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     

Phaseshifting of the photoperiodic flowering response rhythm in Pharbitis nil by red-light pulses

The control by light of the flowering response rhythm in the short-day plant Pharbitis nil Choisy cv. Violet was examined by giving a single pulse of light at various times between 1 and 6 h after a 24-h light period. When the first circadian cycle of the rhythm was monitored, it was found that a pulse of red light given at 1, 2 or 3 h into a 72-dark period caused a 1-h delay of the phase of the response rhythm, while a pulse at 6 h caused a 2-h delay. These results support the hypothesis that, when red-light pulses are given at hourly intervals, they are as effective as continuous light in preventing the onset of dark timing because they repeatedly return the rhythm to the circadian time at which it is apparently suspended in continuous light. The perception of and response to continuous light and red-light pulses are also briefly discussed.     

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

The low chlorophyll content of cotyledons of Pharbitis nil grown for 24 h in far-red light (FR) or at 18° C in white light from fluorescent lamps (WL) allows spectrophotometric measurement of phytochrome in these tissues. The (A) measurements utilize measuring beams at 730/802 nm and an actinic irradiation in excess of 90 s. The constancy of the relationship between phytochrome content and sample thickness confirms that, under these conditions of measurement, a true maximum phytochrome signal was obtained. These techniques have been used to follow changes in the form and amount of phytochrome during an inductive dark period for flowering. Following exposure to 24h WL at 18° C with a terminal 10 min red (R), P_fr was lost rapidly in darkness and approached zero in less than 1 h; during this period there was no change in the total phytochrome signal. Following exposure to 24 h FR with a terminal 10 min R, P_fr approached zero in 3 h, and the total phytochrome signal decreased by about half. The relevance of these changes to photoperiodic time measurement is discussed.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

Inheritance of photoperiod-induced flowering in three photoperiodic lines of Aeschynomene americana L.

Summary Induction of flowering by photoperiod was studied in the parental, F₁, F₂, and reciprocal backcross generations of crosses between three photoperiod-responsive Aeschynomene americana L. lines. Generation means appeared additive. Analysis with Mather and Jinks' scaling tests showed little or no epistasis and indicated that an additive-dominance model was adequate. Partitioning components of variation revealed that nearly all variation was additive genetic with dominance and environmental variation negligible. An additive genetic model with two loci, each with two alleles and all alleles having equal net effect, was tested using Power's partitioning method. Results demonstrated that the model fit the data and that there is a major additive genetic system controlling flowering in these crosses, with minor genetic and environmental influences present. Selection for flowering at a desired day length should be feasible.Accepted by A.R. HallauerFlorida Agricultural Experiment Station, Journal Series No. 9251     

A kinetic analysis of the facultative photoperiodic response in Amaranthus retroflexus L.

The ontogenetic change taking place in the facultative photoperiodic response of A. retroflexus to inductive short-day (SD) conditions was studied by exposing plants to continuous induction after different initial exposures to long-days (LD), and comparing the kinetics of their developmental responses (cumulative number of plants with reproductive apices, flowering stage, and height of the apical dome). As the plants progressed from emergence to autonomous flowering (i.e., in non-inductive conditions), their response to continuous induction became progressively more rapid. Reproductive development was initiated following a progressively shorter lag-phase after the start of induction, but its subsequent rate remained unchanged. Until the onset of reproductive development, the undifferentiated upper part of the shoot apex (apical dome) elongated much more rapidly in SD than in LD. However, in both cases reproductive development was initiated when the apex had elongated to about the same extent, after which its elongation accelerated considerably, but to similar rates in both photoperiods. The data indicate that progress towards reproductive development takes place in inductive (SD), as well as in non-inductive (LD) photoperiods, but one cycle of the latter is as effective as 0.20–0.25 of a cycle of the former. —Plants induced at different stages in ontogeny started to change their subsequent branching pattern (ratio of leafy to leafless branches) as soon as induction was delayed beyond autonomous flowering.Abbreviations LD long-days - SD short-days - RGR Relative Growth Rate     

Changes in gene expression in the leaf of Lolium temulentum L. Ceres during the photoperiodic induction of flowering

Unifoliated plants of Lolium temulentum L. Ceres were induced to flower by a unique 24-h long day (LD) consisting of the extension of the regular 8-h short day (SD) (400 mol photons·m^–2·s^–1, fluorescence + incandescence) with incandescence at 10–15 mol photonsm ^–2·s^–1. The polyadenylated-RNA complement of leaf blade tissues was analysed at 4-h intervals during the photoperiod extension in LD vs. SD, by using two-dimensional polyacrylamide gel electrophoresis to resolve in-vitro-translated products. Of the 991 spots that were analysed, none appeared or disappeared during the inductive cycle, i.e. no qualitative effect of floral induction was detected, at any time. Sixty-eight spots were found whose intensity was influenced by lengthening of the photoperiod; 50 of them, i.e. ca. 5% of the population analysed, were affected before the end of the extension period and were thus potentially related to floral induction. Many of these RNAs were not quantitatively constant during a 24-h cycle in SD. Seven of them oscillated according to the light-on and the light-off signals, among which three seemed to be controlled by phytochrome since their relative amount increased under the standard light conditions but decreased under incandescence even faster than in darkness. The large majority of other RNAs varied with a timing that was not clearly driven by the alternation of light and darkness, indicating that genes related to the biological clock may be especially sensitive to the lengthening of the photoperiod. Furthermore, seven spots were observed that underwent a phase-shift in LD, which consisted, for six of them, of a phase advance of 4–8 h. The steady-state level of CAB mRNA was analysed because the CAB gene family (encoding the chlorophyll a/b-binding proteins of the light-harvesting complexes) is known to be controlled both by the biological clock and phytochrome. In SD, the level was high in the light and low in darkness; the fluctuation was conducted by a circadian rhythm. When plants were exposed to the inductive LD, the peak of mRNA accumulation that was expected according to the endogenous rhythmicity was abolished, possibly because of the change in light quality during the LD extension.Abbreviations CAB chlorophyll a/b-binding proteins of the light-harvesting complexes - 2D two-dimensional - LD(s) longday(s) - LDP(s) long day plant(s) - SD(s) short day(s) - SDP(s) short day plant(s) This work was supported by the University of Liège through the Action de Recherche Concertée (# 88/93-129). Some analyses were performed with the collaboration of Dr. H. Ougham, Institute of Grassland and Environmental Research, Aberystwyth, UK. The authors also want to thank Dr. F. Cremer (Max Planck Institute for Plant Breeding, Köln, Germany) for critical discussion of the results.     

Shifts in the semidian rhythm of flowering do determine night-break timing and critical night length in Pharbitis nil

There is a semidian (≈12 h) rhythm in the flowering response of the short-day plant Pharbitis nil Choisy following 90 min exposure to either far-red light/darkness or a temperature drop (27 °C to 12 °C) given at various times in constant conditions before an inductive dark period. This semidian rhythmic response to the temperature-drop pretreatments in the light is also evident through the inductive dark period without change of phase. Furthermore, those pretreatments which increase flowering also advance the time of maximum sensitivity to red light (R) interruptions of the dark period by up to 1.5 h and shorten the critical night length. Conversely, pretreatments which reduce flowering delay the time of maximum R inhibition by up to 1.5 h and increase the critical night length by the same amount. However the phase of a circadian rhythm of flowering response had no effect on either the time of maximum R inhibition or the critical night length. Thus, the semidian rhythm determines both the time of maximum R inhibition and the critical night length in Pharbitis. Received: 8 November 1997 / Accepted: 7 January 1998     

Simultaneous inhibition of translocation of photosynthate and of the floral stimulus by localized low-temperature treatment in the short-day plant Pharbitis nil

There is considerable evidence to indicate that the floral stimulus moves along with photosynthate in the phloem. In the present study the effect of cooling a localized region of the stem of the short-day plant Pharbitis nil Chois. on translocation was investigated. This low-temperature treatment simultaneously inhibited translocation of photosynthate and of the floral stimulus, thus further supporting the idea that the floral stimulus is transported concurrently with assimilates in the phloem.     

The facultative photoperiodic response in the reproductive development of Amaranthus retroflexus L.: Changes in the dose response during ontogeny

The first inductive (short-day; SD) cycle advanced the initiation of reproductive development, while additional SD cycles progressively reduced the lag phase between the start of induction and initiation. The sensitivity to SD increased during ontogeny in long-days (LD) until even the requirement for the first SD cycle disappeared at the onset of autonomous flowering. In photo-induced plants, the postinitiation rate of elongation of the apex was accelerated as the SD dose was increased, but was progressively slower as the start of induction was delayed closer to autonomous flowering, approaching asymptotically the rate of non-induced controls. The inflorescences were branched in plants growing continuously in LD and unbranched in those growing continously in SD. The subsequent branching of the inflorescence could be repressed by SD at any time prior to autonomous flowering, and the degree of repression increased with the induction dose. After the initial SD cycle, 1–2 additional SD could induce the loss of apical dominance, causing excessive elongation and leaf production in the subjacent branches. Further increase in the SD dose inhibited this elongation by accelerating the transformation of the apices of these branches to the reproductive state.Abbreviations LD long day(s) - SD short day(s) - ContSD continuous short day(s) - RGR relative growth rate     

Changes in cotyledon mRNA during floral induction of Pharbitis nil CV. Violet

Floral induction in seedlings of Pharbitis nil Choisy cv. Violet, with one cotyledon removed, was manipulated by applying various photoperiodic treatments to the remaining cotyledon. Populations of polyadenylated RNA from treated cotyledons were examined to identify messages specifically involved in floral induction. The RNA was translated in vitro using a wheat-germ system, and the resulting translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Substantial qualitative and quantitative differences were found between mRNA from cotyledons of seedlings kept in continuous light (non-induced) and of seedlings given a 16-h dark period (induced). In contrast, inhibition of flowering with a night-break resulted only in one detectable, quantitative difference in mRNA.Abbreviations CL continuous light - kDa kilodalton - NB 16 h darkness+10 min red-light break, 8 h into the dark period - poly(A)⁺ RNA polyadenylated RNA (isolated by binding to a cellulose oligodeoxythymidine affinity column) - SD short day (16 h dark) - SDP short-day plant - SDS sodium dodecyl sulfate     

The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

A circadian clock measures photoperiodic time in the male lizard Anolis carolinensis

Summary Photoperiod plays an important role in controlling the annual reproductive cycle of the male lizard Anolis carolinensis. The nature of photoperiodic time measurement in Anolis was investigated by exposing anoles to 3 different kinds of lighting paradigms (resonance, T cycles, and night breaks) to determine if photoperiodic time measurement involves the circadian system. Both the reproductive response and the patterns of entrainment of the activity rhythm were assessed. The results show that the circadian system is involved in photoperiodic time measurement in this species and that a discrete photoinducible phase resides in the latter half of the animals' subjective night. Significantly, the ability of the circadian system to execute photoperiodic time measurement is crucially dependent on the length of the photoperiod. Resonance, T cycle and night break cycles utilizing a photoperiod 10–11 h in duration reveal circadian involvement whereas these same cycles utilizing 6 or 8 h photoperiods do not.Abbreviation CRPP circadian rhythm of photoperiodic sensitivity     

Influences of plant hormones on photoperiodic flowering in Pharbitis nil: re-evaluation by the perfusion technique

Influences of plant hormones on photoperiodic flowering in Pharbitis nil, var. Violet was re-evaluated by assaying them with a newly developed perfusion technique which can directly treat mesophyll cells with sample solution. Gibberellin A₃ promoted the flowering response and indole-3-acetic acid, trans-zeatin and abscisic acid inhibited it when they were perfused immediately before an inductive dark treatment. The promotion or inhibition of flowering was not or hardly observed when solutions containing these plant hormones were applied by the dropping method to surface of cotyledons or plumules of the assay plants. The detection of clear flower-promoting and -inhibiting effects of the plant hormones may be due to the improved efficiency of incorporation of applied substances into plant tissue in the perfusion technique.     

Genes for dwarfing and photoperiod flowering response inPharbitis nil choisy

Dwarfing and sensitivity to the duration of a single inductive dark period for flowering ofPharbitis nil in F₂ progeny of a cross between the tall strain Tendan, and the dwarf, Kidachi appear to be controlled by the alleles at two independent loci. Progeny of a similar cross between the tall strain Violet and the dwarf Kidachi at F₂ and F₃ also showed single locus segregation for tall: dwarf plants. In this cross, differences in photoperiodic response could be identified in F₃ families but they were not simply inherited. There was some evidence of difficulties with classification of the F₂ plants, but also, the flowering of the F₁ between the two less sensitive strains Tendan and Violet indicated complex inheritance of their photoperiodic response. Complementary dominant alleles at three independent loci may be necessary for flowering in even shorter dark periods with the sensitive strain Kidachi. The dwarf strain Kidachi has a reduced gibberellin (GA) content (Barendse and Lang 1972), it flowers in a short dark period without terminal flowering, and it responds positively to GA application both for flowering and growth. However, since control of dwarfing and photoperiodic sensitivity can be separated genetically, there is no strick link between the gibberellin responsiveness of Kidachi for its growth and flowering. Despite the complexity of flowering genetics in Violet×Kidachi, a short-dark-period-sensitive, terminal flowering and tall F₇ line was obtained in a pedigree previously held heterozygous for the dwarf: tall character but not selected for flowering time. Thus, flowering in a short dark period can also be obtained in the presence of the non-dwarfing allele from strain Violet, again demonstrating genetic independence.     

Environmental effects on circadian rhythms in photosynthesis and stomatal opening

Persistent circadian rhythms in photosynthesis and stomatal opening occurred in bean (Phaseolus vulgaris L.) plants transferred from a natural photoperiod to a variety of constant conditions. Photosynthesis, measured as carbon assimilation, and stomatal opening, as conductance to water vapor, oscillated with a freerunning period close to 24 h under constant moderate light, as well as under light-limiting and CO₂-limiting conditions. The rhythms damped under constant conditions conducive to high photosynthetic rates, as did rates of carbon assimilation and stomatal conductance, and this damping correlated with the accumulation of carbohydrate. No rhythm in respiration occurred in plants transferred to constant darkness, and the rhythm in stomatal opening damped rapidly in constant darkness. Damping of rhythms also occurred in leaflets exposed to constant light and CO₂-free air, demonstrating that active photosynthesis and not simply light was necessary for sustained expression of these rhythms. This is CIWDPB Publication No. 1142 This research was supported by National Science Foundation grant BSR 8717422 (C.B.F.) and a U.S. Department of Agriculture training grant to Stanford University (T.L.H.).     

Comparison of gibberellin-like substances from cotyledons and phloem exudate collected from cotyledons of Pharbitis nil in relation to photoperiodic flowering

Gibberellin (GA)-like substances were analyzed in extracts from cotyledons and phloem exudate collected from cotyledons in photoinduced and vegetative seedlings of the short-day plant Pharbitis nil Chois. var. Violet, using high performance liquid chromatography (HPLC) and the dwarf rice bioassay, to see whether any specific GA-like substances were transported from the photoinduced cotyledons via phloem. Cotyledon extracts exhibited five peaks of free GA-like activity in HPLC, whereas only one or two active peaks were detected in phloem exudate extracts. The level of free GA-like activity was considerably lower in phloem exudate than in the cotyledons. In five out of six analyses of cotyledons and phloem exudate, there were substantially higher levels of free GA-like substances in photoinduced plants. Conjugated GA-like substances were present in much higher levels than free GA-like substances in the cotyledon extracts but the levels were not influenced by daylength. In phloem exudate extracts there was no conjugated GA-like substances. The free GA-like substances that are transported via phloem cochromatographed with GA_5/20 and GA₁₉ on HPLC. These were significantly higher in photoinduced plants and thus could have some influence on the photoperiodically-induced flowering in P. nil.     

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.     

The photoperiodic clock in blackheaded buntings (Emberiza melanocephala) is mediated by a self-sustaining circadian system

Three experimental protocols were employed to clarify whether the circadian system is involved in photoperiodic time-measurement in the blackheaded bunting, Emberiza melanocephala. In a single-pulse paradigm, one 8-h light pulse was delivered at different times to groups of birds across three days of constant darkness (DD). Photoperiodic induction, as measured by a rise in plasma luteinizing hormone (LH), showed clear circadian rhythmicity. The second experiment examined the LH responses in birds exposed to lighting cycles using a Nanda-Hamner type of protocol and confirmed full photostimulation under 6L:30D. The third experiment measured the time of the first photo-induced rise in LH in birds subjected to 30 h of continuous light following entrainment under short days (6L:18D). This experiment aimed to identify the position of the photoinducible phase ( _i). LH first rose at hour 18 following dawn indicating that _i lies in the middle of the day. Plasma concentrations of melatonin were also measured under 6L:18D and 6L:30D light cycles as another physiological marker of the circadian system in buntings. The pattern of melatonin secretion under these LD cycles showed properties consistent with the driving oscillator being circadian in nature. It is concluded that the circadian pacemaker driving the photoinducible rhythm in blackheaded bunting is strongly self-sustaining and free-runs under constant conditions.     

Participation of polyamines in the flowering of the short-day plant Pharbitis nil

The effect of the exogenous application of polyamines on the flowering induction of the short-day plant Pharbtis nil was investigated. Putrescine, spermidine and spermine applied on the cotyledons of 4-day seedlings had no significant effect on the flowering of this plant under conditions of full induction caused by a 16-hour-long inductive night. Under the conditions of partial induction caused by a 13-hour-long subinductive night, polyamines inhibit or stimulate flowering, depending on the time of application. Also, inhibitors of the biosynthesis of polyamines influenced the flowering process. Analysis of endogenous polyamines revealed significant fluctuations in their content in cotyledons during an inductive night, as well as under continuous light conditions. Particularly large changes occurred in spermidine and spermine levels. The putrescine level in induced seedlings was lower than in non-induced ones. However, induced seedlings contained a higher level of spermine and spermidine. The highest spermidine and spermine levels were observed at the 8th h of the night, although the total concentration of spermine during photoinduction was always 2–3 times lower than that of spermidine. A break in the inductive night, leading to a complete inhibition of flowering, had caused significant changes in the polyamine level by the end of the night. The results suggest that the flowering induction of Pharbitis nil took place at a low putrescine level and increased spermidine and spermine levels.