Similarity between Cytokinin and Blue Light Inhibition of Cucumber Hypocotyl Elongation

The cytokinin benzyladenine inhibited endogenous hypocotyl elongation in intact etiolated seedlings of cucumber (Cucumis sativus L.). In hypocotyl segments, the inhibitory effect of benzyladenine on growth was clearly detectable in the presence of indoleacetic acid. Fusicoccin-induced elongation was unaffected by the presence of cytokinin. The effect of cytokinin on elongation of the segments was determined by measuring changes in fresh weight, a linear function of extension growth. The effect of benzyladenine on hypocotyl growth was at least as large in segments prepared from red-light-grown seedlings as in those from seedlings grown in total darkness. A comparison was made between the inhibitory effects of cytokinin and blue light. The use of the calcium chelator ethyleneglycol-bis(β-aminoethyl ether)-N, N′-tetraacetic acid indicated that calcium ions are required for manifestation of benzyladenine-induced inhibition.     

Effect of light on ethylene production and hypocotyl growth of soybean seedlings

The apical 1-cm hypocotyl of dark-grown `Clark' soybean (Glycine max [L.] Merr.) seedlings produced ethylene at rates of 7 to 11 nanoliters per hour per gram when attached to the cotyledons. Such physiologically active rates occurred prior to the deceleration of hypocotyl elongation caused by the temperature of 25 C.

Daily exposure of the etiolated seedlings to red light promoted hypocotyl elongation and prevented its lateral swelling. Red light treatment also caused a 45% decrease in ethylene production. Far red irradiation following the red treatment reversed the red effects, suggesting that the ethylene intervenes as a regulator in the phytochrome control of `Clark' soybean hypocotyl growth at 25 C.

     

Response of excised Avena coleoptile segments to red and far-red light

Summary In seeking a simple, red light-promoted straight growth test in which phytochrome assays may be conducted without interference by protochlorophyll, the response of excised Avena coleoptile segments to red and far-red light was re-examined. The elongation of apical (non-decapitated) segments is promoted by a brief exposure to red light, and this effect is almost completely nullified by an immediately subsequent exposure to far-red light. Although growth promotion by red light occurs in distilled water alone, the effect is greater on a medium consisting of 0.02 M phosphate buffer, pH 6.2 to 6.4, with 1 to 2% sucrose. Over the pH range 4.5 to 7.4, dark-growth decreases with increasing pH, but the absolute increment brought about by red light is nearly constant. Elongation appears to be entirely the result of increased cell size.Contrary to previous reports, similar results can be obtained with subapical (decapitated) coleoptile segments, although the absolute magnitude of the response is reduced.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commision.     

Photomorphogenetic responses to UV radiation and short-term red light in lettuce seedlings

Effects of red light (R), far-red light (FR) and UV radiation on growth and greening of lettuce seedlings (Latuca sativa L., cv. Berlinskii) have been investigated. UV-B and UV-C inhibited hypocotyl elongation and stimulated cotyledonary growth. R in combination with UV-B and UV-C partly eliminated these effects, but FR increased those and reversed the R effect. Chlorophyll accumulation was inhibited by UV-B and UV-C. In comparison with cotyledonary growth, R strengthened the UV inhibitory effect, and FR reversed this effect of R. Thus, UV and phytochrome system modify the effects of each other on hypocotyl and leaf growth in lettuce seedlings depending on the level of active phytochrome formed.     

Arabidopsis Mutants Lacking Blue Light-Dependent Inhibition of Hypocotyl Elongation

We have isolated a new class of photomorphogenic mutants in Arabidopsis. Hypocotyl elongation is not inhibited in the mutant seedlings by continuous blue light but is inhibited by far red light, indicating that these mutations are phenotypically different from the previously isolated long hypocotyl (hy) mutants. Complementation analysis indicated that recessive nuclear mutations at three genetic loci, designated blu1, blu2, and blu3, can result in the blu mutant phenotype and that these mutants are genetically distinct from other long hypocotyl mutants. The BLU genes appear to be important only during seedling development because the blu mutations have little effect on mature plants, whereas hypocotyl elongation and cotyledon expansion are altered in seedlings. The genetic separation of the blue and far red sensitivities of light-induced hypocotyl inhibition in the blu and hy mutants demonstrates that two photosensory systems function in this response.     

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Inhibition of stem elongation in cucumis seedlings by blue light requires calcium

The effects of blue light and calcium on elongation of hypocotyl segments of Cucumber (Cucumis sativa L. cv Burpee's Pickler) were studied. Cucumber seedlings grown in dim red light showed a rapid decline in the rate of hypocotyl elongation when irradiated with high intensity (100 micromoles per square meter per second) blue light. In intact, 4-day-old seedlings the inhibition began within 2 minutes after the onset of blue-light irradiation and reached a maximum of approximately 55% within 4 minutes. Hypocotyl segments cut from 4-day-old seedlings also showed an inhibition of elongation in response to blue light when segments were floated on aqueous buffer and exposed to blue light for 3 hours. In the presence of 2 micromolar indole-3-acetic acid, blue light caused a 50% inhibition of elongation. Buffering free calcium in the incubation medium with 0.1 millimolar ethylene glycol bis(-aminoethyl ether)- N,N,N′,N′-tetraacetic acid eliminated the blue-light inhibition of segment elongation. Several experiments confirmed a specific requirement for calcium for the blue-light-induced inhibition of segment elongation. Treating segments with 0.2 micromolar fusicoccin abolished the inhibition of elongation by blue light as did buffering the medium at pH 4. Adding 1 millimolar ascorbate to incubation medium also eliminated the inhibition of segment elongation caused by blue light. Several compounds implicated in cell-wall redox reactions alter the magnitude of the blue-light-induced inhibition. The activity of peroxidase isolated from the cell-wall free space of cucumber hypocotyls was inhibited by ascorbate and low pH. The results are consistent with the hypothesis that blue light inhibits elongation by inducing an increase in cell-wall peroxidase activity and implicate calcium ions in the response to blue light.     

Role of Cell Wall Calcium in Red Light- inhibited Elongation of Hypocotyls of Etiolated Phaseolus radiatus

When the hypocotyl segments of Phaseolus radiatus L. were incubated in CaC12 (1 mmol/L) medium, the cell wall calcium was increased over threefold more than those incubated in a Ca2+ -free medium. However, red light inhibited elongation of the hypocotyl was 20% to 25% both in the medium with or without Ca2 + . The amount of calcium removed from the wall by ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) ( 1 to 10 mmol/L) was 58.13 % to 75.33%, which offset the red light-inhibited elongation of the hypocotyl by 61.29% to 87.1%. Moreover, treatment with the channel blocker, verapamil ( 10 to 100 μ mol/L), wall calcium was the same as that of the darkness control, by which the red light-inhibited growth was also offset. La3 + ( 100 to 1 000 μmol/L) had no effect on wall calcium as compared to hypocotyl segments treated with red light alone, but eliminated the inhibitory effect of red light. Treatment with the calcium ionophore, A23187 (10 to 100 μmol/L), red light-inhibited elongation was abolished by 66.67% to 142.45% while wall calcium was reduced by 24.53% to 42.81%. In addition, calmodulin antagonist chlorpromazine (1 to 10 μmol/L) also counter acted the red light-induced elongation inhibition. These data indicated that exogenous Ca2+ was involved in the red light-inhibition effect, but that' did not mean that Ca2 + was not required. Perhaps Ca2 + in the wall itself was sufficient for red light-induced inhibition of hypocotyl elongation. The role of wall calcium might be quite complex, it not only acted as a signal of influx Ca2 + from the Ca2 + pool, but also played a regulatory role in the cell wall.     

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; )     

Relationship between IAA-induced elongation growth and plasma membrane NADH oxidase in soybean (Glycine max Merr.) hypocotyls

A relationship between the activity of NADH oxidase of the plasma membrane and the IAA-induced elongation growth of hypocotyl segments in etiolated soybean (Glycine max Merr.) seedlings was investigated. The plasma membrane NADH oxidase activity increased in parallel to IAA effect on elongation growth in hypocotyl segments. Actually, NADH oxidase activity was stimulated 3-fold by 1 u,M IAA, and the elongation rate of segments was stimulated 10-fold by 10 iM IAA. The short-term elongation growth kinetics, however, showed that the IAA-induced elongation of hypocotyl segments was completely inhibited by plasma membrane redox inhibitors such as actinomycin D and adriamycin, at 80 μM and 50 μM respectively. In addition, 1 mM actinomycin D inhibited the IAA-stimulated NADH oxidase activity by about 80%. However, adriamycin had no effect on NADH oxidase activity of plasma membrane vesicles. Based on these results, the plasma membrane redox reactions seemed to be involved in IAA-induced elongation growth of hypocotyls, and the redox component responding to IAA was suggested to be NADH oxidase.     

Aging progression involving dwarfism and its acceleration by red light in bean hypocotyls

The effect of red light on the aging progression of the bean (Phaseolus vulgaris L.) hypocotyl segment unit was examined in relation to dwarfism using Kentucky Wonder (tall) and Masterpiece (dwarf) varieties. In both plants, red light promoted the elongation of younger zones and inhibited that of mature zones. The zone exhibiting maximum elongation was shifted to the younger zones by red light irradiation regardless of the plant type, but its extent was greater in the dwarf than in the tall. Thus, red light hastens both the beginning of elongation in the younger portion and its termination in the mature portion of the hypocotyl, particularly of the dwarf plant. These red light responses in each zone of both the tall and dwarf hypocotyl units were reversed by subsequent exposure to far red light regardless of the duration and intensity of red light, thus indicating that the hastened aging progression of the hypocotyl by red light is mediated by phytochrome. However, there is no difference in the rate of decay of Pfr between the tall and dwarf hypocotyls.     

Green light stimulates early stem elongation, antagonizing light-mediated growth inhibition

During the transition from darkness to light, the rate of hypocotyl elongation is determined from the integration of light signals sensed through the phototropin, cryptochrome, and phytochrome signaling pathways. In all light conditions studied, from UV to far-red, early hypocotyl growth is rapidly and robustly suppressed within minutes of illumination in a manner dependent upon light quality and quantity. In this study, it is shown that green light (GL) irradiation leads to a rapid increase in the growth rate of etiolated Arabidopsis seedlings. GL-mediated growth promotion was detected in response to constant irradiation or a short, single pulse of light with a similar time course. The response has a threshold between 10(-1) and 10(0) micromol m(-2), is saturated before 10(2) micromol m(-2) and obeys reciprocity. Genetic analyses indicate that the cryptochrome or phototropin photoreceptors do not participate in the response. The major phytochrome receptors influence the normal amplitude and timing of the GL response, yet the GL response is normal in seedlings grown for hours under constant dim-red light. Therefore, phytochrome activation enhances, but is not required for, the GL response. Seedlings grown under green, red, and blue light together are longer than those grown under red and blue alone. These data indicate that a novel GL-activated light sensor promotes early stem elongation that antagonizes growth inhibition.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.     

Growth and morphological responses to different UV wavebands in cucumber (Cucumis sativum) and other dicotyledonous seedlings

We examined the influence of short-term exposure of different UV wavebands on the fine-scale kinetics of hypocotyl growth of dim red light-grown cucumbers (Cucumis sativus L.) and other selected dicotyledonous seedlings to evaluate: (1) whether responses induced by UV-B radiation (280-320 nm) are qualitatively different from those induced by UV-A (320-400 nm) radiation, and (2) whether different wavebands within the UV-B elicit different responses. Responses to brief (30 min) irradiations with 3 different UV wavebands all included transient inhibition of elongation during irradiation followed by wavelength specific responses. Irradiations with proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm) induced inhibition of hypocotyl elongation within 20 min of onset of irradiation, while UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation with a lag of 1-2 h. The response to short wavelength UV-B was persistent for at least 24 h, while the response to long wavelength UV-B lasted only 2-3 h. The UV-A treatment induced reductions in elongation rates of approximately 6-9 h following exposure followed by a continued decline in rates for the following 15-18 h. Short wavelength UV-B also induced positive phototropic curvature in both cucumber and Arabidopsis seedlings, and this response was present in nph-1 mutant Arabidopsis seedlings defective in normal blue light phototropism. Reciprocity was not found for the response to short wavelength UV-B. The short wavelength and long wavelength UV-B responses differed in dose-response relationships and both short wavelength responses (phototropic curvature and elongation inhibition) increased sharply at wavelengths below 300 nm. These results indicate that different photosensory processes are involved in mediating growth and morphological responses to short wavelength UV-B (280-300 nm), long wavelength UV-B (essentially 300-320 nm) and UV-A. The existence of two separate types of hypocotyl inhibition responses to UV-B, with one that depends on the intensity of the light source, provides alternate interpretations to findings in other studies of UV-B induced photomorphogenesis and may explain inconsistencies between action spectra for inhibition of stem growth.     

Reversal of 5-fluorodeoxyuridine-caused growth inhibition in intact and excised etiolated hypocotyls of Sinapis alba L. by thymidine

Summary The elongation growth in etiolated, intact seedlings and excised hypocotyl segments of Sinapis alba is inhibited by FdUrd in the same fashion, and in either case there is a direct correlation between FdUrd concentration and inhibition of elongation growth. Removal of the roots reduced elongation; however, the percentage inhibition by FdUrd remained the same. Therefore, the growth inhibition by FdUrd is not a consequence of root growth inhibition.The growth inhibition in excised hypocotyls cultured on synthetic media is inversely proportional to the size of the segments, and of the seedlings from which they are taken. Elongation of the smaller segments is more sensitive to FdUrd than that of the larger ones. Anatomical observations showed that the inhibition of growth elongation by FdUrd in the hypocotyl segments is due to inhibition of cell elongation, and not of cell division. Root formation is inhibited in all isolated segments.The growth inhibition by FdUrd could be reversed by dThd but not by uridine, and this reversibility depended upon the FdUrd concentration. When FdUrd inhibition is partial (up to 10^-7M) relatively high dThd concentration (up to 100 fold) are required for complete reversal; when inhibition is maximal relatively lower dThd concentrations effect a complete or near-complete reversal. Irreversible, unspecific effects of FdUrd were not found.These experiments confirm that DNA synthesis is involved in cell elongation of the hypocotyls even when the apical meristem and roots are removed, and that the growth inhibition by FdUrd is not a nonspecific, toxic effect.Abbreviations FdUrd 5-fluorodeoxyuridine - dThd thymidine     

PHOTOINHIBITION OF ELONGATION GROWTH OF ROOTS IN RICE SEEDLINGS

Effects of white, blue, red and far-red lights on the elongationgrowth of intact primary roots in rice seedlings were investigated.White light inhibited elongation of root cells. Blue light inhibitedboth cell elongation and cell multiplication, but red lightinhibited only cell elongation. The effect of far-red lightwas almost the same as that of the red. The lights exerted;the same effects on the growth of primary roots irrespectiveof age of the seedlings. The inhibitory effects of the lightswere also observed when the root of the deshooted seedlingswas irradiated, but not when only the shoot of the seedlingswas irradiated. It was inferred that it is not the shoot butthe root which responds to the light in this phenomenon. DCMUhardly affected root growth at a concentration so high as toinhibit strongly photosynthesis in the shoot. The possibilitythat photosynthesis participates directly in the photoinhibitionof root growth in rice seedlings was excluded. ¹ Present address: Tohoku National Agricultural Experiment Station,Morioka     

Influence of cobalt on soybean hypocotyl growth and its ethylene evolution

Development of dark-grown “Clark” soybean (Glycine max [L.] Merr.) seedlings is abnormal at 25 C but normal at 20 and 30 C. At 25 C, hypocotyls swell and fail to elongate normally; lateral root formation and seedling ethylene evolution are enhanced.

Co²⁺ promoted hypocotyl elongation of etiolated “Clark” soybean seedlings by 28% when grown at 25 C. The same growth-promoting concentration reduced hypocotyl thickness and primary root elongation by 28 and 43%, respectively. Co²⁺ inhibited ethylene production both of intact seedlings and of apical 1-centimeter hypocotyl segments with attached epicotyls and cotyledons by 65 and 60%, respectively. These results suggest that Co²⁺ exerts its effects on the hypocotyl growth by inhibiting ethylene production, and also confirm our previous conclusion that abnormal ethylene production at 25 C is responsible for the inhibition of hypocotyl elongation and for its swelling.