Tree Physiology 25, 1101–1108 © 2005 Heron Publishing—Victoria, Canada
Shoot water status and ABA responses of transgenic hybrid larch Larix kaempferi × L. decidua to ectomycorrhizal fungi and osmotic stress ANA RINCÓN,1 OUTI PRIHA,1 MARIE-ANNE LELU-WALTER,2 MAGDA BONNET,3 BRUNO SOTTA3 and FRANÇOIS LE TACON1,4 1
Unité Mixte de Recherches 1136, Interactions Arbres-Microorganismes, INRA Centre de Nancy, 54280 Champenoux, France
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UR 588, Unité Amélioration, Génétique et Physiologie Forestières (UAGPF), Équipe C.A.M.B.I.U.M., INRA, Centre de Recherches d’Orléans, avenue de la Pomme de Pin, Ardon, BP 206 19, 45166 Olivet cedex, France
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Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, UMR CNRS 7632, Université Pierre et Marie Curie, le Raphaël, 3 rue Galilée, 94200 Ivry sur Seine, France
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Corresponding author (
[email protected])
Received August 16, 2004; accepted January 15, 2005; published online July 4, 2005
Summary It has been postulated that osmotic effects on plant tissue are mediated by abscisic acid (ABA). Hybrid larch (Larix kaempferi (Lambert) Carr. × L. decidua Mill.) plantlets, transformed with the ABA-inducible wheat Em promoter associated with the Gus reporter gene, were axenically inoculated with two ectomycorrhizal fungi: Cenococcum geophilum Fr., considered tolerant to water stress, and Laccaria bicolor (Marie) Orton, considered less tolerant to drought. The mycorrhizal and non-mycorrhizal transgenic plantlets were subjected to osmotic stress by adding polyethylene glycol (PEG) to the culture medium. In the presence of PEG, L. bicolor and C. geophilum reduced shoot water potential and turgor potential, but increased host osmotic potential. Treatment of plantlets with PEG induced a significant increase in endogenous ABA concentrations. Laccaria bicolor and C. geophilum behaved similarly and significantly decreased the ABA response of plantlets to PEG treatment. Moreover, inoculation with either fungus regulated the ABA response of the plantlets even when the fungus was separated from the host by a cellophane sheet that prevented mycorrhiza formation. Although the wheat Em promoter was inducible in larch plantlets, it was not regulated by endogenous ABA. Induction of the wheat Em promoter in larch plantlets depended on organ type, with maximum induction in the root apex. Induction of the Em promoter was significantly decreased by mycorrhizal inoculation. Keywords: Cenococcum geophilum, Em promoter, GUS activity, Laccaria bicolor, osmotic potential, water potential.
Introduction Mycorrhizal associations have positive, negative or neutral effects on plant–water relations (Augé 2001). Improved plant drought tolerance as a result of association with mycorrhizal fungi has often been reported (Allen 1991, Smith and Read 1997) and has been attributed to mechanisms such as enhanced
water uptake, maintenance of a higher stomatal conductance, improved osmotic adjustment and improved nutritional status in mycorrhizal plants (Dosskey et al. 1991, Guehl et al. 1992, Morte et al. 2001). Several studies have shown that water, osmotic and turgor potentials of mycorrhizal plants differ from those of nonmycorrhizal plants. Nevertheless, mycorrhizal effects on plant–water relations are often subtle, transient and complex. For example, at the end of a drought period, the difference in leaf water potential between mycorrhizal and non-mycorrhizal Pinus halepensis Mill. seedlings was about 0.4 MPa, whereas osmotic potential was unchanged (Morte et al. 2001). Mycorrhizal fungi also affect the hormonal balance of plants (Slankis 1973, Barker and Tagu 2000). Increased abscisic acid (ABA) concentrations are usually reported in plants subjected to water stress, and it has been proposed that these changes in ABA concentration act either as a signal that induces osmotic adjustment, or directly on stomatal regulation. The association with mycorrhizal fungi can affect endogenous ABA concentrations in plants (Coleman et al. 1990, Danneberg et al. 1992, Goicoechea et al. 1997). A modification of internal ABA concentrations could differentially activate ABAresponsive genes and induce changes in mRNA synthesis, giving rise to an adaptive response (Skriver and Mundy 1990, Campalans et al. 1999). Despite numerous studies, the combined effects of water stress and mycorrhizal infection on ABA concentrations in roots and shoots remain unclear. Moreover, there have been relatively few studies on the possible role of ABA in ectomycorrhizas under drought conditions. One objective of this work was to gain insight into the roles of ABA and ectomycorrhizas in response to osmotic stress. We took two approaches to determine changes in ABA concentration in response to osmotic stress. The first was to use a monoclonal anti-ABA antibody to measure ABA concentration in tissues directly. The second was to measure the expression of the wheat Em promoter associated with the β-glucur-
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onidase (Gus) reporter gene in transgenic larch (Martin et al. 1992). In wheat, the Em gene is regulated by ABA and osmotic stress at the transcriptional and posttranscriptional levels (Williamson and Quatrano 1988, Bostock and Quatrano 1992). Numerous studies have shown that ectomycorrhizal fungi release substances having a growth-regulating or hormonal effect on the development of their host (Slankis 1973, Ek et al. 1983, Gay et al. 1994, Karabaghli-Degron et al. 1998). Consequently, another objective of this work was to determine whether ectomycorrhizal fungi affect ABA production by their host during osmotic stress through diffusible metabolites even without direct contact between the fungus and its host. Study of the expression of the Em gene could also help elucidate the possible role of ectomycorrhizal fungi in the regulation of proteins involved in membrane water-binding capacity (Bray 1993). Expression of the Em gene occurs mainly during the late stages of seed development (Hughes and Galau 1989). Late embryogenesis abundant (Lea) genes are a large and diverse group of genes expressed at high levels during the late stages of seed development. Five major groups of LEA proteins have been described. The Em genes belong to Group I of the Lea genes (Vicient et al. 2000). The amount of Em mRNA increases during maturation of wheat seeds and the Em gene is also expressed in somatic embryos and vegetative tissues subjected to ABA or osmotic stress (Hetherington and Quatrano 1991). Although the precise function of the EM and LEA proteins remains unclear, they are believed to play a role in desiccation tolerance (Galau et al. 1987) and in the modification of the hydrophilic properties of plant tissues.
Fungal material and inoculation Two ectomycorrhizal fungi Laccaria bicolor (Marie) Orton strain S238N, considered semi-tolerant to drought (Mexal and Reid 1973, Coleman et al. 1989), and Cenococcum geophilum Fr. strain 147.54 (Centraal Bureau voor Schimmelcultures, The Netherlands), considered tolerant to water stress, were cultivated on Pachlewski agar medium (Pachlewski and Pachlewska 1974) at 25 °C for 4 weeks. To determine the effects of the fungi on the host, roots were either directly associated with the fungal strains by placing four plugs of actively growing mycelium (collected at the edge of the colonies) on the root elongation zone (the first mycorrhizas appeared 2 weeks after inoculation), or four plugs of actively growing mycelium were placed on the root elongation zone, but separated from the root by a cellophane membrane to prevent root colonization and mycorrhiza formation. Osmotic stress Five weeks after inoculation, the plantlets were transferred with their cellophane sheet to new petri dishes filled with autoclaved glass beads (3 mm in diameter) and liquid MSG medium (sucrose reduced to 1 g l – 1 ). Osmotic stress was imposed by adding 30% (w/v) polyethylene glycol 3350 (PEG) (Sigma-Aldrich, St. Louis, MO) to the liquid culture medium. The osmotic potential of the medium was measured with a vapor pressure osmometer (Wescor 5500, Logan, UT). The osmotic potential was 0.06 MPa in the control treatment without PEG and –1.5 MPa in the treatment with PEG. Roots were covered with a filter paper humidified with the corresponding medium. The petri dishes with the plantlets were maintained in a horizontal position. The PEG osmotic stress was applied for 24 h.
Materials and methods Experimental design and sampling Plant material Clonal plantlets of hybrid larch (Larix kaempferi (Lambert) Carr.) × L. decidua Mill.) were obtained by in vitro regeneration from the transformed pro-embryogenic cell line 69.3INRA (Lelu et al. 1994a, 1994b, 1994c, Lelu and Pilate 2000). This transformed cell line harbors the wheat ABA-inducible Em promoter associated with the Gus reporter gene (Marcotte et al. 1988, Levée et al. 1997). After regeneration, plantlets were transferred to petri dishes (140 mm diameter) containing 60 ml of modified Murashige and Skoog agar medium (MSG) (Murashige and Skoog 1962) modified according to Lelu et al. (1994a) (inositol 1.45 g l –1 , glutamine 1.45 g l –1 , sucrose 1 g l – 1 ). Plantlets were separated from the culture medium by a cellophane sheet. To avoid desiccation, roots were covered by a moist filter paper and two cotton rolls were placed in each petri dish to prevent water accumulation. The petri dishes containing the plantlets were inclined 10° from the vertical. The bottom half of each petri dish was covered with aluminum foil to prevent exposure of the roots to direct light. Plants were grown in a climate chamber with a 16-h photoperiod of 50 W m – 2 provided by fluorescent lamps and a constant day/night temperature of 20/24 °C.
There were 10 treatments each with 14 replicates (one clonal plantlet per replicate): uninoculated control (C), inoculation with L. bicolor (Lb–M), inoculation with C. geophilum (Cg–M), inoculation with L. bicolor without root colonization (Lb–NM) and inoculation with C. geophilum without root colonization (Cg–NM). Uninoculated control + PEG (C+PEG), inoculation with L. bicolor + PEG (Lb–M+PEG), inoculation with L. bicolor without root colonization + PEG (Lb–NM +PEG), inoculation with C. geophilum + PEG (Cg–M+PEG), and inoculation with C. geophilum without root colonization + PEG (Cg–NM+PEG). After 24 h of osmotic stress treatment, the plantlets were harvested and the following measurements made: dry mass (roots and shoots separately), shoot water potential (Ψwp), shoot osmotic potential (Ψπ), GUS activity (root apex, roots, young needles and old needles separately), ABA content (roots and shoots separately) and total protein content (root apex, roots, young needles and old needles separately). Determination of shoot water and osmotic potentials We measured Ψwp and Ψπ on five plantlets. After measuring Ψwp with a Scholander pressure chamber, shoot samples were
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immediately frozen in liquid nitrogen and stored at – 80 °C. To measure Ψπ, frozen shoots were thawed for 20 min at room temperature and their cell sap extracted. After measuring Ψwp, shoot samples were introduced into a syringe, immediately frozen in liquid nitrogen and kept at –80 °C. To measure the osmotic potential (Ψπ), the shoots were thawed for 20 min at room temperature and their cell sap extracted from the syringe. A 10-µl sample was measured for osmotic potential with a vapor pressure osmometer (Wescor 5500, Logan, UT). Shoot turgor pressure (Ψτ) was calculated as the difference between Ψwp and Ψπ.
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Statistical analysis Data were subjected to analysis of variance (ANOVA) with two factors (osmotic stress and inoculation) using Statview 5.0 software (SAS Institute, Cary, NC). Significant differences in means (P ≤ 0.05) were compared by Fisher’s test. If the interaction between osmotic stress and inoculation was significant, the effect of inoculation was tested separately for –PEG and +PEG treatments, and the effect of PEG was tested separately for each inoculation treatment. Results
Sample freezing and storage
Effect of fungal inoculation on larch dry mass
Five plantlets were separated into four subsamples: young needles, old needles, root apex (1 cm) and roots without apex. The subsamples were immediately frozen in liquid nitrogen and stored at –80 °C for determination of protein content and GUS activity.
Plantlet shoot and root dry masses were significantly increased by inoculation with L. bicolor and C. geophilum, provided that mycorrhiza formation was prevented by a cellophane membrane (NM treatment) (Figure 1).
Protein content The frozen subsamples were ground with mortar and pestle in extraction buffer (50 mM phosphate buffer, pH 7.0, 10 mM DTT, 1 mM Na2EDTA, 0.1% SDS and 0.1% Triton X-100) and 1% PVP. Each extract was split into two, and one portion was used to determine protein content. Proteins were precipitated with acetone at –20 °C for 1 h. After removing the acetone by evaporation, proteins were suspended in distilled water and quantified with a BIORAD DC protein assay kit (based on the Lowry procedure; Lowry et al. 1951).
Water relations Shoot Ψwp and Ψπ were measured 24 h after applying the osmotic stress treatment. In the absence of PEG, Ψwp was not significantly affected by fungal inoculation (Figure 2A), whereas in the presence of PEG, plantlets mycorrhizal (M) with L. bicolor had a significantly more negative Ψwp than the uninoculated control (Figure 2A). In the absence of PEG, inoculation with C. geophilum (M and NM), but not Laccaria bicolor, reduced Ψπ of non-stressed plantlets (Figure 2B). In the presence of PEG, Ψπ was significantly more negative in control plantlets than the M and NM
Determination of β-glucuronidase (GUS) activity Enzyme activity of the Gus gene product was assayed in the remaining portion of each extract with 4-methylumbelliferylβ-D-gluconide (MUG) as substrate (Jefferson 1987, Martin et al. 1992). At the end of the 20-min enzymatic reaction, the fluorescent product (4-methylumbelliferone, MU) was measured by excitation at 365 nm and emission at 455 nm with a TKO 100 mini-Fluorimeter (Hoefer, TKO 100-115V). We expressed GUS activity as pmol min – 1 mg – 1 protein. Quantification of abscisic acid Abscisic acid was quantified in shoots and roots of four plantlets per treatment. After lyophilization and determination of dry mass, samples were ground to powder in liquid nitrogen. The ABA extraction, purification, quantification by ELISA and identification of immunoreactive molecules were carried out as previously described (Julliard et al. 1994, Kraepiel et al. 1994). A monoclonal anti-ABA antibody (LPDP 229, Université Pierre & Marie Curie, Paris, France) labeled with peroxidase-conjugated goat antibody to mouse immunoglobulins (Sigma) was used. The ABA concentration was determined five times for each sample and expressed as pmol –1 . mg DM
Figure 1. Effects of ectomycorrhizal inoculation on shoot and root dry mass of transgenic larch plantlets. Abbreviations: Lb–M = mycorrhizal plantlets with L. bicolor; Cg–M = mycorrhizal plantlets with C. geophilum; Lb–NM = plantlets inoculated with L. bicolor, but non-mycorrhizal (roots separated from the fungus by a cellophane membrane); and Cg–NM = plantlets inoculated with C. geophilum, but non-mycorrhizal. Error bars represent the standard error of the means within each treatment. Different letters indicate significant differences among inoculation treatments according to Fisher’s test (P = 0.05). Shoots and roots were statistically analyzed separately.
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inoculation treatments with L. bicolor and the NM inoculation treatment with C. geophilum (Figure 2B). In the absence of PEG, Ψτ was not significantly affected by fungal inoculation (Figure 2C). In the presence of PEG, inoculation with L. bicolor (both M and NM), but not C. geophilum, significantly reduced Ψτ (Figure 2C). Abscisic acid concentration The PEG treatment induced highly significant increases in ABA concentrations in both shoots and roots (Figures 3A and 3B, respectively). In the absence of PEG, ABA concentrations in shoots and roots were not significantly affected by fungal inoculation (Figure 3A). In the presence of PEG, however, inoculation with either L. bicolor or C. geophilum sharply reduced root ABA concentration, even when mycorrhiza formation was prevented (Figure 3B). Activity of GUS Treatment with PEG had no significant effect on GUS activity (P = 0.674) (Figure 4A), and there was no correlation between tissue ABA concentration and GUS activity (data not shown). In contrast, fungal inoculation significantly reduced GUS activity (P < 0.011), even when mycorrhiza formation was prevented (Figure 4B). Expression of GUS varied with plant organ (P = 0.001), being highest in the root apex and significantly lower in mature root tissue. The activity of GUS in young needles was also high (Figure 4C). However, GUS expression in young needles was significantly decreased by inoculation with either of the ectomycorrhizal fungi (Figure 4D). Down-regulation of GUS expression in inoculated plants was observed whether or not mycorrhiza formation was allowed. In roots, GUS expression was decreased by C. geophilum in both the M and NM treatments, but was unaffected by inoculation with L. bicolor (Figure 4E).
Discussion
Figure 2. Effects of fungal inoculation and osmotic stress on water status of hybrid larch shoots. (A) Water potential, (B) osmotic potential and (C) turgor potential. Samples treated with (+PEG) and without polyethylene glycol (–PEG) were analyzed separately. Abbreviations: Lb–M = mycorrhizal plantlets with L. bicolor; Cg–M = mycorrhizal plantlets with C. geophilum; Lb–NM = plantlets inoculated with L. bicolor, but non-mycorrhizal (roots separated from the fungus by a cellophane membrane); and Cg–NM = plantlets inoculated with with C. geophilum, but non-mycorrhizal. Different letters indicate significant differences among treatments according to Fisher’s test (P ≤ 0.05). For each inoculation treatment, an asterisk indicates significant differences between –PEG and +PEG according to Fisher’s test (P ≤ 0.05). Error bars represent SE within each treatment.
Interactions between mycorrhizal fungi and the water relations of plants subjected to osmotic stress are complex. Our shortterm (24 h) osmotic stress treatment lowered the Ψπ of uninoculated control plantlets, without affecting either Ψwp or Ψτ. There was a strong interaction between osmotic stress and fungal inoculation. In the absence of osmotic stress, fungal inoculation, with or without mycorrhiza formation, had little effect on host plant Ψwp. In plantlets subjected to osmotic stress, fungal inoculation, with or without mycorrhiza formation, led to a more negative shoot Ψwp. In contrast, fungal inoculation reduced the effect of osmotic stress on Ψπ, but not sufficiently to compensate for the negative effect of the ectomycorrhiza formation on Ψwp. Consequently, in response to osmotic stress, Ψτ reached zero or became slightly negative in ectomycorrhizal plantlets. A decrease in Ψwp has sometimes been reported in arbuscular mycorrhizal plants compared with non-mycorrhizal plants and has been related to effects on the threshold for stomatal closure during drought (Augé 2001). However, such a mechanism cannot explain our results with
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Figure 3. Effects of inoculation and osmotic stress on abscisic acid concentration ([ABA]) in shoots (A) and roots (B) of hybrid larch plantlets. Abbreviations: Lb–M = mycorrhizal plantlets with L. bicolor; Cg–M = mycorrhizal plantlets with C. geophilum; Lb–NM = plantlets inoculated with L. bicolor, but non-mycorrhizal (roots separated from the fungus by a cellophane membrane); and Cg–NM = plantlets inoculated with C. geophilum, but non-mycorrhizal. For each polyethylene glycol (PEG) treatment, different letters indicate significant differences among inoculation treatments by the Fisher test (P = 0.05). For each inoculation treatment, asterisks indicate significant differences between means in treatments without (–PEG) and with PEG (+PEG) by Fisher’s test (P = 0.05). Error bars represent the standard error of the means within each treatment.
plantlets in a water-saturated atmosphere. In our in vitro experiments, both L. bicolor and C. geophilum inoculation had negative effects on Ψwp and Ψτ of larch plantlets, even though they enhanced osmotic adjustment in response to osmotic stress. The beneficial role of ectomycorrhizas on plantlets subjected to water stress has to be attributed to other mechanisms
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such as more effective scavenging of soil water. Under natural conditions, the external mycelium extends several centimeters from the root, thereby improving soil–root contact. Nevertheless, some authors have suggested that ectomycorrhizas increase plant water stress by stimulating transpiration (Pallardy et al. 1995). The literature indicates that L. bicolor and C. geophilum behave differently under water stress. Several studies have shown that pure cultures of C. geophilum are more resistant to water stress or osmotic stress than other ectomycorrhizal fungi (Mexal and Reid 1973, Theodorou 1978, Coleman et al. 1989). Drought tolerance of C. geophilum may result from its ability to adjust osmotically during water stress (Coleman et al. 1989). Pigott (1982) showed that C. geophilum mycorrhizas survived long drought periods and could become dominant after drought. Associations of roots with this fungus could be an advantage for the host in post-drought periods. However, in our in vitro conditions, under 24-h osmotic stress, C. geophilum did not differ significantly from L. bicolor in modifying the water status of larch. Moreover, in the absence of osmotic stress, C. geophilum induced a slight reduction in plantlet osmotic potential compared with both uninoculated control plantlets and plantlets inoculated with L. bicolor. It has often been suggested that osmotic stress responses are mediated by ABA. However, several studies indicate that responses to osmotic stress cannot always be explained solely by changes in ABA concentrations (Skriver and Mundy 1990, Giraudat et al. 1994, Perks et al. 2002). Osmotic stress induced a significant increase in endogenous ABA concentrations in our larch plantlets and, in roots, this change in ABA concentration was partly controlled by the associated ectomycorrhizal fungi. Laccaria bicolor and C. geophilum behaved similarly, both significantly decreasing the ABA response of larch roots to osmotic stress. Moreover, both fungi regulated the ABA response of larch even when separated from the host by a cellophane sheet. Thus it appears that in response to an osmotic stress, L. bicolor and C. geophilum altered the root ABA concentration of their host by the production of diffusible compounds of low molecular weight. Augé (2001) reported an effect of arbuscular symbiosis on host ABA concentration, and several studies have shown decreased ABA concentrations as a result of mycorrhizal symbiosis (Coleman et al. 1990, Duan et al. 1996, Ebel et al. 1997, Goicoechea et al. 1997). Several hundred genes are known to be inducible by exogenous ABA (Skriver and Mundy 1990). Some of these genes may prove useful as tools in the investigation of factors involved in ABA induction (Giraudat et al. 1994). As reported by others, we found that the monocot (wheat) Em promoter is inducible in the gymnosperm larch as indicated by the activity of the associated GUS reporter gene product. Induction of the wheat Em promoter in larch depended on organ type, with maximum induction occurring in the root apex and young needles. Induction of the Em promoter was unaffected by changes in endogenous ABA concentrations. The absence of a response to ABA may be explained by the low concentration of endogenous ABA compared with the high concentration of exogenous ABA used in studies showing Em gene induction by
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Figure 4. Effects of osmotic stress (A), fungal inoculation (B) and organs (C, D, E) on β-glucuronidase (GUS) activity (determined by the production of 4-methylumbelliferone (MU)). Because the osmotic stress treatment had no influence on GUS activity (A), the effect of fungal inoculation (B) was analyzed in control and osmotically stressed samples combined. Similarly, the organ effect (C) was analyzed on control and osmotically stressed and non-mycorrhizal and mycorrhizal plants combined. Because the effect of fungal inoculation was significant as was the interaction with plant organs, expression of GUS activity was analyzed separately in young needles (D) and roots (E) according to the inoculation treatments. Abbreviations: C = control; Lb–M = mycorrhizal with L. bicolor; Cg–M = mycorrhizal with C. geophilum; Lb–NM = non-mycorrhizal L. bicolor; and Cg–NM = nonmycorrhizal C. geophilum. In each graph, different letters indicate significant differences among treatments according to the Fisher test (P = 0.05). Error bars represent the standard error of the means within each treatment.
ABA. The absence of a correlation between GUS activity and endogenous ABA concentrations of larch plantlets tissues negates the hypothesis that ABA is responsible for the observed variation in Em gene expression in vivo. Mycorrhizal inoculation significantly decreased Em induction as indicated by GUS activity. Both ectomycorrhizal fungi, irrespective of whether they are able to form mycorrhizas, decreased GUS expression in both roots and shoots, and markedly so in young needles, although L. bicolor did not significantly decrease GUS expression in roots. The fungi evidently acted through diffusible elicitors as discussed with reference to their effects on tissue ABA content. The postulated diffusible elicitors were absorbed by the roots and then transported to the shoot where they regulated transcription of the Em promoter. The significance of such gene regulation by ectomycorrhizas remains unknown. The LEA proteins are postulated to maintain membrane structures by sequestering ions and binding water. The establishment of ectomycorrhizal symbiosis changes the membrane structure of both partners. For example, changes in fungal hydrophobicity occur during mycorrhizal establishment (Tagu et al. 1996, 2002), and these modifications could be associated with modifications in host hydrophylic properties. Under our experimental conditions, the association of larch with L. bicolor or C. geophilum did not improve plantlet growth, whereas the presence of the fungi separated from the
roots by a cellophane membrane did. Similar results, showing no effect of mycorrhizal infection or even a negative effect during the early stages of infection, have been reported previously (e.g., Sands and Theodorou 1978), and several explanations have been given. Fungal infection affects host growth as a result of at least three processes having opposite consequences: the drain of carbon from the host to the fungus, the transfer of mineral elements from the medium to the host through the fungus, and the production of fungal hormonal compounds able to stimulate host growth. Limiting light conditions, as sometimes occurs under in vitro conditions, may allow mycorrhiza formation, resulting in a carbon drain to the fungus, without a compensating effect on host biomass (Dosskey et al. 1990, 1991). During this initial phase, the carbon cost may not be compensated by improved mineral nutrition in a medium where the nutrients are readily available or through the action of diffusible fungal metabolites. Ectomycorrhizal fungi synthesize various diffusible compounds, including plant hormones (Barker and Tagu 2000), which stimulate root ramification and elongation and also host shoot growth. Stimulation of larch plantlet growth by inoculation with mycorrhizal fungi, when mycorrhiza formation was prevented by a cellophane membrane may result from the action of fungal auxin or other diffusible elicitors as shown for Norway spruce in similar in vitro experiments (Karabaghli-Degron et al. 1998, Rincón et al. 2003).
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HYBRID LARCH, ECTOMYCORRHIZAL FUNGI AND OSMOTIC STRESS Acknowledgments The authors thank Dr. Annegret Kohler and Joëlle Gérard for their helpful assistance. This work was supported by a European research contract (No. FAIR-BM-98-5091, Marie Curie Fellowship) and by the Academy of Finland. References Allen, M.F. 1991. The ecology of mycorrhiza. Cambridge University Press, Cambridge, U.K., 184 p. Augé, R.M. 2001. Water relations, drought and VA mycorrhizal symbiosis. Mycorrhiza 11:3–42. Barker, S.J. and D. Tagu. 2000. The roles of auxins and cytokinins in mycorrhizal establishment. J. Plant Growth Regul. 19:144–154. Bostock, R.M. and R.S. Quatrano. 1992. Regulation of Em gene expression in rice. Interaction between osmotic stress and abscisic acid. Plant Physiol. 98:1356–1363. Bray, E.A. 1993. Molecular responses to water deficit. Plant Physiol. 103:1035–1040. Campalans, A., R. Messeguer, A. Goday and M. Pagès. 1999. Plant responses to drought, from ABA signal transduction events to the action of the induced proteins. Plant Physiol. Biochem. 37: 327–340. Coleman, M.D., C.S. Bledsoe and W. Lopushinsky. 1989. Pure culture response of ectomycorrhizal fungi to imposed water stress. Can. J. Bot. 67:29–39. Coleman, M.D., C.S. Bledsoe and B.A. Smith. 1990. Root hydraulic conductivity and xylem sap levels of zeatin riboside and abscisic acid in ectomycorrhizal Douglas fir seedlings. New Phytol. 115: 275–284. Danneberg, G., C. Latus, W. Zimmer, B. Hundeshagen, H.J. Schneider-Poetsch and H. Bothe. 1992. Influence of vesicular–arbuscular mycorrhiza on phytohormone balances in maize (Zea mays L.). J. Plant Physiol. 141:33–39. Dosskey, M.G., R.G. Linderman and L. Boersma. 1990. Carbon-sink stimulation of photosynthesis in Douglas fir seedlings by some ectomycorrhizas. New Phytol. 115:269–274. Dosskey, M.G., L. Boersma and R.G. Linderman. 1991. Role of photosynthate demand of ectomycorrhizas in the response of Douglas fir seedlings to drying soil. New Phytol. 117:327–334. Duan, X., D. Neuman, J. Reiber, C. Green, A. Saxton and R. Augé. 1996. Mycorrhizal influence on hydraulic and hormonal factors implicated in the control of stomatal conductance during drought. J. Exp. Bot. 47:1541–1550. Ebel, R.C., X. Duan, D.W. Still and R.M. Augé. 1997. Xylem sap abscisic acid concentration and stomatal conductance of mycorrhizal Vigna unguiculata in drying soil. New Phytol. 135:755–761. Ek, M., P.O. Ljungquist and E. Stenström. 1983. Indole-3-acetic acid production by mycorrhizal fungi determined by gas chromatography–mass spectrometry. New Phytol. 94:401–407. Galau, G.A., N. Bijaisoradat and D.W. Hughes. 1987. Accumulation kinetics of cotton late embryogenesis-abundant mRNAs and storage protein mRNAs: co-ordinate regulation during embryogenesis and the role of abscisic acid. Develop. Biol. 123:198–212. Gay, G., L. Normand, R. Marmeisse, B. Sotta and J.C. Debaud. 1994. Auxin overproducer mutants of Hebeloma cylindrosporum Romagnési have increased mycorrhizal activity. New Phytol. 128: 645–657. Giraudat, J., F. Parcy, N. Bertauche, F. Gosti, J. Leung, P.C. Morris, M. Bouvier-Durand and N. Vartanian. 1994. Current advances in abscisic acid action and signalling. Plant Mol. Biol. 26:1557–1577. Goicoechea, N., M.C. Antolín and M. Sánchez-Díaz. 1997. Gas exchange is related to the hormone balance in mycorrhizal or nitrogen-fixing alfalfa subjected to drought. Physiol. Plant. 100: 989–997.
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Guehl, J.M., J. Garbaye and A. Wartinger. 1992. The effects of ectomycorrhizal status on plant–water relations and sensitivity of leaf gas exchange to soil drought in Douglas fir (Pseudotsuga menziesii) seedlings. In Mycorrhizas in Ecosystems. Eds. D.J. Read, D.H. Lewis, A.H. Fitter and I.J. Alexander. CAB International, Cambridge, U.K., pp 322–332. Hetherington, A.M. and R.S. Quatrano. 1991. Mechanisms of action of abscisic acid at the cellular level. New Phytol. 119:9–32. Hughes, D.W. and G.A. Galau. 1989. Temporally modular gene expression during cotyledon development. Genes Develop. 3: 358–369. Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5:387–405. Julliard, J., R. Maldiney, B. Sotta, E. Miginiac, L. Kherhoas and J. Einhorn. 1994. HPLC–ELISA and MS as complementary techniques to study plant hormone metabolites. Analysis 22:483–489. Karabaghli-Degron, C.B., B. Sotta, M. Bonnet, G. Gay and F. Le Tacon. 1998. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the fungus Laccaria bicolor. New Phytol. 140:723–733. Kraepiel, Y., P. Rousselin, B. Sotta, L. Kerhoas, J. Einhorn, M. Caboche and E. Miginiac. 1994. Analysis of phytochrome- and ABAdeficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia. Plant J. 6:665–672. Lelu, M.A. and G. Pilate. 2000. Transgenics in Larix. In Molecular Biology of Woody Plants. Vol. 2. Eds. M. Jain and S.C. Minocha. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 119–134. Lelu, M.A., C. Bastien, K. Klimaszewska, C. Ward and P.J. Charest. 1994a. An improved method for somatic plantlet production in hybrid larch (Larix × leptoeuropaea): Part 1. Somatic embryo maturation. Plant Cell Tissue Organ Cult. 36:107–115. Lelu, M.A., C. Bastien, K. Klimaszewska and P.J. Charest. 1994b. An improved method for somatic plantlet production in hybrid larch (Larix × leptoeuropaea): Part 2. Control of germination and plantlet development. Plant Cell Tissue Organ Cult. 36:117–127. Lelu, M.A., K. Klimaszewska and P.J. Charest. 1994c. Somatic embryogenesis from immature and mature zygotic embryos and from cotyledons and needles of somatic plantlets of Larix. Can. J. For. Res. 24:100–106. Levée, V., M.A. Lelu, L. Jouanin and D. Cornu. 1997. Agrobacterium tumefaciens-mediated transformation of hybrid larch (Larix kaempferi × L. decidua) and transgenic plant regeneration. Plant Cell Rep. 16:680–685. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275. Marcotte, W.R., C.C. Bayley and R.S. Quatrano. 1988. Regulation of a wheat promoter by abscisic acid in rice protoplast. Nature 335: 454–457. Martin, T., R.-V. Wöhner, S. Hummel, L. Willmitzer and W.B. Frommer. 1992. The GUS reporter system as a tool to study plant gene expression. In GUS Protocols. Using the GUS Gene as a Reporter of Gene Expression. Ed. S.R. Gallagher. Academic Press, New York, pp 23–43. Mexal, J. and C.P.P. Reid. 1973. The growth of selected mycorrhizal fungi in response to induced water stress. Can. J. Bot. 51: 1579–1588. Morte, A., G. Díaz, P. Rodríguez, J.J. Alarcón and M.J. SánchezBlanco. 2001. Growth and water relations in mycorrhizal and non mycorrhizal Pinus halepensis plants in response to drought. Biol. Plant. 44:263–267.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
1108
RINCÓN ET AL.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15:473–497. Pachlewski, R. and J. Pachlewska. 1974. Studies on symbiotic properties of mycorrhizal fungi on pine (Pinus sylvestris) with the aid of the method on mycorrhizal synthesis in pure culture agar. For. Res. Inst., Warsaw, Poland, 139 p. Pallardy, S.G., J. Èermák, F.W. Ewers, M.R. Kaufmann, W.C. Parker and J.S. Sperry. 1995. Water transport dynamic in trees and stands. In Resource Physiology of Conifers: Acquisition, Allocation and Utilization. Eds. W.K. Smith and W.K. Hinckley. Academic Press, London, pp 301–389. Perks, M.P., J. Irvine and J. Grace. 2002. Canopy stomatal conductance and xylem sap abscisic acid (ABA) in mature Scots pine during a gradually imposed drought. Tree Physiol. 22:877–883. Pigott, C.D. 1982. Fine structure of mycorrhiza formed by Cenococcum geophilum Fr. on Tillia cordata Mill. New Phytol. 92: 501–512. Rincón, A., O. Priha, B. Sotta, M. Bonnet and F. Le Tacon. 2003. Comparative effect of auxin transport inhibitors on rhizogenesis and mycorrhizal establishment of spruce seedlings inoculated with Laccaria bicolor. Tree Physiol. 23:785–791. Sands, R. and C. Theodorou. 1978. Water uptake of mycorrhizal roots of radiata pine seedlings. Aust. J. Plant Physiol. 5:301–309.
Skriver, K. and J. Mundy. 1990. Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503–512. Slankis, V. 1973. Hormonal relationships in mycorrhizal development. In Ectomycorrhizae. Eds. G.C. Marks and T.T. Kozlowski. Academic Press, New York, pp 231–298. Smith, S.E. and D.J. Read. 1997. Mycorrhizal symbiosis. Academic Press, Cambridge, U.K., 605 p. Tagu, D., B. Nasse and F. Martin. 1996. Cloning and characterization of hydrophobins-encoding cDNAs from the ectomycorrhizal basidiomycete Pisolithus tinctorius. Gene 168:93–97. Tagu, D., R. Marmeisse, Y. Baillet et al. 2002. Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum. Eur. J. Histochem. 46:23–29. Theodorou, C. 1978. Soil moisture and the mycorrhizal association of Pinus radiata D. Don. Soil Biol. Biochem. 3:33–37. Vicient, C.M., G. Hull, J. Guilleminot, M. Devic and M. Delseny. 2000. Differential expression of the Arabidopsis genes coding for Em-like proteins. J. Exp. Bot. 51:1211–1220. Willamson, J.D. and R.S. Quatrano. 1988. ABA-regulation of two classes of embryo-specific sequences in mature wheat embryos. Plant Physiol. 86:208–215.
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