Ultrastructure of Chenopodium rubrum L. apices during the effect of fusicoccin and photoperiodic induction

Plant transition from vegetative to reproductive development is associated with ultrastructural changes in stem apices. Those seen in Chenopodium rubrum L. under the influence of fusicoccin in many ways resemble those induced by a short-day treatment favourable to flowering. This suggests that fusicoccin can play a definite (physiological) role in plant development.     

The Pattern of Reproductive Development in Chenopodium rubrum L.

Individual plants of Chenopodium rubrum were given differentnumbers of inductive cycles in a 12 h photoperiod and the patternof reproductive development was analysed after 40 d of growth.At least 2 inductive cycles are required to form any determinatereproductive organs and at least 12 cycles are required fornormal reproductive development. Individuals given a singleinductive cycle display a loss of apical dominance at thosenodes formed immediately after the treatment without the subsequentformation of any floral structures. Plants given between 2 and12 mductive cycles display both determinate reproductive organsand indeter minate vegetative shoots. The pattern of reproductivedevelopment on such plants depends upon the number of cyclesrelative to the developmental age of newly initiated primordia.It is suggested that the early events of floral induction mayinvolve a radical decrease in the ratio of auxin to cytokinin.     

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light     

Photoperiod and the Determination of Potential Seed Number in Chenopodium rubrum L.

Individuals from two latitudinal populations of Chenopodiumrubrum, a short-day annual, were induced in two inductive photoperiods,15 h and 12 h, to examine the dynamics of reproductive developmentthat determine the potential number of seeds produced. The northernpopulation (50° N) is induced in both photoperiods, whilethe southern population (34° N) is induced only in the 12h photoperiod. Individuals were given either 2, 6, 10 or continuousinductive cycles and dissected at intervals after the startof inductive treatments to determine the rates of initiationand differentiation of primordia on the main axis and selectedaxillary buds. Initial reproductive data indicated that the duration of reproductivedevelopment among individuals of the northern population, whengrown in the longer photoperiod, was 25 per cent greater, butthe number of seeds was increased by a factor of 46. Likewisethe duration of reproductive development among individuals ofthe southern population, when grown in the same photoperiodas the northern population, was 50 per cent longer and the numberof seeds was increased by a factor of 66. Dissection of reproductively-developing individuals revealedthat induction leads to a stimulation in the rate of initiationof primordia followed by a complete inhibition coincident withthe differentiation of terminal floral structures. The timingof this stimulation-inhibition process on each axillary buddepends upon its age relative to the timing of induction. Thuscertain primordia on individuals prematurely removed from inductivetreatments escape floral differentiation and remain vegetative.The total number of floral primordia (potential number of seeds)is determined early in reproductive development by (1) the numberof axillary buds at the start of induction, (2) the stimulatedrate of initiation of primordia after induction, and (3) therate of differentiation of induced primordia. Among individuals of the northern population, the longer inductivephotoperiod leads to a greater stimulation in the rate of primordialinitiation and a decrease in the rate of floral differentiation,which together lead to the production of more primordia. Likewiseamong individuals of the southern population in the shorterphotoperiod, the rate of organ initiation is similar to thatof the northern population in the same photoperiod, but therate of floral differentiation is lower, leading to the initiationof many more primordia. The effects of photoperiod on seed number are discussed in termsof physiological and ecological criteria of optimality. Sincenatural induction occurs in the longest, physiologically-'sub-optimarphotoperiods possible, and this leads to the greatest productionof seeds, it is suggested that the critical photoperiod is amore meaningful focus of interpretation than the physiologically‘optimal’ photoperiod. Of the factors influencing potential seed number, the most significantdifference between the two latitudinal populations is the responseof the rate of floral differentiation to the photoperiod ofinduction. Thus potential seed number in natural populationsis intimately related to the prevailing photoperiods throughthe rates of developmental events. Selection for changes inor maintenance of a particular reproductive ecology must bemediated through developmental responses. Limitations on the potential growth rate of plants are discussedin terms of the ratio of meristematic cells to the total cellpopulation in the plant. Thus the number of growing axillarybuds greatly contributes to the potential growth rate, and thedegree of correlative inhibition is interpreted as a cost ofselection for vertical growth among terrestrial plants competingfor light. By means of a simple model the cost of correlativeinhibition is also discussed in terms of potential seed number.Reproduction is seen as a release from the developmental constraintsrequired by plant form and a stimulation of growth that leadsto a very high production of potential seeds.     

Production of betalains by suspension cultures of Chenopodium rubrum L.

Red-violet cell suspension cultures of Chenopodium rubrum were found to accumulate the betacyanins amaranthin, celosianin and betanin and the betaxanthins vulgaxanthin I and vulgaxanthin II. Under a 16-h daylight regime the cells accumulated 0.3–0.4% betacyanins on a dry mass basis after 2–3 weeks of cultivation on the growth medium. Experiments to define a production medium for betacyanins failed with this habituated line. The accumulation could however be increased up to 1% or 100 mg betacyanins/1 by feeding tyrosine and by adaptation of the inoculum size to the nutrient concentration.     

Phytochrome control of hypocotyl extension in light-grown Chenopodium rubrum

The regulation of hypocotyl extension in light-grown Chenopodium rubrum L. seedlings by light analogous to dense vegetation canopy shade has been monitored. Hypocotyl extension was controlled by both the quantity and quality of the actinic light. At the higher of the two background photon fluence rates which were used (10.0 μmol m⁻²s⁻¹ in the 400–700 nm waveband), increasing the proportion of phytochrome calculated to exist as Pfr resulted in greater inhibition of growth. At the lower photon fluence rate (1.0 μmol m⁻²s⁻¹ in the 400–700 nm waveband), a biphasic response was observed in which minimum inhibition was observed at intermediate photoequilibria. Although photosynthesis was not directly involved in the photomorphogenetic responses, it did play an indirect quantitative role in determining the response.     

The role of phytochrome in photoperiodic time measurement and its relation to rhythmic timekeeping in the control of flowering in Chenopodium rubrum

Summary To follow changes in the status of phytochrome in green tissue and to relate these changes to the photoperiodic control of flowering, we have used a null response technique involving 1.5-min irradiations with mixtures of different ratios of R and FR radiation.Following a main photoperiod of light from fluorescent lamps that was terminated with 5 min of R light, the proportion of P_fr in Chenopodium rubrum cotyledons was high and did not change until the 3rd hour in darkness; at this time, P_fr disappeared rapidly. When the dark period began with a 5-min irradiation with BCJ or FR light to set the proportion of P_fr low P_fr gradually reappeared during the first 3 h of darkness and then disappeared again.The timing of disappearance of P_fr is consistent with the involvement of phytochrome in photoperiodic time measurement. Reappearance of P_fr after an initial FR irradiation explains why FR irradiations sometimes fail to influence photoperiodic time measurement or only slightly hasten time measurement. A R light interruption to convert P_r to P_fr delayed, the timer by 3 h but only for interruptions after and not before the time of P_fr disappearance. Such 5-min R-light interruptions did not influence the operation of the rhythmic timekeeping mechanism. Continuous or intermittent-5 min every 1.5 h-irradiations of up to 6 h in duration were required to rephase the rhythm controlling flowering. A skeleton photoperiod of 6 h that was began and terminated by 5 or 15 min of light failed to rephase the rhythm.The shape of the curves for the rhythmic response of C. rubrum to the length of the dark period are sometimes suggestive of clocks operating on the principle of a tension-relaxation mechanism. Such a model allows for separate timing action of a rhythm and of P_fr disappearance over the early hours of darkness. Separate timing action does not, however, preclude an interaction between the rhythm and phytochrome in controlling flowering.Abbreviations FR far-red - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - R red - BCJ photographic ruby-red irradiation A grant in aid of research from the National Research Council of Canada to B. G. Cumming is gratefully acknowledged.     

Root-shoot correlation linked with photoperiodic floral induction inChenopodium rubrum L.

Inhibition of root growth was observed inChenopodium rubrum under photoperiodic conditions inducing flowering. That this inhibition is mediated by the cotyledons was shown directly by the effect of their excision, which changes the responsiveness of the roots to photoperiodic treatment. On the other hand, decapitation did not lead to such an effect. Some evidence is put forward suggesting that changes in IAA may be involved in these correlations. The existence of two different mechanisms of photoperiodic action in flowering and in root growth is proposed to explain these differences.     

Photoperiodic control of cytokinin transport and metabolism in Chenopodium rubrum

Cytokinin (CK) levels in the short-day plant Chenopodium rubrum L. are known to fluctuate diurnally. The aim of this work was to investigate if the diurnal changes are brought about by changes in transport and/or metabolism of CKs. The effect of photo-period on cytokinin transport was studied by analysing CK concentrations in root, leaf and apical exudates, respectively, under constant light (CL), a 12-h photoperiod (DL) inductive for flowering, DL in which darkness was interrupted at the end of hour 6 by 15 min red light (R), or by 15 min R followed by 30 min far-red irradiation (R/FR). The concentrations of cytokinins (zeatin, zeatin riboside, isopentenyladenine, isopen-tenyladenosine) in all three types of exudates were significantly higher in the first 12-h period after the end of 12 h darkness than in CL. The R break almost fully negated the effect of darkness and its effect was reversed by FR, showing the involvement of phytochrome in the regulation of CK transport. In the next 12-h interval, i.e. 12–24 h after the end of darkness, the CK level remained high in the leaf exudate only, but to a much lower extent than in the previous 12 h. The highest CK concentration (increase by 108%) was observed in apical exudates during inductive darkness. A comparison of the CKs present in the individual exudates indicates that those arriving at the apical part are derived mostly from leaves with varying contributions by the xylem. The metabolism of applied [³H]-zeatin riboside (ZR) was studied using HPLC separation of the metabolites. Metabolism was found to be very rapid and different glucosides, adenine and adenosine were the main metabolites after 12 h incubation with labelled ZR in all regimes tested. The only metabolite that seems to be under photoperiodic control is ZR-5′-monophosphate. It is as yet not clear if photoperiod controls the phosphorylation or dephosphorylation reaction. The activity of the main cytokinin degradative enzyme, cytokinin oxidase, did not change during the photoperiodic regimes tested.     

Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium rubrum L.

Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L. The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes.     

Development of the shoot apex ofChenopodium rubrum L. after photoperiodic induction in the cotyledon stage

Development of the shoot apex up to floral differentiation was investigated in the short-day plantChenopodium rubrum. The changes occurring in the apex from energence until full opening of the cotyledons (Figs 1–4), development during photoperiodic induction (Figs. 5–8), as well as the resulting floral differentiation (Figs. 9–10) are described. It was aimed at excluding the influence of plastochron changes on the interpretation of ontogeny of the shoot apex. For that reason two planes of longitudinal sections and two plastochron stages were compared. In young plants zonation does not become fully evident prior to floral differentiation. The anatomical structure of the shoot apex does not change substantially during the first two inductive cycles which proved to be obligatory under the given experimental conditions. The changes occurring during two further inductive cycles correspond to the total activation of the meristems as manifested by the growth and branching of the apex preceeding floral differentiation proper.     

Electrical membrane potential and resistance in photoautotrophic suspension cells of Chenopodium rubrum L.

On photoautotrophically grown, suspension-cultured cells of Chenopodium rubrum L. the electrical potential difference V _mand the electrical resistance across plasmalemma and tonoplast have been measured using one or two intracellular micro-electrodes. In a mineral test-medium of 5.8 mM ionic strength V _mvalues between 100 and 250 mV, 40% thereof between 170 and 200 mV, and a mean value (±S.E.M.) of 180.6±3.4 mV have been recorded. The average membrane input resistance R _mwas 269±36 M, corresponding to an average membrane resistivity r _mof 3.0 m². V _mand r _mare sensitive to light, temperature, and addition of cyanide, suggesting the presence of an electrogenic hyperpolarizing ion pump, and are ascribed essentially to the plasmalemma. A hexose-specific saturable electrogenic membrane channel is identified through a decrease of V _mand r _mupon addition of hexoses. The hexoseconcentration-dependent depolarization V _msaturates at 92 mV and returns half-saturating concentrations (apparent k _mvalues) of 0.16 mM galactose, 0.28 mM glucose, and 0.48 mM fructose. The magnitude of V _mand r _mwell agrees with pertinent data from mesophyll cells in situ (where only V _mdata are available) and from photoautotrophic lower plant cells. However, V _mis markedly higher than reported for heterotrophically grown suspension cells of different higher plants (with which r _mdata have not been reported so far). It is concluded from the present study and a companion paper on water transport (Büchner et al., Planta, in press) that photoautotrophically grown Chenopodium suspension cells closely resemble mesophyll cells as to cell membrane transport properties.Abbreviations V _m membrane potential(mV) - R _o input resistance () - R _m membrane input resistance () - r _m specific resistance (resistivity) of the membrane (m²)     

The Shoot Apex as a Marker of the Responsivity to Photoperiodic Treatment Inducing Flowering of Chenopodium rubrum L.

Two maxima in flowering response to one inductive dark period of 13 h were found in the short day plant Chenopodium rubrum within three weeks of cultivation under continuous illumination either in vitro or in vivo. These maxima correlated with the number of leaf primordia and their relation to the size of the apical meristem. The first maximum in flowering responsivity corresponded with the stage when primordia of the second leaf pair had not yet overtopped the apical meristem, the second one when the primordia of the fourth leaf overgrew the meristem. Maximum responsivity to flowering reached by a mother plant was reflected in explants derived from it. The above morphological markers of responsiveness to floral induction were not linked to plant age and/or to general growth habit. The explants flowered only when part of the stem was present.     

Biological control of Chenopodium album L. in Europe

Ascochyta caulina (P. Karst) v.d. Aa and v. Kest is aplant pathogenic fungus which is specific to Chenopodium albumL. It has been suggested as a potential mycoherbicide to this weed,which is important and wide spread in arable crops throughout Europe. Toinvestigate its potential as a biocontrol agent, the fungus has beentested in glasshouse and field experiments. Formulations containingdifferent combinations of A. caulina conidia, the phytotoxinsfrom the fungus and low doses of herbicides have been tested.Significant improvement in the efficacy of the fungus was achieved inglasshouse trials with an aqueous formulation containing PVA(0.1% v/v), Psyllium (0.4% w/v), Sylgard 309(0.1% v/v), nutrients and conidia (5 ×10⁶/ml). The extracellular, hydrophilic phytotoxinsproduced by A. caulina were purified and their structuresdetermined. The main toxin, named ascaulitoxin, was characterised as theN²--D-glucopyranoside of the unusual bis-aminoacid2,4,7-triamino-5-hydroxyoctandioic acid. Two other toxins proved to betrans-4-amino-D-proline and the aglycone of ascaulitoxin. Thesetoxins have shown promising herbicidal properties. Field trials haveinvestigated the performance of A. caulina conidia applied atdifferent developmental stages of C. album either as a singletreatment or combined with sub-lethal doses of herbicides or with thefungal phytotoxins. With the available formulation, favourable weatherconditions are needed to obtain infection in the field. The efficacy ofthe strain of A. caulina used so far has proved to beinadequate to justify its development as a bioherbicide. This isprobably due to its low virulence.     

Floral and growth responses in Chenopodium rubrum L. to an extract from flowering Nicotiana tabacum L.

Flowering of Chenopodium rubrum seedling plants was obtained in continuous light after application of fractions of a partially purified extract from leaves of flowering Maryland Mammoth tobacco (Nicotiana tabacum). The stage of flowal differentiation was dependent on the age of the Chenopodium plants used for the bioassay. Apices of plants treated with the extract at the age of four or seven days showed an advanced branching of the meristem or the beginning of formation of a terminal flower; treatment with the extract of plants 12 d old resulted in rapid formation of flower buds in all assay plants. Non-treated control plants kept in continuous light remained fully vegetative. The effects of the extract on flowering were associated with pronounced growth effects. Floral differentiation was preceeded by elongation of the shoot apex. Extension of all axial organs occurred, while growth of leaves, including leaf primordia, was inhibited. The pattern of growth after application of the flower-inducing substance(s) did not resemble the effects of the known phytohormones, but showed some similarities to growth changes resulting from photoperiodic induction of flowering.     

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et␣al. (1995, Biochem J 311:805–813) and LaFayette et␣al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions. Received: 11 July 1996 / Accepted: 24 August 1996     

The Negative Response of Photoperiodic Floral Induction in Chenopodium rubrum L. to Preceding Growth

In Chenopodium rubrum there exists a correlation between theage of the seedlings and the effectiveness of photoperiodicinduction. The younger the plants the more effective was photoperiodictreatment. In three-day-old seedlings one short day was sufficientto promote incomplete flowering, while two short days broughtabout 100 per cent flowering. With six-, eight-, and ten-day-oldplants exposed to two or three short days quantitative differenceswere observed in the earliness of flowering and the percentageof flowering plants. The effects of continuous light and ofshort days with a light break preceding the inductive treatmentwere compared. The results obtained indicate that the inhibitoryeffect of plant age cannot be attributed solely to the appearanceof inhibitors under continuous light but changes of growth patternin plants of different age should also be taken into consideration. The inhibition of RNA synthesis in shoot apices brought aboutby 6-azauridine resulted also in a flowering stimulation, providedthat the inhibitor was applied one or two days prior to inductionand the inductive process itself remained undisturbed. Thisstimulation was accompanied by inhibition of vegetative growthand by a decrease of RNA concentration in the cytoplasm as estimatedcytophotometrically. The competition between growth of vegetative organs and floraldifferentiation affects the response to inductive treatment.The suppression of growth can result in enhancement of flowering.