Reevaluation of the Subunit Molecular Weights of Soybean 11S Globulin

The acidic and basic subunits are the main constituents of soybean 11S globulin. Each of these two subunits consists of three major polypeptides of similar size. The molecular weights of the acidic and basic subunits have been previously estimated to be 37,000 and 22,000, respectively, by SDS-polyacrylamide gel electrophoresis (Catsimpoolas et al, J. Set Food. Agric., 22, 448 (1971)). Reevaluation of the molecular weights by 6 m guanidine gel chromatography gave the values of 28,000 and 18,000, respectively. These are supported by results of equilibrium sedimentation in the same solvent. The previously reported values seem to have been overestimated, especially for the acidic subunits. The overestimations seem to be related to the high percentage of acidic amino acids, which causes the conformation of the SDS-protein polypeptide complexes of these subunits to deviate from those of proteins usually employed as standards for molecular weight estimations.     

Subunit Structure of Sesame 13S Globulin

On SDS-polyacrylamide gel electrophoresis, sesame seed 13S globulin was separated into three intermediary subunits termed IS₁ IS₂ and IS₃. Following a treatment with 0.2M 2-mercaptoethanol, the globulin was separated into three acidic subunits termed AS₁ AS₂ and AS₃, and four basic subunits termed BS₁ BS₂, BS₃ and BS₄. Two dimensional SDS-gel electrophoresis before and after treatment with 0.2 M 2-mercaptoethanol revealed that IS₁ was composed of two combinations of acidic and basic subunits, these being S₁ and BS₂, and AS₂ and BS₂. IS₂ was found to be composed of AS₃ and BS₁, and IS₃ was composed of AS₂ and BS₃, and AS₂ and BS₄. These combinations were consistent with the reactivity of each subunit to a fluorescent thiol reagent. The amino acid compositions were similar among the three acidic subunits and also among the four basic subunits. However, between the acidic and basic subunits, there were great differences in the amino acid composition, especially in the amount of glutamic acid.     

Association of Phosphatidylcholine with Soybean 11S Globulin

To address the multiplicity of aromatic ring hydroxylation dioxygenases, we used PCR amplification and denaturing gradient gel electrophoresis (DGGE). The amplified DNA fragments separated into five bands, A to E. Southern hybridization analysis of RHA1 total DNA using the probes for each band showed that band C originated from a couple of homologous genes. The nucleotide sequences of the bands showed that bands A, C, and E would be parts of new dioxygenase genes in RHA1. That of band B agreed with the bphA1 gene, which was characterized previously. That of band D did not correspond to any known gene sequences. The regions including the entire open reading frames (ORFs) were cloned and sequenced. The nucleotide sequences of ORFs suggested that the genes of bands A,C, and E may respectively encode benzoate, biphenyl, and polyhydrocarbon dioxygenases. Northern hybridization indicated the induction of the gene of band A by benzoate and biphenyl, and that of the gene of band C by biphenyl and ethylbenzene, supporting the above notions. The gene of band E was not induced by any of these substrates. Thus the combination of DGGE and Southern hybridization enable us to address the multiplicity of the ring hydroxylation dioxygenase genes and to isolate some of them.     

Homology between the Acidic Subunits of Soybean 11S Globulin

The formaldehyde dehydrogenase (EC 1.2.1.1) from the yeast Pichia pastoris IFP 206 was purified to homogeneity. The enzyme had a molecular weight of 84,000 daltons and was composed of two identical subunits of a molecular weight of 39,000 daltons. The N-terminal end of the subunits is blocked. The protein showed 6,3 free -SH groups per mole and 12,5 in the presence of NAD⁺. Enzyme stability was increased by addition of glycerol during the purification.

The enzyme activity is NAD⁺ and glutathione dependent. The reaction product is S formylglutathione. The presence of an S-formylglutathione hydrolase (EC 3.1.2.12) in the cell free extract was detected. The formaldehyde dehydrogenase showed an optimum pH of 7.9 and an optimum temperature of 47°C. The activation energy was 3.2 kcal/mol. The Michaelis constants for NAD⁺ and S-hydroxymethyl glutathione were respectively 0.24 mM and 0.26 mM.     

A Micro-method for the Measurement of Gel Properties of Soybean 11S Globulin

We examined the primary structure of α-amylase produced by Bacillus subtilis var. amylosacchariticus by isolation and characterization of CNBr fragments of the enzyme. By solubilization and precipitation in a buffer, the fragments were first fractionated into two. The soluble fraction was fractionated by Bio-Gel P-30, and three fragments were obtained. The insoluble fraction was fractionated by SP-Sephadex C-25 and further purified by Bio-Gels, and five fragments were isolated. Amino acid sequences near the N- and C-terminus were determined with the eight CNBr fragments. By matching the sequences with those of methionine-containing tryptic peptides, alignment of the eight CNBr fragments was determined.     

Polymerization of Soybean 11S Globulin Due to Reactions with Peroxidizing Linoleic Acid

Soybean 11S globulin was polymerized by incubating with peroxidizing linoleic acid. The molar ratio of the acidic subunits to the basic subunits of 11S globulin decreased with the elapse of the incubation time. The acidic subunits were lost faster and formed polymers more easily than the basic subunits. The acidic and basic subunits in 11S globulin were fractionated by DEAE-Sephadex gel chromatography. Each of the acidic and basic subunits was allowed to react with peroxidizing linoleic acid individually. The results also showed that the acidic subunits formed polymers faster than the basic subunits. Both succinylated and acetylated 11S globulins were also submitted to the incubation with peroxidizing linoleic acid. The polymerization of the modified protein was suppressed by masking ε-amino groups.     

Isolation and Some Physico-chemical Properties of the Acidic Subunits of Soybean 11S Globulin

Four kinds of acidic subunits and three kinds of basic subunits of 11S globulin were separated by polyacryl amide gel electrophoresis in the urea system. The four acidic subunits designated as A₁, A₂, A₃ and A₄ (R_m=0.35, 0.40, 0.46 and 0.56 respectively) were isolated by stepwise elution followed by repeating gradient elution with DEAE-Sephadex A-50 in the presence of 6 m urea at 5°C.

Subsequently, some physico-chemical properties of the subunits were determined. For example, N-terminal amino acids were determined as phenylalanine for both A₁ and A₂ and as leucine (or isoleucine) for both A₃ and A₄ by the DNP-amino acid method. The molecular weights of A₁, A₂ and A₃ were shown as 37,000 and 45,000 for A₄ by SDS-gel electrophoresis. The amino acid compositions of the acidic subunits were roughly similar to each other, but some remarkable differences were observed in the content of basic amino acids (lysine, histidine and arginine), serine and proline.     

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.     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.     

Determination of Sulfhydryl and Disulfide Contents of Soybean 11S Globulin and Their Change by lyophilization

Sulfhydryl (SH) and disulfide (SS) contents of soybean 11S globulin (var. Raiden) were determined by means of Ellman’s reagent. The amounts of surface SH, internal SH, and SS bonds of 11S globulin (not lyophilized) were 10.3, 4.6, and 17 mol/mol protein, respectively. On the other hand, when the 1 IS globulin was lyophilized, the surface and internal SH diminished to 5 and 3.6 mol/mol protein, respectively, and the SS bonds increased to 20.1 mol/mol protein. The result from sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested the possibility that the newly formed SS bonds probably existed in each constituent subunit of 1 IS globulin and/or between the intermediary subunits which exist by nature. Differences and similarities between our result and those obtained by various workers were also discussed.     

NADP-Dependent Phenylacetaldehyde Dehydrogenase for Degradation of Phenylethylamine in Arthrobacter globiformis

The role of endogenous gibberellin (GA) in the flowering of the short-day plant, Pharbitis nil, was investigated by using uniconazole, which is a specific inhibitor of GA biosynthesis. Both the endogenous GA level and flowering response decreased with increasing concentration of uniconazole applied via the roots. The strongest inhibition of flowering was observed when uniconazole was applied one day before a 15-h dark treatment. The inhibition by uniconazole was overcome by an application of GAs to the plumules, the order of effectiveness of the endogenous GAs in P. nil being GA₁ ≧GA₂₀>GA₁₉≧GA₄₄>GA₅₃»GA_H. This is the first report of the correlation between the endogenous GA level and flowering response in P. nil. It was found that endogenous GAs were required for the flowering of P. nil during or just after the dark period.     

Effects of diurnal and bidiurnal photoperiodic treatments on mitosis in Pharbitis

Abstract

Photoperiodic effect on mitotic activity of buds from dwarf Pharbitis has been analyzed. No significant differences in mitotic activity were found in plants grown under long days or diurnal (24 h) light break photoperiodic treatments. Differences in both mitotic activity and flowering were seen in plants subjected to diurnal short days, bidiurnal (48 h) short days, or bidiurnal short days with light breaks. An elevation of mitotic activity occurs in plants grown in bidiurnal photoperiodic treatments compared to diurnal treatments. The differences in mitotic activity of buds, both vegetative and floral, seem to indicate that both phytochrome and light effect on an endogenous rhythm influence meristematic activity. Also, the extended dark period of a bidiurnal short day enhances both mitosis and flowering.     

Isolation and Partial Characterization of Heat-denatured Products of Soybean 11S Globulin and Their Analysis by Electrophoresis

Heat denaturation of soybean 11S globulin was examined at 70° and 100°C in phosphate buffer (pH 7.6), at 0.01 and 0.5 ionic strength. Gel electrophoresis (Davis system) indicated that heat-denatured soybean 11S globulin contained two major components (buffer-soluble form). But they were not identified at 70°C-0.5 ionic strength. Gel filtration followed by SDS-gel electrophoresis showed that the major components were composed of a monomer and at least three of kinds of oligomers containing only an acidic subunit. Gel filtration of the precipitate formed at 100°C at 0.5 ionic strength gave two peaks. SDS-gel electrophoresis indicated that the first peak contained aggregates of highly polymerized subunits, and the second peak contained a monomer of basic subunit and seven kinds of oligomers with various proportions of basic subunits to an acidic subunit.     

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.     

The Relationship Between Flowering Induction and the Level of Adenosine Triphosphate in Pharbitis nil

A study has been made on the changes of ATP and protein content in cotyledons and apices of Pharbitis nil after flowering induction. Protein content of the cotyledons which have just got through the induction is 68% higher than that of the control, but the difference trends to disappear there after. The. difference of protein content between the induced and uninduced apices is not so obvious in the first three days after induction, but quite evident on the fourth day (30% higher in the induced apices) suggesting that there is some relationship between protein metabolism and flowering induction both in the cotyledons and in the apices. Just after the seedlings have been induced, ATP content of the cotyledons is getting much (134%) higher than that of the control and the tendency is retained towards the fourth day after induction. Generally ATP content in apices is one order of magnitude higher than that in cotyledons. Although ATP content in the apices is only slightly higher than that of the control soon after induction, it gains quite a lot in the second day until the fifth day the end of our experiment. In the third day after induction ATP level in the apices reaehs to the maximum (20.6×10-2 μmol/g, apices) which is 37% higher than that of the control. The results show that flowering induction is bound to be followed by increase of proteins and ATP both in apices and in cotyledoms. It also. shows both formation of the stimulus in induced cotyledons and evocation in the apices might be all concerned in expression of some genes and synthesis of new RNA and protein. According to the maximum peak of ATP in the apices and cotyledons appeared in 3rd to 4th day after induction, it seems that the inductive effect both in the cotyledons and apices might continue for some time under the following uninduced condition.     

Involvement of cyclic GMP in phytochrome-controlled flowering of Pharbitis nil

Light is one of the most important environmental factors influencing the induction of flowering in plants. Light is absorbed by specific photoreceptors – the phytochromes and cryptochromes system – which fulfil a sensory and a regulatory function in the process. The absorption of light by phytochromes initiates a cascade of related biochemical events in responsive cells, and subsequently changes plant growth and development.

Induction of flowering is controlled by several paths. One is triggered by the guanosine-3′:5′-cyclic monophosphate (cGMP) level. Thus, the aim of our study was to investigate the role of cGMP in phytochrome-controlled flowering.

It is best to conduct such research on short-day plants because the photoperiodic reactions of only these plants are totally unequivocal. The most commonly used plant is the model short-day plant Pharbitis nil.

The seedlings of P. nil were cultivated under special photoperiodic conditions: 72-h-long darkness, 24-h-long white light with low intensity and 24-h-long inductive night. Such light conditions cause a degradation of the light-labile phytochrome. Far red (FR) treatment before night causes inactivation of the remaining light-stable phytochrome. During the 24-h-long inductive darkness period, the total amount of cGMP in cotyledons underwent fluctuations, with maxima at the 4th, 8th and 14th hours. When plants were treated with FR before the long night, fluctuations were not observed. A red light pulse given after FR treatment could reverse the effect induced by FR, and the oscillation in the cGMP level was observed again.

Because the intracellular level of cGMP is controlled by the opposite action of guanylyl cyclases (GCs) and phosphodiesterases (PDEs), we first tested whether accumulation of the nucleotide in P. nil tissue may be changed after treatment with a GC stimulator or PDE inhibitor.

Accumulation of the nucleotide in P. nil cotyledons treated with a stimulator of cGMP synthesis (sodium nitroprusside) was markedly (approximately 80%) higher. It was highest in the presence of dipyridamole, whereas 3-isobutyl-1-methylxanthine did not significantly affect cGMP level.

These results show that the analysed compounds were able to penetrate the cotyledons’ tissue, and that they influenced enzyme activity and cGMP accumulation.

FR light applied at the end of the 24-h-long white light period inhibited flowering. Exogenous cGMP added on cotyledons could reverse the effect of FR, especially when the compound was applied in the first half of the long night. Flowering was also promoted by exogenous application of guanylyl cyclase activator and phosphodiesterase inhibitors, and in particular dipyridamole.

The results obtained suggest that an endogenous cGMP system could participate in the mechanism of a phytochrome-controlled flowering in P. nil.     

Capacity of cytochrome and alternative path in coupled and uncoupled root respiration of Pisum and Plantago

A critical duration of darkness must be exceeded for the photoperiodic induction of flowering in short-day plants. This requires detection of the light/dark transition at dusk and the coupling of this information to a time-measuring system.
Lowering the P_fr/P_tot, ratio photochemically at the end of the day did not accelerate the onset of dark timing in Pharbitis nil Choisy cv. Violet. Time-measurement was initiated when, with no change in spectral quality, the irradiance fell below a threshold value. Thus, if the light/dark transition at dusk is sensed by a reduction in P_fr, this reduction can be achieved as rapidly through thermal reactions as through photochemical ones. When given at hourly intervals during a 6-h extension of a 24-h main light period in white light, pulses of red light were as effective as continuous red light in delaying the onset of timing; pulses every 2 or 3 h were less effective. The effectiveness of intermittent red light indicates that phytochrome is the photoreceptor and the requirement for frequent exposures suggests that P_fr is lost rapidly in the dark. However, the red light pulses could not be reversed by far-red light, which argues against this hypothesis. An alternative explanation is that the perception of light as being continuous occurs only when "new" P_fr is regenerated sufficiently frequently.
The nature of the coupling of the dusk signal to the time-measuring system is discussed and it is suggested that the effect of each red light pulse is to delay the phase of the photoperiodic rhythm by 1–3 h.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.