Trop. Agr. Develop. 52（1）：17－26，2008

Absorption and Distribution of Na+ and Some Ions in Seedlings of vitiense H. Wendl. ex Benth. & Hook. f. under Salt Stress

Hiroshi EHARA1*, Hiroyuki SHIBATA1, Wikanya PRATHUMYOT1, Hitoshi NAITO2, Takashi MISHIMA1, Marika TUIWAWA3, Alivereti NAIKATINI3 and Isaac ROUNDS3

1 Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu 514-8507, Japan 2 College of Life Science, Kurashiki University of Science and The Arts, 2640 Nishinoura, Tsurajima-cho, Kurashiki 712-8505, Japan 3 Institute of Applied Science, The University of the South Pacific, Suva,

Abstract Na+, K+, Ca2+, Mg2+ and Cl- concentrations in different parts and some physiological characteristics under NaCl treatment in solution culture were determined to investigate the salt resistance of Metroxylon vitiense (H. Wendl.) H. Wendl. ex Benth. & Hook. f. that belongs to the same genus as sago palm (M. sagu Rottb.). Seedlings at the 11th to 13th leaf stages were used for the treatment of 342mM (2%) NaCl for 31 days in a phytotron at 30ºC and 75% relative humidity under natural sunlight in mid-summer in central Japan. The Na+ and Cl- concentrations increased in the roots, petioles and leaflets with the NaCl treatment, while, the K+ concentration did not change in the top parts. The Ca2+ and Mg2+ concentrations increased in the leaflets, especially at lower and higher leaf positions, respectively. Leaf emergence and dry matter weight of the top parts were not affected by the NaCl treatment, whereas the transpiration rate decreased. It was, therefore, considered that salt avoidance to maintain the water status in the leaves by restricting the transpiration is important for the salt resistance of M. vitiense. The changes in the Ca2+ concentration can be considered to play a role in adequate absorption of K+, which would enable the plant to maintain osmotic adjustment in the leaflets. Key Words: Metroxylon palm, Na+ concentration, NaCl treatment, salt avoidance, salt resistance, transpiration rate

peaty soils where almost no other crops can grow Introduction without drainage or soil improvement (Sato et al., 1979; The Metroxylon are starch-producing Flach, 1977; Jong, 1995). This palm species stores a palms. The genus Metroxylon that is distributed in large amount of starch in the trunk, approximately Southeast Asia, Melanesia, Micronesia and Polynesia 300kg (dry wt.) per palm (Ehara, 2006) and has long consists of two sections, Metroxylon (Eumetroxylon) been utilized like banana and taro (Barrau, 1959; and Coelococcus (Beccari, 1918; Rauwerdink, 1986). Takamura, 1990). Sago palm is still an important Sago palm (M. sagu Rottb.) that is the only species in staple food in some areas of Southeast Asia and the section Metroxylon (although monophyly of this Papuasia (Ehara et al., 2000). This palm is one of the section remains uncertain) is distributed in Thailand, most important crops not only for subsistence Malaysia, Indonesia, the Philippines, Papua New economy but also for rural development in swampy Guinea and the Solomon Islands. Five species have areas of the tropics because its carbohydrate (sago been recognised within the section Coelococcus that starch) can be further processed into various basic raw represents the eastern half in the distribution of the materials for food, animal feed and for industrial uses. genus Metroxylon as follows: M. amicarum (H. Wendl.) Sago starch could be utilized for the producing bio- Becc. in the Federated States of Micronesia and the plastics and bio-fuels. The sago (palm starch extracted Marshall Islands; M. salomonense (Warb.) Becc. in the from the pith inside of trunk) of the other Metroxylon Solomon Islands and Vanuatu; M. warburgii (Heim) spcecies in the section Coelococcus has been used as an Becc. in Vanuatu, Fiji and Samoa; M. vitiense (H. emergency food in recent decades, when major crops Wendl.) H. Wendl. ex Benth. & Hook. f. in Fiji, and M. such as taro or yam had been damaged by cyclones or paulcoxii McClatchey (M. upoluense Becc.) in Samoa other hazards (Ehara et al., 2003). Presently, Metroxylon (Dowe, 1989; McClatchey, 1999). palms are mainly used as housing materials in the Sago palm grows mainly in swampy, alluvial and countries of the South and West Pacific. On the other

Received May 30, 2007 hand, M. vitiense (H. Wendl.) H. Wendl. ex Benth. & Accepted Sept. 22, 2007 Hook. f. that is distributed in Fiji is often harvested to * Corresponding author obtain young buds (shoot apex) is used as vegetable Kurimamachiya-cho, Tsu 514-8507, Japan [email protected] like bamboo shoots (sprouts). 18 Trop. Agr. Develop. 52 (1) 2008

Since sago palm and closely related species are renewed once a week. distributed and can grow in brackish water areas near Three seedlings at the 11th to 13th leaf stages (the the coast, these are considered to be salt- 12th to 14th leaves were emerging) were used each for resistant. Previously, we observed the presence of an the NaCl treatments, in addition to three other seedlings avoidance mechanism of salt stress in sago palm and at the same leaf stages as those in the treatments, as M. warburgii (Ehara et al., 2006; 2007; 2008). The low controls. The culture solution containing 342mM NaCl, Na+ concentration maintained in the leaflets was corresponding to 2% NaCl, was used in the NaCl attributed to Na+ storage mainly in roots, while a large treatments. Based on our previous findings showing amount of Na+ observed into the roots was stored in that sago palm can grow under 342mM NaCl for 30 the cortex. On Viti Levu in Fiji, M. vitiense grows days (Ehara et al., 2006), this concentration of NaCl beside a Bruguiera gymnorphiza (mangrove) forest was employed in the present experiment. One hundred which is generally considered to be preferably millilitre of culture solution with or without NaCl was distributed at a concentration of 1.5% NaCl in water. supplied for the treatments and controls, respectively, However, there is no information about the growth from July 9 to August 9, 2004. The culture solution was response to salt stress in M. vitiense. renewed every day to avoid excess NaCl accumulation In the present study, we investigated the absorption over the assumed concentration. of Na+, K+, Ca2+, Mg2+ and Cl- and their distribution in the leaflets and petioles at different leaf positions and Transpiration and chlorophyll concentration in the in different parts of roots as well as some physiological leaflets characteristics of M. vitiense under NaCl treatment for The whole pot including the plant, medium and analysing salt resistance. In addition, X-ray micro- culture solution was weighed every day to determine analysis was conducted to observe the distribution of the amount of transpiration of each plant per day. Each Na and K in the roots. pot was tightly sealed with a plastic sheet during the measurement of the amount of transpiration. The Materials and Methods changes in the pot weight during the night were Plant materials and NaCl treatment determined five times during the NaCl treatment to were collected from Navua on Viti Levu in estimate the cuticular transpiration rate. Fiji in early September 2003. Fertilised and well- Chlorophyll concentration in the leaflets at all the developed fruits were selected and used. They were leaf positions was measured by the method of treated physically to remove the coat tissues and Mackinney (1941). Five leaflet disks (0.25cm2 one the cleaned were put, one each, in a plastic each) were punched out from each of right and left half- beaker filled with 100ml water and the beakers were leaflets and were used for chlorophyll extraction with placed in a dark air-conditioned room at 30ºC, as 80% acetone. The chlorophyll content was expressed described in previous reports (Ehara et al., 1998; 2001; as the content per unit leaf area. 2006). The germinated seeds were transplanted one each to a plastic pot (1/10000a) filled with vermiculite Ion concentrations in plants and Kimura B culture solution containing (μM) 36.5 The treated and control plants were sampled after

(NH4)2SO4, 54.7 MgSO4, 18.3 KNO3, 36.5 Ca(NO3)2, the NaCl treatment and were washed in distilled water

18.2 KH2PO4 and 3.9 FeO3 (Baba and Takahashi, 1958). by the following procedures. The plants were separated The initial pH of the culture solution was adjusted to into three parts, leaflets, petioles (including rachis:

5.5 using KOH and H2SO4 before the addition of the petioles hereafter) and roots. The roots were divided culture solution. The Kimura B culture solution is into small roots, and large roots with stele and cortex used for plants that grow under submerged conditions (epidermis, exodermis, suberized sclerenchyma cells: and is often adjusted to pH 5.5 for paddy crops for cortex hereafter). Identification of the large and small instance. Therefore, we selected the pH 5.5 for a series roots was performed according to the method of Nitta of experiments on the salt resistance of Metroxylon et al. (2002) as follows: large roots, about 6 to 11 mm in palms. The pots were placed in a phytotron at 30ºC diameter and small roots, less than 6 mm in diameter. and 75% relative humidity (RH) under natural sunlight. The separated samples were dried at 80ºC and ground Water was added every day, according to the amount into a powder and were reduced to ash in a furnace for of solution consumed, and the culture solution was cation and anion extraction with HNO3. The cation and Ehara et al.: Salt resistance of Metroxylon vitiense 19 anion concentrations in the leaflets and petioles at each the end of the NaCl treatment. The values of the dry leaf position, and in the roots were determined using weight of the roots were lower in the treated plants an ion chromatograph with a conductivity detector than in the control plants, though there were no (Shimazu CDD-6A, IC-C3 and IC-A1, Japan). significant differences in the dry weight of the leaflets and petioles at each leaf position. The effect of the X-ray micro-analysis of Na and K in large roots NaCl treatment on the total dry matter increment A portion in the region above the extension zone during the experiment was small. of the large roots of the treated plants was soaked in liquid nitrogen after sampling and kept at -70ºC. The Ion concentrations in different plant parts frozen root samples were freeze-dried and prepared as Roots, leaflets and petioles at different leaf positions transverse sections (3mm in thickness). The transverse The Na+ concentration in the leaflets, petioles and sections were coated with gold ion (4mA for 1min.) roots is shown in Fig. 2. The Na+ concentration and used for scanning electron microscopy (SEM) increased in all the parts and at all the leaf positions observation and X-ray micro-analysis with an X-ray with the NaCl treatment. There were no distinctive probe micro-analyzer (JAOL JXA-8900R, Japan). differences in the Na+ concentration in both the A statistical analysis was performed using NCSS leaflets and petioles of the treated plants, regardless of 2001 (Number Cruncher Statistical Systems). Statistical the difference in the leaf position except for the analysis for the dry matter weight, ion concentrations uppermost leaf. In the treated plants of sago palm and and the chlorophyll content in the leaflets was not M. warburgii, the Na+ concentration in the petioles was performed for the uppermost and the following leaf higher at lower leaf positions than at higher leaf positions (except for the leaflet of the 13th leaf of the positions and the increase in the Na+ concentration in control plants) because there were less than three the leaflets was small especially at higher leaf replications depending on the difference in the leaf positions, that is, in active leaves (Ehara et al., 2006; stage of each plant at the onset of the NaCl treatment. 2007). It appears that sago palm and M. warburgii maintained a low Na+ concentration in the leaflets by Results and Discussion storing Na+ in the roots and petioles, especially at One or no leaf emerged during the NaCl treatment lower positions (Ehara et al., 2006; 2007; 2008). Our in both the control plants and treated plants. Fig. 1 current results were different from the previous data shows the dry matter weight of the leaflets and in the other species. The Na+ concentration in the petioles at different leaf positions and of the roots at petioles increased with the NaCl treatment even at

Fig. 1 Dry matter weight of roots, leaflets and petioles at different leaf positions under NaCl treatment. White and black bars indicate the leaflets and petioles, respectively. Bar with oblique lines indicates unexpanded leaf. Horizontal lines indicate the standard deviation (n=3). Asterisk indicates a significant difference between the control and treated plants at the 0.05 probability level, according to the T-test. 20 Trop. Agr. Develop. 52 (1) 2008

Fig. 2 Na+ concentration in roots, leaflets and petioles at different leaf positions under NaCl treatment. Bars and symbols are the same as those in Fig. 1.

Fig. 3 K+ concentration in roots, leaflets and petioles at different leaf positions under NaCl treatment. Bars and symbols are the same as those in Fig. 1. higher leaf positions and that in the leaflets increased the leaflets increased with the NaCl treatment in some in M. vitiense. This may be due to the difference leaves, especially those at lower positions (Fig. 4). The between M. vitiense and the other species in the ability Ca2+ concentration in the petioles did not change, to store Na+ in the roots and the petioles at lower leaf regardless of the NaCl treatment and the difference in positions. The petioles stored Na+ in M. vitiense, as the leaf position. The effect of the NaCl treatment on evidenced by the apparently low Na+ concentration in the Mg2+ concentration in the three parts was small the leaflets compared with that in the petioles. (Fig. 5), though the Mg2+ concentration in the leaflets Fig. 3 shows the K+ concentration in each part. increased with the NaCl treatment in some leaves at The K+ concentration decreased in the roots with the higher positions. The Cl- concentration increased with NaCl treatment, unlike in both leaflets and petioles at the NaCl treatment in all the parts and at all the leaf all the leaf positions and was much higher in the positions and was higher in the petioles than in the treated plants than in the control plants at some leaf leaflets (Fig. 6). positions. The K+ concentration in the leaflets and The pattern of the Na+ concentration in the petioles tended to be higher at higher leaf positions leaflets and petioles at different leaf positions in the than at lower leaf positions in both the control and treated plants of M. vitiense was different from that in treated plants. In contrast, the Ca2+ concentration in sago palm and M. warburgii, and the tendency in the Ehara et al.: Salt resistance of Metroxylon vitiense 21

Fig. 4 Ca2+ concentration in roots, leaflets and petioles at different leaf positions under NaCl treatment. Bars and symbols are the same as those in Fig. 1.

Fig. 5 Mg2+ concentration in roots, leaflets and petioles at different leaf positions under NaCl treatment. Bars and symbols are the same as those in Fig. 1.

Fig. 6 Cl- concentration in roots, leaflets and petioles at different leaf positions under NaCl treatment. Bars and symbols are the same as those in Fig. 1. 22 Trop. Agr. Develop. 52 (1) 2008 difference in the K+ concentration at different leaf there were distinct differences in growth response to positions between M. vitiense and the other species salt stress between the species in the genus Metroxylon. was the same (Ehara et al., 2006; 2007; 2008). Based on the results of the changes in the Na+ and K+ Different parts of roots concentrations with the NaCl treatment, the ratio of K+ Since the root system of sago palm consists of two to Na+ in the leaflets and petioles at higher leaf types of roots (Nitta et al., 2002), we investigated the positions was relatively higher, that is, lower at lower Na+ concentration in both types of roots. As it was leaf positions in the treated plants of M. vitiense. This difficult to separate the cortex and stele in the small tendency was different from that observed in sago roots because of their size, the small roots were not palm and M. warburgii. The difference in the ratio of divided into two parts. The ion concentration in K+ to Na+ in the leaves at different positions in the different parts of the roots is shown in Fig. 7. The Na+ treated plants of sago palm and M. warburgii was large, and Cl- concentrations increased with the NaCl compared with that in M. vitiense, because the Na+ treatment in the small roots and both the cortex and concentration in the leaves was lower at higher leaf stele of the large roots. In the large roots, the Na+ and positions ant the K+ concentration was higher at higher Cl- concentrations tended to be lower in the stele than leaf positions. In some species, plant growth is not in the cortex in the treated plants, although the affected when the K+ concentration in plant is differences in both Na+ and Cl- concentrations between maintained under the NaCl treatment (Greenway, them were no significant. This tendency was different 1962a; 1962b; Greenway et al., 1965; Munns et al., from that in the other species. Sago palm showed 1983; Jeschke et al., 1985; Yeo and Flowers, 1986). The comparatively low Na+ and Cl- concentrations in the K+ concentration in the top parts did not decrease, stele of the large roots (Ehara et al., 2008), while M, regardless of the leaf position in M. vitiense as in the warburgii showed a low Na+ concentration in the stele case of sago palm and M. warburguii (Ehara et al., (Ehara et al., 2007). It thus appears that in M. vitiense, 2006; 2007; 2008). Our previous and current results relatively large amounts of Na+ and Cl- were transferred strongly support the assumption that salt resistance is from the roots to the top parts, compared with the related to the exclusion of K+ by Na+ absorption in the other species. leaf blade (Greenway, 1962a; 1962b; Munns et al., 1983; The K+ concentration decreased with the NaCl Jeschke et al., 1985; Yeo and Flowers, 1986). We had treatment in only the small roots (Fig. 7). Although suggested in the previous study that K+ accumulation the K+ concentration tended to be lower in the stele than might be associated with osmotic adjustment in sago in the cortex, the difference was not significant between palm (Ehara et al., 2006). Yoneta et al. (2006) also them, in contrast to the tendency in the case of M. suggested that K+ is important for osmotic adjustment warburgii which displayed a higher K+ concentration in under NaCl stress in sago palm. Based on these the cortex than in the stele (Ehara et al., 2007). The results, K+ may play a role in osmotic adjustment, Ca2+ and Mg2+ concentrations did not change with the especially at higher leaf positions, that is, in the most NaCl treatment in any parts of the roots. active leaves. Fig. 8 shows the Na and K distribution in the large On the other hand, the effect of the NaCl roots of the treated plants, based on X-ray micro- treatment on the Ca2+ concentration in M. vitiense was analysis. The distribution density of Na was slightly different from that in sago palm (Ehara et al., 2008). lower in the stele than in the cortex and a similar The Ca2+ concentration in the leaflets increased with tendency was observed for the K distribution density. the NaCl treatment, especially in the leaves at lower The results of the Na+ and K+ concentrations in each positions in M. vitiense. Jacoby and Hanson (1985) part of the large roots (slightly lower Na+ and K+ reported that the presence of Ca2+ decreased the Na+ concentrations in the stele than in the cortex) reflected influx to plant cell and consequently attenuated the Na+ the gradation in the distribution densities of Na and K, damage. According to Ben-Hayin et al. (1987), Ca2+ is even in the absence of significant differences in the important for adequate K+ absorption and growth Na+ and K+ concentrations between the cortex and under saline conditions. The increase in the Ca2+ stele. The distribution of Na in the large roots of the concentration in the treated plants can be considered treated plants in M. vitiense was apparently different to play an important role in the resistance under saline from that in sago palm (Ehara et al., 2008). In the case conditions in M. vitiense. These results indicated that of sago palm, the distribution of Na was the largest in Ehara et al.: Salt resistance of Metroxylon vitiense 23

the inner region of the cortex near the stele. In this region, the endodermis consists of a uniseriate cylinder of cortical cells surrounding the central vascular region, adjacent to the pericycle. Endodermal cells are typically characterized by the deposition of a band of suberin or lignin in their primary walls, referred to as Casparian stripe, which forms a barrier against non-selective passage of water through the endodermis (Rudall, 2007). It is likely that the region with the endodermis is able to trap some of the excess influx of elements, even of Na, into the large roots in sago palm. However, the difference in the Na+ and K+ concentrations between the stele and cortex of the large roots was not significant in the treated plants of M. vitiense. Based on the current results, there may be an inter-specific difference in the structural adaptation of the physiological response to salt stress.

Transpiration rate and chlorophyll content Since the present experiment was conducted in the phytotron under natural sunlight, therefore transpiration varied daily because of the daily changes in the light conditions, although air temperature and humidity were controlled. Thus, the amount of transpiration per unit time, homely the transpiration rate was represented by a five-day moving average (Fig. 9). The transpiration rate started to decrease with the NaCl treatment after 1 day and it was less than 20% of the control plants after 5 days. Previously, we had observed that the transpiration rate decreased with the NaCl treatment in sago palm and M. warburgii (Ehara et al., 2006; 2007; 2008). The transpiration rate during the night was measured to estimate the cuticular transpiration rate. The difference in the estimated cuticular transpiration rate between the control and treated plants was distinct after 12 days. The value of the lowest transpiration rate of the treated plants at the final day was close to that of the highest estimated cuticular transpiration rate of the control plants at 26 days. It was considered that the stomatal Fig. 7 Ion concentrations in different parts of the roots aperture was small under salt stress. The decrease in under NaCl treatment. Vertical lines indicate the standard deviation (n=3). Different letters in the the stomatal aperture leads to a decrease in the figure indicate significant differences in different photosynthetic rate. However, the remarkable decrease parts within the control and treated plants at the 0.05 in the transpiration rate with the NaCl treatment probability level, according to the Tukey-Kramer presumably enabled the plant body to maintain the test. Asterisks indicate a significant difference in each part between the control and treated plants at water status by restricting water loss in this species. the 0.05 probability level, according to the T-test. Salt resistance in reaction to salt stress involves mechanisms of either salt tolerance or salt avoidance. In addition to ion toxicity, salinity stress decreases the soil water potential, which can result in plant dehydration 24 Trop. Agr. Develop. 52 (1) 2008

Fig. 8 Electronmicrograph of a transverse section of a large root of a treated plant and Na and K distribution, based on X-ray probe micro-analysis. The lower figures indicate the Na and K distribution in the red rectangle showed in the upper electronmicrograph. S: stele, C: cortex, Ep: epidermis, En: endodermis. △ indicates the endodermis. Crack is found in the electronmicrograph because the root samples were freeze-dried to avoid the movement of Na after the sampling.

stress induced by salinity (Johnson, 1991). In the case of Metroxylon palms, restricting the transpiration can be considered to be one of the avoidance responses to salt stress, that is, physiological adaptation associated with the decrease in the stomatal aperture, for maintaining a relatively lower Na+ concentration in the leaflets. Fig. 10 shows the chlorophyll content per unit leaf area at different leaf positions in both the control and treated plants. There was no significant difference in the chlorophyll content at the 9th to 13th leaf positions between the control and treated plants. The chlorophyll content tended to decrease in the treated plants, though the probability level of the difference between the control and treated plants, according to the T-test, was larger than 0.08 even in the 8th leaf. The leaf chlorophyll content at the lowest two leaf positions was very low compared with that of the other leaves in the treated plants because of the senescence of the lowest Fig. 9 Changes in transpiration rate (upper) and estimated two leaves. The seventh leaf of the treated plants which cuticular transpiration rate under NaCl treatment. Open and closed symbols indicate the control and treated was not suitable for chlorophyll extraction was not plants, respectively. Data corrected to mean value and used. According to Lack and Evans (2001), Mg is standard deviation of a five-day moving average (n=3). important for chlorophyll because a chlorophyll Ehara et al.: Salt resistance of Metroxylon vitiense 25

the Laboratory of Crop Production and Ecology, Mie University. We are indebted to Mr. Shoji Nakamura, Faculty of Engineering, Mie University for his technical support to conduct X-ray micro-analysis. The present study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (B1-14405035), to whom we express our gratitude.

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