Biomechanical study of the effect of a controlled bending on tomato stem elongation: local strain sensing and spatial integration of the signal

In a previous paper it has been demonstrated that tomato stems, submitted to a controlled basal bending, had a reduced terminal primary elongation, indicating mechanosensing and intra plant signalling. The 'intensity' of the growth response, as measured by the time to recover an elongation rate similar to the control, varied hugely between plants. However, no relation was found between the intensity of this response and the mechanical variables characterizing the global mechanical state of the stem. In this paper, a local analysis of mechanical state of each bent stem is performed in the context of beam theory. The spatial distributions of local variables all along the stem (curvature, bending moment, strains and stresses) are established. The validity of hypotheses underlying the mechanical analysis is demonstrated. To investigate the relationships between the mechanical stimulus and the growth response, a novel biomechanical analysis based on spatial integration of the mechanical stimulus is presented. It revealed that the mechanosensing is local and scattered through the stem and that the variability of the growth response is only explained by the integrals of the longitudinal strain field.     

Genetic Dissection of the Relative Roles of Auxin and Gibberellin in the Regulation of Stem Elongation in Intact Light-Grown Peas

Exogenous gibberellin (GA) and auxin (indoleacetic acid [IAA]) strongly stimulated stem elongation in dwarf GA1-deficient le mutants of light-grown pea (Pisum sativum L.): IAA elicited a sharp increase in growth rate after 20 min followed by a slow decline; the GA response had a longer lag (3 h) and growth increased gradually with time. These responses were additive. The effect of GA was mainly in internodes less than 25% expanded, whereas that of IAA was in the older, elongating internodes. IAA stimulated growth by cell extension; GA stimulated growth by an increase in cell length and cell number. Dwarf lkb GA-response-mutant plants elongated poorly in response to GA (accounted for by an increase in cell number) but were very responsive to IAA. GA produced a substantial elongation in lkb plants only in the presence of IAA. Because lkb plants contain low levels of IAA, growth suppression in dwarf lkb mutants seems to be due to a deficiency in endogenous auxin. GA may enhance the auxin induction of cell elongation but cannot promote elongation in the absence of auxin. The effect of GA may, in part, be mediated by auxin. Auxin and GA control separate processes that together contribute to stem elongation. A deficiency in either leads to a dwarfed phenotype.     

STEM ELONGATION OF XANTHIUM PLANTS PRESENTED IN TERMS OF RELATIVE ELEMENTAL RATES

Vegetative Xanthium plants grown under noninductive conditions were marked along the stem with India ink and photographed during three successive days. The relative elemental rates of stem elongation [d(dX/dt)/dX] were estimated for 18 plants between 15 and 18 plastochrons. On the average, only the 8.0 cm terminal part of the stem was elongating in this group of plants. Young internodes were elongating at constant relative elemental rates ([d(dX/dt)/dX] was about 0.2 days^–1); nodal portions of the stem beteween two young internodes were not elongating. Internodes longer than 2 cm displayed an acropetal pattern of elongation in which the basal part of an internode stopped elongating and matured first and the apical portion last. The pattern of elongation of the stem could be best approximated to a set of cascading waterfalls with declining plateaus in the direction of the water flow. The acropetal pattern of individual internode elongation observed in Xanthium was similar to those reported for Helianthus and Phaseolus internode growth.     

Thigmomorphogenesis: The response of plant growth and development to mechanical stimulation

Summary When young plants of Hordeum vulgare. Bryonia dioica. Cucumis sativus. Phaseolus vulgaris. Mimosa pudica. and Ricinus communis. were given a gentle mechanical stimulus by rubbing the internodes for about 10 s once or twice daily, elongation was significantly retarded. Plants of Cucurbita pepo Pisum sativum and Triticum aestivum did not exhibit any such response. The initial response to rubbing was very rapid, elongation stopping less than 3 min after application of the stimulus. When the stimulus was discontinued after 7 days, elongation accelerated, reaching a normal or supernormal rate within 3 or 4 days. Mechanical stimulation also affected aspects of growth and development other than stem elongation. In Mimosa pudica, flower bud production was retarded, as was the growth of the tendrils, leaves, and petioles in Bryonia dioica. It is suggested that this response be called thigmomorphogenesis, and that it represents an adaptation designed to protect plants from the stresses produced by high winds and moving animals. Some evidence indicates that thigmomorphogenesis may be mediated by ethylene.     

The role of nodal regions in growth of Xanthium (Compositae) stem

Vegetative Xanthium plants grown under noninductive conditions were marked with India ink along the stem and photographed at three consecutive time intervals. Relative elemental rates (d[dX/dt]/dX) of stem elongation were estimated from displacement of marks during stem elongation. Young internodes elongated with constant relative elemental rates of 0.2 day^-1. Older internodes displayed an acropetal pattern of elongation in which the basal segments of an internode stopped elongating first and the apical portion last. Nodal regions elongated with very small relative elemental rates of 0.05 day^-1. Rates decreased as the age of the internodes and nodes increased and stopped shortly after Leaf Plastochron Index 9.     

THE EFFECT OF LIGHT ON THE CELLULAR COMPONENTS OF POLARIZED GROWTH IN BEAN INTERNODES

First internodes of light-grown bean seedlings exposed to supplementary red and far-red light and those of dark-grown seedlings were sectioned and studied to determine the effects of irradiation on the cellular components of polarized growth. Cell counts and measurements of epidermis, cortex, and pith are given. Increased length of internodes of far-red-treated plants was caused by both increased rate and increased duration of cell elongation. The effect of far-red light is interpreted as a reversal of the accelerating effect of light upon cell maturation. It is suggested that investigations of the mechanism of the red, far-red response of stems be concerned with the processes involved in cell elongation. In darkness, rate and duration of cell division as well as rate and duration of cell elongation were greater than in any of the irradiated plants, indicating that only part of the photocontrol of stem elongation is mediated through the red, far-red system.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

The biophysical basis of elongation growth in internodes of deepwater rice

Partial submergence induces rapid internodal elongation in deepwater rice (Oryza sativa L., cv Habiganj Aman II). We measured in vivo extensibility, tissue tension, hydraulic conductance and osmotic potential in the region of cell elongation in the uppermost internode. The in vivo extensibility of the internode, measured by stretching of living tissue with a custom-made constant stress extensiometer, rose rapidly following submergence of the plant. Both the elastic (E_el) and plastic (E_pl) extensibility increased when growth of the internode was induced. The submerged internode displayed tissue tension (elastic outward bending of longitudinally split internode sections); in air-grown control internodes, no such bending occurred. The hydraulic conductance, estimated from the kinetics of tissue shrinkage in 0.5 molar mannitol and subsequent swelling in distilled water, was not changed by submergence. The osmotic potential, measured with a dew-point hygrometer using frozen-thawed tissue, was only 18% less negative in the submerged internode than in the air-grown control. This indicates that osmoregulation takes place in rapidly elongating rice internodes. We suggest that the rapid expansion of the newly formed internodal cells of submerged plants is controlled by the yielding properties (E_pl) of the cell walls. Experiments with excised stem sections indicate that gibberellin is involved in increasing the E_pl of the elongating cell walls.     

Laser micromarking technique in studying the negative gravitropism in pea stem

A laser micromarking technique on plant epidermis was developed to study how a plant can reduce the stress in bending behavior by controlling the growth and morphogenesis. The negative gravitropism in a pea seedling (Pisum sativum L.) was discussed based on the time-dependent displacement of laser marking points which were formed by spatially-selective laser ablation of the cuticle layer that covers the outer surface of a plant. The elongation of the stem in the horizontal direction was remarkable in the first half of the gravitropism. The elongation percentages of the stem length between laser-marking points at around upper surface, middle, and bottom surface were evaluated to be 2.57, 4.87, and 7.70%, respectively. The characteristic feature of the stem bending in gravitropism is the elongation even at the upper surface region, that is, inside of the bending. This is a different feature from cantilever beams for structural materials like metals and polymers, where the compression of the upper surface and elongation of the bottom surface are caused by bending. Another laser micromarking technique was developed to improve the resolution of a dot-matrix pattern by fluorescent material transfer to a plant through a masking film with a micro-hole matrix pattern. Similar time-dependent displacement behavior was observed for a fluorescent dot-marked stem showing a feedback control loop in the mechanical optimization. These results suggested that plants solve the problem of the stress in stem bending through growth. The laser micromarking is an effective method for studying the mechanical optimization in plants.     

Peroxidase Ontogeny in a Dwarf Pea Stem as Affected by Gibberellin and Decapitation

The ontogeny of peroxidase activity and isoenzyme pattern wasinvestigated in the stem of dwarf pea plants. Peroxidase activityper unit soluble protein was a given internode is highest inthe youngest growth stage, drops during elongation, remainsconstant upon cessation of growth, and increase at senescence.The lower the internode on the stem the higher is its peroxidaseactivity. These developmental differences are already apparentat the youngest growth stage of the internodes adn increaseduring elongation. Several anodic and five cathodic isoperoxidasesare apparent after starch gel electrophoresis. This patternis constant for all internodes at all growth stages, but therelative importance of particular isoenzymes changes with time. Gibberellic acid (GA₃) treatment causes greatly elongated internodes,decreased soluble protein, and inhibition of the rise in peroxidaseactivity within 4–8 h. Application of GA₃ to young internodesleads to a persistent depression in peroxidase activity, whiletreated older internodes suffer only a temporary depression.GA₃ causes no qualitative changes in the isoenzyme pattern butproduces some quantitative alterations in internodes in whichits influence on peroxidase activity is persistent. Decapitation of untreated and GA₃-treated dwarfs has littleinfluence on internode elongation, causes an increase in peroxidaseactivity, especially in the upper internodes, and alters therelative activity of particular isoenzymes. By contrast, decapitationinhibits elongation of young internodes in genetically tallpea plants.     

Photocontrol of stem elongation in plant neighbourhoods: effects of photon fluence rate under natural conditions of radiation

Abstract. We investigated the effects of photon fluence rate on internode elongation in fully de-etiolated plants growing under sunlight. Our goal was to find out whether perception by the stems of fluence rate changes related to canopy density may be involved in the modulation of internode growth in canopies formed by plants of similar stature (e.g. crop stands). Using Datura ferox L. and Sinapis alba L. seedlings growing under natural radiation, we found that internode elongation is promoted by localized shading. This effect was observed with internodes receiving light with a high (>0.9) or a low (0.3) red (R) to far-red (FR) photon ratio. Selective removal of the different wavebands from the light impinging on the internodes showed that part of the response to fluence rate is due to photons in the R + FR range. The blue (B) component, most likely acting through a specific photoreceptor, also inhibited elongation. However, changes in the fluence rate of B light did not have detectable effects on the response of the internodes to R:FR ratio. Fibre-optic studies and measurements with integrating-cylinder sensors in even-aged populations of seedlings showed that both the quality and quantity of radiation received by the stems are profoundly influenced by changes in canopy density. When density is very low (leaf area index = LAI ≥ 1) only the R:FR ratio is reduced, due to FR reflected from nearby leaves. In the LAI range of 1 to 2, though a large proportion of the leaf area is still receiving full sunlight, the photon fluence rate at the stem level drops dramatically. These results suggest that in even-aged populations of LAI > 1 elongation growth is promoted by the low R:FR ratio and the reduced fluence rate. Perception of these two factors at the stem level may elicit morphological adaptations in the canopy before the onset of severe competition among neighbours for the resource of light.     

Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures

We have shown previously that ethylene, which accumulates in the air spaces of submerged stem sections of rice (Oryza sativa L. cv “Habiganj Aman II”), is involved in regulating the growth response caused by submergence. The role of gibberellins in the submergence response was studied using tetcyclacis (TCY), a new plant growth retardant, which inhibits gibberellin biosynthesis. Stem sections excised from plants that had been watered with a solution of 1 micromolar TCY for 7 to 10 days did not elongate when submerged in the same solution or when exposed to 1 microliter per liter ethylene in air. Gibberellic acid (GA₃) at 0.3 micromolar overcame the effect of TCY and restored the rapid internodal elongation in submerged and ethylene-treated sections to the levels observed in control sections that had not been treated with TCY. The effect of 0.01 to 0.2 micromolar GA₃ on internodal elongation was enhanced two- to eight-fold when 1 microliter per liter ethylene was added to the air passing through the chamber in which the sections were incubated. GA₃ and ethylene caused a similar increase in cell division and cell elongation in rice internodes. Thus, ethylene may cause internodal elongation in rice by increasing the activity of endogenous GAs. In internodes from which the leaf sheath had been peeled off, growth in response to submergence, ethylene and GA₃ was severely inhibited by light.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

Antagonistic and Synergistic Interactions between Ancymidol and Gibberellins in Shoot Growth of Cucumber (Cucumis sativus L. )

Effects of the plant growth retardant, ancymidol, on the growthand morphology of the shoot system of cucumber (Cucumis sativusL. ) were investigated. Ancymidol inhibited stem elongation,reducing both number and length of internodes. Reduction inleaf area, attributable to a reduction in both cell size andnumber, was accompanied by an increase in chlorophyll per unitarea. The retardant decreased apical dominance and delayed anthesis.Gibberellic acid fully reversed ancymidol-induced inhibitionof stem elongation, internode length and production, and leafexpansion. GA_4/7 and ancymidol gave a synergistic promotionof stem elongation by increasing elongation of younger internodesand increasing internode production. Synergistic promotion ofpetiole elongation by this combination was also observed. Ancymidol,applied 7 d previously either to the shoot or root, severelyreduced the level of gibberellin-like activity in bleeding sapcollected from decapitated plants.     

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 role of the leaves in the regulation of internode elongation in Phaseolus vulgaris

Time-course patterns of leaf and internode elongation were studied in bean plants. Each leaf started its main elongation period when the leaf below reached half of its final length. The onset of leaf unfolding was nearly synchronous with the initiation of the elongation of the subjacent internode. Excision of young leaves increased the rate of stem elongation as a result of an earlier unfolding of the next upper leaves and the concomitant advancement in the elongation of their subjacent internodes. IAA or NAA (1% in lanolin) suppressed the enhancement effects of leaf excision on leaf and internode elongation. The excision of a young leaf increased the final length of internodes located below it, and at the same time decreased the final length of the internodes located above the excised leaf. The reduction was greater the younger the internode. Differences in internode elongation after leaf excision were related to changes during internode ontogenesis in their relative response to the availability of assimilates on the one hand, and on the other hand to hormonal factors transported acropetally from the young leaves to the growing internodes.     

Changes in mitogen-activated protein kinase activity occur in the maize pulvinus in response to gravistimulation and are important for the bending response

The maize (Zea mays L.) pulvinus was used as a model system to study the signalling events that lead to differential growth in response to gravistimulation in plants. The pulvinus functions to return tipped plants to vertical via differential elongation of the cells on its lower side. By performing immunokinase assays using total soluble protein extracts and an antibody against mammalian ERK1, a mitogen‐activated protein kinase (MAPK)‐like activity was assayed in pulvini halves harvested at various time points after tipping. We detected a reproducible alternation of higher levels of activity occurring between the upper and lower halves of the pulvinus between 75 and 180 min after tipping, with a sustained increase in the upper half occurring at the end of the time‐course. This timing roughly corresponds to the presentation time for maize (i.e. the amount of time that the plant needs to be tipped before it is committed to bend), which occurs between 2 and 4 h. Treatment of maize stem explants with an inhibitor of MAPK activation, U0126, led to a reduction in the activity of this kinase, as well as an almost 65% reduction in bending as measured at 20 h. Rinsing out of the inhibitor resulted in recovery of both bending and kinase activity. It is possible that changes in MAPK activity in the gravistimulated pulvinus are part of a signalling cascade that may help to distinguish between minor perturbations in plant orientation and more significant and long‐term changes, and may also help to determine the direction of bending.     

Growth and molecular responses to long‐distance stimuli in poplars: bending vs flame wounding

Inter‐organ communication is essential for plants to coordinate development and acclimate to mechanical environmental fluctuations. The aim of this study was to investigate long‐distance signaling in trees. We compared on young poplars the short‐term effects of local flame wounding and of local stem bending for two distal responses: (1) stem primary growth and (2) the expression of mechanoresponsive genes in stem apices. We developed a non‐contact measurement method based on the analysis of apex images in order to measure the primary growth of poplars. The results showed a phased stem elongation with alternating nocturnal circumnutation phases and diurnal growth arrest phases in Populus tremula × alba clone INRA 717‐1B4. We applied real‐time polymerase chain reaction (RT‐PCR) amplifications in order to evaluate the PtaZFP2, PtaTCH2, PtaTCH4, PtaACS6 and PtaJAZ5 expressions. The flame wounding inhibited primary growth and triggered remote molecular responses. Flame wounding induced significant changes in stem elongation phases, coupled with inhibition of circumnutation. However, the circadian rhythm of phases remained unaltered and the treated plants were always phased with control plants during the days following the stress. For bent plants, the stimulated region of the stem showed an increased PtaJAZ5 expression, suggesting the jasmonates may be involved in local responses to bending. No significant remote responses to bending were observed.     

Control of Internode Length in Pisum sativum (Further Evidence for the Involvement of Indole-3-Acetic Acid)

The effects of altered endogenous indole-3-acetic (IAA) levels on elongation in garden pea (Pisum sativum L.) plants were investigated. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) were applied to elongating internodes of wild-type and mutant lkb plants. The lkb mutant was included because elongating lkb internodes contained 2- to 3-fold less free IAA than those of the wild type. In the wild type, TIBA reduced both the IAA level and internode elongation below the site of application. Both TIBA and HFCA strongly promoted the elongation of lkb internodes and also raised IAA levels above the application site. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) also markedly increased internode elongation in lkb plants and virtually restored petioles and tendrils to their wild-type length. In contrast, treatment of wild-type plants with TIBA, HFCA, or 2,4-D caused little or no increase in elongation above the application site. The ethylene synthesis inhibitor aminoethoxyvinylglycine also increased stem elongation in lkb plants, and combined application of HFCA and aminoethoxy-vinylglycine restored lkb internodes to the wild-type length. It is concluded that the level of IAA in wild-type internodes is necessary for normal elongation, and that the reduced stature of lkb plants is at least partially attributable to a reduction in free IAA level in this mutant.     

Invertase Activity, Carbohydrate Metabolism and Cell Expansion in the Stem of Phaseolus vulgaris L.

In the stem of Phaseolus vulgaris L. the specific activity ofacid invertase was highest in the most rapidly elongating internode.Activity of the enzyme was very low in internodes which hadcompleted their elongation, in young internodes before the onsetof rapid elongation, and in the apical bud. From shortly afterits emergence from the apical bud the elongation of internode3 was attributable mainly to cell expansion. Total and specificactivities of acid invertase in this internode rose to a maximumat the time of most rapid elongation and then declined. Transferof plants to complete darkness, or treatment of plants withgibberellic acid (GA₃), increased the rate of internode elongationand final internode length by stimulating cell expansion. Bothtreatments rapidly increased the total and specific activitiesof acid invertase in the responding internodes; peak activitiesof the enzyme occurred at the time of most rapid cell expansion. In light-grown plants, including those treated with GA₃, rapidcell and internode elongation and high specific activities ofacid invertase were associated with high concentrations of hexosesugar and low concentrations of sucrose. As cell growth ratesand invertase activities declined, the concentration of hexosefell and that of sucrose rose. In plants transferred to darkness,stimulated cell elongation was accompanied by a rapid decreasein hexose concentration and the disappearance of sucrose, indicatingrapid utilization of hexose. No sucrose was detected in theapical tissues of light-grown plants. The results are discussed in relation to the role of acid invertasein the provision of carbon substrates for cell growth. Key words: Cell expansion, Acid invertase, Hexose, Sucrose, Phaseolus