RNA synthesis in embryo axes of germinating pea seeds

RNA synthesis during germination was investigated by labelingpea embryo axes or seedling roots with radioactive uridine oradenosine. The results indicated that all RNA species of pre-rRNAs(ribosomal precursor RNAs), rRNAs, heterodisperse-type RNA and4–5S low molecular weight RNA were synthesized from the6th to 64th hour of the period examined. At the very early stageof germination, some conspicuous labeling of the heterodisperse-typeRNA was observed after pulse-labeling. There was no great differencein the labeling patterns of various RNA species with regardto other later stages. When embryo axes were labeled for 1 hrwith ³H-adenosine from the 16th hour, about 25% of the labeledwhole cell RNA was retained on the membrane filter. The ratioof labeled poly(A)-containing RNA, however, decreased as germinationproceeded. The poly (A)-containing RNA sedimented heterodisperselywith a mean value of about 20S in a sucrose density gradient;this size-distribution did not vary throughout germination. (Received January 16, 1979; )     

Regulation of diamine oxidase activity in germinating pea seeds

Diamine oxidase present in the cotyledons of germinating pea seeds is induced by putrescine, spermidine and ornithine. Auxins inhibit enzyme synthesis in cotyledons only in the presence of embryo. Cycloheximide inhibits the synthesis of the cotyledon enzyme but has no effect on the embryo enzyme. 5-Fluorouracil inhibits the synthesis of both cotyledon and embryo enzymes.     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

Gibberellin-like activity in cotyledons and embryonic axes during pea seed germination

Endogenous gibberellin-like activity was determined in dry pea seeds (Pisum sativum cv. Bördi), in cotyledons and axes of germinating pea seeds and also in excised cotyledons and axes. During the first two days of pea seed germination, neither the embryonic axes nor the cotyledons show a mutual influence on gibberellin activity, but this appears after 72–96 h of germination. The gibberellin-like activity m cotyledons and axes of germinating seeds increased during the same period, but it decreased in isolated axes and excised cotyledons.     

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.     

RNA synthesis in germinating embryonic axes of soybean and wheat

The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.     

Analysis of RNase isozymes in germinating pea cotyledons by polyacrylamide-gel electrophoresis

In germinating pea cotyledons, at least three different RNase-isozymescan be detected by column chromatography (6). Polyacrylamidegel electrophoresis analysis in the present study revealed thepresence of five bands, with at least two being associated withheavily stained protein bands in reference gels. Assays carriedout in the presence of the nuclease inhibitor diethylpyrocarbonate(DEPC) showed that these bands were most likely artificial andnot ribonucleases (RNases). The nature of a fast-running componentwith the staining behavior of RNase, which neither can be inhibitedby DEPC nor is associated with a heavily stained protein band,is discussed. It may be a small molecule in the range of 20,000daltons. This band was also present in the cotyledons of dryseeds. When transfer RNA was used as a substrate in the assay,the band with the lowest mobility degraded this substrate veryrapidly. (Received January 23, 1980; )     

Fructose 2,6-bisphosphate and the regulation of pyrophosphate-dependent phosphofructokinase activity in germinating pea seeds

The activity of pyrophosphate: d-fructose-6-phosphate-1-phosphotransferase (EC 2.7.1.90, PPi-PFK) in cotyledons and sprouts of germinating pea seeds (Pisum sativum cv Alaska or Green Arrow) increases rapidly during the first 2 to 3 days after imbibition and then declines to a lower activity. The reaction toward fructose 1,6-bisphosphate formation is activated greatly by fructose 2,6-bisphosphate (fru 2,6-P₂); however, the sensitivity of the enzyme's activity to fru 2,6-P₂ activation changes during germination.     

Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds

Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination.     

Starch phosphorylase enzymes in developing and germinating pea seeds

Phosphorylase has been fractionated during development and germination of seeds of smooth and wrinkled-seeded peas. The total phosphorylase levels have been compared. In addition, a number of other pea tissues and other legumes have been examined. Some kinetic properties of the two enzymes present have been measured. Both enzymes have been further purified by affinity chromatography on Sepharose 4B-starch columns and by sequential gel filtration in the absence and presence of amylopectin. The MW and sub-unit structures of the two enzymes have been examined and their possible roles discussed.     

Xyloglucan oligosaccharide alpha-L-fucosidase activity from growing pea stems and germinating nasturtium seeds.

[14C]Fucose-labelled xyloglucan (XG) was synthesized from tamarind seed XG by incubating it with GDP-[14C]fucose plus solubilized pea fucosyltransferase, and [14C]fucose-labelled XG nonasaccharide was prepared from the parent hemicellulose by partial hydrolysis with fungal cellulase. alpha-L-Fucosidase activity was readily detected in crude enzyme extracts of growing regions of etiolated pea stems (Pisum sativum) and in cotyledons of germinating nasturtium seedlings (Tropaeolum majus) using the fucosylated XG-nonasaccharide as substrate. Both enzymes showed little activity against intact fucosylated XG and they were totally inactive against p-nitrophenyl-alpha-L-fucoside. Auxin treatment of pea stems, which greatly increased the activity of endo-1,4-beta-glucanases that hydrolyse XG in apical growing regions, failed to result in a similar increase in XG-nonasaccharide alpha-fucosidase activity. However, germination of nasturtium seed, which resulted in a large increase in endo-1,4-beta-glucanase (XG-ase) activity in the cotyledons, was accompanied by comparable increases in XG-alpha-fucosidase activity.     

Arginine catabolism in the cotyledons of developing and germinating pea seeds

Arginine is the predominant free amino acid in the cotyledons of developing seeds of Pisum sativum L. cv Marzia. Breakdown of arginine was measured by injecting l-[guanido-¹⁴C]arginine into detached cotyledons. Cotyledons of developing seeds showed a low rate of ¹⁴CO₂ evolution whereas a much higher rate of ¹⁴CO₂ evolution was measured from cotyledons of seeds 4 days after the onset of germination. The activities of the catabolic enzymes arginase, urease, and ornithine aminotransferase were measured throughout development and germination. Arginase and ornithine aminotransferase were present at an early stage of development. Urease activity appeared later as the seeds started to desiccate. During germination, all three enzymes were present. The different course of activity of these enzymes indicates that they are controlled separately.     

Biosynthesis of willardiine and isowillardiine in germinating pea seeds and seedlings

The synthesis of the pyrimidinyl amino acids willardiine and isowillardiine was studied in vivo and in vitro. Uracil derivatives stimulate the biosynthesis of both compounds; the free base is the most effective. Significant incorporation of [2-(14)C]uracil and [6-(14)C]orotate into willardiine and isowillardiine was found. Incorporation of [6-(14)C]orotate was substantially decreased in the presence of uracil, and to a lesser extent by uridine and UMP. [3-(14)C]Serine was incorporated into the alanine side chain of the two uracilylalanines but not into the ring. The effect of a number of uracil analogues and inhibitors of pyrimidine metabolism was examined. Some were shown to stimulate the biosynthesis; the most noticeable effects were obtained with 6-azauracil and 2-thiouracil. Attempts to obtain extracts capable of synthesizing the uracilylalanines from uracil and serine were unsuccessful, but weak activity was observed when serine was replaced by O-acetylserine.     

Ethanolamine incorporation into membranes of pea embryonic axes during germination

Changes in the ethanolamine pool of the embryonic axes of pea seeds exposed to different temperatures during imbibition and germination were followed. The ethanolamine pool decreased except during imbibition at 25°. Label from ethanolamine was incorporated almost entirely into phosphatidylethanolamine with incorporation into phosphatidylcholine being observed only after imbibition and germination at 25°. The incorporation of ethanolamine was apparently less sensitive to temperature than that of choline and glycerol, previously reported. Preliminary results also show an effect of the imbibition temperature on some of the membrane proteins, but most did not seem to be affected.     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Adenylate metabolism of embryonic axes from deteriorated soybean seeds

RNA and protein syntheses in axes excised from dry soybean (Glycine max L.) seeds at different levels of deterioration were assayed. Low rates of protein synthesis in slightly deteriorated seeds were not due to losses in ribosomal or soluble fraction activities. However, the lowered rates of RNA and protein syntheses of deteriorated seeds were associated with reduced ATP content of the tissues. Adenine and adenosine conversions to ATP were reduced in deteriorated axes, and these reductions were reflected in reduced incorporation of these compounds into RNA.     

RNA polymerase activity in germinating onion seeds

Frozen sections of endosperm cut from dry unimbibed onion seed were immersed in an aqueous solution of tritium labelled triphosphate; nucleolar RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyltransferase E.C. 2.7.7.6) activity was detected by autoradiography after soaking for 10–15 min in the solution of the radioactive nucleotide. Throughout germination, activity appears to be mainly confined to the nucleolus with chromatin incorporation being very low or non-existent. In the embryo, in contrast to the endosperm, chromatin activity is initiated after 1 hr presoaking, while the nucleolus displays a lag of several hours. No incorporation could be detected in vivo before 18 hr.