Leaf gas exchange and solute accumulation in the halophyte
Sal
6
adora persica
grown at moderate salinity
Albino Maggio
a, Muppala P. Reddy
b, Robert J. Joly
a,*
aDepartment of Horticulture and Landscape Architecture,1165 Horticulture Building,Purdue Uni6ersity,West Lafayette,
IN 47907-1165, USA
bCentral Salt and Marine Chemical Research Institute,Waghawadi Road,Bha
6angar364002, India
Received 26 July 1999; received in revised form 4 February 2000; accepted 8 February 2000
Abstract
The domestication of halophytes has been proposed as a strategy to expand cultivation onto unfavorable land. However, halophytes mainly have been considered for their performance in extremely saline environments, and only a few species have been characterized in terms of their tolerance and physiological responses to moderately high levels of salinity.Sal6adora persicais an evergreen perennial halophyte capable of growing under extreme conditions, from
very dry environments to highly saline soils. It possesses high potential economic value as a source of oil and medicinal compounds. To quantify its response to salinity,S.persicaseedlings were exposed to 200 mM NaCl for 3 weeks, and growth, leaf gas exchange and solute accumulation were measured. The presence of NaCl induced a 100% increase in fresh weight and a 30% increase in dry weight, relative to non-salinized controls. Increases in fresh weight and dry weight were not associated with higher rates of net CO2assimilation, however. Analysis of ion accumulation
revealed thatS.persica leaves accumulated Na+ as a primary osmoticum. The concentration of Na+ in leaves of
salinized plants was 40-fold greater than that measured in non-salinized controls, and this was associated with
significant reductions in leaf K+ and Ca2+ concentrations. In addition, a significant accumulation of proline,
probably associated with osmotic adjustment and protection of membrane stability, occurred in roots of salinized plants. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Halophyte; Ion accumulation; Proline; Salt tolerance; Stomatal conductance; Water relations
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1. Introduction
In arid and semiarid regions of the world, irrigation, high evaporation and inadequate water management each contribute to increased soil
salinity, one of the world’s oldest and most seri-ous agricultural problems (Tanji, 1990). It has been estimated that high soil salinity brought about by mismanaged irrigation systems under-mines the yield productivity of at least one-third of the world’s irrigation lands (Reeve and Fire-man, 1967). Faced with a declining base of arable farmland (O’Leary, 1984) and increasing demand * Corresponding author. Tel.: +1-765-4946997.
E-mail address:[email protected] (R.J. Joly).
for food, fiber and energy (Janick, 1999), two broadly-defined strategies have emerged to extend the salinity range that would permit acceptable crop yields. These are: (a) the genetic manipula-tion of common crop species for increased salt tolerance; and (b) the utilization of naturally salt-tolerant species (halophytes) in irrigated and non-irrigated agriculture. Although the first generation of transgenic plants produced during the last decade has contributed to our understanding of the physiological mechanisms for salt tolerance (Nelson et al., 1998; Yeo, 1998), the functional biology underlying salt tolerance in plants is still poorly understood.
The direct domestication of halophytes repre-sents an alternative approach to expand cultiva-tion onto unfavorable land, and it can be envisioned as a strategy complementary to the genetic engineering of salt tolerance in glyco-phytes. Halophytic species have been extensively studied and have been proposed as potential plant crops (O’Leary, 1984; Glenn et al., 1999). Unfor-tunately, they generally have been considered for their growth performance in extremely saline envi-ronments, and only a few species have been thor-oughly characterized in terms of their tolerance and physiological responses to moderate or high levels of salinity, which may be more representa-tive of irrigated agriculture (O’Leary, 1984; Glenn et al., 1999). Therefore, for most halophytic spe-cies, their potential economic use as cultivated plants and their adaptability to agronomic condi-tions has not been fully assessed.
In this study we investigated the physiological response ofSal6adora persica(L.) exposed to 200 mM NaCl, a concentration that can be considered well below the threshold tolerance for this species. S. persica is an evergreen perennial halophyte belonging to the family Sal6adoraceae, and it is considered an important crop plant for marginal coastal areas of arid and semiarid regions, where the salt concentration of the soil would inhibit the growth of most other crops (Zodape and In-dusekhar, 1997).S.persicacan tolerate salinity up to 635 mM, and it performs well in temperatures up to 45°C in presence of annual rainfalls varying from 200 to 1000 mm. Moreover, it exhibits re-markable flexibility since it can be cultivated in
saline and non-saline soils with various textures from sandy to clay soils. Therefore, it can be considered a drought and salt tolerant species (Zodape and Indusekhar, 1997). In addition to its ecological function in counteracting the advance-ment of desertification as an effective soil binder (Zodape and Indusekhar, 1997), S. persica has considerable economic significance. Seeds fromS. persica plants contain 35 – 40% of oil with high percentages of lauric and myristic acid, which are valuable in the detergent industry (Makwana et al., 1988). Furthermore, plant extracts have recog-nized antifungal, antibacterial and antiviral prop-erties and have a variety of medicinal uses (Zodape and Indusekhar, 1997). The objective of this study was to evaluate the growth ofS.persica in a highly saline environment, as defined by the Food and Agriculture Organization (FAO) (Rhoades et al., 1992). Based on preliminary ob-servation, we hypothesized that seedling growth would be enhanced in the presence of 200 mM NaCl. Here we report on the growth performance of S. persica seedlings exposed to NaCl in a hydroponic culture system for 3 weeks. We also have evaluated leaf gas exchange and solute distri-bution in an attempt to explain the physiological basis of the hypothesized growth stimulation.
2. Material and methods
2.1. Cultural conditions
S.persica(L.) seeds were sown in flats contain-ing a 2:2:1 (v/v/v) mixture of perlite, peat moss, and top soil and kept on benches in the horticul-ture greenhouse at Purdue University under
28°C daytime maximum and 23°C nighttime minimum temperatures. One week after germina-tion, 60 plants of uniform size were selected and transferred to 80-l plastic tubs containing aerated Hoagland solution (Hoagland and Arnon, 1950) in a growth chamber. Chamber temperature was 2591°C, and photosynthetic photon flux density (PPFD) at the top of the canopy was 400 mmol m−2 per s during 12-h photoperiods. Following
each and grown for an additional 3 weeks either in the presence or absence of NaCl. Salt was increased gradually during the initial 3 days of treatment to a final concentration of 200 mM. All the experiments described below were performed using 5-week-old seedlings.
2.2. Gas exchange measurements
After 3 weeks at 200 mM NaCl, net CO2
assim-ilation rate (A), stomatal conductance (gs) and
transpiration (E) were measured on the first and second uppermost expanded leaves of six plants for each treatment using a CIRAS-1 Portable Photosynthesis System (PP Systems, Haverhill, MA). Measurements were performed at three dif-ferent levels of PPFD (200, 700 and 2200 mmol m−2 per s) using a 20-W halogen light source
supplied with the CIRAS-1 system. Single intact leaves were clamped in the leaf chamber, and a first a set of measurements at 200mmol m−2 per
s was recorded after the photosynthetic rate had stabilized (3 min). Subsequently, the light in-tensity was increased to 700mmol m−2per s, and
a second set of measurements was recorded on the same leaf after it had reached a new steady-state. The third set of measurements at 2200mmol m−2
per s was recorded with the same procedure. Temperature and RH during gas exchange evalu-ation were 2592°C and 4595%, respectively. The CO2 concentration in the chamber was 350 –
370 ml l−1.
2.3. Leaf water relations
Water potential (Cw) was measured using a
pressure chamber (PMS Instruments Co., Corval-lis, OR) on the same leaves used to determine gas-exchange rates. After water potential was measured, leaves were frozen in liquid nitrogen in sealed polyethylene bags. Leaf samples were later thawed and then crushed while still in their sealed plastic bags to extract cell sap. The osmotic po-tential of the sap (Cp) was measured using a
Wescor Model 5100C vapor pressure osmometer (Wescor Inc., Logan, UT). Water content was determined from a bulk sample of five leaves from the same plants used for Cw and Cp
measure-ments. Leaf and root fresh weights were measured immediately following the collection of leaf gas exchange and Cwdata. Dry weight of all leaves,
except the single leaf sampled for Cp, was
ob-tained following drying in a mechanical convec-tion oven at 80°C for 48 h. Prior to drying, stomatal frequency was obtained as the number of stomata per mm2of leaf surface by both direct
light microscopic observation of peeled epidermal strips and by examination of nail polish replicas, using a 40× objective.
2.4. Solute analyses and proline determination
Concentration of Na+, K+, Ca2+ and Mg2+
were analyzed in roots and shoots of ten plants per each treatment by inductively coupled plasma (ICP) atomic emission spectrometry, after perchloric acid digestion of 0.3 g of ground dry tissue. Proline was determined according to the method of Troll and Lindsley (1955). The osmotic pressure (P) attributable to each of the measured ion concentrations (ci) was calculated according
toP=RTci(Nobel, 1991), whereRis the
univer-sal gas constant and T is temperature, and then expressed as a percentage contribution to total measured Cp.
3. Results
Shoot growth, leaf water potential and osmotic potential measured inS.persicaseedling grown in salinized and non-salinized solutions are summa-rized in Table 1. Both fresh weight and dry weight were significantly higher in salt-treated plants. The presence of 200 mM NaCl was associated with an approximate doubling of plant fresh weight and a 30% increase in dry weight, relative to non-salinized controls. Cw and Cp were each
Table 1
Growth and water relations parameters in non-salinized and salinizedS.persicaplantsa
Turgor
DW(leaves+roots) Cp
FW(leaves+roots) DW/FW Leaf water Cw
Treatment
content (MPa)
(g)
(g) (MPa) (MPa)
(mg g DW−1)
0.3690.09 6.5490.75 1.3790.11
Control 0.2090.02 4.990.33 −1.2090.14 −1.6890.06
(non-salinized)
NaCl 12.0091.05** 1.7390.10** 0.1490.02* 6.990.37** −1.7090.05* −2.0890.01** 0.3090.05 (200 mM)
aValues are means of six plants9S.E.
* Significant treatment difference atPB0.05, according to Student’st-test. ** Significant treatment difference atPB0.01, according to Student’s t-test.
Fig. 1. Net CO2assimilation rate (A), stomatal conductance (B) and transpiration (C) of developed leaves of non-salinized (shaded bars) and salinized (open bars) S. persica plants at increasing photosynthetic photon flux density (PPFD). Values are means of six plants9S.E.
medium increased the succulence of the shoots, as indicated by the low dry weight to fresh weight ratio and the high leaf water content (Table 1).
In contrast to the significant differences mea-sured in water relations parameters, the presence of NaCl in the nutrient solution did not significantly affect leaf gas exchange rates (Fig. 1).A,gsandE
were each slightly higher in leaves of salinized plants at each of three PPFDs, but the mean values were not statistically different from controls at any given PPFD (Fig. 1). Net CO2 assimilation rate
increased linearly with increasing photon flux be-tween 200 and 2200 mmol m−2
per s for both salinized and non-salinized seedlings, however. To assess whether the presence or absence of NaCl in the nutrient solution had affected the number of stomata per unit of leaf area, we analyzed epider-mal leaf strips under a light microscope. No differ-ences in stomatal frequency were found (data not shown).
Ion accumulation in both leaves and roots ofS. persica seedlings differed greatly between treat-ments (Fig. 2). The concentration of Na+in leaves
of salinized plants was 40-fold greater than that measured in non-salinized controls. Roots of salin-ized seedlings also accumulated significantly higher Na+
than their corresponding controls, but final concentrations were much lower than those mea-sured in leaves. The concentrations of K+, Ca2+
and Mg2+ were each approximately 50% lower in
leaves of salinized plants, and the differences were each statistically significant (Fig. 2A). With the exception of Na+, no significant differences in ion
Fig. 2. Ion concentration in leaves (A) and roots (B) of non-salinized and salinizedS.persicaplants. Values are means of four plants9S.E. ** Significant treatment difference at PB0.01, according to Student’st-test.
4. Discussion
Fresh weight and dry mass production of S. persica seedlings were greater when they were grown at 200 mM NaCl than under non-salinized conditions (Table 1). This concentration is consid-ered optimal for the growth of many halophytes, including Aster, Salicornia and Atriplex species (Ungar, 1991). Although the presence of NaCl is rarely an obligate requirement for growth of halo-phytes (Flowers, et al., 1977), the absence of salt in the nutrient solution strongly inhibited growth in S. persica. Because net CO2 assimilation rate
was unaffected by NaCl (Fig. 1), the lower dry mass accumulation in absence of salt may reflect greater energy loss through respiration and/or a shift from anabolic to catabolic processes (Flow-ers et al., 1977; Ungar, 1991). Rates of leaf gas exchange were similar in salinized and non-salin-ized plants, and they were in the same range as those reported forLimonium californicum (Wood-ell and Mooney, 1970). The lack of a positive correlation between photosynthetic rate and dry matter production has been reported in several other halophytes grown at increasing salt concen-trations below that considered optimal for growth, as shown in A6icennia marina (Ball and Farquhar, 1984), Scirpus robustus and Spartinia foliosa (Pearcy and Ustin, 1984). The molecular and physiological basis for growth inhibition in absence of salt is currently unknown, although a metabolic involvement of Na+, in addition to its
osmotic role, has been proposed to explain its requirement for growth (Flowers et al., 1977).
Succulence of S. persica increased upon 200 mM NaCl treatment. Succulence is a common characteristic in halophytes, which seems to occur within a range of salt concentrations optimal for growth (Short and Colmer, 1999). Succulence minimizes the toxic effects of excessive ion accu-mulation and has been reported to be associated with accumulation of osmotically active solutes for maintenance of cell turgor pressure (Lu¨ttge and Smith, 1984).
Analysis of ion accumulation and partitioning revealed that S. persica, similar to most halo-phytes (Lu¨ttge and Smith, 1984), accumulated Na+ as a primary osmoticum. Approximately
Fig. 3. Proline concentration in leaves and roots of non-salin-ized and salinnon-salin-izedS.persicaplants. Values are means of four plants9S.E. ** Significant treatment difference at PB0.01, according to Student’st-test.
87% of the sodium was localized in the leaves, and it was associated with a decreased K+ content.
The mean concentration of Na+ measured in
leaves of salinized plants (320 mmol l−1) was
estimated to contribute 35% of the Cp whereas
the contribution of K+ was approximately 6%.
These results are consistent with those reported for Suaeda maritima (Flowers et al., 1977), Sal -icornia europea(Ungar, 1991) and for the succu-lent halophyte Disphyma australe (Neales and Sharkey, 1981). Further, they are in agreement with the general view that, at least in glycophytes, high sodium concentration interferes with intra-cellular K+
accumulation presumably by compet-ing for the same sites of influx (Niu et al., 1995). Patterns of Na+ and K+ distribution between
roots and shoots similar to those shown in Fig. 2 have been reported for several other halophytes, including Pucciniella personis (Ungar, 1991) and Spergularia marina (Cheeseman and Wickens, 1986).
The presence of exclusion mechanisms operat-ing at the roots of halophytes has been proposed to explain the occurrence of a lower Na+
concen-tration in roots (Stelzer and La¨uchli, 1978). In some cases, those mechanisms are associated with secondary exclusion systems in the leaves (e.g. salt glands) (Gorham, 1987). Compelling evidence for Na+ extrusion in halophytes exists. Physiological
and biochemical data support the possibility that a Na+
/H+ antiporter operating at the plasma
membrane mediates Na+extrusion into the
extra-cellular space (Glenn et al., 1999). Although such a mechanism may exist in S. persica, our results cannot differentiate between intracellular and ex-tracellular Na+because measurements were made
on a whole-root basis. We note, however, that export of Na+ only to the extracellular space
could not adequately explain the observation that root Na+ concentration was lower than that of
the external medium, unless Na+
uptake had been partially excluded or Na+ had been actively
ex-pelled from the roots to the medium. We recog-nize, however, that our measurements of ion contents in roots may underestimate actual values due to the effect of dilution by xylem water.
The concentrations of Ca2+ and Mg2+ were
similar in roots of salinized and non-salinized
plants. However, the concentration of each of these ions was significantly lower in leaves, sug-gesting a Na+-related interference with Ca2+ and
Mg2+ transport to the shoot. Similar results have
been reported by Richardson and McKell (1980) for Atriplex canescens and by Le Saos (1976) for Cochlearia anglica.
Once Na+ and Cl– have entered in the plant,
they usually are compartmentalized in the vac-uole, and compatible solutes are produced in the cytoplasm to counteract the increased osmolality of the vacuole. Accumulation of proline as a compatible solute has been reported for several halophytes, although the increase of the proline pool upon salt treatment does not seem to be a general phenomenon (Stewart and Lee, 1974). In this respect, S. persica behaves like Ruppia mar -itima, where the proline pool approximately dou-bled upon exposure to 150 mM (Stewart and Lee, 1974). However, due to the dilution effect of xylem sap discussed above, the real concentration of cellular proline may have been underestimated. Therefore the contribution of proline to cellular osmotic adjustment in roots cannot be conclu-sively assessed. It is interesting to note that the increase in proline concentration in S. persica occurred primarily in the roots of salinized plants. In addition to its role as an osmoticum, several other functions have been attributed to proline, including stabilizing cellular membranes or acting as a stress signal (Maggio et al., 1997). Recent work with genetically engineered tobacco and Arabidopsis plants has shown that a small yet constitutive overproduction of proline or other compatible solutes can enhance adaptation (Kishor et al., 1995; Hayashi et al., 1997). There-fore, increased proline in roots of S. persica may be part of a complex adaptive response that may involve the preservation of root integrity and functionality as a key component.
Overall, our results confirm the hypothesized improved growth performance of S. persica in a highly saline environment. The results do not provide a mechanistic basis for the observed growth stimulation, however. Increased biomass was not associated with either higher rates of net CO2 assimilation or higher leaf turgor.
candidate species for cultivation in areas where salinity cannot be kept within acceptable limits by leaching or other salinity management techniques (Rhoades et al., 1992). The observed growth stim-ulation by 200 mM NaCl suggests that longer-term field trials would be justified to evaluate the growth potential of S. persica in areas where it may be conceivable to reuse second-generation drainage water for irrigation (Rhoades et al., 1992).
Acknowledgements
M.P.R. was supported by a CSIR New Delhi Raman Research Fellowship. Publication number 16 046 of the Purdue University Office of Agricul-tural Research Programs.
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