Matsumoto, Hideaki

Yamaya, Tomoyuki

Okayama University Digital Information Repository

ACCUMULATION OF ASPARAGINE IN NaCl-STRESSED BARLEY SEEDLINGS

Okayama University Scientific Achievement Repository

Ber. Ohara Inst. Iandw. Biol.， Okayama Univ. 19 : 181-188 (1989) ACCUMULATION OF ASP ARAGINE IN N aCl-STRESSED BARLEY SEEDLINGS *Tomoyuki Y AMA YA and Hideaki MATSUMOTO INTRODUCTION 181 Within the leaves of many plants subjected to moderate or severe water-or salt stre路， one striking change in nitrogen metabolism is the accumulation of free proline (Hanson and Hitz 1982， Tully et al. 1979， WynJones and Storey 1978). Recent observations with tomato suspension cultures adapted to water st町民 alsoshowed the importance of proline in osmotic adjustment， since the cells adapted to water stress showed a 300・foldincrease in proline pool which represents over 50% of the total free amino acids (Rhodes et al. 1986). The elevated proline pool is primarily the consequence of a 10・foldelevated rate of proline synthesis via the glutamate pathway (Rhodes et al. 1987). Although the function of the adaptive increase in the proline pool is not yet clear， itcould be an osmoticum， an enzyme/membrane protectant， or a nontoxic store of reduced nitrogen (Hanson and Hitz 1982， Rhodes et al. 1986). Metabolic changes in other free amino acids， such as increase in GABA， alanine， valine (Hanower and Brzozowska 1975， Rhodes et al. 1986)， have been shown under the water/salt stressed conditions， but these changes are small compared to the changes in the proline pool. However， we found that the asparagine pool is increased markedly in barley seedlings at the primary leaf stage when the plants were treated with 50mM N aCl in a hydroponic culture. Herein， we discuss the physio-logical function of asparagine accumulated by the NaCl・stress.MA TERIALS AND METHODS Plant Mate'YI，αls Seeds of barley (Hordeum vulgare L. cv. Kikaihadaka) were ger-minated in aerated water for 12h at room temperature. The germinated seeds were planted on a plastic net in a 6l plastic container with 4l of a Research Institute for Bioresources， Okayama University， Chuo 2-20-1， Kurashiki， Okayama 710， ]apan Received May 27， 1988. ・Presentadres : Department of Agricult町aIChemistry， Faculty of Agriculture， Tohoku University， 1-1 Amamiyamachi-Tsutsumidori， Sendai， Miyagi 981， ]apan This work was suported in part by Grants-in-Aid for Scientific Research from the Ministry of Education， Science and Culture of ]apan (No. 61129005 and 62480052). 182 T. Yamaya and H. Matsumoto half strength nutrient solution containing 2mM KN03 and 0.5mM (NH4)2 S04 as the nitrogen source， as described previously (Matsumoto et al. 1980). The seedlings were grown for about 10 days in a Tabai refrigerat-ed incubator LNC-130 at 240C. The seedlings were illuminated with fluorescent lamps giving about 100μmol/m2.s during a 12・hphotoperiod. Extractionα:nd isolation 01α:minoαcids Barley roots and leaf blades (0.5g fresh weight) were incubated in 20ml of an 80% ethanol solution for a few days at room temperature for the extraction of amino acids. The tissues were then homogenized in the ethanol solution with a mortar and pestle， filtered through a Toyo filter paper No. 6， washed with 10ml of 80% ethanol， and then the filtrate was evaporated to dryness in a rotary evaporator under vacuum at 40oC. The dried extract was dissolved in 1ml of water and then applied to a Dowex 50W H+ (200-400 mesh) ion-exchange column (0.6 x 4cm) equilibrated with water. The column was washed with 30ml of water to remove sugars and organic acids. The amino acids were eluted with 20ml of 2N NH40H. Each sample was rotary evaporated to dryness， dissolved in 0.5ml of water， and centrifuged at 16，000rpm with a Sakuma M-150 microcentrifuge. The supernatant fraction was used for derivatization of amino acids. Isolation 01 aminoαcids fiγom勾le慨 SゆThe leaf sheath of barley seedlings was cut approximately 10mm above the endosperm with a sharp razor blade. The exudate from the cut end was collected with a Pasteur pipet during a 1.5・hperiod and designat-ed as a xylem sap. The volume of the xylem sap was measured with a micro-pipet and then ethanol (to give a final concentration of 75%) was added. Amino acids were isolated from the ethanol extract as described above. Anαlysis 01 aminoαcids Amino acids isolated from the ethanol extracts were first derivat-ized with phenylisothiocyanate to synthesize phenylthiocarbamy-amino acid derivatives and the derivatives were then analyzed with a reversed-phase high performance liquid chromatography (HPLC) as described elsewhere (Yamaya and Matsumoto 1988). Isolatio:η01叩:cuolefiγom leafbladeドoto争lastsProtoplasts were isolated from barley leaf blades and purified as described previously (Yamaya and Oask 1988). Asparagine accumulation in NaCl-str巴ssedbarley 183 Vacuoles were isolated by the methods of Foster et aL (1987) with minor modifications. The purified protoplasts (about 1mg chlorophyll) were pelleted by centrifugation at 300g for 5min. Vacuoles were released from these protoplasts by the addition of 0.9ml O.1M K2HP04・HCl(pH 7. 5) and O.1ml of mannitol medium consisting of 0.5M mannitol， 2mM dithiothreitol (DTT)， 2mM ethylenediaminetetraacetate (EDTA)， 2mM CaCl2， 0.2% bovine serum albumin (BSA)， 25mM N・2・hydroxyethylpiperazine-N -2・ethanesulfonicacid (Hepes)-KOH (pH 7.5). After 5min at 25'C， 1ml of the mannitol medium was added and loaded onto Ficoll gradients consisting of 4ml steps of 1， 5， 10% (w/v) Ficoll・400in 25mM Hepes-KOH (pH 7.5) containing O.4M mannitol， 1mM CaCl2， 1mM DTT， 1mM EDT A， and 0.1 % BSA. The gradients were centrifuged at 26，000rpm (100，000g) for 45min at 4'C in a SW・27.1rotor in a Beckman model L5-75 ultracentrifuge. Purified vacuoles at the 1/5% Ficoll inter-face were removed with a Pasteur pipet. Other assays Chlorophyll content (Arnon 1949) and α-mannosidase (Boller and Kende 1979) were determined based on published procedures. RESUL TS AND DISCUSSION Aminoαcid tool sizes inγvots， xylem sap，αnd leal blades 01 bαγley seedlings undeγNαCl stγ四SThe major amino acids in the roots without NaCl stress were glutamate， asparagine and glutamine ; they occupied about a half of the total free amino acids (Table'l). 1n a 50mM N aCl stress condition， the asparagine pool was increased about 4-fold within a day and the aspar-tate pool was increased 4.4 to 6・foldwhen compared to the 0 time control (Table 1). Other amino acids， such as glutamate， alanine， and serine， were also increased a few-fold. Analysis of the amino acid pool size in xylem sap obtained from unstressed bar1ey revealed that the glutamine was the largest pool accounting for 32% of the total amino acids detected in the sap， followed by asparagine which accounted for about 20% (Table 2). This indicates that these amides were the main amino acids as the form of transport from root to top of the barley seedlings. By the N aCl treatment， the asparagine pool was increased 2.8 to 4.5・foldwithin two days to repre-sent about a half of the total amino acids. On the other hand， the glutamine pool was rather decreased after the stress treatment. Some other minor amino acids， such as alanine， ornithine， and proline， were also increased. It is worthy to note that glutamate， one of the major amino acids in root tissues， and aspartate， increased several fold by the 184 T. Yamaya and H. Matsumoto TABLE 1 Effect of 50mM NaCl on pool size of free amino acids in roots o Time Day 1 Day 2 Amino acid (μmol/g fresh weight root) Aspartate (Asp) 0.168 0.7 43( 4.42) 1.008(6.00) Glutamate (Glu) 0.687 2.178(3.17) 1.342(1.95) Serine (Ser) 0.312 0.697(2.24) 0.641(2.06) Asparagine (Asn) 0.623 2.610(4.19) 2.381(3.82) Glycine (Gly) 0.058 ND ND Glutamine (Gln) 0.360 0.659(1.83) 0.547(1.52) Homoserine (Hser) 0.030 0.080(2.70) 0.040(1.34) Histidine (His) 0.035 0.062(1.75) 0.063(1.76) γ-amino butyrate (GABA) 0.023 0.145(6.39) 0.061(2.25) Threonine (Thr) 0.152 0.341(2.23) 0.344(2.26) Alanine (Ala) 0.146 0.914(6.26) 0.685(4.69) Arginine (Arg) 0.021 0.102(4.83) 0.091(4.33) Proline (Pro) 0.029 0.068(2.35) 0.065(2.22) Tyrosine (Tyr) 0.057 0.095(1.66) 0.098(1.71) Valine (Val) 0.231 0.407(1.76) 0.361(1.56) Methionine (Met) ND 0.012 0.012 Isoleucine (Ileu) 0.180 0.230(1.28) 0.247(1.38) Leucine (Leu) 0.168 0.200(1.19) 0.245(1.46) Phenylalanine (Phe) 0.064 0.103(1.61) 0.105(1.63) Orinithine (Orn) 0.063 0.076(1.21) 0.072(1.15) Lysine (Lys) 0.094 0.139(1.49) 0.159(1.70) ND : not detected Parentheses indicate ratio of pool size compared to 0 time. stress in roots， but were hardly detected in xylem sap obtained either from control or NaCl-stressed samples. Thus， these acidic amino acids were probably not transported directly from roots through xylem flux. Itshould be noted that NaCl treatment strongly inhibited the exudation from the xylem， and no sap can be collected after a 5・daytreatment with 50mM NaCl or after 2 days with 200mM NaCl. In leaf blades without NaCl stress， the major amino acids were glutamate， asparagine， and alanine (Table 3). Unlike in roots or xylem sap， the leaf sheath contained a rather small glutamine pool， which suggests rapid metabolism of glutamine transported from roots. As was seen in roots and in xylem sap， the asparagine pool in leaf blades was also expanded about 2-3 fold by the NaCl stress to represent about one third of the total amino acids within one or two days. By the treatment， free pools of serine， alanine， glutamine， and GABA were also increased but not as much as the accumulation of asparagine. The aspartate and glutamate pools had essentially the same size before and after the treatment. The proline pool was also unchanged by the treatments. Asparagine accumulation in NaCl-stressed barley 185 TABLE 2 Effect of 50mM NaCl on pool size of free amino acids in xylem sap o Time Day 1 Day 2 Amino acid (pmol/μ1 of xylem sap) Asp 27 8(0_30) 94(3.42) Glu 80 98(1.23) 178(2.22) Ser 630 935(1.49) 1，200(1.91) Asn 1，709 4，790(2.80) 7，707(4.51) Gly ND ND ND Gln 2，850 1，850(0.65) 2，494(0.88) Hser 41 60(1.46) 43(1.04) His 84 104(1.24) 138(1.65) GABA 68 50(0.74) 99(1.44) Thr 519 429(0.83) 507(0.98) Ala 189 619(3.28) 441(2.34) Arg 173 389(2.25) 374(2.16) Pro 22 63(2.86) 72(3.25) Tyr 113 71(0.62) 120(1.06) Val 786 675(0.86) 949(1.21) Met 17 23(1.37) 27(1.59) IIeu 398 232(0.58) 341(0.86) Leu 286 156(0.54) 206(0.72) Phe 151 77(0.51) 123(0.81) Om 57 181(3.20) 255(4.50) Lys 776 590(0.76) 776(1.00) ND : not detected Parentheses indicate ratio of pool size compared to 0 time. When the bar1ey seedlings were grown in 0.5mM CaC12 alone (with-out a nitrogen source)， the pool size of asparagine was increased 1.9・and0.8・foldin roots and in leaf blades， respectively both in the presence and absence of 200mM NaCl (data not shown). Thus， the supply of nitrogen into the medium caused the large accumulation of asparagine in roots and leaf blades. A mino acid comρosition inρrotoρlasts and vacuoles Compartmentation of amino acids was investigated by measuring their pool sizes in protoplasts and in isolated vacuoles， which can be considered to be a storage pool， from barley leaf blades with or without NaCl stress. To determine the distribution of each amino acid in the vacuole， the actual amount of amino acids was divided by α-mannosidase activity which is the marker enzyme of the vacuole (Boller and Kende 1979， Foster et al. 1987).However， since the recovery of the vacuole based on the yield ofα-mannosidase activity from the protoplast was only 5-7%， the results may not represent a true compartmentation. 186 T. Yamaya and H. Matsumoto TA8LE 3 Effect of 50mM NaCl on pool size of free amino acids in leaf blades o Time Amino acid Day 1 Day 2 (μmol/g fresh weight leaf) Asp 0.367 0.184(0.50) 0.375(1.02) Glu 1.921 1.769(0.92) 1.634(0.85) Ser 0.639 1.076(1.68) 1.312(2.05) Asn 1.281 2.605(2.03) 3.933(3.07) Gly 0.175 0.670(3.83) 0.019(0.11) Gln 0.243 0.423(1.7 4) 0.417(1.71) Hser 0.045 0.019(0.41) 0.042(0.93) His ND ND ND GABA 0.191 0.266(1.39) 0.402(2.11) Thr 0.328 0.372(1.14) 0.639(1.95) Ala 0.951 0.990(1.04) 1.151( 1.21) Arg 0.052 0.063(1.20) 0.116(2.21) Pro 0.052 0.046(0.89) 0.082(1.59) Tyr 0.081 0.1 02(1.26) 0.158(1.96) Val 0.352 0.404(1.15) 0.598(1.70) Met ND 0.021 ND Ileu 0.217 0.243(1.12) 0.376(1. 7 4)Leu 0.143 0.140(0.98) 0.231(1.62) Phe 0.119 0.152(1.27) 0.252(2.12) Orn 0.048 0.037(0.76) 0.085(1.76) Lys 0.276 0.265(0.96) 0.341(1.24) ND : not detected Parentheses indicate ratio of pool size compared to 0 time As shown in Table 4， asparagine， aspartate， glutamine， isoleucine and leucine were mostly located in the vacuole. The value of over 100% probably means further metabolism of these amino acids during the isolation of the vacuole. On the other hand， proline， alanine， GABA， glutamate， serine， and ornithine were detected in small amounts in the vacuole， which indicates that these amino acids were located in the cytosol and other compartments， such as chloroplasts and mitochondria. At the primary leaf stage of barley seedlings， N aCl stress first enhanced the asparagine pool in roots， xylem sap and leaf blades， but not proline pool which has been reported by many groups (Hanson and Hitz 1982， Rhodes et al. 1986， Tully et al. 1979， WynJones and Storey 1978). Assimilation of inorganic nitrogen is not affected by a short period of salt or water stress (Hanson and Hitz 1982). Our data are in agreement with their finding， since the total amino acid pool in roots， xylem sap， and leaf blades was increased by the N aCl treatment. At this stage material in the endosperm of the barley seedlings is stil left over， i. e.， growth of the plants is heterotrophic in the carbon status. A continuous Asparagine accumulation in N aCI-stressed barley 187 TABLE 4 Localization of amino acids in vacuole isolated from leaf blade protoplasts of barley seedings treated with or without NaCI stress Control 200mM NaCI Amino acid (% in vacuole isolated from protoplasts) Asp 206 171 Glu 29 85 Ser 75 58 Asn 166 77 Bly ND 58 Gln 104 128 Hser 88 ND GABA 27 39 Thr 77 ND Ala 25 61 Arg 109 53 Pro 38 68 Tyr 62 ND Val 157 80 I1eu 143 101 Leu 88 169 Phe 81 38 orn 38 26 Lys 135 55 ND : not detected in either protoplast or vacuole His and Met were not detected. Relative distribution of each amino acid in vacuoles was obtained by comparing its amount in protoplasts on a basis of a-monnosidase activity. supply of inorganic nitrogen and sufficient amounts of carbon skeleton could allow the synthesis of amino acids in roots. Because strong inhibi-tion of xylem exudation was observed in the plants treated with NaCI， exce部 amountsof amino acids synthesized by the stre岱 edplant could be converted to asparagine as a storage form of nitrogen in roots. This asparagine was then transported to the top of the plants through the xylem stream. Les accumulation of asparagine with no exogenous supply of nitrogen and its vacuolar localization in the leaf blades also support our consideration on the function of increased asparagine as a storage form of nitrogen. Large accumulation of proline was not observed in our experiment when barley seedlings at the primary leaf stage were tested under the salt stressed conditions. Tomato suspension cultures adapted to water stre岱， which are also heterotrophic growth conditions similar to that in our stage of barley seedlings accumulate proline more than a half of total amino acid pools (Rhode et al. 1986). This large proline pool is suggested to work as an osmoregulator， but proline synthesis was not 188 T. Yamaya and H. Matsumoto stimulated by N aCl stress in our plants. The barley seedling under stress at this stage could not survive. The severe inhibition of xylem flux and the lack of osmoregulator synthesis could be the main reason for the poor survival of our plants. SUMMARY Free pools of amino acids in roots， xylem sap， and leaf blades of barley (Hordeum vulgare L. cv. Kikaihadaka) seedling at the primary leaf-stage， treated with or without 50mM NaCl， were determined by a reversed-phase high performance liquid chromatography. The NaCl・stre田 causeda marked increase in asparagine pool which accounted for 28， 48， and 32% of total free amino acids in roots， xylem sap， and leaf blades， respectively， when three-week old seedlings were treated for 2 days. Accumulation of proline was small compared to that of asparagine in the seedings. More than 75% of the asparagine was detected in vacuoles isolated from protoplasts of the leaf blades. REFERENCES Arnon， D.1. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24 : 1-15. Boller， T.and Kende， H. 1979. Hydrolytic enzymes in the central vacuole of plant cels. Plant Physiol. 63 : 1123-1132. Foster， Jι，Cress Jr.， W.D.， Wright， S.F. and H白s，J.L. 1987. Intracellular localization of 出eneurotoxin 2，4・diaminobutyricacid in Lathyrus sylvent:市L.leaf tisue. Plant Physiol. 83 : 900-904. Hanower， P.and Brzozowska， J. 1975. Infuence d'un choc osmotique sur la composition des feuilles de cottonier en acides amines libres. Phytochemisty 14 : 1691-1694. Hanson， A.D. and Hitz， W. D. 1982. Metabolic responses of mesophytes to plant water deficits. Annu. Rev. Plant Physiol. 33 : 163-203. Matsumoto， H.， Teraoka， K. and Kawasaki， T. 1980. Repression of nitrate reductase in cucumber leaves caused by calcium deficiency. Plant Cell Physiol. 21 : 183-191. Rhodes， D.， Handa， S.and Bressan， R.A. 1986. Metabolic changes associated with adapta-tion of plant cells to water stress. Plant Physiol. 82 : 890-903. Tully， R.E.， Hanson， A.D. and Nelsen， C.E. 1979. Proline accumulation in water-stressed barley leaves in relation to translocation and the nitrogen budget. Plant Physiol. 63 : 518-523. WynJones， Rιand Storey， R. 1978. Salt stress and comparative physiology in the gramineae. 1. Glycinebetaine and proline accumulation in two salt-and water.stressed barley cultivars. Aust. J. Plant Physiol. 5 : 817-829. Yamaya， T. and Oaks， A. 1988. Distribution of two isoforms of glutamine synthetase in bundle shea出 andmesophyll cells of corn leaves. Physiol. Plant. 72 : 23-28. Yamaya， T. and Matsumoto， H. 1988. Analysis of phenylthiocarbamyl剖 ninoacids at pico・molelevel by high performance liquid chromatography and application to plant materials. Soil Sci. Plant Nutr. 34 : 297-302. 

ACCUMULATION OF ASPARAGINE IN NaCl-STRESSED BARLEY SEEDLINGS

Abstract

Similar works

Full text

Available Versions

Okayama University Digital Information Repository

Okayama University Scientific Achievement Repository