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Generation of poly-β-hydroxybutyrate from externally provided acetate in rice root
Plant Physiology and Biochemistry 50 (2012) 35e43
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Research article
Generation of poly-b-hydroxybutyrate from externally provided acetate in rice root Hirohisa Tsuda* Laboratory of Plant Nutrition and Fertilizer, Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 August 2011 Accepted 26 September 2011 Available online 6 October 2011
During the investigation of the metabolism of 14C-acetate or 14C-succinate in rice seedlings, an unknown organic acid (X) with a high specific radioactivity was detected in 10,000 g 30 min precipitate-fraction of rice roots. The X was hardly extracted by 0.1 N-H2SO4 boiling, but was extracted by 0.5 N-KOH boiling. The X was co-chromatographed with several known organic acids, and the radioactive peak of the X matched b-hydroxybutyric acid (b-hydroxybutyrate). The radioactive X and b-hydroxybutyrate were then heated with concentrated H2SO4. The radioactivity and the titration value were completely converted to crotonic acid. Thus, it was concluded that the X was b-hydroxybutyrate, and the original form of this acid was presumed to be poly-b-hydroxybutyrate (PHB). Then rice root incubated with 2-14C-acetate was extracted with hot-ethanol, ethanol/ether, and hot-chloroform. Approximately 10% of the radioactivity absorbed was detected in the chloroform fraction. The chloroform fraction was co-precipitated with authentic PHB by the addition of acetone/ether, and almost all the radioactivity was co-precipitated with the PHB. The radioactive co-precipitate was then heated with 0.5 N-NaOH, and chromatographed. The radioactivity of b-hydroxybutyrate plus crotonic acid almost matched that of the co-precipitate before alkaline-hydrolysis. Hence the radioactive co-precipitate was confirmed to be PHB. In wheat and radish seedlings, 2-14C-acetate was also assimilated into PHB. It is concluded that externally provided acetate was rapidly converted to PHB in higher plants. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Acetate Alkaline-hydrolysis b-Hydroxybutyrate Metabolism PHB Rice
1. Introduction Compartmentation of organic acids of the TCA cycle in plant roots had been investigated by many researchers supplying radioactive acetate (14C-acetate) exogenously [1e4]. We also intended to investigate the existence of physically sequestered organic acidpools in rice roots. Before starting this experiment, establishment of the extraction method of organic acids in rice root was necessary, because a satisfactory method for extraction of total organic acids from plant tissue was yet to be described. Pucher et al. [5] had used a direct ether Soxhlet extraction method of dried plant tissues acidified with 4 N-H2SO4. Maclennan et al. [1] adopted ethanol extraction for organic acids from corn roots, whereas, Jacobson and Ordin [6] described the following: “acetate was not present in the free form in the barley seedling roots. The free acetate was present only in small amounts. The “bound” acetate was insoluble since it remains in the residue after extraction of ground root material with
* Present address: 1-13-63, Tatsuda, Kumamoto City, Kumamoto 861-8006, Japan. Tel./fax: þ81 96 339 8706. E-mail address: [email protected]. 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.09.019
water. It is, however readily hydrolyzed by treatment with 0.1 N-H2SO4 on the steam bath for several hours.” According to the description of Jacobson and Ordin [6], the rice root material was first extracted with distilled water and then 0.1 N-H2SO4 on a water bath at 90e100 C for 3 h, respectively. Indeed, only small amounts of organic acids, especially acetate, were extracted by the hot-water extraction, but a larger amount of acetate was extracted by the ensuing 0.1 N-H2SO4 treatment of the residue. In addition to the 0.1 N-H2SO4 extraction, this paper describes treatment of the residue of 0.1 N-H2SO4 extraction with 0.5 N-KOH (or NaOH), revealing that still large amounts of organic acids including acetate were extracted. Thus, the 2 step extraction (0.1 N-H2SO4 and then 0.5 N-KOH (or NaOH)) method was adopted for further organic acids extraction. For the first purpose (compartmentation of organic acids), rice seedlings were cultured with 14C-labeled acetate or succinate for 6 h, the roots were then ground and fractionated into 300 g 4 min precipitate (ppt), 10,000 g 30 min ppt, and supernatant. These 3 subcellular fractions were then extracted with the above 2 step method and analyzed for organic acids on silica gel chromatography. In the 10,000 g 30 min ppt-fraction of the 2,3-14C-succinate absorption group, an unknown organic acid (X) which had a surprisingly higher specific activity, was detected
36
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
between fumarate and succinate on the silica gel column chromatography (similar tendency was seen also in the 2-14C-acetate absorption group). The X was hardly extracted by 0.1 N-H2SO4, but easily extracted by the second alkaline extraction. This paper deals with the identification and quantification of this unknown X and its original form, and the occurrence of this X in other plants (wheat and radish). 2. Results 2.1. Pre-examination for extraction methods of organic acids from rice roots Table 1 shows the results of a stepwise extraction of acetate and some organic acids from 1 g of dried rice roots. Unknown mix means the mixture of unknown acids which flowed out before the acetate fraction on the silica gel chromatography. Acetate was extracted only a little by hot-water, but much was extracted with hot 0.1 N-H2SO4, confirming the description of Jacobson and Ordin [6]. Pucher et al.’s method (direct ether Soxhlet extraction of sample mixed with 4 N-H2SO4) [5], and 1 N-H2SO4 extraction were also compared and almost similar extraction patterns to hot0.1 N-H2SO4 extraction were observed (data not shown). Table 1 also shows that still larger amount of organic acids can be extracted by alkaline treatment of the residue of 0.1 N-H2SO4 extraction. Thus the 2 step extraction, 0.1 N-H2SO4 and 0.5 N-NaOH (or KOH), was adopted for the following organic acid extraction from plant materials. 2.2. Distribution of radioactivity in organic acids of three subcellular fractions Table 2 shows the radioactivity (cpm) and titration value (ml) of main organic acid fractions including unknown mix and unknown X in three subcellular fractions (300 g ppt, 10,000 g ppt, and supernatant) after 6 h-absorption of 2-14C-acetate or 2,3-14C-succinate in rice roots. Two-step extraction (0.1 N-H2SO4 and 0.5 N-KOH) was adopted for 300 g ppt and 10,000 g ppt. It was predicted that the majority of free organic acids was in the supernatant (sup). Thus the sup fraction was first extracted with ether liquid-Soxhlet apparatus at pH2 for only free organic acids, and then the residue was heated with 0.1 N-H2SO4 and 0.5 N-KOH. As shown in Table 2, most of radioactivity from 2-14C-acetate and/or 2,3-14C-succinate was distributed to known organic acids of the TCA cycle in the supernatant fractions. These acids in sup fractions were not extracted completely by pH2-Soxhlet extraction, but the remaining half was extracted by additional 0.1 N-H2SO4 and 0.5 N-KOH treatment. In the particle fractions (300 g ppt and 10,000 g ppt), only a small amount of the radioactivity was detected in the 0.1 N-H2SO4-extraction groups. However, in 0.5 N-KOH extraction of 10,000 g fraction groups (both from
Table 1 Extraction of organic acids from dried rice root.a Organic acids Extractionb
Unknown
Acetate
Fumarate (formate)
Succinate
Oxalate (glycolate)
Hot-water 0.1 N-H2SO4 0.5 N-KOH
0.92 1.03 8.49
1.54 36.2 72.6
1.12 4.03 39.7
1.14 0.55 17.1
2.36 0.39 11.2
a Contents of organic acids were expressed as titration value (ml) by 0.01 N-NaOH. b Freeze-dried rice root (1 g) was extracted with hot-water, the residue was extracted with 0.1 N-H2SO4, and then the residue was extracted with 0.5 N-KOH, successively.
2-14C-acetate- and 2,3-14C-succinate-absorption), extremely high radioactivities were detected in the unknown mix and unknown X fraction. The unknown mix was a mixture of several acids which eluted earlier than acetate on the silica gel chromatography. The unknown X detected between fumarate and succinate seemed to be a single peak (acid) and the amount as an acid (titration value) was very small. Thus it was speculated that the X might be important in organic acid metabolism, and so compartmentation experimentation of organic acids ceased, and effort was given to the identification of this X. 2.3. Co-chromatography of the X with authentic organic acids The unknown X which had a high radioactivity was flushed out with distilled water from the stainless planchettes (see Materials and methods). The aliquot was mixed with several authentic organic acids and was examined by silica gel chromatography to search an organic acid whose peak of titration value (by 0.01 N-NaOH) matches the peak of 14C-count. The followings were contained in the authentic acids tested: propionic acid, n-butyric acid, acetoacetic acid, a-ketoglutaric acid, glyoxalic acid, formic acid, fumaric acid, lactic acid, succinic acid, b-hydroxybutyric acid. As shown in Fig. 1(1.1), the radioactive peak of the unknown X matched the peak of titration value of b-hydroxybutyric acid (b-hydroxybutyrate). 2.4. Conversion of b-hydroxybutyrate to crotonic acid It is known that b-hydroxybutyrate is dehydrated with concentrated (conc.) H2SO4 and is converted to crotonic acid [7]. This reaction was adopted to confirm that the unknown X was b-hydroxybutyrate. An aliquot of radioactive X was mixed with known amount of b-hydroxybutyrate and heated in conc. H2SO4. The reaction product was separated by silica gel chromatography. Fig. 1(1.2) shows that the peak of titration value was moved from b-hydroxybutyrate to crotonic acid, and the peak of radioactivity completely coincided with that of crotonic acid. In this experiment, the recovery rate of crotonic acid from b-hydroxybutyrate was 51.4%, and that of radioactivity was 39.8%. Thus, it seemed that almost all the radioactivity in the b-hydroxybutyrate fraction converted to the crotonic acid fraction. From the above experiments, it was concluded that the unknown radioactive X was b-hydroxybutyrate. 2.5. Elimination of the possibility of bacterial contamination The unknown X (b-hydroxybutyrate) was hardly extracted with hot 0.1 N-H2SO4 but easily extracted (hydrolyzed) with hot alkaline solution (0.5 N-KOH) from rice roots. Thus the original form of this acid was presumed to be poly-b-hydroxybutyrate (PHB) in the literature [8,9]. To eliminate the possibility of bacterial contamination,100 mg/ml of chloramphenicol, which had a broad anti-bacterial spectrum, was added during isotope-uptake in the next experiment. In the first isotope-absorption (cell fractionation) experiment, 1 mM of carrier organic acid (Na-acetate or 2Na-succinate) was added. It seemed that these high concentrations of organic acids might have perturbed the natural metabolism and PHB-accumulation might be the result of abnormal metabolism in rice roots. Thus, in the next experiment, only radioactive acetate (2-14C-acetate with 0.0165 mM carrier as commercially purchased) was added in the absorption medium and the absorption time was 1hr instead of 6 h. For each experimental group (with or without chloramphenicol), 300 pieces of rice seedling were used. Organic acid analysis was performed for whole rice roots without cell fractionation. NaOH was used for alkaline-hydrolysis instead of KOH. The results are summarized in Table 3. The majority of
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43 Table 2 Distribution of radio activity to major organic acids of each subcellular fractions after absorption of a
b 14
Organic acids (upper ; titration value, lower ;
14
C-acetate and
37
14
C-succinate for 6 h.
C-count)
Group
Fraction & extraction
Unknown
Acetate
Fumarate (formate)
X
Succinate
Malate
Citrate
Acetate absorption group
300 g ppt 0.1 N-H2SO4
0.6 1240
1.9 3030
0.82 518
0.28 292
0.46 322
0.36 973
0.36 993
300 g ppt 0.5 N-KOH
1.48 6860
ADc 1320
5.44 491
0.86 832
1.76 135
0.28 163
0.12 180
10,000 g ppt 0.1 N-H2SO4
0.28 763
0.7 1150
0.48 238
0.18 535
0.41 133
0.8 205
0.17 882
10,000 g ppt 0.5 N-KOH
0.63 39,200
ADc 1350
1.71 2590
0.36 6390
0.61 947
0.32 212
0.19 176
Sup pH2
1.2 13,300
1.06 5280
1.28 14,600
1.12 16,600
0.88 9760
1.08 29,600
0.58 17,500
Sup H2SO4 & KOH
2.22 1990
7.32 16,100
7.4 4760
0.58 2340
26.38 21,600
1.08 16,700
0.8 14,200
300 g ppt 0.1 N-H2SO4
0.42 397
ADc 533
1.22 233
0.34 479
0.38 63
trd trd
trd trd
300 g ppt 0.5 N-KOH
0.94 4490
9.76 302
2.06 180
0.24 3090
0.96 247
0.6 151
trd trd
10,000 g ppt 0.1 N-H2SO4
0.3 1030
0.72 2020
0.58 629
0.14 2110
0.21 142
0.37 480
0.26 270
10,000 g ppt 0.5 N-KOH
1.05 90,500
1.09 1810
1.9 2300
0.66 93,400
0.81 1610
0.49 228
0.35 185
Sup pH2
1.1 7260
5.62 3450
1.14 5920
0.24 13,100
1.3 14,200
3.1 32,900
1.38 4270
Sup H2SO4 & KOH
1.56 11,800
7.26 14,300
5.2 10,800
0.56 5100
22.06 34,600
1.92 9510
1.26 3850
Succinate absorption group
a b c d
Upper; upper column; titration value (ml). Lower; lower column; 14C-count (cpm). AD; abnormal data. tr; trace.
organic acids in the TCA cycle were extracted by 0.1 N-H2SO4. In 0.5 N-NaOH extract of 0.1 N-H2SO4-extraction residue, high radioactivities were observed in the unknown mix fractions and b-hydroxybutyrate fractions without great difference between the chloramphenicol-added group and the chloramphenicol-free group. Thus, it was concluded that b-hydroxybutyrate extracted with 0.5 N-KOH (or 0.5 N-NaOH) was rice root origin and was not bacterial contamination. It seemed that the high radioactivity in the unknown mix detected after the 0.5 N-NaOH treatment was probably crotonic acid, because it had been shown that alkaline-hydrolysis of PHB produced a mixture of b-hydroxybutyrate and crotonic acid [9]. Thus in the next step, direct-extraction of PHB was adopted for plant materials incubated with 14C-acetate. 2.6. Generation of PHB from 2-14C-acetate in various plant materials Table 4 shows the incorporation of 14C into PHB fraction, ethanol extract-fraction and ethanol/ether fraction in various plant materials incubated with 2-14C-acetate for 1 h. The conditions of incubation with 2-14C-acetate and analyzed part (a.p.) of the plant materials were as following (see also Table 4): 1) incubation; intact rice seedlings (100 pieces) in the light, a.p.; roots 2) incubation; intact rice seedling (100 pieces) in the dark, a.p.; roots, 3) incubation; rice roots (100 pieces) in the dark, a.p.; roots, 4) incubation;
rice leaves (100 pieces) in the light, a.p.; leaves, 5) incubation; intact wheat seedlings (50 pieces) in the light, a.p.; roots, 6) incubation; intact radish seedlings (50 pieces) in the light, a.p.; roots. The details of the data are shown in Table 4. The amounts of 14C incorporated by plant materials were calculated from differences in isotope counts in incubation medium before- and after-absorption. The count was expressed as dpm. Isotope incorporation to each fraction was expressed as dpm and/or percentage of isotope count absorbed by plant materials. The hot-ethanol fraction presumably contains almost all of the free organic acids, a small part of amino acids, chlorophyll, ethanol-soluble proteins. The ether/ethanol fraction probably contains lipids. It was expected that the main component of the chloroform fraction was PHB. Indeed, as shown in the Table 4, almost all (88.6e105.8%) of the 14C-counts in the chloroform fractions were co-precipitated with authentic PHB. Furthermore, as shown in the next section, when the radioactive co-precipitates were hydrolyzed with 0.5 N-NaOH, the radioactivity of the precipitate was quantitatively retrieved as the radioactivity of crotonic acid plus b-hydroxybutyrate. Thus, it was clearly shown that almost all the radioactivity in the chloroform fraction was that of PHB generated from 14C-acetate. Other points of this experimental result are described below. (1) Comparison between light- and dark-culture; distributions of radioactivity to ethanol fraction, ether/ethanol fraction, chloroform fraction and PHB precipitate were 14.6%, 0.453%, 9.15%, and
38
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
Fig. 1. Silica gel chromatography patterns. (1.1) Agreement of the peaks: the radioactivity of the X and the titration value of b-hydroxybutyric acid. (1.2) Conversion of the radioactive X and b-hydroxybutyric acid to crotonic acid. In this experiment, the recovery rate of crotonic acid from b-hydroxybutyric acid was 51.4% and the recovery rate of radioactivity was 39.8%. The effluent was collected every 2 ml. (1.3) Silica gel chromatography of alkaline-hydrolysate of PHB-coprecipitate. A; chromatography from rice sample, B; chromatography from wheat sample.
8.11%, respectively in the roots of intact rice seedlings cultured with 14 C-acetate in the light; whereas, in the dark culture, the distributions were 13.1%, 0.705%, 8.33%, and 8.25%, respectively. Thus apparent differences in the distribution of absorbed 14C-acetate to each fraction, especially to PHB precipitate, were not evident between light- and dark-culture. (2) Radioactivity in rice roots; 14 C-acetate absorption by excised rice roots was almost half (22.4%) of the absorption by intact rice (42.9%). The relative assimilations of 14 C-acetate to ethanol fraction, ether/ethanol fraction, chloroform fraction and PHB precipitate were 27.0%, 1.41%, 14.4% and 13.2%, respectively. These values were almost double those of intact rice. (3) Radioactivity in rice leaves; 14C-acetate absorption by excised rice leaves was very low (7.0%) compared to the intact rice seedlings. The isotope distribution to PHB precipitate was 9.53%, i.e. 14 C-acetate absorbed by excised rice leaves was assimilated to PHB as well as in the case of rice roots. This result is also an important supporting evidence that PHB is generated in rice, because it is difficult to consider that there would be significant bacterial growth on the leaf portion of the rice. (4) Radioactivity in wheat; 14 C-acetate absorption by intact wheat seedlings was repeated twice. The assimilation rates to PHB from 14C-acetate were 12.3% and 15.9%, respectively and these values were higher than the assimilation to ethanol fraction (10.6%, and 9.2%). (5) Radioactivity in radish; although the assimilation rate was lower (1.77%) compared to rice or wheat, it was demonstrated that 14C-acetate was assimilated to PHB also in a dicotyledon plant, namely radish. 2.7. Confirming that the radioactivity in the co-precipitate was PHB origin
Fig. 1(1.3A and B), respectively. Fig. 1(1.3A and B) shows that the radioactivity after hydrolysis of PHB-precipitates by 0.5 N-NaOH appeared as two peaks of crotonic acid and b-hydroxybutyric acid on silica gel chromatography. Table 5 shows the 14C-counts of crotonic acid fraction, b-hydroxybutyrate fraction and PHB precipitate before alkaline-hydrolysis. The recovery rates of isotope counts as sum of crotonic acid and b-hydroxybutyrate were 83.9% and 91.3% of PHB precipitates before hydrolysis in rice and in wheat, respectively. Almost all the isotope count of the PHB precipitate was converted to crotonic acid and b-hydroxybutyrate. These results clearly show that the radioactivity co-precipitated with authentic PHB has come from PHB generated from 14C-acetate, and almost all radioactivity in the above chloroform fraction is from PHB as well. 2.8. Time course study The rice seedlings incubated with 14C-acetate for 1 h were transferred to isotope-free medium, and 0, 1, 3, and 6 h after the transfer, roots and leaves were analyzed for radioactivity after separation for ethanol fraction, ether/ethanol fraction, chloroform fraction, and PHB precipitate. The results are shown in Table 6. In the roots, the peak of radioactivity in the ethanol fraction was at 0 h, and was reduced by half at 3 h. In the chloroform fraction (and PHB precipitate), the peak was at 0 h, but the reduction rate was slower compared to the ethanol fraction. In the ether/ethanol fraction, the peak was at 3 h. In the leaves, the peaks of isotope count were 3 h, 3 h, and 1 h in the ethanol fraction, the ether/ethanol fraction and the chloroform fraction, respectively. 2.9. Infrared spectrum of PHB from rice roots and wheat roots
Aliquots of co-precipitates from the rice root and the wheat root were hydrolyzed by 0.5 N-NaOH and separated by silica gel chromatography. Results in rice and wheat are shown in
Infrared spectrum of PHB fraction (Fig. 2B) obtained from rice root incubated with Na-acetate was identical to that of authentic
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43 Table 3 Organic acid contents in rice roots after 1 h-2-14C-acetate absorption with or without chloramphenicol. (Upper column; titration value (ml), lower column; 14 C-count (cpm)). Organic acids
Unknown mix Acetic acid Pyruvic acid Fumaric acid Formic acid
b-Hydroxybutyric acid Succinic acid Aconitic acid Oxalic acid Glycolic acid Malic acid Citric acid Isocitric acid Total cpm
With chloramphenicola 0.5 N-NaOH extb
0.1 N-H2SO4 ext
0.5 N-NaOH ext
0.99 7720 7.82 64,900 0.08 520 0.25 3390 1.49 2800 0.13 4780 0.65 26,900 1.01 139,000 1.44 5560 1.18 3280 2.66 57,700 7.35 68,100 1.27 1090 385,740
7.87 38,500 20.67 27,300 0.27 430 3.15 610 12.01 2430 0.49 49,500 6.43 2920 1.96 10,100 3.4 1000 1.32 650 2.39 4180 2.96 5000 1.32 240 142,860
0.86 8810 8.27 33,100 0.15 1050 0.34 2440 1.55 2900 0.13 17,000 0.65 26,600 1.28 93,900 1.22 4360 1.04 2610 2.71 62,800 6.79 58,600 1.98 930 315,100
7.86 50,100 21.28 22,800 0.27 260 2.38 1710 15.37 1980 0.59 63,100 7.31 1760 2.18 6280 3.71 1000 1.3 530 2.74 3870 2.9 3240 1.38 160 156,790
Origin
Sample
Count
Rice
PHB-coprecipitate (before hydrolysis) a. Crotonic acid fraction b. b-hydroxybutyric acid fraction aþb PHB-coprecipitate (before hydrolysis) a. Crotonic acid fraction b. b-hydroxybutyric acid fraction aþb
3.85 1.52 1.71 3.23 9.51 4.02 4.66 8.68
Wheat
C-count was expressed as cpm. C-count was expressed as dpm. c As shown in Fig. 1(1.3A and B), hydrolysate of PHB-coprecipitate forms two 14C peaks of crotonic acid and b-hydroxybutyric acid.
PHB obtained from Bacillus megaterium (Fig. 2A). The spectrum of PHB fraction from wheat root (Fig. 2C) was also identical to that of B. megaterium. 3. Discussion The purpose of this communication is to describe evidence that externally provided Na-2-14C-acetate was assimilated to poly-bhydroxybutyrate (PHB) at a very high ratio in plant roots. The evidence will be discussed as follows. 3.1. Identification of PHB In the course of the investigation of the metabolism of C-acetate or 14C-succinate in rice roots, an unknown organic acid
was detected between fumarate and succinate on silica gel column chromatography. This organic acid was not extracted by conventional 0.1 N-H2SO4 boiling, but was first extracted by an incidental hot-0.5 N-KOH boiling. This radioactive acid was co-chromatographed with several authentic organic acids and the radioactive peak matched the peak of titration value of b-hydroxybutyrate. The radioactive acid and authentic b-hydroxybutyrate were then heated in conc. H2SO4 which resulted in the conversion of the radioactivity and the titration value to crotonic acid. Therefore, it was concluded that the unknown radioactive acid was b-hydroxybutyrate. The original form of this acid was presumed to be poly-bhydroxybutyrate (PHB), because the acid was not extracted by dilute acid but extracted by alkaline-hydrolysis. However, the possibility that the PHB found in rice root in this experiment might be from bacteria, was considered. This possibility, however, could be excluded by the addition of chloramphenicol, which had a broad anti-bacterial spectrum, in the culture media. There was little difference in 14C-counts of b-hydroxybutyrate generated from 14 C-acetate between the chloramphenicol-added group and the chloramphenicol-free group. As the next step, direct-extraction of PHB from plant materials which incorporated 14C-acetate was conducted. A simplified method was used which was a modification of Lemoigene’s method [10]: 1) hot-ethanol extraction (twice), 2) ethanol/ether (3/1, v/v) extraction (twice), and hotchloroform extraction (3 times). The hot-chloroform fraction was then co-precipitated with authentic PHB by the addition of 5 volume of acetone/ether (3/1, v/v) in a refrigerator, and almost all the radioactivity was co-precipitated with authentic PHB. The radioactive co-precipitate was then heated with 0.5 N-NaOH, and chromatographed. The radioactivity of b-hydroxybutyrate plus crotonic acid almost matched that of the co-precipitate before alkaline-hydrolysis. Hence the radioactive substance in the precipitate was confirmed to be PHB.
Table 4 Assimilation of 2-14C-acetate into ethanol, ether/ethanol, chloroform and PHB fraction after 1 h, absorption in various plant materials; dpm (% of total Part anal.b
14
Rice (L) Rice (D) Rice root (D) Rice leaf (L) Wheat 1 (L) Wheat 2 (L) Radish (L)
Root Root Root Leaf Root Root Root
1.05 1.26 0.547 0.172 1.27 1.37 0.409
a b c d e
C absorbed (%c)
108 108 108 108 108 108 108
(42.9) (51.6) (22.4) (7.02) (48.5) (52.7) (18.1)
EtOH fr.d 1.53 1.65 1.47 2.84 1.34 1.26 1.04
100.0 39.5 44.4 83.9 100.0 42.3 49.0 91.3
a 14
Chloramphenicol was added to the culture medium at a final concentration of 100 mg/ml from 1 h before 14C-acetate absorption to the end of the absorption. b After the 14C-acetate absorption, root part of the rice seedlings was extracted with 0.1 N-H2SO4 for 3 h, and then the residue was extracted with 0.5 N-NaOH for 2 h.
Plants (L or D)a
% 104 cpma 104 104 104 104 dpmb 104 104 104
b 14
a
14
Table 5 14 C-counts of PHB-coprecipitates and their hydrolysates.c
Without chloramphenicol
0.1 N-H2SO4 extb
39
107 107 107 106 107 107 107
EtOH/Ether fr.d (14.6) (13.1) (26.9) (16.5) (10.6) (9.20) (25.4)
4.75 8.89 7.72 2.42 6.45 5.33 1.56
Plant materials used for 14C-acetate absorption. L; absorbed in the light. D; absorbed in the dark. Plant part analyzed. Percentage of 14C-acetate incorporated in the plant materials. fr; fraction. 14 C-count co-precipitated with the authentic PHB from B. megaterium.
105 105 105 105 105 105 105
(0.452) (0.706) (1.41) (1.41) (0.51) (0.388) (0.381)
Chloroform fr.d 9.61 1.05 7.86 1.55 1.57 2.30 7.91
106 107 106 106 107 107 105
(9.15) (8.33) (14.4) (9.01) (12.4) (16.8) (1.93)
14
C absorbed).
PHB precipitatee 8.52 1.04 7.20 1.64 1.56 2.18 7.23
106 107 106 106 107 107 105
(8.11) (8.25) (13.2) (9.53) (12.3) (15.9) (1.77)
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H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
Table 6 Time course study of assimilation of 14C-acetate to ethanol fraction, ethanol/ether fraction, chloroform fraction and PHB precipitate.a dpm at hours after transferring to isotope-free medium Extraction (part analyzed)
0h
EtOH (root) EtOH (leaf) EtOH/Ether (root) EtOH/Ether (leaf) Chloroform (root) Chloroform (leaf) PHB precipitate (root) PHB precipitate (leaf)
1.53 1.78 4.75 4.61 9.61 2.62 8.52 NDb
1h
107 106 105 104 106 105 106
1.16 2.83 5.53 8.43 9.20 3.06 8.46 ND
3h
107 106 105 104 106 105 106
0.91 3.86 8.89 1.38 8.21 2.29 7.18 ND
6h
107 106 105 105 106 105 106
0.64 3.36 2.74 1.33 7.70 1.59 7.13
107 106 105 105 106 105 106
ND
a The rice seedlings incubated with 14C-acetate for 1 h were transferred to isotope-free medium, and 0, 1, 3, and 6 h after transferring, roots and leaves were analyzed separately. 100 pieces were used for each group. b ND; not done.
The existence of PHB in rice root and wheat root was also confirmed by infrared absorption spectrum analysis. For this experiment, rice and wheat were cultured in 10 mM Na-acetate for 18 h before harvesting, because PHB contents in rice or wheat seemed to be a small amount in normal culture condition. Results clearly showed that the IR-absorption spectra of PHB fractions from rice root
and wheat root matched that of authentic PHB from B. megaterium, respectively. The spectra were also identical to the IR-spectra of PHB from Chlorogloea fritschii and Rhodopseudomonas spheroides presented by Carr [11]. Recently Vogel et al. [12] reported IR-absorption spectrum in a blend of PHB with PLA (poly-L-lactic acid), and nonoverlapping bands for bacterial PHB were identified at 1724, 1279, 1057, 980, 897, 826 and 516 cm1. As shown in Fig. 2, these 7 bands are all detectable in IR-spectra of PHB from rice and wheat. 3.2. Physiological role of PHB in plants Poly-b-hydroxybutyrate (PHB) was first discovered by Lemoigene in 1927 as a major component of B. megaterium. The same component was later found in many other bacteria including blue green algae (C. fritschii) [8,11,13e15]. There was a considerable literature on the properties and metabolism of PHB in bacteria until the 1960s, and accumulated evidence suggested that it functioned as an intracellular reserve of carbon and energy for many bacterial species [16]. This thinking seems to be unchanged to the present [17]. However, from the 1980s, PHB has attracted added attention as one of the bio-degradable plastics in the chemical industry [18], and transgenic plants in which bacterial PHB-synthesis-genes are introduced have been generated for PHB production [19,20], but even in a recent paper on acetate metabolism in Arabidopsis, PHB has not appeared as a member of acetate metabolism [21]. With knowledge of the existence of PHB in rice root, the present author recalled immediately a reductive photo-assimilation of acetate in purple bacteria [14,22]. Thus, the 14C-acetate-uptake experiment in rice was conducted in the dark and in the light. However, as shown in Table 4, no difference in PHB assimilation from 14C-acetate was observed between these conditions. Furthermore, the PHB was synthesized from 14C-acetate at a high rate by only the root of rice in the dark. PHB seems to be synthesized from acetate even in the absence of light in rice. The very high specific radioactivity in b-hydroxybutyrate extracted by alkaline-hydrolysis (Tables 2 and 3) suggests two possibilities: 1) PHB pool might be very small in normal culture conditions of rice. 2) The acetate which was absorbed externally, might be converted to PHB without being diluted by the acetate pool in plant cells. The time course study (Table 6) showed that the decrease of assimilated PHB in the root was slower than that of ethanol-soluble fraction. However, from this experiment, it cannot be concluded that metabolism of PHB is slower than other free organic acids, because one cannot exclude the PHB synthesized from other organic acids via acetate. A pulse experiment (e.g. 1e10 min isotope incorporation and transferring to isotope-free medium) will be needed. In the first experiment by which synthesis of PHB from acetate or succinate was shown in rice, a fairly high concentration of acetate (1 mM) was added in the culture medium. Thus it was thought that the PHB-accumulation might be the result of abnormal metabolism of acetate. However, as shown in Table 4, even in the case of lower concentration of acetate (0.02 mM), PHB was synthesized at a high ratio in rice, wheat, and also in radish although to a lesser extent. This suggests that PHB might be of considerable biological interest, for example, it might function as a hub-compound in organic acid metabolism more than merely as a carbon reserve in higher plants. A general scheme of acetate metabolism in plant cells is shown in Fig. 3 in which the biosynthetic pathway from acetate to PHB is added by the dotted line, although the intermediate portion is a black box at present. 3.3. Possible enzymes involved in PHB formation in plants
Fig. 2. Infrared spectra of PHBs from B. megaterium, rice root and wheat root. A; B. megaterium, B; rice root, C; wheat root.
In the bacterium Alcaligenes eutrophus, PHB is synthesized from acetyl CoA by the consecutive reaction of three
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
41
4. Materials and methods 4.1. Plant materials
Fig. 3. Acetate metabolism in plant cells. PGA; 3-phosphoglycerate, G-6-P; glucose-6phosphate, F-6-P; fructose-6-phosphate.
Seeds of rice (O. sativa L., cv. Koshihikari) and wheat (Triticum aestivum L, cv. Fujimi) were germinated on paper soaked with tap water for 2e3 days at room temperature. After germination, the seedlings were grown on a plastic net floating in tap water for 2 or 3 weeks under natural light in a green house at 25 C or in an environmentally controlled room (approximately 10,000 lux of white light, 16 h photoperiod (7:00e23:00) at 25 C). Nutrients (medium I for the rice and medium II for the wheat) were added 1 week after germination. The compositions were as following: Medium I (Kasugai’s medium): (NH4)2SO4; 1 mM, NaH2PO4; 0.5 mM, KCl; 1 mM, CaCl2; 1 mM, MgCl2; 1 mM, FeCl3$6H2O; 1 ppm, pH5.5. Medium II (Hoagland’s medium): KNO3; 2.5 mM, Ca(NO3)2; 2.5 mM, MgSO4; 1 mM, KH2PO4; 0.5 mM, FeCl3$6H2O; 1 ppm, pH5.5. Seeds of radish (Raphanus sativus var. sativus) were grown in sand culture supplied with medium II after germination. 4.2. Extraction of organic acid from rice root
enzymes: 3-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, which are encoded by the phbA, phbB, and phbC, respectively [23]. Of the three enzymes only 3-ketothiolase is found in higher plants in which it is involved in the synthesis of mevalonate [19,24]. However, there has been no information about the occurrence of the other two enzymes in higher plants. To find the rice genes whose sequences are similar to the phbB, and/or phbC, the author then performed homology search of NCBI database using BLASTP program. As a consequence, no rice gene which was similar to the phbC, was detected. Whereas, the phbB was found to show a high homology with a rice gene (gene name; Os12g0242700 (Oryza sativa Japonica group)) at amino acid sequence level. A putative protein encoded by this gene is b-keto acyl carrier protein reductase [EC.1.1.1.100], involved in fatty acid synthesis [25]. Although it is not clear yet whether this gene encodes a protein having a real acetoacetyl-CoA reductase activity, enzymes involved in PHB formation in plants may overlap in part with enzymes involved in fatty acids synthesis.
In the preliminary experiments, several methods were tested. 1) distilled water extraction on a water bath for 3 h at 90e100 C. 2) 0.1 N-H2SO4 extraction on a water bath for 3 h at 90e100 C [6]. 3) 1 N-H2SO4 extraction on a water bath for 3 h at 90e100 C. 4) 0.5 N-KOH extraction on a water bath for 2 h at 90e100 C. 5) stepwise extraction of 1), 2) and 4). 6) direct ether Soxhlet extraction of sample mixed with 4 N-H2SO4 [5]. In each extraction from 1) to 5), 1 g of freeze-dried rice root sample was immersed in 50 ml of extraction solution in a round-bottom flask (100 ml) with a Liebig condenser. In the latter experiments using radioactive tracer, the two-step extraction of 0.1 N-H2SO4 and 0.5 N-NaOH (or 0.5 N-KOH) was adopted, and fresh root materials were used unless otherwise stated. Each extracted-solution was then adjusted to pH1, and further extracted with ether liquid-Soxhlet extractor (50 ml water and 50 ml ethyl ether) for 1 week. Extracted organic acids were captured with 20 ml of 0.1 N-NaOH, neutralized with 0.1 N-H2SO4 using a phenol red indicator, dried up on a water bath at 80e90 C and transferred to the next procedure for organic acids separation.
3.4. Future issues
4.3. Separation of organic acids
The experimental results shown in this communication are only one issue in the study of PHB-physiology in higher plants. Future issues are outlined below. (1) Elucidation of PHB-synthesis and -depolimerization pathways in higher plants, and their participant enzymes (and genes). (2) localization of PHB in plant cells: In the first incorporation experiment of 14C-labeled organic acids, PHB was detected mainly in 10,000 g 30 min ppt-fraction in which mitochondria, plastid etc. are contained. However the data were from 6 h-incorporation and 1 mM Na-acetate or 2Na-succinate addition. PHB localization in higher plants under normal physiological conditions should be investigated. In many microorganisms [17] or higher plants introduced bacterial PHB-synthesizing genes [20], inclusion bodies which contain PHB have been observed. When a high concentration of acetate is provided externally, similar inclusion bodies may be formed also in plant cells. (3) Whether or not PHB is synthesized from acetate in other plants especially in Arabidopsis, a model experimental plant. (4) PHB synthesis from monomers other than acetate in plants (e.g. organic acids, glucose). (5) Physico-chemical properties of PHB from plant materials.
Silica gel column chromatography by Bulen et al. [26] was used for organic acids fractionation. Silica gel was prepared from Mallinckrodt’s silicic acid by removal of the fine particles through repeated suspension in distilled water and decantation until approximately one third of original material was removed. The remaining coarse fraction was dried in an oven at 100 C for 24 h, and stored in a closed container. A glass-wool plug is placed in the bottom of the chromatographic tube (12 mm 25 cm). Eight gram of the above silica gel was mixed with 5.5 ml of 0.5 N-H2SO4, was slurried in 60e70 ml chloroform and added to the chromatographic tube with the aid of a glass rod. This procedure gives a uniformly packed column of 14.5 cm long. The mixture of standard organic acids (Na-salts, 10e100 mg) or organic acid fraction from plant materials were dissolved in 0.5 ml of 0.5 N-H2SO4, and were mixed quickly and thoroughly with 1 g of silica gel. The resulting powder containing samples was adhered to absorbent cotton and transferred to the top of the column quantitatively. Development of this survey column proceeded by the addition of a series of n-butyl alcohol/chloroform (v/v) solvents: 100 ml of 5/95, 135 ml of 15/85, 100 ml of 25/75, 300 ml of 35/65, and 100 ml of 50/50. Each solvent
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H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
mixture was equilibrated against 0.5 N-surfuric acid by shaking the two phases in separatory funnel and passing the solvent layer through dry filter paper to remove suspended water droplets. The effluent was captured every 4 ml (or 2 ml) in a test tube using a fraction collector. One ml of distilled water was added to each individual fraction and was titrated by addition of standard 0.01 N-NaOH using a phenol red indicator. Before the examination of samples from plant materials, standard organic acids were tested by Soxhlet extraction and succeeding Bulen’s silica gel chromatographic analysis, and it was confirmed that these acids were quantitatively retrieved. However, peaks of fumaric acid/formic acid and oxalic acid/glycolic acid were overlapped and could not be separated clearly.
4.7. Conversion from b-hydroxybutyrate to crotonic acid Commercial b-hydroxybutyrate-Na (Kokusan Chem., Japan) was purified by silica gel chromatography. The b-hydroxybutyrate fractions titrated with 0.01 N-NaOH were collected (16.4 ml ¼ 0.164 mmol), mixed with the radioactive X fractions (5160 cpm) and evaporated on a water bath at 80e90 C. The mixture was heated with 10 ml of concentrated H2SO4 in a bottom-round flask (100 ml) with a Liebig condenser on a water bath at 100 C for 15 min. The solution was cooled by addition of appropriate amount of ice, then saturated with NaCl and shaken with ethyl ether in a separation funnel. The ether phase was collected and evaporated. The remains of evaporation were analyzed by the silica gel chromatography.
4.4. Uptake experiments of radioactive organic acids After 2 or 3 weeks of cultivation, the seedlings (rice or wheat) were harvested from the culture net, the hull was removed from each seedling, and washed thoroughly with tap water. The seedlings were transferred to a 100 or 200 ml beaker containing culture medium including 2-14C-acetate or 2,3-14C-succinate, and were cultivated under 10,000 lux white light in an environmentally controlled room or natural light in a green house at 25 C for hours indicated. The beaker was wrapped in aluminum foil so the roots were in the dark during absorption experiment. Contents and specific activity of the radioactive compounds are described for each experiment. After isotope-absorption, the seedlings were washed with tap water thoroughly, wiped with paper towel to remove excess water and used for ensuing analysis. 4.5. Cell fractionation The uptake conditions of labeling compounds by rice seedlings in this fractionation experiment, are as follows. To a 100 ml beaker containing100 ml of culture medium (medium I) including labeling compound, 250 pieces of 3 weeks old rice seedlings were transferred and cultured for 6 h in a green house at 25 C. Labeling compounds supplied to the rice seedlings: 2-14C-acetate-Na (specific activity; 740 MBq/mmol), 0.74 MBq/100 ml; 2,3-14C-succinate-2Na (specific activity; 389 MBq/mmol), 0.74 MBq/100 ml. As a carrier, 1 mM of nonlabeling Na-acetate or 2Na-succinate was added to the above culture medium, respectively. Thus the start concentration of acetate was 1.01 mM and that of succinate was 1.02 mM, respectively. For each experimental group, 3 beakers were used. After absorption of labeling compounds, cell fractionation was performed based on the description by Beevers [27]. The root part of the rice seedling (750 pieces) was ground down by a mortar in 100 ml of 1/20 M-phosphate buffer (pH6.9) containing 0.7 M mannitol. The resultant soup was passed through 4 files of gauze and centrifuged at 300 g for 4 min (300 g precipitate (ppt) fraction). The supernatant was then centrifuged at 10,000 g for 30 min (10,000 g ppt-fraction), and the remaining supernatant (sup fraction) was also collected. The each fraction was washed once by resuspension in the homogenizing buffer and subsequent centrifugation. Thus these 3 subcellular fractions were used for further analysis. 4.6. Measurement of radioactivity An aliquot of water phase from each silica gel chromatography fraction titrated (neutralized) by 0.01 N-NaOH was transferred to stainless tray (planchette), dried up with an infrared lamp, and count of 14C was measured by a gas flow counter. The radioactivities were expressed as cpm. Radioactivity of solvent-extraction fraction was measured by a liquid scintillation counter and expressed as cpm or dpm.
4.8. Extraction and separation of poly-b-hydroxybutyrate from plant materials Poly-b-hydroxybutyrate fraction (PHB) was extracted by the method for bacterial PHB [10,28] with some modifications. The uptake conditions of labeling compounds by plant materials in this experiment, were as follows. To a 200-ml beaker containing 100-ml of culture medium (medium I or II) including labeling compound, 3 weeks old rice seedlings (100 pieces) or wheat seedlings (50 pieces) were transferred and cultured for 1 h in an environmentally controlled room (approximately 10,000 lux of white light at 25 C). The beaker was wrapped by aluminum foil to keep the roots in the dark. The dark condition of whole plantbody was made by lights-out. Ten days old radish seedlings (50 pieces) were cultured in a 50-ml beaker containing 50-ml absorption solution in the above controlled room. Labeling compounds supplied to the plant materials: 2-14C-acetate-Na (specific activity; 740 MBq/mmol), 3.7 MBq/100 ml for rice and wheat, 3.7 MBq/50 ml for radish. Thus the start concentrations of acetate were 0.05 mM for rice and wheat, and 0.1 mM for radish. After isotope-uptake, plant materials were washed quickly with tap water and extracted successively twice with 50 ml hotethanol (ethanol fraction), twice with 50 ml ethanol/ether (v/v, 3/1) (ethanol/ether fraction), and finally three times with 50 ml hot-chloroform (chloroform fraction). The above fractions were filtrated with dry filter paper (Whatmann G3). To the chloroform fraction, approximately 10 mg of authentic PHB from B. megaterium (kindly supplied by Dr. N. Tsukagoshi; The University of Tokyo) was added to serve as a carrier and heated to dissolve. After reducing the volume of chloroform by evaporation, 5 volumes of acetone/ether (v/v; 3:1) were added and cooled in a refrigerator. PHB was deposited as a white precipitate. The precipitate was air-dried and weighed. An aliquot was used for isotope counting (an aliquot of solid PHB was first dissolved in Soluene TM (Packard L.T.D) and then dissolved in scintillation fluid). The procedure of this dissolving/precipitation was repeated three times, and the radioactivity of PHB per mg was reached a plateau. The quantity of PHB originally contained in each experimental group of plant materials was expected to be far less than 10 mg from the titration value of b-hydroxybutyrate (see Table 3). Thus PHB amount (14C-count) synthesized from 14 C-acetate in plant materials was calculated according to the following equation:
PHB added ðmgÞ PHB synthesized dpm ¼ count of A dpm A ðmgÞ A; aliquot of precipitate. PHB added; PHB from B. megaterium which was added to the chloroform fraction as a carrier.
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
4.9. Confirmation of PHB by alkaline-hydrolysis Another aliquot of the above radioactive precipitate (PHB) was heated with 10 ml of 0.5 N-NaOH in a bottom-round flask (100 ml) with a Liebig condenser on a water bath at 100 C for 60 min. After neutralization, the hydrolysate was separated by silica gel chromatography. The radioactivities (dpm or cpm) of crotonic acid fraction and b-hydroxybutyrate fraction were counted by liquid scintillation counter (Packard L.T.D) and compared with the count (dpm or cpm) of precipitate (PHB) before the alkaline-hydrolysis. 4.10. Infrared spectrum of PHB from rice root and wheat root Two weeks old seedlings of rice and wheat were cultured in each culture medium containing non-labeled Na-acetate at a final concentration of 10 mM for 18 h (PM6:00eAM12:00) in a green house at 25 C. The root part of these seedlings (rice; 100 g, wheat; 65 g) was washed thoroughly by running tap water and then successively extracted twice with 200 ml of hot-ethanol, twice with 200 ml of ethanol/ether(3/1; v/v), and three times with 200 ml of hotchloroform. The chloroform fraction was evaporated to a smaller volume to which approximately 5 volumes of acetone/ether (3/1; v/v) were added and cooled in a refrigerator to precipitate the PHB. The precipitate was collected by a filter paper and dried. Dissolving in chloroform and re-precipitation by acetone/ether was twice repeated. Approximately 1 mg of precipitate was obtained from rice root and wheat root, respectively. The white grayish dried precipitates were ground with KBr powder to make KBr-disk and were examined by Infrared spectroscopy (IRA-type2; Nihonbunko, Japan). As a control of PHB, PHB from B. megaterium was used. Note This paper was prepared based on the research carried out in the master course of The University of Tokyo, Department of Agricultural Chemistry [29]. Most of the contents of this paper were presented at the Japanese Society of Plant Physiologists [30]. Acknowledgments The author thanks Dr. Satoshi Mori (Emeritus Professor, The University of Tokyo) for critical reading of the manuscript. References [1] D.H. Maclennan, H. Beevers, J.L. Harley, Compartmentation of acids in plant tissues, Biochem. J. 89 (1963) 316e327. [2] S.H. Lips, H. Beevers, Compartmentation of organic acids in corn roots. I. Differential labeling of 2 malate pools, Plant Physiol. 41 (1966) 709e712. [3] S.H. Lips, H. Beevers, Compartmentation of organic acids in corn roots. II. The cytoplasmic pool of malic acid, Plant Physiol. 41 (1966) 713e717. [4] B.T. Steer, H. Beevers, Compartmentation of organic acids in corn roots. III. Utilization of exogenously supplied acids, Plant Physiol. 42 (1967) 1197e1201.
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[5] G.W. Pucher, A.J. Wakeman, H.B. Vickery, Organic acids in plant tissues modifications of analytical methods, Industr. Engng. Chem. (Anal., ed.) 13 (1941) 244e246. [6] L. Jacobson, L. Ordin, Organic acid metabolism and ion absorption in roots, Plant Physiol. 29 (1954) 70e75. [7] J.H. Law, R.A. Slepecky, Assay of poly-b-hydroxybutyric acid, J. Bacteriol. 82 (1961) 33e42. [8] W.G.C. Forsyth, A.C. Hayward, J.B. Roberts, Occurrence of poly-b-hydroxybutyric acid in aerobic gram-negative bacteria, Nature 182 (1958) 800e801. [9] G.A.F. Ritchie, E.A. Dawes, The non-involvement of acyl-carrier protein in poly-b-hydroxybutyric acid biosynthesis in Azotobacter beijerinckii, Biochem. J. 112 (1969) 803e805. [10] M. Lemoigne, Études sur l’autolyse microbienne origine de l’acide beoxybutyrique formé par autolyse, Ann. Inst. Pasteur 41 (1927) 148e165. [11] N.G. Carr, The occurrence of poly-b-hydroxybutyrate in the blue-green alga Chlorogloea fritschii, Biochim. Biophys. Acta 120 (1966) 308e310. [12] C. Vogel, E. Wessel, H.W. Siesler, FT-IR imaging spectroscopy of phase separation in blends of poly(3-hydroxybutyrate) with poly(L-lactic acid) and poly(3-caprolactone), Biomacromolecules 9 (2008) 523e527. [13] M. Doudoroff, R.Y. Stanier, Role of poly-b-hydroxybutyric acid in the assimilation of organic carbon by bacteria, Nature 183 (1959) 1440e1442. [14] R.Y. Stanier, M. Doudoroff, R. Kunisawa, R. Contopoulou, The role of organic substrates in bacterial photosynthesis, Proc. Natl. Acad. Sci. USA 45 (1959) 1246e1260. [15] H.G. Schlegel, G. Gottschalk, R.V. Bartha, Formation and utilization of poly-bhydroxybutyric acid by Knallgas bacteria (Hydrogenomonas), Nature 191 (1961) 463e465. [16] E.A. Dawes, D.W. Robbins, Some aspects of the endogenous metabolism of bacteria, Bacteriol. Rev. 28 (1964) 126e149. [17] D. Jendrossek, Polyhydroxyalkanoate granules are complex subcellular organelles (Carbonosomes), J. Bacteriol. 191 (2009) 3195e3202. [18] Y. Poirier, Green chemistry yields a better plastic, Nat. Biotechnol. 17 (1999) 960e961. [19] Y. Poirier, D.E. Dennis, K. Klomparens, C. Somerville, Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants, Science 256 (1992) 520e523. [20] C. Nawrath, Y. Poirier, C. Somerville, Targeting of polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation, Proc. Natl. Acad. Sci. USA 91 (1994) 12760e12764. [21] M. Lin, D.J. Oliver, The role of acetyl-coenzyme A synthetase in Arabidopsis, Plant Physiol. 147 (2008) 1822e1829. [22] H. Gaffron, Über den Stoffwechsel der schwefelfreien Purpurbakterien, Biochem. Z. 260 (1933) 1e17. [23] A. Steinbüchel, H.G. Schlegel, Physiology and molecular genetics of poly(bhydroxyalkanoic acid) synthesis in Alcaligenes eutrophus, Mol. Microbiol. 5 (1991) 535e542. [24] R. Croteau, T.M. Kutchan, N.G. Lewis, Natural products (secondary metabolites). in: B.B. Buchanan, W. Gruissem, R.L. Jones (Eds.), Biochemistry & Molecular Biology of Plants. American Society of Plant Physiologists, Maryland, 2000, pp. 1250e1318. [25] M. Fisher, J.T.M. Kroon, W. Martindale, A.R. Stuitje, A.R. Slabas, J.B. Rafferty, The X-ray structure of Brassica napus b-keto acyl carrier protein reductase and its implications for substrate binding and catalysis, Structure 8 (2000) 339e347. [26] W.A. Bulen, J.E. Varner, R.C. Burrell, Separation of organic acids from plant tissues, Anal. Chem. 24 (1952) 187e190. [27] H. Beevers, Respiratory Metabolism in Plants. Harper & Row/John Weatherhill, Inc, New York, Evanston & London/Tokyo, 1961, pp. 130e143. [28] N. Tsukagoshi, K. Arima, Poly-b-hydroxybutyrate, Kobunshi 16 (1967) 1185e1191 (in Japanese). [29] H. Tsuda, Generation of poly-b-hydroxybutyrate in rice root, The master thesis abstracts, Department of Agricultural Chemistry, The University of Tokyo, 1970, pp. 49e50 (in Japanese). [30] H. Tsuda, Generation of poly-b-hydroxybutyrate in rice root, in: Proceedings of Annual Meeting and Symposium, The Japanese Society of Plant Physiologists, vol. 11, 1970, p. 100 (in Japanese).
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Research article
Generation of poly-b-hydroxybutyrate from externally provided acetate in rice root Hirohisa Tsuda* Laboratory of Plant Nutrition and Fertilizer, Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 August 2011 Accepted 26 September 2011 Available online 6 October 2011
During the investigation of the metabolism of 14C-acetate or 14C-succinate in rice seedlings, an unknown organic acid (X) with a high specific radioactivity was detected in 10,000 g 30 min precipitate-fraction of rice roots. The X was hardly extracted by 0.1 N-H2SO4 boiling, but was extracted by 0.5 N-KOH boiling. The X was co-chromatographed with several known organic acids, and the radioactive peak of the X matched b-hydroxybutyric acid (b-hydroxybutyrate). The radioactive X and b-hydroxybutyrate were then heated with concentrated H2SO4. The radioactivity and the titration value were completely converted to crotonic acid. Thus, it was concluded that the X was b-hydroxybutyrate, and the original form of this acid was presumed to be poly-b-hydroxybutyrate (PHB). Then rice root incubated with 2-14C-acetate was extracted with hot-ethanol, ethanol/ether, and hot-chloroform. Approximately 10% of the radioactivity absorbed was detected in the chloroform fraction. The chloroform fraction was co-precipitated with authentic PHB by the addition of acetone/ether, and almost all the radioactivity was co-precipitated with the PHB. The radioactive co-precipitate was then heated with 0.5 N-NaOH, and chromatographed. The radioactivity of b-hydroxybutyrate plus crotonic acid almost matched that of the co-precipitate before alkaline-hydrolysis. Hence the radioactive co-precipitate was confirmed to be PHB. In wheat and radish seedlings, 2-14C-acetate was also assimilated into PHB. It is concluded that externally provided acetate was rapidly converted to PHB in higher plants. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Acetate Alkaline-hydrolysis b-Hydroxybutyrate Metabolism PHB Rice
1. Introduction Compartmentation of organic acids of the TCA cycle in plant roots had been investigated by many researchers supplying radioactive acetate (14C-acetate) exogenously [1e4]. We also intended to investigate the existence of physically sequestered organic acidpools in rice roots. Before starting this experiment, establishment of the extraction method of organic acids in rice root was necessary, because a satisfactory method for extraction of total organic acids from plant tissue was yet to be described. Pucher et al. [5] had used a direct ether Soxhlet extraction method of dried plant tissues acidified with 4 N-H2SO4. Maclennan et al. [1] adopted ethanol extraction for organic acids from corn roots, whereas, Jacobson and Ordin [6] described the following: “acetate was not present in the free form in the barley seedling roots. The free acetate was present only in small amounts. The “bound” acetate was insoluble since it remains in the residue after extraction of ground root material with
* Present address: 1-13-63, Tatsuda, Kumamoto City, Kumamoto 861-8006, Japan. Tel./fax: þ81 96 339 8706. E-mail address: [email protected]. 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.09.019
water. It is, however readily hydrolyzed by treatment with 0.1 N-H2SO4 on the steam bath for several hours.” According to the description of Jacobson and Ordin [6], the rice root material was first extracted with distilled water and then 0.1 N-H2SO4 on a water bath at 90e100 C for 3 h, respectively. Indeed, only small amounts of organic acids, especially acetate, were extracted by the hot-water extraction, but a larger amount of acetate was extracted by the ensuing 0.1 N-H2SO4 treatment of the residue. In addition to the 0.1 N-H2SO4 extraction, this paper describes treatment of the residue of 0.1 N-H2SO4 extraction with 0.5 N-KOH (or NaOH), revealing that still large amounts of organic acids including acetate were extracted. Thus, the 2 step extraction (0.1 N-H2SO4 and then 0.5 N-KOH (or NaOH)) method was adopted for further organic acids extraction. For the first purpose (compartmentation of organic acids), rice seedlings were cultured with 14C-labeled acetate or succinate for 6 h, the roots were then ground and fractionated into 300 g 4 min precipitate (ppt), 10,000 g 30 min ppt, and supernatant. These 3 subcellular fractions were then extracted with the above 2 step method and analyzed for organic acids on silica gel chromatography. In the 10,000 g 30 min ppt-fraction of the 2,3-14C-succinate absorption group, an unknown organic acid (X) which had a surprisingly higher specific activity, was detected
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H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
between fumarate and succinate on the silica gel column chromatography (similar tendency was seen also in the 2-14C-acetate absorption group). The X was hardly extracted by 0.1 N-H2SO4, but easily extracted by the second alkaline extraction. This paper deals with the identification and quantification of this unknown X and its original form, and the occurrence of this X in other plants (wheat and radish). 2. Results 2.1. Pre-examination for extraction methods of organic acids from rice roots Table 1 shows the results of a stepwise extraction of acetate and some organic acids from 1 g of dried rice roots. Unknown mix means the mixture of unknown acids which flowed out before the acetate fraction on the silica gel chromatography. Acetate was extracted only a little by hot-water, but much was extracted with hot 0.1 N-H2SO4, confirming the description of Jacobson and Ordin [6]. Pucher et al.’s method (direct ether Soxhlet extraction of sample mixed with 4 N-H2SO4) [5], and 1 N-H2SO4 extraction were also compared and almost similar extraction patterns to hot0.1 N-H2SO4 extraction were observed (data not shown). Table 1 also shows that still larger amount of organic acids can be extracted by alkaline treatment of the residue of 0.1 N-H2SO4 extraction. Thus the 2 step extraction, 0.1 N-H2SO4 and 0.5 N-NaOH (or KOH), was adopted for the following organic acid extraction from plant materials. 2.2. Distribution of radioactivity in organic acids of three subcellular fractions Table 2 shows the radioactivity (cpm) and titration value (ml) of main organic acid fractions including unknown mix and unknown X in three subcellular fractions (300 g ppt, 10,000 g ppt, and supernatant) after 6 h-absorption of 2-14C-acetate or 2,3-14C-succinate in rice roots. Two-step extraction (0.1 N-H2SO4 and 0.5 N-KOH) was adopted for 300 g ppt and 10,000 g ppt. It was predicted that the majority of free organic acids was in the supernatant (sup). Thus the sup fraction was first extracted with ether liquid-Soxhlet apparatus at pH2 for only free organic acids, and then the residue was heated with 0.1 N-H2SO4 and 0.5 N-KOH. As shown in Table 2, most of radioactivity from 2-14C-acetate and/or 2,3-14C-succinate was distributed to known organic acids of the TCA cycle in the supernatant fractions. These acids in sup fractions were not extracted completely by pH2-Soxhlet extraction, but the remaining half was extracted by additional 0.1 N-H2SO4 and 0.5 N-KOH treatment. In the particle fractions (300 g ppt and 10,000 g ppt), only a small amount of the radioactivity was detected in the 0.1 N-H2SO4-extraction groups. However, in 0.5 N-KOH extraction of 10,000 g fraction groups (both from
Table 1 Extraction of organic acids from dried rice root.a Organic acids Extractionb
Unknown
Acetate
Fumarate (formate)
Succinate
Oxalate (glycolate)
Hot-water 0.1 N-H2SO4 0.5 N-KOH
0.92 1.03 8.49
1.54 36.2 72.6
1.12 4.03 39.7
1.14 0.55 17.1
2.36 0.39 11.2
a Contents of organic acids were expressed as titration value (ml) by 0.01 N-NaOH. b Freeze-dried rice root (1 g) was extracted with hot-water, the residue was extracted with 0.1 N-H2SO4, and then the residue was extracted with 0.5 N-KOH, successively.
2-14C-acetate- and 2,3-14C-succinate-absorption), extremely high radioactivities were detected in the unknown mix and unknown X fraction. The unknown mix was a mixture of several acids which eluted earlier than acetate on the silica gel chromatography. The unknown X detected between fumarate and succinate seemed to be a single peak (acid) and the amount as an acid (titration value) was very small. Thus it was speculated that the X might be important in organic acid metabolism, and so compartmentation experimentation of organic acids ceased, and effort was given to the identification of this X. 2.3. Co-chromatography of the X with authentic organic acids The unknown X which had a high radioactivity was flushed out with distilled water from the stainless planchettes (see Materials and methods). The aliquot was mixed with several authentic organic acids and was examined by silica gel chromatography to search an organic acid whose peak of titration value (by 0.01 N-NaOH) matches the peak of 14C-count. The followings were contained in the authentic acids tested: propionic acid, n-butyric acid, acetoacetic acid, a-ketoglutaric acid, glyoxalic acid, formic acid, fumaric acid, lactic acid, succinic acid, b-hydroxybutyric acid. As shown in Fig. 1(1.1), the radioactive peak of the unknown X matched the peak of titration value of b-hydroxybutyric acid (b-hydroxybutyrate). 2.4. Conversion of b-hydroxybutyrate to crotonic acid It is known that b-hydroxybutyrate is dehydrated with concentrated (conc.) H2SO4 and is converted to crotonic acid [7]. This reaction was adopted to confirm that the unknown X was b-hydroxybutyrate. An aliquot of radioactive X was mixed with known amount of b-hydroxybutyrate and heated in conc. H2SO4. The reaction product was separated by silica gel chromatography. Fig. 1(1.2) shows that the peak of titration value was moved from b-hydroxybutyrate to crotonic acid, and the peak of radioactivity completely coincided with that of crotonic acid. In this experiment, the recovery rate of crotonic acid from b-hydroxybutyrate was 51.4%, and that of radioactivity was 39.8%. Thus, it seemed that almost all the radioactivity in the b-hydroxybutyrate fraction converted to the crotonic acid fraction. From the above experiments, it was concluded that the unknown radioactive X was b-hydroxybutyrate. 2.5. Elimination of the possibility of bacterial contamination The unknown X (b-hydroxybutyrate) was hardly extracted with hot 0.1 N-H2SO4 but easily extracted (hydrolyzed) with hot alkaline solution (0.5 N-KOH) from rice roots. Thus the original form of this acid was presumed to be poly-b-hydroxybutyrate (PHB) in the literature [8,9]. To eliminate the possibility of bacterial contamination,100 mg/ml of chloramphenicol, which had a broad anti-bacterial spectrum, was added during isotope-uptake in the next experiment. In the first isotope-absorption (cell fractionation) experiment, 1 mM of carrier organic acid (Na-acetate or 2Na-succinate) was added. It seemed that these high concentrations of organic acids might have perturbed the natural metabolism and PHB-accumulation might be the result of abnormal metabolism in rice roots. Thus, in the next experiment, only radioactive acetate (2-14C-acetate with 0.0165 mM carrier as commercially purchased) was added in the absorption medium and the absorption time was 1hr instead of 6 h. For each experimental group (with or without chloramphenicol), 300 pieces of rice seedling were used. Organic acid analysis was performed for whole rice roots without cell fractionation. NaOH was used for alkaline-hydrolysis instead of KOH. The results are summarized in Table 3. The majority of
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43 Table 2 Distribution of radio activity to major organic acids of each subcellular fractions after absorption of a
b 14
Organic acids (upper ; titration value, lower ;
14
C-acetate and
37
14
C-succinate for 6 h.
C-count)
Group
Fraction & extraction
Unknown
Acetate
Fumarate (formate)
X
Succinate
Malate
Citrate
Acetate absorption group
300 g ppt 0.1 N-H2SO4
0.6 1240
1.9 3030
0.82 518
0.28 292
0.46 322
0.36 973
0.36 993
300 g ppt 0.5 N-KOH
1.48 6860
ADc 1320
5.44 491
0.86 832
1.76 135
0.28 163
0.12 180
10,000 g ppt 0.1 N-H2SO4
0.28 763
0.7 1150
0.48 238
0.18 535
0.41 133
0.8 205
0.17 882
10,000 g ppt 0.5 N-KOH
0.63 39,200
ADc 1350
1.71 2590
0.36 6390
0.61 947
0.32 212
0.19 176
Sup pH2
1.2 13,300
1.06 5280
1.28 14,600
1.12 16,600
0.88 9760
1.08 29,600
0.58 17,500
Sup H2SO4 & KOH
2.22 1990
7.32 16,100
7.4 4760
0.58 2340
26.38 21,600
1.08 16,700
0.8 14,200
300 g ppt 0.1 N-H2SO4
0.42 397
ADc 533
1.22 233
0.34 479
0.38 63
trd trd
trd trd
300 g ppt 0.5 N-KOH
0.94 4490
9.76 302
2.06 180
0.24 3090
0.96 247
0.6 151
trd trd
10,000 g ppt 0.1 N-H2SO4
0.3 1030
0.72 2020
0.58 629
0.14 2110
0.21 142
0.37 480
0.26 270
10,000 g ppt 0.5 N-KOH
1.05 90,500
1.09 1810
1.9 2300
0.66 93,400
0.81 1610
0.49 228
0.35 185
Sup pH2
1.1 7260
5.62 3450
1.14 5920
0.24 13,100
1.3 14,200
3.1 32,900
1.38 4270
Sup H2SO4 & KOH
1.56 11,800
7.26 14,300
5.2 10,800
0.56 5100
22.06 34,600
1.92 9510
1.26 3850
Succinate absorption group
a b c d
Upper; upper column; titration value (ml). Lower; lower column; 14C-count (cpm). AD; abnormal data. tr; trace.
organic acids in the TCA cycle were extracted by 0.1 N-H2SO4. In 0.5 N-NaOH extract of 0.1 N-H2SO4-extraction residue, high radioactivities were observed in the unknown mix fractions and b-hydroxybutyrate fractions without great difference between the chloramphenicol-added group and the chloramphenicol-free group. Thus, it was concluded that b-hydroxybutyrate extracted with 0.5 N-KOH (or 0.5 N-NaOH) was rice root origin and was not bacterial contamination. It seemed that the high radioactivity in the unknown mix detected after the 0.5 N-NaOH treatment was probably crotonic acid, because it had been shown that alkaline-hydrolysis of PHB produced a mixture of b-hydroxybutyrate and crotonic acid [9]. Thus in the next step, direct-extraction of PHB was adopted for plant materials incubated with 14C-acetate. 2.6. Generation of PHB from 2-14C-acetate in various plant materials Table 4 shows the incorporation of 14C into PHB fraction, ethanol extract-fraction and ethanol/ether fraction in various plant materials incubated with 2-14C-acetate for 1 h. The conditions of incubation with 2-14C-acetate and analyzed part (a.p.) of the plant materials were as following (see also Table 4): 1) incubation; intact rice seedlings (100 pieces) in the light, a.p.; roots 2) incubation; intact rice seedling (100 pieces) in the dark, a.p.; roots, 3) incubation; rice roots (100 pieces) in the dark, a.p.; roots, 4) incubation;
rice leaves (100 pieces) in the light, a.p.; leaves, 5) incubation; intact wheat seedlings (50 pieces) in the light, a.p.; roots, 6) incubation; intact radish seedlings (50 pieces) in the light, a.p.; roots. The details of the data are shown in Table 4. The amounts of 14C incorporated by plant materials were calculated from differences in isotope counts in incubation medium before- and after-absorption. The count was expressed as dpm. Isotope incorporation to each fraction was expressed as dpm and/or percentage of isotope count absorbed by plant materials. The hot-ethanol fraction presumably contains almost all of the free organic acids, a small part of amino acids, chlorophyll, ethanol-soluble proteins. The ether/ethanol fraction probably contains lipids. It was expected that the main component of the chloroform fraction was PHB. Indeed, as shown in the Table 4, almost all (88.6e105.8%) of the 14C-counts in the chloroform fractions were co-precipitated with authentic PHB. Furthermore, as shown in the next section, when the radioactive co-precipitates were hydrolyzed with 0.5 N-NaOH, the radioactivity of the precipitate was quantitatively retrieved as the radioactivity of crotonic acid plus b-hydroxybutyrate. Thus, it was clearly shown that almost all the radioactivity in the chloroform fraction was that of PHB generated from 14C-acetate. Other points of this experimental result are described below. (1) Comparison between light- and dark-culture; distributions of radioactivity to ethanol fraction, ether/ethanol fraction, chloroform fraction and PHB precipitate were 14.6%, 0.453%, 9.15%, and
38
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
Fig. 1. Silica gel chromatography patterns. (1.1) Agreement of the peaks: the radioactivity of the X and the titration value of b-hydroxybutyric acid. (1.2) Conversion of the radioactive X and b-hydroxybutyric acid to crotonic acid. In this experiment, the recovery rate of crotonic acid from b-hydroxybutyric acid was 51.4% and the recovery rate of radioactivity was 39.8%. The effluent was collected every 2 ml. (1.3) Silica gel chromatography of alkaline-hydrolysate of PHB-coprecipitate. A; chromatography from rice sample, B; chromatography from wheat sample.
8.11%, respectively in the roots of intact rice seedlings cultured with 14 C-acetate in the light; whereas, in the dark culture, the distributions were 13.1%, 0.705%, 8.33%, and 8.25%, respectively. Thus apparent differences in the distribution of absorbed 14C-acetate to each fraction, especially to PHB precipitate, were not evident between light- and dark-culture. (2) Radioactivity in rice roots; 14 C-acetate absorption by excised rice roots was almost half (22.4%) of the absorption by intact rice (42.9%). The relative assimilations of 14 C-acetate to ethanol fraction, ether/ethanol fraction, chloroform fraction and PHB precipitate were 27.0%, 1.41%, 14.4% and 13.2%, respectively. These values were almost double those of intact rice. (3) Radioactivity in rice leaves; 14C-acetate absorption by excised rice leaves was very low (7.0%) compared to the intact rice seedlings. The isotope distribution to PHB precipitate was 9.53%, i.e. 14 C-acetate absorbed by excised rice leaves was assimilated to PHB as well as in the case of rice roots. This result is also an important supporting evidence that PHB is generated in rice, because it is difficult to consider that there would be significant bacterial growth on the leaf portion of the rice. (4) Radioactivity in wheat; 14 C-acetate absorption by intact wheat seedlings was repeated twice. The assimilation rates to PHB from 14C-acetate were 12.3% and 15.9%, respectively and these values were higher than the assimilation to ethanol fraction (10.6%, and 9.2%). (5) Radioactivity in radish; although the assimilation rate was lower (1.77%) compared to rice or wheat, it was demonstrated that 14C-acetate was assimilated to PHB also in a dicotyledon plant, namely radish. 2.7. Confirming that the radioactivity in the co-precipitate was PHB origin
Fig. 1(1.3A and B), respectively. Fig. 1(1.3A and B) shows that the radioactivity after hydrolysis of PHB-precipitates by 0.5 N-NaOH appeared as two peaks of crotonic acid and b-hydroxybutyric acid on silica gel chromatography. Table 5 shows the 14C-counts of crotonic acid fraction, b-hydroxybutyrate fraction and PHB precipitate before alkaline-hydrolysis. The recovery rates of isotope counts as sum of crotonic acid and b-hydroxybutyrate were 83.9% and 91.3% of PHB precipitates before hydrolysis in rice and in wheat, respectively. Almost all the isotope count of the PHB precipitate was converted to crotonic acid and b-hydroxybutyrate. These results clearly show that the radioactivity co-precipitated with authentic PHB has come from PHB generated from 14C-acetate, and almost all radioactivity in the above chloroform fraction is from PHB as well. 2.8. Time course study The rice seedlings incubated with 14C-acetate for 1 h were transferred to isotope-free medium, and 0, 1, 3, and 6 h after the transfer, roots and leaves were analyzed for radioactivity after separation for ethanol fraction, ether/ethanol fraction, chloroform fraction, and PHB precipitate. The results are shown in Table 6. In the roots, the peak of radioactivity in the ethanol fraction was at 0 h, and was reduced by half at 3 h. In the chloroform fraction (and PHB precipitate), the peak was at 0 h, but the reduction rate was slower compared to the ethanol fraction. In the ether/ethanol fraction, the peak was at 3 h. In the leaves, the peaks of isotope count were 3 h, 3 h, and 1 h in the ethanol fraction, the ether/ethanol fraction and the chloroform fraction, respectively. 2.9. Infrared spectrum of PHB from rice roots and wheat roots
Aliquots of co-precipitates from the rice root and the wheat root were hydrolyzed by 0.5 N-NaOH and separated by silica gel chromatography. Results in rice and wheat are shown in
Infrared spectrum of PHB fraction (Fig. 2B) obtained from rice root incubated with Na-acetate was identical to that of authentic
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43 Table 3 Organic acid contents in rice roots after 1 h-2-14C-acetate absorption with or without chloramphenicol. (Upper column; titration value (ml), lower column; 14 C-count (cpm)). Organic acids
Unknown mix Acetic acid Pyruvic acid Fumaric acid Formic acid
b-Hydroxybutyric acid Succinic acid Aconitic acid Oxalic acid Glycolic acid Malic acid Citric acid Isocitric acid Total cpm
With chloramphenicola 0.5 N-NaOH extb
0.1 N-H2SO4 ext
0.5 N-NaOH ext
0.99 7720 7.82 64,900 0.08 520 0.25 3390 1.49 2800 0.13 4780 0.65 26,900 1.01 139,000 1.44 5560 1.18 3280 2.66 57,700 7.35 68,100 1.27 1090 385,740
7.87 38,500 20.67 27,300 0.27 430 3.15 610 12.01 2430 0.49 49,500 6.43 2920 1.96 10,100 3.4 1000 1.32 650 2.39 4180 2.96 5000 1.32 240 142,860
0.86 8810 8.27 33,100 0.15 1050 0.34 2440 1.55 2900 0.13 17,000 0.65 26,600 1.28 93,900 1.22 4360 1.04 2610 2.71 62,800 6.79 58,600 1.98 930 315,100
7.86 50,100 21.28 22,800 0.27 260 2.38 1710 15.37 1980 0.59 63,100 7.31 1760 2.18 6280 3.71 1000 1.3 530 2.74 3870 2.9 3240 1.38 160 156,790
Origin
Sample
Count
Rice
PHB-coprecipitate (before hydrolysis) a. Crotonic acid fraction b. b-hydroxybutyric acid fraction aþb PHB-coprecipitate (before hydrolysis) a. Crotonic acid fraction b. b-hydroxybutyric acid fraction aþb
3.85 1.52 1.71 3.23 9.51 4.02 4.66 8.68
Wheat
C-count was expressed as cpm. C-count was expressed as dpm. c As shown in Fig. 1(1.3A and B), hydrolysate of PHB-coprecipitate forms two 14C peaks of crotonic acid and b-hydroxybutyric acid.
PHB obtained from Bacillus megaterium (Fig. 2A). The spectrum of PHB fraction from wheat root (Fig. 2C) was also identical to that of B. megaterium. 3. Discussion The purpose of this communication is to describe evidence that externally provided Na-2-14C-acetate was assimilated to poly-bhydroxybutyrate (PHB) at a very high ratio in plant roots. The evidence will be discussed as follows. 3.1. Identification of PHB In the course of the investigation of the metabolism of C-acetate or 14C-succinate in rice roots, an unknown organic acid
was detected between fumarate and succinate on silica gel column chromatography. This organic acid was not extracted by conventional 0.1 N-H2SO4 boiling, but was first extracted by an incidental hot-0.5 N-KOH boiling. This radioactive acid was co-chromatographed with several authentic organic acids and the radioactive peak matched the peak of titration value of b-hydroxybutyrate. The radioactive acid and authentic b-hydroxybutyrate were then heated in conc. H2SO4 which resulted in the conversion of the radioactivity and the titration value to crotonic acid. Therefore, it was concluded that the unknown radioactive acid was b-hydroxybutyrate. The original form of this acid was presumed to be poly-bhydroxybutyrate (PHB), because the acid was not extracted by dilute acid but extracted by alkaline-hydrolysis. However, the possibility that the PHB found in rice root in this experiment might be from bacteria, was considered. This possibility, however, could be excluded by the addition of chloramphenicol, which had a broad anti-bacterial spectrum, in the culture media. There was little difference in 14C-counts of b-hydroxybutyrate generated from 14 C-acetate between the chloramphenicol-added group and the chloramphenicol-free group. As the next step, direct-extraction of PHB from plant materials which incorporated 14C-acetate was conducted. A simplified method was used which was a modification of Lemoigene’s method [10]: 1) hot-ethanol extraction (twice), 2) ethanol/ether (3/1, v/v) extraction (twice), and hotchloroform extraction (3 times). The hot-chloroform fraction was then co-precipitated with authentic PHB by the addition of 5 volume of acetone/ether (3/1, v/v) in a refrigerator, and almost all the radioactivity was co-precipitated with authentic PHB. The radioactive co-precipitate was then heated with 0.5 N-NaOH, and chromatographed. The radioactivity of b-hydroxybutyrate plus crotonic acid almost matched that of the co-precipitate before alkaline-hydrolysis. Hence the radioactive substance in the precipitate was confirmed to be PHB.
Table 4 Assimilation of 2-14C-acetate into ethanol, ether/ethanol, chloroform and PHB fraction after 1 h, absorption in various plant materials; dpm (% of total Part anal.b
14
Rice (L) Rice (D) Rice root (D) Rice leaf (L) Wheat 1 (L) Wheat 2 (L) Radish (L)
Root Root Root Leaf Root Root Root
1.05 1.26 0.547 0.172 1.27 1.37 0.409
a b c d e
C absorbed (%c)
108 108 108 108 108 108 108
(42.9) (51.6) (22.4) (7.02) (48.5) (52.7) (18.1)
EtOH fr.d 1.53 1.65 1.47 2.84 1.34 1.26 1.04
100.0 39.5 44.4 83.9 100.0 42.3 49.0 91.3
a 14
Chloramphenicol was added to the culture medium at a final concentration of 100 mg/ml from 1 h before 14C-acetate absorption to the end of the absorption. b After the 14C-acetate absorption, root part of the rice seedlings was extracted with 0.1 N-H2SO4 for 3 h, and then the residue was extracted with 0.5 N-NaOH for 2 h.
Plants (L or D)a
% 104 cpma 104 104 104 104 dpmb 104 104 104
b 14
a
14
Table 5 14 C-counts of PHB-coprecipitates and their hydrolysates.c
Without chloramphenicol
0.1 N-H2SO4 extb
39
107 107 107 106 107 107 107
EtOH/Ether fr.d (14.6) (13.1) (26.9) (16.5) (10.6) (9.20) (25.4)
4.75 8.89 7.72 2.42 6.45 5.33 1.56
Plant materials used for 14C-acetate absorption. L; absorbed in the light. D; absorbed in the dark. Plant part analyzed. Percentage of 14C-acetate incorporated in the plant materials. fr; fraction. 14 C-count co-precipitated with the authentic PHB from B. megaterium.
105 105 105 105 105 105 105
(0.452) (0.706) (1.41) (1.41) (0.51) (0.388) (0.381)
Chloroform fr.d 9.61 1.05 7.86 1.55 1.57 2.30 7.91
106 107 106 106 107 107 105
(9.15) (8.33) (14.4) (9.01) (12.4) (16.8) (1.93)
14
C absorbed).
PHB precipitatee 8.52 1.04 7.20 1.64 1.56 2.18 7.23
106 107 106 106 107 107 105
(8.11) (8.25) (13.2) (9.53) (12.3) (15.9) (1.77)
40
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
Table 6 Time course study of assimilation of 14C-acetate to ethanol fraction, ethanol/ether fraction, chloroform fraction and PHB precipitate.a dpm at hours after transferring to isotope-free medium Extraction (part analyzed)
0h
EtOH (root) EtOH (leaf) EtOH/Ether (root) EtOH/Ether (leaf) Chloroform (root) Chloroform (leaf) PHB precipitate (root) PHB precipitate (leaf)
1.53 1.78 4.75 4.61 9.61 2.62 8.52 NDb
1h
107 106 105 104 106 105 106
1.16 2.83 5.53 8.43 9.20 3.06 8.46 ND
3h
107 106 105 104 106 105 106
0.91 3.86 8.89 1.38 8.21 2.29 7.18 ND
6h
107 106 105 105 106 105 106
0.64 3.36 2.74 1.33 7.70 1.59 7.13
107 106 105 105 106 105 106
ND
a The rice seedlings incubated with 14C-acetate for 1 h were transferred to isotope-free medium, and 0, 1, 3, and 6 h after transferring, roots and leaves were analyzed separately. 100 pieces were used for each group. b ND; not done.
The existence of PHB in rice root and wheat root was also confirmed by infrared absorption spectrum analysis. For this experiment, rice and wheat were cultured in 10 mM Na-acetate for 18 h before harvesting, because PHB contents in rice or wheat seemed to be a small amount in normal culture condition. Results clearly showed that the IR-absorption spectra of PHB fractions from rice root
and wheat root matched that of authentic PHB from B. megaterium, respectively. The spectra were also identical to the IR-spectra of PHB from Chlorogloea fritschii and Rhodopseudomonas spheroides presented by Carr [11]. Recently Vogel et al. [12] reported IR-absorption spectrum in a blend of PHB with PLA (poly-L-lactic acid), and nonoverlapping bands for bacterial PHB were identified at 1724, 1279, 1057, 980, 897, 826 and 516 cm1. As shown in Fig. 2, these 7 bands are all detectable in IR-spectra of PHB from rice and wheat. 3.2. Physiological role of PHB in plants Poly-b-hydroxybutyrate (PHB) was first discovered by Lemoigene in 1927 as a major component of B. megaterium. The same component was later found in many other bacteria including blue green algae (C. fritschii) [8,11,13e15]. There was a considerable literature on the properties and metabolism of PHB in bacteria until the 1960s, and accumulated evidence suggested that it functioned as an intracellular reserve of carbon and energy for many bacterial species [16]. This thinking seems to be unchanged to the present [17]. However, from the 1980s, PHB has attracted added attention as one of the bio-degradable plastics in the chemical industry [18], and transgenic plants in which bacterial PHB-synthesis-genes are introduced have been generated for PHB production [19,20], but even in a recent paper on acetate metabolism in Arabidopsis, PHB has not appeared as a member of acetate metabolism [21]. With knowledge of the existence of PHB in rice root, the present author recalled immediately a reductive photo-assimilation of acetate in purple bacteria [14,22]. Thus, the 14C-acetate-uptake experiment in rice was conducted in the dark and in the light. However, as shown in Table 4, no difference in PHB assimilation from 14C-acetate was observed between these conditions. Furthermore, the PHB was synthesized from 14C-acetate at a high rate by only the root of rice in the dark. PHB seems to be synthesized from acetate even in the absence of light in rice. The very high specific radioactivity in b-hydroxybutyrate extracted by alkaline-hydrolysis (Tables 2 and 3) suggests two possibilities: 1) PHB pool might be very small in normal culture conditions of rice. 2) The acetate which was absorbed externally, might be converted to PHB without being diluted by the acetate pool in plant cells. The time course study (Table 6) showed that the decrease of assimilated PHB in the root was slower than that of ethanol-soluble fraction. However, from this experiment, it cannot be concluded that metabolism of PHB is slower than other free organic acids, because one cannot exclude the PHB synthesized from other organic acids via acetate. A pulse experiment (e.g. 1e10 min isotope incorporation and transferring to isotope-free medium) will be needed. In the first experiment by which synthesis of PHB from acetate or succinate was shown in rice, a fairly high concentration of acetate (1 mM) was added in the culture medium. Thus it was thought that the PHB-accumulation might be the result of abnormal metabolism of acetate. However, as shown in Table 4, even in the case of lower concentration of acetate (0.02 mM), PHB was synthesized at a high ratio in rice, wheat, and also in radish although to a lesser extent. This suggests that PHB might be of considerable biological interest, for example, it might function as a hub-compound in organic acid metabolism more than merely as a carbon reserve in higher plants. A general scheme of acetate metabolism in plant cells is shown in Fig. 3 in which the biosynthetic pathway from acetate to PHB is added by the dotted line, although the intermediate portion is a black box at present. 3.3. Possible enzymes involved in PHB formation in plants
Fig. 2. Infrared spectra of PHBs from B. megaterium, rice root and wheat root. A; B. megaterium, B; rice root, C; wheat root.
In the bacterium Alcaligenes eutrophus, PHB is synthesized from acetyl CoA by the consecutive reaction of three
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
41
4. Materials and methods 4.1. Plant materials
Fig. 3. Acetate metabolism in plant cells. PGA; 3-phosphoglycerate, G-6-P; glucose-6phosphate, F-6-P; fructose-6-phosphate.
Seeds of rice (O. sativa L., cv. Koshihikari) and wheat (Triticum aestivum L, cv. Fujimi) were germinated on paper soaked with tap water for 2e3 days at room temperature. After germination, the seedlings were grown on a plastic net floating in tap water for 2 or 3 weeks under natural light in a green house at 25 C or in an environmentally controlled room (approximately 10,000 lux of white light, 16 h photoperiod (7:00e23:00) at 25 C). Nutrients (medium I for the rice and medium II for the wheat) were added 1 week after germination. The compositions were as following: Medium I (Kasugai’s medium): (NH4)2SO4; 1 mM, NaH2PO4; 0.5 mM, KCl; 1 mM, CaCl2; 1 mM, MgCl2; 1 mM, FeCl3$6H2O; 1 ppm, pH5.5. Medium II (Hoagland’s medium): KNO3; 2.5 mM, Ca(NO3)2; 2.5 mM, MgSO4; 1 mM, KH2PO4; 0.5 mM, FeCl3$6H2O; 1 ppm, pH5.5. Seeds of radish (Raphanus sativus var. sativus) were grown in sand culture supplied with medium II after germination. 4.2. Extraction of organic acid from rice root
enzymes: 3-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, which are encoded by the phbA, phbB, and phbC, respectively [23]. Of the three enzymes only 3-ketothiolase is found in higher plants in which it is involved in the synthesis of mevalonate [19,24]. However, there has been no information about the occurrence of the other two enzymes in higher plants. To find the rice genes whose sequences are similar to the phbB, and/or phbC, the author then performed homology search of NCBI database using BLASTP program. As a consequence, no rice gene which was similar to the phbC, was detected. Whereas, the phbB was found to show a high homology with a rice gene (gene name; Os12g0242700 (Oryza sativa Japonica group)) at amino acid sequence level. A putative protein encoded by this gene is b-keto acyl carrier protein reductase [EC.1.1.1.100], involved in fatty acid synthesis [25]. Although it is not clear yet whether this gene encodes a protein having a real acetoacetyl-CoA reductase activity, enzymes involved in PHB formation in plants may overlap in part with enzymes involved in fatty acids synthesis.
In the preliminary experiments, several methods were tested. 1) distilled water extraction on a water bath for 3 h at 90e100 C. 2) 0.1 N-H2SO4 extraction on a water bath for 3 h at 90e100 C [6]. 3) 1 N-H2SO4 extraction on a water bath for 3 h at 90e100 C. 4) 0.5 N-KOH extraction on a water bath for 2 h at 90e100 C. 5) stepwise extraction of 1), 2) and 4). 6) direct ether Soxhlet extraction of sample mixed with 4 N-H2SO4 [5]. In each extraction from 1) to 5), 1 g of freeze-dried rice root sample was immersed in 50 ml of extraction solution in a round-bottom flask (100 ml) with a Liebig condenser. In the latter experiments using radioactive tracer, the two-step extraction of 0.1 N-H2SO4 and 0.5 N-NaOH (or 0.5 N-KOH) was adopted, and fresh root materials were used unless otherwise stated. Each extracted-solution was then adjusted to pH1, and further extracted with ether liquid-Soxhlet extractor (50 ml water and 50 ml ethyl ether) for 1 week. Extracted organic acids were captured with 20 ml of 0.1 N-NaOH, neutralized with 0.1 N-H2SO4 using a phenol red indicator, dried up on a water bath at 80e90 C and transferred to the next procedure for organic acids separation.
3.4. Future issues
4.3. Separation of organic acids
The experimental results shown in this communication are only one issue in the study of PHB-physiology in higher plants. Future issues are outlined below. (1) Elucidation of PHB-synthesis and -depolimerization pathways in higher plants, and their participant enzymes (and genes). (2) localization of PHB in plant cells: In the first incorporation experiment of 14C-labeled organic acids, PHB was detected mainly in 10,000 g 30 min ppt-fraction in which mitochondria, plastid etc. are contained. However the data were from 6 h-incorporation and 1 mM Na-acetate or 2Na-succinate addition. PHB localization in higher plants under normal physiological conditions should be investigated. In many microorganisms [17] or higher plants introduced bacterial PHB-synthesizing genes [20], inclusion bodies which contain PHB have been observed. When a high concentration of acetate is provided externally, similar inclusion bodies may be formed also in plant cells. (3) Whether or not PHB is synthesized from acetate in other plants especially in Arabidopsis, a model experimental plant. (4) PHB synthesis from monomers other than acetate in plants (e.g. organic acids, glucose). (5) Physico-chemical properties of PHB from plant materials.
Silica gel column chromatography by Bulen et al. [26] was used for organic acids fractionation. Silica gel was prepared from Mallinckrodt’s silicic acid by removal of the fine particles through repeated suspension in distilled water and decantation until approximately one third of original material was removed. The remaining coarse fraction was dried in an oven at 100 C for 24 h, and stored in a closed container. A glass-wool plug is placed in the bottom of the chromatographic tube (12 mm 25 cm). Eight gram of the above silica gel was mixed with 5.5 ml of 0.5 N-H2SO4, was slurried in 60e70 ml chloroform and added to the chromatographic tube with the aid of a glass rod. This procedure gives a uniformly packed column of 14.5 cm long. The mixture of standard organic acids (Na-salts, 10e100 mg) or organic acid fraction from plant materials were dissolved in 0.5 ml of 0.5 N-H2SO4, and were mixed quickly and thoroughly with 1 g of silica gel. The resulting powder containing samples was adhered to absorbent cotton and transferred to the top of the column quantitatively. Development of this survey column proceeded by the addition of a series of n-butyl alcohol/chloroform (v/v) solvents: 100 ml of 5/95, 135 ml of 15/85, 100 ml of 25/75, 300 ml of 35/65, and 100 ml of 50/50. Each solvent
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H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
mixture was equilibrated against 0.5 N-surfuric acid by shaking the two phases in separatory funnel and passing the solvent layer through dry filter paper to remove suspended water droplets. The effluent was captured every 4 ml (or 2 ml) in a test tube using a fraction collector. One ml of distilled water was added to each individual fraction and was titrated by addition of standard 0.01 N-NaOH using a phenol red indicator. Before the examination of samples from plant materials, standard organic acids were tested by Soxhlet extraction and succeeding Bulen’s silica gel chromatographic analysis, and it was confirmed that these acids were quantitatively retrieved. However, peaks of fumaric acid/formic acid and oxalic acid/glycolic acid were overlapped and could not be separated clearly.
4.7. Conversion from b-hydroxybutyrate to crotonic acid Commercial b-hydroxybutyrate-Na (Kokusan Chem., Japan) was purified by silica gel chromatography. The b-hydroxybutyrate fractions titrated with 0.01 N-NaOH were collected (16.4 ml ¼ 0.164 mmol), mixed with the radioactive X fractions (5160 cpm) and evaporated on a water bath at 80e90 C. The mixture was heated with 10 ml of concentrated H2SO4 in a bottom-round flask (100 ml) with a Liebig condenser on a water bath at 100 C for 15 min. The solution was cooled by addition of appropriate amount of ice, then saturated with NaCl and shaken with ethyl ether in a separation funnel. The ether phase was collected and evaporated. The remains of evaporation were analyzed by the silica gel chromatography.
4.4. Uptake experiments of radioactive organic acids After 2 or 3 weeks of cultivation, the seedlings (rice or wheat) were harvested from the culture net, the hull was removed from each seedling, and washed thoroughly with tap water. The seedlings were transferred to a 100 or 200 ml beaker containing culture medium including 2-14C-acetate or 2,3-14C-succinate, and were cultivated under 10,000 lux white light in an environmentally controlled room or natural light in a green house at 25 C for hours indicated. The beaker was wrapped in aluminum foil so the roots were in the dark during absorption experiment. Contents and specific activity of the radioactive compounds are described for each experiment. After isotope-absorption, the seedlings were washed with tap water thoroughly, wiped with paper towel to remove excess water and used for ensuing analysis. 4.5. Cell fractionation The uptake conditions of labeling compounds by rice seedlings in this fractionation experiment, are as follows. To a 100 ml beaker containing100 ml of culture medium (medium I) including labeling compound, 250 pieces of 3 weeks old rice seedlings were transferred and cultured for 6 h in a green house at 25 C. Labeling compounds supplied to the rice seedlings: 2-14C-acetate-Na (specific activity; 740 MBq/mmol), 0.74 MBq/100 ml; 2,3-14C-succinate-2Na (specific activity; 389 MBq/mmol), 0.74 MBq/100 ml. As a carrier, 1 mM of nonlabeling Na-acetate or 2Na-succinate was added to the above culture medium, respectively. Thus the start concentration of acetate was 1.01 mM and that of succinate was 1.02 mM, respectively. For each experimental group, 3 beakers were used. After absorption of labeling compounds, cell fractionation was performed based on the description by Beevers [27]. The root part of the rice seedling (750 pieces) was ground down by a mortar in 100 ml of 1/20 M-phosphate buffer (pH6.9) containing 0.7 M mannitol. The resultant soup was passed through 4 files of gauze and centrifuged at 300 g for 4 min (300 g precipitate (ppt) fraction). The supernatant was then centrifuged at 10,000 g for 30 min (10,000 g ppt-fraction), and the remaining supernatant (sup fraction) was also collected. The each fraction was washed once by resuspension in the homogenizing buffer and subsequent centrifugation. Thus these 3 subcellular fractions were used for further analysis. 4.6. Measurement of radioactivity An aliquot of water phase from each silica gel chromatography fraction titrated (neutralized) by 0.01 N-NaOH was transferred to stainless tray (planchette), dried up with an infrared lamp, and count of 14C was measured by a gas flow counter. The radioactivities were expressed as cpm. Radioactivity of solvent-extraction fraction was measured by a liquid scintillation counter and expressed as cpm or dpm.
4.8. Extraction and separation of poly-b-hydroxybutyrate from plant materials Poly-b-hydroxybutyrate fraction (PHB) was extracted by the method for bacterial PHB [10,28] with some modifications. The uptake conditions of labeling compounds by plant materials in this experiment, were as follows. To a 200-ml beaker containing 100-ml of culture medium (medium I or II) including labeling compound, 3 weeks old rice seedlings (100 pieces) or wheat seedlings (50 pieces) were transferred and cultured for 1 h in an environmentally controlled room (approximately 10,000 lux of white light at 25 C). The beaker was wrapped by aluminum foil to keep the roots in the dark. The dark condition of whole plantbody was made by lights-out. Ten days old radish seedlings (50 pieces) were cultured in a 50-ml beaker containing 50-ml absorption solution in the above controlled room. Labeling compounds supplied to the plant materials: 2-14C-acetate-Na (specific activity; 740 MBq/mmol), 3.7 MBq/100 ml for rice and wheat, 3.7 MBq/50 ml for radish. Thus the start concentrations of acetate were 0.05 mM for rice and wheat, and 0.1 mM for radish. After isotope-uptake, plant materials were washed quickly with tap water and extracted successively twice with 50 ml hotethanol (ethanol fraction), twice with 50 ml ethanol/ether (v/v, 3/1) (ethanol/ether fraction), and finally three times with 50 ml hot-chloroform (chloroform fraction). The above fractions were filtrated with dry filter paper (Whatmann G3). To the chloroform fraction, approximately 10 mg of authentic PHB from B. megaterium (kindly supplied by Dr. N. Tsukagoshi; The University of Tokyo) was added to serve as a carrier and heated to dissolve. After reducing the volume of chloroform by evaporation, 5 volumes of acetone/ether (v/v; 3:1) were added and cooled in a refrigerator. PHB was deposited as a white precipitate. The precipitate was air-dried and weighed. An aliquot was used for isotope counting (an aliquot of solid PHB was first dissolved in Soluene TM (Packard L.T.D) and then dissolved in scintillation fluid). The procedure of this dissolving/precipitation was repeated three times, and the radioactivity of PHB per mg was reached a plateau. The quantity of PHB originally contained in each experimental group of plant materials was expected to be far less than 10 mg from the titration value of b-hydroxybutyrate (see Table 3). Thus PHB amount (14C-count) synthesized from 14 C-acetate in plant materials was calculated according to the following equation:
PHB added ðmgÞ PHB synthesized dpm ¼ count of A dpm A ðmgÞ A; aliquot of precipitate. PHB added; PHB from B. megaterium which was added to the chloroform fraction as a carrier.
H. Tsuda / Plant Physiology and Biochemistry 50 (2012) 35e43
4.9. Confirmation of PHB by alkaline-hydrolysis Another aliquot of the above radioactive precipitate (PHB) was heated with 10 ml of 0.5 N-NaOH in a bottom-round flask (100 ml) with a Liebig condenser on a water bath at 100 C for 60 min. After neutralization, the hydrolysate was separated by silica gel chromatography. The radioactivities (dpm or cpm) of crotonic acid fraction and b-hydroxybutyrate fraction were counted by liquid scintillation counter (Packard L.T.D) and compared with the count (dpm or cpm) of precipitate (PHB) before the alkaline-hydrolysis. 4.10. Infrared spectrum of PHB from rice root and wheat root Two weeks old seedlings of rice and wheat were cultured in each culture medium containing non-labeled Na-acetate at a final concentration of 10 mM for 18 h (PM6:00eAM12:00) in a green house at 25 C. The root part of these seedlings (rice; 100 g, wheat; 65 g) was washed thoroughly by running tap water and then successively extracted twice with 200 ml of hot-ethanol, twice with 200 ml of ethanol/ether(3/1; v/v), and three times with 200 ml of hotchloroform. The chloroform fraction was evaporated to a smaller volume to which approximately 5 volumes of acetone/ether (3/1; v/v) were added and cooled in a refrigerator to precipitate the PHB. The precipitate was collected by a filter paper and dried. Dissolving in chloroform and re-precipitation by acetone/ether was twice repeated. Approximately 1 mg of precipitate was obtained from rice root and wheat root, respectively. The white grayish dried precipitates were ground with KBr powder to make KBr-disk and were examined by Infrared spectroscopy (IRA-type2; Nihonbunko, Japan). As a control of PHB, PHB from B. megaterium was used. Note This paper was prepared based on the research carried out in the master course of The University of Tokyo, Department of Agricultural Chemistry [29]. Most of the contents of this paper were presented at the Japanese Society of Plant Physiologists [30]. Acknowledgments The author thanks Dr. Satoshi Mori (Emeritus Professor, The University of Tokyo) for critical reading of the manuscript. References [1] D.H. Maclennan, H. Beevers, J.L. Harley, Compartmentation of acids in plant tissues, Biochem. J. 89 (1963) 316e327. [2] S.H. Lips, H. Beevers, Compartmentation of organic acids in corn roots. I. Differential labeling of 2 malate pools, Plant Physiol. 41 (1966) 709e712. [3] S.H. Lips, H. Beevers, Compartmentation of organic acids in corn roots. II. The cytoplasmic pool of malic acid, Plant Physiol. 41 (1966) 713e717. [4] B.T. Steer, H. Beevers, Compartmentation of organic acids in corn roots. III. Utilization of exogenously supplied acids, Plant Physiol. 42 (1967) 1197e1201.
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