response through induction of a larger lateral gradient of the raphanusanins in the hypocotyls by the phototropic stimulus.     

Inhibition of Gravitropism in Etiolated Radish Hypocotyls by Chloramphenicol

The hypocotyls of radish seedlings can grow at an almost normalrate when the roots are immersed in 10^–4 M chloramphenicol(CAP) although there is a reduction in the length of the roots.However, when 4-d-old seedlings are placed horizontally, thehypocotyls of those growing in water bend completely uprightwithin 3 h, whereas those growing in CAP react slowly or notat all. The elongation rate of these gravitropically stimulatedhypocotyls is about 0.87 and 0.72 mm h^–1 in water andin CAP, respectively, and both these values are higher thanthose of unstimulated seedlings, where a rate of 0.29 mm h^–1occurred in both water and CAP treatments. The effect of CAPon gravitropism occurs only when CAP is applied to seedlingsin the first 24 h after sowing. At the cell level the main characteristicof the hypocotyls of CAP-treated seedlings not showing geocurvatureis the absence of starch in the cell layer surrounding the vasculartissues. From a comparison between the presence of starch andgravitropic reaction it is suggested that amyloplasts containedin endodermal cells are the receptors of the gravitational stimulus. Raphanus sativus, radish, gravitropism, amyloplasts, chloramphenicol     

PHOT2介导拟南芥下胚轴向光弯曲调节子的筛选与鉴定

向光素(PHOT1和PHOT2)功能冗余调节单侧强蓝光诱导的拟南芥(Arabidopsis thaliana)黄化苗下胚轴向光弯曲表现功能冗余,限制了人们对PHOT2信号转导机制的深入研究。通过化学诱变剂甲基磺酸乙酯(EMS)诱变拟南芥phot1突变体,避开PHOT1基因的干扰,寻找PHOT2下游信号分子。研究结果表明,已成功筛选到1株遗传稳定的下胚轴向蓝光不弯曲突变体。遗传分析结果显示,该突变体可能是PHOT2下游信号分子突变,将其命名为p2sa1(phototropin2 signaling associated1)。用100μmol·m–2·s–1强蓝光单侧照射,phot1p2sa1下胚轴向光弯曲缺失,呈现phot1phot2双突变的表型,然而phot1p2sa1在强蓝光下叶绿体避光正常,明显不同于phot1phot2。实验证实P2SA1可能位于PHOT2的下游,参与调节PHOT2介导的拟南芥下胚轴向光弯曲反应。     

Phototropism in Vaucheria geminata II. The mechanism of bending and branching

The mechanism of phototropic bending and branching in Vaucheriageminata was analyzed. Using the half-side-illumination methodit was shown that phototropic bending is initiated by the formationof a new growth center on the illuminated side of the apicaldome of the tube, not by the difference in growth rate betweenthe lighted and shaded sides of the tip. Branching was alsoinitiated when the part proximal to the apical dome was illuminated.Blue light was effective for branching as it was for phototropicbending. The refractive indices measured at the growing tipand an area other than the tip were 1.36 and 1.34 respectively.A hyaline patch resembling the hyaline cap, which appeared priorto the onset of tip-growth in the apical dome, was invariablyformed shortly before the initiation of a new branch at theilluminated locus. (Received December 20, 1974; )     

Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin

The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca²⁺ concentration. Stimulation was marked at low, nonsaturating Ca²⁺ concentrations and decreased increasing Ca²⁺, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca²⁺. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca²⁺, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.     

Phototropism: Some History,Some Puzzles,and a Look Ahead

After over a century of progress, phototropism research still presents some fascinating challenges.Though few and far between, phototropism studies through 1937 established a number of important principles. (1) Blue light is the active spectral region. (2) The phototropic stimulus is perceived by the coleoptile tip, and the consequences of the stimulation progress down into the growing region. (3) Lateral transport of auxin mediates the curvature response. (4) The reciprocity law holds for first positive curvature, whereas second positive curvature is time dependent. (5) Red light treatment had a major effect on phototropic sensitivity. Later studies established the following. (6) Seven blue light receptors (cryptochromes, phototropins, and three F-box proteins) were identified and characterized. (7) A flavin was established as the photoreceptor chromophore for all seven. (8) The chromophore domain, designated the LOV domain (for light, oxygen, or voltage), carries out a unique photochemistry. (9) LOV domains must be truly ancient chromophore domains. There remain some puzzles. The fluence-response threshold level for first positive curvature is far below that for phototropin photochemistry. Likewise, the fluence-response threshold level for the red light effect on coleoptile phototropism is far below those for phytochrome phototransformation. Cytological effects of red light are also very insensitive compared with the physiological effects of red light. What is the mechanism allowing for this extraordinary photosensitivity? How is phototropin specificity controlled? What are the functions of the phytochrome kinase substrate proteins in both phytochrome and phototropin responses? What mechanism leads to lateral auxin transport? Finally, are LOV domain proteins true photoreceptors in all of the bacteria in which they occur? If so, what is their biological function?Even in the ancient world, astute observers noted that plants could turn to face the sunlight. What was originally designated heliotropism for plants that followed the sun eventually became divided into two distinct response categories: solar tracking (the real heliotropism), a repetitive and completely reversible turgor-driven process; and phototropism, an irreversible directional growth response determined by light direction. Over the past 200 years, a large number of brilliant biologists, including Julius Sachs (1864), Charles Darwin (1881), Frits Went (1928), and Kenneth Thimann (Went and Thimann, 1937) have applied their talents to examining and elucidating the mechanisms accounting for both of these responses. The entire history of phototropism and solar tracking parallels and is intertwined with that for a number of other blue light responses, found not just in higher plants but in bryophytes, ferns, algae, fungi, and, most recently bacteria. For a detailed account of this history, see Briggs (2006).Progress in research on blue light-activated processes over the last half century was severely hampered by a lack of knowledge of the relevant blue light receptors. By contrast, the discovery and initial characterization of a red/far-red-reversible phytochrome (Butler et al., 1959) nurtured an enormous and sophisticated body of knowledge about these photoreceptors: their structure, their chromophores, their photophysics and photochemistry, and the extraordinary signal transduction networks that they rule. Meanwhile, although a huge and scattered body of knowledge on blue light responses in plants and fungi accumulated, there was scarcely a clue to what the photoreceptor(s) might be, and competing hypotheses abounded. Attention focused on the physical and physiological characterization of responses to blue light and, eventually, biochemical investigations of intriguing in vitro photochemistry. It was only with the advent of modern molecular genetics and its extraordinary capabilities that research on blue light photobiology finally began to catch up with phytochrome photobiology 20 years ago and the first blue light receptor, cryptochrome1 (cry1), became identified (Ahmad and Cashmore, 1993).This review will go back to some of the puzzles of earlier years, a few over 100 years old, and provide a glimpse of the status of these puzzles today. They arose from studies of response kinetics, action spectroscopy, interactions between blue and red regions of the visible spectrum, and discrepancies between in vivo and in vitro results. Although the focus will be on higher plant phototropism, several other blue light responses will contribute to the discussion. No effort will be made to be comprehensive. Any effort here would be redundant with that of three recent excellent reviews on phototropism (Sakai and Haga, 2012; Christie and Murphy, 2013; Hohm et al., 2013) and on LOV (for light, oxygen, or voltage) domain photochemistry (Losi and Gärtner, 2011, 2012). The article concludes with a look at the unexpected role of LOV domains in prokaryotes.     

Phototropism, Adaptation, and the Light-Growth Response of Phycomyces

Phototropic bending can be initiated without the transient changes in growth speed that characterize a light-growth response. The conditions required are a change from a symmetric to an asymmetric illumination pattern while the cell receives a constant radiant flux. Phototropism is thus basically a steady state process. It cannot be founded on differential light-growth responses as in Blaauw's theory. A possible model system for the unequal partition of growth during steady bending is discussed. The fact that light-growth responses show adaptation while phototropic bending does not follows from the different natures of the two responses.     

Turgor Pressure and Phototropism in Sinapis alba L. Seedlings

Rich, T. C. G. and Tomos, A. D. 1988. Turgor pressure and phototropismin Sinapis alba L. seedlings.—J. exp. Bot 39: 291-299. Phototropic responses were studied in light-grown mustard hypocotyls.Phototropism was induced by adding 0.27 µmol m^–2s^–1 unilateral blue light to a background of low pressuresodium (SOX) lamp light. Curvatures of some 6° from thevertical were reached by 60 min, the curvature rate between20 min and 60 min being 0.14° min^–1. From the axialgrowth rate and tissue geometry the local growth rates of illuminatedand shaded sides of the hypocotyl were calculated to be 1.5and 4.5 µmin^–1 respectively. Turgor pressures ofexpanding cells in control plants and in the shaded and illuminatedsides of the blue light illuminated hypocotyls were measuredto be 0.40-0.55 MPa with a pressure probe. No changes in turgorpressure were observed on initiation of curvature. The decayof pressure in the cells of non-transpiring plants followingexcision indicated that the yield stress threshold of the tissuemay be as low as 0.1 MPa. These results indicate that the phototropicgrowth response in this tissue is not mediated by changes inturgor pressure. Key words: Sinapis alba L., phototropism, turgor pressure     

Phototropism in Dunaliella and its application in a harvesting device.

Summary The influence of wavelength, light intensity and algal concentration on the phototropism of Dunaliella sp. is described. A practical device for harvesting the alga, based in this effect is shown.     

Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid

Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction.     

Polymorphism of linoleic acid (cis-9, cis-12-octadecadienoic acid) and alpha-linolenic acid (cis-9, cis-12, cis-15-octadecatrienoic acid)

Crystallization and polymorphic properties of linoleic acid (cis-9, cis-12-Octadecadienoic acid) (LA) and alpha-linolenic acid (cis-9, cis-12, cis-15-Octadecatrienoic acid) (alpha-LNA) have been studied by optical microscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The DSC analyses presented three polymorphs in LA, and two polymorphs in alpha-LNA. The XRD patterns of the higher- and lower-temperature forms in LA and alpha-LNA showed orthorhombic O'(//)+O-like and O'(//) subcell, which were similar to those of alpha- and gamma-forms of mono-unsaturated fatty acids, respectively. From the solvent crystallization of LA and alpha-LNA in acetonitrile, single crystals of the higher temperature polymorphs have been obtained. The crystal habits of truncated rhombic shape were also similar to those of alpha-forms of the mono-unsaturated fatty acids. The enthalpy and entropy values of fusion and dissolution of the alpha-forms of LA, alpha-LNA and oleic acid showed that the two values decreased with increasing number of the cis-double bond.     

Role of cis-12-oxo-phytodienoic acid in tomato embryo development

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

Phototropism and Local Adaptation in Phycomyces Sporangiophores

Phototropic responses to unilateral ultraviolet stimuli were studied to determine whether the response of one side of the cell is affected by the previous exposure of the opposite side to ultraviolet. It has been found that the direction of bending is not parallel to the stimulus direction, but is along a straight line rotated 17° clockwise from the stimulus direction. This deviation indicates that the photoreceptors may be in a state of continual clockwise rotation. If before the stimulus the cell is exposed briefly to ultraviolet and rotated through 90°, the response is not along the 17° line, but is deviated a greater or lesser amount, depending on whether the 90° rotation is clockwise or counterclockwise. This difference is evidence that the first ultraviolet exposure leaves a persistent patch of light-adapted receptors and the shaded part of the cell remains dark adapted. The phototropic stimulus straddles the edge between light- and dark-adapted regions, and the differing responses of the two regions affects the direction of phototropic bending. A phototropic mechanism is proposed which combines the features of local adaptation and photoreceptor rotation.     

Effect of cis-, trans-diamminedichloroplatinum(II) and DBP on human serum albumin

Both isomers of diamminedichloroplatinum(II) bind to albumin and induce the formation of the albumin dimer (MW approximately 140 kDa). The trans isomer exhibits a much greater tendency to induce a protein dimerization than the cis isomer. Under similar experimental conditions, the phosphonic derivative of diammineplatinum(II) (DBP) does not induce any dimer formation. The amount of bound complex per mol of human serum albumin (HSA, for an incubation time of 7 days) was found to be 6, 10.5 and 1 mol for cis-, trans-DDP and DBP, respectively. The relative fluorescence intensity of platinum-bound HSA decreases to about 55% for cis-DDP, 45% for trans-DDP and to 85% for DBP when compared to the complex-free protein, suggesting that the binding occurs in the proximity of the Trp214 residue. The structural studies (CD) have shown that only DDP-isomers cause the distinct modification of HSA native structure (alpha-helical content). Pt(II) complexes binding to HSA affect the affinity of HSA towards heme and bilirubin. High excess of DDP prevents the heme and bilirubin binding, while DBP affects this binding much less effectively due to the low amount of the protein-bound complex. Reactions of platinum complexes with albumin are believed to play an important role in the metabolism of this anticancer drug. The minor effect of DBP on HSA may indicate that the toxicity of the phosphonate analog is much lower than toxicities of DDP isomers, most likely due to kinetic reasons.     

Role of K+ and Cl- in Osmotic Adjustment in Roots and Hypocotyls of Intact Mung Bean Seedlings

It was confirmed that osmotic adjustment occurred in young intactmung bean (Vigna mungo (L.) Hepper) seedlings exposed to highosmotic pressure stress. Root growth was not affected by osmoticpressure of less than 200 mOsra in the external solution, althoughhypocotyl growth was conspicuously reduced. Under this moderateosmotic stress, intracellular K⁺ concentration, [K⁺]_i, increaseddramatically during the osmotic adjustment in all the regionsof the root, but the intracellular Cl^– concentration,[Cl^–]_i, increased only in the aged mature region of theroot (28–33 mm from the root tip). About a half of theintracellular osmotic pressure in the aged mature region ofthe root could be ascribed to the contributions of [K⁺]_i and[Cl^–]_i, but in the hypocotyl, [K⁺]_i only contributed slightlyto the osmotic adjustment. (Received June 18, 1986; Accepted August 26, 1986)