Characterization of Solute Efflux from Dehydration Injured Soybean (Glycine max L. Merr) Seeds

Soybean (Glycine max L. Merr) seeds lose their tolerance of dehydration between 6 and 36 hours of imbibition. Soybean axes and cotyledons were excised 6 hours (tolerant of dehydration) and 36 hours (susceptible) after commencing imbibition and subsequently dehydrated to 10% moisture. Kinetics of the efflux of potassium, phosphate, amino acid, sugar, protein, and total electrolytes were compared in the four treatments during rehydration. Only slight differences were observed in the kinetics of solute efflux between the two cotyledon treatments dehydrated at 6 and 36 hours suggesting that the cotyledons may retain their tolerance of dehydration at this stage of germination. Several symptoms of injury were observed in the axes dehydrated at 36 hours. An increase in the initial leakage of solutes during rehydration, as quantified by the y-intercept of the linear regression line for solute efflux between 2 and 8 hours suggests an increased incidence of cell rupture. An increase in the rate of solute efflux (slope of regression line between 2 and 8 hours) from fully rehydrated axes was observed in comparison to axes dehydrated at 6 hours. The Arrhenius activation energy for potassium, phosphate, and amino acid efflux decreased and for protein remained unchanged. Both observations indicate an increase in membrane permeability in dehydration-injured tissue. Increasing the H⁺ concentration of the external solution increased K⁺ efflux from both control and dehydrated/rehydrated samples, increased sugar efflux from axes at 6 hours imbibition but decreased sugar efflux from axes at 36 hours imbibition, indicating changes in membrane properties during germination. The dehydration treatment did not alter the pattern of the pH response of axes dehydrated at 6 or 36 hours but did increase the quantity of potassium and sugar efflux from dehydration injured axes. These results are interpreted as indicating that dehydration of soybean axes at 36 hours of imbibition increased both the incidence of cell rupture during rehydration and altered membrane permeability of the rehydrated tissue.     

Activation of acid growth in germinating horse chestnut seeds

The validity of the acid-growth hypothesis is proved for the case of cell elongation initiation in germinating seeds of horse chestnut (Aesculus hippocastanum L.), the embryo axes of which are known to extend during the first stages of germination only by cell elongation. During seed imbibition, H⁺-ion excretion was firstly low; it increased several times prior to radicle emergence and was maintained at a high level during growth initiation and further cell elongation. Cell wall acidification and radicle emergence were enhanced in the presence of 0.02 mM fusicoccin, thus indicating the involvement of the plasma membrane H⁺-ATPase in the execution of acid growth. The presence of this enzyme and its activator (14-3-3 protein) in microsomal fractions obtained from radicles and hypocotyls of the embryo axes during and after initiation of cell elongation was demonstrated immunochemically. It is supposed that the initiation of cell elongation at early germination occurs via the activation of the plasma membrane H⁺-ATPase and results in the acidification of cell walls, leading to their higher extensibility, in accordance with the hypothesis of acid growth.     

High Energy Charge as a Requirement for Axis Elongation in Response to Gibberellic Acid and Kinetin during Stratification of Acer saccharum Seeds

The growth potential of embryonic axes of Acer saccharum Marsh. increased during moist storage at 5 C but not at 20 C. During the period of increasing growth potential, the oxygen consumption of the axes remained constant. It was possible to distinguish three phases of the stratification-germination process at 5 C with respect to response of the axis to gibberellic acid and kinetin. From 0 to 10 days the growth regulators had no effect on elongation; from 10 to 60 days axis elongation was stimulated; and between day 60 and day 75, when germination had begun, the growth substances were inhibitory. The adenylate energy charge remained low (0.15) in axes of dry dormant seeds but increased to 0.78 following imbibition of water and 10 days of moist storage at 5 C. This phenomenon was not specifically related to low temperature stratification, since a rapid increase in the energy charge of the axes also occurred following imbibition and moist storage at 20 C. The excised axes would elongate in response to the growth substances only when a high energy charge (approximately 0.8) was maintained.     

On the Role of Stored mRNA in Protein Synthesis in Embryonic Axes of Germinating Vigna unguiculata Seeds

Polyadenylated (poly A⁺) RNAs were prepared from both dry and incubated embryonic axes of Vigna unguiculata seeds and were translated by a wheat germ translation system. Analysis with gel electrophoresis and fluorography showed that translation products of poly A⁺ RNA from dry embryonic axes were nearly the same as those from 2-hour incubated axes but somewhat different from those of 4- to 24-hour incubated axes, and that translation products remained almost unchanged between the 4- and 24-hour stages of postimbibition. The results indicate the possibility that the stored mRNA (poly A⁺ RNA from dry embryonic axes) directs the protein synthesis required for early stages of germination. This is supported by comparison of the in vitro translation products of poly A⁺ RNAs with those of polysomal RNAs. Experiments with α-amanitin, a specific inhibitor of RNA polymerase II (J. Jendrisak 1980 J Biol Chem 255: 8529-8533), suggested that the synthesis of some of the stored mRNA species is resumed as early as 4 hours after the onset of imbibition.     

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.     

Dehydration Injury in Germinating Soybean (Glycine max L. Merr.) Seeds

The sensitivity of soybean (Glycine max L. Merr. cv Maple Arrow) seeds to dehydration changed during germination. Seeds were tolerant of dehydration to 10% moisture if dried at 6 hours of imbibition, but were susceptible to dehydration injury if dried at 36 hours of imbibition. Dehydration injury appeared as loss of germination, slower growth rates of isolated axes, hypocotyl and root curling, and altered membrane permeability. Increased electrolyte leakage due to dehydration treatment was observed only from isolated axes but not from cotyledons, suggesting that cotyledons are more tolerant of dehydration. The transition from a dehydration-tolerant to a dehydration-susceptible state coincided with radicle elongation. However, the prevention of cell elongation by osmotic treatment in polyethylene glycol (−6 bars) or imbibition in 20 micrograms per milliliter cycloheximide did not prevent the loss of dehydration tolerance suggesting that neither cell elongation nor cytoplasmic protein synthesis was responsible for the change in sensitivity of soybean seeds to dehydration. Furthermore, the rate of dehydration or rate of rehydration did not alter the response to the dehydration stress.     

Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ⁵- and Δ⁶-elongases of Marchantia polymorpha as well as the Δ⁶-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ⁹-, Δ⁶-C₁₈, and Δ⁵-C₂₀ LC-PUFAs, whereas only Δ⁹- and Δ⁶-C₁₈ LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C₂₀ fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C₂₀ substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.     

Differential response of PCNA and Cdk-A proteins and associated kinase activities to benzyladenine and abscisic acid during maize seed germination

The proliferating cell nuclear antigen (PCNA) is a protein factor required for processive DNA synthesis that is associated with G(1) cell cycle proteins. It has been demonstrated previously that, in germinating maize (Zea mays) embryonic axes, PCNA forms protein complexes with two Cdk-A proteins (32 and 36 kDa) and with a putative D-type cyclin. These complexes exhibit protein kinase activity on histone H1 and on the maize homologue of the pRB (retinoblastoma) protein. Flow cytometry has been used to study the influence of the phytohormones benzyladenine (BA) and abscisic acid (ABA) on cell cycle advancement during maize germination. It was found that, while BA accelerates the passage of cells from G(1) to G(2), ABA delays cell cycle events so that most cells seem to remain in G(1). The amounts of PCNA and Cdk-A proteins also vary according to the hormone treatment. In embryonic axes, PCNA increases rapidly during early germination in BA, compared with a gradual increase in water, while ABA treatment had only a marginal effect. However, of the two Cdk-A proteins, the 32 kDa protein is strongly reduced after 15 h of imbibition in water while this occurs later when axes are imbibed in BA or ABA. The PCNA-associated protein kinase activity in the BA and ABA treatments falls after 3 h of imbibition compared with activity in the control; however, while kinase activity in the BA treatment continues to decline during imbibition, it remains relatively constant until 24 h of imbibition in the ABA treatment. By contrast, a p13(Suc1)-associated Cdk-A kinase is activated after 15 h of imbibition under all treatments, particularly in ABA. These results suggest that, in maize, ABA delays the germination process by affecting cell cycle advancement, stopping cells mostly in a G(1) state.     

The Fate of l-Phenylalanine Fed to Germinating Pea Seeds, Pisum sativum (L.) var. Alaska, during Imbibition

Radioactive l-phenylalanine-l-¹⁴C or -U-¹⁴C was fed to pea seeds during imbibition. More than 95% was imbibed. Less than 1% of the radioactivity was respired as CO₂. Of the radioactivity taken into the embryos, 80% was still in the cotyledons by 3 days. About half of this was unchanged phenylalanine: 5% free, 10 to 20% in soluble proteins, 1 to 6% in cell wall proteins, and 14% released by mild acid hydrolysis. No other radioactive amino acid was found. About 0.3% of the radioactivity was identified as free caffeic, ferulic, and coumaric acids or their glycosides, and a further 5% was released by mild acid hydrolysis into a phenolic acid fraction. About half of the radioactivity in the cotyledons was lost in the fractionation procedures.     

Pyrimidine metabolism in cotyledons of germinating alaska peas

Cotyledons from Pisum sativum L. cv. Alaska seeds were excised 12, 36, 108, 132, and 156 hours after imbibition in aerated distilled water. They were then incubated under aseptic conditions for 6 hours in solutions containing either uridine-2-¹⁴C or orotic acid-6-¹⁴C. Uridine was more extensively degraded to ¹⁴CO₂ at all germination stages than was orotate, and these rates remained essentially constant at each stage. Incorporation of each compound into RNA increased about 2-fold from the 12th to the 156th hour, although the total RNA present decreased slightly over this interval. Paper chromatography of soluble labeled metabolites produced from orotate showed that the capacity to metabolize this pyrimidine increased markedly as germination progressed. Radioactivity in uridine-5′-P, uridine diphosphate-hexoses, and uridine diphosphate increased most, while smaller or less consistent increases in uridine, uracil, uridine triphosphate, and an unidentified UDPX compound were also observed. The data suggest that orotate metabolism was initially limited by orotidine-5′-phosphate pyrophosphorylase or by 5-phosphoribosyl-1-pyrophosphate. Incorporation of uridine into RNA appeared to be limited at the earliest germination periods by conversion of uridine-5′-P to uridine diphosphate. Thus, during the 1st week of germination the orotic acid pathway and a salvage pathway converting uridine into RNA become activated.     

Embryonic protein composition and synthesis during imbibition of white lupin seeds: effect of low temperatures

White lupin seed imbibition under low temperature conditions (8 °C) and their subsequent effects on embryonic protein composition and synthesis were investigated. The response to low temperatures was accompanied by changes in polypeptide composition and synthesis. The embryonic axes labelled in vivo with (³⁵S)-methionine retained their capacity to synthesize proteins during imbibition at 8 °C. The synthesis of some polypeptides was increased during low temperature treatment as compared to that at 25 °C. These cold-induced polypeptides were essentially detected in a molecular mass range from 15 to 35 kDa and a pI range from 6.3 to 8.7.     

Effect of calmodulin antagonists on auxin-induced elongation

Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of α-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of ³H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation.     

In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle

Fat body, ovaries without their surrounding connective tissue, and ovarian connective tissue of the ovoviviparous cockroach Leucophaea maderae were incubated in vitro. Incorporation of ¹⁴C-labelled amino acids into proteins by all three tissues was investigated quantitatively and qualitatively during oöcyte maturation (corpora allata active), immediately after ovulation, and during the gestation period (corpora allata inactive). Proteins in the tissues and in the incubation media were processed separately. The qualitative analysis involving disk electrophoretic separation of Ringer soluble tissue proteins or of proteins in the incubation media and subsequent autoradiography of the dried gels showed that all three tissues synthesize the same 26 proteins in all stages of the sexual cycle. An additional fraction is produced by the ovarian connective tissue only. All three tissues synthesize proteins at a higher rate during oöcyte maturation than during gestation.     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

LABELING WITH 14C AMINO ACIDS OF ALBUMIN-LIKE PROTEIN BY RAT LIVER RIBONUCLEOPROTEIN PARTICLES

Ribonucleoprotein particles were prepared by treatment of rat liver microsomes with detergents and high concentrations of KCl. They were active in incorporating ¹⁴C amino acids into protein when incubated with cell sap together with ATP, GTP, and a system to regenerate the triphosphates. The albumin of the incubation mixture, soluble at 105,000 g, and that of the fraction released by ultrasonication of the particles were studied by immunoelectrophoresis in agar gel. When the ribonucleoprotein particles were incubated with cell sap the immunological precipitation lines formed with antiserum to rat serum albumin were highly radioactive as tested by autoradiography. After zone electrophoresis on cellulose acetate, two immunologically reactive albumins were obtained which differed in their electrophoretic mobility from rat serum albumin. Labeled albumin, when purified on DEAE-cellulose columns, retained its radioactivity as tested by autoradiography following immunoelectrophoresis. On cellulose acetate this purified albumin showed an electrophoretic mobility higher than that of rat serum albumin.     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Sites of synthesis of plasma proteins in the foetal rat

1. The foetal rat of 16 or more days incorporates ¹⁴C-labelled amino acids into all the demonstrable plasma protein fractions in vivo. 2. Slices of foetal rat liver incubated in vitro incorporate ¹⁴C-labelled amino acids into the main plasma protein fractions, including the foetal-specific `post-albumin'. 3. Slices of placenta are unable to incorporate ¹⁴C-labelled amino acids into plasma proteins in vitro. 4. Liver slices from maternal rats incubated in vitro incorporate ¹⁴C-labelled amino acids into plasma proteins. The presence of post-albumin cannot be demonstrated after incubation. 5. Liver slices from foetal rats, but not from adult rats, contain demonstrable amounts of haemoglobin into which ¹⁴C-labelled amino acids are incorporated.