JXB Advance Access published online on May 17, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm102
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Lotus tenuis tolerates the interactive effects of salinity and waterlogging by ‘excluding’ Na+ and Cl– from the xylem
1School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, WA, Australia
2CRC for Plant-based Management of Dryland Salinity, The University of Western Australia, Crawley 6009, WA, Australia
3School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
* To whom correspondence should be addressed. E-mail: tdcolmer{at}cyllene.uwa.edu.au
Received 22 March 2007; Accepted 17 April 2007
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Abstract |
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Salinity and waterlogging interact to reduce growth of poorly adapted species by, amongst other processes, increasing the rate of Na+ and Cl– transport to shoots. Xylem concentrations of these ions were measured in sap collected using xylem-feeding spittlebugs (Philaenus spumarius) from Lotus tenuis and Lotus corniculatus in saline (NaCl) and anoxic (stagnant) treatments. In aerated NaCl solution (200 mM), L. corniculatus had 50% higher Cl– concentrations in the xylem and shoot compared with L. tenuis, whereas concentrations of Na+ and K+ did not differ between the species. In stagnant-plus-NaCl solution, xylem Cl– and Na+ concentrations of L. corniculatus increased to twice those of L. tenuis. These differences in xylem ion concentrations, which were not caused by variation in transpiration between the two species, contributed to lower net accumulation of Na+ and Cl– in shoots of L. tenuis, indicating that ion transport mechanisms in roots of L. tenuis were contributing to better ‘exclusion’ of Cl– and Na+ from shoots, compared with L. corniculatus. Root porosity was also higher in L. tenuis, due to constitutive aerenchyma, than in L. corniculatus, suggesting that enhanced root aeration contributed to the maintenance of Na+ and Cl– ‘exclusion’ in L. tenuis exposed to stagnant-plus-NaCl treatment. Lotus tenuis also had greater dry mass than L. corniculatus after 56 d in NaCl or stagnant-plus-NaCl treatment. Thus, Cl– ‘exclusion’ is a key trait contributing to salt tolerance of L. tenuis, and ‘exclusion’ of both Cl– and Na+ from the xylem enables L. tenuis to tolerate, better than L. corniculatus, the interactive stresses of salinity and waterlogging.
Key words: Aerenchyma, Cl–, Lotus corniculatus, Lotus tenuis (Lotus glaber), Na+, Philaenus, root porosity, salinity, waterlogging, xylem
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Lotus tenuis (Waldst. & Kit., syn. Lotus glaber; Kirkbride, 2006) is more salt and waterlogging tolerant than the more widely grown Lotus corniculatus (Schachtman and Kelman, 1991; Rogers et al., 1997a; Striker et al., 2005; Teakle et al., 2006). Previous work comparing waterlogging and salt tolerance in L. tenuis and L. corniculatus found that L. tenuis had a higher proportion of aerenchyma in roots under flooded conditions (Striker et al., 2005), accumulated less shoot Cl– under high NaCl concentrations, and also accumulated less shoot Na+ and Cl– under combined salt and waterlogging treatments (Teakle et al., 2006). Here we report the results of a study to determine if low shoot Cl– and Na+ concentrations are due to lower xylem ion concentrations in L. tenuis than those in L. corniculatus, under salt and waterlogging treatments.
The combination of salinity and waterlogging is a severe stress for most plant species. In comparison with well-aerated conditions, hypoxia generally increases the rate of transport of Na+ and Cl– to the shoot, where these ions can accumulate to toxic levels (Barrett-Lennard, 2003). Waterlogging reduces oxygen availability in roots, which reduces ATP production (Greenway and Gibbs, 2003). This presumably decreases the energy available to maintain H+ gradients across the plasma membrane and tonoplast generated by H+-ATPase activity, and required for the active secondary transport of Na+ and Cl– essential for ‘exclusion’ from the shoots (Greenway and Munns, 1980; Barrett-Lennard, 2003; Munns, 2005). Consequently, waterlogging-induced ATP deficits might be expected to increase the concentrations of Na+ and Cl– in the xylem sap of plants growing under saline conditions (Barrett-Lennard, 2003). Regulating xylem solute composition (mainly Na+, Cl–, and K+) is important for determining shoot ion concentrations (Watson et al., 2001), and hence salt tolerance. Such regulation has been termed ion ‘exclusion’ from the shoots (Munns, 2002).
Despite the importance of ion transport for salt tolerance, few studies have measured ion concentrations in the xylem of intact, transpiring plants. Such measurements are experimentally difficult, due to the large negative pressures in xylem vessels and tracheids (Steudle, 2000; de Boer and Volkov, 2003). The methods used for collecting xylem sap to measure ion concentrations include using a pressure bomb (Cabot et al., 2005), collecting root exudates (Jeschke and Pate, 1991), and using root perfusion systems (Lacan and Durand, 1996). These methods can yield useful comparative data within an experiment, but the ion concentrations measured may not accurately reflect those of the transpiration stream of intact plants (Watson et al., 2001; Malone et al., 2002). An ion-selective electrode combined with a xylem pressure probe has been used to measure K+ concentrations in the xylem of intact, transpiring plants (Wegner and Zimmermann, 2002), but this very specialized technique would require additional specific electrodes to measure Na+ and Cl–. Xylem-feeding insects, such as meadow spittlebugs (Philaenus spumarius), have been used to collect xylem sap (insect excreta) from plants, and the concentrations of a range of ions can be readily measured using ion chromatography (Watson et al., 2001; Gong et al., 2006; Hall et al., 2006). The spittlebugs feed on the xylem at the full hydraulic tension of the transpiration stream (Malone et al., 1999), and ion concentrations in the excreta, with the exception of NH4+, do not differ from those in xylem (Ponder et al., 2002).
In the present study, xylem ion concentrations were measured using xylem-feeding spittlebugs, as this technique has been used successfully in studies of salt-stressed wheat (Watson et al., 2001), rice (Gong et al., 2006), and Arabidopsis thaliana (Hall et al., 2006). To our knowledge, this technique has not previously been used to measure xylem ion concentrations of plants under combined salt and waterlogging stress. As shoot tissues accumulate Na+ and Cl–, growth is likely to become progressively inhibited (Munns et al., 1995). Therefore, we also evaluated shoot ion concentrations and dry mass production over 56 d of treatments in the two contrasting Lotus species in response to the interactive effects of salinity and waterlogging.
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Materials and methods |
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Plant material and growth conditions
Scarified seeds of L. tenuis (cv. Chaja) and L. corniculatus (cv. San Gabriel) were washed with 0.04% (w/v) NaHClO and then thoroughly rinsed with deionized (DI) water. Seeds were imbibed for 3 h in aerated 0.5 mM CaSO4 in darkness, then placed on a mesh screen over aerated 10% concentration nutrient solution and kept in darkness for 3 d. The full-strength nutrient solution consisted of macronutrients (mM), 0.5 KH2PO4, 3.0 KNO3, 4.0 Ca(NO3)2, 1.0 MgSO4; and micronutrients (µM), 37.5 FeNa3EDTA, 23.0 H3BO3, 4.5 MnCl2, 4.0 ZnSO4, 1.5 CuSO4, and 0.05 MoO3. After 3 d, seedlings were transferred to 25% nutrient solution still on mesh and exposed to light. After a further 4 d, individual seedlings were transplanted to 50% nutrient solution and another 7 d later the solution was changed to full concentration. Nutrient solutions were changed weekly and topped up with DI water as required. Each treatmentxspecies combination was represented by three replicate pots in a completely randomized block design where blocks were designated according to position in a glasshouse or controlled environment room (facility used depended on the experiment).
Four treatments were imposed 28 d after imbibition and these were: an aerated control (0 mM NaCl); a saline treatment (aerated, 200 mM NaCl); a stagnant non-saline treatment (non-aerated, 0 mM NaCl); and the combination of stagnant-plus-saline (non-aerated, 200 mM NaCl). NaCl was added in daily 50 mM increments until the final concentration of 200 mM. The next day hypoxia was imposed in all pots assigned to stagnant and stagnant-plus-NaCl treatments by bubbling with N2 gas until the dissolved O2 level was less than approximately 10% of air-saturated solution, so as to give a hypoxic pretreatment and thus avoid subsequent ‘anoxic shock’ (Gibbs and Greenway, 2003). The following day the nutrient solution in these pots was changed to a stagnant deoxygenated (i.e. anoxic) 0.1% (w/v) agar solution. This method simulates the decrease in dissolved O2 and increase in ethylene that occur under waterlogged conditions (Wiengweera et al., 1997).
Experiment 1. Xylem ion concentrations of intact plants
Seeds were germinated as described above but in a growth cabinet (18–20 °C, 60% relative humidity, 12 h day/night with maximum PAR 500 µmol m–2 s–1). Seedlings were transplanted into 3.0 l plastic pots painted black and held in holes in the lid using non-absorbent cotton wool. After transplanting, plants were grown in a controlled temperature room at 20/15 °C (day/night) and PAR approximately 120 µmol m–2 s–1 with a 16 h photoperiod for 21 d, before being transferred to a Weiss growth cabinet (20/15 °C 12 h day/night with PAR 300 µmol m–2 s–1 and 60–80% relative humidity).
Xylem fluid was collected using P. spumarius (meadow spittlebugs). These insects were collected from June to August 2005 at a site near Devil's Dyke in East Sussex, UK (Grid reference 265100) and caged as per Malone et al. (2002), except that they were maintained for up to 14 d in the laboratory on thyme plants. Seven days after treatments commenced (35 d after imbibition), the insects were caged onto the stems of two plants from each replicate pot of L. tenuis and L. corniculatus. On one plant, the cages were positioned at the internodal region between the two oldest leaves on the main stem, while on the second plant the cage was positioned between the two youngest leaves on the main stem. Each cage (one spittlebug per cage) consisted of a Technicon plastic tube plus lid (1.5 ml, Kartell Plastics, Cambridge, UK), with two cuts made down each side of the tube to fit the plant stem. The insects were caged on the stems for 24 h and excreta was collected from the bottom of the cage using a pipette. Samples of <50 µl were not used for analysis due to the possible influence of evaporation on ion concentrations (Malone et al., 2002). Xylem fluid was collected at weekly intervals (24 h each collection period) during the 28 d treatment period and stored in Eppendorf tubes at –20 °C before analyses. Na+ and K+ were measured as per Malone et al. (2002) using ion chromatography (Dionex, DX120, Surrey, UK), and Cl– was measured using a Chloride electrode (WTW Cl 501, Weilheim, Germany) with 2% 5 M NaNO3 as an ionic strength adjuster.
Experiment 2. Whole plant transpiration
Transpiration was measured for plants in a phytotron (20/15 °C day/night with average PAR at midday during the experimental period of 1163 µmol m–2 s–1). Fourteen days after imbibition, seedlings were transplanted to 4.5 l plastic pots wrapped in aluminium foil. Transpiration was measured 7 d after treatment commenced (35 d after imbibition) and then weekly (for 24 h intervals) for 28 d to coincide with measurements of xylem ion concentrations in Experiment 1. Individual plants (three replicates) were sealed with Parafilm in 250 ml conical flasks containing the same nutrient solution and respective treatments as in Experiment 1. For aerated treatments, flasks were gently bubbled with humidified air and consequential water loss estimated from flasks without plants (three replicates). All flasks were equilibrated for at least 10 min before initial mass was recorded, and then flasks were reweighed after 24 h and the plants removed for analysis. Roots and stem bases were rinsed gently in DI water and blotted dry. Leaf area (Li-Cor LI-3100, Lincoln, NE, USA) and root, shoot, and leaf fresh mass were recorded before all samples were oven-dried for 3 d at 70 °C.
Experiment 3. Ion uptake and growth response
Shoot ion concentrations and plant biomass were measured over an 8-week period to assess the responses of the two Lotus species to salt and waterlogging treatments. Seedlings were transplanted into 4.5 l pots wrapped in aluminium foil and plants were grown in a controlled environment room (20/15 °C, 12 h day/night, average relative humidity 78%, average maximum PAR 686 µmol m–2 s–1). Four harvests were taken: (i) start of treatment, 28 d after imbibition; (ii) after 14 d of treatment; (iii) after 28 d of treatment; and (iv) after 56 d of treatment. For each harvest, three plants per replicate were combined for measurements to minimize the influence of plant to plant variability, and the results were expressed on a per plant basis. For all harvests, roots and stem bases were gently rinsed in DI water and blotted dry. Root and shoot fresh mass were recorded and samples oven-dried for 3 d at 70 °C. Na+, K+, and Cl– concentrations were measured in dried shoot samples (100 mg) that had been ground to a fine powder and extracted with HNO3 (10 ml, 0.5 M) by shaking for 48 h in darkness at 30 °C. Diluted extracts were analysed for Na+, K+ (Jenway PFP7 flame photometer, Essex, UK), and Cl– (Buchler-Cotlove Chloridometer 662201, Fort Lee, NJ, USA). For the third and fourth harvests, roots were separated into tap root, laterals, and, where applicable (i.e. stagnant treatments only), newly formed laterals at or near to the stem base. A subsample of these roots was taken for root porosity measurements by assessing buoyancy before and after vacuum infiltration (Raskin, 1983; Thomson et al., 1990). Newly formed lateral roots, about 8 cm in length and closest to the stem base, were removed from one plant per pot to evaluate the extent of any aerenchyma. Transverse sections were cut approximately 4 cm from the root tip, mounted onto glass slides, and viewed under a microscope (Olympus BH-2, Tokyo, Japan); photographs were taken using a digital camera.
Statistical analyses and calculations
All statistical analyses used Genstat for Windows 8th Edition (Genstat software, VSN International, Hemel Hempstead, UK). Residuals were checked for normality and homogeneity, and no transformations were necessary. General analysis of variance (ANOVA) was used to check for overall significant differences and interactions between species and treatments. As xylem fluid was collected weekly from the same plants to measure changes in xylem ion concentrations over time, the effect of ‘time’ was analysed using a repeated measures ANOVA (Webster and Payne, 2002). The treatment means were tested for significant differences between species using paired t-tests or orthogonal contrasts, depending on the complexity of the data set. Unless otherwise stated, the significance level was P 
0.05.
Rates of ion transport from root to shoot were calculated from changes in shoot ion content and root dry mass between 14 d and 28 d of treatment, according to Williams (1948). ‘Exclusion’ of Na+ and Cl– was calculated from xylem ion concentrations using the equation used for Na+ in Colmer et al. (2005).
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Results |
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Xylem ion concentrations
Ion concentrations were measured in the xylem, using xylem-feeding spittlebugs, to determine if differences in shoot Na+, K+, and Cl– concentrations were due to differential delivery to the shoot. The spittlebugs fed well on L. tenuis and L. corniculatus plants from all treatments, producing volumes of excreta that varied from <10 µl to about 1500 µl in 24 h, and this was the same for all treatments. No samples <50 µl were used for analyses. There was no difference in ion concentrations in the xylem sap collected from insects caged on proximal or distal regions of the stem on either species for any of the treatments; therefore, means of values from these two sampling positions are presented.
Xylem Cl– concentrations increased significantly (up to 10-fold) by treatment with 200 mM NaCl (Fig. 1). After 7 d of NaCl treatment, both L. tenuis and L. corniculatus had relatively high xylem Cl– concentrations of 15 mM and 23 mM, respectively. However, in subsequent weeks, the xylem Cl– concentrations decreased by about one-third, indicating possible down-regulation of Cl– uptake. For both species, stagnant-plus-NaCl treatment increased xylem Cl–, and again concentrations were highest after 7 d and then decreased in subsequent weeks. Lotus tenuis had significantly less (
50%) xylem Cl– than L. corniculatus in NaCl treatment, and the difference between the species was even greater for stagnant-plus-NaCl treatment. For example, at 28 d, Cl– was >3-fold higher in the xylem of L. corniculatus compared with L. tenuis, i.e. Cl– ‘exclusion’ was higher in L. tenuis compared with L. corniculatus (Table 1).
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Treatment with NaCl also increased xylem Na+ concentrations for both species, by up to 10-fold above those in the absence of salt (Fig. 2). There was only a significant difference between the species in xylem Na+ for the stagnant-plus-NaCl treatment at 21 d and 28 d, when values for L. corniculatus were twice those in L. tenuis. Na+ ‘exclusion’ was therefore higher for L. tenuis in stagnant-plus-NaCl treatment compared with L. corniculatus, although there was no difference between the species in Na+ ‘exclusion’ under aerated NaCl treatment, in contrast to Cl– (Table 1).
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Stagnant treatment reduced the xylem K+ concentration of L. corniculatus to less than half of that in the aerated treatment for the first 21 d, whereas for L. tenuis, xylem K+ in the stagnant treatment was not significantly different from that in the aerated controls (Fig. 3). Lotus tenuis had about a 2-fold higher xylem K+ concentration than L. corniculatus for the first 21 d of stagnant treatment. In addition, L. tenuis had a significantly higher xylem K+ concentration than L. corniculatus for the stagnant-plus-NaCl treatment for measurements taken after 7 d. Surprisingly, xylem K+ was not reduced by NaCl treatment compared with non-saline aerated controls for either species, even though xylem concentrations of Na+ were increased by up to 10-fold by NaCl (Fig. 2).
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Xylem ion concentrations are a function of the rate of loading of ions into the transpiration stream and the water flux through the xylem. Whole plant transpiration was measured weekly for L. tenuis and L. corniculatus in NaCl and stagnant treatments for 28 d to coincide with the times when measurements of xylem ion concentrations were taken. NaCl and stagnant-plus-NaCl treatments decreased transpiration rates by about half for both species, relative to non-saline controls (Fig. 4). After 28 d, L. tenuis had significantly higher transpiration rates than L. corniculatus for all treatments except stagnant-plus-NaCl.
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Shoot ion concentrations
NaCl treatment increased shoot Cl– significantly for both species compared with non-saline controls, and the increase was greater for L. corniculatus than for L. tenuis (Table 2). After 56 d, the stagnant-plus-NaCl treatment had more than tripled the shoot Cl– concentration of L. corniculatus compared with the aerated NaCl treatment. In contrast, L. tenuis shoot Cl– was similar for both NaCl and stagnant-plus-NaCl treatments, thus at 56 d of stagnant-plus-NaCl treatment the Cl– concentration in the shoots of L. corniculatus was five times that in L. tenuis.
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NaCl treatment also significantly increased shoot Na+ concentrations; from <20 µmol g–1 DM to >700 µmol g–1 DM after 56 d treatment for both species (Table 2). However, the combination of salinity and root-zone O2 deficiency had a different effect on the two species. In L. corniculatus, shoot Na+ concentrations were increased by up to 4-fold from the stagnant-plus-NaCl treatment. In contrast, in L. tenuis, shoot Na+ concentrations were similar for both NaCl and stagnant-plus-NaCl treatments; so, after 56 d the shoot Na+ of L. corniculatus was about 3-fold higher than that of L. tenuis in stagnant-plus-NaCl treatment.
While stagnant conditions in the absence of salinity had no significant effect on shoot Na+ and Cl– concentrations (Table 2), shoot K+ was reduced in L. corniculatus by as much as 50% during the first 28 d (data not shown), although by 56 d the K+ concentration recovered to levels similar to those in aerated controls (Table 2). The stagnant treatment, compared with the aerated control, had no effect on the shoot K+ concentration of L. tenuis. NaCl treatment reduced shoot K+ concentrations by about 30% of aerated controls for both species. The combined stagnant-plus-NaCl treatment further reduced shoot K+ initially to about 50% of the aerated NaCl values for both species (data not shown). However, shoot K+ concentration recovered somewhat for L. tenuis so that at 56 d stagnant-plus-NaCl treatment the value was about 30% higher than in L. corniculatus (Table 2).
Net ion transport and ion ratios
Net rates of ion transport between 14 d and 28 d of treatment were calculated from changes in shoot ion content and root dry mass (Table 3). For all treatments, L. tenuis had a higher net transport rate of K+ than L. corniculatus; at the most extreme, L. corniculatus failed to increase its shoot K+ in waterlogged saline conditions over the period of measurement. The net transport of Na+ for L. tenuis increased from 305 µmol g–1 root DM d–1 in aerated NaCl to 358 µmol g–1 root DM d–1 in the stagnant-plus-NaCl treatment. In contrast, the net transport of Na+ for L. corniculatus decreased by nearly half in stagnant-plus-NaCl compared with the aerated NaCl treatment. This was associated with only a small increase in dry mass for stagnant-plus-NaCl treatment between 14 d and 28 d. Similar trends to those described above for Na+ were observed for Cl– net transport.
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Ratios of K+/Na+ and Cl–/Na+ for NaCl treatments were calculated from xylem and shoot ion concentrations and compared with ratios calculated from estimates of net transport. The ratios of Cl–/Na+ for the whole shoot and net transport were similar for both species in aerated and stagnant-plus-NaCl treatment, and these were at least half the xylem ratio (Table 4). Lotus corniculatus had a 2-fold higher xylem Cl–/Na+ ratio than L. tenuis for the aerated NaCl treatment but were similar for stagnant-plus-NaCl. For L. corniculatus, the xylem Cl–/Na+ ratio was similar in both aerated NaCl and stagnant-plus-NaCl treatments, as xylem concentrations of Na+ and Cl– were increased by stagnant-plus-NaCl treatment (compared with the aerated NaCl treatment) by similar proportions (Figs 1 and 2).
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K+/Na+ ratios were more variable between species and treatments than the Cl–/Na+ ratios. In general, the ratios were higher in L. tenuis than in L. corniculatus (Table 4). For example, in stagnant-plus-NaCl treatment, the xylem K+/Na+ ratio of L. tenuis was four times that of L. corniculatus. Similarly, the whole shoot and net transport values for L. tenuis were about twice those of L. corniculatus.
Root porosity
For all treatments, L. tenuis had greater porosity in its tap root, lateral roots, and newly formed laterals than in those of L. corniculatus (Table 5). These differences between the two species were most significant in aerated treatments as roots of L. tenuis produced aerenchyma constitutively whereas those of L. corniculatus did not (data not shown). Consequently, L. tenuis had more than twice the porosity of L. corniculatus in tap roots and >40% higher root porosity in the laterals for aerated treatments (Table 5). Stagnant treatments increased root porosity in both genotypes and, although the increases were greater for L. corniculatus than for L. tenuis, the porosity of roots of L. tenuis after 4 weeks of stagnant conditions always exceeded that in L. corniculatus (Table 5). The roots of L. tenuis should suffer less damage from waterlogging than those of L. corniculatus, as in L. tenuis aerenchyma was present prior to the onset of stagnant treatment, whereas in L. corniculatus the roots only formed aerenchyma in response to oxygen deficiency.
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Plant growth
Root and shoot dry mass were similar for both species and all treatments over the first 4 weeks of the experiment (data not shown), but after 2 months treatment, significant differences were measured between the two species (Table 2). Shoot dry mass of L. tenuis at 56 d in aerated NaCl treatment was 19% higher than for L. corniculatus and almost 5-fold higher for the stagnant-plus-NaCl treatment. Interestingly, the shoot dry mass was not reduced by the non-saline stagnant treatment compared with the aerated control for either species, even after 56 d, indicating that both species are tolerant to waterlogging. Significant differences between the species for root dry mass were only measured in the stagnant-plus-NaCl treatment after 56 d, when L. tenuis root dry mass was more than twice that of L. corniculatus (Table 2).
The growth data indicate a strong interaction between waterlogging and salinity, as dry mass was reduced more by the stagnant-plus-NaCl treatment than the sum of the reductions in NaCl and stagnant treatments alone. For example, after 56 d treatment, L. corniculatus shoot dry mass per plant was reduced by 8 g by NaCl and 1.2 g by stagnant treatment, compared with aerated controls, to give a total of 9.2 g. However, the stagnant-plus-NaCl treatment reduced shoot dry mass of L. corniculatus by 14.2 g compared with the aerated control. In comparison, for L. tenuis the stagnant-plus-NaCl treatment reduced shoot dry mass by 10.1 g compared with the aerated control. These results clearly demonstrate that the combined stresses of salinity and waterlogging have a significant interactive effect to severely reduce plant growth, even for relatively tolerant species such as L. tenuis.
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Discussion |
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Lotus tenuis (Chaja) has higher tolerance to salinity and waterlogging than L. corniculatus (San Gabriel). Lotus tenuis had 18% higher shoot dry mass than L. corniculatus under saline conditions, and this was five times higher when in stagnant-plus-saline solution, even though there was no significant difference in biomass when in aerated non-saline solution (Table 2). The greater tolerance to the interactive effects of salinity and waterlogging in L. tenuis was associated with lower xylem and shoot Na+ and Cl– concentrations, and with (constitutive) aerenchyma in the roots. Thus, the present findings support the hypothesis of Barrett-Lennard (2003) that maintenance of Na+ and Cl– ‘exclusion’ in saline-plus-waterlogged environments is required for tolerance to these adverse conditions.
Lotus tenuis had lower xylem Cl– concentrations than L. corniculatus under aerated saline treatment, as well as lower concentrations of both Na+ and Cl– in the xylem for the stagnant-plus-NaCl treatment. Xylem ion concentrations are a function of ion loading and transpiration volume (de Boer and Volkov, 2003), and decreased rates of transpiration can lead to greater concentrations of solutes in xylem fluid (Jackson, 2002). As transpiration rates did not differ between the two Lotus species for the combined stagnant-plus-NaCl treatment (Fig. 4), we therefore conclude that the lower shoot concentrations of Na+ and Cl– (Table 2) measured for L. tenuis are a direct result of lower xylem concentrations (Figs 1, 2). Similar results have been reported showing that transpiration rates cannot explain differences in shoot ion concentrations between other closely related species that differ in salt tolerance (e.g. Triticum turgidum and T. aestivum; Watson et al., 2001; Arabidopsis thaliana and Thellungiella halophila; Volkov et al., 2004). Therefore, the present results confirm predictions by Barrett-Lennard (2003) that concentrations of Na+ and Cl– in the xylem can regulate shoot Na+ and Cl– concentrations under salt and waterlogging stresses.
Xylem ion concentrations have, to our knowledge, never previously been measured directly for transpiring, intact plants under combined salinity and waterlogging. Using direct measurements of xylem ion concentrations, the xylem Cl– of L. tenuis was found to be about half that of L. corniculatus for aerated NaCl treatment, and up to a third less for stagnant-plus-NaCl treatment (Fig. 1). In one of the few previous studies that estimated xylem Cl–, the concentration for the halophyte Atriplex amnicola was calculated from measurements of expressed sap from cut shoots under pressure, with values of about 30 mM after 21 d treatment with 50 mM NaCl plus hypoxia (Galloway and Davidson, 1993). This concentration is more than three times the xylem Cl– of L. tenuis, measured in the present experiments. Collecting sap from a cut stem under pressure could overestimate xylem ion concentrations compared with those of intact, transpiring plants as used in the present study. Although A. amnicola is a halophytic plant that can accumulate large concentrations of Cl– and Na+ in the shoots, the interactive effects of salinity and hypoxia did cause large fluxes of Cl– and Na+ to the shoot, and also reduced growth (Galloway and Davidson, 1993). A similar interactive effect of salinity and root-zone oxygen deficiency on reducing growth was also observed in the present study, particularly for L. corniculatus.
Minimizing concentrations of Na+ and Cl– in the xylem would have contributed to the low shoot concentrations of these ions in L. tenuis when grown in stagnant-plus-saline conditions. The means by which plants restrict transport of salt to the shoots is known as Na+ and Cl– ‘exclusion’ (Munns, 2002), and this could be controlled by a number of root processes (see reviews for Na+, Tester and Davenport, 2003; Munns, 2005; and for Cl–, White and Broadley, 2001). Na+ ‘exclusion’ is an important determinant of salt tolerance for many crop species, for example, wheat (Watson et al., 2001; Davenport et al., 2005), barley (Flowers and Hajibagheri, 2001), and rice (Gong et al., 2006). Cl– ‘exclusion’ has also been linked to salt tolerance in species in the Papilionaceae and Rutaceae, such as Trifolium (Winter, 1982; Rogers et al., 1997b), Medicago (Sibole et al., 2003), Lupinus (Van Steveninck et al., 1982), Glycine (Luo et al., 2005), and Citrus (Romero-Aranda et al., 1998; Moya et al., 2003). In the present study, L. tenuis had from a 2–3-fold lower xylem Cl– concentration, and hence greater Cl– ‘exclusion’, than L. corniculatus for both aerated NaCl treatment and stagnant-plus-NaCl, while greater Na+ ‘exclusion’ in L. tenuis was only found for the combined stagnant-plus-NaCl treatment (Table 1). The greater Cl– ‘exclusion’ of L. tenuis could result from: (i) less unidirectional influx into roots (e.g. via regulation of passive Cl– influx through outward-rectifying Cl– channels; Skerrett and Tyerman, 1994); (ii) reduced loading of Cl– into the xylem (e.g. by retrieval of Cl– into root xylem parenchyma cells), (iii) greater accumulation in root vacuoles (e.g. via Cl–/H+ symporters; Pantoja et al., 1989); and/or (iv) greater efflux of Cl– from roots (e.g. via depolarization-activated channels; White and Broadley, 1999; Roberts, 2006). While the exact mechanism of Cl– ‘exclusion’ is still unclear, the results of the present study have clearly shown that this is an important trait contributing to salt tolerance in Lotus species.
A further notable difference between the two species was the ability of L. tenuis to maintain its selectivity for K+ over Na+ under root-zone oxygen deficiency. After 4 weeks of exposure to NaCl, the ratios of K+/Na+ calculated for the whole shoots of L. tenuis were about double those of L. corniculatus (Table 4). The stagnant-plus-NaCl treatment widened the difference between the two species; K+/Na+ ratios in the xylem of L. tenuis were four times those in L. corniculatus, while the net transport K+/Na+ ratio for L. tenuis was 40 times that of L. corniculatus. Since K+ is an essential element, maintenance of its concentration in the cytoplasm is an important component of salt tolerance (Maathuis and Amtmann, 1999). However, this has to be combined with an ability to prevent the build up of toxic concentrations of Na+ (and Cl–) while allowing sufficient accumulation of these elements in the vacuoles for osmotic adjustment. There is evidence that Na+ can substitute for K+ (Flowers and Lauchli, 1983; Maathuis et al., 1996), but an active role for Na+ in the metabolism may be restricted to some halophytes (Flowers and Dalmond, 1992) and C4 plants (Ohta et al., 1988). The data suggest that selectivity for K+ over Na+ is a contributory factor in the greater tolerance of L. tenuis over L. corniculatus to the interactive effects of salinity and root-zone oxygen deficiency.
Differences in the transport rates of Na+ and Cl– from roots to shoots under combined waterlogging and salinity could be linked to differences in O2 availability in roots. Lotus tenuis had higher root porosity than L. corniculatus for all treatments due to constitutive aerenchyma (Table 5). Similar results were reported by Striker et al. (2005), who found that the proportion of constitutive aerenchyma in roots of L. tenuis (Chaja) was 11% higher than for L. corniculatus (San Gabriel). In the combined stagnant-plus-NaCl treatment, Na+ was >3-fold higher in L. corniculatus shoot (Table 2) and twice as high in the xylem (Fig. 2), compared with L. tenuis. This large increase in Na+ for L. corniculatus was probably caused by disruption of those Na+ transport processes in the root cells dependent on H+ gradients across membranes, as these gradients are maintained by H+-ATPase activity that would be impaired due to hypoxia (Barrett-Lennard, 2003). Possible Na+ transporters that would be impaired include Na+/H+ antiporters such as SOS1 (Shi et al., 2002), NHX (Li et al., 2006), and CHX transporters (Hall et al., 2006). It is likely that greater aerenchyma in roots of L. tenuis contributed to the maintenance of O2 supply, and thus better maintenance of energy-dependent transport processes.
In addition to transport processes at the root level contributing to ion ‘exclusion’, other processes such as xylem retrieval and phloem recirculation have also been proposed to determine shoot Na+ and Cl– concentrations (Munns, 2002; Tester and Davenport, 2003). For both Lotus species, retrieval of Na+ or Cl– along the shoot xylem was not significant, as xylem ion concentrations measured at the stem base versus the distal stem region did not differ. Phloem recirculation is also unlikely to have contributed to the differences in shoot Na+ and Cl– concentrations measured for L. tenuis and L. corniculatus. For NaCl-treated plants, the Cl–/Na+ ratio was higher in the xylem compared with the shoot for both Lotus species (Table 4), which indicates that more Cl– relative to Na+ is re-circulated from shoots back to the roots. However, the difference between the xylem and whole shoot Cl–/Na+ ratios was greater for L. corniculatus than for L. tenuis. This suggests that L. corniculatus re-circulates a greater proportion of the Cl– initially delivered to the shoot via the xylem, despite having higher shoot Cl– concentrations, compared with L. tenuis. Therefore, it is unlikely that differences in phloem re-circulation of Cl– are contributing to the lower shoot Cl– concentrations measured in L. tenuis. Ion ‘exclusion’ from shoots is a more likely mechanism contributing to low shoot concentrations, as it would take less energy for a plant initially to ‘exclude’ more Na+ and Cl– from shoots than to recycle it from shoots to roots for storage in root vacuoles and/or future efflux from roots. Furthermore, high fluxes of Na+ and/or Cl– in the phloem have been found to be negatively correlated with salt tolerance in barley (Munns et al., 1986) and maize (Lohaus et al., 2000). While the present results do indicate that there is greater phloem transport of Cl– compared with Na+ for both species (Jeschke et al., 1995), the results of the present study agree with other recent studies (Hall et al., 2006) that shoot concentrations are mainly determined by loading in the root xylem, as L. tenuis does not re-circulate more Cl– than L. corniculatus.
In summary, this study compared a salt- and waterlogging-tolerant cultivar of L. tenuis with a less tolerant cultivar of L. corniculatus and found that control of xylem loading of Cl– to the shoot contributes to salt tolerance in L. tenuis. When salinity and waterlogging stresses were combined, minimizing entry to the shoot (via low xylem concentrations) of both Na+ and Cl– contributed to lower shoot Na+ and Cl–, and in turn the 5-fold greater shoot dry mass measured in L. tenuis compared with L. corniculatus. L. tenuis also had a higher K+/Na+ selectivity than L. corniculatus. Higher root porosity (due to constitutive formation of aerenchyma) allows O2 to continue to reach waterlogged roots, enabling L. tenuis to maintain energy-dependent transport processes influencing entry of Cl– and Na+. This study has demonstrated the interactive adverse effects of salinity and waterlogging stresses on plants, and has shown that ‘excluding’ Na+ and Cl– from the xylem contributes to greater tolerance to these combined stresses.
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NLT is on a GRDC PhD scholarship with supplementary funding provided by the CRC Salinity and The AW Howard Memorial Trust.
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|---|
Barrett-Lennard E. The interaction between waterlogging and salinity in higher plants: causes, consequences and implications. Plant and Soil (2003) 253:35–54.[CrossRef][Web of Science]
Cabot C, Garcia MC, Sibole JV. Relationship between xylem ion concentration and bean growth responses to short-term salinisation in spring and summer. Journal of Plant Physiology (2005) 162:327–334.[CrossRef][Web of Science][Medline]
Colmer TD, Munns R, Flowers TJ. Improving salt tolerance of wheat and barley: future prospects. Journal of Experimental Agriculture (2005) 45:1425–1443.[CrossRef]
Davenport R, James R, Zakrisson-Plogander A, Tester M, Munns R. Control of sodium transport in durum wheat. Plant Physiology (2005) 137:807–818.
de Boer AH, Volkov V. Logistics of water and salt transport through the plant: structure and functioning of the xylem. Plant, Cell and Environment (2003) 26:87–101.[CrossRef]
Flowers TJ, Dalmond D. Protein-synthesis in halophytes—the influence of potassium, sodium and magnesium in vitro. Plant and Soil (1992) 146:153–161.[CrossRef][Web of Science]
Flowers TJ, Hajibagheri M. Salinity tolerance in Hordeum vulgare: ion concentrations in root cells of cultivars differing in salt tolerance. Plant and Soil (2001) 231:1–9.[CrossRef][Web of Science]
Flowers TJ, Lauchli A. Sodium versus potassium: substitution and compartmentation. Encyclopedia of Plant Physiology (1983) 15:651–681.
Galloway R, Davidson NJ. The response of Atriplex amnicola to the interactive effects of salinity and hypoxia. Journal of Experimental Botany (1993) 44:653–663.
Gibbs J, Greenway H. Mechanisms of anoxia tolerance in plants. I. Growth survival and anaerobic catabolism. Functional Plant Biology (2003) 30:1–47.[CrossRef][Web of Science]
Gong H, Randall D, Flowers TJ. Silicon deposition in the root reduces sodium uptake in rice (Oryza sativa L.) seedlings by reducing bypass flow. Plant, Cell and Environment (2006) 29:1970–1979.[CrossRef][Medline]
Greenway H, Gibbs J. Mechanisms of anoxia tolerance in plants. II. Energy requirements for maintenance and energy distribution to essential processes. Functional Plant Biology (2003) 30:999–1036.[CrossRef][Web of Science]
Greenway H, Munns R. Mechanisms of salt tolerance in non-halophytes. Annual Review of Plant Physiology (1980) 31:149–190.[Web of Science]
Hall D, Evans A, Newbury H, Pritchard J. Functional analysis of CHX21: a putative sodium transporter in Arabidopsis. Journal of Experimental Botany (2006) 57:1201–1210.
Jackson M. Long-distance signalling from roots to shoots assessed: the flooding story. Journal of Experimental Botany (2002) 53:175–181.
Jeschke WD, Pate JS. Cation and chloride partitioning through xylem and phloem within the whole plant of Ricinus communis L. under conditions of salt stress. Journal of Experimental Botany (1991) 42:1105–1116.
Jeschke W, Klagges S, Hilpert A, Bhatti A, Sarwar G. Partitioning and flows of ions and nutrients in salt-treated plants of Leptochloa fusca L. Kunth. I. Cations and chloride. New Phytologist (1995) 130:23–35.[CrossRef][Web of Science]
Kirkbride JH. The scientific name of narrow-leaf trefoil. Crop Science (2006) 46:2169–2170.
Lacan D, Durand M. Na+–K+ exchange at the xylem/symplast boundary. Its significance in the salt sensitivity of soybean. Plant Physiology (1996) 110:705–711.[Abstract]
Li WYF, Wong FL, Tsai SN, Phang TH, Shao G, Lam HM. Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. Plant, Cell and Environment (2006) 29:1122–1137.[CrossRef][Medline]
Lohaus G, Hussmann M, Pennewiss K, Schneider H, Zhu JJ, Sattelmacher B. Solute balance of a maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching. Journal of Experimental Botany (2000) 51:1721–1732.
Luo Q, Bingjun Y, Liu Y. Differential selectivity to chloride and sodium ions in seedlings of Glycine max and G. soja under NaCl stress. Journal of Plant Physiology (2005) 162:1003–1012.[Web of Science][Medline]
Maathuis FJM, Amtmann A. K+ nutrition and Na+ toxicity: the basis of cellular K+/Na+ ratios. Annals of Botany (1999) 84:123–133.
Maathuis FJM, Verlin D, Smith FA, Sanders D, Fernandez J, Walker N. The physiological relevance of Na+-coupled K+-transport. Plant Physiology (1996) 112:1609–1616.[Abstract]
Malone M, Herron M, Morales M. Continuous measurement of macronutrient ions in the transpiration stream of intact plants using the meadow spittlebug coupled with ion chromatography. Plant Physiology (2002) 130:1436–1442.
Malone M, Watson R, Pritchard J. The spittlebug Philaenus spumarius feeds from mature xylem at the full hydraulic tension of the transpiration stream. New Phytologist (1999) 143:261–271.[CrossRef][Web of Science]
Moya JL, Gomez-Cadenas A, Primo-Millo E, Talon M. Chloride absorption in salt-sensitive Carrizo citrange and salt-tolerant Cleopatra mandarin citrus rootstocks is linked to water use. Journal of Experimental Botany (2003) 54:825–833.
Munns R. Comparative physiology of salt and water stress. Plant, Cell and Environment (2002) 25:239–250.[CrossRef][Medline]
Munns R. Genes and salt tolerance: bringing them together. New Phytologist (2005) 167:645–663.[CrossRef][Web of Science][Medline]
Munns R, Fisher D, Tonnet M. Na+ and Cl– transport in the phloem from leaves of NaCl-treated barley. Australian Journal of Plant Physiology (1986) 13:757–766.[Web of Science]
Munns R, Schachtman DP, Condon AG. The significance of a two-phase growth response to salinity in wheat and barley. Australian Journal of Plant Physiology (1995) 13:143–160.
Ohta D, Matsui J, Matoh T, Takahashi E. Sodium requirement of monocotyledonous C4 plants for growth and nitrate reductase activity. Plant Cell Physiology (1988) 29:1429–1432.
Pantoja O, Dainty J, Blumwald E. Ion channels in vacuoles from halophytes and glycophytes. FEBS Letters (1989) 255:92–96.[CrossRef][Web of Science]
Ponder KL, Watson RJ, Malone M, Pritchard J. Mineral content of excreta from the spittlebug Philaenus spumarius closely matches that of xylem sap. New Phytologist (2002) 153:237–241.[CrossRef][Web of Science]
Raskin I. A method for measuring leaf density, thickness and internal gas. HortScience (1983) 18:698–699.[Web of Science]
Roberts SK. Plasma membrane anion channels in higher plants and their putative functions in roots. New Phytologist (2006) 169:647–666.[CrossRef][Web of Science][Medline]
Rogers ME, Noble CL, Halloran GM, Nicolas ME. Selecting for salt tolerance in white clover (Trifolium repens): chloride ion exclusion and its heritability. New Phytologist (1997b) 135:645–654.[CrossRef][Web of Science]
Rogers ME, Noble CL, Pederick RJ. Identifying suitable forage legume species for saline areas. Australian Journal of Experimental Agriculture (1997a) 37:639–645.[CrossRef][Web of Science]
Romero-Aranda R, Moya JL, Tadeo FR, Legaz F, Primo-Millo E, Talon M. Physiological and anatomical disturbances induced by chloride salts in sensitive and tolerant citrus: beneficial and detrimental effects of cations. Plant, Cell and Environment (1998) 21:1243–1253.[CrossRef]
Schachtman DP, Kelman WM. Potential for Lotus germplasm for the development of salt, aluminium and manganese tolerant pasture plants. Australian Journal of Agricultural Research (1991) 42:139–149.[CrossRef][Web of Science]
Shi HZ, Quintero FJ, Pardo JM, Zhu JK. The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport in plants. The Plant Cell (2002) 14:465–477.
Sibole JV, Cabot C, Poschenrieder C, Barcelo J. Efficient leaf partitioning, an overriding condition for abscisic acid-controlled stomatal and leaf growth resposes to NaCl salinization in two legumes. Journal of Experimental Botany (2003) 54:2111–2119.
Skerret M, Tyerman SD. A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots. Planta (1994) 192:295–305.[Web of Science]
Steudle E. Water uptake by roots: effects of water deficit. Journal of Experimental Botany (2000) 51:1531–1542.
Striker GG, Insausti P, Grimoldi AA, Ploschuk EL, Vasellati V. Physiological and anatomical basis of differential tolerance to soil flooding of Lotus corniculatus L. and Lotus glaber Mill. Plant and Soil (2005) 276:301–311.[CrossRef][Web of Science]
Teakle NL, Real D, Colmer TD. Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis. Plant and Soil (2006) 289:369–383.[CrossRef][Web of Science]
Tester M, Davenport R. Na+ tolerance and Na+ transport in higher plants. Annals of Botany (2003) 91:503–527.
Thomson CJ, Armstrong W, Waters I, Greenway H. Aerenchyma formation and associated oxygen movement in seminal and nodal roots of wheat. Plant, Cell and Environment (1990) 13:395–403.[CrossRef]
Van Steveninck RFM, Van Steveninck ME, Stelzer R, Lauchli A. Studies on the distribution of Na and Cl in two species of lupin (Lupinus luteus and Lupinus angustifolius) differing in salt tolerance. Physiologia Plantarum (1982) 56:465–473.[CrossRef]
Volkov V, Wang B, Dominy PJ, Fricke W, Amtmann A. Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana, possesses effective mechanisms to discriminate between potassium and sodium. Plant, Cell and Environment (2004) 27:1–14.[Medline]
Watson R, Pritchard J, Malone M. Direct measurement of sodium and potassium in the transpiration stream of salt-excluding and non-excluding varieties of wheat. Journal of Experimental Botany (2001) 52:1873–1881.
Webster R, Payne W. Analysing repeated measurements in soil monitoring and experimentation. European Journal of Soil Science (2002) 53:1–13.[CrossRef][Web of Science]
Wegner L, Zimmermann U. On-line measurements of K+ activity in the tensile water of the xylem conduit of higher plants. The Plant Journal (2002) 32:409–417.[CrossRef][Web of Science][Medline]
White PJ, Broadley MR. Chloride in soils and its uptake and movement within the plant: a review. Annals of Botany (2001) 88:967–988.
Wiengweera A, Greenway H, Thomson CJ. The use of agar nutrient solution to simulate lack of convection in waterlogged soils. Annals of Botany (1997) 80:115–123.
Williams RF. The effects of phosphorus supply on the rates of intake of phosphorus and nitrogen and upon certain aspects of phosphorus metabolism in gramineous plants. Australian Journal of Scientific Research (1948) B1:332–361.
Winter E. Salt tolerance of Trifolium alexandrinum L. II. Ion balance in relation to its salt tolerance. Australian Journal of Plant Physiology (1982) 9:227–237.[Web of Science]
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