Original Paper

Mangrove , Bruguiera gymnorrhiza and Its Salt Environment for Growth*

Shigeru KAro**

Mangrove are distributed along coastal areas and estuarine areas of the sub-tropical and tropical worlds which include Okinawa, Japan. These are called halophytes which possess peculiar physiological mechanisms for salt control. Characteristic distribution of mangrove plants is observed as conditioned by tidal regime and salinity. Bruguiera gymnorrhiza (Japanese name: Ohirugi) is one of the mangrove species and its distribution in a mangrove forest is confined to the areas that are not strongly affected by sea water. In this study, Bruguiera gymnorrhiza was compared under different NaCl conditions of culture solution. The inor- ganic ions (anions and cations) and organic acids in leaves, rhizophores and roots parts in each cultured sample were analyzed, respectively. Bruguiera gymnorrhiza grew very well under F-20 culture condi- tion (0.6% NaCl) like a natural growth in the mangrove forest. Leaf size was large and leaf color also was a healthy green. On the contrary, at high salinity condition (F-100, 3% NaCl) the leaf of Bruguiera gymnorrhiza was small and thick. These thick leaves contained high amount of Na+ and Cl- ions. After culturing, ion components of culture solution were also analyzed. The data indicated that K+, PO43-, and NO3- ions in culture solution were decreased or disappeared by absorption for plant growth. These three elements are essential elements for plant growth. Nat, and Cl- ions were also absorbed and trans- located to upper parts, then these ions were stored in the leaves. Furthermore, leaves gradually increase succulences and finally these leaves will fall down because of NaCl abandonment from plant body. Pro- duction of organic acids depends on salinity condition. All leaves, rhizophores and roots contained high amount of oxalic and malic acids. These dicarboxylic acids are important for osmotic regulation of halophytes.

produce viviparous seeds. This seed matures 1.INTRODUCTION completely on the tree and may germinate in its locality or may distribute to other areas Mangrove plants are one of the typical halo- following the sea water current movement. phytes distributed in river mouth and sea shore Mangrove forest areas of areas of sub-tropical and tropical regions in the countries are decreasing. Reduction is due to world. Distribution of mangrove plants has many reasons, foremost of which are: produc- been reported by H. Barth.1) Six species of tion of charcoal for fuel, development of fish mangrove plants have been identified in the and shrimp ponds, logging and ultimately re- Nansei islands, Okinawa, Japan and distribut- gional development needs for new housing ing an area of approximately 400 ha. Char- areas. Preservation of the environment and acteristic of many mangrove plants is the fact reforestation of mangrove areas as energy that they can grow very well within wide ranges sources is going on. Extension of young of salinity which can go up to a maximum of mangrove trees and method of tree planting are approximately 3% NaCl. Notable are those important for reforestation of destroyed man- belonging to the which grove forests. *Study on the Salt Tolerance Mechanism of Halophytes (2) **NODAI Research Institute , Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156, Japan ―397― 398 Bull. Soc. Sea Water Sci. Jpn. Vol.46 No.6 (1992)

In this paper, we report results of our studies Table-1 Ion components and concentrations

on optimal growth conditions for viviparous in standard culture solution.

seeds of Bruguiera gymnorrhiza as well as our

investigation on salinity level in cultured solu-

tions for the efficient production of young

mangrove trees.

2. MATERIALS AND METHODS

2.1 Mangrove plant and cultivation method

Bruguiera gymnorrhiza L. Lam (Japanese

name: Ohirugi) was used in the experiment.

Viviparous seeds of this mangrove species were

collected from Iriomote-jima island of Okinawa

prefecture, Japan. Bruguiera gymnorrhiza was cultivated in a 1/2,000 a Wagner-pot and with

air continuously supplied through the bottom Table-2 NaCl concentration in culture solution. of the pot.

Cultivation conditions were as follows: tem-

perature was set at 30•Ž for daytime and 25•Ž for nighttime; light and dark condition was 12

hr & 12 hr, with light supplied by 50,000 lux

florescent lamp; relative humidity was con-

trolled at 50-70% in a plant growth cabinet

(Koito Inc.). Standard culture solution used was the designated condition for No. 1 and No.

2 of Otsuka liquid fertilizer (Otsuka Chemical Inc.) as shown in Table-1. The other condi- *Otsuka liquid fertilizer No . 1+No. 2. **Deionized water tion was prepared D.W., F-20, F-50, F-80 and .

F-100 (Table-2): F-20 contains 0.6% NaCl in

solution; F-50 contains 1.5% NaCl in solution; Shimadzu Inc.). Conditions were as follows:

F-80 contains 2.4% NaCl in solution; F-100 5 mM HNO3 solution as eluent and IC-Cl as contains 3.0% NaCl in solution. Culture solu- column for monovalent cations (Na+, NH4+ and tion was changed every 2 weeks for 4 months K+) analysis,(electroconductivity about 2,500 and the ion concentration of the final culture μScm-1);eluent for divalent cations (Mg2+and solution was analyzed. Internode number, a Ca2+) was 40 mM tartaric acid and 20 mM ethyl-

enediamine solution (electroconductivity about plant growth indicator, was counted as 2-week intervals for 4 months. 800μScm-1)and IC-Cl was used as column,

2.2 Preparation of sample for analysis eluent for anions (F-, Cl-, NO2-, PO43-, Br-,

Plants cultured for 4 months were used for NO3-and SO4-)was 1 mM p-hydroxybenzoic the experiment. Leaves, rhizophore and roots acid and 1.1 mM N,N-diethylethanolamirie solu- of each plant were separated and washed care- tion(electroconductivity about 120μScm-1)and fully with deionized water (D.W.), especially IC-AI was used as column. the root parts. Each sample was cut and 2.2.2 0rganic acids analysis homogenized for 5 min in a blender with D.W. Apart of the homogenate was freezed-dried-

The homogenate was analyzed for inorganic After butyl esterification, the sample was ana- ions and organic acids content. lyzed for organic acids content by gas chromatog-

2.2.1 Inorganic ion analysis raphy (GC-7AG, Shimadzu Inc.). The gas

Inorganic ions (cations and anions) of the chromatograph was equipped with a flame homogenate were analyzed by ion chromato- ionization detector (FID)and 2 m×3 mm graph (Shimadzu Ion Chromatograph IC-6A, (inner diameter)glass column packed with S.Kato: Study on the Salt Tolerance Mechanism of Halophytes 399

Table-3 Internode number of Bruguiera gymnorrhiza (Ohirugi)by various culturalconditlons.

Table-4 Ion concentrations in culture solutions taken in the last 2 weeks of 4 months culture period of Brugttiera gymnorrhiza.

*B: standard solution ,**U: 2 weeks culture solution. (unit: ppm)

Reoplex 400 on 80-100 mesh Chromosorb produced 3.9 intemodes. Growth rate of the AW-DMCS. The column temperature was held stretching parts was inferior and each leaf area at 40℃ for 1 min, then programmed at 5℃/min was small. Only the main root developed up to 210℃. under D. W. condition and absorptional fine

root could hardly be recognized、The leaves

3. RESULTS were also small. In sharp contrast, plants

grown under F-20 conditions produced many 3.1 Cultivation of Bruguiera gymnorrhiza internodes with development of many main and

under several culture conditions hair roots. Also, the leaf area was wide. Roots

Internode number was counted at every 2-week of plants grown under F-100 conditions were interval for 4 months until harvest. Internode mostly main root. Also, some buds of vivi- number of Bruguiera gymnorrhiza is shown in parous seeds withered after new leaf develop- Table-3; these results are true total internode ment. On the other hand, plants grown under number of 10 viviparous seeds. Bruguiera F-20 conditions developed many main roots and gymnorrhiza internode number under D. W. fine roots (hair roots). The leaves were big and condition was anaverage of 3.4. Bruguiera a healthy green, while some plants developed gymnorrhiza grown under F-20 conditions pro- some branches. Formation of main root of duced 7.6 internodes. Internode number grad- F-100 condition was recognized much than root ually decreased with an increase in NaCl con- of D. W. condition, and development of fine centration in the culture solution. Bruguiera root was inferior to that F-20 condition. Inter- gymnorrhiza grown under F-100 conditions node number and leaf areas gradually decreased 400 Bull. Soc. Sea Water Sci. Jpn. Vol.46 No.6 (1992)

Fig.-1 Ion concentratlon in Bruguiera gymnorrhiza leaf.

Fig.-2 Ion concentration in Bruguiera gymmrrhiza rhizophore.

with increasing NaCl concentration in culture 3.2 Inorganic ion concentration in harvested

solution. But, leaf thickness gradually in- plants creased. Results of ion analysis for the Bruguiera

Ion components and their concentrations in gymnorrhiza plant are shown in Figs。-1 to 3. the final culture solution are shown in Table-4. The main cation distributed in the tissue was After culturing plants for 4 months, the ion Na+, while K+, Mg2+ and Ca2+ cations followed components of the culture solution show a closely. The main anion distributed in the

simllar trend for .5) Na+, K+, tissues was Cl-. But, SO42- was also detected

Ca2+, C1- and SO42- ions were detected in D. W. in very low concentration levels in the tissues.

These results suggest that these ions passed Na+ concentration in the leaf showed an increase through the root and viviparous seed tissues concomitant with the increase in NaCl concentra-

into the D. W. Concentrations of K+, NO3-, tion in the culture solution. Na+ concentration and PO43-, which are essential elements for plant in the leaf under F-100 conditions was 22.80 meq growth, typically decreased under F-20 condi- and this was the highest level attained among tions. The same trend was absorbed under the culture conditions. K+ concentration under

F-50 conditions. Ratio of each ion decrease in F-20 conditions was 5.80 meq and was higher than the solution gradually decreased with increasing in other conditions. As for the ratio of Na/K in

NaCl concentration in the culture solution. the leaf, F-0 condition indicated the lowest S. Kato: Study on the Salt Tolerance Mechanism of Halophytes 401

Fig.-3 Ion concentration in Bruguiera gymnorrhiza root.

(2.23) and F-100 condition was 5.83. Na/K the NaCl concentration in the culture solution: ratio in Bruguiera gymnorrhiza leaf was higher F-0 conditions produced only 1.65 meq; on the than that of Kandelia candel.5) Concentration other hand, F-100 conditions produced 37.89 of distributed Mg2+ in the leaf was within 3.00 meq and this value was approximately 23 times meq to 4.20 meq. And Ca2+ concentration was that of F-0 conditions. K+ concentration within 1.15 meq to 1.86 meq. Both divalent indicated high concentration at F-0 and F-20 cation concentrations in the leaf of Bruguiera conditions. K+ concentration under F-50, F-80 gymnorrhiza were higher than that found in and F-100 conditions gradually decreased. Na/ Kandelia candel leaf.5) The main anion distri- K ratio in the root under F-0 conditions regis- buted in the leaf was Cl-, and SO42- was also tered the lowest (0.17), while F-100 was 6.34. detected. This SO42- concentration increased This Na/K value was approximately 37 times slightly according to NaCl concentration in the that of F-0 condition. Mg2+, and Ca2+ levels culture solution. Cl- concentration under F-20 in the root were found to be lower than in the conditions was 9.50 meq (i.e., the lowest) and leaf and rhizophore under all growth conditions F-100 condition produced the highest concen- tested. Mg2+ concentration in the root was tration (25.50 meq). lower than Ca2+ concentration. The main anion The main cation distributed in the rhizophore distributed in the root was Cl-, and SO42- was was Nat and its concentration gradually in- also detected. Cl- concentration under F-100 creased according to NaCl concentration in the conditions was the highest, 40.31 meq. culture solution; F-100 condition indicated 22.67 3.3 Organic acid content in cultured plants meq. K+ concentration in the rhizophore under Many halophytes regulate osmosis in the F-50 conditions was 4.72 meq. K+ concentra- plant body to maintain normal physical mech- tion under F-20 conditions was 3.58 meq. Na/ anisms. Regulation under conditions of excess K ratio in the rhizophore under D. W. conditions absorbed cations may be obtained through the was 2.78 and the other conditions gradually use of organic acids and other organic sub- increased with NaCl concentration in the culture stances.3) In this study, organic acids (diacids solution. Na/K ratio under F-100 conditions and triacids) which are osmosis control sub- was 7.08. Mg2+ and Ca2+ cations in the rhizo- stances in the plants were analyzed. Analysis phore were found to be lower than in the leaf results of Bruguiera gymnorrhiza leaves and roots under all growth conditions tested. The main are shown in Figs.-4 and 5. F-20 condition anion distributed in the rhizophore was Cl-, produced the best growth and the concentration and SO42- was also detected. of oxalic acid also was the highest concentration, The main cation distributed in the roots as compared with a higher concentration than nutrient absorption organ was Nat. Concen- that of other growth conditions. Oxalic acid, tration of Nat gradually increased according to and malic acid concentration in the leaf were 402 Bull. Soc. Sea Water Sci. Jpn. Vol.46 No.6 (1992)

Fig.-4 Organic acid concentration in Bruguiera gymnorrhiza leaf.

Fig.-5 Organic acid concentration in Bruguiera gymnorrhiza root. found to be higher than the other organic acids. water and regular tide of sea water. Salt Oxalic acid and malic acid in the root were concentration ranges from 0% up to sea water present at concentrations higher than the other NaCl concentration level. These mangrove organic acids. forest areas are regularly affected by the daily sea water tide. Salt concentrations were fixed in this 4. DISCUSSION study. Distribution area of Bruguiera gymnor- rhiza is from middle to upper areas which are 4.1 Relationship of NaCl concentration and affected by sea water through river to mangrove growth of Bruguiera gymnorrhiza forest; perhaps F-20 condition as the best Results of this study on the growth of Bru- growth culture was similar to water component guiera gymnorrhiza for 4 months under several condition of this Bruguiera gymnorrhiza of culture conditions showed F-20 conditions pro- mangrove plants area. Essential elements for duced the best plant growth. The NaCl con- plant growth such as K+, NH4+, NO3- and centration at this level (F-20) is 0.6%. Streams PO43- correspond to K, N and P. After the in natural mangrove forest areas usually bring culture period, ion components decreased in in fresh water from upper river areas (moun- the F-20 condition, which is the best growth tains). Salt concentration of these mangrove culture condition. As these components were areas is very changeable with the inflow of fresh gradually decreasing, it was observed that the S. Kato: Study on the Salt Tolerance Mechanism of Halophytes 403

NaCl concentration was increasing in the culture in the distribution area of mangrove species solution. These results suggest that absorbed under natural ecosystem. Bruguiera gymnor- essential elements, possibly NaCl, suppressed rhiza root is able to control as much as possible other cations and anions which were essential ion absorption. Usually, K+ was absorbed for growth of Bruguiera gymnorrhiza. Bruguiera under high salt condition. The cation K+ is gymnorrhiza might preferentially absorb K+ for important for bio-synthesis of starch, opening normal growth. Distribution of Bruguiera and closing of stomata, oxidative phosphoryla- gymnorrhiza in the natural mangrove forests is tion, metabolism of protein, and is related to not directly affected by tidal sea water and they pH control and osmosis adjustment in the cell. are not found in fringe areas of mangrove But, physiological action of Na+ in the plants forests. And, thick growth area of this man- not well understood at present. Mangrove grove species is found in the inner areas of plants are classified as C3 plant group and these mangrove forest and the upper stream area. C3 plants need twice as much water as C4 The experimental F-20 condition (0.6% NaCl) plants for normal growth. Mangrove plants of this study may be similar to NaCl and other can absorb nutrients in water from low nutri- ion components in natural conditions. If level ents condition of growth environment and control (high and low) of culture solution transport these to leaf while water is transpired corresponded to tidal sea water, further optimum to the air. Absorbed nutrients remain in the growth and the best NaCl concentration for leaf, where their concentration gradually in- Bruguiera gymnorrhiza growth will be con- creases. Part of the rhizophore is a pathway firmed. route for absorbed ions and water into the leaf. 4.2 Inorganic ion components in harvested Bruguiera gymnorrhiza in this rhizophore part Bruguiera gymnorrhiza plants showed a tendency to accumulate these ions. Physiological mechanism of halophytes is These results indicate that rhizophore may be studied in several conditions. Generally, ad- useful as a preserving mechanism for unneces- justment to high salt conditions by halophytes sary excess ions for normal physiological pro- are as follows: (1) control of Na+ inflow at root cesses of the plant. cell, (2) once entered ions from the root, excess Total cations in Bruguiera gymnorrhiza roots salts are discharged from root cells, (3) absorbed were higher than in the leaf. This result shows excess salts accumulate in roots and stems for that at first, absorbed ions might accumulate regulation of salt accumulation into the leaf, (4) in the root and then necessary amounts of each translocated excess salts until full storage in salt ion were transported through the rhizophore to hair of leaf and excreted out of plant body by the leaf for normal physiological plant processes. disruption of hair root, (5) excess salts excreted And, this process in Bruguiera gymnorrhiza is from salt glands of leaf surface, (6) excess salts more advanced than in Kandelia candel.5) storage in vacoule cell of mesophyll cell and After ion transport into the leaf, the transloca- increase in cell water for osmosis adjustment, (7) tion of ions from leaf to the root and ion ac- excess cations are neutralized by organic acids cumulation in the root needs to be studied and some amino acids. Halophytes respond further. with a combination of these methods for osmosis The Cl- ion is the pair anion of absorbed control. cations. Physiological actions of this absorbed The main distributed cation in the leaf was Cl- are important for oxygen generation of Nat, which entered root cell through membrane photosynthetic process, and participation to and translocated into leaf of Bruguiera gymnor- dehiscent water with Mn of micro nutrients.2,4) rhiza. Na+ concentration in Kandelia candel Absorbed inorganic ions (NO3-, PO43-, SO42-) leaf was higher than Bruguiera gymnorrhiza are utilized for plant composition. These leaf.5) The Na+ concentration was 1.40 times absorbed inorganic ions, however, cannot re- in F-20, 1.49 times in F-50 and 1.57 times in spond to pH changes, control of osmotic pres- F-80 conditions. These results show that dif- sure and control of turgor pressure in the cell. ference in growth condition of mangrove plants And they do not have an equivalent molar ratio in natural ecosystem is reflected in the difference against absorbed cations. When halophytes 404 Bull. Soc. Sea Water Sci. Jpn. Vol.46 No.6 (1992) grow under high salinity conditions, these in Plants," R. C. Staple and G. H. Toenniessen plants stimulate synthesis of organic acids for (ed.), pp. 125-150, John Wiley and Sons, New neutralization of excess cations. Results of York (1984) Kandelia candel cultivation showed that oxalic 4) S. Izawa, R. L. Heath and G. Hind, Biochem. acid and malic acid increased in the roots and Biophys. Acta, 180, 388 (1969) 5) S. Kato, Bull. Soc. Sea Water Sci. Jpn., 46, 89 leaves with a corresponding increased NaCl (1992) concentration in the culture solution.5) A relationship between organic acid content and NaCl concentrations was supposed from Kan- 和 文 要 旨 delia candel cultivation.5) On the other hand, マ ング ロー ブ植物,オ ヒル ギ と塩環 境 for Bruguiera gymnorrhiza, the relationship between organic acid content and NaCl concen- 加藤 茂 trations was not so clear. But, Bruguiera gym- マ ングロー ブ植物 は,熱 帯 ・亜熱帯 の海 岸線や河 口・ norrhiza also has the same process for absorbed 河 川流域 の定 期的 に海 水 あるいは汽水 の流入す る地域 excess cations in that the amount of organic に分布 し生育 してい る植物群 であ る.マ ングロー ブ林 acids depended on NaCl concentration in the culture solution. The mechanism of neutraliza- の構成樹種 の一種 であ るオ ヒル ギは,マ ングロー ブ林 tion of absorbed excess cations with organic が直接海 水 の影響 を受 け る林 先端域 にはほ とん ど分布 acids for these two species of mangrove plants が見 られない種 であ る.オ ヒルギ栽培試 験 の結果,F- is recognized. 20区 す なわ ち0.6%NaCl濃 度 におけ る生育は 自然 マ ングロー ブ林 での生育 の よ うに最 も良好 であ った. ACKNOWLEDGMENTS 平均 的な海水塩分 濃度 であ る3%NaClのF-100区 で の生育 状況は,発 根は認 め られ たが葉 の伸長展 開は I express special thanks to Dr. Jiro Sugi, Emeritus Professor of Tokyo University of 劣 り,葉 面積は小 さ くそ の葉 も厚 さを増 していた.オ Agriculture, for valuable suggestions and dis- ヒルギ栽 培液 中のNaCI濃 度上 昇 とともにNa+イ オ cussions on this research. I also thank Mr. ンお よびCl-イ オ ンの植物体 へ の吸収 移行 が増加 し植 Yukio Yaguchi for plant cultivation. I have to 物体 内蓄 積濃度 も増 加 した.オ ヒルギ の葉 内お よび根 thank Salt Science Foundation, Japan, for 内に分布 したお もな有 機酸 は,シ ュウ酸 と リンゴ酸 で partly supporting this research. あ った.こ れ ら有機酸 類は,過 剰 カチ オ ンと塩 を形成 して浸透圧調 節を行 って いる ことが 推察 され た.オ ヒ REFERENCES ルギ の幼苗生 産を行 うには,約0.6%NaClの 塩分環

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(1985) (Received Jun. 29, 1992) 3) U. Luttge and A. S. Smith, "Salinity Tolerance