Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

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.     

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。     

Pea seedling growth and development regulated by cotyledons and modified by irradiance

Growth and development of hydroponically grown pea seedlings ( Pisum sativum L. cv. Alaska) were measured using stem and root length as well as number of leaves and lateral roots. The growth was dependent on the presence of cotyledons and was modulated by the irradiance. All plants were grown in a full nutrient solution. If grown at low irradiance (73 μmol m^-2s^-1) they depended more and for a longer time on the cotyledons than plants grown at high irradiance (220 μmol m^-2s^-1). Low irradiance caused stem elongation but decreased root length and number of lateral roots as compared to plants grown at high irradiance. The dark respiration of the leaves was measured as oxygen uptake. In plants grown at the low irradiance, excision of the cotyledons caused the rate of oxygen uptake to increase by a factor of three, and the increase was sensitive to cyanide. Decotyledonized plants showed a high respiration rate and a diminished leaf growth for their entire life cycle. CO₂ fixation also increased in decotyledonized pea seedlings grown at either irradiance. The mobilization of food reserves from the seeds was positively correlated to seed dry weight, but only if the plants were grown at 73 μmol m^-2s^-1. Increasing dry weight of the seed enhanced top growth, whereas root growth was depressed, so that top and root responds differently with regard to that part of growth which depends on mobilization of reserves from the seed.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl-transferase (FTase) gene was examined using transgenic expression of the β-glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Changes in Phospholipid Composition of a Winter Wheat Cultivar during Germination at 2 C and 24 C

Evaluation of various solvent systems for lipid extraction of wheat Triticum aestivum L. cv. Rideau seeds showed that boiling 2-propanol followed by the Bligh-Dyer procedure was the most efficient method, with respect to lipid yield and ability to inactivate lipolytic enzymes. Ten phospholipids were identified in dry seeds; the major components being phosphatidylcholine, lysophosphatidylcholine, N-acyl lysophosphatidyl-ethanolamine, N-acylphosphatidylethanolamine, and phosphatidylethanolamine. After growth for 1 week (2 C) or 31 hours (24 C), the proportions of phosphatidylethanolamine + lysophosphatidic acid and phosphatidic acid increased, lysophosphatidylcholine decreased, and the remaining phospholipids showed little change. At 5 weeks (2 C) or 72 hours (24 C), the seedlings showed 5-fold increases in the proportion of phosphatidic acid largely at the expense of phosphatidylcholine, small decreases in N-acyl lysophosphatidylethanolamine and N-acylphosphatidylethanolamine, and significant increases in lysophosphatidylcholine. The changes in phosphatidic acid and phosphatidylcholine are interpreted as being partially due to increasing phospholipase D activity during germination. In general, the phospholipid composition was similar in morphologically equivalent seedlings grown at 2 C or 24 C. The increased membrane content in seedlings grown at 2 C does not reflect any preferential synthesis of individual phospholipids.     

Fatty acid changes in Hibiscus esculentus tissues during growth

Lipids were isolated from roots, stems, cotyledons, leaves, buds, flowers, pods and seeds of okra (Hibiscus esculentus) at different stages of plant growth from germination to seed formation and their fatty acid compositions analysed. The lipid contents of roots and stems were 1–3%, cotyledons 3.7–9%, leaves 2.5–5.1% and seeds 2.2–20.2%. Palmitic, linoleic and linolenic were the main fatty acids present in all tissues at all stages, but their relative proportions varied. Cyclopropene fatty acids (CFA) were present at some stages in roots and seeds. In the roots their formation coincided with bud formation (35 days after sowing) and their content reached a maximum (12.8%) seven days after flowering. CFA were present in maturing seeds from 31 days after flowering and occurred as dihydro derivatives throughout. Dihydro derivatives of the CFA were absent in all other tissues. Heptadecenoic acid was present (0.4–1.3%) in root lipids at all stages and in the stem lipids (0.4–1.2%) in the initial stages and after flowering.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Activation and Inactivation of Alcohol Dehydrogenase in Germinating Pea Cotyledons

Alcohol dehydrogenase (E.C.1.1.1.1.) activity increases markedly in the germinating pea cotyledon in the first 2 days. The activity was not suppressed by the administration of actinomycin D, 6-methylpurine, DL-p-fluorophenylalanine, and D-chloramphenicol. The compounds rather depressed the decrease of alcohol dehydrogenase activity in cotyledons after 3 days of germination. The alcohol dehydrogenase activity in ungerminated pea seeds was activated by treatment with sodium lauryl sulphate, sodium dioctyl sulfosuccinate, dithiothreitol, 2-mercaptoethanol and NADH. The inhibitory effect caused by the extract from 7 day-old cotyledons was diminished markedly in the presence of dithiothreitol and 2-mercaptoethanol, as well as by addition of bovine serum albumin. If dithiothreitol was added to the extraction medium, the enzyme activity from older cotyledons was greatly enhanced.     

Characterizing the interaction between fire ants (Hymenoptera: Formicidae) and developing soybean plants

This research characterizes the interaction between the fire ants Solenopsis invicta Buren and developing soybean plants. Phagostimulant studies showed that fire ant foraging on soybean seeds increased once the seeds imbibed water. During seedling development over a 5-d germination period, fire ant foraging shifted from the stem/cotyledons to the roots, despite continual increases in fresh weights for each region, and the fact that stem/cotyledon tissue contained the majority of food reserves. Carbohydrate analysis showed that although 2-d-old seedlings had higher concentrations of phagostimulant carbohydrates, especially sucrose, than tissues of mature plants, all tissues analyzed had enough of these sugars to induce a phagostimulant response. Fire ant association with seeds/seedlings germinated in soil resulted in reduced seedling vigor, as determined by a doubling of seedling emergence time, a threefold increase in malformed seedlings, and visible damage to cotyledons. Seeds germinated and grown to mature plants in association with fire ants, allocated 43% more assimilate into pods, but produced 28% less root dry matter, 11% less total dry matter, and there was an 81% reduction in the number of root nodules compared with control plants. We propose that reduced root development and inhibitions of nodule formation would be major yield limiting factors under field conditions. This work demonstrates that fire ant damage to soybeans is not limited to seedling establishment and that more research should be directed at the subterranean activities of the fire ant.     

GUS expression in Arabidopsis directed by 5′ regions of the pea metallothionein-like gene PsMT A

Upstream sequences (including the first seven codons) of a metallothionein (MT)-like gene from pea, PsMT A, were fused to GUS and introduced into Arabidopsis. High-level GUS expression was detected in the roots of plants grown on MS medium, except in regions proximal to the root apex. There was precise delineation of the root-shoot boundary. In soil-grown plants there was low GUS expression and this was absent from the more mature regions of the roots. In the aerial tissues of soil-grown plants, GUS expression was restricted to hydathodes, stipules, expanding cotyledons and the following senescent tissues: leaves, cotyledons, petals, sepals, filaments, stigmas, nectaries and siliques. A 298 bp region was shown to be required for GUS expression in roots but not for expression in vegetative aerial tissues of plants grown on MS medium. This region contains predicted ethylene-responsive elements (EREs) but similar patterns of GUS expression were detected in etr1 seedlings. GUS expression was significantly higher in roots exposed to 500 nM copper, but this increase was small in proportion to expression in roots exposed to 50 nM copper.     

Immunohistochemical localization of apyrase during initial differentiation and germination of pea seeds

Localization of the 49-kDa apyrase (ATP diphosphohydrolase, EC3.6.1.5; DDBJ/EMBL/GenBank BAB40230) was investigated during early stages of germination of pea (Pisum sativum L. var. Alaska) at the organ, tissue, cellular, and sub-cellular level using light-microscopical immunohistochemistry. Whole mount tissues were immuno-reacted with anti-APY1 serum, pre-immune serum or anti-actin antibody for control. Antigen to the anti-APY1 serum was not detected until 16 h after sowing (26 h after start of imbibition), when the antigen was detected throughout the tissue, especially in the epidermis and cortex. At 35 h after sowing, the younger regions including the root tip and the tip of the stele were more strongly stained than the control. Both, epidermal and cortical cells of the epicotyl and root tip were stained. The stain was mainly localized in the cytoplasm and around nuclei in the apical meristem and the root tip, while vacuoles and cell walls were not stained. At 62 h, there was major staining in the plumule, hook, and elongating regions of the epicotyl and in the region between cotyledons and the epicotyl. After 84 h, lateral root primordia were stained. The pre-immune serum showed virtually no staining while the anti-actin antibody reacted solely with the cytoplasm. Since the antigen to the anti-APY1 serum was primarily found in the cytoplasm and around nuclei in elongating and differentiating tissues and labeling declined in mature tissues, it is suggested that apyrases may play a role in growth and development of tissues, for example, lateral roots.     

Soybean Lipoxygenase L-4, a Component of the 94-kilodalton Storage Protein in Vegetative Tissues: Expression and Accumulation in Leaves Induced by Pod Removal and by Methyl Jasmonate

A lipoxygenase L-4 gene was isolated from a soybean genomiclibrary. The amino acid sequence of lipoxygenase L-4 is highlyhomologous with the partial amino acid sequence of the 94-kDavegetative storage protein, vsp94, found in paraveinal mesophyllcells in the leaves of depodded soybean plants. No L-4 expressionwas observed in maturing seeds. The L-4 gene is highly expressedin the vegetative tissues of young seedlings, including cotyledons,hypocotyls, roots and primary leaves. L-4 expression followedthe same pattern as lipoxygenase activity in cotyledons peaking3 to 5 days after germination, and returning to a basal levelby 9 days after germination. L-4 gene expression was low inthe roots, stems and leaves of 10-week-old plants. Exposureof 4-week-old plants to atmospheric methyl jasmonate increasedL-4 mRNA in leaves. Continuous pod removal from 7-week-old plantsover a 2 week period resulted in dramatic accumulation of L-4mRNA in leaves. Accumulation of the L-4 protein and three otherlipoxygenase fractions in the leaves of depodded plants wasdemonstrated by ion exchange chromatography. These results indicatethat lipoxygenase L-4 is a component of vsp94. (Received May 31, 1993; Accepted August 9, 1993)     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

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.     

Effect of hexavalent chromium on the growth and physiological and biochemical parameters on Brassica oleracea L. var. acephala DC

In order to determine the toxic effect of chromium Cr(VI) on the seed germination, the root and shoot length, the root-cotyledonary leaves, the fresh and dry weight in eight-day-old seedlings Brassica oleracea L. var. acephala DC (kale) were treated with various concentrations of Cr in the growth medium. The accumulation of chromium in the tissues was determined in the cotyledons and the roots of the kale seedlings. High rate of Cr uptake was observed in the roots. But the organs could not accumulate large amount Cr. The effect of Cr on B. oleracea var. acephala was evaluated by changes in chlorophyll a, b, lipid peroxidation, proline, ascorbate, protein carbonyl groups, non-protein thiols and peroxidase activity. There were significant decreases in chlorophylls a, b content of the plants treated with Cr. Chromium treated kale seedlings had higher lipid peroxidation and the protein carbonyl groups in cotyledonary leaves than the roots. The changes refer to toxic effects of Cr. There were increases in the non-protein thiol, the total ascorbate, and proline content in the cotyledons and the roots of the seedlings grown on the media containing 0.1 and 0.15 mM Cr. The guaiacol peroxidase activity was higher in the roots of the seedlings than their cotyledons.     

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 effects of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis

The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2- and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.