Intact Pea Seedlings Research Articles

Aminoaldehyde dehydrogenase activity during wound healing of mechanically injured pea seedlings

Aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.19) is an enzyme that, in association with amine oxidase, participates in polyamine catabolism. In plants, the enzyme is well characterized in pea seedlings. In this study, we used etiolated and light-grown pea seedlings as model plants to evaluate the possible AMADH role in response to stress caused by mechanical damage. In the beginning, the activity distribution of AMADH, amine oxidase and peroxidase in organs of 7-day-old intact pea seedlings was analyzed. To perform mechanical damage, stems of 10-day-old seedlings were each divided into four segments of equal length. The top (=fourth) segments were then longitudinally cut with a lancet. During healing, the injured segments and their control counterparts were harvested in 1-day intervals and analyzed for activity of the above enzymes, polyamine and 4-aminobutyrate (GABA) concentrations. The injury elicited increases in AMADH, amine oxidase and peroxidase activities in both etiolated and green seedlings, accompanied by parallel increases in putrescine, cadaverine, spermidine and GABA content. Histochemical experiments allowed visualization of increased AMADH activity in cross sections obtained from the injured stem segments. The activity was localized in cortical parenchyma and epidermal cells adjacent to the wound site in spatial correlation with an intensive lignification. In the control seedlings, AMADH activity or lignification in these tissues could not be visualized. Thus, we conclude that, in plants, AMADH may participate in processes of adaptation to stress events caused by mechanical injury, which involve polyamine catabolism, GABA production and lignification.

The effect of fluridone and flurochloridone on the incidence of albinism in pea (Pisum sativum) and on the abscission of leaves of privet (Ligustrum vulgare)

The effect of fluridone (or flurochloridone), the inhibitor of carotenoid, chlorophyll and abscisic acid synthesis, on the abscission of Ligustrum vulgare leaves was investigated. Both forms of fluridone inhibited the abscission of petioles when they were applied as a 1.0% concentration in lanolin to the leaf blade. Fluridone was capable of inhibiting abscission even when it was applied to the petiole as late as 7 hours after the blade was cut off. Fluridone applied in lanolin to the apical part of intact pea seedlings or on the cut surface after decapitation of the epicotyl caused albinism of the stipules. The degree of albinism decreased according to the concentration of applied flurodine (from the highest – 0.5% to the lowest – 0.03%) and was higher in intact than in decapitated plants. Albinism also appeared in pea seedlings grown from seeds swollen in differently concentrated solutions of flurochloridone, particularly in the basal part of the shoots. Complete albinism occurred only in plants cultivated from seeds swollen in high concentrations (0.06–0.12%). The lowest concentration (0.007%) stimulated root growth of the pea seedlings, but inhibited epicotyl growth.

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.

The Short-term Growth Stimulation Induced by External Supply of IAA in Internodes of Intact Pea Seedlings

Indole-3-acetic acid (IAA) mixed with lanolin was applied to a defined region of the top elongating internode (third internode, initially 21–24 mm long) of intact pea (Pisum sativum L. cv. Alaska) seedlings, and the effects of applied IAA on growth of internode zones were investigated. Experiments were conducted with plants grown in red light (R), with supplementary use of plants grown in white light (W). Growth of the internode was first characterised without IAA application. Internode zones elongated at an identical and constant rate per unit length until each zone sharply ceased elongation at a certain distance from the apex. The elongation rate was similar even between R-grown and W-grown plants, the major difference being an earlier cessation of zone elongation in W-grown plants. Following IAA application, the growth rate of zones increased (maximally about two times) but, after a few hours, it declined to the pre-stimulation rate. This short-term growth stimulation (SGS) was observed when IAA was applied directly to the zone monitored for growth and when IAA was applied to an apically separated region. It was demonstrated that SGS was caused not because radial growth was enhanced in place of elongation, nor because energy became limiting. Based on the kinetic differences observed by applying IAA to different regions of the internode and also on the data obtained by applying IAA again at the post-stimulation phase, two independent regulatory elements could be resolved for SGS: (1) reduction of the tissue responsiveness for IAA, and (2) reduction of IAA supply to the target tissue. These regulations are considered to operate in response to increases in the tissue content of IAA.

Expression of a Ribosomal Protein Gene in Axillary Buds of Pea Seedlings

Axillary buds of intact pea seedlings (Pisum sativum L. cv Alaska) do not grow and are said to be dormant. Decapitation of the terminal bud promotes the growth of these axillary buds, which then develop in the same manner as terminal buds. We previously showed that unique sets of proteins are expressed in dormant and growing buds. Here we describe the cloning, sequencing, and expression of a cDNA clone (pGB8) that is homologous to ribosomal protein L27 from rat. RNA corresponding to this clone increases 13-fold 3 h after decapitation, reaches a maximum enhancement of about 35-fold after 12 h, and persists at slightly reduced levels at later times. Terminal buds, root apices, and elongating internodes also contain pGB8 mRNA but fully expanded leaflets and fully elongated internodes do not. In situ hybridization analysis demonstrates that pGB8 mRNA increases in all parts of the bud within 1 h of decapitation. Under appropriate conditions, growing buds can be made to stop growing and become dormant; these buds subsequently can grow again. Therefore, buds have the capacity to undergo multiple cycles of growth and dormancy. RNA gel blots show that pGB8 expression is reduced to dormancy levels as soon as buds stop growing. However, in situ hybridization experiments show that pGB8 expression continues at growing-bud levels in the apical meristem for 2 d after it is reduced in the rest of the bud. When cultured stems containing buds are treated with indoleacetic acid at concentrations >/=10 mum, bud growth and expression of pGB8 in the buds are inhibited.

Embryonal growth-correlative effects in pea seedlings as reflected in the level of endogenous gibberellin-like and cytokinin-like substances

The level of endogenous gibberellin-like and cytokinin-like substances was estimated in the plumule and the root of 2-to 3-day-old intact pea seedlings, in the plumule of seedlings from which the radicle was cut off, and in the root of seedlings from which the plumule was removed. An increase in the level of gibberellin-like substances and a decrease in the level of cytokininlike substances in the plumule as a consequence of radicle amputation were observed within 48 h. Plumule amputation resulted in a decreased level of both gibberellin-like and cytokinin-like substances in the root in the same period.

Characteristics of nitrate uptake into seedlings of pea (Pisum sativum L. cv. Feltham First). Changes in net NO−3 uptake following inoculation with Rhizobium and growth in low nitrate concentrations

Abstract Nitrate uptake into intact pea seedlings (Pisum sativum L. cv. Feltham First) grown in hydroponic culture has been investigated. Following inoculation with Rhizobium leguminosarum a twofold increase in net nitrate uptake was observed. Changes in morphological characteristics following inoculation were found to decrease the effective area available for absorption.There was a two‐fold decrease in net nitrate uptake into intact seedlings grown in the presence of N compared with N free media. In the former case net nitrate uptake appeared to stall at regular intervals. In both cases only the initial rates of nitrate uptake were found to be responsive to the external nitrate concentration. The results are discussed in terms of current models for the regulation of NO−3 uptake by higher plants.

The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.)

Plastids were separated from extracts of pea (Pisum sativum L.) roots by sucrose-density-gradient centrifugation. The incubation of roots of intact pea seedlings in solutions containing 10 mM KNO3 resulted in increased plastid activity of nitrite reductase and to a lesser extent glutamine synthetase. There were also substantial increases in the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. No other plastid-located enzymes of nitrate assimilation or carbohydrate oxidation showed evidence of increased activity in response to the induction of nitrate assimilation. Studies with [1-(14)C]-and [6-(14)C]glucose indicated that there was an increased flow of carbon through the plastid-located pentose-phosphate pathway concurrent with the induction of nitrate assimilation. It is suggested that there is a close interaction through the supply and demand for reductant between the pathway of nitrite assimilation and the pentose-phosphate pathway located in the plastid.

The Morphogenetic Response of Intact Pea Seedlings With Respect to Translocation and Metabolism of Root-applied Auxin

The effects of 2,4-D, NAA and IAA on intact pea seedlings were studied under standardized conditions: The auxins were applied via the roots of one week old etiolated seedlings in a concentration of 10-4 M for 24 hours, followed by another culture period with a light/dark rhythm of 16/8 hours. This pulse application effects a morphogenetic response depending on the auxins applied: 2,4-D inhibits the natural development of the seedlings totally (swelling of tissue and irregular root formation), while neither NAA nor IAA exhibit these drastic effects on morphogenesis. This different morphogenetic response was related to different behaviour in translocation and metabolism of the root-applied auxins. The 2,4-D system is characterized by high mobility and low metabolic accessibility of the free molecule. On the other hand the NAA and IAA molecules are metabolically inactivated and immobilized within the roots, showing that the herbicidal effects after the 2,4-D pulse depend on the endogenous available amount of free unmetabolized auxin under these conditions. This in vivo-syslem using intact seedlings is discussed as a model system for studying auxin-specific long-term effects on the biochemical level.

Light-enhanced perception of gravity in stems of intact pea seedlings

Dark-grown, 6-d-old pea seedlings (Pisum sativum L. cv. Alaska) do not respond gravitropically to brief (approx. 3 min) horizontal presentations, but seedlings given a pulse of red light (R) 16-24 h earlier respond to such stimuli by vigorous curvature of the epicotyl. With continuous horizontal stimulation (approx. 100 min), the kinetics and extent of the gravitropic response are almost identical in irradiated and dark-control plants. Prior R thus increases graviperception without altering the rate-limiting steps underlying the generation of curvature. This effect of R on graviperception develops slowly; seedlings studied only a few hours after R show differences in the kinetics of the gravitropic response, but not in presentation time. Neither the kinetics nor the extent of gravitropic curvature should be used as criteria for establishing changes in primary processes in gravitropism.

The influence of small direct electric currents on the transport of auxin in intact plants

When a d.c. potential of 9.0 V was applied to the stem of intact pea seedlings (Pisum sativum L. cv. Meteor and cv. Alderman) via 10 mM KCl-soaked filter paper electrodes placed ca. 50 mm apart the stem passed a steady current of 15-20 μA (resistance ca. 100 kΩ cm(-1)). The basipetal transport of [1-(14)C]IAA applied to the apical bud was completely inhibited over the portion of the stem through which current flowed and (14)C-labelled compounds accumulated in the vicinity of the upper electrode. The inhibition of transport was independent of the polarity of the applied potential. The basipetal transport of IAA in the stem above the electrode was not affected.Labelled auxin accumulated at the upper electrode both as unchanged IAA and as a compound tentatively identified as indol-3yl-acetyl aspartic acid (IAAsp). These compounds were only slowly remobilised when the current was interrupted. However, the ability of the transport system to move freshly-applied IAA was rapidly and fully restored when the potential was removed. No injury to the plant was detected after maintaining a current flow for up to 72 h. No leakage of (14)C-labelled compounds into the KCl solution bathing the electrodes was detected.

Rapid Metabolism of Propylene by Pea Seedlings

Propylene uptake by intact pea seedlings (Pisum sativum L. cv. Alaska) was easily detected using standard gas chromatographic techniques suggesting rapid metabolism. Comparative studies with highly purified (14)C(3)H(6) and (14)C(2)H(4) under aseptic conditions verified that propylene was rapidly metabolized and indicated that some aspects of its metabolism were similar to that of ethylene since (14)C(3)H(6), like (14)C(2)H(4) (Beyer, Nature 1975, 255: 144-147), was oxidized to (14)CO(2) and incorporated into water-soluble tissue metabolites. However, (14)C(2)H(6) was metabolized at a substantially faster rate and unlike (14)C(2)H(4) the rate of (14)C(3)H(6) tissue incorporation exceeded its rate of oxidation to (14)CO(2). In addition the neutral (14)C-metabolites derived from (14)C(3)H(6) were chromatographically distinct from those formed from (14)C(2)H(4).

Transport of Abscisic Acid‐2‐C‐14 in Intact Pea Seedlings

AbstractWhen abscisic acid‐2‐C‐14 (AbA‐2‐C‐14), 1 μg in 5 μl 40% ethanol, is applied to the apical bud of light‐grown pea seedlings, C‐14 is translocated downwards only in very small amounts and does not enter the root. In contrast to this, C‐14 from indoleacetic acid‐C‐14 (IAA‐2‐C‐14) applied in the same manner is translocated to the root where it accumulates. When AbA‐2‐C‐14 is injected to the stem tissue at the apical bud, more labelled material is transported downwards than after application to the surface. Application of AbA‐2‐C‐14 to an expanded leaf results in considerable accumulation of C‐14 in the growing apical parts and in the lateral roots.

Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid

The application of 2,3,5-triiodobenzoic acid (TIBA, 10 mg·g(-1) in lanolin) to the stem of intact pea seedlings (Pisum sativum L.) inhibited the basipetal transport of (14)C from indoleacetic acid-1-(14)C (IAA-1-(14)C) applied to the apical bud, but not the transport of (14)C in the phloem following the application of IAA-1-(14)C or sucrose-(14)C to mature foliage leaves. It was concluded that fundamentally different mechanisms of auxin transport operate in these two pathways.When TIBA was applied at the same time as, or 3.0 h after, the application of IAA-1-(14)C to the apical bud, (14)C accumulated in the TIBA-treated and higher internodes; when TIBA was applied 24.0 h before the IAA-1-(14)C, transport in the stem above the TIBA-treated internode was considerably reduced. TIBA treatments did not consistently influence the total recovery of (14)C, or the conversion of free IAA to indoleaspartic acid (IAAsp). These results are discussed in relation to the possible mechanism by which TIBA inhibits auxin transport,.Attention is drawn to the need for more detailed studies of the role of the phloem in the transport of endogenous auxin in the intact plant.

Pathways of auxin transport in the intact pea seedling (Pisum sativum L.)

When small colonies of the pea aphid [Acyrthosiphon pisum (Harris)] were established on the stem of Meteor Dwarf Pea seedlings (Pisum sativum L.), (14)C was found in the honeydew 4.5 h after applying IAA-1-(14)C to a fully-expanded foliage leaf. In contrast, no activity was found in the honeydew or aphids 4.5 h after the application of IAA-1-(14)C to the intact apical bud even though the internode upon which the aphids were feeding contained high levels of (14)C. The lack of radio-activity in aphids feeding on stems to which IAA-1-(14)C was applied via the apical bud was found not to be influenced by the internode position or by the transport interval allowed (up to 24 h).Radioactivity derived from either foliar or apical applications of IAA-1-(14)C was not transported through stem tissues killed by heat treatment. Xylem function was shown not to be impared by the heat treatment employed.It was concluded that the long-distance transport of IAA from the apical bud of intact pea seedlings does not take place in the phloem sieve tubes involved in the transport of metabolites from foliage leaves, or in the non-living tissues of the xylem.

Auxin and Ribonucleic Acid Synthesis in Pea Stem Tissue as Studied by Deoxyribonucleic Acid-Ribonucleic Acid Hybridization

The ability of auxin to alter gene transcription in pea (Pisum sativum L.) stem tissues has been investigated by means of DNA/RNA hybridization-competition techniques. In order to obtain reproducible hybridization with total nucleic acid preparations from plants it was found necessary to remove interfering substances, probably polysaccharides; this was accomplished by methoxyethanol extraction and precipitation with cetyltrimethylammonium bromide. When purified in this fashion, plant nucleic acids could be made to form hybrids which showed both species specificity and high thermal stability.No change in hybridizable RNA of stem sections in response to auxin could be detected over a 2- to 24-hour period, regardless of the auxin level employed. In contrast, when large doses of auxin were applied to intact pea seedlings, definite changes in the hybridizable RNA of stem tissue were detected both 8 and 24 hours after treatment. Many of the 2,4-D-induced species of RNA which were present at 24 hours were also present at 8 hours. Sections apparently lack a factor needed for the manifestation of the auxin effect on RNA synthesis.Since the hybridization assay employed does not measure all cellular RNA, it is still possible that certain RNA species may be synthesized in sections in response to auxin. However, the auxin promotion of cell elongation in such sections is clearly not associated with changes in hybridizable RNA such as have been reported for several hormonal responses in animal systems.

Separation of Effects of Auxin and Ethylene in Pea Roots

WE have treated intact pea seedlings (Pisum sativum L. var. Alaska) with four synthetic auxins and found inhibition of primary root elongation, stimulation of radial swelling, proliferation of massed lateral roots and faster evolution of ethylene. Exposure to exogenous ethylene had the same effects, except that lateral root development was normal.

Effects of Growth Retardants on Biosynthesis of Gibberellin Precursors in Root Tips of Peas, Pisum sativum L.1

Abstract Pea root tips were pre-incubated for 2 hr in the presence of Alar, Amo–1618, or Cycocel after which a quantity of 2-14C-mevalonic acid was added to the medium. The incorporation of mevalonic acid subsequent to decarboxylation into 3 gibberellic acid precursors, trans-geranylgeraniol, kauren-19-ol, and kauren-19-al was ascertained by paper and thin layer chromatography. The amount incorporated into a fourth acidic fraction containing kaurenoic acid was also estimated. In the presence of Alar, the amount of radioactivity in trans-geranylgeraniol increased while that in kauren-19-al decreased simultaneously; the radioactivity in the acidic and kauren-19-ol fractions remained unchanged. Amo-1618 at 125 ppm induced an accumulation of radioactive acidic materials and concurrent decreases in the other 3 fractions. With Cycocel, the incorporation of 14C in kauren-19-ol and kauren-19-al remained unchanged while that in the acidic fraction increased and trans-geranylageraniol decreased. Amo-1618 was 100-fold more effective than Cycocel in altering the levels of GA precursors. The Alar concentration required to affect the biosynthesis of GA precursors was the same as that which inhibited epicotyl elongation of intact pea seedlings. The findings support the hypothesis that these growth retardants disrupt GA biosynthesis which in turn affects subapical meristematic activity, thereby inhibiting internodal elongation.

The transport and metabolism of 14C-labelled indoleacetic acid in intact pea seedlings

Part of the IAA-I- or IAA-2-(14)C applied at low concentrations to the apices of intact, light-grown dwarf pea seedling was transported unchanged to the root system The calculated velocity of transport in the stem was 11 mm per hour. In the root the label accumulated in the developing lateral root primordia.A large proportion of the applied IAA was converted by tissues of the apical bud, stem and root to indole-3-acetyl-aspartic acid (IAAsp). This compound was not transported. In addition evidence was obtained for the formation of IAA-protein complexes in the apex and roots, but not in the fully-expanded internodes.Large quantities of a decarboxylation product of IAA, tentatively indentified as indole-3-aldehyde (IAld), and several minor metabolites of IAA, were detected in extracts of the roots and first internodes, but not in the above-ground organs exposed to light. These compounds were readily transported through stem and root tissues. Together, the decarboxylation of IAA and the formation of IAAsp operated to maintain a relatively constant level of free IAA-(14)C in the root system.

The Relation between Cell-wall and Protein Synthesis in Dwarf Pea Plants treated with Gibberellic Acid

The amount of protein and soluble nitrogen present in expanding internodes of intact dwarf pea seedlings, was investigated at different times after treatment of plants with gibberellic acid (GA). There was a marked increase in rate of protein synthesis following GA treatment, but the rate of synthesis did not keep pace with internode expansion, so that the amount of protein per unit length fell. The rate of cell-wall synthesis was also increased, and, in contrast to protein, the amount of cell wall per unit length remained approximately constant during internode expansion. It is suggested that the increased rate of cell-wall synthesis which follows GA treatment is mediated by a change in protein metabolism. The amount of soluble nitrogen present in expanding internodes was also increased. There was little effect of GA upon the protein content of internodes which were almost fully expanded at the time of treatment.