Planta
Planta (1982) 155:204-211
9 Springer-Verlag 1982
Histochemical localization of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase in seeds and shoots of Triticum Marcjanna Bartkiewicz and Halina Sierakowska Institute of Biochemistry and Biophysics, Academy of Sciences, Rakowiecka 36, PL-02-532 Warszawa, Poland
Abstract. The activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common substrate, p-nitrophenyl thyrnidine 5'-phosphate and its histochemical analogue, AS-BInaphthyl thymidine 5'-phosphate, were determined with the aid of relatively specific inhibitors, NAD and 2',3'-cAMP, respectively. These inhibitors were utilized to reexamine wheat (Triticum aestivum L. cv. Mironovska 808) seeds and 3-5-d old shoots for the occurrence and histochemical localization of nucleotide pyrophosphatase, and to establish the localization of cyclic nucleotide phosphodiesterase. Nucleotide pyrophosphatase is a cytoplasmic enzyme found to be particularly active in the coleoptile epidermis and hypodermis, leaf mesophyll, as well as in developing fibres and phloem. Cyclic nucleotide phosphodiesterase is also a cytoplasmic enzyme active in the shoot vascular bundles, particularly the xylem, and in the seed. Within the seed it is highly active in the crushed cell layer adjacent to the scutellum and in endosperm cells adjacent to the aleurone layer. Within the embryo, cyclic nucleotide phosphodiesterase is most active in epithelial cells adjacent to the crushed cell layer, the suspensor, radicle and root-cap, as well as in the pro-vascular tissues of the scutellum. Key words: Cytochemistry - Nucleotide phosphodiesterase (cyclic) - Nucleotide pyrophosphatase - TritiCUgYt.
Introduction Nucleotide pyrophosphatase, purified from potato tubers (Kornberg and Pricer 1950; Razzell 1968 ; Ashton and Polya 1975; Kole et al. 1976), cleaves not only the pyrophosphate linkage in nucleotide pyrophosphates, e.g., NAD + or ATP, but also the phosphodiester linkage in aryl esters of nucleoside 5'-phos-
0032-0935/82/0155/0204/$01.60
phates (Razzell 1968; Kole et al. 1976). This ability to hydrolyze c~-naphthyl and AS-BI-naphthyl nucleoside Y-phosphates has already been exploited to devise a procedure for its histochemical localization in higher plant tissues by the simultaneous diazo coupling technique (Sierakowska et al. 1978; Gahan et al. 1979). Unfortunately, this procedure did not take into account reports of another plant enzyme, cyclic nucleotide phosphodiesterase, which also hydrolyzed the p-nitrophenyl ester of nucleoside Y-phosphate (Shinshi et al. 1976) and bis-p-nitrophenylphosphate (Ashton and Polya 1975). Studies subsequently undertaken (to be reported elsewhere) confirmed and extended the findings of Ashton and Polya (1975) that potato tubers contain two activities, nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase, which are able to cleave (at significantly different rates) the following types of linkages: a) pyrophosphate in nucleotide pyrophosphates and b) phosphodiester in aryl esters of nucleoside 3'-phosphates, nucleoside Y-phosphates, arylphosphate and orthophosphate as well as in cyclic 2I,Y - and 3',5'-nucleoside phosphates. Nucleotide pyrophosphatase hydrolyzes preferentially the nucleotide pyrophosphate linkage, exhibiting relatively less activity toward the phosphodiester linkage, particularly in cyclic nucleotides, while cyclic phosphodiesterase cleaves preferentially the phosphodiester bond in cyclic nucleotides and synthetic esters of nucleotides. The ability of both enzymes to cleave the c~-naphthyl and AS-BI-naphthyl thymidine 5'-phosphates is of major concern, since the activity localized with the aid of these substrates has been attributed solely to nucleotide pyrophosphatase (Sierakowska et al. 1978; Gahan et al. 1979). This is all the more disturbing in that cyclic nucleotide phosphodiesterase has been found in many higher plants (Lin and Varner 1972; Brewin and Northcote 1973; Vandepeute et al.
M. Bartkiewicz and H. Sierakowska : Localization of nucleotide pyrophosphatase in Triticum
1973; Shinshi et al. 1976; Matsuzaki and Hashimoto 1981), whereas there is no conclusive evidence for nucleotide pyrophosphatase activity outside the potato tuber. Nucleotide pyrophosphatase activity reported for other plant species (Roberts 1959 ; Clayton and Hanselman 1960; Razzell 1966) may be attributed equally well to cyclic nucleotide phosphodiesterase, since both enzymes exhibit similarities not only in specificity, but in many other properties, e.g., pH optimum and cation requirements. This paper represents an effort to distinguish between the activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common synthetic substrate with the aid of relatively specific inhibitors. Moreover, it utilizes these inhibitors to reexamine the occurrence and histochemical localization in wheat tissues of nucleotide pyrophosphatase as well as to determine the localization of cyclic nucleotide phosphodiesterase.
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The p-nitrophenol liberated from p-nitrophenyl thymidine 5'-phosphate was assayed according to Razzell and Khorana (1961). To prevent turbidity with wheat germ homogenate, the incubation was terminated after 30 min by addition of 0.6 ml cooled water and, after rapid centrifugation0 0.5 ml supernatant was mixed with 0.1 ml 1 M NaOH and read at 400 rim. Cytochemistry. Serial sections of shoots were incubated for 30 min and like sections of seeds for 60 rain at 37 ~ C in 0.1 M acetate buffer p H 5.2, 5 m M AS-BI-naphthyl thymidine 5'-phosphate, 10 mM ethylenediaminetetraacetic acid (EDTA), and 0.2% w/v filtered Fast Garnet GBC with a) no additions; b) 1 or 2 m M N A D + ; 3 or 5 mM 2',3'-cAMP. The standard controls were sections incubated in the absence of substrate. Additional controls were preincubated for 30 min at 37 ~ C in excess 0.1 M acetate buffer, pH 5.2, and, after rinsing, incubated in the whole medium. After incubation, the sections were rinsed in distilled water, fixed for 10 min in 4% formalin, rinsed in water again and mounted in Farrants' medium.
Results
Biochemical assays. NAD + and 2',31-cAMP were seMaterial and methods Material p-Nitrophenyl thymidine Y-phosphate was purchased from Sigma Chemical Co. (St. Louis, Mo., USA), while AS-BI-naphthyl thymidine 5'-phosphate was synthesized, as previously described (Sierakowska et al. 1978). All other reagents were commercial preparations. Nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase from potato tubers were isolated according to the procedure of Bartkiewicz et al. (in preparation). Unfixed frozen sections were prepared from soaked Triticum sp. seeds and, unless otherwise specified, from shoots of 3- or 5-d old seedlings, t0-12 mm from the base, according to Gahan et al. (1967). Methods Assays of enzyme activities. All incubations were carried out for 15 or 30 rain at 37 ~ C. The effects of NAD + and 2',3'-cAMP on the activities of purified potato tuber nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase were assayed in 0.0%0.1 ml 0.1 M Tris-acetate buffer, pH 6.0, containing 5 m M p-nitrophenyl thymidine 5'-phosphate or AS-BI-naphthyl thymidine 5'-phosphate, the enzyme and a) no additives; b) 0.5-5 m M N A D + ; c) 0.5-5 m M T,3'-cAMP. The effects of NAD + and 2',3'-cAMP on nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase activities of wheat germ and coleoptile homogenates were assayed in 0.1 ml 0.1 M Tris-acetate buffer, pH 4.0-7.5, containing 5 m M p-nitrophenyl thymidine Y-phosphate, 10 m M EDTA, either 0.025 ml 1.5% imbibed germ homogenate or 0.025 ml 2% 3-d old coleoptile homogenate, and a) no additives; b) 2 mM N A D + ; c) 5 mM 2',T-cAMP. The AS-BI-naphthyl liberated from AS-BI-naphthyl thymidine-5'-phosphate was assayed by terminating the incubation with sequential addition, to 0.1 ml incubation medium, of 0.5 ml ethanol, 1 ml 0.2 M acetate buffer pH 5.2, and 0.25 ml filtered aqueous Fast Garnet GBC, 7.5 mg/ml. After 10 rain 0.25 ml 40% trichloroactic acid (TCA) was added and the dye extracted with 1.5 ml toluene and read at 540 nm.
lected as potentially specific inhibitors of potato tuber nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase, respectively, on the basis of differences in enzymes affinities toward these substrates. The Km values for NAD + for nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase were 0.2 and 1.6 raM, respectively, whereas those for 2',3'-cAMP were 0.9 and 0.4 raM, respectively. Figure 1 illustrates the effects of NAD + and 2/,31-cAMP on the cleavage of p-nitrophenyl and ASBI-naphthyl thymidine 5r-phosphates by potato tuber nucleotide pyrophosphatase and cyclic nucteotide phosphodiesterase, purified over 500-fold and 6,000-fold, respectively. NAD + is obviously an effective selective inhibitor; at 2 mM it inhibits over 85% of the nucleotide pyrophosphatase activity and less than 10% of the cyclic nucleotide phosphodiesterase activity against either substrate (Fig. l a and c). 2',3'-cAMP is somewhat less effective and less selective, and at 5 mM it inhibits cyclic nucleotide phosphodiesterase activity about 90%, but nucleotide pyrophosphatase activity 50% with AS-BI-napththyl thymidine Y-phosphate (Fig. 1 d). With the p-nitrophenyl substrate (Fig. 1 b), it inhibits cyclic nucleotide phosphodiesterase activity by about 75% and nucleotide pyrophosphatase activity by about 20%. These relatively specific inhibitors of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase from potato tubers were subsequently applied to assays of activity against aryl esters of nucleotide5'-phosphates in homogenates of various wheat tissues in order to examine sensitivity to the inhibitors and thus identify the nature of the activity. Figure 2 illustrates the effects of addition of 2 mM
206
M. Bartkiewicz and H. Sierakowska: Localization of nucleotide pyrophosphatase in Triticum
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Fig. l a - & The effects of N A D + (a, e) and 2',3'-cAMP (b, d) on the activities of purified potato tuber nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase activities against p-nitrophenyl thymidine 5'-phosphate (a, b) and AS-BI-naphthyl thymidine Y-phosphate (e, d). nucleotide pyrophosphatase; . . . . 9 . . . . 9 . . . . cyclic nucleotide phosphodiesterase
Fig. 2a, b. The effects of N A D + and 2',3"-cAMP on the activity of wheat germ (a) and coleoptile (b) homogenate against p-nitrophenyl thymidine 5'-phosphate at pH 4.0-7.5. no additives; --- o o --with 2 mM "NAD § ; . . . . 9 . . . . 9 . . . . with 5 m M 2',3'-cAMP
NAD-- and 5 mM 2',3'-cAMP on the activities of wheat germ and coleoptile homogenates against pnitrophenyl thymidine 5'-phosphate at pH values 4.0-7.5. Figure 2a shows that the activity of wheat germ homogenate against p-nitrophenyl thymidine 5'-phosphate at pH 5.0 is reduced in the presence of 5 mM T,3'-cAMP to 35% of the control value and remains insensitive to 2 mM NAD § By contrast, the homogenate of wheat coleoptile (Fig. 2b) under similar conditions exhibits, at pH 5.0 in the presence of 5 mM 2',3'-cAMP, only 11% inhibition and, in the presence of 2 mM NAD § as much as 62% inhibition. The homogenate of wheat shoots (not shown) resembles that of the coleoptile, but is somewhat more sensitive to the presence of 2',3'-cAMP and less sensitive to the presence of NAD § being inhibited at pH 5.0 by 17% and 58%, respectively. Since the results in Fig. 2 indicate a relatively broad pH optimum for the homogenate activities against p-nitrophenyl thymidine 5'-phosphate
(4.5-6.0) and optimal inhibitory effects of both NAD + and T,3'-cAMP at pH values 4.5-5.5, analogous experiments with AS-BI-naphthyl thymidine 5'-phosphate, the histochemical substrate, were performed at pH 5.2, optimal both from the point of selective inhibition and the histochemical reaction. Under these conditions the activity of the wheat germ homogenate was inhibited 47% by 5 mM 2',3'-cAMP and 22% by 2 mM NAD +. The activity of coleoptiles was inhibited 29% by 5 mM 2',3'-cAMP and 77% by 2 mM NAD + The response of wheat germ activity clearly corresponds to that of potato cyclic nucleotide phosphodiesterase, while that of wheat Shoot, and, even more so of wheat coleoptile, corresponds to the behavior of potato nucleotide pyrophosphatase (Fig. 1). This indicates that the germ tissues contain mainly cyclic nucleotide phosphodiesterase and the wheat seedlings mainly nucleotide pyrophosphatase, which predominates in the shoot and particularly in the coleoptile.
Fig. 3a-c. Section of imbibed Triticum scutellum (S) and embryo incubated for 60 min with AS-BI-naphthyl thymidine 5'-phosphate. X indicates false positive reaction in controls devoid of substrate. Bar=0.2 mm. Magnification x 50. a No additives. Good cytoplasmic reaction in all cells of the rootcap (RT), radicle (R) and coleorhiza (CO). Note particularly high activity in the pro-vascular tissue (PV), suspensor (SU) and epithelial layer (E), especially at ends adjacent to heavily reacted crushed cell layer (arrows). b with l mM NAD +. Reaction essentially as in a. e with 5 mM 2',Y-cAMP. Lack of activity at sites responding in a Fig. 4a-e. Section of imbibed Triticum seed incubated for 60 rain with AS-BI-naphtbyl thymidine Y-phosphate. X is a false positive reaction. Bar=20 gm. Magnification x 390. a No additives. Granular cytoplasmic product is seen in cells of the aleurone layer (A) and starchy endosperm adjacent to the aleurone layer (arrows, E). b with 1 mM NAD +. Response essentially as in a. e with 5 mM T,Y-cAMP. Lack of activity at sites responding in a
208
M. Bartkiewiczand H. Sierakowska: Localizationof nucleotidepyrophosphatasein Triticum
Table 1. Times of incubation with AS-BI-naphthyl thymidine 5'-phosphate at 37~ C required for observable deposition of reaction product in imbibed Triticurn seeds Tissue
Time (rain)
Endosperm aleurone layer endosperm cells adjacent to aleurone layer inner endosperm cells crushed cell layer
15 5 45 1
Embryo scutellum epithelial cells adjacent to the crushed cell layer apical cells pro-vascular tissue suspensor coleorhiza rootcap radicle plumule coleoptile
5 10 10 5 15 10 10 45 45
Cytochemistry. Having shown the presence in wheat germ of an activity resembling cyclic nucleotide phosphodiesterase, and in wheat shoots of one resembling nucleotide pyrophosphatase, we proceeded to check these findings at the histochemical level and to examine the localization pattern of both activities in wheat tissues, with the aid of a common substrate and the inhibitors. Figures 3 and 4 illustrate the localization pattern of sections of imbibed wheat seeds reacted against AS-BI-naphthyl thymidine 5'-phosphate alone and in the presence of either NAD § or 2',3'-cAMP. The reactions against AS-BI-naphthyl thymidine 5'-phosphate of the scutellum and embryo (Fig. 3a) and of the endosperm (Fig. 4a) are evaluated in Table 1 in terms of the minimum incubation time necessary for the detectable deposition of the precipitate. The intracellular activity is normally confined to the cytoplasm, as reported previously (Gahan et al. 1979). Figures 3b and 4b show that incubation of similar sections in the presence of 1 mM NAD + has no appreciable effect on any of the active sites shown in Figs. 3a and 4a and listed in Table 1. NAD + ( ~ 3 raM) likewise exerted no effect. By contrast, sections incubated in the presence of 5 mM 2',3'-cAMP (Figs. 3c and 4c) show complete lack of activity at all responding sites shown in Figs. 3 ~ b and 4a-b; a similar effect is observable with 3 mM 2',3"-cAMp. These results indicate that the activity against AS-BInaphthyl thymidine 5'-phosphate observable in the embryo and scutellum (Fig. 3a), and in the endosperm (Fig. 4a), is due to cyclic nucleotide phosphodiesterase.
Figure 5 presents sections of 5-d old wheat shoot, and Fig. 6 of 3-d old coleoptile, reacted against ASBI-naphthyl thymidine Y-phosphate alone (Figs. 5a and 6 a) or in the presence of either N A D § (Figs. 5 b and 6b) or 2',3'-cAMP (Figs. 5c and 6c). The localization in Figs. 5a and 6a corresponds to that described by Sierakowska et al. (1978) and shows activity in the following tissues of the leaves and coleoptile, in order of diminishing intensity: a) leaf: fibres, xylem, mesophyll, phloem, abaxial epidermis, adaxial epidermis; b) coleoptile : outer epidermis, hypodermis, xylem, phloem, cortex, inner epidermis and bundle sheath. Addition of 1 m M N A D § to the incubation medium causes the disappearance of nearly all activity from the less active sites and partial inhibition of the more active ones, with obviously less inhibition in the xylem (see Figs. 5b and 6b). NAD § at 2 mM inhibits the overall activity even more effectively, again with least inhibition in the xylem. The localization pattern in the presence of 5 mM 2',3'-cAMP (Figs. 5c and 6c) differs from that in Figs. 5a and 6a in that there is about 50% reduction in overall activity with an appreciably greater decrease in activity in the xylem. A similar, though smaller, decrease in overall activity is obtained in the presence of 3 m M 2',3'-cAMP. These results indicate that all tissues of the leaves and coleoptile, with the exception of the vascular tissue, reveal the presence of only nucleotide pyrophosphatase. Within the vascular bundles there is also appreciable cyclic nucleotide phosphodiesterase activity, particularly in the xylem, where it surpasses nucleotide pyrophosphatase activity. Figure 7 gives more detailed information on these two activities in the vascular tissue. Incubation in the absence of inhibitors (Fig. 7a) results in the deposition of considerably more reaction product in the xylem than in the phloem region. Addition of 1 mM NAD + to the incubation medium (Fig. 7b) leads to inhibition of about two-thirds of the phloem activity and of approximately one-third of the xylem activity, whereas addition of 5 mM 2',3'-cAMP gives the reverse effect (Fig. 7 c). The deposition of reaction product in the xylem and phloem become comparable, and it appears that the xylem activity is inhibited by over two-thirds, and the phloem activity appreciably less. This indicates the presence in the xylem and phloem of both activities (see Discussion), with predominance in the phloem of nucleotide pyrophosphatase and in the xylem of cyclic nucleotide phosphodiesterase. Control sections of both seed and shoot incubated without substrate were negative. Seed and shoot sections preincubated in buffer exhibited no change in localization pattern or amount of product, indicating
Fig. 5a-c. Section of 5-d old wheat shoot incubated for 30 min with AS-BI-naphthyl thymidine 5'-phosphate. Bar = 0.2 mm. Magnification x 55. a no additives. Relatively inactive coleoptile (C), highly active first leaf mesophyll and moderately active second leaf mesophyll. Note the particularly heavily reacted fibres (F) and vascular bundIes (VB). b with 1 mM NAD +. Striking decrease in response of leaf mesophylI and of fibres (F). Note the appreciable activity of the vascular bundles (VB), particularly the xylem (J(Y) which has gained relative prominence, e with 5 mM 2",Y-cAMP. Moderate decrease in mesophyll and fibre (F) activity with relatively greater decrease in xylem (XY) activity Fig. 6a-c. Section of 3-d old coleoptile incubated for 30 min with AS-BI-naphthyl thymidine 5'-phosphate. Bar=40 gm. Magnification x 200. a no additives. Highly responding epidermis (E) and hypodermis (H) with relatively less active cortex and inner epidermis (IE). b with 1 mM NAD+. Uniformly greatly diminished response in all tissues, c with 5 mM 2',Y-cAMP. Reaction essentially as in a but of somewhat diminished intensity
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M. Bartkiewicz and H. Sierakowska : Localization of nucleotide pyrophosphatase in Triticum
Fig. 7a-c. Vascular bundle of first leaf of 5-d old wheat shoot incubated for 30 min with AS-BI-naphthyl thymidine 5'-phosphate. Bar= 10 gin. Magnification • 450. a no additive. Highly active xylem (XY) region with somewhat less activity in the phloem (P). b with 1 mM NAD +. Greatly diminished response of the phloem (P) and moderately decreased response of the xylem (XI/). e with 5 mM 2',3'-cAMP. Greatly diminished response of the xylem (XY) and moderately diminished response of the phloem (P)
no appreciable loss of either cyclic nucleotide phosphodiesterase or nucleotide pyrophosphatase activites due to enzyme diffusion. Discussion
The foregoing results show that, by taking advantage of the relative specificities of NAD § and 2',3'-cAMP as inhibitors of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase activities against aryl esters of nucleoside 5'-phosphates, it is possible to detect both activities in wheat tissues and to distinguish between them at the histochemical level. The selectivity of inhibitors is not sufficient to fully exclude the possibility that the active sites attributed to nucleotide pyrophosphatase contain some cyclic nucleotide phosphodiesterase activity, and vice versa. Nonetheless, the results in Figs. 1 and 2 justify the adoption of the following criteria for evaluating the relative amounts of nucleotdie pyrophosphatase and cyclic nucleotide phosphodiesterase activities in tissue sections : 1. Insensitivity to 2 mM NAD § and almost complete inhibition by 2-5 mM 2',3'-cAMP indicates cyclic nucleotide phosphodiesterase activity. 2. Nearly complete inhibition by NAD § and about 50% inhbition by 2',3'-cAMP indicates nucleotide pyrophosphatase activity. 3. Approximately 50% inhibition by NAD + and about 75% inhibition by 2',3"-cAMP indicates equal contribution by both enzymes, and deviations toward 1 or 2 point to predominance of either cyclic nucleotide phosphodiesterase or nucleotide pyrophosphatase, respectively. On this basis we conclude that nucleotide pyrophosphatase occurs in the coleoptile and leaves, where it is found in the cytoplasm of all cells, and is particu-
larly active in the coleoptile epidermis and hypodermis, as well as in the leaf mesophyll and developing fibers. The relative activity of nucleotide pyrophosphatase in wheat shoot tissues is essentially as reported previously (Sierakowska et al. 1978), except that the major part of the xylem and a minor part of the phloem activities of the coleoptile and leaves are due to cyclic nucleotide phosphodiesterase activity. There appears to be a correlation between the metabolic activity of the tissue and its nucleotide pyrophosphatase content. The activity of the mesophyll becomes apparent when the leaf adopts its role, and that of the fiber-forming cells is coincidental with fiber formation, diminishing upon its completion. Similarly, the activity of the coleoptile epidermis decreases with the coleoptile's loss of protective function and aging. Cyclic nucleotide phosphodiesterase, on the other hand, in addition to its vascular activity in the coleoptile and the leaves, occurs in the seed. In fact, all seed activity against AS-BI-naphthyl thymidine Y-phosphate, described by Gahan et al. (1979), and attributed to nucleotide pyrophosphatase, must now be ascribed to cyclic nucleotide phosphodiesterase. This includes the cytoplasmic activity in the aleurone layer and adjacent endosperm, the crushed cell layer, the scutellum with its pro-vascular elements, and the embryo. Particularly high levels of the enzyme appear to be associated with ceils mobilizing their content, e.g., endosperm, and with vascular functions. With respect to inhibitors, it may be noted that inhibition of wheat homogenate activities appears to be less effective than that quantitatively observed in tissue sections. This may be due to two factors. First, homogenate assays involve more tissue, with more enzyme(s) potentially acting on the inhibitors and rendering them less effective. Second, the histochemical
M. Bartkiewicz and H. Sierakowska: Localization of nucleotide pyrophosphatase in pattern discloses sites o f relatively high activity of, m o s t likely, a single enzyme and the full extent of its inhibition, w i t h o u t registering b a c k g r o u n d activity o f the other enzyme, whereas h o m o g e n a t e assays give the sum o f b o t h activities and their median inhibition. A n o t h e r aspect of our procedure concerns the rates of cleavage of the synthetic substrate by nucleotide p y r o p h o s p h a t a s e and cyclic nucleotide p h o s p h o diesterase. The relative rates of cleavage o f AS-BIn a p h t h y l thymidine-5'-phosphate and the natural substrates are indicative o f the accuracy with which the synthetic substrate reflects the physiological activities o f the two enzymes. Cyclic nucleotide phosphodiesterase f r o m p o t a t o cleaves A S - B I - n a p h t h y l thymidine 5'-phosphate at a rate c o m p a r a b l e to that for 3',5'-cAMP, its natural substrate, while the p o t a t o nucleotide p y r o p h o s p h a t a s e cleaves the A S - B I - n a p h thyl derivative at 13% o f the rate for N A D § its p r e s u m e d natural substrate (in preparation). Hence, the histochemical substrate adequately reflects the activity o f cyclic nucleotide phosphodiesterase, and, to a lesser degree, that o f nucleotide p y r o p h o s p h a t a s e . Consequently, assuming that the rates o f hydrolysis for the p o t a t o enzymes are applicable to wheat activities, c o m p a r i s o n of the physiological activities o f nucleotide p y r o p h o s p h a t a s e and cyclic nucleotide phosphodiesterase in a wheat section m a y require multiplying 8-fold the observed activity of nucleotide p y r o p h o s p h a t a s e . This would indicate that wheat shoots contain m o r e nucleotide p y r o p h o s p h a t a s e activity than is histochemically observed and that nucleotide p y r o p h o s p h a t a s e m a y actually p r e d o m i n a t e even in the shoot xylem, where the cyclic nucleotide phosphodiesterase appears to prevail. Cyclic nucleotide phosphodiesterase activity p r o b a b l y really predominates only in the seed. The limitations o f the foregoing technique, stemming f r o m the use o f a c o m m o n substrate and imperfect selectivity o f the inhibitors, are quite obvious. However, similarities in specificity, p H optima, ionic requirements, and non-diffusibility o f nucleotide p y r o p h o s p h a t a s e and cyclic nucleotide phosphodiesterase limit the possibilities o f devising alternative cytochemical procedures by the simultaneous coupling azo dye technique. Moreover, there is no other histochemical m e t h o d for nucleotide p y r o p h o s p h a tase; while the m e t h o d for cyclic nucleotide p h o s p h o diesterase in plants (Payne and Bal 1974) is a multistep procedure with inherent d o u b t s as to the localization o f the final p r o d u c t (Pearse 1968, pp. 538, 570). Used for onion tissues without exogenous nucleotidases, it p r o b a b l y demonstrates tissue p h o s p h o m o n o esterases; while with a d d e d nucleotidases, it exhibits nuclear localization, quite likely due to the a d s o r p t i o n o f a free-floating reaction p r o d u c t (Moses et al. 1966).
Triticum
211
We are deepIy indebted to Dr. D. Shugar for his continued guidance and helpful discussion, to Dr. Peter B. Gahan for advice and criticism of the manuscript, and to Dr. Matgorzata Zan-Kowalczewska for help with some phases of this investigation. This investigation was carried out as Project 09.7 of the Polish Academy of Sciences.
References Ashton, A.R., Polya, G.M. (1975) Higher-plant cyclic nucleotide phosphodiesterases, Resolution, partial purification and properties of three phosphodiesterases from potato tuber. Biochem. J. 149, 329-339 Brewin, N.J., Northcote, D.H. (1973) Partial purification of a cyclic AMP phosphodiesterase from soybean callus. Isolation of a non-dialysable inhibitor, Biochim. Biophys. Acta 320, 104-122 Clayton, R.A., Hanselman, L.M. (1960) Tobacco nucleotide pyrophosphatases. Arch. Biochem. Biophys. 87, 161 166 Gahan, P.B., McLean, J., Kalina, M., Sharma, W. (1967) Freezing sectioning of plant tissues: the technique and its use in plant histochemistry. J. Exp. Bot. 18, 151-159 Gahan, P.B., Sierakowska, H., Dawson, A.L. (1979) Nucleotide pyrophosphatase activity in dry and germinated seeds of Triticurn, and its relationship to general acid phosphatase activity. Planta 145, 159-166 Kole, R., Sierakowska, H., Shugar, D. (1976) Novel activity of potato nucleotide pyrophosphatase. Biochim. Biophys. Acta 438, 540 550 Kornberg, A., Pricer, W.E. (1950) Nucleotide pyrophosphatase. J. Biol. Chem. 182, 763-778 Lin, P.P., Varner, J.E. (1972) Cyclic nucleotide phosphodiesterase in pea seedlings. Biochim. Biophys. Acta 276, 454-474 Matsuzaki, H., Hashimoto, Y. (198I) Purification and characterization of acid phosphodiesterases of cultured tobacco cells. Agric. Biol. Chem. 45, 1317-1325 Moses, H.L., Rosenthal, A.S., Beaver, D.L., Schuffman, S.S. (1966) Lead ion and phosphatase histochemistry. II. The effect of adenosine triphosphate hydrolysis by lead ion on the histochemical localization of adenosine triphosphatase activity. J. Histochem. Cytochem. 14, 702-710 Payne, J.F., Bal, A.K. (1974) Histochemical detection of cyclic nucleotide phosphodiesterase activity in germinating onion seed. Plant Sci. Lett. 2, 319-330 Pearse, A.G.E. (1968) Histochemistry, theoretical and applied, vol. 1. J. and A. Churchill, London Razzell, W.E., Khorana, H.G. (1961) Studies on polynucleotides. Enzymic degradation. Some properties and mode of action of spleen phosphodiesterase. J. Biol. Chem. 236, 1144-1149 Razzell, W.E. (1966) Plant tissue phosphodiesterase activities. Biochem. Biophys. Res. Commun. 22, 243 247 Razzell, W.E. (1968) Polynucleotidase activity of animal and plant tissue phosphodiesterases. Can. J. Biochem. 46, 1-7 Roberts, D.W.A. (1959) The hydrolysis of diphosphopyridine nucleotide by juice expressed from wheat leaves. J. Biol. Chem. 234, 655-657 Shinshi, H., Miwa, M., Kato, K., Noguchi, M., Matsushima, T., Sugimura, T. (1976) A novel phosphodiesterase from cultured tobacco cells. Biochemistry 15, 2185-2190 Sierakowska, H., Gahan, P,B., Dawson, A.L. (1978) The cytochemical localization of nucleotide pyrophosphatase activity in plant tissues using naphthyl esters of thymidine-5'-phosphate. Histochem. J. 10, 679 693 Vandepeute, J., Huffaker, R.C., Alvarez, R. (1973) Cyclic nucleotide phosphodiesterase activity in barley seeds. Plant Physiol. 52, 278-282 Received 20 January; accepted 31 March 1982
Planta (1982) 155:204-211
9 Springer-Verlag 1982
Histochemical localization of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase in seeds and shoots of Triticum Marcjanna Bartkiewicz and Halina Sierakowska Institute of Biochemistry and Biophysics, Academy of Sciences, Rakowiecka 36, PL-02-532 Warszawa, Poland
Abstract. The activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common substrate, p-nitrophenyl thyrnidine 5'-phosphate and its histochemical analogue, AS-BInaphthyl thymidine 5'-phosphate, were determined with the aid of relatively specific inhibitors, NAD and 2',3'-cAMP, respectively. These inhibitors were utilized to reexamine wheat (Triticum aestivum L. cv. Mironovska 808) seeds and 3-5-d old shoots for the occurrence and histochemical localization of nucleotide pyrophosphatase, and to establish the localization of cyclic nucleotide phosphodiesterase. Nucleotide pyrophosphatase is a cytoplasmic enzyme found to be particularly active in the coleoptile epidermis and hypodermis, leaf mesophyll, as well as in developing fibres and phloem. Cyclic nucleotide phosphodiesterase is also a cytoplasmic enzyme active in the shoot vascular bundles, particularly the xylem, and in the seed. Within the seed it is highly active in the crushed cell layer adjacent to the scutellum and in endosperm cells adjacent to the aleurone layer. Within the embryo, cyclic nucleotide phosphodiesterase is most active in epithelial cells adjacent to the crushed cell layer, the suspensor, radicle and root-cap, as well as in the pro-vascular tissues of the scutellum. Key words: Cytochemistry - Nucleotide phosphodiesterase (cyclic) - Nucleotide pyrophosphatase - TritiCUgYt.
Introduction Nucleotide pyrophosphatase, purified from potato tubers (Kornberg and Pricer 1950; Razzell 1968 ; Ashton and Polya 1975; Kole et al. 1976), cleaves not only the pyrophosphate linkage in nucleotide pyrophosphates, e.g., NAD + or ATP, but also the phosphodiester linkage in aryl esters of nucleoside 5'-phos-
0032-0935/82/0155/0204/$01.60
phates (Razzell 1968; Kole et al. 1976). This ability to hydrolyze c~-naphthyl and AS-BI-naphthyl nucleoside Y-phosphates has already been exploited to devise a procedure for its histochemical localization in higher plant tissues by the simultaneous diazo coupling technique (Sierakowska et al. 1978; Gahan et al. 1979). Unfortunately, this procedure did not take into account reports of another plant enzyme, cyclic nucleotide phosphodiesterase, which also hydrolyzed the p-nitrophenyl ester of nucleoside Y-phosphate (Shinshi et al. 1976) and bis-p-nitrophenylphosphate (Ashton and Polya 1975). Studies subsequently undertaken (to be reported elsewhere) confirmed and extended the findings of Ashton and Polya (1975) that potato tubers contain two activities, nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase, which are able to cleave (at significantly different rates) the following types of linkages: a) pyrophosphate in nucleotide pyrophosphates and b) phosphodiester in aryl esters of nucleoside 3'-phosphates, nucleoside Y-phosphates, arylphosphate and orthophosphate as well as in cyclic 2I,Y - and 3',5'-nucleoside phosphates. Nucleotide pyrophosphatase hydrolyzes preferentially the nucleotide pyrophosphate linkage, exhibiting relatively less activity toward the phosphodiester linkage, particularly in cyclic nucleotides, while cyclic phosphodiesterase cleaves preferentially the phosphodiester bond in cyclic nucleotides and synthetic esters of nucleotides. The ability of both enzymes to cleave the c~-naphthyl and AS-BI-naphthyl thymidine 5'-phosphates is of major concern, since the activity localized with the aid of these substrates has been attributed solely to nucleotide pyrophosphatase (Sierakowska et al. 1978; Gahan et al. 1979). This is all the more disturbing in that cyclic nucleotide phosphodiesterase has been found in many higher plants (Lin and Varner 1972; Brewin and Northcote 1973; Vandepeute et al.
M. Bartkiewicz and H. Sierakowska : Localization of nucleotide pyrophosphatase in Triticum
1973; Shinshi et al. 1976; Matsuzaki and Hashimoto 1981), whereas there is no conclusive evidence for nucleotide pyrophosphatase activity outside the potato tuber. Nucleotide pyrophosphatase activity reported for other plant species (Roberts 1959 ; Clayton and Hanselman 1960; Razzell 1966) may be attributed equally well to cyclic nucleotide phosphodiesterase, since both enzymes exhibit similarities not only in specificity, but in many other properties, e.g., pH optimum and cation requirements. This paper represents an effort to distinguish between the activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common synthetic substrate with the aid of relatively specific inhibitors. Moreover, it utilizes these inhibitors to reexamine the occurrence and histochemical localization in wheat tissues of nucleotide pyrophosphatase as well as to determine the localization of cyclic nucleotide phosphodiesterase.
205
The p-nitrophenol liberated from p-nitrophenyl thymidine 5'-phosphate was assayed according to Razzell and Khorana (1961). To prevent turbidity with wheat germ homogenate, the incubation was terminated after 30 min by addition of 0.6 ml cooled water and, after rapid centrifugation0 0.5 ml supernatant was mixed with 0.1 ml 1 M NaOH and read at 400 rim. Cytochemistry. Serial sections of shoots were incubated for 30 min and like sections of seeds for 60 rain at 37 ~ C in 0.1 M acetate buffer p H 5.2, 5 m M AS-BI-naphthyl thymidine 5'-phosphate, 10 mM ethylenediaminetetraacetic acid (EDTA), and 0.2% w/v filtered Fast Garnet GBC with a) no additions; b) 1 or 2 m M N A D + ; 3 or 5 mM 2',3'-cAMP. The standard controls were sections incubated in the absence of substrate. Additional controls were preincubated for 30 min at 37 ~ C in excess 0.1 M acetate buffer, pH 5.2, and, after rinsing, incubated in the whole medium. After incubation, the sections were rinsed in distilled water, fixed for 10 min in 4% formalin, rinsed in water again and mounted in Farrants' medium.
Results
Biochemical assays. NAD + and 2',31-cAMP were seMaterial and methods Material p-Nitrophenyl thymidine Y-phosphate was purchased from Sigma Chemical Co. (St. Louis, Mo., USA), while AS-BI-naphthyl thymidine 5'-phosphate was synthesized, as previously described (Sierakowska et al. 1978). All other reagents were commercial preparations. Nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase from potato tubers were isolated according to the procedure of Bartkiewicz et al. (in preparation). Unfixed frozen sections were prepared from soaked Triticum sp. seeds and, unless otherwise specified, from shoots of 3- or 5-d old seedlings, t0-12 mm from the base, according to Gahan et al. (1967). Methods Assays of enzyme activities. All incubations were carried out for 15 or 30 rain at 37 ~ C. The effects of NAD + and 2',3'-cAMP on the activities of purified potato tuber nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase were assayed in 0.0%0.1 ml 0.1 M Tris-acetate buffer, pH 6.0, containing 5 m M p-nitrophenyl thymidine 5'-phosphate or AS-BI-naphthyl thymidine 5'-phosphate, the enzyme and a) no additives; b) 0.5-5 m M N A D + ; c) 0.5-5 m M T,3'-cAMP. The effects of NAD + and 2',3'-cAMP on nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase activities of wheat germ and coleoptile homogenates were assayed in 0.1 ml 0.1 M Tris-acetate buffer, pH 4.0-7.5, containing 5 m M p-nitrophenyl thymidine Y-phosphate, 10 m M EDTA, either 0.025 ml 1.5% imbibed germ homogenate or 0.025 ml 2% 3-d old coleoptile homogenate, and a) no additives; b) 2 mM N A D + ; c) 5 mM 2',T-cAMP. The AS-BI-naphthyl liberated from AS-BI-naphthyl thymidine-5'-phosphate was assayed by terminating the incubation with sequential addition, to 0.1 ml incubation medium, of 0.5 ml ethanol, 1 ml 0.2 M acetate buffer pH 5.2, and 0.25 ml filtered aqueous Fast Garnet GBC, 7.5 mg/ml. After 10 rain 0.25 ml 40% trichloroactic acid (TCA) was added and the dye extracted with 1.5 ml toluene and read at 540 nm.
lected as potentially specific inhibitors of potato tuber nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase, respectively, on the basis of differences in enzymes affinities toward these substrates. The Km values for NAD + for nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase were 0.2 and 1.6 raM, respectively, whereas those for 2',3'-cAMP were 0.9 and 0.4 raM, respectively. Figure 1 illustrates the effects of NAD + and 2/,31-cAMP on the cleavage of p-nitrophenyl and ASBI-naphthyl thymidine 5r-phosphates by potato tuber nucleotide pyrophosphatase and cyclic nucteotide phosphodiesterase, purified over 500-fold and 6,000-fold, respectively. NAD + is obviously an effective selective inhibitor; at 2 mM it inhibits over 85% of the nucleotide pyrophosphatase activity and less than 10% of the cyclic nucleotide phosphodiesterase activity against either substrate (Fig. l a and c). 2',3'-cAMP is somewhat less effective and less selective, and at 5 mM it inhibits cyclic nucleotide phosphodiesterase activity about 90%, but nucleotide pyrophosphatase activity 50% with AS-BI-napththyl thymidine Y-phosphate (Fig. 1 d). With the p-nitrophenyl substrate (Fig. 1 b), it inhibits cyclic nucleotide phosphodiesterase activity by about 75% and nucleotide pyrophosphatase activity by about 20%. These relatively specific inhibitors of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase from potato tubers were subsequently applied to assays of activity against aryl esters of nucleotide5'-phosphates in homogenates of various wheat tissues in order to examine sensitivity to the inhibitors and thus identify the nature of the activity. Figure 2 illustrates the effects of addition of 2 mM
206
M. Bartkiewicz and H. Sierakowska: Localization of nucleotide pyrophosphatase in Triticum
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Fig. l a - & The effects of N A D + (a, e) and 2',3'-cAMP (b, d) on the activities of purified potato tuber nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase activities against p-nitrophenyl thymidine 5'-phosphate (a, b) and AS-BI-naphthyl thymidine Y-phosphate (e, d). nucleotide pyrophosphatase; . . . . 9 . . . . 9 . . . . cyclic nucleotide phosphodiesterase
Fig. 2a, b. The effects of N A D + and 2',3"-cAMP on the activity of wheat germ (a) and coleoptile (b) homogenate against p-nitrophenyl thymidine 5'-phosphate at pH 4.0-7.5. no additives; --- o o --with 2 mM "NAD § ; . . . . 9 . . . . 9 . . . . with 5 m M 2',3'-cAMP
NAD-- and 5 mM 2',3'-cAMP on the activities of wheat germ and coleoptile homogenates against pnitrophenyl thymidine 5'-phosphate at pH values 4.0-7.5. Figure 2a shows that the activity of wheat germ homogenate against p-nitrophenyl thymidine 5'-phosphate at pH 5.0 is reduced in the presence of 5 mM T,3'-cAMP to 35% of the control value and remains insensitive to 2 mM NAD § By contrast, the homogenate of wheat coleoptile (Fig. 2b) under similar conditions exhibits, at pH 5.0 in the presence of 5 mM 2',3'-cAMP, only 11% inhibition and, in the presence of 2 mM NAD § as much as 62% inhibition. The homogenate of wheat shoots (not shown) resembles that of the coleoptile, but is somewhat more sensitive to the presence of 2',3'-cAMP and less sensitive to the presence of NAD § being inhibited at pH 5.0 by 17% and 58%, respectively. Since the results in Fig. 2 indicate a relatively broad pH optimum for the homogenate activities against p-nitrophenyl thymidine 5'-phosphate
(4.5-6.0) and optimal inhibitory effects of both NAD + and T,3'-cAMP at pH values 4.5-5.5, analogous experiments with AS-BI-naphthyl thymidine 5'-phosphate, the histochemical substrate, were performed at pH 5.2, optimal both from the point of selective inhibition and the histochemical reaction. Under these conditions the activity of the wheat germ homogenate was inhibited 47% by 5 mM 2',3'-cAMP and 22% by 2 mM NAD +. The activity of coleoptiles was inhibited 29% by 5 mM 2',3'-cAMP and 77% by 2 mM NAD + The response of wheat germ activity clearly corresponds to that of potato cyclic nucleotide phosphodiesterase, while that of wheat Shoot, and, even more so of wheat coleoptile, corresponds to the behavior of potato nucleotide pyrophosphatase (Fig. 1). This indicates that the germ tissues contain mainly cyclic nucleotide phosphodiesterase and the wheat seedlings mainly nucleotide pyrophosphatase, which predominates in the shoot and particularly in the coleoptile.
Fig. 3a-c. Section of imbibed Triticum scutellum (S) and embryo incubated for 60 min with AS-BI-naphthyl thymidine 5'-phosphate. X indicates false positive reaction in controls devoid of substrate. Bar=0.2 mm. Magnification x 50. a No additives. Good cytoplasmic reaction in all cells of the rootcap (RT), radicle (R) and coleorhiza (CO). Note particularly high activity in the pro-vascular tissue (PV), suspensor (SU) and epithelial layer (E), especially at ends adjacent to heavily reacted crushed cell layer (arrows). b with l mM NAD +. Reaction essentially as in a. e with 5 mM 2',Y-cAMP. Lack of activity at sites responding in a Fig. 4a-e. Section of imbibed Triticum seed incubated for 60 rain with AS-BI-naphtbyl thymidine Y-phosphate. X is a false positive reaction. Bar=20 gm. Magnification x 390. a No additives. Granular cytoplasmic product is seen in cells of the aleurone layer (A) and starchy endosperm adjacent to the aleurone layer (arrows, E). b with 1 mM NAD +. Response essentially as in a. e with 5 mM T,Y-cAMP. Lack of activity at sites responding in a
208
M. Bartkiewiczand H. Sierakowska: Localizationof nucleotidepyrophosphatasein Triticum
Table 1. Times of incubation with AS-BI-naphthyl thymidine 5'-phosphate at 37~ C required for observable deposition of reaction product in imbibed Triticurn seeds Tissue
Time (rain)
Endosperm aleurone layer endosperm cells adjacent to aleurone layer inner endosperm cells crushed cell layer
15 5 45 1
Embryo scutellum epithelial cells adjacent to the crushed cell layer apical cells pro-vascular tissue suspensor coleorhiza rootcap radicle plumule coleoptile
5 10 10 5 15 10 10 45 45
Cytochemistry. Having shown the presence in wheat germ of an activity resembling cyclic nucleotide phosphodiesterase, and in wheat shoots of one resembling nucleotide pyrophosphatase, we proceeded to check these findings at the histochemical level and to examine the localization pattern of both activities in wheat tissues, with the aid of a common substrate and the inhibitors. Figures 3 and 4 illustrate the localization pattern of sections of imbibed wheat seeds reacted against AS-BI-naphthyl thymidine 5'-phosphate alone and in the presence of either NAD § or 2',3'-cAMP. The reactions against AS-BI-naphthyl thymidine 5'-phosphate of the scutellum and embryo (Fig. 3a) and of the endosperm (Fig. 4a) are evaluated in Table 1 in terms of the minimum incubation time necessary for the detectable deposition of the precipitate. The intracellular activity is normally confined to the cytoplasm, as reported previously (Gahan et al. 1979). Figures 3b and 4b show that incubation of similar sections in the presence of 1 mM NAD + has no appreciable effect on any of the active sites shown in Figs. 3a and 4a and listed in Table 1. NAD + ( ~ 3 raM) likewise exerted no effect. By contrast, sections incubated in the presence of 5 mM 2',3'-cAMP (Figs. 3c and 4c) show complete lack of activity at all responding sites shown in Figs. 3 ~ b and 4a-b; a similar effect is observable with 3 mM 2',3"-cAMp. These results indicate that the activity against AS-BInaphthyl thymidine 5'-phosphate observable in the embryo and scutellum (Fig. 3a), and in the endosperm (Fig. 4a), is due to cyclic nucleotide phosphodiesterase.
Figure 5 presents sections of 5-d old wheat shoot, and Fig. 6 of 3-d old coleoptile, reacted against ASBI-naphthyl thymidine Y-phosphate alone (Figs. 5a and 6 a) or in the presence of either N A D § (Figs. 5 b and 6b) or 2',3'-cAMP (Figs. 5c and 6c). The localization in Figs. 5a and 6a corresponds to that described by Sierakowska et al. (1978) and shows activity in the following tissues of the leaves and coleoptile, in order of diminishing intensity: a) leaf: fibres, xylem, mesophyll, phloem, abaxial epidermis, adaxial epidermis; b) coleoptile : outer epidermis, hypodermis, xylem, phloem, cortex, inner epidermis and bundle sheath. Addition of 1 m M N A D § to the incubation medium causes the disappearance of nearly all activity from the less active sites and partial inhibition of the more active ones, with obviously less inhibition in the xylem (see Figs. 5b and 6b). NAD § at 2 mM inhibits the overall activity even more effectively, again with least inhibition in the xylem. The localization pattern in the presence of 5 mM 2',3'-cAMP (Figs. 5c and 6c) differs from that in Figs. 5a and 6a in that there is about 50% reduction in overall activity with an appreciably greater decrease in activity in the xylem. A similar, though smaller, decrease in overall activity is obtained in the presence of 3 m M 2',3'-cAMP. These results indicate that all tissues of the leaves and coleoptile, with the exception of the vascular tissue, reveal the presence of only nucleotide pyrophosphatase. Within the vascular bundles there is also appreciable cyclic nucleotide phosphodiesterase activity, particularly in the xylem, where it surpasses nucleotide pyrophosphatase activity. Figure 7 gives more detailed information on these two activities in the vascular tissue. Incubation in the absence of inhibitors (Fig. 7a) results in the deposition of considerably more reaction product in the xylem than in the phloem region. Addition of 1 mM NAD + to the incubation medium (Fig. 7b) leads to inhibition of about two-thirds of the phloem activity and of approximately one-third of the xylem activity, whereas addition of 5 mM 2',3'-cAMP gives the reverse effect (Fig. 7 c). The deposition of reaction product in the xylem and phloem become comparable, and it appears that the xylem activity is inhibited by over two-thirds, and the phloem activity appreciably less. This indicates the presence in the xylem and phloem of both activities (see Discussion), with predominance in the phloem of nucleotide pyrophosphatase and in the xylem of cyclic nucleotide phosphodiesterase. Control sections of both seed and shoot incubated without substrate were negative. Seed and shoot sections preincubated in buffer exhibited no change in localization pattern or amount of product, indicating
Fig. 5a-c. Section of 5-d old wheat shoot incubated for 30 min with AS-BI-naphthyl thymidine 5'-phosphate. Bar = 0.2 mm. Magnification x 55. a no additives. Relatively inactive coleoptile (C), highly active first leaf mesophyll and moderately active second leaf mesophyll. Note the particularly heavily reacted fibres (F) and vascular bundIes (VB). b with 1 mM NAD +. Striking decrease in response of leaf mesophylI and of fibres (F). Note the appreciable activity of the vascular bundles (VB), particularly the xylem (J(Y) which has gained relative prominence, e with 5 mM 2",Y-cAMP. Moderate decrease in mesophyll and fibre (F) activity with relatively greater decrease in xylem (XY) activity Fig. 6a-c. Section of 3-d old coleoptile incubated for 30 min with AS-BI-naphthyl thymidine 5'-phosphate. Bar=40 gm. Magnification x 200. a no additives. Highly responding epidermis (E) and hypodermis (H) with relatively less active cortex and inner epidermis (IE). b with 1 mM NAD+. Uniformly greatly diminished response in all tissues, c with 5 mM 2',Y-cAMP. Reaction essentially as in a but of somewhat diminished intensity
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M. Bartkiewicz and H. Sierakowska : Localization of nucleotide pyrophosphatase in Triticum
Fig. 7a-c. Vascular bundle of first leaf of 5-d old wheat shoot incubated for 30 min with AS-BI-naphthyl thymidine 5'-phosphate. Bar= 10 gin. Magnification • 450. a no additive. Highly active xylem (XY) region with somewhat less activity in the phloem (P). b with 1 mM NAD +. Greatly diminished response of the phloem (P) and moderately decreased response of the xylem (XI/). e with 5 mM 2',3'-cAMP. Greatly diminished response of the xylem (XY) and moderately diminished response of the phloem (P)
no appreciable loss of either cyclic nucleotide phosphodiesterase or nucleotide pyrophosphatase activites due to enzyme diffusion. Discussion
The foregoing results show that, by taking advantage of the relative specificities of NAD § and 2',3'-cAMP as inhibitors of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase activities against aryl esters of nucleoside 5'-phosphates, it is possible to detect both activities in wheat tissues and to distinguish between them at the histochemical level. The selectivity of inhibitors is not sufficient to fully exclude the possibility that the active sites attributed to nucleotide pyrophosphatase contain some cyclic nucleotide phosphodiesterase activity, and vice versa. Nonetheless, the results in Figs. 1 and 2 justify the adoption of the following criteria for evaluating the relative amounts of nucleotdie pyrophosphatase and cyclic nucleotide phosphodiesterase activities in tissue sections : 1. Insensitivity to 2 mM NAD § and almost complete inhibition by 2-5 mM 2',3'-cAMP indicates cyclic nucleotide phosphodiesterase activity. 2. Nearly complete inhibition by NAD § and about 50% inhbition by 2',3'-cAMP indicates nucleotide pyrophosphatase activity. 3. Approximately 50% inhibition by NAD + and about 75% inhibition by 2',3"-cAMP indicates equal contribution by both enzymes, and deviations toward 1 or 2 point to predominance of either cyclic nucleotide phosphodiesterase or nucleotide pyrophosphatase, respectively. On this basis we conclude that nucleotide pyrophosphatase occurs in the coleoptile and leaves, where it is found in the cytoplasm of all cells, and is particu-
larly active in the coleoptile epidermis and hypodermis, as well as in the leaf mesophyll and developing fibers. The relative activity of nucleotide pyrophosphatase in wheat shoot tissues is essentially as reported previously (Sierakowska et al. 1978), except that the major part of the xylem and a minor part of the phloem activities of the coleoptile and leaves are due to cyclic nucleotide phosphodiesterase activity. There appears to be a correlation between the metabolic activity of the tissue and its nucleotide pyrophosphatase content. The activity of the mesophyll becomes apparent when the leaf adopts its role, and that of the fiber-forming cells is coincidental with fiber formation, diminishing upon its completion. Similarly, the activity of the coleoptile epidermis decreases with the coleoptile's loss of protective function and aging. Cyclic nucleotide phosphodiesterase, on the other hand, in addition to its vascular activity in the coleoptile and the leaves, occurs in the seed. In fact, all seed activity against AS-BI-naphthyl thymidine Y-phosphate, described by Gahan et al. (1979), and attributed to nucleotide pyrophosphatase, must now be ascribed to cyclic nucleotide phosphodiesterase. This includes the cytoplasmic activity in the aleurone layer and adjacent endosperm, the crushed cell layer, the scutellum with its pro-vascular elements, and the embryo. Particularly high levels of the enzyme appear to be associated with ceils mobilizing their content, e.g., endosperm, and with vascular functions. With respect to inhibitors, it may be noted that inhibition of wheat homogenate activities appears to be less effective than that quantitatively observed in tissue sections. This may be due to two factors. First, homogenate assays involve more tissue, with more enzyme(s) potentially acting on the inhibitors and rendering them less effective. Second, the histochemical
M. Bartkiewicz and H. Sierakowska: Localization of nucleotide pyrophosphatase in pattern discloses sites o f relatively high activity of, m o s t likely, a single enzyme and the full extent of its inhibition, w i t h o u t registering b a c k g r o u n d activity o f the other enzyme, whereas h o m o g e n a t e assays give the sum o f b o t h activities and their median inhibition. A n o t h e r aspect of our procedure concerns the rates of cleavage of the synthetic substrate by nucleotide p y r o p h o s p h a t a s e and cyclic nucleotide p h o s p h o diesterase. The relative rates of cleavage o f AS-BIn a p h t h y l thymidine-5'-phosphate and the natural substrates are indicative o f the accuracy with which the synthetic substrate reflects the physiological activities o f the two enzymes. Cyclic nucleotide phosphodiesterase f r o m p o t a t o cleaves A S - B I - n a p h t h y l thymidine 5'-phosphate at a rate c o m p a r a b l e to that for 3',5'-cAMP, its natural substrate, while the p o t a t o nucleotide p y r o p h o s p h a t a s e cleaves the A S - B I - n a p h thyl derivative at 13% o f the rate for N A D § its p r e s u m e d natural substrate (in preparation). Hence, the histochemical substrate adequately reflects the activity o f cyclic nucleotide phosphodiesterase, and, to a lesser degree, that o f nucleotide p y r o p h o s p h a t a s e . Consequently, assuming that the rates o f hydrolysis for the p o t a t o enzymes are applicable to wheat activities, c o m p a r i s o n of the physiological activities o f nucleotide p y r o p h o s p h a t a s e and cyclic nucleotide phosphodiesterase in a wheat section m a y require multiplying 8-fold the observed activity of nucleotide p y r o p h o s p h a t a s e . This would indicate that wheat shoots contain m o r e nucleotide p y r o p h o s p h a t a s e activity than is histochemically observed and that nucleotide p y r o p h o s p h a t a s e m a y actually p r e d o m i n a t e even in the shoot xylem, where the cyclic nucleotide phosphodiesterase appears to prevail. Cyclic nucleotide phosphodiesterase activity p r o b a b l y really predominates only in the seed. The limitations o f the foregoing technique, stemming f r o m the use o f a c o m m o n substrate and imperfect selectivity o f the inhibitors, are quite obvious. However, similarities in specificity, p H optima, ionic requirements, and non-diffusibility o f nucleotide p y r o p h o s p h a t a s e and cyclic nucleotide phosphodiesterase limit the possibilities o f devising alternative cytochemical procedures by the simultaneous coupling azo dye technique. Moreover, there is no other histochemical m e t h o d for nucleotide p y r o p h o s p h a tase; while the m e t h o d for cyclic nucleotide p h o s p h o diesterase in plants (Payne and Bal 1974) is a multistep procedure with inherent d o u b t s as to the localization o f the final p r o d u c t (Pearse 1968, pp. 538, 570). Used for onion tissues without exogenous nucleotidases, it p r o b a b l y demonstrates tissue p h o s p h o m o n o esterases; while with a d d e d nucleotidases, it exhibits nuclear localization, quite likely due to the a d s o r p t i o n o f a free-floating reaction p r o d u c t (Moses et al. 1966).
Triticum
211
We are deepIy indebted to Dr. D. Shugar for his continued guidance and helpful discussion, to Dr. Peter B. Gahan for advice and criticism of the manuscript, and to Dr. Matgorzata Zan-Kowalczewska for help with some phases of this investigation. This investigation was carried out as Project 09.7 of the Polish Academy of Sciences.
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