CHANGES IN GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE ACTIVITY IN SHOOT APICAL MERISTEMS OF BRASSICA CAMPESTRIS DURING TRANSITION TO FLOWERING

An assay system has been developed for the histochemical determination of glyceraldehyde 3-phosphate dehydrogenase (G3PD) activity to indicate glycolytic pathway capacity during evocation in the shoot apical meristem of Brassica campestris L. G3PD activity was differentially distributed in a zonate pattern within the meristems at the vegetative, the transition, and the floral stages. The activity of G3PD changed in all apical zones of evoked apices, but especially in the central and the peripheral zones of apices at the prefloral stage. In the prefloral stage of development heavy enzyme activity was localized in island-like areas within the peripheral zone. These results indicate that 1) the capacity of glycolysis fluctuates during evocation, 2) during floral evocation the capacity of the glycolytic pathway parallels the capacity of the citric acid cycle and the electron transport system only at the prefloral stage, and 3) G3PD activity marks incipient floral primordia. It is proposed that the enzymic marking of an incipient floral primordium indicates the end of evocation in Brassica.     

Histochemical Study of Photoperiodic and Low Temperature Effects on Floral Induction of Spinacia oleracea

Shoot apices of Spinacia oleracea plants have been induced toflower either by: (a) subjecting leaves to 24 h long day, or(b) exposure to a short photoperiod but displaced by 8 h (displacedshort day) in the usual 24 h short-day cycle, or (c) exposureto low temperature (5 °C) during the dark period of thenormal short day. A quantitative cytochemical assay of pentosephosphate pathway activity during floral induction indicatesan approximate doubling of the rate of activity when comparedto that of vegetative apices (short day) (21 °C). Exposure to either low temperature, or a displaced short photoperiodstimulates pentose phosphate pathway activity in the shoot apexin a manner similar to that seen by long-day induction. Thischange in metabolic activity is accompanied by changes in theshape of the shoot apex which resembles that seen at an earlystage during floral induction. Spinacia oleracea, pentose phosphate pathway, shoot apex, glucose-6-phosphate dehydrogenase, floral induction, chilling, displaced short day     

HISTOCHEMICAL STUDY OF ENZYME ACTIVITY IN THE SHOOT APICAL MERISTEM OF BRASSICA CAMPESTRIS DURING TRANSITION TO FLOWERING. IV. GLUCOSE-6-PHOSPHATASE

Glucose-6-phosphatase (G6P) activity was determined in fresh-frozen, cryostat sections in the shoot apical meristem of Brassica campestris L. Enzymatic activity was differentially distributed in a zonate pattern in the vegetative meristem, but not in the transition and floral meristem. Vegetative apices showed a heterogenous localization with the highest activity in the central zone and the pith-rib meristem zone. At the early transition stage of development, G6P activity in the peripheral zone increased slightly. At the late transitional (prefloral) stage, G6P activity was not localized within the peripheral zone in island-like areas of activity. This is the first demonstration of G6P in shoot apical meristem at the vegetative, transition, and floral stage. The results indicate that G6P activity 1) is an accompanying event of evocation, but 2) does not mark incipient floral primordia. G6P may play an important role in the maintenance of glucose-6-phosphate homeostasis in an evoked shoot apical meristem.     

The relationship between the activities of the pentose phosphate pathway and glycolysis during early stages of floral induction in spinach

Summary A quantitative cytochemical study was made of fructokinase, glucokinase, and fructokinase (both PFK-ATP and PFK-PP+F-2:6-P) activities in shoot apices of 4-week old Spinacia oleracea. The rates of activity of these enzymes in the central zone of the shoot apex of plants kept on a short day regime were compared with those from plants transferred from a range of timing up to 24 h to a continuous light regime when floral induction occurred. A mechanism is suggested explaining how no measurable change in activities of the enzymes assayed could still account for the availability of adequate levels G-6-P as substrate for pentose pathway activity which is almost doubled early on in cells of the central zone of shoot apices induced to flower.     

Increased Pentose Phosphate Pathway Activity Linked to Floral Induction in Apices of Spinacia oleracea during Short Days

A quantitative cytochemical study of glucose-6-phosphate dehydrogenaseactivity as a marker of the pentose phosphate pathway (PPP)was made on shoot apices of Spinacia oleracea kept either continuouslyin short days for up to eight weeks or transferred for a singleperiod of 20 h to continuous light after between three and eightweeks in short days. By the time the apices kept in continuousshort days showed morphological changes relating to the floralstate, the PPP activity was already elevated. From four weeksonwards, the apices were more readily induced to the floralstate as evidenced by the increased PPP activity. In addition,the level of PPP activity achieved in the short-day apices asthey progressed to the floral state was as great as that observedin the apices induced to flower by a 20-h day. Spinacea oleracea, pentose phosphate pathway, shoot apex, cytochemistry, glucose-6-phosphate dehydrogenase, floral induction     

Changes in cytokinin activities before,during and after floral initiation in Polianthes tuberosa

The contents of endogenous cytokinin in tuberose corms (Polianthes tuberosa) at vegetative, early floral initiation, and flower development stages were investigated. We also determined the influence of exogenous cytokinin treatment on the corm apex at three different growth stages in relation to floral initiation and development in tuberose. The exogenous cytokinin effectively induced floral initiation and development, especially at the early floral initiation and flower development stages. Endogenous cytokinins were higher in early floral initiation and development stages in comparison to the vegetative stage. During floral initiation stage, the zeatin and dihydrozeatin increased significantly, while the cytokinins, zeatin riboside, dihydrozeatin riboside, ⁶N-(δ²-isopentenyl) adenine, and ⁶N-(δ²-isopentenyl) adenine riboside at consistently low levels. The increase of cytokinin levels in tuberose corms during floral induction suggests a role for cytokinins in tuberose apex evocation. Moreover, these results indicate that cytokinins seem to promote the development of flower buds rather than inducing flowering in tuberose.     

The relationship between the activities of the pentose phosphate pathway and glycolysis during early stages of floral induction in spinach

A quantitative cytochemical study was made of fructokinase, glucokinase, and fructokinase (both PFK-ATP and PFK-PP + F-2:6-P) activities in shoot apices of 4-week old Spinacia oleracea. The rates of activity of these enzymes in the central zone of the shoot apex of plants kept on a short day regime were compared with those from plants transferred from a range of timing up to 24 h to a continuous light regime when floral induction occurred. A mechanism is suggested explaining how no measurable change in activities of the enzymes assayed could still account for the availability of adequate levels G-6-P as substrate for pentose pathway activity which is almost doubled early on in cells of the central zone of shoot apices induced to flower.     

Changes in Apical Growth and Phyllotaxis on Flowering and Reversion in Impatiens balsamina L.

When plants of Impatiens balsamina L were subjected to 5 shortdays and then re-placed in long days, they began to form a terminalflower and then reverted to vegetative growth at this terminalshoot apex The onset of flowering was accompanied by an increasein the rate of initiation of primordia, an increase in the growthrate of the apex, a change in primordium arrangement from spiralto whorled or pseudo-whorled, a lack of internodes, and a reductionm the size at initiation of the primordia and also of the stemfrusta which give rise to nodal and internodal tissues On reversion,parts intermediate between petals and leaves were formed, followedby leaves, although in reverted apices the size at initiationand the arrangement of primordia remained the same as in thefloweing apex The apical growth rate and the rate of primordiuminitiation were less in the reverted apices than in floral apicesbut remained higher than in the original vegetative apex Sincethe changes in apical growth which occur on the transition toflowering are not reversed on reversion, the development oforgans as leaves or petals is not directly related to the growthrate of the apex, or the arrangement, rate of initiation orsize at initiation of primordia Impatiens balsamina L, flower reversion, evocation, phyllotaxis, shoot meristem     

RNA Synthesis in the Apex of Sinapis alba in Transition to Flowering

RNA synthesis in the 1.5 mm apex during floral evocation ofSinapis alba (a long-day plant) was measured using a double-labellingtechnique to compare precursor incorporation in evoked and vegetativeapices, followed by analysis by gel electrophoresis and oligo(dT)-cellulosechromatography. In plants induced to flower by exposure to asingle long day, higher levels of RNA synthesis showed a two-phasepattern, the first increase starting very early in evocation,from 10 h after the start of the long day. This early extrasynthesis is of rRNA, and then also sRNA.² Subsequently higherlevels of RNA synthesis, especially rRNA and 5S RNA, are shownfrom 36 h. At no time was such additional synthesis of the RNAfraction retained on oligo(dT)-cellulose detected. The experimentwas repeated with plants subjected to a single displaced shortday, a treatment which also induces flowering, and similar higherRNA synthesis found in evoked apices. These results were comparedwith those obtained in two non-inductive treatments which resultin some features of floral evocation: a single short day athigh intensity light, and a single treatment with benzyladenine.Neither gave rise to additional RNA synthesis, and thus theobserved high levels of synthesis of rRNA and sRNA seem to beassociated with other features of floral evocation.     

Mitotic activity in wheat shoot apical meristems: effect of dissection to expose the apical dome

An efficient method, less laborious than histological procedures, is described to screen relatively large numbers of shoot apices for mitotic activity. Mitotic activity of shoot apices of Triticum aestivum L. was observed by differential interference contrast (DIC) microscopy of apices infiltrated with a clearing fluid (chloral hydrate/phenol/lactic acid/dibutylphthalate/benzyl benzoate). Serial optical sections were viewed through entire vegetative apical domes and floral primordia. In vegetative shoots, mitotic cells were observed throughout the light and dark cycles of plants maintained in either 8 or 16 h photoperiods. Mitotic activity was lower in the dark phase and increased through the light cycle in both photoperiods. Cells in L1 and L2 layers at the summit of the apex were mitotically active and contributed to the developing shoot and floral structures. Thus, cells in L2 at the summit of vegetative apices are valid targets for transformation leading subsequently to modified germ line cells. Dissections to expose apices for DNA delivery inhibited mitotic activity; recovery periods greater than 48 h would be needed for restoration of normal activity. This suggests that a period of recovery from dissection would be beneficial for attempts at integrative transformation of apical cells.     

QUANTITATIVE STUDY OF NUCLEIC ACIDS AND PROTEINS IN THE SHOOT APEX OF SINAPIS ALBA DURING TRANSITION FROM THE VEGETATIVE TO THE REPRODUCTIVE CONDITION

Quantitative changes in DNA, histone, RNA, and total protein have been measured in meristematic cells during floral evocation.² A single 22-hr, long-day exposure induced two-month-old vegetative plants of Sinapis alba to flower. Periodic collections of shoot apices were made and stained with Schiff's reagent (DNA), azur B (RNA), alkaline fast green (histone), and naphthol yellow S (total protein). The two-wavelength method was used for DNA and histone measurements and the one-wavelength, two-area procedure was chosen for RNA and total protein determinations. The DNA and histone amounts per cell decreased to a minimum value 34 hr after treatment, and most of the nuclei shifted from 4C to 2C values. DNA and histone quantities paralleled each other from 34–46 hr, after which time the histone values continued to increase and the DNA values decreased. The RNA values increased rapidly after treatment as did the total protein quantities, after a slight decrease at 34 hr concurrent with the 4C to 2C cell population shift. The significance of these events is discussed in relation to the changes which were previously described in the shoot apex of Sinapis in transition to flowering.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.     

In vitro modification of spinach plasmalemma thickness

Floral induction in the long day plant spinach (Spinacia oleracea) has been shown to be accompanied by a thickening of plasmalemma. This change was observed at early evocation, in both shoot apices and leaves, as well as after inducing GA₃ treatment. To get further information on this thickening, plasma membranes from spinach leaves were isolated, in the present study, using aqueous two phase partitioning and the effect of variousin vitro treatments on their thickness was investigated. The average plasmalemma thickness was unaffected by Na⁺ and K⁺ ions. It was increased upon the effect of either Ca²⁺ or gibberellic acid. A thickening of plasmalemma was also observed when plasma membranes from vegetative plants were incubated with a cytosolic preparation from photoinduced plants. The results were discussed in relation with the plasmalemma modifications previously reported in spinach.     

Glucose-6-Phosphate Dehydrogenase and NADPH Redox Regulates Cardiac Myocyte L-Type Calcium Channel Activity and Myocardial Contractile Function

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca²⁺ currents (I_Ca-L) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I_Ca-L and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca²⁺ channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I_Ca-L were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of −80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO₂ and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I_Ca-L amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I_Ca-L by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca²⁺ channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

The effect of Ca²⁺-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca²⁺ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10⁻⁴ M), or staurosporine (10⁻⁷ M), indicating that Ca²⁺-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10⁻⁷ M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc.     

Long-day induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex

One challenge for plant biology has been to identify floral stimuli at the shoot apex. Using sensitive and specific gas chromatography-mass spectrometry techniques, we have followed changes in gibberellins (GAs) at the shoot apex during long day (LD)-regulated induction of flowering in the grass Lolium temulentum. Two separate roles of GAs in flowering are indicated. First, within 8 h of an inductive LD, i.e. at the time of floral evocation, the GA(5) content of the shoot apex doubled to about 120 ng g(-1) dry weight. The concentration of applied GA(5) required for floral induction of excised apices (R.W. King, C. Blundell, L.T. Evans [1993] Aust J Plant Physiol 20: 337-348) was similar to that in the shoot apex. Leaf-applied [(2)H(4)] GA(5) was transported intact from the leaf to the shoot apex, flowering being proportional to the amount of GA(5) imported. Thus, GA(5) could be part of the LD stimulus for floral evocation of L. temulentum or, alternatively, its increase at the shoot apex could follow import of a primary floral stimulus. Later, during inflorescence differentiation and especially after exposure to additional LD, a second GA action was apparent. The content of GA(1) and GA(4) in the apex increased greatly, whereas GA(5) decreased by up to 75%. GA(4) applied during inflorescence differentiation strongly promoted flowering and stem elongation, whereas it was ineffective for earlier floral evocation although it caused stem growth at all times of application. Thus, we conclude that GA(1) and GA(4) are secondary, late-acting LD stimuli for inflorescence differentiation in L. temulentum.     

Effects of exogenous calcium ions on tip growth,intracellular Ca2+ concentration,and actin arrays in hyphae of the fungus Saprolegnia ferax

The maintenance of growth of hyphae of Saprolegnia ferax was dependent on the presence of external Ca²⁺ and the growth rate increased with increased external Ca²⁺ up to 5 × 10⁻² m Ca²⁺. When Ca²⁺ was greater than 5 × 10⁻² m, growth rates decreased. Internal membrane-associated Ca²⁺ was localized with chlortetracycline. Internal Ca²⁺ became depleted in hyphae grown in the absence of Ca²⁺ and was increased in hyphae grown in high concentrations of Ca²⁺, showing that internal Ca²⁺ can be modulated by external Ca²⁺. However, the range of the internal change was not as great as the range of external concentration used, indicating that the hyphae are capable of regulating Ca²⁺ in the presence of a large concentration gradient. In the absence of external Ca²⁺, growth can occur for a limited time through use of internal Ca²⁺. The actin cytoskeleton was altered in hyphae grown in both high and low Ca²⁺. Hyphae grown in 10⁻³ m Ca²⁺ had more actin in their apical network and peripheral plaques of actin were further from the apex than in more slowly growing hyphae in 10⁻¹ m and 0 Ca²⁺. The tips of hyphae growing in low Ca²⁺ also had a tendency to swell, giving these hyphae irregular shapes. Ca²⁺ is known to affect cell wall rigidity and the consistency of actin gels, two factors that can be expected to affect hyphal growth. External Ca²⁺ does play a role in hyphal growth possibly directly by acting on the cell wall and indirectly by altering internal Ca²⁺, thus affecting the actin cytoskeleton and possibly other growth processes.     

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     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.