Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.Abbreviations 9-HFCA 9-hydroxyfluorenecarboxylic acid - NPA naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - IAA indole-3-acetic acid     

Induction of hydrotropism in clinorotated seedling roots of Alaska pea,Pisum sativum L.

Roots of the agravitropic pea (Pisum sativum L.) mutantageotropum show positive hydrotropism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradlent. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropically and their weak hydrotropic response results from a counteracting effect of gravitropism.     

Light-enhanced perception of gravity in stems of intact pea seedlings

Dark-grown, 6-d-old pea seedlings (Pisum sativum L. cv. Alaska) do not respond gravitropically to brief (approx. 3 min) horizontal presentations, but seedlings given a pulse of red light (R) 16–24 h earlier respond to such stimuli by vigorous curvature of the epicotyl. With continuous horizontal stimulation (approx. 100 min), the kinetics and extent of the gravitropic response are almost identical in irradiated and dark-control plants. Prior R thus increases graviperception without altering the rate-limiting steps underlying the generation of curvature. This effect of R on graviperception develops slowly; seedlings studied only a few hours after R show differences in the kinetics of the gravitropic response, but not in presentation time. Neither the kinetics nor the extent of gravitropic curvature should be used as criteria for establishing changes in primary processes in gravitropism.     

Hydrotropism and its interaction with gravitropism in roots

We have studied hydrotropism and its interaction with gravitropism in agravitropic roots of a pea mutant and normal roots of peas (Pisum sativum L.) and maize (Zea mays L.). The interaction between hydrotropism and gravitropism in normal roots of peas or maize were also examined by nullifying the gravitropic response on a clinostat and by changing the stimulus-angle for gravistimulation. Depending on the intensity of both hydrostimulation and gravistimulation, hydrotropism and gravitropism of seedling roots strongly interact with one another. When the gravitropic response was reduced, either genetically or physiologically, the hydrotropic response of roots became more unequivocal. Also, roots more sensitive to gravity appear to require a greater moisture gradient for the induction of hydrotropism. Positive hydrotropism of roots occurred due to a differential growth in the elongation zone; the elongation was much more inhibited on the moistened side than on the dry side of the roots. It was suggested that the site of sensory perception for hydrotropism resides in the root cap, as does the sensory site for gravitropism. Furthermore, an auxin inhibitor, 2,3,5-triiodobenzoic acid (TIBA), and a calcium chelator, ethyleneglycol-bis-(-aminoethylether)-N,N,N,N- tetraacetic acid (EGTA), inhibited both hydrotropism and gravitropism in roots. These results suggest that the two tropisms share a common mechanism in the signal transduction step.     

Callose deposition during gravitropism ofZea mays andPisum sativum and its inhibition by 2-deoxy-D-glucose

In etiolated corn (Zea mays L.) and etiolated pea (Pisum sativum L.) seedlings, a gravitropic stimulation induces the deposition of callose. In the corn coleoptiles this occurs within 5 min of gravity stimulation, and prior to the beginning of curvature. Both gravitropic curvature and callose deposition reach their maxima by 12 h. Within the first 2 h more callose is deposited on the upper (concave) side, but after 2–3 h, this deposition pattern is reversed. An inhibitor of protein glycosylation, 2-deoxy-d-glucose (DDG), inhibits callose production and considerably retards gravitropic bending in both species of plants. Mannose can relieve the inhibition of gravitropic bending by DDG. The pea mutant Ageotropum, which does not respond to gravity when etiolated, also fails to produce callose in response to a gravitic stimulus. These correlations indicate that callose deposition may be a biochemical component of gravitropism in plant shoots.Abbreviation DDG 2-deoxy-d-glucose     

The role of the epidermis and cortex in gravitropic curvature of maize roots

In order to determine the role of the epidermis and cortex in gravitropic curvature of seedling roots of maize (Zea mays L. cv. Merit), the cortex on the two opposite flanks was removed from the meristem through the growing zone; gravitropic curvature was measured with the roots oriented horizontally with the cut flanks either on the upper and lower side, or on the lateral sides as a wound control. Curvature was slower in both these treatments (53° in 5 h) than in intact roots (82°), but there was no difference between the two orientations in extent and rate of curvature, nor in the latent time, showing that epidermis and cortex were not the site of action of the growth-regulating signal. The amount of cortex removed made no difference in the extent of curvature. Curvature was eliminated when the endodermis was damaged, raising the possibility that the endodermis or the stele-cortex interface controls gravitropic curvature in roots. The elongation rate of roots from which just the epidermis had been peeled was reduced by 0.01 mM auxin (indole-3-acetic acid) from 0.42 to 0.27 mm h^-1, contradicting the hypothesis that only the epidermis responds to changes in auxin activity during gravistimulation. These observations indicate that gravitropic curvature in maize roots is not driven by differential cortical cell enlargement, and that movement of growth regulator(s) from the tip to the elongating zone is unlikely to occur in the cortex.Abbreviations df degrees of freedom - IAA indole-3-acetic acid     

Hydrotropism in roots: sensing of a gradient in water potential by the root cap

Roots of the agravitropic pea (Pisum sativum L.) mutant, ageotropum, responded to a gradient in water potential as small as 0.5 MPa by growing toward the higher water potential. This positive response occurred when a sorbitol-containing agar block was unilaterally applied to the root cap but not when applied to the elongation region. Unilateral application of higher concentrations of sorbitol to the elongation region caused root curvature toward the sorbitol source, presumably because of growth reduction on the water-stressed side. The control blocks of plain agar applied to either the root cap or the elongation region did not cause significant curvature of the roots. These results demonstrate that hydrotropism in roots occurs following perception of a gradient in water potential by the root cap.     

Action of malformin A1 on gravitropic curvature in primary roots of maize (Zea mays L.)

Malformins, a small family of cyclic pentapeptides, are active plant growth regulators isolated from the fungusAspergillus niger. We purified malformin A₁ from the crude malformin A mixture, and studied its action in the gravitropic response of maize roots. Intact primary roots that had been pretreated vertically with malformin A₁ were placed in a humidified box in the horizontal position. Positive curvature (downward) was inhibited in the pretreated roots compared with the control. In addition, we measured the lateral transport of IAA in primary roots. Roots pretreated with malformin A, did not show asymmetric distribution of IAA between the upper and lower sides of the elongation zone. Malformin A, also stimulated ethylene production in maize root segments. Our results had suggested that malformin A₁ might inhibit the lateral transport of IAA across the roots from the upper to the lower side because of an increased level of ethylene. Therefore, we placed more IAA on the upper side at the initial phase of gravistimulation. These results were consistent with malformin A₁-pretreated roots showing inhibited positive gravitropic curvature.     

A novel mutant with modified tropic responses in<Emphasis Type="Italic"> Pisum sativum</Emphasis> L.

A single-gene recessive mutant which displays increased phototropic and gravitropic responses has been isolated in Pisum sativum L. cv. Torsdag and is provisionally named mtr-1, for its modified tropic response. Mutant plants attain a greater degree of bending during both phototropic and gravitropic induction due to an extension of the curvature phase. In addition to their increase in tropic curvature, mutant plants have longer and narrower leaves as mature plants, attenuated blue-light-induced ion flux responses, and lower levels of PsPK5 mRNA (a PHOT1 orthologue). Possible causes of these effects are discussed.     

Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.     

Ethylene involvement in the over-expression of Fe(III)-chelate reductase by roots of E107 pea [Pisum sativum L. (brz, brz)] and chloronerva tomato (Lycopersicon esculentum L.) mutant genotypes

Recently, ethylene was reported to be involved in the regulation of Fe(III)-chelate reducing capacity by cucumber (Cucuinis sativus L.) roots. Here, we studied the effect of two ethylene inhibitors, aminooxyacetic acid (AOA) and cobalt, on the Fe(III) reducing capacity in roots of mutant genotypes [E107 pea [Pisum sativum L. (brz, brz)] and chloronerva tomato (Lycopersicon esculentum L.) that exhibit high rates of Fe(III)-chelate reduction and excessive iron accumulation. The ethylene inhibitors, AOA and cobalt, markedly inhibited Fe(III)-chelate reducing capacity in roots of both genotypes. Over-expression of root Fe(III) reductase activity by both mutants appears to be related to ethylene. Possibly, both mutants are genetically defective in their ability to regulate root ethylene production. The large inhibitory effect of both ethylene inhibitors on Fe(III)-chelate reducing capacity in roots of the mutant tomato genotype, chloronerva, disputes the contention that the nicotianamine-Fe(II) complex is the repressior of the gene responsible for Fe(III)-chelate reductase activity, as previously suggested by others. However, since nicotianamine shares the same biosynthetic precursor as ethylene, i.e. S-adenosyl methionine, nicotianamine may affect Fe(III)-chelate reductase activity in dicot and non-grass monocot roots by influencing ethylene biosynthesis.     

Effects of Ethylene on the Kinetics of Curvature and Auxin Redistribution in Gravistimulated Roots of Zea mays

We tested the involvement of ethylene in maize (Zea mays L.) root gravitropism by measuring the kinetics of curvature and lateral auxin movement in roots treated with ethylene, inhibitors of ethylene synthesis, or inhibitors of ethylene action. In the presence of ethylene the latent period of gravitropic curvature appeared to be increased somewhat. However, ethylene-treated roots continued to curve after control roots had reached their final angle of curvature. Consequently, maximum curvature in the presence of ethylene was much greater in ethylene-treated roots than in controls. Inhibitors of ethylene biosynthesis or action had effects on the kinetics of curvature opposite to that of ethylene, i.e. the latent period appeared to be shortened somewhat while total curvature was reduced relative to that of controls. Label from applied ³H-indole-3-acetic acid was preferentially transported toward the lower side of stimulated roots. In parallel with effects on curvature, ethylene treatment delayed the development of gravity-induced asymmetric auxin movement across the root but extended its duration once initiated. The auxin transport inhibitor, 1-N-naphthylphthalamic acid reduced both gravitropic curvature and the effect of ethylene on curvature. Since neither ethylene nor inhibitors of ethylene biosynthesis or action prevented curvature, we conclude that ethylene does not mediate the primary differential growth response causing curvature. Because ethylene affects curvature and auxin transport in parallel, we suggest that ethylene modifies curvature by affecting gravity-induced lateral transport of auxin, perhaps by interfering with adaptation of the auxin transport system to the gravistimulus.     

Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model

According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legué et al. 1994, Physiol Plant 91: 559–566). After stimulation at 90°C or 135°, the transformed root tips curve, but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.Abbreviations DEZ distal elongation zone - ELISA enzymelinked immunosorbent assay - T-DNA DNA transferred from Agrobacterium rhizogenes to the plant genome This work was supported by the Centre National d'Etudes Spatiales.     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Root growth regulation and gravitropism in maize roots does not require the epidermis

We have earlier published observations showing that endogenous alterations in growth rate during gravitropism in maize roots (Zea mays L.) are unaffected by the orientation of cuts which remove epidermal and cortical tissue in the growing zone (Björkman and Cleland, 1988, Planta 176, 513–518). We concluded that the epidermis and cortex are not essential for transporting a growth-regulating signal in gravitropism or straight growth, nor for regulating the rate of tissue expansion. This conclusion has been challenged by Yang et al. (1990, Planta 180, 530–536), who contend that a shallow girdle around the entire perimeter of the root blocks gravitropic curvature and that this inhibition is the result of a requirement for epidermal cells to transport the growth-regulating signal. In this paper we demonstrate that the entire epidermis can be removed without blocking gravitropic curvature and show that the position of narrow girdles does not affect the location of curvature. We therefore conclude that the epidermis is not required for transport of a growth-regulating substance from the root cap to the growing zone, nor does it regulate the growth rate of the elongating zone of roots.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.     

On the function of pea flower feeding by Bruchus pisorum

Pea flower feeding by adult pea weevils, Bruchus pisorum (L.) (Coleoptera: Bruchidae), with special emphasis on nectar feeding, was investigated in a series of laboratory experiments. Male and female adults robbed nectar from flowers of the garden and field pea, Pisum sativum L., and females which fed on the nectar, petals, and female organs of pea flowers lived significantly longer than those denied food and water and those that fed on water only. The results of other experiments suggested that pea flower qualities other than pollen influenced the reproductive success of female B. pisorum. It is hypothesized that pollen seeking B. pisorum effected cross-pollination in the wild progenitor of the modern-day autogamous pea, and adult pea weevils of both sexes rob pea nectar to obtain a readily available source of energy to sustain flight.