Thigmomorphogenesis: the effect of mechanical perturbation on plants

Thigmomorphogenetic responses occur in many environmental settings. The most pronounced effects are found under conditions of extremely high rates of turbulent wind or water flow. However, it is an ubiquitous phenomenon, since mechanical perturbations are to be encountered under all but the most stringent laboratory conditions. Our present understanding of these phenomena is the result of studies at the ecological, anatomical, physiological, biochemical, biophysical and molecular biological levels.This contribution is dedicated to Art Galston, who gave me (M.J.) the freedom to begin my exploration of thigmophysiology.     

Stem pithiness in tomato plants: The effect of water stress and the role of abscisic acid

The pith parenchyma in tomato ( Lycopersicum esculentum ) stems was found to be disrupted in response to water stress (stem pithiness). The process of the degradation starts at the upper part of the stem and proceeds downwards as the stress is prolonged. The damage to the stem tissue was found to be irreversible upon rehydration of the stressed plants. Scanning electron microscopy revealed that the protoplast of the affected cells is disrupted first, followed by degradation of the cell wall.
Application of abscisic acid (ABA) induced pithiness in non-stressed plants and also enhanced the effect of a short period of dehydration. Kinetin, although causing severe wilting, did not induce pithiness. However, when given after a short period of water stress or within the period of stress, kinetin enhanced pithiness development.
In the course of the stress, ABA levels in the upper part of the stem and in the young leaves were higher than the levels found in the lower parts of the plant shoot. The increase in ABA levels was detected before any sign of pithiness.
It is suggested that ABA might be the triggering agent of the cellular degradation process initiated by water stress.     

Thigmomorphogenesis: The relationship of mechanical perturbation to elicitor-like activity and ethylene production

An extracellular solution obtained from bean ( Phaseolus vulgaris L. cv. Resistant Cherokee Wax) stems induced phytoalexin-like substance and ethylene production in a soybean [ Glycine max (L.) Merr. cv. Wayne] cotyledon bioassay. The elicitor-like activity for phytoalexin formation and ethylene production was increased by mechanical perturbation of bean stems. Moreover, the application of extracted or known elicitors to bean plants mimicked the effect of mechanical perturbation (i.e., inhibition of stem elongation and enhancement of radial growth). The effects of extract when applied exogenously, on elicitor-like activity in the bioassay as well as stem thickening were decreased by aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis. These results suggest that elicitor-like substances which are formed in response to mechanical perturbation contribute to the thigmomorphogenesis.     

Thigmomorphogenesis: The induction of callose formation and ethylene evolution by mechanical perturbation in bean stems

Mechanical perturbation by rubbing of the first internode of 11–12 day old plants of Phaseolus vulgaris L. cv. Cherokee wax induces the rapid deposition of callose in the cells of phloem and other tissues. Callose deposition begins immediately after mechanical perturbation, and shows a minor transient peak 1.5 h, and a major peak 6 h later. The callose gradually disappears and is gone after 3 days. If the stems are perturbed every day, the amount of callose decreases by day 2 but then gradually increases again through day 12. Both the top and bottom of the internode produce callose in response to mechanical perturbation. The evolution of ethylene in response to mechanical perturbation begins after 1 h, peaks at 2–3 h and is gone by 5–6 h. A spray of 10⁻² M 2-deoxy-D-glucose (DDG) completely blocks stem thickening, callose deposition and ethylene evolution due to mechanical perturbation. DDG at 10⁻⁵ to 10⁻⁴ M blocks callose production in mechanically perturbed stem segments and increases ethylene evolution from unperturbed stem segments to greater levels than those obtained by mechanically perturbed segments. It is concluded that mechanical perturbation of bean stems tissue induces deposition of callose more rapidly than it induces evolution of ethylene and that DDG can block both processes.     

Thigmomorphogenesis: The role of ethylene in the response of Pinus taeda and Abies fraseri to mechanical perturbation

Ethylene production was monitored for 48 h in two half-sibs of Pinus taeda L. grown in the greenhouse and given mechanical perturbation (MP) by flexing; and for 22 h in Abies fraseri (Pursh) Poir, grown in the field and exposed to wind-mediated MP. Both species produced a peak of ethylene 18 h after MP. Seedlings of P. taeda exposed to MP for the duration of the growing season (preconditioned) produced less ethylene compared to non-MP controls, with a peak production at 8 h. One half-sib which responded to MP by an increase in radial growth produced 16 times more ethylene than another half-sib which had no significant change in radial growth. Preconditioned A. fraseri produced no significant quantities of ethylene after MP. The production of wound ethylene appears to be different from MP-induced ethylene. When an ethylene-generating solution was applied to P. taeda seedlings, it mimicked many of the morphological and mechanical characteristics of MP seedlings. The putative role of ethylene in the thigmomorphogenetic response is addressed.     

Immunolocalization of mannitol dehydrogenase in celery plants and cells.

Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.     

Computational investigation of the effect of thermal perturbation on the mechanical unfolding of titin I27

The emergence of single-molecule force measurement experiments has facilitated a better understanding of protein folding pathways and the thermodynamics involved. Computational methods such as steered molecular dynamics (SMD) simulations are helpful in providing atomistic level information on the unfolding pathways. Recent experimental studies have showed that combinations of single-molecule experiments with traditional methods such as chemical and/or thermal denaturation yield additional insights into the folding phenomenon. In this study, we report results from extensive computations (a total of about 60 SMD simulations with a total length of about 0.4 μs) that address the effect of thermal perturbation on the mechanical stability of the I27 domain of the protein titin. A wide range of temperatures (280-340 K) were considered for the pulling, which was done at both constant velocity and constant force using SMD simulations. Good agreement with experimental data, such as for the trends in changes in average force and the maximum force with respect to the temperature, was obtained. This study identifies two competing pathways for the mechanical unfolding of I27, and illustrates the significance of combining various techniques to examine protein folding.     

Ethyl methanesulfonate treatment of celery plants affects the expression pattern of CEL I endonuclease

CEL I enzyme from celery, as a member of S1 family of nucleases, is known for its high specific activity in recognition and cleavage of base-substitution mismatches on heteroduplex DNA molecules. Despite valuable applications of the enzyme in mutation screening studies, little is known about its function at cellular level. In the present study, we investigated the pattern of CEL I expression in ethyl methanesulfonate (EMS) treated celery plants. An abnormal growth pattern along with wide and clear lesions were observed on the treated plants. A considerable increase in the level of CEL I protein happened in vegetative and generative parts of EMS-treated plants compared with controls. Despite such induction, the enzyme is not expected to be involved in DNA repair during EMS treatment due to the absence of any known nuclear localization signal in the deduced sequence of CEL I protein. Considering the fact that CEL I orthologs are induced during programmed cell death, the high expression of CEL I upon EMS treatment could be due to the stress and necrotic cell death created by the treatment.     

The involvement of gibberellin 20-oxidase genes in phytochrome-regulated petiole elongation of Arabidopsis

Long day (LD) exposure of rosette plants causes rapid stem/petiole elongation, a more vertical growth habit, and flowering; all changes are suggestive of a role for the gibberellin (GA) plant growth regulators. For Arabidopsis (Arabidopsis thaliana) L. (Heynh), we show that enhancement of petiole elongation by a far-red (FR)-rich LD is mimicked by a brief (10 min) end-of-day (EOD) FR exposure in short day (SD). The EOD response shows red (R)/FR photoreversibility and is not affected in a phytochrome (PHY) A mutant so it is mediated by PHYB and related PHYs. FR photoconversion of PHYB to an inactive form activates a signaling pathway, leading to increased GA biosynthesis. Of 10 GA biosynthetic genes, expression of the 20-oxidase, AtGA20ox2, responded most to FR (up to a 40-fold increase within 3 h). AtGA20ox1 also responded but to a lesser extent. Stimulation of petiole elongation by EOD FR is reduced in a transgenic AtGA20ox2 hairpin gene silencing line. By contrast, it was only in SD that a T-DNA insertional mutant of AtGA20ox1 (ga5-3) showed reduced response. Circadian entrainment to a daytime pattern provides an explanation for the SD expression of AtGA20ox1. Conversely, the strong EOD/LD FR responses of AtGA20ox2 may reflect its independence of circadian regulation. While FR acting via PHYB increases expression of AtGA20ox2, other GA biosynthetic genes are known to respond to R rather than FR light and/or to other PHYs. Thus, there must be different signal transduction pathways, one at least showing a positive response to active PHYB and another showing a negative response.     

Thigmomorphogenesis: membrane lipid and protein changes in bean plants as affected by mechanical perturbation and Ethrel

When bean plants (Phaseolus vulgaris L. cv. Red Cherokee Bush) are mechanically perturbed by rubbing, their stem elongation is inhibited and the stem thickness increases. The decrease in cell elongation and the increase in lateral cell divisions, which are partially responsible for this syndrome, were correlated with a decrease in the tree fatty acids and in the phospholipids of the membranes of microsomal fractions of first and second internodes of mechanically stimulated plants. This was true even though only the first internode was mechanically stimulated. Of the microsomal free fatty acids, mechanical perturbation induced an increase in myristic acid and large decreases in stearic, oleic, linoleic and linolenic acids. It also reduced the unsaturated:saturated ratio of the fatty acids. It induced a decrease in phosphatidyl choline but an increase in phosphatidyl ethanolamine. When the fatty acids were cleaved from the microsomal phospholipids, mechanical perturbation caused only a slight decrease in the unsaturated:saturated ratio and no significant changes in the double bond index. Mechanical perturbation induced an increase in the total microsomal protein and of membrane-associated latent IDPase. However, the activity of membrane-associated KCN-insensitive NADPH cytochrome-c reductase was decreased by mechanical perturbation. Treatment of the first internode with exogenous Ethrel produced results that were very similar in all respects to those obtained by mechanical perturbation. The factors inducing hardening against frost and drought, as achieved by mechanical perturbation and Ethrel treatment, are not only related to sterols or the polar head-groups of phospholipids but may also be related to the protein components, and all may have an effect on the fluidity of a bilayer membrane model. These data support the hypothesis that ethylene mediates thigmomorphogenesis and that mechanical perturbation of the first internode results in the acropetal transport of a translocatable thigmomorphogenetic factor.     

The effect of the substance exin on development of microbial infections and isolation of ethylene in plants

Effects of Exin on infection of tomato, potato, and cabbage plants with Pseudomonas solanacearum and Erwinia carotovora and a fungus Sclerotium rolfsii were studied. The treatment of infected plants with Exin caused no significant effect on the development of the disease. Treatment with streptomycin as a standard for comparison completely inhibited the growth of these microorganisms. Pretreatment with Exin one to eight days before infecting inhibited the development of diseases. The numbers of tomato and potato plants damaged among those infected with P. solanacearum were lower by 10 and 35% respectively. In field experiments (350 plants per variant), treatment with Exin decreased the development of wilt caused by S. rolfsii and P. solanacearum and rot caused by E. carotovora. Treatment with Exin activated the release of ethylene for not less than 30 days. Possible mechanisms of the effects of Exin are discussed.     

The involvement of ethylene in liatris corm dormancy

Corms of liatris (L. spicata, cv. Callilepsis) show a seasonal dormancy, being most active in the November harvest and least active in June. Storage of dormant corms at 3 °C for about 9 weeks resulted in a complete break of dormancy. This was accompanied by a sharp temporal increase in their rate of ethylene production, which was more pronounced in the buds than in the parenchyma tissue. Application of ethylene to the corms in the form of ethrel solution increased both ethylene production rate and sprouting. The ethylene-forming activity from ACC, measured both in vivo and in vitro, was higher in corms producing more ethylene. However, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) of the corms was inversely related to their ethylene production rate. Ethylene thus seems to be involved in the dormancy control of liatris corms, and its production is apparently regulated mainly by the activity of the membranous ethylene-forming system.     

Internode length in Pisum. The involvement of ethylene with the gibberellin-insensitive erectoides phenotype

The gene lk in peas ( Pisum sativum L.) confers the erectoides phenotype. This phenotype possesses much reduced internode and petiole lengths and is practically insensitive to applied GA₁, compared with Lk plants. Application of the ethylene synthesis inhibitor, aminoethoxyvinylglycine (AVG), resulted in increased internode elongation and increased GA-sensitivity in lk plants, but not in the Lk line, L53. The ethylene-releasing compound, ethephon, had the reverse effect when applied to the Lk line, L58, reducing internode length and GA-sensitivity. Ethylene production was higher in lk segregates than in Lk segregates under the conditions used, and the shoot anatomy of lk segregates was consistent with these higher ethylene levels.
These results suggest that the phenotypic effects of gene lk may be due, at least in part, to increased ethylene production in erectoides plants. However, AVG application to lk plants did not produce true phenocopies of comparable lk plants and ethephon application to Lk plants did not produce the erectoides phenotype. Further work is therefore required to determine whether the effect of the gene lk on ethylene production is the primary action of this gene or merely a secondary consequence.     

The involvement of ethylene in the coiled sprout disorder of potato

Application of low concentrations of ethylene gas to the sprouts of Arran Pilot elicited many of the morphological and anatomical symptoms of the coiled sprout disorder. Similar effects were observed using 2-chloroethyl-phosphonic acid (Ethrel). Obstruction of coil-prone etiolated sprouts led to enhanced ethylene production for a period of two to three days but this pattern was not shown by non-coil-prone sprouts.     

The role of ethylene in the flowering response of bulbous plants

Ethylene induces the flower formation and stimulates the flower-bud development of some bulbous plants exposed to the gas when the apex is in the vegetative state. For iris bulbs cv. Ideal maximum responses have been found after exposure to 5 ppm for 8 h; lower concentrations, shorter exposure periods and, depending on seasonal conditions, low temperatures during gas treatment, gave intermediate responses. The effects are opposite to the ethylene induced flower-bud blasting which occurs when bulbous plants are exposed to the gas after completion of the flower formation. Dry storage of the bulbs in atmospheres containing 5% CO₂ reduces the temperature-enhanced flower formation, suggesting a possible effect of endogenous ethylene. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22 1984 at Liblice, Czechoslovakia.     

A kinetic analysis of hyponastic growth and petiole elongation upon ethylene exposure in Rumex palustris

Background and Aims

Complete submergence is an important stress factor for many terrestrial plants, and a limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris is one such species and manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment. This study aimed to investigate the kinetics of ethylene-induced leaf hyponasty and petiole elongation.

Methods

Leaf hyponasty and petiole elongation was studied using a computerized digital camera set-up followed by image analyses. Linear variable displacement transducers were used for fine resolution monitoring and measurement of petiole growth rates.

Key Results

We show that submergence-induced hyponastic growth and petiole elongation in R. palustris can be mimicked by exposing plants to ethylene. The petiole elongation response to ethylene is shown to depend on the initial angle of the petiole. When petiole angles were artificially kept at 0°, rather than the natural angle of 35°, ethylene could not induce enhanced petiole elongation. This is very similar to submergence studies and confirms the idea that there are endogenous, angle-dependent signals that influence the petiole elongation response to ethylene.

Conclusions

Our data suggest that submergence and ethylene-induced hyponastic growth and enhanced petiole elongation responses in R. palustris are largely similar. However, there are some differences that may relate to the complexity of the submergence treatment as compared with an ethylene treatment.     

The effect of core destabilization on the mechanical resistance of I27

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.