Regulation in tobacco callus of enzyme activities of the ... - Springer Link

Planta

Planta (1986)168:408-413

9 Springer-Verlag1986

Regulation in tobacco callus of enzyme activities of the nicotine pathway II. The pyridine-nucleotide cycle R. Wagner, F. Feth and K.G. Wagner* Arbeitsgruppe Enzymologie, Gesellschaft ffir Biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig, Federal Republic of Germany

Abstract. In tobacco callus, the induction of nicotine synthesis, which stimulates enzyme activities of the ornithine-methylpyrroline route (see the preceding paper), also leads to marked changes in the enzyme activities of the pyridine-nucleotide cycle. This cycle provides the metabolite (probably nicotinic acid) for condensation with methylpyrroline to produce nicotine. The activities of eight enzymes of the pyridine-nucleotide cycle and of quinolinic-acid phosphoribosyltransferase, the anaplerotic enzyme, were determined by high-performance liquid chromatography assays. The distinct changes of their activities upon induction of nicotine synthesis lead to the following conclusions: i) nicotinic acid is the relevant metabolite which is provided by the pyridine-nucleotide cycle and consumed for nicotine synthesis, ii) The enhancement of the nicotinic-acid pool arises in two ways, by synthesis of N A D and degradation via nicotinamide mononucleotide and by a direct route from nicotinic-acid mononucleotide (NaMN) which is degraded by a glycohydrolase with a rather high Km value. Such a Km value prevents the complete depletion of the N a M N pool. Key words: Callus culture (nicotine pathway) - Nicotiana (nicotine pathway) - Nicotine biosynthesis - Pyridine nucleotide cycle - Pyridine nucleotide glycohydrolase Quinolinic acid phosphoribosyltransferase. * To whom correspondence should be addressed Abbreviations: HPLC = high-performance liquid chromatogra-

phy; NAD-PPase:NAD-pyrophosphatase; NaMN-ATase= nicotinic-acid mononucleotide (NaMN) adenylyltransferase; NaMN-GHase = NaMN-glycohydrolase; Na-PRTase = nicotinic-acid phosphoribosyltransferase; NMN-ATase=nicotinamide mononucleotide (NMN) adenylyltransferase; NMNGhase- =NMN-glycohydrolase; PMT =putrescine methyltransferase; Qa-PRTase = quinolinic acid phosphoribosyltransferase

Introduction In the preceding paper (Feth et al. 1986) a callus tissue derived from Nicotiana tabacurn cv. Samsun was described which displays a strong induction of nicotine synthesis upon reduction of the medium auxin concentration. It was further shown that this nicotine synthesis is provided for by an increase in the activities of putrescine methyltransferase (PMT) and methylputrescine oxidase (MPO) which produces methylpyrroline. The control is most stringent for the enzyme leading from primary to secondary metabolism (PMT) and less stringent for the subsequent enzyme (MPO). N-Methylpyrroline is converted to nicotine by condensation with a metabolite of the pyridinenucleotide cycle, which is most probably nicotinic acid. This suggestion is, however, not proven, as the condensing enzyme has not been characterized (Waller and Dermer 1981). In previous work in our laboratory the enzymes of the pyridine-nucleotide cycle (Wagner and Wagner 1985; Wagner et al. 1986) and of quinolinic-acid phosphoribosyltransferase (Qa-PRTase; Wagner and Wagner 1984) have been characterized by the application of activity assays developed for determination of the enzymatic product by high-performance liquid chromatography (HPLC). In the present work, these methods were used to follow the different enzyme activities after induction of nicotine biosynthesis in the tobacco callus described. The investigation was performed in order to answer the following questions, i) Are the changes of the enzyme activities consistent with a flux of metabolites in the direction of nicotinic acid? ii) By which routes is the pool of nicotinic acid replenished? iii) How are, in general, the different enzyme activities coordinated for a cyclic metabolic route which has to serve both for primary metabolism (provid-

R. Wagner et al. : Enzymeactivitiesof the nicotinepathway. II

409 1984), for the determinationof nicotinic-acid mononucleotide (NaMN), the product of the Qa-PRTase. The procedureis illustrated in Fig. I and the chromatographic conditions are described in its legend.

Z 0 Z

Results and discussion

l.,tl ii

8

tm

f

0 2

4 6 8 min

Fig. 1. Determination of the enzymatic activity for Qa-PRTase by HPLC. A 5-gm Merck Lichrosorb NH2 column (25 cm long, 4 m m diameter) and a guard column filled with Vydac TM201 RP, 3 0 4 4 p.m (Machery and Nagel, Dfiren, F R G ) were used. The chromatogram was developed by isocratic elution with 54mmo1-I 1 KHzPO4 (pH4.5) at a flow rate of 2 m l . m i n - 1 . Detection was at 259 nm with 0.008 absorption units at full scale (AUFS); 25 gl were injected. Retention time for N a M N was 5.3 rain. Quinolinic acid which is bound to the column has to be eluted after each run with 0.3 mol.1 -x KHzPO4 (pH 4.5)

ing NAD) and secondary metabolism (producing nicotine)? Materials and methods Materials and buffers. Origin and growth of the tobacco calli derived from N. tabacum Var. Samsun have been described in the preceding work (Feth et al. 1986). Protein extraction and the application of disposable PD 10 columns was performed as described by Wagner and Wagner (1984) using buffer (A): 100 mmol. 1-1 2-(N-morpholino)propanesulfonic acid (MOPS)-NaOH (pH7.4), 5 m m o l . l - l M g C 1 2 , 10mmol-1 x dithioerythritol (DTE) and 0.1 mmol. 1- 1 ethylenediaminetetraacetic acid (EDTA). For determination of the phosphoribosyltransferases the calli were dried by lyophyllization without freezing (Wagner et al. 1986).

Enzyme assays. The assays for the several enzymaticreactions of the pyridine-nucleotidecycle,the buffers used, the substrate concentrations and the HPLC procedures to quantify the enzymic products have been described previously (Wagner and Wagner 1985; Wagner et al. 1986). An improved HPLC assay was developed, based on anion-exchangechromatographyinstead of reversed-phase chromatography(Wagner and Wagner

Variations of the enzyme activities during nicotine induction and callus growth. The activities of nine enzymatic reactions were determined by H P L C methods (Wagner and Wagner 1985; Wagner et al. 1986). The individual enzyme steps are illustrated in Fig. 2, which shows the formation of N a M N , the anaplerotic step, and eight enzymatic transformations of the six-membered pyridine-nucleotide cycle (Wagner et al. 1986). The reverse reactions for two steps have also been followed; these are the NMN-adenylyltransfer which is the reversal of the pyrophosphorylytic cleavage of NAD, and NaMN-glycohydrolysis which is the reversal of the phosphoribosyltransfer onto nicotinic acid. The enzyme activities were determined in three individual series of experiments; every point in the growth cycle of the different series corresponds to a four-week-old callus which was divided and 20-g portions of it were used as inoculum of agar with growth and induction medium, respectively. The results are shown for two selected enzymes (Fig. 3) and average values are listed for all enzymes in Table 1. Although in the three series of experiments performed the individual data showed slight variations, their qualitative features were the same. Thus the data shown from one experiment are typical. Most of the enzymes (Table 1) displayed activities which increased in the induction medium. The strongest stimulation occurred for Qa-PRTase, the enzyme feeding the pyridine-nucleotide cycle. Its induction factor was almost three when average values are compared; however, on several days of the callus growth (Fig. 3A) the activity was enhanced 5- to 10-fold. The NAD-pyrophosphatase (NAD-PPase), which cleaves N A D to N M N , was also stimulated up to twofold; the same holds for NaMN-glycohydrolase (NaMN-GHase), the bypass enzyme which transforms N a M N directly to nicotinic acid (data not shown). Three enzymes (Table 1) were reduced in activity upon stimulation of nicotine production. These were NMN-adenylyltransferase (NMN-ATase) which transforms N M N back to NAD, NAD-synthetase and nicotinamidase. The strongest reduction was obtained with NMN-ATase which showed a decrease relative to the growth medium of more than four fold on several days (Fig. 3 B).

410

R. Wagner et al. : Enzyme activities of the nicotine pathway. II Qo

1, ~

A

~ NoMN ~

y

No

NaAD

'T

E .L o [I

N

NAD

Fig. 2. Schematic presentation of the pyridine-nucleotide cycle and its enzymatic steps. Qa, quinolinic acid; NaMN, nicotinic acid mononucleotide; NaAD, nicotinic-acid adenine dinucleotide; NMN, nicotinamide mononucleotide; N, nicotinamide; Na, nicotinic acid; (1) Qa-phosphoribosyltransferase; (2) NaMN-adenylyltransferase; (3) NAD-synthetase; (4) NMNadenylyltransferase; (5) NAD-pyrophosphatase; (6) NMN-glycohydrolase; (7) nicotinamidase; (8) Na-phosphoribosyltransferase; (9) NaMN-glycohydrolase. The figure also illustrates the regulation of the pyridine-nucleotide cycle upon induction of nicotine synthesis. Thick arrows show the enzymatic steps which are increased in activity; for further explanation see text

10

20 days

30

15 B

10 'T 0~ E

NaMN/NMN-transforming isoenzymes. In Table 1, two pairs of enzymes are listed which transform either N a M N or N M N . In our previous work (Wagner and Wagner 1985; Wagner et al. 1986) we suggested that in each case the same enzyme entity could accept both substrates. The changes obtained upon nicotine induction, however, displayed dissimilarities. On the one hand the glycohydrolases behaved very similarly with an induction factor of about 1.25 indicating that in this case a single enzyme could be responsible. On the other hand the adenylyltransferases behaved differently. The NaMN-specific activity was slightly enhanced whereas the activity of N M N - A T a s e was strongly reduced. This is indicative of the presence of isoenzymes with different substrate selectivity which may be regulated differently upon the induction of nicotine synthesis.

Influence of nicotinic acid of the medium. Murashige-Skoog medium contains 0 . 5 m g nicotinic acid per liter. As nicotinic acid is a constituent of the pyridine-nucleotide cycle its influence on nicotine production and enzyme activities should be determined. In Table 1 the values in parenthesis are derived from an experiment in which nicotinic acid was omitted in both the growth and induction medium. This experiment was repeated once; the

.x o_

0

10

2O days

}0

Fig. 3A, B. Activities of Qa-PRTase (A) and NMN-ATase (B) in the growth cycle of tobacco calli; (o) growth medium, (o) induction medium. Every point in the figure represents a separate experiment, i.e. a separate inoculum of 20 g (FW) callus tissue at the start of the growth cycle. Enzyme activities were determined as described in Material and methods

data of both series showed the same qualitative features with only slight variations. Omission of nicotinic acid revealed a slight reduction of growth in both media and a slightly higher protein content (per g FW) which resulted in general in lower values of the specific enzyme activities (Table 1). The only exception was N M N GHase. Nicotine production was also slightly reduced in nicotinic-acid-free medium (Table 1). On the other hand the changes of enzyme activities generated by nicotine induction were very similar to those obtained in the medium containing nicotinic acid. The induction factors (Table 1) were

R. Wagner et al. : Enzyme activities of the nicotine pathway. II

41 t

Table 1. Specific activities (pkat-mg-a) and nicotine content (~tmol-g-1 DW) of the calli in the growth and induction medium. The values are mean values averaged throughout the growth cycle (35 days with six or seven determinations). The values in parentheses are from an experiment performed with the omission of nicotinic acid from the medium. Induction factors are obtained by dividing the values of the induction medium by those of the growth medium. Induction factor based on NAD-synthetase is obtained from the mean values of the induction factors by dividing by mean value (0.6) of NAD-synthase Growth medium

Qa-PRTase NaMN-ATase NMN-ATase NAD-synthetase NAD-PPase NaMN-GHase NMN-GHase Nicotinamidase Na-PRTase Nicotine

2.6 (2.2) 88 (68) 8.8 (2.0) 31 (23) 2,850 (2,150) 65 (30) 73 (89) 150 (123) 71 (65) 0.23 (0.24)

Induction medium

7.1 (4.8) 97 (84) 3.5 (1.3) 21 (11) 4,030 (2,630) 83 (45) 89 (119) 86 (91) 110 (75) 1.54 (1.0)

very much the same. The only obvious large difference was for nicotinic-acid phosphoribosyltransferase (Na-PRTase). However, this enzyme is also the only one which showed a higher variance when experiments with different calli were compared. Thus the difference shown in Table 1 for this enzyme may not be meaningful. The nicotinic acid content of the 100 ml medium used for callus growth corresponds to about 0.4 gmol. The nicotine content of the whole callus at day 2 (compare Fig. 1 of the preceding paper) was 0.65 gmol in the induction medium relative to 0.25 gmol in the growth medium. This indicates that the relatively low amount of nicotinic acid in the medium should be consumed very early in the growth phase (provided it is available by diffusion) and should affect enzyme activities only in this early phase. A comparison of the early effects with and without nicotinic acid on the enzyme activities is difficult to interpret although the data for N a M N - A T a s e and NAD-PPase seem to indicate an earlier induction in the absence of nicotinic acid.

Strategy of enzyme induction. The induction factors of the different enzymes (Table 1) indicate stimulation and reduction of individual activities. The activities are based on 1 mg of protein of the extract. In a previous report we have shown (Wagner and Wagner 1985) that NAD-synthetase is a rather constitutive enzyme, whose activity did not change appreciably in tissues with marked differences in their capacity for nicotine production. If one relates the induction factors to the NAD-synthetase activity (Table 1, last column) one can make the following suggestions. The enzymes NMN-ATase, nicotinamidase and of course NAD-synthetase can

Induction factor

Induction factor based on NAD-synthetase

2.7 1.1 0.4 0.7 1.4 1.3 1.2 0.6 1.6 6.7

4.1 1.9 0.9 i 2.2 2.3 2.1 1.1 -

(2.2) (1.2) (0.65) (0.5) (1.2) (1.5) (1.3) (0.7) (1.15) (4.2)

be considered to be constitutive enzymes whose activity is not enhanced upon induction of nicotine synthesis; Na-PRTase can also be included for the reasons discussed above. Four enzymes have an induction factor of about two; these are N a M N ATase, NAD-PPase, N a M N - G H a s e and N M N GHase, whereas Qa-PRTase has an induction factor of about 4. These data can be interpreted as follows (Fig. 2): Qa-PRTase is the most critical enzyme, i.e. it is the bottleneck for the production of N A D and nicotine. This is also obvious upon comparison of the absolute enzyme activities (Table 1) and is in accord with previous suggestions (Saunders and Bush 1979; Mann and Byerrum 1974). Apart from regulation at this point, four other enzymes are co-regulated although less stringently. These are NaMN-ATase, NAD-PPase and N M N - G H a s e which operate to enhance the flux from N a M N via N A D in the direction of nicotinamide and nicotinic acid. On the other hand, the activity of N a M N - G H a s e , which opens the direct way from N a M N to nicotinic acid, is also enhanced. One should remember that N a M N - G I t a s e has a rather high Km value for N a M N (4 mmol.1-a) (Wagner et al. 1986); this ensures that the pool of N a M N is not completely depleted and a threshold amount is maintained for N A D production. A high rate of nicotine production requires an enhanced activity of the pyridine-nucleotide cycle. This is performed by stimulation of the anaplerotic enzyme, Qa-PRTase, which produces a larger pool of N a M N , and a consumption of the latter by two fluxes, a direct route to nicotinic acid and a second indirect one via N A D and N M N . This again indicates that it is the metabolite nicotinic acid which is provided by the pyridine-nucleotide cycle and

412


mp

-0.5

i200

l

g

10 - - 0 . 4

PMT

7 "~3 150

-0.3

2 o o

0 rll

_Y Ix 100

-0.2

Qo-P RTase

"7

E :k

50

10

20

30

days

Fig. 4. A comparison of enzyme activities and pool sizes during induction of nicotine production in tobacco calli. The values are total activities of the whole calli and total methylputrescine and nicotine content of the calli. The data of putrescine methyltransferase (PMT) (o), methylputrescine (nap) (n) and nicotine (ni) (B) are from the preceding paper (Feth et al. 1986). The data for Qa-PRTase (e) are from Fig. 3A

consumed by condensation with methylpyrroline to give nicotine. Previous results on labeled-NAD feeding also pointed to nicotinamide or nicotinic acid as possible precursors (Yasumatsu and Murayama 1973). The concerted action of the two routes of the nicotine pathway. In Fig. 4 the activity of the main control enzymes of both routes of the nicotine pathway are presented together with the pool values of methylputrescine and nicotine. It is obvious that the stimulation of these enzyme activities occurs rather early in the growth cycle with Qa-PRTase slightly in advance of PMT. The increase of the pool of methylputrescine correlates well with the increased activity of the coordinating enzyme (PMT). The enzyme activities are integral values related to the whole growing callus (this is to correct for the dilution due to growth). The data of Fig. 4 show that the enzyme activities increase strongly during the early growth phases; this can be interpreted to indicate that induction of nicotine

synthesis is generated by a stimulation of protein synthesis of the control enzymes. The decline of the enzyme activities during the later stages may indicate that these enzymes undergo a turnover and are degraded again when enzyme synthesis comes to a halt. Methylputrescine is obviously not affected by a turnover, as its pool is maintained although the activity of PMT declines. It is also interesting that the course of the methylputrescine pool roughly parallels that of nicotine, although their absolute values are very different. We have no data on the pool sizes of methylpyrroline and nicotinic acid, or on the activity of the condensing enzyme. The latter step could have a final bottleneck function in nicotine production. The rather late, strong increase in the nicotine content could indicate a different time scale for the induction of this enzyme. Conclusions. The present callus induction system is certainly a special system and may not be totally identical to the situation when tobacco root tissue differentiates and expresses the enzymes for nicotine production. Furthermore, the enzyme activities have been determined in vitro, i.e. in protein extracts, and this does not take into account that enzyme activities may be confined to cellular compartments. For example, the high pyrophosphorylic activity which cleaves N A D should not be in the same compartment as the main pool of NAD, otherwise it would be difficult to envisage how a functional N A D pool could be maintained. With these reservations the presented data from the callus system, nevertheless, illustrate the peculiar regulatory phenomenon of a secondaryproduct pathway which is bifurcated and closely connected with the primary metabolism. The data show that both routes are strictly regulated and obviously tuned to each other. For the route leading to methylpyrroline it is the activity of PMT which is very stringently controlled, whereas with the pyridine-nucleotide cycle it is the anaplerotic enzyme, Qa-PRTase. However, in both routes, further enzyme activities are co-regulated although in a less stringent way. The pyridine-nucleotide system has two possible routes leading to nicotinic acid, the metabolite which is most likely used for condensation with methylpyrroline. One enzymatic route takes advantage of the fact that N A D is involved in a rather rapid turnover, as the enhanced nicotine production implies stimulation of synthesis and degradation of NAD. The second route is a direct one using one enzymatic step which transforms N a M N to nicotinic acid.


413

We are grateful to Dr. V. Wray for linguistic advice, to Mrs. H. Starke for typing the manuscript and to Mrs. C. Lippelt and Mrs. K. Wagner for preparing the graphs. This work has been supported by the Fonds der Chemischen Industrie.

acid phosphoribosyltransferase in tobacco. Phytochemistry 23, 1881-1883 Wagner, R., Wagner, K.G. (1985) The pyridine-nucleotidecycle in tobacco. Enzyme activities for the de-novo synthesis of NAD. Planta 165, 532-537 Wagner, R., Feth, F., Wagner, K.G. (1986) The pyridine-nucleotide cycle in tobacco. Enzyme activities for the recycling of NAD. Planta 16/, 226-232 Waller, G.R., Dermer, O.C. (1981) Enzymology of alkaloid metabolism in plants and microorganisms. In: The biochemistry of plants, vol. 7, pp. 317M02, Stumpf, P.K., Conn, E.E., eds. Academic Press, New York London Yasumatsu, N., Murayama, T. (1973) Effect of 2,4-D upon the incorporation of pyridine nucleotides into nicotine by excised root cultures of N. rustica. Bull. Hatano Tobacco Exp. Stn. 73, 225-232

References Feth, F., Wagner, R., Wagner, K.G. (1986) Regulation in tobacco callus of enzyme activities of the nicotine pathway. I. The route ornithine to methylpyrroline. Planta 168, 402~407 Mann, D.F., Byerrum, R.U. (1974) Activation of the de novo pathway for pyridine nucleotide biosynthesis prior to ricinine biosynthesis in castor beans. Plant Physiol. 53, 603-609 Saunders, J.W., Bush, L.P. (1979) Nicotine biosynthetic enzyme activities in Nicotiana tabaeum L. Genotypes with different alkaloid levels. Plant Physiol. 64, 236-240 Wagner, R., Wagner, K.G. (1984) Determination of quinolinic

Received 17 February; accepted 8 April 1986