Glomus intraradices causes differential changes ... - Wiley Online Library

RESEARCH New Phytol. (2000), 148, 177–186

Glomus intraradices causes differential changes in amino acid and starch concentrations of in vitro strawberry subjected to water stress C I N T A H E R N A! N D E Z - S E B A S T I A' "*, G U Y S A M S O N", P I E R R E - Y V E S B E R N I E R#, Y V E S P I C H E! $    Y V E S D E S J A R D I N S" "Centre de Recherche en Horticulture, CRH, Envirotron, UniversiteT Laval, Ste-Foy, QueT bec, G1K 7P4, Canada #Centre Forestier des Laurentides, CFL, ForeV t Canada, Ste-Foy, QueT bec, G1K 7P4, Canada $Centre de Recherche en Biologie ForestieZ re, CRBF, Pavillon Ch. E. Marchand, UniversiteT Laval, Ste-Foy, QueT bec, G1K 7P4, Canada Received 7 December 1999 ; accepted 15 May 2000  The effect of colonization of tissue-cultured strawberry (Fragariaiananassa Duch. cv. Kent) plantlets in vitro by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol) (PEG)-8000-induced water stress was investigated. The plantlets were inoculated axenically and co-cultured with the AMF for 4 wk, then transferred to 15% PEG-8000 solutions for 4, 8 and 12 h. Relative water content, water potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of the intraradical phase of the AMF. However, distinct differences in PEG-induced changes in amino acid content were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of aspartic acid, serine, threonine, amino-N-butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG-induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism. Key words : acclimation, arbuscular mycorrhizal fungus, asparagine, Glomus intraradices, osmotic stress, poly(ethylene glycol), strawberry, tripartite culture.

 A major problem that restricts the widespread commercial use of micropropagation is the low *Author for correspondence (Apartat de Correus, 76, 43520 Roquetes, Tarragona, Spain; e-mail cintah!tinet.fut.es). Abbreviations: AMF, arbuscular mycorrhizal fungus; ANBA, amino-N-butyric acid; Fjph\Fph, photochemical efficiency of photosynthesis or quantum yield of PSII electron transport; gs, leaf stomatal conductance to diffusion of water; MWM, minimal White‘ s medium; PEG, poly(ethylene glycol); RWCleaf, relative water content of the leaf; RWCplantlet, relative water content of the whole plantlet; Ψw, water potential; Ψπ, osmotic potential.

survival rate of plantlets produced in vitro during acclimation ex vitro, which is due to a high transpirational water loss (Dı! az-Pe! rez et al., 1995b). Wang et al. (1993), and Azco! n-Aguilar & Barea (1997) reported that the acclimation success rate might be improved by the colonization of plants produced in vitro with arbuscular mycorrhizal fungi (AMF). These authors showed a beneficial effect of AMF even though root colonization occurred after the transfer of plantlets in vitro to conditions ex vitro. It is therefore possible that AMF colonization before transplantation might be appropriate to improve the

178

RESEARCH C. HernaT ndez-SebastiaZ et al.

tolerance of plants produced in vitro to water stress during acclimation (Herna! ndez-Sebastia' et al., 1999). Elmeskaoui et al. (1995) developed an efficient method for the inoculation of strawberry plantlets in vitro with Glomus intraradices in an axenic tripartite culture system, involving the co-culture of plants produced in vitro, AMF and root organs. Using this system, Herna! ndez-Sebastia' et al. (1999) showed an increase in the relative water content (RWC) of strawberry plantlets colonized by this AMF under well watered conditions. This effect was related to an increased water content in mycorrhizal root systems, whereas root osmotic potentials and root dry weights remained similar to those measured in nonmycorrhizal controls. Dı! az-Pe! rez et al. (1995a) reported that higher RWC in leaves of micropropagated apple plants were correlated with improved resistance to transplantation stress, although the mechanism involved is still unknown. In the present study we investigated the influence of AMF on the response of strawberry plantlets produced in vitro to a controlled and reproducible water deficit. This was achieved by exposing mycorrhizal and nonmycorrhizal plantlets to a short-term (4–12 h) osmotic stress induced by poly(ethylene glycol) (PEG)-8000. Transfer of the mycorrhizal plantlets from the tripartite culture systems to the PEG solutions resulted in the loss of the external AMF mycelium, which remained trapped in the culture medium. Therefore the mycorrhizal response to the induced osmotic stress was due mostly to the fungal tissue within the roots, which is referred to below as the intraradical phase. In this context, our objectives were (1) to determine whether basic water relations during water deficits were improved in mycorrhizal plantlets in vitro (e.g. whether roots and leaves of micropropagated mycorrhizal strawberry plantlets had higher water contents than nonmycorrhizal ones) and (2) to evaluate the involvement of the intraradical phase of the associated AMF on the accumulation of free amino acid and starch content after PEG-induced water stress.

   Fungal inoculum Spores of Glomus intraradices Schenck & Smith (DAOM 197198 ; Biosystematic Research Center, Ottawa, Canada) were obtained from an arbuscular mycorrhizal carrot root-organ culture maintained on minimal White’s medium (MWM) (Be! card & Fortin, 1988). (See Chabot et al. (1992) for a complete description of the growth of G. intraradices in this system.) To inoculate new roots, fungal chlamydospores were extracted from the growth medium with a scalpel (size 11). Manipulations were performed

under sterile conditions and with a stereomicroscope (i40). Carrot root-organ cultures were obtained by inoculating surface-sterilized slices of a carrot tap root with Agrobacterium rhizogenes strain HRI (from Dr D. Tepfer, INRA, Versailles, France). To obtain bacteria-free root cultures, roots transformed with Ri T-DNA, which show a negative geotropism that is absent from nontransformed roots, were excised and treated with antibiotics (Balaji et al., 1994). Routine maintenance of the root cultures was performed as described by Be! card & Fortin (1988).

Plant material Strawberry plantlets (Fragariaiananassa Dutch cv. Kent) were initiated, multiplied and rooted in vitro as described by Desjardins et al. (1987) and Herna! ndez-Sebastia' et al. (1999). Tripartite culture in vitro The preparation of rooted strawberry plantlets and inoculation with G. intraradices were achieved with the tripartite in vitro culture system described by Elmeskaoui et al. (1995) and Herna! ndez-Sebastia' et al. (1999). In brief, nine rooted strawberry plantlets were co-cultivated with the fungus for 4 wk in polycarbonate Magenta2 containers (Magenta Corp., Chicago, IL, USA). These containers had been precolonized by AMF carrot root-organ cultures showing extensive extraradical mycelial development and chlamydospore production. The tripartite cultures were maintained at 25mC, with a 16-h photoperiod (100 µmol m−# s−") and at a CO concentration of # 0n4% (see Elmeskaoui et al. (1995) for details of CO # feeding). This system typically produced root colonization levels in strawberry of 20–25% after 3 wk (Herna! ndez-Sebastia' et al., 1999). PEG-induced water stress conditions in vitro After 1 month of co-culture, plantlets were removed from the growth boxes and placed individually in test tubes containing 15 ml of liquid minimal medium MWM and either 0% (controls) or 15% PEG-8000 (from BDH). To avoid possible phytotoxic effects (Jackson, 1962 ; Jacomini et al., 1988) caused by the direct contact of PEG on plant tissues, plantlet roots were placed in a dialysis membrane (Spectra Por 7, PM 1000, 18 mm diameter ; Spectrum Medical Industries, Los Angeles, CA, USA), closed with two knots at one extremity to form an envelope and filled with 1 ml of liquid MWM. As preliminary trials had shown no effect of the dialysis membrane on the mineral content of nonstressed plantlets, nonstressed plantlets (0% PEG) were placed directly into liquid MWM. Furthermore, the

RESEARCH Water stress in mycorrhizal strawberry plantlets in vitro use of dialysis membrane bags did not interfere with the main goal of our experiment, which was to compare the susceptibilities of mycorrhizal and nonmycorrhizal plantlets to osmotic stress. The osmotic potential Ψπ (measured by a vapour-pressure osmometer (Wescor 5500 ; Wescor, Logan, UT, USA)) of the 15% PEG solution at pH 5n5 was k0n33 MPa (SD 0n04) and for 0% PEG k0n07 MPa (SD 0n01). Deionized nanopure water has a theoretical value of 0 MPa. However, we measured values of k0n06 MPa (SD 0n02), which is considered an inherent error of the instrument. It is interesting to note that PEG does not behave in accordance with van‘ t Hoff‘ s law (Steuter et al., 1981). The water potential for a given PEG solution is related to molarity but not linearly, varying with the molecular size of the PEG. Thus, Steuter et al. (1981) suggested that water potential in PEG might be controlled primarily by the matrix forces of the ethylene oxide subunits of the polymer. The measures of PEG Ψπ already presented are given only as a reference and probably underestimate the real matrix forces of such solutions. Plants were incubated for 4, 8 or 12 h under Cool White fluorescent light (200 µmol m−# s−") at 50% rh. Experimental design and statistical analysis A factorial experiment (2i2i3) was conducted in four complete randomized blocks. Factors were inoculation (G. intraradices and control), PEG concentration (0% and 15%) and sampling time (4, 8 and 12 h). The experimental unit was one strawberry plantlet inside a test tube. There were three replicates of the experimental unit for destructive measurements and 12 for nondestructive measurements. ANOVAs and orthogonal contrasts were conducted on the variables to be described with the SuperAnova statistical program (Abacus Concepts Inc., Berkeley, CA, USA). Linear and quadratic contrasts of the time course and the PEG interactions are not shown because of their lack of significance with regard to our hypotheses. Degrees of freedom for residual errors in the ANOVA tables were 59 for foliar water contents (RWCleaf) and 24 for all other variables. Amino acid concentrations in the 4-h treatment were analysed as a 2i2 (inoculation and PEG concentration) factorial ANOVA. There were nine degrees of freedom for the error term. To satisfy the assumptions of the ANOVA test, the homogeneity of the variances was checked with Levine’s test and a residual graphic analysis. When variances were not homogeneous, the variables were either log-transformed (log(xj1)) or raised to the power of two (x#), depending on the distribution of residuals. Where the homogeneity of the variances was not acceptable after mathematical transformation, we applied the equivalent of nonparametrical statistics, in the form of the rank

179

transformation of means in the parametric ANOVA test (Rank(x, Allrows) from the SuperANOVA program ; Conover, 1980). In this case, an assumption of normal distribution of the errors is not required. In all analyses, the independence of the errors in the ANOVA test was satisfied by randomized sampling for each treatment. Means in italics in the tables are the transformed values used in the ANOVA tests. Means in the original scale are presented after detransformation. Rank values cannot be shown as detransformed means ; means before rank transformation are therefore shown to indicate their biological range. To avoid autocorrelation, the level of significance was fixed for every table by dividing P l 0n05 by the number of variables measured. Water status measurements Leaf conductance for water vapour diffusion (gs) was measured on the abaxial surface of the leaf with a diffusion porometer (AP4 ; Delta-T Devices, Cambridge, UK). All gs measurements inside a given block (36 samples per block, 12 samples at each hour of the timing course) were made in 10 min. The RWC values of detached leaves (RWCleaf) and whole plantlets (RWCplantlet) were calculated as follows : RWC (%) l 100i(f. wtkd. wt)\(t. wtkd. wt) Turgid weights (t.wt) were measured on whole plantlets or detached leaves (with petiole cut twice under water to avoid cavitation) that had been resaturated for 12 h in deionized water, in darkness and at room temperature. Leaf water potential, Ψw, was measured with a pressure chamber (Plant Moisture Stress (PMS) Instrument Co., Corvallis, OR, USA) at a pressure rate of 0n6 MPa min−". Osmotic potentials of root sap extracts (Ψπ,root) or leaf sap extracts (Ψπ,leaf), collected as described by Herna! ndez-Sebastia' et al. (1999), were measured with a vapour pressure osmometer (Wescor 5500). Measurements were transformed from mmol kg−" of water (or mosmol kg−") to MPa by using the van‘ t Hoff empirical relation Ψπ l kCiRT, where C is the concentration of the solution (moles of solute kg−" of water), i is an ionization constant (assumed to be 1), R is the gas constant (0n00831 kg MPa mol−" K−") and T is 310 K. Determination of fluorescence of Chl a Chl a fluorescence of intact leaves, maintained under their growth conditions of photon flux density and temperature, was determined with a Plant Efficiency Analyzer fluorimeter (Hansatech Ltd, King’s Lynn, UK). Chl a fluorescence induction kinetics were induced by a 1 s flash of red light (c. 4000 µmol m−# s−", centred at 650 nm) fired 5 s after the leaves had

180


been placed in darkness. Fluorescence yields corresponding to the maximal (Fph) levels were immediately estimated by the instrument, whereas the Fj yield was measured 2 ms after the onset of the excitation light. The difference between the Fph and Fj levels corresponds to the amplitude of the thermal phase of variable fluorescence (Strasser et al., 1995). In preliminary assays, in leaves of strawberry plantlets produced under identical conditions, we observed the presence of a strong linear relation (R# l 0n992) between the ratio Fjph\Fph (where Fjph l FphkFj) and the apparent quantum yield of CO -supported O evolution. # # Extraction and analysis of free amino acids After 4 h of PEG-induced water stress, free amino acids present in leaf or root tissues were extracted in methanol : chloroform : water (12 : 5 : 3, v\v\v), with a slightly modified version of the technique described by Paquin & Lechasseur (1979). Samples of freshly harvested leaves or roots (50–100 mg) were frozen in liquid nitrogen and powdered in a cold mortar and pestle. The resulting material was placed in 1n5-ml microtubes (prefrozen in liquid nitrogen) with an 800 µl aliquot of methanol : chloroform : water (12 : 5 : 3, v\v\v). The microtubes were vortex-mixed and incubated at 65mC for 20 min, kept on ice for at least 1 h and centrifuged (13600 g) for 25 min at 4mC to separate out the particles. The supernatant fraction (c. 600 µl) was transferred to new microtubes. The extraction was repeated three times for each pellet and the supernatants were pooled in the same microtube. The extracted pellets were kept in methanol at k80mC before use for starch content analysis. To separate the chloroform fraction from the methanol :water fractions in the methanol : chloroform : water supernatant, chloroform : water (4 : 1, v\v) was added to 15 vol. of methanol : chloroform : water, then vortex-mixed and centrifuged at 10400 g for 10 min. The supernatant of the methanol :water fraction was transferred to clean microtubes, freeze-dried in a speed-vacuum and stored at k80mC before analysis. Freeze-dried extracts were dissolved in 100 µl of lithium buffer at pH 2n2. Sulphosalicylic acid (2n5 mg) was added to precipitate proteins. The solution was centrifuged (1600 g) for 5 min and the supernatant was filtered through a 0n2-µm nylon mesh before injection into an HPLC analyser. A 20–80 µl sample of deproteinized supernatant was charged on a cation-exchange column of particle size of 8 µm with an elution gradient of five citrate– lithium buffers as described in the manual of the standard LKB Alpha Plus 4151 amino acid analyser with a Hewlett Packard 3392A integrator. Amino acid analyses were performed by ion-exchange chromatography on sulphonated polystyrene resin linked to divinylbenzene.

Starch analysis Pellets kept at k80mC in pure methanol after the extraction of free amino acids (which also extracted soluble sugars) were used to quantify the insoluble starch content. The pellets were dried and resuspended in 750 µl of 0n02 M KOH, homogenized on a vortex and heated at 80mC for 10 min. The microtubes were cooled and the reaction was neutralized by adding 250 µl of 0n1 M citrate buffer at pH 4n2. Citrate buffer was prepared with 31n5 ml of 0n1 M citric acid monohydrate, 18n5 ml of 0n1 M trisodium citrate dihydrate and 50 ml of nanopure water. The starch suspension was incubated with 25 units ml−" of amyloglucosidase (EC 3.2.1.3) from Aspergillus (A-3042 ; Sigma Chemical Co., St Louis, MO, USA) for 3 h in a water bath at 55mC. Microtubes were cooled and centrifuged (10 400 g) at room temperature for 10 min ; the hydrolysed glucose was assessed with a YSI 2700 Select Sugar Analyser (Yellow Springs Instruments Co., Yellow Spring, OH, USA), with a glucose membrane coupling-enzyme immobilized under membrane. Quantitative hydrolysis of the starch to glucose under these conditions was indicated by the complete hydrolysis of a 10-mg starch standard. Glucose present in the commercial enzyme amyloglucosidase was measured and subtracted from the results.  Water relations The presence of the intraradical phase of G. intraradices did not significantly modify the water status of the strawberry plantlets under our experimental conditions. The RWC of leaves or whole plantlets (RWCleaf, RWCplantlet), the water potential (Ψw), the osmotic potentials (Ψπ,leaf, Ψπ,root) and the stomatal conductance (gs) did not differ significantly between mycorrhizal and nonmycorrhizal plantlets. As expected, exposure to PEG, either as a main factor or in interaction with the treatment time, significantly (P l 0n0001) modified all the water status variables measured (Table 1). Means of RWCleaf and Ψw for the significant interactions between the two main factors PEG concentration (Pg) and sampling time (T ) (PgiT ) are shown in Fig. 1. Exposure to 15% PEG-8000 progressively dehydrated the plantlets during the duration of the experiment. This was indicated by marked decreases in Ψw and in the RWCleaf of plantlets (Fig. 1). In the plot of the interactions PgiT, only means of the two inoculation treatments (control (without G. intraradices) and mycorrhizal) are presented because their respective means at both 0% and 15% were not significantly different. After 4 h of induced osmotic stress, some plantlets started to wilt. The water

RESEARCH Water stress in mycorrhizal strawberry plantlets in vitro

181

Table 1. (a) Significance levels of the ANOVA test for the water relations parameters and the chlorophyll fluorescence measurements ; (b) means of the factor Pg (exposure to PEG ) for the relative water content of whole plantlets (RWCplantlet) and detached leaves (RWCleaf), stomatal conductance (gs ), leaf water potential (Ψw), osmotic potential of leaves (Ψπ,leaf) and roots (Ψπ,root) and photochemical efficiency of photosynthesis (Fjph\Fph) of in vitro mycorrhizal and nonmycorrhizal strawberry plantlets exposed to PEG-8000 at 4, 8 and 12 h (a) Source of variation

df

RWCplantlet

RWCleaf

gs

Ψ

w

Ψπ

PEG (Pg) Inoculation (I ) Time course (T) PgiI PgiT IiT PgiIiT Error

1 1 2 1 2 2 2 df l

** ns * ns ns ns ns 59

** ns ** ns * ns ns 59

** ns ns ns ns ns ns 126

** ns ** ns * ns ns 59

* ns ns ns ns ns ns 59

(b) PEG-8000 (%) 0 15 SE

Level of significance in the ANOVA

RWCplantlet (%)

RWCleaf (%)

gs (mm s−")

Ψ (MPa)

106 27.50 41 9.50 1.56

92 8448.4 61 3691.7 285.5

9.96 1.04 1.19 0.34 0.03

k0.66 53.94 k2.63 18.50 1.81

w

Ψπ

,leaf

,leaf

(MPa) k1.08 2.64 k1.43 2.76 0.03

Ψπ

,root

* ns ns ns ns ns ns 59

Ψπ

,root

Fjph\Fph ** ns ** ns * ns ns 59

(MPa)

Fjph\Fph

k0.34 2.14 k0.61 2.39 0.05

0.333 0.111 0.255 0.065 0.07

Significant PgiT interactions are plotted in Fig. 1. Transformed means used by the ANOVA test are shown in italic numbers, as well as their corresponding SE values of the difference between these means. The mathematical transformations were : on the logarithmic scale for gs and Ψπ ; on the power of two (x#) scale for RWCleaf and Fjph\Fph and as rank values for RWCplantlet and Ψw. ns, not significant ; *, P 0.008 ; **, P 0.0001. To avoid autocorrelation the significance level was fixed at P 0.008(0.05\6).

status of nonstressed plantlets (0% PEG), whether mycorrhizal or nonmycorrhizal, did not alter significantly throughout the experiment (Fig. 1). The water potential means for the PgiT interaction (Fig. 1b) support the results observed for RWCleaf (Fig. 1a). At all time points during the PEG-induced water stress, the mean dry weights of whole plantlets (0n065 g, SE 0n003 g) and detached leaves (6n1 mg, SE 0n4 mg) were similar in mycorrhizal and nonmycorrhizal plantlets. Fresh weights differed significantly between PEG-stressed (0n242 g, SE 0n26 g) and nonstressed (0n570 g, SE 0n26 g) whole plantlets.

by the mycorrhizal status of the plantlets. Both mycorrhizal and nonmycorrhizal plantlets under well watered conditions had a mean Fjph\Fph ratio of 0n333, which did not differ significantly between sampling time points. Under PEG-induced stress, the Fjph\Fph ratio significantly (P l 0n0001) decreased from 0n318 after 4 h to 0n237 after 8 h, and to 0n195 after 12 h. A strong linear relation between Fjph\Fph and the quantum yield of O evolution from # strawberry leaves in vitro under saturating CO # concentrations was also observed (results not shown). Composition and concentration of free amino acids in leaves

Fluorescence measurements of Chl a Root colonization by G. intraradices did not alter the quantum efficiency of photosynthetic electron transport as estimated by the Fjph\Fph fluorescence parameter (Table 1). This parameter corresponds to the ratio between the amplitude of the slow induction phase Fjph over the maximal fluorescence yield Fph. However, we found a highly significant effect of the PEG-induced water stress on photosynthetic efficiency (Table 1). This effect increased with time (significant PgiT interaction) and was not altered

On the basis of the water potential values already shown, 8 and 12 h of exposure to PEG-induced water stress were harmful to the plantlets. Therefore free amino acid composition and concentration were determined only for material sampled after 4 h of water stress. The presence of the intraradical phase of G. intraradices had a marked effect on the concentration of the three principal amino acids found in leaves after PEG-induced water stress (PgiI in Table 2), where asparagine, glutamic acid and aspartic acid

9764

Significant PgiI interactions are shown in Fig. 2. *, P 0.05 ; **, P 0.001. ANBA, amino-N-butyric acid. †Arithmetic mean of all amino acids found, used to calculate percentages, including traces of isoleucine, ornithine and β-isobutyric acid (not shown).

18 0.09 16 0.09 28 0.1 88 0.4 36 0.4 71 0.5 23 0.8 868 2 266 2 233 3 791 4 353 4 1315 5 1382 17 9012 53

626 8

47541† 44 45 66 198 187 226 398 1133 857 1204 1796 1870 2395 8013

3694

ns ns ns ns ns ns * ns ns ns ns ns

Cys Lys Arg GABA

ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

25004

accounted for 78% of the total amino acid pool. In the absence of PEG, the leaf concentration of asparagine (53% of the total pool) was much higher in mycorrhizal (69n5 mmol kg−" d. wt) than in nonmycorrhizal (12n8 mmol kg−" d. wt) (Fig. 2). The opposite was observed after treatments with 15% PEG, in which leaf asparagine content in nonmycorrhizal plantlets (22 mmol kg−" d. wt) was almost twice that observed in mycorrhizal plantlets (c. 10 mmol kg−" d. wt) (Fig. 2). Glutamic acid (Glu) almost tripled its concentration in leaves of mycorrhizal plantlets after exposure to 15% PEG (Fig. 2). However, leaves from G. intraradices-colonized plantlets at 0% PEG had significantly lower levels of glutamic acid than did noncolonized ones. Aspartic acid levels were similar between well watered mycorrhizal and nonmycorrhizal plantlets. A significant increase in aspartic acid was observed in leaves of stressed mycorrhizal plants produced in vitro. Within treatments, the total amino acid concentrations in leaves did not differ significantly (Table

* * ns

Fig. 1. Combination of the levels of PEG-8000 (0% and 15%) and the time course (4, 8 and 12 h) for the variables for which the interaction PgiT was significant in Table 1. 0% PEG, closed circles ; 15% PEG, open circles. (a) RWCleaf. Means plotted are in the power-transformed scale as considered in the ANOVA. Their corresponding means are shown as percentages. The SE of the ANOVA test was 495. Error bars plotted refer to the SE for variability between treatments. (b) Ψw. Means plotted are the transformed rank values. Means before transformation are given for orientation on the biological levels found. The SE of the ANOVA test was 3n14. Error bars are the SE values for variability between treatments.

ns ns ns

12

ns ns ns

8 Time (h)

ns * ns

4

ns * *

0

* ns **

–3.22MPa 10

ns ns *

–2.78MPa

20

1 1 1 8

–1.89MPa

PEG (Pg) Inoculation (I ) PgiI Error Mean of all (n l 12) (µmol kg−" d. wt) SE Percentage of total AAs

30

Phe

40

His

–0.87MPa

50

Val

–0.64MPa

Gly

(b) –0.47MPa

60 Rank of Ψw

12

Thr

8

ANBA

4

Ala

70

Gln

0

Ser

43% 2

Asp

65% 4

Glu

71%

Asn

RWC 2leaf × 103

6

df

90%

Total amino acids

90% 8

Sources of variation

(a) 96%


10

Table 2. Significance of the ANOVA test for the composition and concentration of free amino acids in the leaves of strawberry plantlets after 4 h of exposure to poly(ethylene glycol) (PEG)-8000, inoculated or not with Glomus intraradices


182

As in leaves, the presence of the intraradical phase of G. intraradices had a marked effect on the concentration of the principal free amino acids in roots (Table 3). After PEG-induced water stress, the total

10243

0.0001. ANBA, amino-N-butyric acid ; GABA, γ-aminobutyric acid.

36 0.3 24 0.3 51 0.4 76 0.4 54 0.7 69 0.8 205 1 111 1 318 2 151 2 103 2 184 3 585 7 9291 75

234 4

41465† 105 113 169 179 276 335 486 602 751 631 879 1082 2798 31264

1635

* ns ** ns ns ns ns ns ns ns ns ns ns ns ns * ns ** *** ns ns

Total Lys Val Cys His Thr Gly GABA

ns ns ns ns ns * ns ns ns * ns * * ns ** ns ns ns ** ns *** ns ns ns ns ns ** 1 1 1 8

Significant PgiI interactions are shown in Fig. 3. *, P 0.05 ; **, P 0.001 ; ***, P †Arithmetic mean of all amino acids found, used to calculate percentages.

Composition and concentration of free amino acids in roots

183

PEG (Pg) Inoculation (I ) PgiI Error Mean of all (n l 12) (µmol kg−" d. wt) SE Percentage of total AAs

2). However, the intraradical colonization by G. intraradices (main factor ‘ inoculation ’, P l 0n0051 ; Table 2) increased the concentration of amino-Nbutyric acid (ANBA) from 672 to 1736 µmol kg−" d. wt in leaves, regardless of the osmotic stress. Independently of the inoculation treatment, PEGinduced stress decreased the concentration of ANBA from 1545 µmol kg−" d. wt at 0% PEG to 862 µmol kg−" at 15% PEG. Intraradical colonization by G. intraradices decreased the levels found of serine in leaves from 1279 µmol kg−" d. wt at 0% PEG to 887 µmol kg−". The osmotic stress increased the concentration of lysine in both mycorrhizal and nonmycorrhizal plantlets similarly. At 0% PEG, the lysine level was 16 µmol kg−" d. wt ; at 15% PEG this increased to 93 µmol kg−" d. wt. Traces of isoleucine, leucine and ornithine were found, representing 0n03%, 0n05% and 0n9% of the total amino acid pool (results not shown in Table 2). Their concentrations were not affected by the applied stress or by the fungus partner.

Ala

Glu

Arg

Asp

ANBA

Asn

Fig. 2. Concentrations of the amino acids asparagine (Asn), aspartic acid (Asp) and glutamine (Glu) in leaves of strawberry plantlets after 4 h of exposure to PEG-8000. Plots correspond to the significant interactions PgiI in Table 2. 0% PEG without AMF colonization, closed columns ; 0% PEG with colonization, grey columns ; 15% PEG without colonization, open columns ; 15% PEG with colonization, hatched columns. Errors bars are the SE values for variability between treatments. The SE of the ANOVA test (after logarithmic transformation of the means) was 0n315 for Asn and 0n088 for Asp. The SE of the ANOVA test for Gln was 1839.

Ser

0

Gln

10

Asp

20

Glu

30

Asn

40

df

50


60

Source of variation

Amino acid concentration (mmol kg–1 d. wt)

70

Table 3. Significance of the ANOVA test for the composition and concentration of free amino acids in roots of strawberry plantlets after 4 h of exposure to poly(ethylene glycol) (PEG)-8000 and inoculated or not with Glomus intraradices

RESEARCH Water stress in mycorrhizal strawberry plantlets in vitro


184

(83)

2.1 Log (starch concentration +1)

(63) (62) (65)

Amino acid concentration (mmol kg–1 d. wt)

90

60

30

Asn

Total amino acids

3

2

1

0

Asp

Ser

ANBA

Ala

Thr

Fig. 3. Concentrations of asparagine (Asn), total amino acids, aspartate (Asp), serine (Ser), amino-N-butyric acid (ANBA), alanine (Ala) and threonine (Thr) found in strawberry roots after 4 h of stress. Plots corresponding to the significant interactions PgiI in Table 3. 0% PEG without AMF colonization, closed columns ; 0% PEG with colonization, grey columns ; 15% PEG without colonization, open columns ; 15% PEG with colonization, hatched columns. Error bars are the SE values for variability between treatments. The SE of the ANOVA test after logarithmic transformation of the means was 0n225 for Asn, 0n156 for the total amino acids, 0n099 for Ser and 0n142 for Ala. The SE for the ANOVA test was 291 for Asp, 251 for ANBA and 81 for Thr.

amino acid concentration increased sevenfold in roots of mycorrhizal plantlets, whereas it decreased in roots of nonmycorrhizal plantlets (Fig. 3). The amino acid asparagine accounted for 75% of the total amino acid pool (Table 3 and Fig. 3). Aspartic acid, serine, ANBA, alanine and threonine, although minor contributors to the total amino acid pool ( 10%), showed similar patterns of PEG-induced changes in mycorrhizal and nonmycorrhizal roots (Fig. 3). The osmotic stress decreased the concentration of glycine from 533 to 137 µmol kg−" d. wt in mycorrhizal and nonmycorrhizal roots. Traces of isoleucine, leucine and ornithine were found, representing respectively 0n3%, 0n08% and 0n01% of the total amino acid pool (results not shown in Table 3). Starch concentration in leaves and roots As in the amino acid analyses, intraradical colonization by G. intraradices significantly affected

(34)

1.7 1.5 1.3

(10)

1.1

(7)

(8)

0.9 0.7 0.5

0

Amino acid concentration (nmol kg–1 d. wt)

1.9

Starch in leaves

Starch in roots

Fig. 4. Starch concentration in leaves and roots of strawberry plants produced in vitro after 4 h of PEGinduced osmotic stress. 0% PEG without AMF colonization, closed columns ; 0% PEG with colonization, grey columns ; 15% PEG without colonization, open columns ; 15% PEG with colonization, hatched columns. Means plotted are those considered for the ANOVA : the logarithmic transformation of the variable ‘ starch concentration ’. The SE of the ANOVA test was 0n185. Error bars are the SE for variability between treatments. Numbers in parentheses are values of detransformed means (10xk1) in µg of glucose mg−" d. wt for orientation about the real biological range of the variable.

root starch concentration (Fig. 4). After 4 h, mycorrhizal roots under PEG-induced stress increased their starch concentration significantly (34 µg of glucose mg−" d. wt) compared with nonstressed mycorrhizal controls (7 µg of glucose mg−" d. wt). In contrast, treatment with PEG did not modify the starch content of nonmycorrhizal roots (Fig. 4). Root colonization by G. intraradices had no effect on the starch content of stressed or unstressed leaves, or on that of well watered roots (Fig. 4). Furthermore, the PEG-induced water stress did not modify the starch level in leaves. Similar conclusions were obtained whether the starch concentration was calculated on the basis of fresh or dry weight. Only dry weight means are indicated in the text and plotted in Fig. 4.  Recently Herna! ndez-Sebastia' et al. (1999) showed that mycorrhizal strawberry plantlets in vitro, produced in a tripartite culture system, had increased root RWC. Following on from this study, we investigated whether this increase in RWC could be an advantage to plants subjected to an osmotic stress that simulated the shock experienced after transfer to conditions ex vitro. Our results indicate that the presence of the intraradical phase of G. intraradices does not improve the water status of host plants under short-term PEG-induced water stress. This might be due to the loss of the extraradical mycelium of the fungus after the transfer of the plantlets from the in vitro growth medium. In addition, the advantages associated with a pre-colonization in vitro

RESEARCH Water stress in mycorrhizal strawberry plantlets in vitro might have been masked by the short duration of this study, in which the extraradical mycelia of the fungus did not have time to develop. Although the water transport ability of ectomycorrhizal hyphae and the associated benefits to host plants have been well established (e.g. Lamhamedi et al., 1992), investigations are needed to determine whether the extraradical mycelium of endomycorrhizal fungi can have a similar role during drought stress. In contrast to the study of Herna! ndez-Sebastia' et al. (1999), we observed here no difference between the RWC of unstressed mycorrhizal and nonmycorrhizal strawberry plantlets. This was due to the new experimental conditions in which the nonstressed (0% PEG) plantlets were soaked in a liquid medium with a water potential close to that of nanopure water. As a consequence, after 4 h the RWC increased by up to 100% (in mycorrhizal and nonmycorrhizal plantlets), masking the initial effect of AMF on root RWC. In plants, asparagine serves as an important nitrogen transport compound whose levels are markedly regulated by light in many plant species (Lam et al., 1998). Although the presence of the intraradical phase of G. intraradices did not influence the effects of PEG-induced water stress on plant water status, the analyses of the main amino acid levels revealed the presence of distinct responses in mycorrhizal and nonmycorrhizal plantlets. Asparagine was the major amino acid that we found in strawberry tissues grown in vitro, accounting for 53% of the total amino acid pool in leaves and 75% in roots. In mycorrhizal plantlets, the PEG-induced water stress caused a large decrease in asparagine in leaves that was mirrored by a marked increase in asparagine in roots. Interestingly, the opposite was observed in nonmycorrhizal plantlets, in which a marked increase in asparagine occurred in leaves balanced by a marked decrease in roots (Figs 2, 3). Our results therefore suggest the presence of an important mobile pool of asparagine that can be translocated from leaves to roots or vice versa, in response to drought stress, depending on the plant’s mycorrhizal status. Our results are also consistent with the idea that asparagine, which is a major component of xylem and phloem saps and tissues (Pacowsky, 1989 ; Auge! et al., 1992 ; Parsons & Baker, 1996), is the main compound involved in nitrogen transport in strawberry plants. High asparagine contents are also common in roots (Auge! et al., 1992), and tissues of many plants accumulate asparagine in response to water deficits (Stewart & Larher, 1980 ; Drossopoulos et al., 1985). Interestingly, however, drought induces increases in asparagine in nonmycorrhizal Rosa roots but not in mycorrhizal roots (Auge! et al., 1992). Results supporting the notion that plants have one or more nitrogen regulatory systems include those from previous studies showing that the levels of

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mRNA for plant nitrogen assimilatory genes such as glutamine synthetase and asparagine synthetase are modulated by carbon and organic nitrogen metabolites (Hsieh et al., 1998). In strawberry leaf and roots tissues, the modulation of the asparagine levels by the intraradical structures of the fungus takes place in opposite directions under well watered and water-stressed conditions. After prolonged stress the increase in asparagine in mycorrhizal roots might be due to different causes. Translocation from the leaves, where their corresponding levels decreased, seems possible, given the mobility of the asparagine in the phloem. However, it should be tested in future experiments whether the asparagine was released by protein breakdown stimulated by the fungal presence or accumulated by synthesis de novo, as a result of indirect changes in the levels of carbon and organic nitrogen metabolites under the influence of the symbiotic in-root compartment. During salinity stress, several amino acids were found to accumulate in both the sink and source tissues, including arginine, asparagine and serine in Coleus blumei (Gilbert et al., 1998). As in our work, a larger proportion of asparagine and less arginine was found in the sink tissue of the salinity-stressed plants and no proline was found to accumulate in either source or sink tissues during the exposure to salinity. Finally, it is noteworthy that the distinct effects of PEG-induced osmotic stress on the asparagine concentration in mycorrhizal and nonmycorrhizal roots are closely reflected by changes in the root starch contents. As for asparagine, the presence of PEG caused a large increase in starch content in mycorrhizal roots. These distinct responses between mycorrhizal and nonmycorrhizal plantlets, whether water-stressed or not, suggest different strategies involving coordinated adjustments to the nitrogen and carbon metabolisms. Obviously, such an adaptive response has a limited efficiency, which would have been overwhelmed by the severity of the PEGinduced osmotic stress. Because this study considered only the effects of the intraradical phase of G. intraradices on PEGinduced water stress, further experiments are needed to test whether or not the presence of the extraradical phase of the fungus might improve the adaptability of micropropagated strawberry plants to drought stress.                C. H. S. thanks Dr Robert Gigue' re (Re! seau de Me! decine Ge! ne! tique du Que! bec, Centre Universitaire de Sante! de l’Estrie, Sherbrooke, Que! bec, Canada) for his assistance in the amino acid analyses, and Andrew Coughlan (CRBF, Universite! Laval, Que! bec Canada) for his final revision of the manuscript. This work has been supported by a PhD fellowship from the Comissionat per a Universitats i Recerca, Generalitat de Catalunya, Spain, and the Ministe' re de l’E! ducation, Que! bec, to C. H. S.

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