Abstract
The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (=Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14 C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 μg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.

Derris indica and Caesalpinia crista are mangrove plants belonging to the Leguminosae family. They are widely distributed on seashores in Southeast Asia and Pacific Islands. Different organs of these plants possess antimycobacterial activity [1] and have been used in folk medicine. Seeds of D. indica contain oils and fatty acids suitable for biodiesel production [2] . These plants grow by seashores with their roots in the sea. They therefore apparently have salt-tolerant properties. These mangrove legumes are interesting from the viewpoint of biotechnology as well as basic plant biology. There have, however, been few physiological studies of these plants.
Nicotinamide is formed as a catabolite of NAD and NADP, and is a key metabolite of pyridine metabolism. In plants, nicotinamide is not directly used for re-synthesis of NAD, as it is in animals [3] , but it is readily deaminated to nicotinic acid and then used for pyridine nucleotide synthesis [4] [5] [6] . As well as the salvage pathway for NAD synthesis, nicotinic acid is used for the synthesis of secondary metabolites. In a few plants, nicotinic acid is a substrate for the synthesis of unique pyridine alkaloids, such as nicotine and ricinine [7, 8] , but most plants produce simple and common secondary products: trigonelline (1-N-methylnicotinic acid) or nicotinic acid N-glucoside ( Figure 1 ) [9, 10] . The biosynthesis of these two compounds is an example of biochemical evolution in plant chemosystematics [11] . Diverse physiological functions of trigonelline have been proposed by several researchers [9] [10] 12] , and one role of trigonelline is as a compatible solute to adjust the osmotic pressure when the plants are affected by salt stress [13] [14] [15] [16] .
As part of our studies of pyridine metabolism in plants, we examined the metabolic fate of [carbonyl-14 C]nicotinamide in leaves of two mangrove legumes in the presence and NPC Natural Product Communications 2011 Vol. 6 No. 12 1835 -1838 absence of NaCl. The short-term effect of NaCl on endogenous trigonelline content was also examined in leaf segments of D. indica.
We examined the changes in 14 C-labelled metabolites, along with the incubation time. Figure 2 shows the results from leaf disks of C. crista. [ 14 C]Nicotinamide, taken up by the disks, was readily converted into nicotinic acid. Within 30 min of administration, radioactivity was found in NaMN, NAD and NADP. This implies that nicotinic acid derived from nicotinamide was salvaged to NaMN by nicotinate phosphoribosyltransferase (EC 171.132.149.166), and was then utilized for pyridine nucleotide synthesis. Radioactivity was also found in trigonelline; the proportion gradually increased with incubation time to 60% of the total radioactivity at 24 h after initiation. In these disks, therefore, nicotinic acid appears to be converted to trigonelline by nicotinic acid N-methyltransferase (trigonelline synthase, EC 171.132.149.166); this pyridine alkaloid was accumulated. Release of 14 CO 2 from [carbonyl- 14 C]nicotinamide was low; less than 1.2% of total activity was found during the time of incubation. Small amounts of radioactivity were found in NMN at 6 h after administration of [ 14 C]nicotinamide.
Although it is difficult to infer the metabolic sequence from these results alone, our data support the functioning of the pyridine nucleotide cycle shown in Figure 1 , which has been reported in other plants [5, [16] [17] .
We examined the metabolic fate of [carbonyl- 14 C]nicotinamide in leaf disks from C. crista and D. indica at 3 h after administration in the presence and absence of NaCl ( Figure 3A,B) . In leaf disks of both species the radioactivity was recovered in nicotinamide, nicotinic acid, NAD/NADP, NaMN and trigonelline. Only a small amount of radioactivity was found in CO 2 . No radioactivity was detected in nicotinic acid glucoside. The activity of pyridine salvage, estimated from the sum of the incorporation rate of radioactivity into NAD, NADP and NaMN, varied from 18% to 22% of the total uptake in leaf disks of both species, and no large difference was caused by NaCl. In contrast, widely differing trigonelline synthesis activity was found in the different species; the incorporation rate in D. indica (42-47% of total uptake) was much greater than in C. crista (15-17%) . No increase in trigonelline synthesis due to NaCl took place, and there was even a slight reduction in activity (88% of control) in D. indica.
The endogenous concentration of trigonelline in leaf disks of D. indica at 3 h after incubation with 0, 250 and 500 mM NaCl was respectively 831.5 ± 53.3, 857.0 ± 22.0 and 893.9 ± 13.7 μg per g dry weight. Cho et al. [13] reported that the trigonelline concentration in leaf tissue of cultivated Glycine max ranged from 64 to 162 μg per g dry weight. Trigonelline concentration in D. indica leaves is consequently 5-12 times higher than in G. max leaves. High concentrations of trigonelline may act as compatible
Pyridine metabolism in mangrove legumes
Natural Product Communications Vol. 6 (12) 2011 1837 solutes in this mangrove legume. Although we found that the concentration of trigonelline increased slightly as a result of 3 h salt stress, these differences were not statistically significant (P > 0.05).
Our observations suggest that trigonelline synthesis in mangrove leaves does not respond quickly to salt stress. However, it is clear that D. indica has a mechanism for producing trigonelline, so that its content in leaves is much greater than in G. max, even if the plant is grown in a saltfree environment. It is, therefore, plausible that the strong trigonelline formation in D. indica is an inherent property of this mangrove legume. 
Experimental

Plant materials and chemicals: Seeds of

Radioactivity incorporation:
Administration of [carbonyl- 14 C]nicotinamide to the leaf disks and analysis of labeled metabolites were essentially the same as in a previous paper [18] . Leaf disks (ca. 100 mg fresh weight), together with 2.0 mL of 30 mM potassium phosphate buffer (pH 5.6) containing 10 mM sucrose, 50 mM sodium ascorbate, and 0, 250 or 500 mM NaCl, were placed in the main compartment of a 30 mL Erlenmeyer flask. The flask was fitted with a glass tube containing a piece of folded filter paper that had been impregnated with 0.1 mL of 20% KOH in the centre well. Each reaction was started by adding 10 µL (37 kBq) of a solution of [carbonyl- 14 C]nicotinamide to the main compartment of the flask. The flasks were incubated in an oscillating water bath at 27ºC. After incubation, the glass tube was removed from the centre well and placed in a 50 mL Erlenmeyer flask containing 10 mL distilled water. At the same time, the leaf disks were harvested and washed with distilled water, frozen with liq. N 2 , and then stored at -80ºC prior to extraction. The filter paper, containing CO 2 trapped as potassium bicarbonate, was soaked overnight in distilled water (10 mL), and aliquots of the resulting solution (usually 0.5 mL) were used for determination of radioactivity. Radioactivity was measured with a liquid scintillation counter (Beckman, type LS 6500, Fullerton, CA, USA).
Analysis of metabolites:
For analysis of 14 C-labelled metabolites, the frozen samples were homogenized using a mortar and pestle with ice-cold 6% perchloric acid (PCA) containing 0.1 mM NAD. NAD was supplemented to prevent the degradation of labeled nucleotides during extraction and analysis, and also for action as endogenous markers in the TLC analysis. The homogenate was centrifuged at 3,000 x g for 7 min, the supernatant was collected, and the precipitate was re-suspended in the same extraction reagent and washed 3 times. The 14 C-labelled metabolites extracted with 6% PCA were combined, neutralized with 20% KOH, and centrifuged briefly (3,000 x g, 7 min) to remove potassium perchlorate as precipitate. The radioactivity of aliquots of supernatant was measured with a liquid scintillation counter. The neutralized fractions were freeze-dried and then redissolved in a small amount of 50%, v/v, ethanol.
The concentrated PCA-soluble metabolites (free nicotinic acid related small molecular weight compounds) were separated by using microcrystalline cellulose TLC sheets (200 × 200 mm, Merck, Darmstadt, Germany). As solvent systems, system I (n-butanol: acetic acid: water, 4:1:2, v/v) and system IV (isobutyric acid: ammonia: water, 660: 17: 330, v/v) as specified by Zheng and Ashihara [19] were used. The distribution of 14 C to individual spots on the TLC sheet was estimated using a bio-imaging analyzer (Type, FLA-2000, Fuji Photo Film Co., Ltd. Tokyo, Japan). The total radioactivity of each fraction was determined by a liquid scintillation counter (Beckman, type LS 6500). Incorporation into individual metabolites was calculated using the total radioactivity values in the PCA-soluble fraction, as measured by a liquid scintillation counter, and the % distribution of radioactivity on TLC plates was obtained from the bio-imaging analyzer.
Effects of NaCl:
To determine the short-term effect of NaCl on the accumulation of trigonelline, leaf disks (ca. 300 mg fresh weight) were incubated with 2 mL of the medium which had been used for the 14 C-tracer experiments, supplemented with 0, 250 or 500 mM NaCl, in a 30 mL Erlenmeyer flask at 27ºC for 3 h. After incubation, the leaf disks were washed with distilled water, boiled in 10 mL of hot water (> 95ºC) for 10 min, and then homogenized using a mortar and pestle. After brief centrifugation (20,000 x g, for 10 min), the water-soluble supernatant fraction was collected and freeze dried. The samples were dissolved in small amounts of distilled water and analyzed by HPLC, as in our previous paper [20] , except that the absorbance was monitored using a Shimadzu Diode Array Detector, type SPD-M10A (Shimadzu Corp., Kyoto, Japan).

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