Effects of temperature on the development of Sinapis alba L. phytochrome-control of nitrate reductase activity at 10°C

Abstract. An analysis of three biochemical parameters during growth of white mustard in continuous white light (WL) has been undertaken at 10°, 15° and 20°C. The time required for anthocyanin and chlorophyll content and nitrate reductase [E.C. 1661 NAD(P)H: nitrate oxidoreductase] activity (NRA) to reach a peak in the cotyledons is shown to be temperature dependent, the rise in chlorophyll content being delayed to a much greater extent than anthocyanin content. In addition, with NRA, there is a significant increase in the level of the peaks with a lowering of temperature.
The NRA in dark-grown plants has been investigated in detail at 10°C. The pre-competence time for this response is increased to 20–24 h, compared with 14 h in seeds grown at 25°C. Other responses are affected far more by the lower temperature; for example, time for 50% loss of photoreversibility of a red (R) pulse in 48-h-old seeds is approximately 13 h, compared with 8 min in seeds grown at 25°C. At 25°C, light treatments during precompetence have been found to increase significantly the effectiveness of a subsequent R pulse on NRA; at 10°C, this effect appears to be almost entirely absent.     

The 'end-of-day' phytochrome control of internode elongation in mustard: kinetics, interaction with the previous fluence rate, and ecological implications

Abstract The ‘end-of-day’ phytochrome control of internode growth was characterized in Sinapis alba, seedlings previously grown under continuous white light for 13 d. The transition from white light to darkness caused a reduction in internode extension rate with a lag of less than 10 min. Following this, extension rate remained almost constant for at least 48 h. i.e. ‘re-etiolation’ was not noticed. The phytochorme controlling the growth processes was stable in the Pfr form. The growth rate of plants receiving a red light pulse, and the growth promotion caused by a far-red light pulse, increased with increasing fluence rate of the previous white light treatment. In far-red treated plants a first growth rate acceleration peaked at 20–30 min after the end of white light, followed by a transient deceleration which led to a growth rate minimum at 40–60 min, followed by a final growth rate recovery yielding a more-or-less steady elevated rate. Pulses establishing different Pfr/P modified the extent, but not the early kinetics, of the growth response. The relative promotion of growth caused by low Pfr/P was limited by darkness as follows: (a), The growth promotion caused by far-red directed to the internode alone was transient. (b), The promotion caused by a reduction of Pfr/P in the whole shoot persisted in darkness for at least 48 h and also persisted if, after a 3–9 h dark period, the plants were returned to continuous white light. In darkness, however, the magnitude of this growth rate promotion decreased with time, particularly when the previous white light fluence rate was low, or the pulse preceding darkness provided the lowest Pfr/P. (c), When compared over the same period in darkness, growth rate was higher in those seedlings in which Pfr/P was reduced during the continuous white light pretreatment than in those ones in which the Pfr/P was only reduced immediately before darkness. It is proposed that in the natural environment, red/far-red signals could be more effective when provided during daytime than at the end of the photoperiod, as both the background growth rate and the relative promotion caused by low Pfr/P are reduced by darkness.     

Differential regulation of phenylalanine ammonia-lyase genes during anthocyanin synthesis and by transfer effect in carrot cell suspension cultures

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyses the first step in the phenylpropanoid pathway and is induced during differentiation and by various stimuli. In carrot ( Daucus carota L. cv. Kurodagasun) suspension culture cells, PAL is slowly induced during anthocyanin synthesis which occurs in a medium lacking 2,4-dichlo-rophenoxyacetic acid and is also induced rapidly and transiently by transferring and diluting cells to fresh medium. Analyses of nucleotide sequences derived from PAL cDNAs revealed that the PAL mRNAs induced by transfer were transcribed from different carrot PAL genes than the PAL mRNAs induced during anthocyanin synthesis. Northern blotting using probes derived from 3'non-coding regions for PAL cDNAs confirmed that different PAL genes were induced during anthocyanin synthesis and after transfer. Induction of different PAL genes occurs in response to differences in the induction trigger.     

The effect of red and far-red light in the high irradiance reaction of phytochrome (hypocotyl growth in dark-grown Sinapis alba L.)

Abstract. Fluence-rate response curves were determined for the inhibition of hypocotyl growth in 54-h-old dark-grown Sinapis alba L. seedlings by continuous or hourly 5 min far-red light irradiation (24 h). Just as in red light ( Heim & Schäfer, 1982 ), a fluence-rate dependence was observed for both kinds of irradiations, even if only 35% of the continuous light effect could be substituted for by hourly far-red pulses. The same total fluence was used for the two different light regimes. Measurements of Pfr and Pfr/Ptot showed a strong fluence-rate dependence under continuous light which only partially paralleled the fluence-rate response curves for the inhibition of the hypocotyl growth. It was concluded that neither spectrophotometrically determined levels of Pfr nor Pfr/Ptot can be the only light-dependent factor controlling hypocotyl lengthening under continuous irradiation.     

Phytochrome effects on stem carbon gain in light-grown mustard seedlings are not simply the result of stem extension-growth responses

Carbon allocation and partitioning were investigated in the first internode of light-grown Sinapís alba L. seedlings exposed to white light (WL) with or without supplementary far-red light (FR). In the internode, supplementary FR increased the rates of extension-growth and the accumulation of radiolabeled carbon (fed through the leaves), reducing sugars (even per unit volume), starch, hemicellulose and cellulose, but had no effect on the levels of sucrose and ammonium oxalate-solubilised cell wall carbohydrates, on invertase activity or on the use of additional sucrose fed through the leaves. In source leaves, supplementary FR had no effect on photosynthesis rates and reduced the accumulation of radiolabeled carbon. Mechanical reduction of stem extension-growth responses to supplementary FR did not affect internode carbohydrate or carbon accumulation responses. Supplementary FR provided only to one leaf had no effect on internode extension growth but increased carbon accumulation in the internode. provided that supplementary FR and radiolabeled carbon were both given to the same leaf. Phytochrome-mediated effects on carbon partitioning are not the mere consequence of internode extension-growth responses. Some additional control point(s) (e.g. leaf-source strength) must be under the direct influence of phytochrome.     

Evidence that a mustard seedling responds to the amount of Pfr and not to the Pfr/Ptotratio

Abstract Phytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) was investigated. Light pre-treated and dark-grown seedlings differing in responsiveness and level of phytochrome (P_tot) were compared. The data obtained support the traditional view that a seedling measures the amount of P_fr. The alternative view that a plant measures the P_fr/P_tot ratio does not seem to be compatible with the data obtained with the mustard seedling.     

Influence of cytokinins and phytochrome on nitrate reductase activity in etiolated leaves of maize

Of the different hormones tested, cytokinins stimulated nitrate-induced nitrate reductase (NR) activity in the dark. The optimal stimulation was obtained at 16 hr and this was sensitive to tungstate, 6-methylpurine and cycloheximide. The cytokinin stimulation of NR activity was further enhanced by brief irradiation with red light, but this effect was not noticed when leaves were exposed to far-red light. Both kinetin and red light, when given together, or given with a darkness interruption, stimulated the NR activity more than with either of them alone.     

Enhancement of Pfr-mediated responses by light pretreatments: persistence of the pretreatment effects

Abstract. In germinating seedlings of Sinapis alba , nitrate reductase activity is under phytochrome control and becomes accessible to phytochrome at about 15 h from sowing. The induction of the enzyme with pulses of light is strongly affected by pretreatments given prior to 15 h, also acting through phytochrome. It is shown that the effects of these pretreatments can persist undiminished for a considerable time (>40 h) but do not alter the pattern of the subsequent responsiveness to P_fr. The nitrate reductase response is compared with other data pertaining to a similar response.     

Photocontrol of the hypocotyl hook opening of Sinapis alba seedlings. Involvement of phytochrome and a high irradiance response

Baumgartner, N. and Fondeville, J. C. 1989. Photocontrol of the hypocotyl hook opening of Sinapis alba seedlings. Involvement of phytochrome and a high irradiance response.
A statistical evaluation of the hypocotyl hook opening (hook opening index) was used for measurement of the hook angle in lots of etiolated Sinapis alba L. cv. Albatros seedlings. Studies of the kinetics for hook opening were carried out in continuous fluorescent white, blue and red light (6, 15 and 40 μmol m^-2s^-1) with 2-day-old dark-grown seedlings. At the beginning of the irradiation period the photoresponse in red light was the opposite to that in blue (low photon fluences). Blue rapidly induced the hook opening (in less than 20 min), while red produced hook tightening (photon fluences up to 70 mmol m^-2), which precedes the normal progressive hook opening. For low fluences, the data were consistent with the involvement of phytochrome and a specific blue light photoreceptor. A phytochrome effect was observed in the hook opening, dependent upon a high irradiance response (HIR). This HIR (like that for the inhibition of the hypocotyl elongation) was characterized by a wavelength response curve with maxima in the blue and far-red regions of the spectrum.     

Time course of signal transduction in phytochrome-mediated anthocyanin synthesis in mustard cotyledons

Abstract. The effectiveness of phytochrome (Pfr) in mediating anthocyanin synthesis in epidermal cells of mustard ( Sinupis alba L.) cotyledons is strongly increased by a light pretreatment of the seedling prior to competence (Mohr et al. , 1979; Johnson, 1980). Here we test the hypothesis that the observed 'sensitivity amplification' is related to a change in the time course of signal transduction from phytochrome to the responsive cell function. It was found that the time course of escape from photoreversibility—considered to reflect the time course of signal transduction—is not changed by a light pretreatment, although the rate of signal transduction increases with age and depends strongly on the level of Pfr.     

Ammonium toxicity: description of the syndrome in Sinapis alba and the search for its causation

In many plant species, prolonged application of ammonium (NH₄⁺) as a source of nitrogen results in physiological and morphological disorders (‘ammonium toxicity’). In the mustard (Sinapis alba L.) seedling we have previously observed particularly severe symptoms of ammonium toxicity in the absence of external nitrate (NO₃^-) or with increasing NH₄⁺/NO₃^- ratios. In the present investigation we have studied the symptoms of this ‘toxicity’in more depth, i.e. at the morphological, plastidic, enzyme and mRNA levels, in an effort to elucidate the causation of the syndrome. It could be confirmed that the syndrome is specific for ammonium and is not caused by a surplus of nitrogen. The syndrome is caused neither by pH changes in the medium nor by non-specific osmotic effects. Furthermore, the syndrome is not causally related to the fact that nitrate reductase (NR; EC 1.6.6.1.) is induced by ammonium. Development of the syndrome requires neither photosynthesis nor intact plastids. Nevertheless, the plastids are severely affected by ammonium application as is anthocyanin synthesis. Enzymes are differently affected. Among the plastidic enzymes, levels of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GPD; EC 1.2.1.13) are strongly reduced and abundance of translatable mRNA of the small subunit of RuBPCase is decreased, whereas nitrite reductase (NIR; EC 1.7.7.1) is not affected. Among extraplastidic enzymes, the level of chalcone synthase (CHS; EC 2.3.1.74) is strongly reduced, the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NADGPD; EC 1.2.1.12) level is unaffected, whereas the isocitrate lyase (ICL; EC 4.1.3.1) level is strongly promoted. The fat → carbohydrate transformation seems to be impaired by ammonium: fat degradation is reduced, starch accumulation is strongly inhibited and the levels of glucose and fructose are decreased. It appears from the present data and from results obtained in a companion study (U. Hecht and H. Mohr, in preparation) that the ammonium toxicity syndrome is detectable as soon as ammonium accumulation occurs in the plant. However, the actual mechanism through which the excess ammonium affects metabolism remains unclear at present.     

Mechanism of phytochrome action in the control of biosynthesis of anthocyanin in Brassica oleracea

-The synthesis of anthocyanin in red-cabbage is very sensitive to control by light, R/FR reversibility being effected by exposures of 5 min duration, The demonstration of this control does not depend upon a preceding irradiation of high-intensity light but depends upon the duration of incubation in darkness subsequent to irradiation. R/FR reversibility is well shown in seedlings kept in darkness for 48 hr after exposure but after 120 hr this reversibility is no longer evident. This is due to the fact that a further synthesis of anthocyanin occurs in unexposed seedlings and in FR and R/FR treated material in the period from 48 to 120 hr but does not occur in the R treatment after 48 hr. Reagents such as n-propanol which are believed to increase membrane permeability, greatly increase anthocyanin synthesis in dark grown material. n-PrOH also reverses the effect of 5 min FR irradiation but, by contrast with R light, does not promote PAL activity. It is concluded that the limitation to synthesis in material unexposed to light is substrate availability at the site of flavonoid biosynthesis, rather than the level of PAL activity. The evidence presented supports the hypothesis that R/FR reversible phytochrome action involves the control of the passage of substrate through a membrane to the site of anthocyanin biosynthesis.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

Cation fluxes in the saps of Sinapis alba during the floral transition

Plants of Sinapis alba L. were induced to flower by either a single long day or a single displaced short day. The levels of three cations. Ca²⁺, Mg²⁺ and K⁺, were measured by atomic absorption spectrophotometry in exudates from roots, leaves and apical stem tips. The export of all three cations out of the root system (root exudate) was increased in induced as compared to non-induced plants. No changes were observed in cation export out of the mature leaves (leaf exudate). The supply of cations to the apical bud (apical exudate) did not originate from the phloem and, so, should mainly be of apoplastic origin. Only the supply of Ca²⁺ to the apical bud was increased, not the supply of Mg²⁺ or K⁺. The increase in Ca²⁺ supply was transient and occurred at about the same time as a conspicuous stimulation of cell division, previously detected in the apical bud.     

Nutrients-mediated shift in temporal expression of phytochrome controlled β-amylase synthesis in mustard (Sinapis alba L.) cotyledons

Abstract. In cotyledons of mustard ( Sinapis alba L.) seedlings grown with distilled water (DW) phytochrome controlled increase in β-amylase (E.C. 3.2.1.2) level takes place at about 42 h after sowing (starting point), while the photoresponse escapes from photoreversibility at 30 h after sowing. The temporal onset of starting point is presumed to be determined by innate process of developmental homeostasis, which is not amenable to influence of environmental factors such as light and nutrients. However, the temporal appearance of onset of phytochrome controlled increase in β-amylase level (starting point) in seedlings grown with Hoagland's nutrient solution (HS) is delayed by 9 h as compared to DW-grown seedlings. Concomitantly, the temporal appearance of the loss of photoreversibility of phytochrome mediated increase in β-amylase level (coupling point) is also delayed by 9 h in HS-grown seedlings. HS does not influence the primary action of phytochrome, the lifetime of components involved in signal chain of above photoresponse and the turnover of β-amylase enzyme. These results indicate that HS-induced temporal shift in onset of starting point of above photoresponse is caused by interaction of nutrients with the process of developmental homeostasis.     

Early detection of neighbour plants by phytochrome perception of spectral changes in reflected sunlight

Abstract We have tested the hypothesis that a plant may detect the presence of a neighboug42r, before being shaded by it, through the perception of the spectral composition of reflected sunlight. Within seedling canopies the red: far-red ratio (R: FR) of the light received by a sensor with a geometry approximating that of a stem was significantly reduced by selective reflection. This effect was observed before any reduction in the amount of photosynthetic light energy received by an individual seedling could be detected. Small green fences of grass, east-west orientated, altered the spectral distribution of the light on the north (sunlit) side of them. Fully illuminated seedlings of Sinapis alba grown on the north side of these green fences produced longer internodes and had a lower leaf: stem dry weight ratio than those grown in front of fences of bleached grasses. A similar redistribution of growth was elicited in seedlings of Chenopodium album, Datura ferox and S. alba growing in full sunlight by exposing plants to additional small quantities of far-red reflected by selective mirrors. These results suggest that the change in the R: FR ratio serves as an early warning signal of oncoming competition.     

Phytochrome control of amino acid synthesis in cotyledons of Sinapis alba

Continuous far red light, acting through phytochrome, stimulates the rate of incorporation of density label into amino acids in the cotyledons of Sinapis alba. It is shown that such stimulation leads to increased incorporation of label into proteins. This has important consequences for experiments in which rates of enzyme synthesis in light treated and dark grown plants are compared by labelling methods. The results of some such experiments are re-evaluated.     

Time-dependent changes in the responsiveness to light of phytochrome-mediated anthocyanin synthesis

Abstract. It is well established that seedlings of mustard ( Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far-red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag- phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.