Correlation between Calmodulin Activity and Gravitropic Sensitivity in Primary Roots of Maize

Recent evidence indicates a role for calcium and calmodulin in the gravitropic response of primary roots of maize (Zea mays, L.). We examined this possibility by testing the relationship between calmodulin activity and gravitropic sensitivity in roots of the maize cultivars Merit and B73 × Missouri 17. Roots of the Merit cultivar require light to be gravitropically competent. The gravitropic response of the Missouri cultivar is independent of light. The occurrence of calmodulin in primary roots of these maize cultivars was tested by affinity gel chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with bovine brain calmodulin as standard. The distribution of calmodulin activity was measured using both the phosphodiesterase and NAD kinase assays for calmodulin. These assays were performed on whole tissue segments, crude extracts, and purified extracts. In light-grown seedlings of the Merit cultivar or in either dark- or light-grown seedlings of the Missouri cultivar, calmodulin activity per millimeter of root tissue was about 4-fold higher in the apical millimeter than in the subtending 3 millimeters. Calmodulin activity was very low in the apical millimeter of roots of dark-grown (gravitropically nonresponsive) seedlings of the Merit cultivar. Upon illumination, the calmodulin activity in the apical millimeter increased to a level comparable to that of light-grown seedlings and the roots became gravitropically competent. The time course of the development of gravitropic sensitivity following illumination paralleled the time course of the increase in calmodulin activity in the apical millimeter of the root. The results are consistent with the suggestion that calmodulin plays an important role in the gravitropic response of roots.     

Phytochrome Control of Another Phytochrome-mediated Process

The phytochrome-mediated attachment of root tips of mung bean (Phaseolus aureus) and barley (Hordeum vulgare) to glass is affected by the prior exposure of hydrated seeds or seedlings to red or far red radiation. Prior irradiation of seeds or seedlings of mung bean with red light promotes attachment, while far red light promotes detachment of root tips. Similar exposure of barley seeds and seedlings to red light accentuates detachment, while far red light accentuates attachment of root tips. Red-far red light reversibility of the pretreatments indicates phytochrome control.     

Light-Regulated Gravitropism in Seedling Roots of Maize

Red light-induced changes in the gravitropism of roots of Zea mays variety Merit is a very low fluence response with a threshold of 10⁻⁹ moles per square meter and is not reversible by far red light. Blue light also affects root gravitropism but the sensitivity of roots to blue is 50 to 100 times less than to an equal fluence of red. In Z. mays Merit we conclude that phytochrome is the sole pigment associated with light-induced changes in root gravitropism.     

Evidence for a phytochrome-mediated phototropism in etiolated pea seedlings

Entirely etiolated pea seedlings (Pisum sativum, L. cv Alaska) were tested for a phototropic response to short pulses of unilateral blue light. They responded with small curvatures resembling in fluence-dependence and kinetics of development a phytochrome-mediated phototropic response previously described in maize mesocotyls. Irradiations from above with saturating red or far-red light, either immediately before or after the unilateral phototropic stimulus, strongly reduced or eliminated subsequent positive phototropic curvature. Only blue light from above, however, entirely eliminated curvature at all fluences of stimulus. It is concluded that the phototropism is primarily a result of phytochrome action.     

Light-enhanced protein synthesis in gravitropically stimulated root caps of corn

Light stimulates gravitropic bending (downward growth) in roots of many cultivars of corn (Zea mays). In this work, using the cultivar Merit, we show that light stimulates protein synthesis in the root cap, with protein levels increasing 1.3 to 1.6 times that recorded for tissues maintained in continuous dark. Light enhances protein levels both in intact caps (attached to the root) and in caps in culture. Protein synthesis is optimal in cultured caps when 1 nanomolar indole-3-acetic acid is included in the culture medium. If cap tissue is illuminated and subsequently returned to the dark, in the 2-hour period following illumination protein levels decline to that observed in dark controls. It is proposed that light-stimulated protein synthesis mediates in part downward bending in roots of these cultivars of corn.     

Osmotic adjustment in Spirulina platensis

Unilateral irradiation of maize (Zea mays L.) seedlings results in a fluence-rate gradient, and hence below saturation, a gradient of the far-red-absorbing form of phytochrome (Pfr). The Pfr-gradients established by blue, red and far-red light were spectrophotometrically measured in the mesocotyl. Based on these Pfr-gradients and the fluence-response curves of phytochrome photoconversion the fluence-rate gradients were calculated. The fluence-rate gradient in the blue (460 nm) was steeper than that in the red (665 nm), which in turn was steeper than that in the far-red light (725 nm). The fluence-rate ratios front to rear were 1:0.06 (460 nm), 1:0.2 (665 nm), and 1:0.33 (725 nm). The assumption that phytochrome-mediated phototropism of maize mesocotyls is caused by local phytochrome-mediated growth inhibition was tested in the following manner. Firstly, the Pfr response curve for growth inhibition was calculated; these calculations were based on measurements of Pfr-gradients and data from red-light-induced phototropism. Secondly, the Pfr response curve for growth inhibition was used as a basis for calculating fluence-response curves for blue-and far-red-light-induced phototropism. Finally, these calculated results were compared with experimental data. It was concluded that the threshold for phytochrome-mediated phototropism of maize mesocotyls reflects the apparent photoconversion cross section of phytochrome whereas the maximal inducable curvature depends on the steepness of the light (Pfr) gradient across the mesocotyl.Abbreviations Pfr far-red-absorbing form of phytochrome - Ptot total phytochrome - Fr far-red light     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin     

The function of phytochrome A

Knowledge of the photoperceptive function of phytochrome A has improved substantially thanks to the availability of mutants lacking phytochrome A and transgenic plants transformed with the PHYA gene in sense or anti-sense orientation. In imbibed seeds, phytochrome A mediates very-low-fluence responses. In etiolated seedlings, phytochrome A mediates very-low-fluence responses, high-irradiance responses under continuous far-red light, responsivity amplification to phytochrome B and red-light enhancement of the phototropic response to blue light. In light-grown seedings, phytochrome A modulates the extent of response to reductions in red/far-red ratio perceived by phytochrome B, perceives daylength extensions and night interruptions affecting flowering, and perceives light treatments resetting endogenous rhythms. Under natural radiation these abilities are manifested during seed germination and seedling de-etiolation under dense canopies or extremely low light fluences, and during early neighbour detection, but other processes await experimental evaluation. Phytochrome A affects growth and development throughout the whole life cycle of angiosperms.     

Membrane permeability changes: A component of phytochrome-mediated anthocyanin synthesis in Sinapis alba

Dark synthesis of anthocyanin in Sinapis alba seedlings is greatly promoted by short treatments with n-propanol. The effect of n-propanol treatment is reversed by subsequent far-red light pulses. Photoreversibility kinetics suggest that n-propanol and red light act in the same way. However, phytochrome measurements and other control experiments suggest that n-propanol treatment does not lead to significant Pfr production, nor does it increase the effectiveness of any ‘dark’ Pfr present in the seedlings. The findings are difficult to explain in terms of Pfr as the sole effector of this phytochrome-mediated response.     

Phenotypic Analysis of a Dwarf Wheat (Triticum aestivum L.) with Altered Phytochrome-Mediated Growth Responses

The influence of the amount of red light relative to far red light (red/far red ratio) on leaf-sheath elongation, leaf length, tillering, assimilate partitioning to shoots and roots, and chlorophyll content in a dwarf wheat (Triticum aestivum L. cv Tibet Dwarf) and seven other dwarf and standard cultivars was determined. All cultivars tested showed far red-stimulated leaf and leaf-sheath elongation except Tibet Dwarf. Tibet Dwarf was also unresponsive to a brief end-of-day exposure to far red light, although the other cultivars exhibited increased leaf-sheath and leaf length. In these cultivars, the effects of an end-of-day 5-min far red exposure were reversible by a subsequent 5-min red light exposure. Shoot/root ratios were higher and tillering was suppressed by increased far red irradiance in all cultivars except Tibet Dwarf. In addition, Tibet Dwarf was less responsive to dark-induced leaf and leaf-sheath elongation (etiolation) and retained chlorophyll in dark-adapted leaves longer than control cultivars. Tibet Dwarf did not differ from the other cultivars in leaf chlorophyll content. Western blot analysis, using an antibody against phytochrome A, showed that dark-grown Tibet Dwarf shoots contained at least twice the amount of detectable phytochrome A protein present in the other wheat cultivars. These results, plus the short, thick-stemmed, dark-pigmented phenotype of Tibet Dwarf suggest the possibility of a mutation in the phytochrome/signal transduction pathway.     

Elongated mesocotyl1, a phytochrome-deficient mutant of maize

To begin the functional dissection of light signal transduction pathways of maize (Zea mays), we have identified and characterized the light-sensing mutant elm1 (elongated mesocotyl1). Seedlings homozygous for elm1 are pale green, show pronounced elongation of the mesocotyl, and fail to de-etiolate under red or far-red light. Etiolated elm1 mutants contain no spectrally active phytochrome and do not deplete levels of phytochrome A after red-light treatment. High-performance liquid chromatography analyses show that elm1 mutants are unable to convert biliverdin IX alpha to 3Z-phytochromobilin, preventing synthesis of the phytochrome chromophore. Despite the impairment of the phytochrome photoreceptors, elm1 mutants can be grown to maturity in the field. Mature plants retain aspects of the seedling phenotype and flower earlier than wild-type plants under long days. Thus, the elm1 mutant of maize provides the first direct evidence for phytochrome-mediated modulation of flowering time in this agronomically important species.     

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

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

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl⁻ and K⁺. The postshrinking volume recovery is achieved by K⁺ and Cl⁻ influxes, with contribution by the proton motive force. External Ca²⁺ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.     

The effect of red and far red light on the desaturation of Fatty acids in barley leaves

Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-¹⁴C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly.     

Desensitization by red and blue light of phototropism in maize coleoptiles

The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that theses desensitization responses contribute to the induction of second positive curvature, a response induced by prolonged irradiation.Abbreviations BL blue light - RL red light CIW-DPB Publication No. 1001     

The influence of plant growth regulators on the growth of the embryonic axes of red- and far-red-treated lettuce seeds

The influence of several plant growth regulators on the growth of the embryonic axes from red- and far-red-(R- and FR-)treated lettuce (Lactuca sativa L., cv. Grand Rapids) seeds was examined; as shown previously, the water potential of the axes from R-treated seeds has been lowered by 3.5–5.6 bars compared to that in axes from FR-treated ones. Kinetin and abscisic acid (ABA), when included in the incubation medium, reduced the elongation of the axes whereas fusicoccin stimulated it; however, these effects were the same in axes of both R- and FR-treated seeds. In contrast, elongation of axes from FR-treated seeds was stimulated by gibberellic acid (GA₃, but elongation of axes from R-treated ones was not affected by this hormone. This latter result indicates that gibberellins may be involved in the phytochrome-mediated growth responses in lettuce axes.When the root caps of the embryos were removed prior to light treatment, R was still able to induce a water-potential decrease in the embryonic axes, indicating that at least a portion of the active Pfr resides in the axis and not the root cap.Abbreviations ABA abscisic acid - FR far red light - GA₃ gibberellic acid - PEG polyethylene glycol - Pfr far-red-absorbing form of phytochrome - R red light     

Correlations between Gravitropic Curvature and Auxin Movement across Gravistimulated Roots of Zea mays

We compared the kinetics of auxin redistribution across the caps of primary roots of 2-day-old maize (Zea mays, cv Merit) seedlings with the time course of gravitropic curvature. [³H] indoleacetic acid was applied to one side of the cap in an agar donor and radioactivity moving across the cap was collected in an agar receiver applied to the opposite side. Upon gravistimulation the roots first curved upward slightly, then returned to the horizontal and began curving downward, reaching a final angle of about 67°. Movement of label across the caps of gravistimulated roots was asymmetric with preferential downward movement (ratio downward/upward = ca. 1.6, radioactivity collected during the 90 min following beginning of gravistimulation). There was a close correlation between the development of asymmetric auxin movement across the root cap and the rate of curvature, with both values increasing to a maximum and then declining as the roots approached the final angle of curvature. In roots preadapted to gravity (alternate brief stimulation on opposite flanks over a period of 1 hour) the initial phase of upward curvature was eliminated and downward bending began earlier than for controls. The correlation between asymmetric auxin movement and the kinetics of curvature also held in comparisons between control and preadapted roots. Both downward auxin transport asymmetry and downward curvature occurred earlier in preadapted roots than in controls. These findings are consistent with suggestions that the root cap is not only the site of perception but also the location of the initial redistribution of effectors that ultimately leads to curvature.