The Relationship Between Flowering Induction and the Changes of some Phosphatase Activities in Pharbitts nil

This paper is dealing with the effects of the photoperiodic induction of flowering on the activities of four phosphatases (DNase, RNase, PDase, and 5’-NDase) in Pharbitis nil. According to our experimental results, the patterns of changes of activity in these four phosphatases after short-day induction can be put into two cases. One is the case of DNase, which activities are decreased altar short-day treatment both in cotyledons and apices indicating that floral induction leads to dropping of DNA metabolism level Another is the case of the other three phosphatases with a very similar but quite different characteristic from that of DNase. In cotyledons, activities of the three phosphatases after induction are higher than those in the non-induced group. In apices, activities are also higher just after induction, and then drop gradually while activities in the non-induced group rise sharply so that both the two contrast curves show a form of “X”. These changes are evidently interrelated with RNA metabolic regulation in the apices after floral induction.     

Changes in ATP in relation to floral induction and initiation in Pharbitis nil

No changes in metabolism of adenosine phosphates as a function of short day induction were detected in cotyledons of Pharbitis nil Chois strain Violet. A gradual increase in ATP level was detected throughout the dark period in plumules. A rapid decline of ATP pool size was observed in induced plumules shortly after floral induction. The decline occurred close to the 14th hour of the dark period, 1 to 1.5 h after the dark period length required for a 90% flowering response, which is thought to be the minimum time required for transport of the floral stimulus (and assimilates) from the induced cotyledons to the plumule. Transport of the major adenylates from the cotyledons was verified using [¹⁴C]-adenine. Estimates of the amount, and rate, of adenylate transport suggest that the cotyledons could be an important source of adenylates to re-establish the ATP pool size in evoked plumules.     

Ethylene and ABA interactions in the regulation of flower induction in Pharbitis nil

Hormones are included in the essential elements that control the induction of flowering. Ethylene is thought to be a strong inhibitor of flowering in short day plants (SDPs), whereas the involvement of abscisic acid (ABA) in the regulation of flowering of plants is not well understood. The dual role of ABA in the photoperiodic flower induction of the SDP Pharbitis nil and the interaction between ABA and ethylene were examined in the present experiments. Application of ABA on the cotyledons during the inductive 16-h-long night inhibited flowering. However, ABA application on the cotyledons or the shoot apices during the subinductive 12-h-long night resulted in slight stimulation of flowering. Application of ABA also resulted in enhanced ethylene production. Whereas nordihydroguaiaretic acid (NDGA) - an ABA biosynthesis inhibitor - applied on the cotyledons of 5-d-old seedlings during the inductive night inhibited both the formation of axillary and of terminal flower buds, application of 2-aminoethoxyvinylglycine (AVG) and 2,5-norbornadiene (NBD) - inhibitors of ethylene action - reversed the inhibitory effect of ABA on flowering. ABA levels in the cotyledons of seedlings exposed to a 16-h-long inductive night markedly increased. Such an effect was not observed when the inductive night was interrupted with a 15-min-long red light pulse or when seedlings were treated at the same time with gaseous ethylene during the dark period. Lower levels of ABA were observed in seedlings treated with NDGA during the inductive night. These results may suggest that ABA plays an important role in the photoperiodic induction of flowering in P. nil seedlings, and that the inhibitory effect of ethylene on P. nil flowering inhibition may depend on its influence on the ABA level. A reversal of the inhibitory effect of ethylene on flower induction through a simultaneous treatment of induced seedlings with both ethylene and ABA strongly supports this hypothesis.     

Influence of the excision of cotyledons on the content of indole-3-acetic acid, growth and flowering of Lupinus albus plants

Excision of both cotyledons of Lupinus albus L. (L. termis Forssk.) seedlings at different morphological stages during the first 8 days after sowing, retarded significantly the growth of 16-day-old seedlings and decreased the content of IAA in the shoots. The effect was more obvious at the early excision (4 days after sowing). A pattern similar to that of control plants with intact cotyledons was attained when cotyledons were excised later than 8 days after sowing. It is believed that the cotyledons control the IAA content of the shoot during the early stages of development. At day 36 flower primordia were observed in controls but not in cotyledon-less plants. At this time the IAA content of the shoot apices and the growth of cotyledon-less plants were the same as in controls except for plants that lost cotyledons 4 days after sowing, which showed similar values at week 11, but did not produce flower primordia. The other cotyledon-less plants produced flower primordia 4–11 days after controls. It is postulated that the cotyledons convey certain factor(s) to the developing axis at an early stage of the development which promote flowering.     

[14C]-Assimilate partitioning in photoperiodically induced seedlings of Pharbitis nil. The effect of benzyladenine

Partitioning of [¹⁴C]-labeled assimilates was studied in relation to photoperiodic floral induction and evocation in one-week-old Pharbitis nil Choisy cv. 'Violet' seedlings. In plants kept under 16 h photoperiods, one 15 h night induced 100% axillary flowering whereas a 24 h night induced both terminal and axillary flowering. A 15 min night break of red light given 8 h after the beginning of the dark period inhibited flowering. Total [¹⁴C]-assimilate distribution among major sinks (plumules + epicotyl and roots + hypocotyl) from a single source cotyledon was unchanged by one inductive night; however, import of [¹⁴C]-assimilates into shoot apices was increased in induced plants compared to vegegative controls. This increase was several-fold in plants subjected to a 24 h night. N⁶-Benzyladenine (BA) application to cotyledons or plumules under non-saturating night lengths increased the number of floral buds per plant without affecting the position of the first floral bud (i.e. the speed of induction). The same treatment caused increased label accumulation in induced apices, while it only slightly affected non-induced ones. The mode of action of BA on flowering through growth stimulation and resulting assimilate mobilization is discussed.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.     

Appearance of peroxidase isozymes in floral‐initiated shoot apices of Pharbitis nil

A novel biochemical assay system for detecting the early stage of flowering is reported. Peroxidase isozymes in shoot apices of Pharbitis nil plants that had been exposed to flower‐inducing or non‐inducing conditions were analyzed by isoelectric focusing in polyacrylamide gels and activity staining for peroxidase. Several isozymes with pH 8.5–8.8 appeared for the first time 7 days after the beginning of short‐day treatment, but not after nightbreak (non‐inducing) treatment. When shoot tips were cultured in vitro, these same isozymes also appeared after short‐day treatment but not after night‐break treatment. The extent of the appearance of these isozymes was reduced by exposure to high or low temperature during the inductive dark period and removal of cotyledons after the inductive dark period. Such treatments also reduced the extent of flowering. The appearance of an isozyme with pH 8.5 was more closely correlated with flowering than that of the other isozymes. From these results, the appearance of this peroxidase isozyme in shoot apices is discussed as a biochemical marker of flowering in intact plants and in cultured shoot tips.     

Protein Synthesis in Cotyledons of Pisum sativum L: I. Changes in Cell-Free Amino Acid Incorporation Capacity during Seed Development and Maturation

The changes in protein content of pea cotyledons have been followed during the period from 9 to 33 days after flowering. Initially protein content increased gradually with a rapid period of deposition occurring between days 21 and 27 after flowering. After the 28th day the rate of accumulation of protein declined as the seed dehydrated and matured. At maturity the pea cotyledon contained approximately 25% protein which was divided into albumins and globulins in the ratio of 1:1.4.     

Flower-Inducing Activity of Phloem Exudate in Cultured Apices from Pharbitis Seedlings

Phloem exudate from cotyledons of photoperiodically inducedPharbitis plants induced flowering in apices excised from non-inducedseedlings and cultured in vitro. The exudate also stimulatedflowering in apices excised from photo-induced seedlings andcultured under long-day conditions. The application of benzoicacid had similar effects. Both the exudate from non-inducedplants and gallic acid suppressed flowering in apices from photo-inducedseedlings. It appears that the phloem exudate contains flower-inducingor flower-suppressing substance(s), depending on the plant materialsfrom which it was collected. (Received August 15, 1989; Accepted May 14, 1990)     

Effect of Photoperiodic Induction on the Protein Synthesis of Cotyledons and the Morphology of Apical Meristem of Pharbitis nil Plant

After photoperiodic induction of Pharbits nil seedlings with two expanded cotyledons byshort day, the changes of protein in cotyledons or in shoot apex were investigated by polyacrylamide gel electrophoresis technique and electron, microscopy respectively Electron microscopicobservation shows that a kind of spherical electron-dense bodies appears in vacuoles of the apical meristem. Change of protein patterns also observed in the cotyledons. The number of basicprotein bands increased from eight in the untreated control to ten in the induced cotyledons, andthe number of, buffer-soluble protein bands increased from ten in the untreated control totwelve in the induced cotyledons. Authors suggest that the appearence of new protein bandsand electron-dense bodies is probably related to gene expression in the induction process ofphotoperiod of Pharbitis.     

Gibberellins are not essential for photoperiodic flower induction of Pharbitis nil

The influence on photoperiodic flowering of (2-chloroethyl)trimethylmmonium chloride (CCC), an inhibitor of gibberellin (GA) biosynthesis, was studied in the short-day plant Pharbitis nil cv. Violet. The cotyledons contained high levels of endogenous bioactive gibberellins, whereas in the plumules and first leaves the levels were low or undetectable. The first leaf responded to a single'dark treatment by inducing flowering when it was 10 mm or wider. Similar seedlings, but without cotyledons, were used as the assay plants to study the effect of CCC on photoperiodic flowering. Treatment with CCC had no effect on flowering of seedlings without cotyledons, although stem elongation was inhibited. By contrast. CCC inhibited flowering of the intact seedlings with cotyledons. Gibberellic acid applied to the shoot apex or to the first leaf promoted flowering in the CCC-treated seedlings without cotyledons. The results indicate thai gibberellins are not essential for the flower induction process in leaves, but that they promote flower initiation and/or later processes in the shoot apices.     

Restricted cell/cell communication in the shoot apex ofSilene coeli-rosa during the transition to flowering is associated with a high mitotic index rather than with evocation

Summary Plants ofSilene coeli-rosa given 5 or more long days (LDs) flowered, even when the LDs were followed by 48 hours of darkness before their return to short days (SDs). The mitotic indices of shoot apices from induced plants shortly after induction were significantly higher than the indices of shoot apices from vegetative plants. Two major mitotic peaks were observed in the shoot apices of plants given 7 long days (LDs) on day 8. One coincided with that reported byFrancis andLyndon (1979).Cell to cell movement was tested in the shoot apices of vegetative and LD treated plants using probes with a molecular size of 749 daltons (fluorescein-hexaglycine) and 847 daltons (fluorescein-leucyl diglutamyl leucine). These probes showed some movement in the shoot apices of both short day (SD) and LD treated plants, but fluorescein-leucyl diglutamyl leucine was immobile in the induced apices of 7 LD plants on day 8 at time intervals which coincided with major mitotic activity in the shoot apex. Symplasmic restriction in the shoot apex was also observed in plants given 8 LDs (i.e., plants not returned to SDs on day 7).In plants that were placed in 48 hours of darkness after the 7 LD treatment or in plants given 5 LDs, there was no strong peak in the mitotic index, even though all these LD treatments resulted in 100% flowering. In such plants no symplasmic restriction was found in the shoot. Thus the symplasmic restriction on day 8 of 7 LD plants is associated with the high mitotic index, but neither of these phenomena is an essential part of the evocation process.Abbreviations F(Glu)₂ L-glutamylglutamic acid conjugated to fluorescein isothiocyanate isomer I (F-) - F(Gly)₆ F-hexaglycine - FLGGL F-leucyl-diglutamyl-leucine - F(PPG)₅ F-the pentamer (propyl-propyl glycine) - LD long day - LDs long days - SD short day - SDs short days     

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.     

High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface.

The kinetics of the appearance at the cell surface of the outer membrane LamB protein after induction were determined by using specific antibodies and radioiodinated protein A as a probe. This was done in two different induction systems. First, LamB protein was induced in a wild-type strain by the simultaneous addition of cyclic AMP and maltose. Second, an operon fusion strain in which the lamB gene is expressed under lac promoter control was used; in this system, LamB protein can be induced by isopropyl-beta-D-thiogalactopyranoside. When uninduced cells were grown in glucose minimal medium, background expression of the lamB gene was found to be ca. 10-fold lower in lac-lamB cells than in wild-type cells. The level of LamB protein present in uninduced wild-type cells could, however, be reduced by supplementing the growth medium with Casamino Acids. After induction, the LamB protein appeared at the cell surface of both strains within a few minutes, and then the LamB level per cell increased linearly. The time lag in cell surface exposure of LamB protein differed slightly under both induction conditions: the LamB protein appeared at the surface of lac-lamB cells within 3 min of induction, whereas in wild-type cells it could not be detected earlier than after 4 to 5 min of induction.     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.     

Synchronisation of cell division in the shoot apex of Silene in relation to flower initiation

In plants of Silene coeli-rosa, induced to flower by 7 LD, synchronisation of cell division in 20 per cent or more of the cells in the shoot apical dome was found on the 8th and 9th days after the beginning of induction, during the plastochron before sepal initiation. Synchronisation was inferred from the changes in the proportions of cells with the 2C and 4C amounts of DNA, and changes in mitotic index and labelling index. From the peaks of mitotic index a cell cycle of 10 h was measured for the synchronised cells, half that of cells in the apices of uninduced plants in short days. The faster cell cycle and synchronisation in the induced plants was associated with a shortening, of both G1 and G2, suggesting two control points, while S and M remained unchanged. These results are compared with those from other plants in which synchronisation occurs at the beginning rather than the end of evocation.Abbreviations LD long day(s) - SD short day(s) - S DNA synthesis phase of cell cycle - G1 pre-S interphase - G2 post-S interphase - M mitosis     

Changes in RNA levels in the shoot apex of Silene during the transition to flowering

Changes in RNA concentration in the shoot apical meristem during induction and the transition to flowering were measured histochemically in Silene coeli-rosa (L.) Godron, a long-day plant. In the apices of plants induced by 7 long days the RNA concentration increased to about 25 per cent higher than in non-induced plants. Three long days did not induce flowering but resulted in a transient rise in RNA concentration. When plants were given long days interrupted by varying numbers of short days successful induction was accompanied by a sustained increase in RNA concentration but those treatments which were not inductive gave only transient increases in RNA. Gibberellic acid had no effect on induction or apical growth rates but increased the RNA concentration by 50 per cent or more in both induced and non-induced plants. Plants induced to flower at 13° C had the same RNA concentration and growth rate at the apex as in non-induced plants at 20° C. Since changes in RNA concentration in the apex could occur without changes in growth rate and without flowering, and induction could occur without a change in RNA concentration or growth rate, it is suggested that the increase in RNA and growth rate which normally occur at the transition to flowering might not be essential for the formation of a flower but may be more closely related to the rapid growth associated with the formation of the inflorescence.Abbreviations LD long day - SD short-day     

Flower-Inducing Activity of Phloem Exudates from Pharbitis cotyledons Exposed to Various Photoperiods

The phloem exudate prepared from the cotyledons of Pharbitisseedlings that had been exposed to a single dark period (oflonger than 10 h) induced flowering in cultured apices excisedfrom non-induced seedlings. The flower-inducing activity ofthe exudate increased as the seedlings were exposed to longerperiods of darkness. The highest activity was associated withthe exudate taken from cotyledons exposed to a single 16-h darkperiod. The activity of the exudate taken from cotyledons exposedto an inductive dark period was clearly reduced by interruptionof the dark period. The addition of exudate taken from threecotyledons to 10 ml of medium resulted in the highest flower-inducingactivity. About 50% of cultured apex explants formed floralbuds, even when the concentration of the exudate was reducedto 0.1 cotyledon equivalents per 10 ml of medium. The flower-inducingactivity of the exudate appeared to be heat-stable. (Received December 13, 1991; )     

营养胁迫诱导的油菜子叶衰老过程中IAA氧化酶和细胞分裂素氧化酶活性的变化

以离体油菜子叶为材料,研究了营养胁迫诱导的子叶衰老过程中吲哚乙酸氧化酶(IAA氧化酶)和细胞分裂素氧化酶活性的变化。在光照条件下,离体子叶在不含任何无机元素的0．8％的琼脂中培养9d后,出现明显的衰老迹象(叶绿素含量下降,丙二醛含量上升),15d时完全死亡。在营养胁迫诱导的衰老过程中,IAA氧化酶和细胞分裂素氧化酶的活性表现出相似的变化趋势,在诱导处理1d时,两种酶的活性均比处理前有明显下降,之后又随着衰老进程逐渐上升。IAA氧化酶活性在诱导处理11d时达到高峰,超出处理前30％以上;比对照高出1倍以上;而细胞分裂素氧化酶活性在诱导处理13d时达到高峰,比对照高出3倍以上,也超过了处理前的水平。衰老过程中IAA氢化酶和细朐分裂素氧化酶活性的上升可能是导致内源激素含量下降的重要原因。     

Flowering Response in Relation to C and N Contents of Pharbitis nil Plants Cultured in Nitrogen-Poor Media

Flowering of seedlings of Pharbitis nil, strains Violet andTendan, cultured in modified White's medium, was promoted bymedium dilution, the critical dark period being shortened byabout 15 min. Dilution of the N source alone was enough to causethe medium-dilution effect. Dilution of the culture medium duringthe day before and on the day of exposure to the dark-period(a total of two days) caused the maximum dilution effect. TheC and N contents of the cotyledons and of the shoot apices changedrapidly in response to medium dilution. In 1/2-strength White'smedium with 1/1,000 strength NO₃^– which was most effectivefor flower promotion, the C-N ratio was highest. In 1/2-strengthmodified White's medium, in which flowering was lowest withthe longest critical dark period, the C-N ratio was lowest.Thus, there is a close relation between flowering response andthe C-N ratio in cotyledons or shoot apices of Pharbitis nil. (Received September 14, 1984; Accepted January 26, 1985)