The distribution of phospholipase D in developing and mature plants

1. The distribution of phospholipase D (phosphatidylcholine phosphatido-hydrolase, EC 3.1.4.4) was examined in the tissues of a number of plants and seeds. 2. The highest activities were found in various swollen storage tissues of certain plants: cabbage, central stalk; cauliflower, flower; celery, swollen leaf stalk; Kohl rabi, swollen stem; carrot, root; pea and marrow, seed. 3. Appreciable activity was retained in pea seeds for at least 1 year after drying. After germination and growth in the dark the total activity present in the cotyledons and also in the whole seedling decreased. 4. In the growing pea seedling (7 days old), about 3% of the total activity was in the plumule, 9% in the root and the remainder in the cotyledons. However, the activity in the root on a dry-weight basis was higher than that in the cotyledons. In both the root and the plumule the activity on a wet- or a dry-weight basis was highest in the growing tip. 5. The activity per dry weight in the roots and aerial parts of pea plants declined to low values as growth continued, but roots struck from cuttings of mature plants showed the same high activity as found in roots from young seedlings with cotyledons attached. 6. The total phospholipids present in the cotyledons of pea seeds were depleted on germination and growth. Of the individual phospholipids, phosphatidylcholine and phosphatidylethanolamine showed the same loss in 11 days as the whole phospholipid fraction, whereas phosphatidylinositol was decreased to a greater extent and cardiolipin and phosphatidylserine were not decreased. There was no increase of phosphatidic acid, as might have been expected if the phospholipids had disappeared through phospholipase D hydrolysis. 7. It is concluded that phospholipase D in plant storage tissues and seeds may be related to the rapid growth involved in their formation rather than being necessary for the utilization of their food reserve substances.     

Changes in the activity of poly(adenosine diphosphate-ribose) polymerase during germination of pea

Poly(adenosine diphosphate-ribose) polymerase activity has been detected in nuclei from pea seedlings. The reaction is a specific polymerization of the adenosine diphosphate-ribose moiety of NAD, giving an average chain length of 2.4 residues. The enzyme shows marked changes in activity during germination: it exhibits a clear peak between 24 and 36 hr of germination, after which the activity declines to the level observed in ungerminated seeds.     

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.     

Proteolytic and trypsin inhibitory activities in extracts of germinating Pisum sativum seeds

A rise in azoglobulytic activity and a fall in trypsin-inhibitory activity has been detected in extracts of germinating pea seeds free from microbial infection. By investigating the subcellular distribution of protease activity, trypsin-inhibitory activity and the effect of the inhibitor on endogenous protease activity, no convincing evidence could be obtained to suggest that the trypsin-inhibitory activity was involved in the regulation of proteases during 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.     

Losses of Alcohol and Alcohol Dehydrogenase Activity in Germinating Seeds

Losses of alcohol, which had accumulated under anaerobic conditions,occurred during the germination of several species of seedswhich could not be attributed to the volatility of the alcohol.It is suggested that utilization of the alcohol by the seedsmay occur. From the seeds, an active alcohol dehydrogenase,which is mainly confined to the cotyledons in pea seeds, canbe extracted. The activity of the enzyme decreases as the cotyledonsgrow older during germination. The properties of the enzymehave been investigated.     

Growth and curvature in seedlings of Pisum sativum and an ageotropic mutant

In an attempt to explain the influence of gravity on the behaviour of ageotropic plant organs, a pea mutant (Pisum sativum ageotropum) and normal pea (Pisum sativum cv. Sabel) were examined. The mutant has a significantly lower germination rate (large seeds: 25%, small seeds: 10%) than normal pea seeds (55%). Removal of testa increased germination dramatically, the values obtained were 63 and 89%, respectively. Immediately after imbibition the mutant from which the testa had been removed, developed more slowly than normal pea seeds; after 28 h the difference in elongation rate between the two types was reversed. When continuously stimulated geotropically in the horizontal position the elongation in the mutant is larger than in the normal pea roots kept in the same position. During a 24 h period starting 48 h after imbibition the mutant root elongated 45.0 mm while the value for the normal pea root was 11.5 mm. The course of the geotropic curvature in roots of the two types has been followed during a period of 24 h. Normal pea roots develop an asymmetry in the extreme root tip region after 30 min of horizontal stimulation. After prolonged stimulation (exceeding 2 h) the asymmetry has disappeared and the curvature distributed over the entire growth region. When roots of normal pea are stimulated continuously at various angles, the optimum angle of geotropic response is 90° with decreasing responses in the order 135° (i.e. the root tip is pointing obliquely upward) and 45°. The presumed ageotropic behaviour of the mutant has only to a certain extent been confirmed in the present study. When stimulated at 135° a slight positive curvature developed; stimulation at 90° and 45° gave a slight negative curvature.     

Protein kinase activity in meristematic root tips of Pisum sativum during germination

Membranes isolated from pea root meristems contain a protein kinase activity and a number of unidentified endogenous substrates. The enzyme activity increases in presence of MgCl₂ and MnCl₂ but not in presence of CaCl₂; the maximum of phosphorylation activity has been observed after 45 s of incubation. This protein kinase activity is partially solubilized by Triton X-100, although no extensive purification has been attempted. The activity falls during the first hours of germination and is detected only in membranes obtained from meristematic tissues. In the same membrane system, protein phosphatase activity could be present; if this data is confirmed then an interesting mechanism of phospho-dephosphorylation of proteins could exist in pea root meristematic membranes. In this case, such a particular membrane system could become very useful for investigating the physiological role of phosphorylations of protein membrane components during cell proliferation and germination.     

Differential inactivation of seed exudate stimulation of Pythium ultimum sporangium germination by Enterobacter cloacae influences biological control efficacy on different plant species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

NAD pattern and NADH oxidase activity in pea (Pisum sativum L.) under cadmium toxicity

Seeds of pea (Pisum sativum L.) were germinated for 5 days by soaking in distilled water or 5 mM cadmium chloride. Compared to the control, cadmium (Cd) caused a reduction in percent germination and embryo growth. Pyridine nucleotide coenzyme concentrations were determined in cotyledons and embryonic axis. Nicotinamide adenine dinucleotide (NADH) oxidase activity was examined. Cd treatment caused a restriction in levels of reduced coenzyme form in the mitochondria and the post-mitochondrial fraction of cotyledons, and embryonic axis. The oxidized coenzyme form has been accumulated by Cd-treated mitochondria of both tissues. It was also found that NADH oxidase activity was stimulated. The relationship between coenzyme levels, seed germination, pea growth, and Cd stress has been reported.     

Measurement of the Water Potential of Intact Plant Tissues2PISUM SATIVUM L.)

Through the use of a microthermocouple psychrometer it has becomepossible to measure the water potential over part of the surfaceof a pea seed, starting about 19 hours after sowing in distilledwater, or later in the case of seeds germinating at lower osmoticpotentials, in both instances until well after radicles haveemerged. It has been shown that the potential of air-dry seedsis well below –6,000 joules/kg but increases rapidly duringimbibition, depending upon the water potential of the germinationmedium. Pea seeds subjected to lower external water potentialsgerminate at lower internal water potentials than they exhibitin distilled water. The water potentials of the seeds decreasejust after radicle emergence till the radicle establishes contactwith its germination medium, possibly as a result of demandfor moisture during that period due to incipient cell elongation.No detectable amounts of osmotically active substances are exudedfrom the seed during germination; the pea seed coat restrictsthe entry of polyethylene glycol molecules into the seed untilemergence of the radicle.     

Thiamine binding and metabolism in germinating seeds of selected cereals and legumes.

The basic characteristics of thiamine metabolism in germinating seeds of maize (Zea mays), oat (Avena sativa), faba bean (Vicia faba) and garden pea (Pisum sativum) are presented with a special emphasis of a possible thiamine storage function of seed thiamine-binding proteins (TBPs). Seeds were germinated for 6 d in the dark. Thiamine-binding activity in seeds decreased during germination by 50% in cereals and by 30% in legumes. The degradation of TBPs was also detected by polyacrylamide gel electrophoresis. The total thiamine content decreased rapidly to 20-40% of the initial value in cereal seeds during first 3 d of germination while in legume seeds thiamine content started changing from the fourth day and dropped by 50% at the sixth day. A composite pattern was found for the changes in thiamine pyrophosphate (TPP) contribution to total thiamine during seed germination. A peak of the coenzyme percentage was usually detected at the second day of germination. Another gain of TPP was often seen toward the sixth day of germination. The activity of thiamine pyrophosphokinase (EC 2.7.6.2) was high in resting legume seeds and did not significantly change during germination. In contrast, the low activity of this thiamine-activating enzyme in cereal seeds progressively increased during germination. Thiamine phosphate synthase (EC 2.5.1.3) was also detected in seeds and was shown to contribute significantly to the balance of thiamine compounds during seed germination.     

Polyadenylation of pea seed RNA at the early stages of germination.

1. The RNA polyadenylating activity was found in embryo axes of dry, as well as imbibed and germinated pea seeds, both in nucleus and cytoplasm. 2. The total enzymatic activity remains unchanged during germination, but the intracellular distribution is altered; the activity in nuclei is increased about four-fold at the expense of the postmitochondrial fraction. 3. Specificity towards RNA primers of the polyadenylating system from pea embryo axes is low. 4. Cordycepin inhibits RNA polyadenylation only when [14C]ATP is used as a nucleotide donor, and has no visible influence on the activity of the system utilizing [14C]oligo(A)-nucleotides. 5. It seems that RNA in the pea embryo axes is polyadenylated by a two-step mechanism: synthesis of oligo(A)-nucleotides, and their addition to RNA.     

Amylase Activities in Attached and Excised Cotyledons and in Embryonic Axes of Pisum sativum L.

Amylase activity increased in attached cotyledons of peas, Pisumsativum L. var. Bördi, only during imbibition and remainedalmost constant up to 96 h after germination, but in excisedcotyledons the activity increased slightly at first then markedly.In contrast, the content of the reducing sugars was higher inattached cotyledons than in excised ones. A similar inverserelationship has been found between the concentration of reducingsugars in axes (both attached and excised) and amylase activity. The leakage from intact seeds contained more reducing sugarsthan the leakage from excised cotyledons, whereas the amountof proteins released from the cotyledons was four times greaterduring imbibition. This increase in amylase activity in excisedcotyledons is not thought to be the result of axis excision,but to be the result of the leakage of sugars from the cotyledonsduring incubation. These results suggest that the concentration of reducing sugarsmay be a factor that regulates amylase activity in vivo in boththe cotyledons and axis during the germination of pea seeds. (Received August 4, 1982; Accepted December 14, 1982)     

Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination

The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling.     

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.