Regulation of root formation by auxin-ethylene interaction in pea stem cuttings

The possibility was investigated that the inhibition of rooting in pea ( Pisum sativum L. cv. Weibull's Marma) cuttings caused by low indol-3yl-acetic acid (IAA) concentrations is due to ethylene produced as a result of IAA treatment. Treatment with 10 uμ IAA reduced the number of roots to about 50% of the control and increased ethylene production in the stem bases by about 20 times the control value during the two first days of treatment. Ethylene-releasing compounds (ethephon and 1-amino-cyclopropane-1-carboxylic acid, ACC), in concentrations giving a similar ethylene release, inhibited rooting to the same extent or more strongly than IAA. These results indicate that IAA-induced ethylene is at least responsible for the negative component in IAA action on root formation in pea cuttings. A higher IAA concentration (100 μ) and indol-3yl-butyric acid efficiently counteracted the negative effect of ethylene on root formation.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.     

Auxin effects on rooting in pea cuttings

Light-grown stem cuttingss of Pisum sativum L. cv. Weibull's Marma were rooted in a nutrient solution. The presence of 10 μ M indolylacetic acid (IAA) in the solution for 24 h or longer periods decreased the number of roots subsequently formed to about 50% of control, provided IAA was present in the solution during any of the 4 first 24 h periods. Treatment for 6 h or shorter periods caused no or small response. IAA did not appreciably change the time needed for root formation, the time course of root appearance or the pattern of root distribution along the basal internode. IAA at 100 μ M usually increased the number of roots although variable results were obtained with this IAA concentration.
The number of roots was strongly increased by treatment with indolylbutyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4-D). None of these or other synthetic auxins decreased the number of roots in suboptimal concentrations. Experiments with 10 μ M IBA showed that stimulation of rooting was obtained only if the auxin was present in the rooting solution for several days. Simultaneous treatment with IAA decreased the stimulating effect of IBA to some extent, whereas no such response was obtained if IAA was combined with 2,4-D.
IAA applied in lanolin to the stem of intact cuttings decreased the number of roots formed. Decapitation and debudding of the cuttings decreased the number of roots formed. If at least 2 leaves were left this decrease was efficiently counteracted by an optimal IAA dose applied to the upper part of the stem. A five times higher dose was less effective, indicating a negative effect on rooting also by IAA applied to the shoots.     

Physiological studies on pea tendrils

When tendrils which have been dark adapted for 3 days are mechanically stimulated, they will only coil appreciably if they are irradiated with light. A spectral response curve suggests that this is a blue light effect. Red or far red light do not modulate this response, but a high intensity flash of blue white light given before the actinic blue light blocks coiling for 30 min. The dark-adapted tendril, which has not been previously rubbed, has the ability to store the tactile sensory information for about 60 min, but loses it if it is not irradiated within 120 min after mechanical perturbation.     

Changes in concentration of endogenous growth inhibitors during growth recovery of dwarf pea seedlings

In order to clarify the role of endogenous growth inhibitors A-2α and A-2β in a dwarf pea plant, red light (emission peak 657 nm) treated, 9-d-old seedlings of dwarf pea (Pisum sativum L. cv. Progress No. 9) were transferred to darkness, and the resulting changes in growth rate and concentrations of A-2α and A-2β were monitored. The growth rate of the epicotyls increased, and the concentration of the inhibitors in the epicotyls decreased, according to sigmoidal time courses. The relationship between the logarithms of the concentration of the inhibitors and the corresponding growth rate was linear. These results suggest that A-2α and A-2β, may play an important role in the growth recovery process of the dwarf pea cultivar after termination of red light irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transport and accumulation of indole-3-acetic acid in pea cuttings under two levels of irradiance

The transport and accumulation of 2-[¹⁴C]-IAA applied to the apex of cuttings of Pisum sativum L. cv. Alaska was greater in cuttings from stock plants grown under 38 W m⁻² than 16 W m⁻². Accumulation of ^14C in the base of the cuttings from the highest level of irradiance was correspondingly more significant. The level of irradiance to the stock plants greatly affected the rate of accumulation, while the light conditions during IAA transport had a minor effect. The amount of IAA reaching the base of the cuttings increased with increasing concentration of IAA in the treatment solution, but the percentage of applied IAA reaching the base decreased.
The relative chromatographic partition of ethanol-extractable ¹⁴C showed that, after 12 h of IAA-transport, the amount of 2-[¹⁴C]-IAA was higher in the base of cuttings from 38 W m⁻² than in those from 16 W m⁻². After a further 12 h of transport the relative amounts of 2-[¹⁴C]-IAA in the two types of cuttings were reduced to the same lower level.
A possible role of an irradiance-mediated difference in the topographic distribution of IAA in the base of pea cuttings on the subsequent adventitious root formation is discussed.     

Interaction of ethylene with indole-3-acetic acid in regulation of rooting in pea cuttings

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated.Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.     

Effect of nitrate on pea sucrose synthase

The in vivo and in vitro nitrate effects on pea (Pisum sativum L.) sucrose synthase (SS) were studied. At the period of plant transition from heterotrophic to autotrophic nutrition, exogenous nitrate (14.2 mM) absorbed in the form of KNO₃ and Ca(NO₃)₂ during 10–20 days activated SS in the roots by 22–100% as compared with plants grown on nitrogen-free medium. Such effect was observed only at plant growing under high light (natural illumination up to 25 klx) and thus their sufficient supplement with sucrose. Under low light (climate-controlled chamber, 2.5 klx), nitrate could not activate SS. In the in vitro experiments, nitrate activated SS exponentially by a dose-dependent mode with the plateau at 3–5 mM, where its activity was increased by 50%. It is supposed that there is a second constituent in SS activation by nitrate, and it carries information about plant carbohydrate status. Possible mechanisms of nitrate-induced SS activation are discussed.     

Light effects on root formation in aspen and willow cuttings

The effect of light on rooting of leafy cuttings of aspen (Populus tremula × tremuloides) and a willow hybrid (Salix caprea × viminalis) was investigated under controlled conditions in water culture. Two levels of irradiance were used, 40 and 8 W m^?2. The lower level gave the best rooting of aspen cuttings, both when applied to the stock plants before the cuttings were taken and when given to the cuttings during the rooting period. Irradiation of the cutting base during the rooting period inhibited rooting almost completely in aspen and decreased the number of roots formed in the Salix hybrid. Net photosynthesis in the cuttings of Salix decreased considerably after excision and increased again after formation of roots. Indirect evidence indicated that photosynthesis was even more affected in aspen cuttings. The possible roles of carbohydrates and inhibitors in the light effects are discussed.     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

Relations between carbohydrate, water status and adventitious root formation in leafy pea cuttings rooted under various levels of atmospheric CO2 and relative humidity

Three levels of atmospheric CO₂ and 2 levels of relative humidity (RH) during the rooting period were tested for their effect on several factors presumed to influence adventitious root formation in leafy pea ( Pisum sativum L. cv. Alaska) cuttings. Compared to normal CO₂ levels (350 μl l⁻¹), neither 1800 nor 675 μl l⁻¹ CO₂ affected the rooting percentage or the number of roots per cutting. However, 1800 μl l⁻¹ CO₂ increased root and shoot dry weight, root length, carbohydrate levels in the base of the cuttings and water potential (Ψ_w) of cuttings compared to normal levels of CO₂. Compared to 87% RH. 55% RH decreased all of the above parameters, including the number of roots per cutting. A polyvinyl chloride antitranspirant (which partially blocks stomata and slows photosynthesis) applied simultaneously with 87% RH increased Ψ_w and root length but lowered all of the other above parameters, compared to 87% RH without antitranspirant. Increasing current photosynthate (products of photosynthetic activity after excision), carbohydrate, or Ψ_w either alone or together was associated with increased root system size but not necessarily with increased rooting percentage or root number. The data are consistent with a hypothesis that the number of roots per cutting increased with increasing current photosynthate and carbohydrate until some other factor became limiting. Also, the effect of Ψ_w on rooting percentage and root number was mediated through its effect on current photosynthate and carbohydrate.     

Amino acid sequence of a Bowman-Birk proteinase inhibitor from pea seeds

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified by ammonium sulfate precipitation, gel filtration, and anion and cation exchange chromatography and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Their molecular weights were determined by electrospray mass spectrometry as 6916, 6807, 7676, 7944, 7848, and 7844 D, respectively, and the sequences of the first 20 N-terminal amino acid residues of these six inhibitors were found to be identical. The complete amino acid sequence of PSTI IVa was determined. This protein comprises a total of 72 residues and has 14 cysteines, all involved in disulfide bridges. Comparison of the sequence of PSTI IVa with those of other leguminous Bowman-Birk type inhibitors revealed that PSTI could be classified as a group III inhibitor, closely related toVicia faba andVicia angustifolia inhibitors.     

The nature of protein differentiation in the growing pea root

Abstract Sections of the growing root of pea (Pisum sativum) have been microdissected into stele, cortex and epidermis. Using labelled amino acids, two dimensional separations employing non-equilibrium pH gel electrophoresis (NEPHGE) and isoelectric focusing (IEF), and silver staining, the complexity of protein differences between the cortex and the stele has been assessed. Analyses commenced as cells in these two tissues appear in the meristem (0.7—1 mm from the tip) and continued up to 30 mm from the tip as they subsequently mature. From the earliest stages at which the cortex and stele can be distinguished and dissected apart the protein patterns differ substantially. However these tissue differences, involving over one third of the detected protein species, are almost all quantitative. Very few qualitative (i.e. tissue specific) proteins were detected. Many proteins also show quantitative stage-specific variation, detected using successively older root segments. In vitro translation studies involving isolated mRNA showed only a very limited stage-specific variation in translated proteins. This supports the notion that translational controls may contribute significantly to the development of these two tissue types.     

Effects of exogenous cytokinins on root formation in pea cuttings

Benzylaminopurine (BAP) or zeatin continuously supplied through the rooting solution to cuttings of pea ( Pisum sativum L. cv. Weibull's Marma), inhibited root formation down to a concentration of 3.10⁻⁹ M . The inhibitory effect of BAP in the concentration range 10⁻⁸–10⁻⁷ M was readily reversible if the cuttings were transferred to solutions without cytokinin after treatment for 1–4 days. A slight increase in the number of roots formed was obtained after treatment with low cytokinin concentrations for 1–2 days. Evidence from microscopic studies of primordia formation indicates that BAP inhibits differentiation of primordia at an early stage in their development. Growth of already formed primordia, or root elongation, was considerably less sensitive to the inhibitory effect of BAP. The results indirectly support the hypothesis that endogenous cytokinins prevent root formation in stems of intact plants and may be of importance for the regulation of rooting in cuttings.     

Relationship between H2O2 and jasmonic acid in pea leaf wounding response

The relationship of H₂O₂ and jasmonic acid (JA) in wound-induced defense response was investigated in the leaves of pea (Pisum sativum L.) plants. The results showed that both wounding and JA treatment led to a significant increase in activities of plasma membrane NADPH oxidase and phenylalanine ammonialyase. However, such an increase was blocked by the pretreatment with plasma membrane NADPH oxidase inhibitors, O ₂ ^? scavengers, or H₂O₂ scavenger, implying that H₂O₂ functions downstream of JA. Furthermore, wounding treatment activated two key enzymes of JA biosynthesis, lipoxygenase and allene oxide synthase, while JA biosynthetic inhibitors impaired the wounding-induced H₂O₂ burst. Thus, it is suggested that H₂O₂ burst depends on JA production in plant wounding response.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Subcellular aspects of the protective effect of spermine against atrazine in pea plants

The action of the exogenously applied tetraamine spermine in reversing the effect of atrazine stress on intact pea plants (Pisum sativum L., cv. Koray) was investigated at the ultrastructural level. The results indicate that atrazine increases cell senescence by lipid peroxidation and loss of unsaturated fatty acids from thylakoid membranes of pea plant chloroplasts. Spermine acts as a polyfunctional effector. It stabilises the molecular composition of the membranes by preventing lipid peroxidation and as a consequence protects the conformation of the thylakoid system and structural integrity of the chloroplasts. Spermine treatment also contributes to the process of neutralisation of the free radicals by peroxisomes.     

Molecular properties of 4-substituted indole-3-acetic acids affecting pea pericarp elongation

Pea (Pisum sativum L.) fruit naturally contain the auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA). However, only 4-Cl-IAA can substitute for the seeds in maintaining pea fruit growth in planta. The importance of the substituent at the 4-position of the indole ring was tested by comparing the molecular properties of 4-X-IAA (X = H, Me, Et, F, or Cl) and their effect on the elongation of pea pericarps in planta. Structure-activity is discussed in terms of structural data derived from X-ray analysis, computed conformations in solution, semiempirical shape and bulk parameters, and experimentally determined lipophilicities and NH-acidities. The size of the 4-substituent, and its lipophilicity are associated with growth promoting activity of pea pericarp, while there was no obvious relationship with electromeric effects.     

Somatic embryogenesis in pea (Pisum sativum L.): effect of explant, genotype and culture conditions

In this study 16 cultivars of pea (Pisum sativum L.) were screened in vitro for the formation of somatic embryos which were dependent on the genotype, culture conditions and explant source used. The cultivars Stehgolt, Maro and Progreta showed the highest tendency to form somatic embryos (c. 25%) while Alaska, Rondo and Ascona did not show any embryo production. Using the cultivar Belman, the highest embryo production was achieved by using nodal explants of shoots (10.6%) and a cotyledon-free embryo as explant source (14.1%) in the light (15.8%) compared to using apices as explants (1.8%) and a seedling as the explant source (9.4%) in the dark (3.3%). Media containing picloram (0.75 mg/litre) followed by BA (1 mg/litre) or kinetin (1 mg/litre), each for four weeks gave the highest somatic embryo production. The development of embryos to whole plants was unreliable and some 90% of the embryos induced did not develop any further, died, recallused or formed secondary embryos. The size of the embryo at separation and the timing of the separation from the original callus were important factors determining success for complete development to whole plant. Regeneration of 184 plants was achieved with ensuing flowering, pod formation and viable seed production from the techniques described.     

Short-chain fatty-acid-induced effects on the cell cycle in root meristems of Pisum sativum

Propionic acid and valeric acid at 1 m M reduced the mitotic index of root meristem cells of Pisum sativum to <1% after 12 h in aerated White's medium. After 12 h exposure to either acid, seedlings transferred to fresh medium resumed their normal mitotic index 12 h after transfer, with a burst of mitosis at 8 h. Exposure times of 8 h to either acid inhibited DNA synthesis, and nuclei released from either propionic or valeric acid inhibition were still unable to resume normal DNA synthesis after 12 h. Neither acid significantly altered the distribution of meristematic cells in G1 and G2 after 12 h. Propionic acid at 1 m M reduced the uptake of [¹⁴C]-leucin but conversion rates to protein were constant regardless of whether any acid was present. Another longer fatty acid, caprylic acid, at 1 m M did not significantly reduce the mitotic index nor did 1 m M benzoic acid, another organic acid. This information suggests that only the short-chain fatty acids, propionic acid and valeric acid, limit progression through the cell cycle by inhibiting DNA synthesis and arresting cells in G1 and G2 in a manner similar to butyric acid, a known cell arresting agent.