Comparison of electric and growth responses to excision in cucumber and pea seedlings. II. Long-distance effects are caused by the release of xylem pressure

Excision of a growing stem causes local wound responses, such as membrane depolarization and growth inhibition, as well as effects at larger distances from the cut. In this study, cucumber hypocotyls were excised 100mm below the hook, so that the growing region was beyond the reach of the wound-induced depolarization (up to 40mm). Even at such a distance, the cut still caused a considerable and rapid drop in the hypocotyl growth rate. This growth response is not a direct wound response because it does not result from the cut-induced depolarization and because it can be simulated by root pressure manipulation (using a pressure chamber). The results indicate that the growth response resulted from the rapid release of the xylem pressure upon excision. To test this conclusion we measured the xylem pressure by connecting a pressure probe to the cut surface of the stem. Xylem pressure (P_x) was found to be +10 to +40kPa in cucumber hypocotyls and -5 to -10 kPa or lower in pea epicotyls. Excision of the cucumber hypocotyl base led to a rapid drop in P_x to negative values, whereas excision in pea led to a rapid rise in P_x to ambient (zero) pressure. These fast and opposite p_x changes parallel the excision-induced changes in growth rate (GR): a decrease in cucumber and a rise in pea. The sign of the endogenous xylem pressure also determined whether excision induced a propagating depolarization in the form of a slow wave potential (SWP). Under normal circumstances pea seedlings generated an SWP upon excision whereas cucumber seedlings failed to do so. When the P_x in cucumber hypocotyls was experimentally inverted to negative values by incubating the cumber roots in solutions of NaCN or n-ethylmaleimide, excision caused a propagating depolarization (SWP). The experiment shows that only hydraulic signals in the form of positive P_x steps are converted into propagating electric SWP signals. These propagating depolarizations might be causally linked to systemic ‘wound’ responses, which occur independently of the short-distance or direct wound responses.     

Slow wave potentials in cucumber differ in form and growth effect from those in pea seedlings

A positive hydraulic signal in the form of a xylem pressure step was applied to the roots of intact seedlings of Cucumis sativus L. and Pisum sativum L. Surface electrodes at three positions along the epicotyl/hypocotyl recorded a propagating depolarization which appeared first in the basal, then the central and sometimes the apical electrode positions and fitted the characteristics of a slow wave potential (SWP). This depolarization differed between pea and cucumber. It was transient in cells of pea epicotyls but sustained in cucumber hypocotyls. It was not associated with a change in cell input resistance in pea epicotyls but preceded an increase in the input resistance of cucumber hypocotyl cells. With the increased xylem pressure the growth rate (GR) of cucumber hypocotyls and pea epicotyls underwent a transient increase, peaking after 5 min. If the depolarization reached the growing upper region, it preceded a sustained decrease in the GR of cucumber hypocotyls but only a transient decrease in the GR of pea epicotyls. A temperature jump in the root medium (heat treatment) induced a steep pressure spike in the xylem of the cucumber hypocotyl which showed similar electric and growth effects as the previously applied, non-injurious pressure steps. We suggest that the observed differences in the electric and growth responses between the species were caused by the closure of ion channels in depolarized cells of cucumber but not pea seedlings.     

A reduced xylem pressure altered the electric and growth responses in cucumber hypocotyls

This study measured the electric and growth responses in excised cucumber hypocotyls and compared them with those in intact seedlings. Root excision (first severing cut) eliminated most of the positive xylem pressure (P_x) in the hypocotyl, caused a rapid, transient drop in the hypocotyl growth rate (GR) and some small, local depolarization near the cut site. Although accompanied by a smaller decrease in P_x, a second, severing cut in the basal hypocotyl caused a decrease in GR which was no longer transient and a depolarization which was increased in both size and extent. These changes were not wound effects because they could be simulated by root incubation in mannitol. The reduced GR recovery occurred also in the absence of electric changes after a second increase in the mannitol concentration incubating the root of intact seedlings. Increased electric sensitivity and altered growth response therefore appear to be two independent examples of physiological changes resulting from a lowered P_x.     

Mannitol inhibits growth of intact cucumber but not pea seedlings by mechanically collapsing the root pressure

The positive xylem pressure (Px) in cucumber hypocotyls is a direct extension of root pressure and therefore depends on the root environment. Solutions of the electrolyte KCl (0-10 osm) reduced the hypocotyl Px transiently (biphasic response), while the Px reduction by mannitol solutions was sustained. The amplitudes of the induced Px reduction depended directly, and the degree of Px restoration after stress release depended indirectly, on the size of the initial positive Px indicating that mannitol released the root pressure by a mechanical rather than osmotic mechanism. Mannitol treatment and other means of root pressure reduction revealed two separate growth responses in the affected cucumber hypocotyls. Only steep Px drops (following root excision or root pressure release in mannitol) directly cause a rapid, transient drop in growth rate (GR). Both rapid and slow (after root incubation in KCN or NEM) decreases in root pressure, however, led to a sustained growth inhibition of cucumber hypocotyls after about 30 min. This delay characterizes the growth response as an indirect consequence of the Px change. Pea seedlings, which lacked root pressure and had a negative Px throughout, showed extremely small changes in epicotyl Px and GR after root incubation in mannitol. It is apparent that the higher sensitivity of cucumber growth to mannitol depended on the presence and release of root pressure.     

Maximum axial root growth pressure in pea seedlings: effects of measurement techniques and cultivars

Values of the maximum axial growth pressure (σ_max) of seedling pea (Pisum sativum L.) roots reported in the literature, obtained using different apparatuses and cultivars, range from 0.3 MPa to 1.3 MPa. To investigate possible reasons for this large range, we studied the effect of apparatus and cultivar on measurements of σ_max in peas. We describe four types of apparatus which can be used to measure axial root growth force and hence σ_max, and used them to measure σ_max in seedling pea roots using cultivar Meteor. Two of these apparatuses were also used to compare σ_max for three pea cultivars (Helka, Meteor and Greenfeast). Both cultivar and apparatus significantly affected σ_max , but there were greater differences between apparatuses than between the three cultivars. Estimating root cross-sectional area from the diameter of cross-sections, rather than from in situ measurements (i.e. measurements made with the root still in place in the apparatus) may explain these differences. This revised version was published online in June 2006 with corrections to the Cover Date.     

The distribution of hydroxyproline-rich glycoprotein mRNAs in shoots of cucumber and pea

The distribution of hydroxyproline-rich glycoprotein (HRGP) mRNAs in the shoots of dark-grown and irradiated cucumber ( Cucumis sativus L. cv. Burpee pickler) and pea ( Pisum sativum L. cv. Alaska) was studied. A cloned genomic DNA fragment encoding carrot ( Daucus carota ) root extensin (pDC5A1) was used to measure HRGP mRNAs from cucumber and pea along the length of dark-grown and irradiated shoots. There was a marked difference in the levels of HRGP mRNAs isolated from apical and basal regions of cucumber. Whereas apical, elongating regions had low levels of HRGP mRNAs, basal regions of the shoot had high levels. Levels of HRGP mRNAs were also compared in shoots of dark-grown and irradiated cucumber. Although light inhibits hypocotyl growth, it had no effect on levels of HRGP mRNAs. There was no gradient in the distribution of HRGP mRNAs along the epicotyl of dark-grown pea. As was the case with cucumber, light did not affect the accumulation of HRGP mRNAs in pea shoots. We conclude that light does not affect elongation by regulating the accumulation of HRGP mRNAs. The gradient of accumulation of HRGP mRNAs along the hypocotyl of cucumber probably reflects differences in cellular differentiation along the shoot.     

Induction of peroxidases in cucumber hypocotyls by wounding and fungal infection

The induction of peroxidases (EC 1.11.1.7) during elicitation of lignification by α-1,4-linked oligogalacturonides in cucumber hypocotyl segments ( Cucumis sativus L. cv, Wisconsin SMR 58) was investigated. The wounding associated with the preparation of hypocotyl segments induced a 19-fold increase in peroxidase activity during the following 72 h. The increase was partially due to an increase in activity of a constitutive peroxidase with a pI of 8.9 and partially due to the expression of new peroxidase isozymes with pIs of 3.8, 5.4, 6.2, 9.1 and 9.4. The oligogalacturonides did not induce any peroxidase activity in addition to the wound-induced activity. These results suggest that either the constitutive peroxidase isozyme (pI 8.9) of intact hypocotyls or some of the wound-induced peroxidases are involved in the oligogalacturonide-induced lignification.
Induction of the peroxidases by wounding was inhibited by cycloheximide. This indicates that they accumulate as a result of de novo protein synthesis. Actinomycin D caused only a modest inhibition of the wound-induction peroxidases, indicating that the process is regulated at the level of translation.
Peroxidase activity increased more rapidly in resistant than in susceptible cucumber hypocotyls after inoculation with the pathogen Cladosporium cucumerinum Ellis & Arthur. The pattern of isozymes which was induced by fungal infection of resistant hypocotyls was similar to the pattern of isozymes induced by wounding. This suggests that similar induction mechanisms may be involved in the two processes.     

Influence of electrolytes on growth, phototropism, nutation and surface potential in etiolated cucumber seedlings

A variety of electrolytes (10-30 mol m-3) increased the relative growth rate of etiolated cucumber (Cucumis sativus L. cv. Burpee's Pickler) hypocotyls by 20-50% relative to water-only controls. The nonelectrolyte mannitol inhibited growth by 10%. All salts tested were effective, regardless of chemical composition or valence. Measurements of cell-sap osmolality ruled out an osmotic mechanism for the growth stimulation by electrolytes. This, and the nonspecificity of the response, indicate that an electrical property of the solutions was responsible for their growth-stimulating activity. Measurements of surface electrical potential supported this reasoning. Treatment with electrolytes also enhanced nutation and altered the pattern of phototropic curvature development. A novel analytical method for quantitating these effects on growth was developed. The evidence indicates that electrolytes influence an electrophysiological parameter that is involved in the control of cell expansion and the coordination of growth underlying tropisms and nutations.     

Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm · s^–1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a slow-wave potential. These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.Abbreviations and Symbols GR growth rate - P_x xylem pressure - R_in cell input resistance - SWP slow wave potential - V_m membrane potential - V_s surface potential This work was supported by grants to D.J.C. from the National Science Foundation and the U.S. Department of Energy.     

The rates of shoot and root growth in intact plants of pea mutants in leaf morphology

Isogenic lines of pea (Pisum sativum L.) with the genetically determined changes in leaf morphology, afila (af) and tendril-less (tl), were used to study the relationship between shoot and root growth rates. The time-course of shoot and root growth was followed during the pre-floral period in the intact plants grown under similar conditions. The af mutation produced afila leaves without leaflets, whereas in the case of the tl mutations, tendrils were substituted with leaflets, and acacia-like leaves were developed. Due to the changes in leaf morphology caused by these mutations, pea genotypes differed in leaf area: starting from day 7, the leaf area was lower in the af plants and larger in the tl plants as compared to the wild-type plants. Such divergence was amplified in the course of plant development and reached its maximum immediately before the transition to flowering. Plants of isogenic lines did not notably differ in stem surface areas. In spite of significant difference in total leaf area, the wild type and tl plants did not differ in leaf dry weight. Starting from leaf 9, the af plants lagged behind two leaflet-bearing genotypes (wild type and tl) in leaf dry weight, whereas stem dry weight was similar in the wild type and tl forms and slightly lower in the af plants. Root dry weights were practically similar in the wild type and tl plants until flowering. The reduction of leaf area in the af plants drastically reduced root dry weight. In other words, the latter index was related to the total weight and total area of leaves and stems. The correlation analysis demonstrated an extremely low relationship between leaf and stem area and dry weight and those of roots early in plant development (when plants develop five to seven leaves). Later, immediately before flowering (nine to eleven leaves), root weight was positively related to leaf weight and area; however, stem area and root weight did not correlate. Thus, in three genotypes (wild type, af, and tl), at the end of their vegetative growth phase, leaf and root biomass accumulated in proportion, independently of leaf area expansion.     

Rapid, bilateral changes in growth rate and curvature during gravitropism of cucumber hypocotyls: implications for mechanism of growth control

Abstract. The growth response of etiolated cucumber ( Cucumis sativus L.) hypocotyls to gravitropic stimulation was examined by means of time-lapse photography and high-resolution analysis of surface expansion and curvature. In comparison with video analysis, the technique described here has five- to 20-fold better resolution; moreover, the mathematical fitting method (cubic splines) allows direct estimation of local and integrated curvature. After switching seedlings from a vertical to horizontal position, both upper and lower surfaces of the stem reacted after a lag of about 11 min with a two- to three-fold increase in surface expansion rate on the lower side and a cessation of expansion, or slight compression, on the upper surface. This growth asymmetry was initiated simultancously along the length of the hypocotyl, on both upper and lower surfaces, and did not migrate basipetally from the apex. Later stages in the gravitropic response involved a complex reversal of the growth asymmetry, with the net result being a basipetal migration of the curved region. This secondary growth reversal may reflect oscillatory and or self-regulatory behaviour of growing cells. With some qualifications, the kinetics and pattern of growth response are consistent with a mechanism involving hormone redistribution, although they do not prove such a mechanism. The growth kinetics require a growth mechanism which can be stimulated by two-to three-fold or completely inhibited within a few minutes.     

Reversion of the apical hook of pea in the dark and its promotion by a red light pulse.

At the developmental stage at which the apical hook passed the 3rd and 4th nodes, dark-grown seedlings of pea ( Pisum sativum L. cv. Progress No.9) opened the hook upright and then formed a new hook above the node nearly in the opposite direction to the previous one. In cv. Alaska, in contrast, many (about 84%) seedlings closed the hook in the original direction after they partially (up to about 110°) opened it at the 3rd node, thus doing a wagging movement, while a small percentage (about 16%) of the seedlings reversed the hook direction. Exposure to red light of cv. Alaska seedlings for 10 min increased the percentage of the hook reversion up to 71% or more. The hook reversion was never observed except when the hook part passed the nodes, suggesting the involvement of the nodes in the phenomenon.     

Inhibition and recovery of growth processes in roots of pisum sativum L. Exposed to 60-Hz electric fields

Roots of Pisum sativum L. were chronically exposed in aqueous inorganic nutrient medium to 60-Hz electric fields between 140 and 490 V/m (growth medium conductivity ～ 0.08 S/m). The growth rate, meristematic mitotic index, and growth rate recovery of the roots were determined. At 140 V/m there was no perturbation in growth rate or mitotic index. At 430 V/m the growth rate and the mitotic index were reduced. The mitotic index had a maximum depression (～ 55% of control), which occurred at 4 h. The depression in growth rate was immediate and constant over time. When roots were exposed to an electric field at 430 V/m for 2 days, the growth rate was depressed by about 40%. When the field was terminated, the growth rate steadily increased and was almost normal after 5 days. At 490 V/m root growth rate was almost completely arrested. According to these results, there is a narrow range of induced membrane potentials that span the range from slightly altered to almost completely arrested growth rates.     

The contribution of fruit photosynthesis to the carbon requirement of cucumber fruits as affected by irradiance, temperature and ontogeny

The photosynthetic contribution of a fruit to its carbon requirement throughout ontogeny and under different growing conditions was quantified in cucumber ( Cucumis sativus L. cv. Corona). In addition, the effects of shading on fruit dry matter accumulation and the diurnal course of the elongation rate were studied. Fruit darkening had no photomorphogenic effect on fruit growth, while the cumulative photosynthetic contribution of a fruit to its own carbon requirement ranged from 1 to 5%. During the day there was always a net CO₂ efflux. The photosynthetic rate per fruit, calculated as the difference between rates of CO₂ exchange in light and dark, increased during fruit ontogeny, while the photosynthetic rate per unit fruit surface area declined. The latter was not dependent on fruit size. The photosynthetic activity per unit surface area of fruits was estimated to be about 20–30% as efficient as that of leaves. The rate of calculated photosynthesis was reduced by 60–65% when the photosynthetically active radiation incident on the fruit decreased from 200 to 50 μmol m⁻² s⁻¹. Temperature (20–30°C) had no pronounced effect on the rate of calculated fruit photosynthesis when fruits of the same developmental stage (temperature sum) were compared. However, the relative photosynthetic contribution of a fruit to its carbon requirement increased when temperature decreased. Moreover, this contribution increased when irradiance increased or fruit growth was reduced by competing fruits. During fruit ontogeny the daily photosynthetic contribution was highest (up to 15%) in young and old fruits, with a small growth rate.     

Biophysics of the growth responses of pea roots to changes in penetration resistance

Two experiments were performed in simplified soil-less systems to study how roots respond to changes in mechanical impedance. In the first the increases in root force and diameter that occur when a pea root was impeded mechanically inside a hole with rigid conical walls were determined. The experiment was performed at 8°C and at 25°C, and the root growth pressures generated were calculated during periods of 12, 24, 48 and 72 hours. The maximum growth pressures generated were approximately the same at both temperatures, although the maximum pressure was achieved approximately twice as quickly at 25°C than at 8°C, being reached within 15–20 hours. In the second set of experiments a new technique was developed to measure simultaneously the elongation rate and the force exerted by the roots of seedlings grown in moist air. A constant force was exerted by a force transducer on a pea radicle using a system of pulleys, and the elongation rate of the pea root was monitored using a linear variable differential transformer (LVDT). The changes in root elongation rate were recorded that occurred in response to increases and decreases in the applied force. Root elongation rate decreased by more than 50% within 30 min of increasing the applied force by 100 mN. A similarly fast, but smaller increase in growth rate occurred when the force was removed. The interpretation of results from both studies will be discussed in terms of a modified form of the Lockhart model of growth.     

Genetics of resistance to Fusarium oxysporum f.sp. cucumerinum races 1 and 2 in cucumber line Wisconsin-2757

The mode of inheritance of resistance to Fusarium oxysporum f.sp. cucumerinum races 1 and 2 in Wisconsin-2757 (WI-2757), a gynoecious cucumber (Cucumis sativus L.), was determined by analysing segregation of F₁, F₂ and BC₁ populations of crosses with susceptible cultivar Straight-8. Resistance to either race 1 or race 2 in WI-2757 was conferred by a single dominant gene. In allelism tests, resistance to either race in WI-2757 was determined by the gene Fcu-1, which also confers resistance in line SMR-18.     

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

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

Internode length in Pisum. Comparison of genotypes in the light and dark

There is a strong relationship across the full range of gibberellin deficient, internode length genotypes ( le, lh, is, na ) between internode length in the dark and in red or white light. Further, the new, more severe allele at the le locus. Ie ^d, is shown to influence growth in the dark as well as in the light. These results suggest that darkeness does not specifically overcome any of the steps blocked by the gibberellin (GA) synthesis genes contrasting with the conclusions drawn by other workers. Supporting this conclusion in relation to the Ie gene are results which show that, at least at certain dosage rates, dark-grown Ie na plants respond better to GA₁ than to GA₂₀ similar to the response previously reported in light grown plants.
The greater response by plants of the nana line NGB1766 ( na ) to GA₁ in the dark than in the light suggests that light may influence internode length by altering GA-sensitivity. These results are discussed in relation to previous views on the control of stem elongation by light.     

Gibberellin-enhanced sugar accumulation in growing subhooks of etiolated Pisum sativum seedlings. Effects of gibberellic acid, indoleacetic acid and cycloheximide on invertase activity, sugar accumulation and growth

The possible involvement of invertase in the action of gibberellic acid (GA) on stimulating sugar accumulation in growing subhooks of Alaska pea ( Pisum sativum L. cv. Alaska) was studied. GA and indoleacetic acid (IAA) stimulated elongation growth to a similar extent. GA, in contrast to IAA, increased the amount of soluble sugars in the subhook. GA substantially increased invertase activity whereas IAA did not. These results suggest that the mode of action of GA and IAA differs, although both stimulate pea subhook growth.
Cycloheximide (CH) inhibited the effect of GA on invertase activity, accumulation of soluble sugars, and elongation growth. Good correlations were found between invertase activity, the amount of soluble sugars and growth. The results suggest that GA-induced enhancement of sugar accumulation in the subhook cells is dependent on increased invertase activity. The sugar accumulated in the subhook may be involved in growth promotion by GA.