Acta Physiol Plant (2007) 29:543–549 DOI 10.1007/s11738-007-0065-y
ORIGINAL PAPER
Osmotic solute responses of mycorrhizal citrus (Poncirus trifoliata) seedlings to drought stress Qiang-Sheng Wu Æ Ren-Xue Xia Æ Ying-Ning Zou Æ Gui-Yuan Wang
Received: 21 December 2006 / Revised: 20 March 2007 / Accepted: 10 April 2007 / Published online: 31 May 2007 Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2007
Abstract This study investigated the accumulation of osmotic solutes in citrus (Poncirus trifoliata) seedlings colonized by Glomus versiforme subjected to drought stress or kept well watered. Development of mycorrhizae was higher under well watered than under drought-stressed conditions. Arbuscular mycorrhizal (AM) seedlings accumulated more soluble sugars, soluble starch and total non-structural carbohydrates in leaves and roots than corresponding non-AM seedlings regardless of soil-water status. Glucose and sucrose contents of well-watered and drought-stressed roots, fructose contents of well-watered roots and sucrose contents of drought-stressed leaves were notably higher in AM than in non-AM seedlings. K+ and Ca2+ levels in AM leaves and roots were greater than those in non-AM leaves and roots, while AM symbiosis did not affect the Mg2+ level. AM seedlings accumulated less proline than non-AM seedlings. AM symbiosis altered both the allocation of carbohydrate to roots and the net osmotic solute accumulations in response to drought stress. It is concluded that AM colonization enhances osmotic solute accumulation of trifoliate orange seedlings, thus providing better osmotic adjustment in AM seedlings, which did not correlate with proline but with K+, Ca2+, Mg2+, glucose, fructose and sucrose accumulation.
Communicated by W. Horst. Q.-S. Wu (&) Y.-N. Zou G.-Y. Wang College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei Province 434025, People’s Republic of China e-mail:
[email protected] Q.-S. Wu R.-X. Xia College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, Hubei Province 430070, People’s Republic of China
Keywords Arbuscular mycorrhizal fungi Citrus Drought stress Osmotic adjustment Solutes Abbreviations AM Arbuscular mycorrhizal AMF Arbuscular mycorrhizal fungi DS Drought-stressed W Leaf water potential RWC Relative water content NSC Total non-structural carbohydrates WW Well-watered
Introduction Citrus is one of the most important commercial fruit crops all over the world, and cultivated in the south and southwest regions of China. However, these regions are subject to water deficiency, which restricts the yield and quality of citrus fruits. Drought stress is a problem for both vegetative and reproductive phases of plant growth (Iannucci et al. 2002; Martinez et al. 2004). Citrus has few and short root hairs in the field and is highly dependent on arbuscular mycorrhizae, because the most common mutualistic symbiosis replaces part of the root-hair functions (Graham and Syvertsen 1985). Previous studies have indicated that inoculation with arbuscular mycorrhizal fungi (AMF) appeared to improve water relations of host plants including citrus under wellwatered (WW) and drought-stressed (DS) conditions (Auge´ 2001, 2004). The possible mechanisms for improved water relations of arbuscular mycorrhizal (AM) plants include direct water uptake and transport via external hyphae
123
544
(Faber et al. 1991; Ruiz-Lozano and Azco´n 1995; Auge´ et al. 2003), regulation of stomatal conductance in response to hormonal signals (Goicoechea et al. 1997a), indirect effects of improved phosphorus nutrition (Nelsen and Safir 1982), and greater enzymatic and non-enzymatic antioxidative capacity and osmotic adjustment in AM plants (Ruiz-Lozano 2003; Porcel et al. 2004; Wu and Xia 2004, 2006; Wu et al. 2006a, 2006b). Although the benefit of colonization by AMF to plant–water relations has been supported by many studies, the debate still continues. For instance, Bryla and Duniway (1997) observed no AMF effects on osmotic adjustment of DS plants. A similar result was also found by Auge´ et al. (1992) and Goicoechea et al. (1997b). AM plants sometimes have shown more osmotic adjustment and sometimes not (Auge´ 2001). Trifoliate orange, a major citrus rootstock used in China, shows less drought resistance but higher cold resistance. The objective of this study was to determine whether AM fungus colonization improves water relations of trifoliate orange seedlings by affecting osmotic adjustment under WW and DS conditions. In a previous study (Wu and Xia 2006), we found that Glomus versiforme inoculation enhanced the osmotic adjustment of Citrus tangerine, and that this correlated with total non-structural carbohydrates, K+, Ca2+ and Mg2+ levels, but not with proline levels. However, the monosaccharide and disaccharide sugars were not determined in these experiments. Another objective of the study was to determine whether our conclusion (Wu and Xia 2006) holds true also for trifoliate orange.
Materials and methods Plant materials and culture Seven day-old non-AM-infected trifoliate orange [Poncirus trifoliata (L.) Raf.] seedlings were used in this study. Six seedlings were grown in a plastic pot (15 · 20 cm) containing 3.37 kg of autoclaved growth substrate (soil:vermiculite:sphagnum, 5:2:1, v/v/v). The substrate had a pH of 5.9, 1.3% organic matter, 30 mg kg–1 available phosphorus, 147 mg kg–1 alkali hydrolysable nitrogen, and 141 mg kg–1 available potassium. Half of the pots received Glomus versiforme (Karsten) Berch (30 g of inoculum placed 5 cm deep) at transplanting. This inoculum contained approximately 2,233 spores and was provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences. Control treatment received no AMF inoculum (30 g of autoclaved growth substrate). The AM and non-AM seedlings were placed in a greenhouse without temperature control from March to September 2004.
123
Acta Physiol Plant (2007) 29:543–549
All seedlings were watered daily until differential water treatments were initiated 97 days after transplanting. Pots with WW and DS seedlings were maintained everyday at 75% (corresponding to –0.09 MPa) or 55% (corresponding to –0.40 MPa) relative soil–water content by gravimetry, respectively. Experimental design The experiment was laid out in randomized complete blocks with two water treatment (WW and DS) and two mycorrhizal treatment (G. versiforme and non-AMF). Each of the four treatments had six replicates. Parameters measured The AM and non-AM seedlings were harvested 80 days after differential water treatments, divided into shoots and roots, and oven-dried at 75C for 48 h. Root samples were cleared with 10% (w/v) KOH solution at 95C for 1 h, stained with 0.05% (v/v) trypan blue in lactic acid for 5 min (Phillips and Hayman 1970), and AM root colonization microscopically observed. The mycorrhizal structures such as entry points, vesicles and arbuscles were calculated for the infected roots. The root colonization by G. versiforme was estimated by the gridline intersection method (Giovanetti and Mosse 1980). Chlorophyll was extracted in 80% (v/v) acetone from 100 mg of fresh leaf sample in the dark at room temperature and was measured at 470, 646 and 663 nm with a UV/ VIS spectrophotometer by the method of Li (2000). About 100 mg of dry samples of leaves and roots were used to extract inorganic ions. The dry samples were incubated in 20 ml distilled water at 100C for 2 h, cooled at room temperature for 30 min, and then filtered. The cations K+, Ca2+ and Mg2+ contents were determined directly using an Atomic Emission Spectrometer (AA670, Shimadzu, Japan). Soluble sugar and soluble starch contents of fresh leaves and roots were determined using the method of described by Wu and Xia (2006). A calibration curve with sucrose was used as a standard. Total non-structural carbohydrates (NSC) were the sum of soluble sugar and soluble starch. Proline content was quantified using the ninhydrin method of Troll and Lindsley (1955). A modified method of Mao et al. (2003) was employed for the extraction of glucose, fructose and sucrose. About 500 mg of fresh samples was homogenized in imidazole– hydrochloric acid (pH 7.0) containing 80% methanol, and then filtered into 10-ml volumetric flasks containing 1 ml 2.5% (w/v) methyl-a-D-glucoside. Five-milliliter extracts were centrifuged at 10,000 · g for 10 min. The 1.0-ml supernatant was freeze-dried, dissolved in 0.5 ml pyridine
Acta Physiol Plant (2007) 29:543–549
anhydrous. Then 0.2 ml hexamethyl disilazane and 0.1 ml trimethylchlorosilane were added. The mixture was shaken at 30C for 3 h before 0.6 ml n-hexane and 0.5 ml of distilled water were added, and was separated phase quietly. The upper phase was used for the GS analysis. Gas chromatography was performed as described by Bartolozzi et al. (1997) with a slight modification. Injector and detector temperatures were 250 and 270C, respectively. The following temperature programme was set: 130C for 1 min, followed by increases from 130 to 152C at 8C min–1, from 152 to 176C at 12C min–1, from 176 to 198C at 16C min–1, from 198 to 238C at 20C min–1, from 238 to 280C at 24C min–1, and finally to 290C for 2 min. Flow-rates of H2, N2 and air were 40, 25 and 400 ml min–1, respectively. Chapiter pressure was 1.03 · 105 Pa. One microlitre of reagents was injected into the chromatograph, with a split ratio of 60:1. Relative water content (RWC) of the fifth fully expanded leaf from the shoot apex was evaluated by the method of Wu and Xia (2006). Leaf water potential (W) was determined using a pressure chamber, as described by Li (2000). Statistical analysis
545 Table 1 Root colonization, entry points, vesicles and arbuscles of AM Poncirus trifoliata seedlings grown under well-watered (WW) or drought-stressed (DS) conditions Water status
AM fungus colonization (%)
Entry points (num cm–1 root)
Vesicles (num cm–1 root)
Arbuscles (num cm–1 root)
WW
36.98a
7.2a
4.5a
10.0a
DS
22.32b
3.6b
2.9a
4.5b
No colonization was found in uninoculated seedlings Means followed by the same letter within a column are not significantly different among treatments at P < 0.05
Table 2 Shoot dry weight, root dry weight and leaf chlorophyll contents of AM or non-AM Poncirus trifoliata seedlings grown under well-watered (WW) or drought-stressed (DS) conditions Water status
AMF status
Shoot dry weight (g plant–1)
Root dry weight (g plant–1)
Chlorophyll content [mg g–1 (f.w.)]
WW
AMF
1.16a
0.49a
2.17a
Non-AMF
1.00b
0.42b
1.98b
AMF
0.89b
0.35c
2.07ab
Non-AMF
0.71c
0.30c
1.75c
**
**
**
DS ANOVA DS
Data were analyzed using 2-factor ANOVA with the Statistical Analysis System (SAS) 8.1 software (SAS Institute Inc., Cary, NC, USA). Probabilities of significant differences were used to test the significance among treatments and interactions. For all characteristics studied significant differences between means were determined using Fisher’s protected least significant difference at P < 0.05.
Results No AM structures were noted in the roots of uninoculated seedlings. AM seedlings were infected by AMF, and colonization, entry points and arbuscles were reduced by drought stress (Table 1). Drought stress had no effect on vesicles. AMF inoculation improved plant biomass, especially shoot dry weight (Table 2). Chlorophyll contents of leaves remained high in AM seedlings under WW and DS conditions compared with non-AM seedlings. Drought stress notably decreased W, but the decrease was larger in non-AM seedlings (–0.53 MPa) than in AM seedlings (–0.44 MPa) (Fig. 1). AM symbiosis significantly increased W of seedlings under WW and DS conditions. AM seedlings showed higher RWC than non-AM seedlings regardless of soil–water status (Fig. 2). However, RWC was not affected by drought stress in both, AM and non-AM seedlings. Interaction of AMF and DS was not exhibited for W and RWC.
AMF
**
*
**
DS · AMF
NS
NS
NS
Means followed by the same letter within a column are not significantly different among treatments at P < 0.05 Data were analyzed with ANOVA. * P < 0.05, ** P < 0.01. NS not significant
Soluble sugar contents of leaves were higher in AM than in non-AM seedlings under WW and DS conditions (Table 3). AM and non-AM roots subjected to drought stress had similar soluble starch contents, while wellwatered AM roots had higher soluble starch contents than non-AM roots. Compared with that of non-AM seedlings, soluble starch contents of AM leaves increased by 43 and 51% and soluble starch contents of AM roots by 27 and 25% under WW and DS conditions, respectively. AMF colonization significantly increased NSC contents of leaves and roots under WW and DS conditions. However, NSC contents of leaves and roots remained unchanged in response to drought stress regardless of AM presence or absence. Drought stress increased proline contents in leaves or roots of both AM and non-AM seedlings compared with WW conditions (Table 3). In roots, there were no significant differences in proline contents of both AM and nonAM seedlings under WW and DS conditions. In leaves, however, AMF colonization significantly decreased proline contents under WW and DS conditions.
123
546
Acta Physiol Plant (2007) 29:543–549
WW
DS
0
(MPa)
-0.2 -0.4
a
-0.6
b
-0.8
c
AMF Non-AMF
-1
d
-1.2
Fig. 1 Leaf water potential (W) in AM or non-AM Poncirus trifoliata seedlings grown under well-watered (WW) or drought-stressed (DS) conditions
AM symbiosis did not affect glucose contents of leaves and fructose contents of leaves and roots, while AM seedlings showed notably higher glucose contents of roots as well as sucrose contents of leaves and roots. Regardless of drought treatments, AMF notably increased K+ levels in leaves and Ca2+ levels in roots (Table 5). Although K+ levels in roots and Ca2+ levels in leaves were higher in AM than in non-AM seedlings, the differences were only significant under DS conditions. Mg2+ levels in leaves and roots were not affected by AM symbiosis regardless of soil–water status.
Discussion AMF
100
a
Non-AMF
ab
bc
c
RWC(%)
90 80 70 60 50 WW
DS
Fig. 2 Leaf relative water content (RWC) of AM or non-AM Poncirus trifoliata seedlings grown under well-watered (WW) or drought-stressed (DS) conditions
Under WW conditions, AM symbiosis significantly reduced glucose contents of leaves as well as fructose contents of leaves, whereas glucose, fructose, and sucrose contents of roots were higher in AM than in non-AM seedlings, respectively (Table 4). Under DS conditions,
Upon exposure to drought stress, higher plants exhibit a series of modulated responses at morphological, anatomical, cellular and molecular levels, and osmotic adjustment is a common response at the cellular level (Xiong and Zhu 2002; Chimenti et al. 2006). Osmotic adjustment has been found to alleviate damage of drought stress and is considered to be a drought-tolerance mechanism in several plant species (Martinez et al. 2004). A number of osmotically active substances including inorganic ions and organic solutes play an important role in osmotic adjustment (Morgan 1984). Our study indicated that drought stress affects inorganic ion (K+, Ca2+, Mg2+) and organic solute (NSC, soluble sugar, soluble starch, proline, glucose, fructose and sucrose) levels in leaves or roots of both, AM and non-AM seedlings, suggesting that osmotic adjustment is a response to drought stress also in citrus seedlings. In addition, AM symbiosis, in part, alters these solute levels in citrus seedlings regardless of soil–water status. For example, AM seedlings grown in DS conditions exhibited higher soluble sugar contents of leaves, soluble starch and NSC
Table 3 Soluble sugar, soluble starch, total non-structural carbohydrate (NSC) and proline contents of AM or non-AM Poncirus trifoliata seedlings grown under well-watered (WW) or drought-stressed (DS) conditions Water status WW DS
AMF status
Soluble sugar (% f.w.)
Soluble starch (% f.w.)
Proline [mg g–1 (f.w.)]
NSC (% f.w.)
Leaf
Leaf
Leaf
Leaf
Root
Root
Root
Root
AMF
8.39b
5.58a
8.94a
6.31a
0.26d
0.20c
17.33a
11.89a
Non-AMF
6.16c
3.22b
6.24bc
4.96b
0.46b
0.24bc
12.40b
8.19b
AMF
9.14a
6.24a
7.97ab
5.20b
0.37c
0.27ab
17.11a
11.44a
Non-AMF
8.21b
5.46a
5.27c
4.17c
0.55a
0.30a
13.47b
9.63b
DS
**
*
NS
**
**
**
NS
NS
AMF
**
*
**
**
**
NS
**
**
DS · AMF
**
NS
NS
NS
NS
NS
NS
NS
ANOVA
Means followed by the same letter within a column are not significantly different among treatments at P < 0.05 Data were analyzed with ANOVA. * P < 0.05, ** P < 0.01. NS not significant
123
Acta Physiol Plant (2007) 29:543–549
547
Table 4 Glucose, fructose and sucrose contents of AM or non-AM Poncirus trifoliata seedlings grown under well-watered (WW) or droughtstressed (DS) conditions Water status
AMF status
Glucose [mg g–1 (f.w.)]
Fructose [mg g–1 (f.w.)]
Sucrose [mg g–1 (f.w.)]
Leaf
Root
Leaf
Root
Leaf
Root
WW
AMF
21.31b
14.81b
23.75b
23.08b
2.14c
1.49c
Non-AMF
25.13a
11.98c
28.70a
18.08c
2.28c
1.29d
AMF
22.32b
20.66a
27.85a
26.58a
5.76a
2.41a
Non-AMF
21.89b
16.37b
25.61ab
25.71ab
5.48b
1.96b
DS
NS
**
NS
**
**
**
AMF DS · AMF
NS *
** NS
NS *
** **
NS *
** NS
DS ANOVA
Means followed by the same letter within a column are not significantly different among treatments at P < 0.05. Data were analyzed with ANOVA. * P < 0.05, ** P < 0.01. NS not significant
Table 5 K+, Ca2+ and Mg2+concentrations in AM or non-AM Poncirus trifoliata seedlings grown under well-watered (WW) or drought-stressed (DS) conditions K+[mg g–1 (d.w.)]
Ca2+[mg g–1 (d.w.)]
Mg2+[mg g–1 (d.w.)]
Leaf
Root
Leaf
Root
Leaf
Root
AMF
21.98a
13.50b
3.58c
3.36c
1.84a
2.14b
Non-AMF
20.92bc
13.27b
3.56c
2.53d
1.97a
2.14b
AMF
21.56ab
14.30a
6.44a
4.38a
1.91a
2.24ab
Non-AMF
20.52c
13.63b
4.83b
3.95b
1.99a
2.37a
DS
NS
*
**
**
NS
**
AMF DS · AMF
* NS
* NS
** **
** NS
NS NS
NS NS
Water status
WW DS
AMF status
ANOVA
Means followed by the same letter within a column are not significantly different among treatments at P < 0.05. Data were analyzed with ANOVA. * P < 0.05, ** P < 0.01. NS not significant
contents of leaves and roots, glucose contents of roots, sucrose contents of leaves and roots, and K+ and Ca2+ levels in leaves and roots, when compared with DS nonAM seedlings. These beneficial responses of solutes to AMF colonization would help to enhance drought tolerance of trifoliate orange seedlings by osmotic adjustment thus protecting and stabilizing macromolecules and structures from damage induced by drought stress (Martinez et al. 2004). AMF colonization kept higher water status (e.g. W and RWC) in trifoliate orange seedlings due to better osmotic adjustment. These observations were in agreement with other pervious investigations with different plants (Abdel-Fattah et al. 2002; Wu and Xia 2004, 2006). It is well-known that proline is a non-protein amino acid and is believed to protect plant tissues against stress by acting as an N-storage compound, osmo-solute, and hydrophobic protectant for enzymes and cellular structures (Madan et al. 1995). Thus, a high proline level may help plants to survive short-time drought and recover from stress
(Sanchez et al. 1998). In the present work, AM leaves had lower proline levels than non-AM leaves when exposed to WW and DS conditions, which may be attributed to either greater drought resistance of AM seedlings or less injury in AM seedlings grown under drought stress conditions. The result agrees with previous reports obtained from Citrus tangerine (Wu and Xia 2006), Zea mays (Ramakrishnan et al. 1988), Lactuca sativa (Ruiz-Lozano and Azco´n 1997) and Glycine max (Porcel et al. 2004). Osmotic adjustment involves the lowering of the osmotic potential due to a net solute accumulation in response to drought stress (Iannucci et al. 2002; Martinez et al. 2004; Martinez-Ballesta et al. 2004; Chimenti et al. 2006). Our results showed that the net accumulations of K+ in roots, Ca2+ and Mg2+ in leaves, glucose in leaves and roots, fructose in leaves, sucrose in leaves and roots were higher in AM than in non-AM seedlings, respectively. According to the finding of Wu and Xia (2006), better osmotic adjustment in AM C. tangerine seedlings
123
548
originated not from proline but from NSC, K+, Ca2+ and Mg2+. NSC consists of soluble starch and soluble sugars including glucose, fructose, sucrose, etc. According to our above-mentioned analysis, the greater net solute accumulation in AM seedlings would help to enhance osmotic adjustment, which did not correlate with proline but with K+, Ca2+, Mg2+, glucose, fructose and sucrose accumulation. AM symbiosis altered the allocation of carbohydrate to roots. The distributed proportions of soluble sugars in WW and DS seedlings and NSC in WW seedlings were higher in AM than in non-AM seedlings. This result is in agreement with Nemec and Guy (1982) and Wu and Xia (2006). In addition, contents of glucose, fructose, and sucrose in WW seedlings and of glucose and sucrose in DS seedlings were higher in the roots of AM seedlings when compared with non-AM seedlings. Thus, AM roots represented greater sinks for carbohydrates than non-AM roots. AM fungi take up glucose from the host plants to use for trehalose synthesis (Shachar-Hill et al. 1995), which is needed for sustaining fungal growth and development. Trehalose, a known mycorrhizal metabolite, is thought to play a role in the protection of membranes and proteins (Schellenbaum et al. 1998). Thus, AM symbiosis appears to strongly compete for root-allocated carbon resulting in an enhanced allocation of carbohydrates to roots for AM growth and development as well as for protection of membranes and proteins. In conclusion, the present study demonstrates that AMF colonization enhances osmotic solute accumulation of trifoliate orange seedlings, thus providing more osmotic adjustment in AM seedlings, which does not correlate with proline but with K+, Ca2+, Mg2+, glucose, fructose and sucrose accumulation. Acknowledgments This work was supported by Ministry of Science and Technology, P.R. China (2002EP090016; 2003EP090018; 2004EP090019) as well as Scientific and Developmental Fund, Yangtze University (39210264).
References Abdel-Fattah GM, Migahed FF, Ibrahim AH (2002) Interactive effects of endomycorrhizal fungus Glomus etunicatum and phosphorus fertilization on growth and metabolic activities of broad bean plants under drought stress conditions. Pak J Biol Sci 5:835–841 Auge´ RM (2001) Water relations, drought and vesicular–arbuscular mycorrhizal symbiosis. Mycorrhiza 11:3–42 Auge´ RM (2004) Arbuscular mycorrhizae and soil/plant water relations. Can J Soil Sci 84:373–381 Auge´ RM, Stodola AJ, Brown MS, Bethlenfalvay GJ (1992) Stomatal response of mycorrhizal colonization and drought acclimation. Physiol Plant 70:175–182 Auge´ RM, Moore JL, Stutz JC, Sylvia DM, Al-Agely AK, Saxton AM (2003) Relation foliar dehydration tolerance of mycorrhizal
123
Acta Physiol Plant (2007) 29:543–549 Phaseolus vulgaris to soil and root colonizatin by hyphae. J Plant Physiol 160:1147–1156 Bartolozzi F, Bertazza G, Bassi D, Cristoferi G (1997) Simultaneous determination of soluble sugars and organic acids as their thrimethylsilyl derivatives in apricot fruits by gas–liquid chromatography. J Chromatogr A 758:99–107 Bryla DR, Duniway JM (1997) Growth, phosphorus uptake, and water relations of safflower and wheat infected with an arbuscular mycorrhizal fungus. New Phytol 136:581–590 Chimenti CA, Marcantonio M, Hall AJ (2006) Divergent selection for osmotic adjustment results in improved drought tolerance in maize (Zea mays L.) in both early growth and flowering phases. Field Crops Res 95:305–315 Faber BA, Zasoske RJ, Munns DN, Shackel K (1991) A method for measuring hyphal nutrition and water uptake in mycorrhizal plants. Can J Bot 69:87–94 Giovannetti M, Mosse B (1980) An evaluation of techniques for measuring vesicular mycorrhizal infection in roots. New Phytol 84:489–500 Goicoechea N, Antolin MC, Sanchez-Diaz M (1997a) Gas exchange is related to the hormone balance in mycorrhizal or nitrogenfixing alfafa subjected to drought. Physiol Plant 100:989–997 Goicoechea N, Antolin MC, Sanchez-Diaz M (1997b) Influence of arbuscular mycorrhizae and Rhizobium on nutrient content and water relations in drought-stressed alfalfa. Plant Soil 192:261–268 Graham JH, Syvertsen JP (1985) Host determinants of mycorrhizal dependency of citrus rootstock seedlings. New Phytol 101:667– 676 Iannucci A, Russo M, Arena L, Fonzo ND, Martiniello P (2002) Water deficit effects on osmotic adjustment and solute accumulation in leaves of annual clovers. Eur J Agron 16:111–122 Li HS (2000) Principles and techniques of plant physiological biochemical experiments. Higher Education Press, Beijing Madan S, Nainawatee HS, Jain RK, Chowdhury JB (1995) Proline and proline metabolising enzymes in in-vitro selected NaCltolerant Brassica juncea L. under salt stress. Ann Bot 76:51–57 Mao DB, Qu ZG, Zhang JS, Tian NY, Zhang WY (2003) Capillary gas chromatography of dissociative sugar in tobacco. Chin J Chromatogr 21:437 Martinez J-P, Lutts S, Schanck A, Bajji M, Kinet J-M (2004) Is osmotic adjustment required for drought stress resistance in the Mediterranean shrub Atriplex halimus L? J Plant Physiol 161:1041–1051 Martinez-Ballesta MC, Martinez V, Carvajal M (2004) Osmotic adjustment, water relations and gas exchange in pepper plants grown under NaCl or KCl. Environ Exp Bot 52:161–174 Morgan JM (1984) Osmoregulation and water stress in higher plants. Annu Rev Plant Physiol 35:299–319 Nelsen CE, Safir GR (1982) Increased drought tolerance of mycorrhizal onions due to improved phosphorus nutrition. Planta 154:407–413 Nemec S, Guy G (1982) Carbohydrate status of mycorrhizal and nonmycorrhizal citrus rootstocks. J Am Soc Hort Sci 107:177– 180 Phillips JM, Hayman DS (1970) Improve procedures for clearing roots and staining parasitic and vesicular–arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–161 Porcel R, Barea JM, Ruiz-Lozano JM (2004) Arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress. J Exp Bot 55:1743–1750 Ramakrishnan R, Jehri BN, Gupta RK (1988) Influence of VAM fungus Glomus caledonius on free proline accumulation in water-stressed maize. Curr Sci 57:1082–1084
Acta Physiol Plant (2007) 29:543–549 Ruiz-Lozano JM (2003) Arbuscular mycorrhizal symbiosis and alleviation of osmotic stress, new perspectives for molecular studies. Mycorrhiza 13:309–317 Ruiz-Lozano JM, Azco´n R (1995) Hyphal contribution to water uptake in mycorrhizal plants as affected by the fungal species and water status. Physiol Plant 95:472–478 Ruiz-Lozano JM, Azco´n R (1997) Effect of calcium application on the tolerance of mycorrhizal lettuce plants to polyethylene glycol-induced water stress. Symbiosis 23:9–21 Sanchez FJ, Manzanares M, de Andres EF, Tenorio JL, Ayerbe L (1998) Turgor maintenance, osmotic adjustment and soluble sugar and proline accumulation in 49 pea cultivars in response to water stress. Field Crops Res 59:225–235 Schellenbaum L, Muller J, Boller T, Wiemken A, Schuepp H (1998) Effects of drought on non-mycorrhizal and mycorrhizal maize: changes in the pools of non-structural carbohydrates, in the activities of invertase and trehalase, and in the pools of amino acids and imino acids. New Phytol 138:59–66 Schellenbaum L, Sprenger N, Schuepp H, Wiemken A, Boller T (1999) Effects of drought, transgenic expression of a fructan synthesizing enzyme and of mycorrhizal symbiosis on growth and soluble carbohydrate pools in tobacco plants. New Phytol 142:67–77
549 Shachar-Hill Y, Preffer PE, Douds D, Osman SF, Doner LW, Ratcliffe RG (1995) Partitioning of intermediary carbon metabolism in versicular–arbuscular mycorrhizal leek. Plant Physiol 108:7–15 Troll W, Lindsely J (1955) A photometric method for the determination of proline. J Biol Chem 215:655–660 Wu QS, Xia RX (2004) Effects of arbuscular mycorrhizal fungi on plant growth and osmotic adjustment matter content of trifoliate orange seedlings under water stress. J Plant Physiol Mol Biol 30:583–588 Wu QS, Xia RX (2006) Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. J Plant Physiol 163:417–425 Wu QS, Xia RX, Zou YN (2006a) Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress. J Plant Physiol 163:1101–1110 Wu QS, Zou YN, Xia RX (2006b) Effects of water stress and arbuscular mycorrhizal fungi on reactive oxygen metabolism and antioxiant production by citrus (Citrus tangerine) roots. Eur J Soil Biol 42:166–173 Xiong L, Zhu J-K (2002) Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ 25:131–139
123
