Effect of Light Irradiation on the D-Alanylglycine Content in Rice Leaf Blades

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1 Plant & Cell Physiol. 24(6): (983) Effect of Light Irradiation on the D-Alanylglycine Content in Rice Leaf Blades Hisashi Manabe and Koji Ohira 2 Aizu Junior College of Fukushima Prefecture, Aizuwakamatsu 965, Japan 2 Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai 980, Japan () The fluctuating pattern of D-alanylglycine content, although the increase and decrease rates were not very high, in the leaf blades of rice seedlings growing in light or dark was related to that of the chlorophyll content in the leaf blades. (2) The D-alanylglycine content in the leaf blades of the chlorophyll-less cultivar was lower than that of the normal one. (3) Photosynthetically assimilated carbon was smoothly incorporated into D-alanylglycine. (4) Exogenous D-alanine was only incorporated into D-alanylglycine when the D-alanine feeding was performed under light. These results suggested that D-alanylglycine synthesis in the leaf blades of rice plants is closely related to the amino acid metabolism accompanying photosynthesis in rice plants. Key words: D-Alanylglycine Conjugated D-alanine Dark treatment Light irradiation Rice leaf blades. D-Alanylglycine has been found in rice plants (Yamauchi et al. 979) but its physiological role in the tissues remains unknown. Our previous paper (Manabe et al. 980) reported that () D-alanylglycine exists in the leaf blades of axenic rice seedlings, (2) it is not detected in the leaf blades of seedlings grown in the dark, but is formed upon light irradiation of the tissues and (3) almost all of the D-alanylglycine in the seedlings exists in the tissues. These results show that D-alanine in D-alanylglycine is a metabolite of the rice plants themselves and suggest that light plays- an important role in the production of D-alanylglycine in the tissues. The present paper discusses the effects of light irradiation on the content and behavior of D-alanylglycine in rice leaf blades. Materials and Methods Plant materials Three cultivars of rice plants, Norin No. 6, Sasanishiki and Oshokukamenoo (chlorophyll-less cv.), were solution-cultured (cf. Manabe and Ohira 980a) in a greenhouse or a growth cabinet as the experiment required. ^COz feeding Five intact rice plants grown in a greenhouse until the early tillering stage were transferred to a chamber containing 0 [id of NaH 4 CO3. At this growth stage, the 9th leaf blade on the main stem was developing from the 8th leaf sheath. 4 CO2 was produced by having the NaH 4 CO3 react with 80% lactic acid (after the treatment, the CO2 concentration in the chamber was ca. %), and the 4 CO2 produced was assimilated photosynthetically for 3 h under sunlight (ca. 3 X 4 lux, 33 G) at 3:00 in the autumn. Thefiveplants in the chamber Downloaded from at Penn State University (Paterno Lib) on March 5, 206 Abbreviations: Ala, alanine; Asp, aspartic acid; Asn, asparagine; GABA, y-amino butyric acid; Glu, glutamic acid; OPA, o-phthalaldehyde; PPO, 2,5-diphenyloxazole; Ser, serine; Thr, threonine. 37

2 38 H. Manabe and K. Ohira light 2 hr > light 5 hr > d dark dark 6 hr Hi 3 hr dark 3 hr light 3 hr Fig. D-Alanine feeding procedure. a, b: Excised leaf blades (ca. 4g) were divided equally and put into two test tubes each containing ml of D-[ 4 C(U)]alanine solution (~ 3 M, 2.5 yuci). The blades were then irradiated with sunlight. As the D-alanine solution was absorbed within min, distilled water was added thereafter. After feeding for 2 h (a) and 5 h (b), the blades were subjected one tube at a time to analysis, c, d: Excised leaf blades (ca. 2 g) were divided equally and put into two test tubes each containing 0.5 ml of D-[ 4 C(U)]alanine solution (~ 3 M,.25 /ici). The blades were then kept in a dark place. After feeding for 3 h (c) and 6 h (d), the blades were subjected one tube at a time to analysis. In this procedure, one-half and most of the D-alanine solution were absorbed by the blades during the 3-h and 6-h feeding, respectively, e: Excised leaf blades (ca. 2 g) were put into a test tube containing ml of D-[ 4 C(U)]alanine solution (~ 3 M, 2.5 /*Ci). The blades were kept in a dark place for 3 h, and they were irradiated with sunlight for 3 h. The blades were then subjected to analysis. were then exposed to air for, 7, 23, 34 and 45 h, respectively, and then lyophilized. Three leaf blades were selected from leaf sheaths of the main stem and immersed in 300 ml of 70% ethanol. The 70% ethanol extract was separated into cationic and non-cationic fractions with a column of Dowex 50W-X8 (H + form) (Manabe and Ohira 980a). The cationic fraction was concentrated and dried with an evaporator, and then dissolved with an appropriate volume of water. Various amino acids in the solution were separated on paper (Toyo Roshi No. 5) by two-dimensional chromatography and the labeled amino acids on the paper were detected by radioautography (Manabe and Ohira 980a). 4 C-D-Alanine feeding Rice plants were grown in a greenhouse until the early tillering stage, then some were cultivated further in the greenhouse for 3 days while others were cultivated in a dark place for 3 days. Two or three leaf blades were selected from leaf sheaths of the main stem of each plant and placed in test tubes containing D-[ 4 C(U)] alanine solution. The D-alanine feeding experiment was carried out in light or dark as shown in Fig.. At the end of an appropriate exposure time, the blades were cut into small pieces and immersed in 300 ml of 70% ethanol. Cationic compounds were fractionated from the 70% ethanol extract using a Dowex 50-X8 (H+ form) column, and amino acids in the cationic fraction were separated by paper chromatography and labeled amino acids on the paper were detected by radioautography. The areas with the labeled D-alanylglycine were cut out from the paper and placed in a vial containing ml of toluene and 50 mg of PPO to measure the radioactivity in the D-alanylglycine using a liquid scintillation spectrometer (Packard, Model 527). Measurement of free amino acid, D-alanylglycine and chlorophyll contents Leaf blades of rice plants (ca. g) were immersed in 200 ml of 70% ethanol. An aliquot of the 70% ethanol extract or its cationic fraction was subjected to high-pressure liquid chromatography (JASCO, TRI ROTAR-III) and the free amino acid and D-alanylglycine contents were measured by the OPA method (Manabe et al. 98). Chlorophyll content in rice leaf blades was determined by the method of Arnon (949). Downloaded from at Penn State University (Paterno Lib) on March 5, 206 Results Changes in chlorophyll and D-alanylglycine contents in rice seedlings Rice seedlings (Norin No. 6) grown in the dark for 8 days were irradiated with light for 2 days, cultivated in the dark for 4 days and irradiated with light for 3 days. Chlorophyll and D-alanylglycine contents in the leaf blades of the seedlings both increased with time after light irradiation of the dark-grown rice seedlings was begun and decreased gradually after the light irradiation was stopped (Fig. 2).

3 Changes in D-alanylglycine content in rice plants 39 Fig. 2 Changes in chlorophyll and D-alanylglycine contents in rice seedlings. Rice seeds (Norin No. 6) were sowed and germinated in running water in the dark at 28 C C. At the 9th day after sowing, running water was replaced with nutrient solution A (Table ), and the seedlings were irradiated with fluorescent light ( 4 lux) for 2 days at 30 C. They were then cultivated in the dark for 4 days at 28 C and they were again irradiated with fluorescent light for 3 days at 30 C Days after sowing Next, the chlorophyll and D-alanylglycine contents were compared with two rice cultivars. As shown in Fig. 3, not only the chlorophyll content (a) but also the D-alanylglycine content (b) in the leaf blades of the chlorophyll-less cultivar (Oshoku-kamenoo) was ca. one-fifth as much as that of the normal cultivar (Norin No. 6). ^GOi-fceding experiment Rice plants (Sasanishiki) were allowed to assimilate 4 CC>2 photosynthetically. Radioactivity in the leaf blades was measured and, as shown in Fig. 4, was detected in the cationic fraction of the ethanol extract. The radioactivity decreased gradually with time throughout the exposure period. To find what amino acids were labeled in the cationic fraction, paper chromatography was done and radioautograms were prepared. In the cationic fraction, D-alanylglycine as well as Glu, Asp, Ala, GABA, Ser, and Glu was labeled predominantly (Table 2). In this experiment, moreover, radioactivity in D-alanylglycine decreased gradually with time throughout the exposure period, while that in other amino acids decreased rapidly. This is why the ranking of D-alanylglycine rose with time when its magnitude of labeling was compared with those of other amino acids (Table 2). 4 C-D-Alanine feeding experiment To observe the labeling pattern of D-alany]glycine in the leaf blades of rice plants (Sasanishiki), 4 C-D-alanine was fed to the blades under light or dark condition. D-Alanylglycine was labeled when the 4 C-D-alanine feeding was performed in the light (a, b and e of Fig. 5), but not in the dark (c and d of Fig. 5). Increase and decrease patterns of D-alanylglycine content in rice leaf blades As the D-alanylglycine content in rice leaf blades is influenced strongly by the presence or absence of light irradiation Table Composition of the nutrient solution A and B Concentration (ppm) Component N P2O5 K 2 O v_jnemicai NH4NO3 NaH 2 PO 4 2H 2 O Solution A 5 Solution B 50 5 MgO CaO Fe Mn B Zn Cu Mo K 2 SO 4 MgCl 2 6H 2 O 5 CaCl 2 2H 2 O Fe-EDTA 2.5 MnCl 2 4H 2 O H3BO ZnSO 4 7H 2 O CuSO 4 5H 2 O 0.0 (NH 4 ) 6 Mo 7 O 2 44H 2 C) Downloaded from at Penn State University (Paterno Lib) on March 5, 206 The ph of the nutrient solution was adjusted to 5.5.

4 40 H. Manabe and K. Ohira Chlorophyll, mg/g fr wt D-Alanylglycine, pmol/g fr wt (a) Norin No. 6 Oshoku-kamenoo Fig. 3 Chlorophyll (a) and D-alanylglycine contents (b) in the leaf blades of two rice cultivars. Normal cultivar (Norin No. 6) and chlorophyll-less cultivar (Oshoku-kamenoo) were cultivated in nutrient solution under 2 h-light ( 4 lux, 30 C)/2 h-dark (28 C) regime for 33 days. Hrs after exposure ( Table 2 Distribution of radioactivity in each amino acid in the cationic fraction of rice leaf blades after feeding 4 CO2 photosynthetically Hours after exposure Magnitude of labeling Glu>Asp>Ala=D-alanylglycine>GABA>Ser>Glu 7 Glu>GABA> D-alanylglycine > Ala 23 Glu> D-alanylglycine >GABA> Ala 34 D-alanylglycine > Glu > Asp = GABA=Ala 45 D-alanylglycine=Asp An aliquot of the cationic fraction of the leaf extract was chromatographed with a filter paper and a radioautogram was made. The magnitude of labeling in each amino acid on the paper was estimated by the blacking degree of the X-ray film. Radioactivity, 0 I H ^f ' L ' ' : ethanol extract Fig. 4 X!0 6 cpm/g c ry wt svity (cpro u 2 hr 3 hr Fig. 5 2 hr 3 hr Downloaded from at Penn State University (Paterno Lib) on March 5, 206 Fig. 4 Distribution of radioactivity in the ethanol extract of rice leaf blades and its cationic fraction after feeding 4 CC>2 photosynthetically. Five rice plants (tillering stage, Sasanishiki) were placed in a chamber filled with 4 CO2, and the 4 CO2 was assimilated photosynthetically for 3 h. The five plants were then exposed to air for, 7, 23, 34 and 45 h, and the leaf blades of the plants were subjected one piece at a time to analysis. Fig. 5 Incorporation of exogenous D-alanine into D-alanylglycine under light or dark condition in rice leaf blades. Excised leaf blades of non-dark-treated (A) or dark-treated rice plants (tillering stage, Sasanishiki) (B) were fed with D-[ 4 C(U)]alanine. For details of D-alanine-feeding procedures and explanation of a, b, c, d and e, see the section on " 4 C-D-Alanine feeding" in Materials and Methods.

5 Changes in D-alanylglycine content in rice plants 4 2 days 3 weeks 3 days ht -» h""+ < 8-5 hr " light Sowing Norin No. 6 Oshoku-kamenoo I D-alanylglycine Hrs after the start of light irradiation (a) or dark treatment (b) Fig. 6 D Time after the start of light irradiation (hr) Fig. 7 Fig. 6 Contents of free amino acids in leaf blades of rice seedlings (Sasanishiki) cultivated in light (a) or dark (b). (a): Rice seedlings grown in Solution A (Table ) under 2 h-light/2 h-dark regime for 3 weeks were cultivated in Solution B (Table ) in the dark for 2 days. They were then irradiated with fluorescent light ( 4 lux) for 8.5 h. (b): Rice seedlings grown in Solution A (Table ) in the dark (28 C) for 3 weeks were cultivated in Solution B (Table ) in the light for 3 days. They were then cultivated in the dark for 8.5 h. Fig. 7 Changes in D-alanylglycine content in rice leaf blades. Rice plants (Norin No. 6 and Oshoku-kamenoo) grown in Solution A (Table ) under 2 h-light ( 4 lux, 30 C)/2 h-dark (28 C) regime for 33 days were successively cultivated under the same light/dark regime for 24 h and the D-alanylglycine content in the leaf blades was measured with time. (Fig. 2), the dark-grown rice plants were irradiated with light or the light-irradiated rice plants were cultivated in the dark, and the increase and decrease patterns of D-alanylglycine content in the leaf blades were observed precisely. Rice seedlings kept in the dark for 2 days (Sasanishiki) were irradiated with light for 8.5 h. D-Alanylglycine, which had disappeared by the previous dark treatment, did not appear with such short-term irradiation (Fig. 6a). However, during the irradiation, increases in Glu and Asp contents and decreases in Ser+Asn and Ala contents were significant (Fig. 6a). When rice seedlings irradiated with light for 3 days (Sasanishiki) were cultivated in the dark for 8.5 h, the Glu content, which had increased by the previous light irradiation, decreased drastically (Fig. 6b). Ala, Ser and Thr contents fluctuated rapidly with time. The D-alanylglycine content decreased very slowly during the dark treatment (Fig. 6b). When rice seedlings (Norin No. 6 and Oshoku-kamenoo) grown under light/dark regime for 33 days were cultivated under 2 h-light/2 h-dark regime for 24 h, the D-alanylglycine content in the leaf blades increased gradually with time after the seedlings were irradiated with light; the increase continued for a few hours after light irradiation was stopped, then decreased gradually (Fig. 7). Downloaded from at Penn State University (Paterno Lib) on March 5, 206 Discussion This study showed that the D-alanylglycine content in rice leaf blades increased when the plants were irradiated with light, but decreased when the irradiation was stopped (Fig. 2); the fluctuating pattern of D-alanylglycine content in rice leaf blades was related to that of the chlorophyll content in the blades (Fig. 2 and 3); the photosynthetically assimilated carbon was metabolized smoothly to D-alanylglycine in the rice leaf blades (Table 2); the transformation of exogenous D-alanine into D-alanylglycine, which takes place when D-alanine is fed to rice leaf

6 42 H. Manabe and K. Ohira blades during the day (Manabe and Ohira 980a), was not observed when D-alanine feeding was carried out in the dark (Fig. 5). These results suggest that D-alanylglycine synthesis in the leaf blades of rice plants is closely related to the amino acid metabolism accompanying photosynthesis in rice plants. As described above, D-alanylglycine content in rice leaf blades is influenced strongly by the presence or absence of light irradiation. However, the increase and decrease rates of D- alanylglycine in the leaf blades after light or dark treatment was low in comparison with those of free amino acids (Fig. 6). Moreover, the D-alanylglycine content in rice leaf blades increased for a while after light irradiation stopped, then decreased gradually (Fig. 7). In addition, labeled D-alanylglycine, which was formed from photosynthetically assimilated 4 CO2 in rice leaf blades, tended to be metabolized very slowly (see the section on "^COvfeeding experiment" in Results). Turnover of D-alanylglycine in rice leaf blades, therefore, appeared to be low. On the other hand, iv-malonyl-d-amino acids are well-known conjugated D-amino acids found in higher plants (Zenk and Scherf 963, Ogawa et al. 973, Ogawa and Sasaoka 976); they are also common metabolites of exogenous D-amino acids in higher plants (Robinson 974). Such JV-malonyl-D-amino acids are considered to be inert in the tissues (cf. Rosa and Neish 968). In fact, a N-malonyl-D-alanine-like substance, which is one of the conjugated D-alanines found in rice plants and also one of the metabolites of exogenous D-alanine in rice leaf blades, is stable in the tissues (Manabe and Ohira 980b). Therefore, conjugated D-alanine found in rice leaf blades might not be very active in the tissues. More detailed observation for the behavior of D-alanylglycine in rice plants is now in progress. The 4 C-D-alanine feeding experiment was carried out in the Faculty of Agriculture, Tohoku University. are grateful to Dr. T. Mae of this University for the usage of labeled compound. References Arnon, D. I. (949) Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: -5. Manabe, H. and K. Ohira (980a) Fate of D- and L-alanine fed to rice leaf blades. SoilSci. Plant Nutr. 26: Manabe, H. and K. Ohira (980b) Metabolism of D-[I- 4 C]alanine fed to rice leaf blades. Soil Sci. Plant Nutr. 26: Manabe, H., M. Yamauchi and K. Ohira (98) Studies on D-amino acids in rice plants: Behaviors of D-alanylglycine in rice seedlings. Plant & Cell Physiol. 22: Ogawa, T., M. Fukuda and K. Sasaoka (973) Occurrence of A^-malonyl-D-alanine in pea seedlings. Biochim. Biophys. Acta 297: Ogawa, T. and K. Sasaoka (976) D-Amino acid in nature. Kagaku to Seibutsu 4: 6-66 (in Japanese). Rosa, N. and A. C. Neish (968) Formation and occurrence of N-malonylphenylalanine and related compounds in plants. Can. J. Biochem. 46: Yamauchi, M., T. Ohashi and K. Ohira (979) Occurrence of D-alanylglycine in rice leaf blades. Plant & Cell Physiol. 20: Zenk, M. H. and H. Scherf (963) D-Tryptophan in hoheren Pflanzen. Biochim. Biophys. Acta 7: We Downloaded from at Penn State University (Paterno Lib) on March 5, 206 (Received March 9, 983; Accepted June 29, 983)

Abstract. Keywords: 2-Acetyl-1-pyrroline, Nutrient Elements, Soilless Conditions, KDML 105. Introduction

Abstract. Keywords: 2-Acetyl-1-pyrroline, Nutrient Elements, Soilless Conditions, KDML 105. Introduction Influence of single nutrient element on 2-Acetyl-1-pyrroline contents in Thai fragrant rice (Oryza sativa L.) cv. Khao Dawk Mali 105 grown under soilless conditions Sakon Monggoot 1, Phumon Sookwong 1,