Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.     

Inhibition of phytochrome synthesis by the transaminase inhibitor, 4-amino-5-fluoropentanoic Acid

Gardner and Gorton (1985 Plant Physiol 77: 540-543) demonstrated that the transaminase inhibitor gabaculine (5-amino-1,3-cyclohexadienyl-carboxylic acid) inhibits the initial synthesis and resynthesis of spectrophotometrically detectable phytochrome in vivo. Another mechanism-based transaminase inhibitor, 4-amino-5-fluoropentanoic acid (AFPA), is examined in this report for its effects on phytochrome synthesis in developing etiolated seedlings. Preemergence treatment with AFPA was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis. These results confirm those with gabaculine, indicating that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein. AFPA was a more effective inhibitor of both chlorophyll and phytochrome synthesis than was gabaculine, suggesting that AFPA may be the preferred tool with which to probe the physiological consequences of the inhibition of phytochrome biosynthesis.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Aspects of phytochrome decay in etiolated seedlings under continuous Illumination

Summary The rate of total phytochrome decay in the dicotyledons Amaranthus caudatus, Mirabilis jalapa and Pisum sativum under continuous illumination with red, incandescent, and blue light depends on the P_FR/P_total maintained by each source. Amaranthus is an exception to this in that there is a deviation from firstorder decay kinetics under continuous illumination with incancdescent light. This deviation is probably not related to the chlorophyll present in the Amaranthus sample since chlorophyll-rich Pisum buds have the same phytochrome decay rate as epicotyl tissue under continuous incandescent light. Reports of a prolonged lag phase before the onset of first-order decay kinetics of phytochrome in Pisum have not been confirmed and the small lag phase observed in the present work can be accounted for by the time required to attain the P_FR/P_total ratio characteristic of blue light in a carotenoid rich tissue. In the monocotyledon, Avena sativa, and perhaps monocotyledons in general, decay rate is maximal at a low P_FR concentration and the decay curve is the same under continuous red, incandescent and blue light. This dicotyledon/monocotyledon difference with respect to saturation of phytochrome decay does not correlate with the other dicotyledon/monocotyledon difference, the presence or absence of dark reverions of P_FR to P_R, since the dicotyledons Amaranthus and Mirabilis that lack reversion still show no saturation of decay. Possible growth control by the P_FR/P_total ratio is discussed in relation to environmental changes in light quality.Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission.     

Induction versus modulation in phytochrome-regulated biochemical processes

The time course of appearance of competence towards phytochrome (P_fr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated formation of anthocyanin (aglycone cyanidin) and NADP-dependent plastidal glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13). The experiments were performed to answer the following question: Does phytochrome act to turn responses on (induction), or — as an alternative — does phytochrome cause an amplification of processes already occurring in absolute darkness albeit at low rates once competence is reached (modulation)? The data show that in the case of GPD, phytochrome causes an amplification of the rate of synthesis once the competence point is reached at approximately 36 h after sowing at 25° C. In the case of anthocyanin, it was found that two distinct points of competence exist (26 h and 39 h after sowing, 25° C). In the case of ‘early anthocyanin’ (competence point at 26 h), synthesis does not occur in darkness without P_fr, while in the case of ‘late anthocyanin’ (competence point at 39 h), phytochrome causes an amplification of a process occurring in complete darkness albeit at a very low rate. It is concluded that in phytochrome-mediated photomorphogenesis, modulation as well as induction of biosynthetic processes plays a role.     

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: Phytochrome

Fluorescence lifetimes of ‘large’ (mol. wt. 120 000) and ‘small’ (mol. wt. 60 000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of ‘large’ phytochrome was significantly shorter than that of ‘small’ phytochrome and its chromophore models. The phytochrome chromophore of P_r form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of ‘small’ phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very high (0.4) at the main absorption band and is negative (−0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of P_r and P_fr forms of phytochrome are essentially identical. The induced ellipticity of ‘large’ rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of ‘small’ phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in ‘large’ phytochrome but not in ‘small’ P_r. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of P_r. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the P_r → P_fr phototransformation have been proposed on the basis of the above results.     

Luxury uptake of magnesium by peas,Pisum sativum

Increasing the magnesium (Mg) concentration of vegetables (biofortification) will often require ‘luxury’ uptake where the whole‐plant concentration of Mg (c_p) is greater than required for maximum yield. Our aim was to quantify some of the physiological factors influencing luxury uptake of Mg to aid subsequent development of agronomic techniques for biofortification. Peas, Pisum sativum, were used as a test species. A sand culture experiment related vegetative growth and c_p for plants grown with a range of Mg and potassium (K) supply rates. We developed a model of Mg uptake including feedback control exerted by c_p. The model was parameterised with results from a solution culture experiment and then used to explore ways to increase luxury uptake of Mg. Feedback control of Mg uptake by c_p was weak. Biomass did not increase if the Mg concentration exceeded 0.11% in the whole plant or 0.13% in the shoots. Values obtained in the field are often larger than this. Our results indicate that luxury uptake of Mg by peas is readily achieved, provided that there is ample supply of Mg²⁺ to the root surfaces. In field soils though, transport of Mg²⁺ to the roots may limit uptake and cation exchange processes restrict the ability of Mg fertilisers to substantially increase Mg uptake. Increasing root growth will usually increase Mg uptake, but c_p may not rise if biomass is also increased.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Phytochrome in seeds of Amaranthus caudatus

Summary Dry seeds of Amaranthus caudatus show little or no photoreversible absorption changes, attributable to phytochrome. During imbibition phytochrome appears in two phases, one immediately after sowing and the second after about 8 hr. Experiments at different temperatures and under continuous illumination with red, far-red and blue light suggest that there are two pools of phytochrome. The first phase in the appearance of phytochrome could be due to the change in optical properties of the sample on hydration or to rehydration of inactive phytochrome, or both. The second phase probably represents phytochrome synthesis. It is absent at 0° and precedes the water uptake associated with germination by some 10 hr. This second pool of phytochrome does not accumulate in red and blue illuminated seeds indicating that the rate of P _fr decay is more rapid than the rate of phytochrome synthesis. The difference spectra of phytochrome in both 2 hr imbibed seeds and 72 hr old seedlings show peaks of absorption at 663 and 735 nm. The presence of P _fr in dark imbibed seeds and the process of inverse reversion of P _r to P _fr in darkness have been demonstrated. The results are discussed in relation to previous hypotheses for the mechanism of photocontrol of Amaranthus seed germination.     

4-amino-5-hexynoic Acid-a potent inhibitor of tetrapyrrole biosynthesis in plants

4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants.     

Some thoughts about the use of predicted values of the state of phytochrome in plant photomorphogenesis research

Abstract Predicted values of photoequilibrium ratios and rates of photoconversion and cycling, calculated from known optical parameters of purified phytochrome and the spectral photon flux distribution of the light sources used, arc often applied in the evaluation of the relationships between the state of phytochrome and the expression of phytochrome-mediated responses. This is commonly done when the state of phytochrome in vivo cannot be determined experimentally. The ‘predicted’ states of phytochrome may be quite different from the actual ones in vivo for several reasons: the particular set of optical parameters of purified phytochrome used in the calculations and the difficulties encountered in correcting the predicted values for the contribution of the non-photochemical reactions (dark reversion, destruction, synthesis), the effects of the optical properties of the tissue (light attenuation, scattering, trapping) on the rate of phytochrome photo-conversion, and the geometrical relationships between irradiated sample and the light source. At present, in many studies, it is not possible to avoid using predicted values of the state of phytochrome. The limitations imposed by the use of ‘predicted’ values in the interpretation of results obtained in plant photomorphogenesis research should be always clearly stated.     

SODIUM AND POTASSIUM IONS AND ACCUMULATION OF LABELLED d-ASPARTATE AND GABA IN CRUDE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

The accumulation of labelled d -aspartate into crude synaptosomal fraction (P₂) prepared from the rat cerebral cortex proceeded by a ‘high affinity’ system (K_m= 15.1 μm The maximal velocity of d -aspartate uptake was higher than that of the ‘high affinity’ component of l -aspartate uptake and almost equal to that of l -glutamate under the same incubation conditions. Negligible metabolism of labelled d -aspartate was observed in the P₂ fraction. These findings are in accord with those which have been reported for rat cerebral cortical slices. The following observations were made on d -aspartate uptake into rat cerebral P₂ fraction. (1) The requirement of sodium is almost absolute and obligatory. (2) The affinity of the carrier for the substrate is increased by increasing sodium concentration in the medium, but the maximal velocity is not altered. (3) It is suggested that sodium ion is co-transported mole for mole with the substrate molecule. (4) Omission of potassium from the medium inhibits the uptake competitively. (5) Ouabain is a competitive inhibitor on the uptake. (6) Whereas thallium, rubidium and ammonium are efficient substitutes for potassium in exhibiting Na–K ATPase activity of the P₂ fraction, the uptake is activated only by rubidium in the absence of potassium. These observations were in common with the uptake of l -aspartate as well as of l - and d -glutamate, but not with GABA uptake. The requirement of sodium for the uptake of d -glutamate was indicated to be higher than that in the uptake of the other amino acids. Mutual inhibitions of the uptake among l - and d -isomers of glutamate and aspartate suggested that a common carrier is involved in the transport. Mechanisms of the transport of these amino acids in the crude synaptosomal fraction were discussed.     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbitis nil Chois

For dark-grown seedlings of Pharbitis nil capacity to flower in response to a single inductive dark period was established by 24 h white, far-red (FR) or ruby-red (BCJ) light and by a skeleton photoperiod of 10 min red (R)-24 h dark-10 min R. FR alone was ineffective without a brief terminal (R) irradiation, confirming that the form of phytochrome immediately prior to darkness is a crucial factor for flowering in Pharbitis. The magnitude of the flowering response was significantly greater after 24 h FR or white light (WL) (at 18° C and 27° C) than after two brief skeleton R irradiations, but the increased flowering response was not attributable to photosynthetic CO₂ uptake because this could not be detected in seedlings exposed to 24 h WL at 18° C. Photophosphorylation could have contributed to the increased flowering response as photosystem I fluorescence was detectable in plants exposed to FR, BCJ, or WL, but there were large differences between flowering response and photosystem I capacity as indicated by fluorescence. We conclude that phytochrome plays a major role in photoresponses regulating flowering. There was no simple correlation between developmental changes, such as cotyledon expansion and chlorophyll formation during the 24-h irradiation period, and the capacity to flower in response to a following inductive dark period. Changes in plastid ultrastructure were considerable in light from fluorescent lamps and there was complete breakdown of the prolamellar body with or without lamellar stacking at 27 or 18° C, respectively, but plastid reorganization was minimal in FR-irradiated seedlings.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing from of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

The Effect of Colored Light on Pigment Synthesis in Cultured Fern Leaf Primordia

The effect of white, blue, yellow, red and far-red light on the quantitative synthesis of the primary and auxilliary photosynthetic pigments in cultured leaf primordia of Osmunda cinnamomea L. is reported. The P₆₆₀ form of the now classical photoreceptor pigment system, phytochrome, has been demonstrated to be active in chlorophyll synthesis in cultured cinnamon fern leaf primordia as shown by red/far-red reversibility of chlorophyll synthesis. Also, it is apparent from the data presented that a blue absorbing pigment (P₄₂₀) is responsible for the extensive accumulation of chlorophylls and carotenoids in these cultured leaves.     

Correlation between far-red absorbing phytochrome and response in phytochrome-mediated anthocyanin synthesis

Abstract Mustard seedlings were light treated at 24 h after sowing (25°C) to induce phytochrome-mediated anthocyanin synthesis in cotyledons and hypocotylar hook. All light treatments were performed within the range of the reciprocity law. The in situ photoconversion kinetics of phytochrome (P_r→ P_fr) were measured under the same light treatment. It was found that between 0.4 and 1.0 relative P_fr level the amount of anthocyanin extracted from the organs at 52 h after sowing was linearily correlated with the amount of P_fr produced at 24 h in cotyledons and hypocotylar hook. It is concluded that an explanation of the fluence response function for red light mediated anthocyanin synthesis in the mustard seedling does not require the concept of active vs. bulk phytochrome.     

Etude du phytochrome detectable,in vivo dans les graines de Cucurbita pepo L. au cours des differentes phases de la germination

Summary In dry gourd seeds all the phytochrome is in the P_fr form. The increase of phytochrome content from the beginning of hydration involves two phases, A and B, in the embryonic axis as well as in the cotyledons. Cycloheximide does not prevent the appearance of P_r during phase A. We assume that P_r is gradually released from an inactive complex. On the other hand phase B is inhibited by cycloheximide; this could mean that a de novo synthesis of P_r occurs.Some experiments indicate that the phytochrome which is localized in the embryonic axis may be involved only in the germinating process.The phytochrome which is synthesized during phase B disappears when the seeds are irradiated with red light, while the original phytochrome does not.According to our data it seems necessary to lay down a new and precise definition of the germination process.     

HIGH AFFINITY UPTAKE OF l-GLUTAMINE IN RAT BRAIN SLICES

—l -Glutamine is taken up into rat brain slices by a specific‘high affinity’uptake system (K_m 52 μm ) which is not influenced by high concentrations of l -glutamate and l -asparagine. The uptake system appears to be associated with cellular structures that do not survive homogenization under conditions which yield synaptosomes. The‘high affinity’uptake of glutamine is dependent on the external sodium ion concentration and can be inhibited by p-chloromercuriphenylsulphonate, amino-oxyacetic acid, ouabain, dibenamine and allylglycine. The effects of several inhibitors indicate that l -asparagine uptake is mediated by a system different from the‘high affinity’system mediating l -glutamine uptake.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Coaction of three factors controlling chlorophyll and anthocyanin synthesis

In a three-factor analysis the rate of chlorophyll a (Chl) accumulation in excised mustard cotyledons was studied as a function of kinetin, light (operating through phytochrome, P _fr) and an excision factor. It was found that the three factors operate additively provided that the P _fr level is high enough. When the P _fr level is below approximately 1 per cent (<0.01) the effectiveness of the excision factor decreases while the effect of kinetin remains additive. The observed additivity is explained by a model where the three factors operate independently through a common intermediate (presumably 5-aminolevulinate) in the biosynthetic chain leading to Chl. With regard to the coaction of the excision factor and phytochrome it is concluded that the production of the excision factor requires the operation of phytochrome (even though saturated at a low P _fr level) while the action of the excision factor is independent of phytochrome. This conclusion was confirmed by experiments in which the rate of light-mediated anthocyanin synthesis was measured in excised mustard cotyledons. The effect of excision in the case of anthocyanin formation differs kinetically from the effect of excision on Chl formation.Abbreviations Chl chlorophyll(ide) a - P _fr far-red absorbing form of phytochrome - P _fr/P _tot ratio at photoequilibrium - RL red light - FR far-red light - GL green light - RG9 light long wavelength far-red light - WL white light