Changes in the phospholipid molecular species composition of soybean hypocotyl and cotyledon after dedifferentiation

The effect of dedifferentiation on the molecular species composition of soybean phospholipids was studied by using hypocotyl, cotyledon and the suspension culture cells established from those organs. Three major phospholipids (phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) and phosphatidylmonomethylethanolamine were composed of twelve molecular species. Major species were 1-palmitoyl-2-linoleoyl, 1-obeoyl-2-linoleoyl, 1-palmitoyl-2-linolenoyl and 1-linoleoyl-2-linoleoyl species. Different proportions of the molecular species were found among the three major phospholipids, but phosphatidylmonomethylethanolamine was composed of the same proportions of the molecular species as those of phosphatidylethanolamine. After dedifferentiation, the 1-palmitoyl-2-linoleoyl species increased in the cell established from hypocotyl. In the cells established from cotyledon, the 1-palmitoyl-2-linolenoyl species increased dramatically. In both cells, the 1-palmitoyl-2-linolenoyl species increased in response to increase in the 2,4-dichlorophenoxyacetic acid concentrations and the progress of cell growth.     

Spiral feedback oscillations of growing hypocotyl with radicle inPisum sativum L.

Cinematographic records of longitudinal growth showed that hypocotyl with radicle inPisum sativum L. undergoes spiral oscillations during growth. This phenomenon can be characterized by the following time-space limits:

1. (1)
   Curvature of the hypocotyl with radicle takes place always in the zone of most rapid elongation (Fig. 2, 8).     
   
   

Dehydration of germinating perennial ryegrass seeds can alter rate of subsequent radicle emergence

Perennial ryegrass (Lolium perenne L.) seeds (caryopses) germinateat or near the soil surface, where water potential can fluctuatewidely. This study examined germination of ‘Del Ray’perennial ryegrass seeds when imbibition was interrupted bydehydration prior to radicle emergence. Seeds were hydratedfor 0 to 40 h (26C), dehydrated at atmospheric water potentialsof –4, –40, –100 and/or –150 MPa for4–168 h, then rehydrated. Germination (radicle elongation 1 mm), seedling growth, solute leakage, and endogenous abscisicacid (ABA) levels were measured. Treatment differences in finalgermination percentage, seedling growth, and solute leakagewere generally not significant. However, the onset of radicleemergence was delayed and the rate of germination slowed whendehydration at –150 MPa was initiated after 36 or 40 hhydration. Slowed germination rates were not observed when dehydrationwas initiated before 36 h, when dehydration occurred at –4MPa, or when dehydration at –150 MPa was preceded by dehydrationat –4 MPa for 24 h. Exogenous abscisic acid (ABA) concentrationsabove 10^–6 M inhibited germination. However, endogenouswhole seed ABA levels declined during imbibition due to leaching,and did not increase during dehydration treatments that delayedgermination. These results illustrate that rate of late-occurringdehydration treatments is critical in determining subsequentgermination response. We propose that seed response to late-occurringdehydration may be of ecological significance in timing radicleemergence to coincide with adequate soil moisture for seedlingestablishment. Key words: Abscisic acid, seed germination, timing     

Isolation and culture of soybean hypocotyl protoplasts

Summary Protoplasts were isolated seedling hypocotyls of soybean (Glycine max), and cultured in both liquid and agarose-solidified, modified K8P medium. Nuclear staining revealed that only 2% of protoplasts lacked a nucleus, 93% contained a single nucleus, and 5% contained more than one. Maximum protoplast yields and subsequent division frequencies, in liquid medium, were obtained from 5 days-old seedlings. Maximum division frequencies (54%) were obtained from hypocotyl protoplasts plated at a density of 5×10⁴ ml⁻¹. Using different osmolality reduction régimes for liquid cultures, hypocotyl protoplasts developed into green, nodular callus, similar to that which has previously given rise to shoot buds in perennialGlycine species. This tissue, however, did not produce shoot buds in soybean. N. H. was supported by a SERC CASE studentship and a postdoctoral fellowship from Shell Research Ltd., Sittingbourne, Kent, UK.     

Organogenic regeneration of soybean from hypocotyl explants

Summary Thirteen soybean genotypes representing maturity groups IV−VI were compared for organogenic responses on three media cultured under two lighting conditions with hypocotyl sections excised from 7-d-old seedlings. All soybean lines responsed by producing adventitious shoots on the acropetal end of the hypocotyl explants, confirming genotype-independence of shoot initiation. Media containing 6-benzyladenine (BA; 5.0–10 μM) induced the greatest numbers of shoots. Histological studies confirmed the adventitious nature of arising shoots by indicative formation of meristematic zones and shoot primordia from parenchymatous tissues of central pith and plumular trace regions of the hypocotyl. Incompletely excised cotyledonary buds also contributed to shoot initiation. Degrees of responses were media-dependent and varied with regard to genotype. Centennial, Epps, and Lyon gave the greatest individual responses. Between cultivars (across all treatments), the regeneration potential (percentage of explants producing meristem-like structures or shoot primordia) 4 wk after initiation ranged from 47 to 75%. Four wk later, regenerative ability (number of shoots produced per responding explant) and regeneration efficiency (number of shoots produced per explant plated) yielded 1.4–7.1 and 1.0–5.0 shoots, respectively. The optimized protocol included initiation on a medium containing 5.0 μM BA for 4 wk, then transfer onto a shoot elongation medium (0.36 μM BA) for 4 wk. For 11 genotypes tested, 66–100% of excised shoots produced roots after 4 wk on media containing 12.5–29.2 μM indole-3-butyric acid. Of 109 regenerants transplanted to soil, 94% survived and no sterility has been observed on those mature enough to flower.     

Activity of soybean lipoxygenase in the absence of lipid hydroperoxide

Soybean lipoxygenase was assayed under conditions such that the concentration of the enzyme was in excess of the concentration of the substrate, arachidonic acid. Under these conditions, the concentration of lipid hydroperoxides present as contaminants in the substrate was negligible relative to the enzyme concentration, and the concentration of lipid hydroperoxide product could be determined accurately. The ferric form of the enzyme was observed to be fully active and to catalyze the oxidation of arachidonic acid at a near-diffusion-controlled rate, 1.4 X 10(7) M-1 s-1 at 0 degree C, at concentrations of lipid hydroperoxides as low as 5% of the enzyme concentration. From this, it can be concluded that the higher oxidation states that would be accessible by oxidation of Fe(III) by hydroperoxide are not required for catalysis by soybean lipoxygenase. Surprisingly, the activation of the ferrous form of the enzyme was also observed at insignificantly low lipid hydroperoxide concentrations. This activation presumably involves oxidation of the ferrous to the ferric form of the enzyme and must be more facile than has hitherto been reported. This result may rationalize previous reports that the ferrous and the ferric forms of the enzyme are both active.     

Induction by gamma-radiation of DNA synthesis in radicle cells of germinating seeds of Pisum sativum l.

In radicle meristem cells of germinating seeds of the pea (Pisum sativum L) before the onset of replicative synthesis of DNA, irradiation with 2-3 krad of gamma-rays induced the incorporation of 3H-thymidine (3H-TdR). Maximum isotope incorporation was noted during the first 2 hours after irradiation. Higher doses of radiation suppressed 3H-TdR incorporation. It was not seen after gamma-irradiation of air-dried seeds, nor after fast-neutron irradiation. The replication inhibitors hydroxyurea and 5-aminouracil had no effect on the gamma-induced incorporation of 3H-TdR, Whereas caffeine and acriflavine inhibited it to some extent. It is suggested that the gamma-radiation-induced incorporation of 3H-TdR in meristem cells during the pre-replicative period may be connected with repair phenomena.     

Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes

Characterization of sugar content and enzyme activity in germinating soybean (Glycine max L. Merrell) seeds led to the discovery of sorbitol accumulating in the axes during germination. The identity of sorbitol was confirmed by relative retention times on high-performance liquid chromatography and gas liquid chromatography and by mass spectra identical with authentic sorbitol. Accumulation of sorbitol in the axes started on day 1 of germination as sucrose decreased and glucose and fructose increased. Sucrose also decreased in the cotyledons, but there was no accumulation of sorbitol, glucose, or fructose. Accumulation of sorbitol and hexoses was highly correlated with increased invertase activity in the axes, but not with sucrose synthase and sucrose phosphate synthase activities. Sucrose synthase activity was relatively high in the axes, whereas the activity of sucrose phosphate synthase was relatively high in the cotyledons. Ketose reductase and aldose reductase were detected in germinating soybean axes, but not in cotyledons. Fructokinase and glucokinase were present in both axes and cotyledons. The data suggest a sorbitol pathway functioning in germinating soybean axes, which allows for the interconversion of glucose and fructose with sorbitol as an intermediate.     

Characterization of post-flooding recovery-responsive enzymes in soybean root and hypocotyl

Soybean exhibits markedly reduced growth and yields under flooding stress. To determine the functional roles of four soybean proteins in post-flooding recovery, the organ/stress specificity and time-dependency of their enzymatic activities were analyzed. Peroxidase activity decreased in root and hypocotyl exposed to flooding and cold stresses, but increased during the post-stress recovery period. In contrast, its activity increased in both root and hypocotyl under drought stress. Acid phosphatase activity was suppressed in root treated with flooding and cold stresses, and slightly increased during the recovery period; however, the opposite profile was observed in hypocotyl. In response to drought stress, it did not change in root, but was decreased in hypocotyl. Beta-ketoacyl reductase activity did not change in root under flooding conditions, but was decreased in hypocotyl, although the activity increased slightly during the recovery period. In addition, it was decreased in both organs under drought and cold stresses, but again increased during the recovery period. Nucleotidylyl transferase activity was increased in root under flooding and drought stresses, but was decreased in hypocotyl. It was decreased in response to cold stress, but exhibited a slight increase during the recovery period. Furthermore, the treatment with jasmonate and salicylate suppressed the activities of peroxidase and acid phosphatase in root and hypocotyl under flooding stress; however, the activity of acid phosphatase increased during the recovery period. Nucleotidylyl transferase activity in root was also elevated by treatment with jasmonate, but gradually decreased during the recovery period. These results suggest that jasmonate-induced changes in nucleotidylyl transferase activity may facilitate soybean root recovery after flooding stress.     

Inhibition of soybean lipoxygenase 1 by N-alkylhydroxylamines

Micromolar concentrations of N-octylhydroxylamine dramatically increase the induction period in the conversion of linoleic acid to 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) catalyzed by soybean lipoxygenase 1. The induction period produced by N-octylhydroxylamine is abolished by 13-HPOD but not by the corresponding hydroxy acid. Addition of a catalytic amount of lipoxygenase to a mixture of 13-HPOD and N-octylhydroxylamine results in consumption of approximately 1 mumol of 13-HPOD/mumol of N-octylhydroxylamine present. These results can be explained by a model in which 13-HPOD oxidizes the enzyme from an inactive ferrous form to an active ferric form, as proposed by previous workers, and N-octylhydroxylamine reduces the enzyme back to the ferrous form. Consistent with this model, the ESR signal at g = 6.1 characteristic of ferric lipoxygenase is rapidly abolished by N-octylhydroxylamine and can be regenerated by 13-HPOD. These results provide additional support for earlier proposals that ferric lipoxygenase is the catalytically active form and also establish a novel method of inhibiting enzymes in this class. The octyl group of N-octylhydroxylamine appears to contribute to binding near the iron, since hydroxylamine and N-methylhydroxylamine do not extend the induction period. In the n-RNHOH series, activity passes through an optimum at R = decyl.     

Primary structure of soybean lipoxygenase L-2

The nucleotide sequence of soybean lipoxygenase-2 cDNA has been determined, and the complete amino acid sequence of the enzyme has been deduced. Limited direct amino acid sequence data for lipoxygenase-2 protein support this assignment and exclude mRNA representing lipoxygenase-1 and -3. Lipoxygenase 2 has a molecular weight of 97,036 and contains 865 amino acid residues, in contrast to the isozymes, lipoxygenase-1 and -3, which are known to contain 838 and 859 amino acid residues, respectively. Despite significant differences in behavior between these three isozymes, the amino acid sequences of lipoxygenase-1 and -3 are 81 and 74% identical to lipoxygenase-2, respectively. A region of 40 amino acid residues containing a cluster of six histidines and two tyrosines, which is highly conserved in all three isozymes, is discussed as a possible iron-binding region.     

Purine nucleotide metabolism of germinating soybean embryonic axes

Isolated soybean (Glycine max L. cv. Kent) embyronic axes metabolized [¹⁴C]glycine to ATP within the 1 hour of imbibition. Radioactivity was not detected in GTP until the 3rd hour. Throughout most of the first 24 hours of germination about 10 to 26 times as much label from [¹⁴C]glycine appears in ATP as GTP. About five times as much [¹⁴C]hypoxanthine and [¹⁴C]inosine was converted into GTP as into ATP in embryonic axes. Two independent pools of IMP appear to be used in purine nucleotide synthesis of soybean axes.