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.     

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Distinct ethylene- and tissue-specific regulation of β-1,3-glucanases and chitinases during pea seed germination

The expression of β-1,3-glucanase (βGlu) and chitinase (Chn) was investigated in the testa, cotyledons, and embryonic axis of germinating Pisum sativum L. cv. `Espresso generoso' seeds. High concentrations of βGlu and Chn activity were found in the embryonic axis. Treatment with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that an early, 4-fold induction of βGlu activity in the embryonic axis during the first 20 h after the start of imbibition is ethylene-independent. This initial increase was followed by a later 4-fold ethylene-dependent induction in the embryonic axis starting at 50 h, which is after the onset of ethylene evolution and after completion of radicle emergence. The βGlu activity in cotyledons increased gradually throughout germination and was ethylene-independent. In contrast, the ethylene-independent Chn activity increased slightly after the onset of radical emergence in the embryonic axis and remained at a constant low level in cotyledons. Immunoinactivation assays and immunoblot analyses suggest that early βGlu activity in the embryonic axis is due to a 54-kDa antigen, whereas late induction is due to a 34.5-kDa antigen, which is likely to be the ethylene-inducible class I βGlu G2 described for immature pea pods. Increases in Chn in the embryonic axis were correlated with a 26-kDa antigen, whereas amounts of the additional 32- and 20-kDa antigens remained roughly constant. Thus, ethylene-dependent and ethylene-independent pathways regulate βGlu and Chn during pea seed germination. The pattern of regulation differs from that of leaves and immature pods, and from that described for germinating tobacco seeds. The functional significance of this regulation and its underlying mechanisms are discussed. Received: 12 January 1999 / Accepted: 22 March 1999     

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.     

Changes in some nitrogenous components during the germination of pea seeds

The changes in major nitrogenous components during the germination of pea seeds have been followed. During the period of rapid axis growth, 3 to 8 days following germination, the nitrogen content of the cotyledons declines rapidly with an accompanying increase of nitrogen in the developing axis. The accumulation of alcohol soluble nitrogen, primarily amino nitrogen, in the cotyledons and axis during germination indicates that the mobilization of nitrogen is facilitated by proteolysis and translocation of the products.     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

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.     

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.     

Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L

During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, γ-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

Effects of gibberellic Acid and gold light on germination, enzyme activities, and amino Acid pool size in a dwarf strain of watermelon

Gibberellic acid (GA₃) promotes and continuous gold light inhibits germination of seeds of a dwarf strain (WB-2) of watermelon [Citrullus lanatus (Thunb.) Matsu. and Nakai]. Osmotic inhibition of germination with mannitol in light-grown seeds of WB-2 was only slightly reversed by GA₃ at the concentrations used, whereas, GA₃ substantially relieved osmotic inhibition in dark-grown seeds.

The effects of GA₃ and gold light on development of catalase and invertase activities and on levels of free amino acids in germinating seeds of WB-2 were examined. Light depressed development of catalase and invertase activity. Levels of free amino acids increased more slowly in embryonic axes of light- than dark-incubated seeds, but in cotyledons higher levels of amino acids were maintained in light-grown seeds. GA₃ accelerated the development of catalase activity in whole embryos and invertase activity in embryonic axes, but did not significantly affect invertase activity in cotyledons during germination. GA₃ had little effect on amino acid pools in cotyledons and embryonic axes.

     

Phospholipids and the Phospholipid Fatty Acids of Germinating Hazel Seeds (Corylus avellana L.)

An analysis of the phospholipids and phospholipid fatty acidsof germinating hazel seeds has been carried out. The phospholipidcontent of the cotyledons and embryonic axes increased duringgermination. Early increases in the relative amounts of phosphatidylglycerol and decreases in the relative amounts of phosphatidylethanolamine occurred in both cotyledons and axes. The laterstages of germination were accompanied by further changes inthe phospholipid composition of the axes. Increases in the degreeof unsaturation of the C₁₈ fatty acids occurred in both tissues.     

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris)

French bean ( Phaseolus vulgaris ) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.     

THE EFFECT OF LIGHT ON THE USE OF THE NITROGEN RESERVES OF GERMINATING SOYBEAN SEEDS

Light affects the mobilization and distribution of several of the storage components of the cotyledons of germinating soybean seeds. The nitrogen content of the cotyledons began to decrease during the first day of germination, continued through day 12 for plants in the light, and day 14 for those in the dark. Cotyledons from both treatments had lost about the same amount of nitrogen by day 14. Plants from both treatments lost about the same amount of dry weight by day 8, but those in the light had taken up nitrogen from the nutrient solution; while those in the dark showed no increase. The plants in the light had higher concentrations of soluble amino nitrogen in the cotyledons than did those in the dark, but the opposite was true for the seedling axis. Aspartate and its amide accounted for half or more of the total free amino acids in all parts of dark-grown plants at 6 and 14 days. In the light-grown plants aspartate and asparagine usually accounted for less than half of the total free amino acids in all plant parts except the cotyledons at 6 and 14 days. Total soluble amino acids were much lower in these plants than those in the dark, excepting the cotyledons.     

Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds

The temporal and spatial distribution of cysteine proteinases (CPRs) was analyzed immunologically and by in situ hybridization to identify the CPRs involved in the initiation of storage-globulin degradation in embryonic axes and cotyledons of germinating vetch (Vicia sativa L.). At the start of germination several CPRs were found in protein bodies in which they might have been stored in the mature seeds. Cysteine proteinase 1 was predominantly found in organs like the radicle, which first start to grow during germination. Cysteine proteinase 2 was also present at the start of germination but displayed a less-specific histological pattern. Proteinase B was involved in the globulin degradation of vetch cotyledons as well. The histological pattern of CPRs followed the distribution of their corresponding mRNAs. The latter were usually detected earlier than the CPRs but the in situ hybridization signals were histologically not as restricted as the immunosignals. Proteolytic activity started in the radicle of the embryonic axis early during germination. Within 24 h after imbibition it had also spread throughout the whole shoot. At the end of germination, newly synthesized CPRs might have supplemented the early detectable CPRs in the axis. In the cotyledons, only the abaxial epidermis and the procambial strands showed proteinase localization during germination. Both CPR1 and CPR2, as well as the less common proteinase B, might have been present as stored proteinases. Three days after imbibition, proteolytic activity had proceeded from the cotyledonary epidermis towards the vascular strands deeper inside the cotyledons. The histochemical detection of the CPRs was in accordance with the previously described histological pattern of globulin mobilization in germinating vetch [Tiedemann J, et al. (2000)]. A similar link between the distribution of CPRs and globulin degradation was found in germinating seeds of Phaseolus vulgaris L. The coincidence of the histological patterns of globulin breakdown with that of the CPRs indicates that at least CPR1, CPR2 and proteinase B are responsible for bulk globulin mobilization in the seeds of the two legumes. Received: 14 February 2000 / Accepted: 16 August 2000     

胚轴对萌发豌豆子叶中淀粉酶活性表达的影响

萌发豌豆的上、下肢轴均能诱导子叶中淀粉酶活性,外源GA和6—BA具有类似胚轴的作用。离体子叶的淀粉酶凝胶电泳只有一条活性极低的酶带,连生子叶中有两条酶带,其中由胚轴诱导新出现了一条活性很高的同工酶带,它的活性受亚胺环己酮的强烈抑制,而受放线菌素D影响不大。推测豌豆子叶中存在淀粉酶的长寿命mRN—A,胚轴和外源激素的作用在于促进mRNA的翻译。     

Dipeptidase development in cotyledons of Cucurbita maxima during germination

The dipeptidase activity of an unpurified soluble extract of the cotyledons of Cucurbita maxima Duch. var. Hubbard remained unchanged during the first 2 days of germination and then increased at a rapid rate during the next 3 days. The dipeptidase activity of two of three lots of seeds required the presence of the embryo axis for maximal dipeptidase activity, whereas the third lot was uninfluenced by the embryo axis. This discrepancy was possibly due to genetic differences. In those seeds which required the presence of the embryo axis for maximal dipeptidase activity, the cytokinin benzyladenine could replace the embryonic axis. When the protein synthesis inhibitor cycloheximide was added to the seeds at the beginning of germination, it inhibited dipeptidase activity of the cotyledons from 26 to 55 per cent, depending of the basis of calculation, at 5 days. When the cycloheximide was added to 3-day-old seedling the inhibition of dipeptidase activity in the cotyledons was almost immediate. The relative hydrolysis of -leucylglycine and glycylglycine were compared after temperature inactivation and purification; the results showed that more than one enzyme was responsible for the dipeptidase activity. The presence of a dialysable dipeptidase inhibitor(s) was demonstrated. Relatively high dipeptidase activity was also found in the roots and shoots.     

Phosphoenolpyruvate carboxykinase in developing pea seeds is associated with tissues involved in solute transport and is nitrogen-responsive

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in developing pea (Pisum sativum) seeds in relation to their nitrogen supply. PEPCK was present throughout development, with the peak of PEPCK protein and activity in the seed coat and cotyledons preceding protein accumulation in the cotyledons. It showed a different developmental pattern from enzymes involved in amino acid metabolism (phosphoenolpyruvate carboxylase, glutamine synthetase and glutamate dehydrogenase). Immunolocalization showed that PEPCK was present in parts of the developing seed that are involved in the transport and metabolism of assimilates. Early in development, it was associated with the inner integument of the ovule, the endospermic cytoplasm and the outer cells of the embryo. In the middle of development, around the peak of activity, PEPCK was abundant at the outer surface of the developing cotyledons, in the embryonic axis and in the vasculature of the seed coat. Later in development, PEPCK was associated with the embryonic leaf primordia and meristem and cortex of the radicle. PEPCK protein was strongly induced in vitro in the seed coat by nitrate, ammonium and asparagine, in the cotyledons by asparagine and in planta by the supply of nitrogen, which led to an increase in asparagine secretion by empty seed coats. It is suggested that PEPCK is involved in the metabolism of nitrogenous solutes in developing pea seeds.     

Embryonic control of isocitrate lyase activity in cotyledons of germinating soybean

The activity of isocitrate lyase (EC 4.1.3.1) in the cotyledons of germinating soybean is controlled by the embryonic axis. Plant growth regulators like gibberellic acid, indole acetic acid and 2,4-dichlorophenoxy acetic acid are able to increase the enzyme activity in cotyledons of whole seedlings but not in dissected cotyledons. The control of induction of the enzyme activity during germination by the embryo could be mediated by the elaboration of kinetin.