Pea Internodes Research Articles

Plant hormone interactions: how complex are they?

Models describing plant hormone interactions are often complex and web-like. Here we assess several suggested interactions within one experimental system, elongating pea internodes. Results from this system indicate that at least some suggested interactions between auxin, gibberellins (GAs), brassinosteroids (BRs), abscisic acid (ABA) and ethylene do not occur in this system or occur in the reverse direction to that suggested. Furthermore, some of the interactions are relatively weak and may be of little physiological relevance. This is especially true if plant hormones are assumed to show a log-linear response curve as many empirical results suggest. Although there is strong evidence to support some interactions between hormones (e.g. auxin stimulating ethylene and bioactive GA levels), at least some of the web-like complexities do not appear to be justified or are overstated. Simpler and more targeted models may be developed by dissecting out key interactions with major physiological effects.

Specificity patterns indicate that auxin exporters and receptors are the same proteins.

A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts.

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.

The Contents of Auxins and Cytokinins in Pea Internodes as Related to the Growth of Lateral Buds

Hormonal control exerted by the apical leaves of different age over the outgrowth of the 1st and 2nd lateral buds was studied in pea seedlings. The endogenous phytohormones in the 1st and in the 2nd internodes and basipetal transport ofindole-3-acetic acid (IAA) and cytokinins were examined in 8-10-day-old plants. Three kinds of plants were under study, completely intact ones and plants that had been either decapitated or partially defoliated on the 7th day of growth. In the internodes, decapitation and partial defoliation led to the following changes (as compared with intact plants): slightly decreased levels of abscisic acid (ABA), unchanged levels of gibberellins (GAs), lowered levels of IAA (up to 2-fold), and significantly enhanced levels of zeatin/zeatin riboside (Z/ZR) (up to 5-fold). The extent of lateral bud inhibition corresponded well to the IAA to Z/ZR ratio in the subtending internode, thus supporting the concept of apical dominance control by the ratio of auxin to cytokinin in stem. The results evidence that apical leaves, the source of basipetally transported IAA, up-regulate IAA contents in internodes, but down-regulate Z/ZR content, which is most likely determined by IAA-induced inhibition of xylem transport of cytokinins from the root (Bangerth, 1994). We found that the estimated levels of ZR phloem transport inversely correlated with the levels of IAA transport. It was proposed that phloem ZR is evidently a xylem ZR reexported via leaves; the latter was shown to be regulated by IAA (Bangerth, 1994). It was found, however, that irrespective of the amount of IAA transported from leaves to roots, the older leafwas responsible for the lower rate of phloem basipetal transport of ZR and lower level of Z/ZR in internodes. These data argue that the apical leaves can additionally regulate the cytokinin status of stem tissues through reexport ofZR depending on the leaf age. Both IAA and Z/ZR contents in internodes were found to be affected by the age and the position of internodes themselves. Possible roles of endogenous ABA and GAs in regulation of IAA and Z/ZR contents in internode tissues are discussed.

Effects of Gibberellin on Cellulose Biosynthesis and Membrane-associated Sucrose Synthase Activity in Pea Internodes

The gibberellic acid (GA 3 )-induced growth of the second internode in 4- to 8-day-old light-grown pea seedlings ( Pisum sativum L. var. Senator) was analysed. During hormone-mediated growth and after cessation of elongation the rate of cellulose biosynthesis was much larger than in the water control. In GA 3 -treated stems the concentration of UDP-glucose was higher than in the control. The specific catalytic activity of the plasma membrane-associated form of the enzyme sucrose synthase was determined in growing and mature stems. In GA 3 -treated internodes enzyme activity was 70 % higher than in the water control. These results indicate that the membrane-bound sucrose synthase may playa role in GA 3 -mediated cellulose biosynthesis by supplying UDP-glucose as substrate.

Axis elongation can occur with net longitudinal orientation of wall microfibrils.

During the initial phases of elongation of pea internodes, oat and rice coleoptiles, oat mesocotyls, soybean hypocotyls and dandelion peduncles, net transverse orientation of cellulose wall microfibrils (Mfs) was found in the outer epidermal wall. This paper demonstrates that in all these axes, with the exception of rice coleoptile, net longitudinal orientation of microfibrils occurs in the outer epidermal wall in portions of the axes that were still elongating at the time of sampling. The timing of the transition to net longitudinal orientation and whether the transition proceeded acropetally or basipetally varied with the type of axis under study. The variability of the relationship between extension and the transition from net transverse to net longitudinal orientation suggests that factors other than extension are important in determining the transition. Layers of longitudinal wall microfibrils may be added to the extending epidermal wall to bolster its tensile strength commensurate with its function during and after extension. Attention is drawn to the parallels between the concept of tissue tension in growing axes and the concept that the epidermis functions as a stressed skin in the support of mature plant parts in primary growth.

Differential Effects of Brefeldin A and Cycloheximide on the Activity of Auxin Efflux Carriers in Cucurbita pepo L.

Summary Brefeldin A (BFA), an inhibitor of Golgi-mediated protein traffic, stimulated net uptake of indole-3-acetic acid (IAA) by etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with a lag of less than 10 min, substantially reduced the rate of efflux of IAA from pre-loaded zucchini tissue, and strongly inhibited polar transport of IAA in zucchini hypocotyl and pea internode (Pisum sativum L.) segments. Pre-treatment of zucchini tissue with BFA (60 min), but not its simultaneous addition to the uptake medium, also greatly increased the FCCP-sensitive uptake of IAA by microsomal vesicles prepared from the pre-treated tissue. In contrast to the effects of BFA, application of the translation inhibitor cycloheximide (CH) did not affect any of the parameters of IAA transport investigated for at least 80 min and did not alter the responses to BFA. Nevertheless, CH rapidly (

Auxin-growth relationships in maize coleoptiles and pea internodes and control by auxin of the tissue sensitivity to auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied 3H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.

Re-Examination of the solute-import hypothesis of gibberellin action in developing pea internodes

Exogenous gibberellic acid (GA 3 ) strongly stimulated the growth of internodes 1, 2 and 3 in light-grown pea seedlings ( Pisum sativum L. var. Senator). In all three internodes, rapid GA 3 -induced elongation was accompanied by a rise in the specific catalytic activity of the sucrolysis enzyme soluble acid invertase. The activity of the second sucrose-metabolizing enzyme, soluble sucrose synthase, was not affected by GA 3 . Cessation of growth was associated with a corresponding decrease in acid invertase and an increase in sucrose synthase activity. In all three internodes, GA 3 -induced growth was associated with an increase in the level of glucose. The osmotic concentration of the tissue sap from the rapidly growing internodes of GA 3 -treated seedlings was as large as in the water control. As a result of GA 3 -treatment, the concentration of osmotica increased in the mature region of the stem. We conclude that GA 3 -induced growth of pea internodes is in part attributable to a hormone-mediated increase in acid invertase activity, which results in the provision of hexoses for energy metabolism, biosynthesis of cell walls and the maintenance of intracellular osmotic concentration during rapid elongation of the stem.

Effects of gibberellin on the cellular dynamics of dwarf pea internode development

This study analysed the dynamics of cell production and extension, and how these were affected by applied gibberellic acid (GA3), during internode development in dwarf peas (Pisum sativum L. cv. Meteor). Image analysis was used to obtain cell number and length data for entire cell columns along the epidermis, the two outermost cortical layers, and the pith, from internode 7, over a time period covering the whole of the internode's growth phase. For a few days following the inception of an internode at the shoot apex, little further growth occurred, and there was no significant effect of GA3 on cell division or cell extension. The subsequent growth of the internode was stimulated more than fourfold by GA3 as a result of the production of more than twice the number of cells, which were twice as long. At least 96.5% of the cells of the mature internode were actually formed within the internode itself during this period of growth, demonstrating that the internode cells themselves represent the morphogenetic site of response to GA3. Mitoses and cell extension occurred along the full length of the internode throughout its development. The daily changes in cell numbers were modelled by the Richards function, and manipulations of the fitted functions to reveal time trends of absolute and specific cell production rates were performed for each stem tissue. The increase in cell numbers in the +GA3 plants was brought about by an increase in the rate of cell production, over a shorter time interval; specific cell production rates declined continuously from initial rapid rates in the +GA3 epidermis and pith, but declined more slowly in the cortex. The control (−GA3) epidermis and cortex cells exhibited a constant specific cell production rate (i.e. purely exponential) for several days. Cell extension rates were calculated so as to compensate for the size-reduction effects of concurrent cell division. These calculations confirmed that `real' cell extension rates were higher in the +GA3 internodes. Models of the cellular controls of internode growth, based on the estimated dynamics of cell division and extension, are discussed.

Gibberellin does not accelerate rates of cell division in the dwarf pea shoot apical meristem

Although GA3 doubled the numbers of cells in dwarf pea internodes, it caused no significant acceleration of cell division rates in the apical meristem, estimated using cell doubling times, mitotic indices, or percentage labelled mitoses data. Increased cell numbers in GA3-treated pea stems must be generated within the extending internodes.

Effect of Cellulose Synthesis Inhibition on Growth and the Integration of Xyloglucan into Pea Internode Cell Walls

2,6-Dichlorobenzonitrile (DCB, 100 mum) inhibited by 80 to 85% the incorporation of [(3)H]glucose into cellulose in stem segments of etiolated pea (Pisum sativum) seedlings. The inhibition lasted for at least 24 h. In the period 1 to 4 h after the excision of the segments, DCB did not influence elongation in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). However, during the period 1 to 24 h after excision, DCB enhanced endogenous and 2,4-D-stimulated elongation by 65 and 34%, respectively. DCB did not affect the incorporation of (3)H from [(3)H]arabinose into xyloglucan, and did not change the ability of the [(3)H]xyloglucan formed in vivo to bind strongly to the cell wall. Therefore, at least 80 to 85% of newly synthesized cellulose was excess to the requirements for tight wall binding of newly synthesized xyloglucan. This conflicts with the hypothesis that xyloglucan is held in the cell wall solely by direct hydrogen bonding to the surfaces of cellulosic microfibrils.

Fusicoccin-Induced Changes in the Translatable RNAs of Etiolated Pea Stem Tissue

The fungal toxin fusicoccin (FC) was found to induce substantial increases in the abundance of specific translatable RNAs over several hours, concomitantly with tissue expansion, in segments of etiolated pea internodes. The FC-induced RNAs encoded some relatively basic polypeptides of MWs 27-5-29-2 kDa, and some relatively acidic polypeptides of MWs 12-5-15-5 kDa. The earliest detectable increases in the FC-induced translation products occurred after 1-5-2-5 h. Auxin treatment caused a different overall pattern of changes in translation product spots, though some induced spots appeared to be common to both treatments. The possible significance of these observations is discussed in relation to other factors known to alter gene expression in plant tissues.

Ultrastructural immunolocalization of developmental proteins in phloem cell wall regions of Pisum Sativum l. shoot tissue

The spatial and temporal distribution of 2 antigenic proteins have been examined in shoot apical and internode tissues of pea seedlings in the context of protein expression during cell differentiation. Monoclonal antibodies were raised against the proteins of interest extracted from pea internode tissue and were employed as cytochemical probes. Goat anti-mouse second antibody conjugated with colloidal gold was used for labelling the proteins. A 32-kDa protein appeared as concentrated aggregations in cell wall areas between phloem parenchyma and sieve elements, and in the cell wall junctions between phloem parenchyma cells. A 47-kDa protein was also observed in the cell walls of the phloem tissue only, but was more dispersed. Both the 32-kDa and the 47-kDa proteins were absent from apical tissue. The observation of such specific localizations and the time of their appearance suggests possible developmental roles for the antigenic proteins.

Effect of Indoleacetic Acid- and Fusicoccin-Stimulated Proton Extrusion on Internal pH of Pea Internode Cells

Using (31)P nuclear magnetic resonance spectroscopy, we followed cytoplasmic and vacuolar pH in pea (Pisum sativum cv Alaska) internode segments during treatment with indoleacetic acid (IAA) or fusicoccin (FC) in continuously perfused, oxygenated buffer. Although IAA and FC induced normal H(+) extrusion, elongation, and glucan synthase activity responses during the measurements, neither the cytoplasmic nor the vacuolar pH showed significant change at any time between 5 minutes and 1 to 3 hours of treatment. Changes in cytoplasmic pH as small as about 0.04 pH unit were detected after treatment with 1-naphthyl acetate. Therefore, cytoplasmic pH changes do not appear to mediate IAA or FC stimulation of H(+) extrusion or other metabolic responses to these effectors.

Interaction between cortical cylinder and epidermis during auxin-mediated growth in pea internodes

The interaction between cortical cylinder (cortex plus vascular tissue) and epidermis during auxin (indole-3-acetic acid, IAA)-induced growth of third internode sections from red light-grown pea seedlings ( Pisum sativum L. cv. Alaska) was investigated. A quantitative comparison of the relative effects of IAA on growth of intact and peeled sections showed that intact segments are nearly 20-fold more sensitive to IAA than peeled cortical cylinders. Tissue tension, determined with the ‘split section test’, was constant during IAA-induced growth of intact sections. Peeled sections also displayed a small amount of tissue tension, which was likewise independent of IAA. The incorporation of myo-[2- 3H(N)]inositol ([ 3H]Ins) into non-cellulosic polysaccharides in the cell walls was stimulated by IAA in both the cortical cylinder and the epidermis by + 70% and + 55%, respectively, after 4 h. A mich higher amount of incorporation was detectable in the epidermis than in the cortical cylinder on a unit weight basis. During a 4-h growth period in IAA the cortical cylinder lost about 50 μg of its initial dry weight per section whereas the epidermis increased in dry weight by about + 24 μg. We conclude that during IAA-induced long-term growth the cortical cylinder (1) provides the driving force for organ growth, (2) responds to IAA by an increase in matrix cell wall synthesis and (3) releases material, some of which is transferred to the attached epidermal cells.

Growth, in Vivo Extensibility, and Tissue Tension in Developing Pea Internodes

The relationship between growth, in vivo extensibility, and tissue tension in the first 3 internodes of 5, 6, and 7 day-old pea plants (Pisum sativum L. cv Alaska), grown under continuous red light was investigated. The upper 15 millimeters of each internode was marked with ink and its elongation growth measured over the next subsequent 8 hours. In vivo extensibility was measured by stretching living tissue at constant force (creep test) in a custom-built extensiometer. Tissue tension was determined by (a) measuring the rate of expansion of the isolated cortical cylinder after adding water and the amount of contraction of the epidermis after peeling, and (b) by use of the ;split section test.' A good correlation between rate of elongation growth, in vivo extensibility, and tissue tension was established. The epidermis peeled from the growing third internode of 7 day-old plants and measured immediately showed a plastic extensibility (E(pl) twice that of peels from nongrowing excised sections. This high E(pl)-value was lost on incubation of the sections in distilled water, and was subsequently restored by incubating the sections in auxin (indole-3-acetic acid). We conclude that the in situ growth of the internodes is a function of tissue-tension, which provides the driving force of organ growth, and the extensibility (E(pl) of the outer epidermal wall, which is in the growing plant in a ;loosened' state. We furthermore suggest that in the intact plant auxin is causally involved in the wall loosening process in the epidermis.

Differential Effect of Auxin on in Vivo Extensibility of Cortical Cylinder and Epidermis in Pea Internodes

The effect of auxin indole-3-acetic acid (IAA) on growth and in vivo extensibility of third internode sections from red light grown pea seedlings (Pisum sativum L. cv Alaska) and the isolated tissues (cortex plus vascular tissue = cortical cylinder, and epidermis) was investigated. Living tissue was stretched at constant force (creep test) in a custom-built extensiometer. In the intact section, IAA-induced increase in total (E(tot)), elastic (E(el)), and plastic (E(pl)) extensibility is closely related to the growth rate. The extensibility of the cortical cylinder, measured immediately after peeling of intact sections incubated for 4 hours in IAA, is not increased by IAA. Epidermal strips, peeled from growing sections incubated in IAA, show a E(pl) increase, which is correlated to the growth rate of the intact segments. The isolated cortical cylinder expands in water; IAA has only a small growth-promoting effect. The extensibility of the cortical cylinder is not increased by IAA. Epidermal strips contract about 10% on isolation. When incubated in IAA, they do not elongate, but respond with an E(pl) increase. The amount of expansion of the cortical cylinder and contraction of the epidermis (tissue tension), measured immediately following excision and peeling, stays constant during IAA-induced growth of intact sections. The results support the hypothesis that IAA induces growth of the intact section by causing an E(pl) increase of the outer epidermal wall. The driving force comes from the expansion of the cortical cylinder which is under constant compression in the intact section.

Rapid auxin-induced stimulation of cell wall synthesis in pea internodes

The effect of auxin (indole-3-acetic acid; IAA) on growth and incorporation of myo-[2-(3)H(N)]inositol ([(3)H]Ins) into noncellulosic polysaccharides in the cell walls of third internode sections from red light-grown pea seedlings (Pisum sativum L. cv. Alaska) was investigated. Intact sections were incubated on [(3)H]Ins for 4 hr to permit uptake of the tracer and then IAA was added. Growth started after a lag phase of 15 min under these conditions. The sections were removed from the tracer and separated into epidermis and cortical cylinder (cortex plus vascular tissue). In the epidermis, IAA-induced stimulation of [(3)H]Ins incorporation started after a lag of 15 min. The amount of incorporation was 15% higher after 30 min and 24% higher after 2 hr than in the control. In the cortical cylinder, IAA-induced stimulation of [(3)H]Ins incorporation started only approximately 1 hr after adding IAA. The ionophore monensin (20 muM) inhibited the IAA-induced growth by 95%. Under these conditions, the IAA-induced stimulation of [(3)H]Ins incorporation and the IAA-induced increase in in vivo extensibility of the sections was almost completely inhibited, although oxygen uptake was unaffected. We suggest that wall synthesis (as represented by [(3)H]Ins incorporation) and wall loosening (increase in in vivo extensibility) are related processes. The results support the hypothesis that IAA induces growth by rapid stimulation of cell wall synthesis in the growth-limiting epidermal cell layer.

Effects of Gibberellin on the Arrangement and the Cold Stability of Cortical Microtubules in Epidermal Cells of Pea Internodes

Effects of Gibberellin on the Arrangement and the Cold Stability of Cortical Microtubules in Epidermal Cells of Pea Internodes