Biochirnica et Biophysiea Acta, 5 6 3 ( 1 9 7 9 ) 2 7 8 - - 2 9 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 9 9 4 7 0


C H R I S T I A N S A N D E R a n d L E E A. H A D W I G E R

Plant Pathology, Washington State University, Pullman, WA 99164 (U.S.A.) (Received N o v e m b e r 21st, 1978)

Key words: t~'satin induction; L-Phenylalanine amrnonia-lyase, 5-Bromodeoxyuridine

Summary The substitution of the base analogue 5-bromodeoxyuridine (BrdU) for thymidine in the DNA of pea pods (Pl'sum sativum) induces or enhances the level of phenylalanine ammonia-lyase {PAL) and also induces the phytoalexin, pisatin, a product of the same metabolic pathway. Cordycepin, a polyadenylate inhibitor at the RNA level, is a potent inhibitor of pisatin synthesis. Kinetic studies on the inhibition of the PAL-pisatin production by hydroxyurea indicate that BrdU must be incorporated into DNA oefore any induction takes place. 5-Iododeoxyuridine is also an inducer while 5-fluorodeoxyuridine is ineffective when applied alone. BrdU is incorporated into the DNA of pea cells and the nuclei undergoes condensation just prior to the detection of the induced responses. Introduction In nature antifungal compounds called phytoalexins are produced in legumes and other plants in response to the presence of plant pathogenic fungi. The de novo production of the isoflavonoid phytoalexin, pisatin, occurs when pea tissue is inoculated with plant pathogenic fungi [1,2,~], treated with DNAspecific compounds [3---5] or exposed to specific physical treatments such as ionizing radiation or ultraviolet light (260 nm) [6,7]. The increase in pisatin synthesis is preceded by large increases in the activity of L-phenylalanine ammonia-lyase (EC (PAL) the first enzyme in the pisatin pathway [4]. One of the initial effects of pisatin-inducing components is on the nucleus of the plant cell [8]. Within the first 15 min after the pea endocarp tissue is * Scientific paper No. 5038, College of Agriculture Research Center Projects 1834 and 1844, Abbreviations: BrdU, 5-bromodeoxyuridine~ IdU, 5-iododeoxyuridine; FdU, 5-fluorodeoxyuridine; PAL, phenylalaninc ammonia-lyase.

279 inoculated with macroconidia of Fusarium solani f. sp. phaseoli, the organization o f plant nuclear fibers is visibly altered by increased condensation [9]. It has been possible to follow the uptake and nuclear localization o f radioactively labeled inducers such as actinomycin D which possesses a DNA-intercalating moiety [8]. Some DNA-specific c o m p o u n d s such as actinomycin D also effect changes in nuclear structures within pea cells and influence the structure and the sedimentation coefficient of chromatin fractions isolated from induced tissue (Hadwiger, L.A., Adams, M.J. and Von Brombsen, S., unpublished data). Whether the pisatin-eliciting action is dependent on a direct effect on the DNA of the producing cells or is due to a secondary effect of the 'DNA-specific c o m p o u n d s ' on other cell structures is n o t clear. Since most of the c o m p o u n d s screened as pisatin inducers have a known potential to effect DNA conformation, DNA or chromatin may be one of the primary target sites. Since compounds applied to cells also come in contact with macromolecules of the membranes, chloroplast mitochondria, etc., it seemed useful to study pisatin induction with a c o m p o u n d which is specifically incorporated into DNA (reviewed by Rutter et al., [ 10] ) w i t h o u t effecting other macromolecules. The thymidine analogue, 5-bromodeoxyuridine (BrdU) appears to meet this criterion. It was thus decided to investigate the effectiveness of BrdU in inducing pisatin and to follow the cellular and nuclear changes p r o m o t e d by this base analog in pea pods. The incorporation of BrdU into the DNA of animal cells is known to cause many RNA-dependent alterations in gene expression [10]. Thus, the endogenous C-type oncornaviruses are activated and induced by the incorporation o f BrdU into the DNA of certain murine cells [11]. Many altered activities of enzymes have been reported in BrdU-substituted cells [10]. Also, biophysical studies have revealed distinct physical changes in BrdU-substituted chromatin [ 12--14]. However, there have been no studies on the induction or suppression of any enzyme activities in plant cells. If phytoalexin induction action is at the DNA level the expected response time for BrdU induction should be longer than that which is observed for inducing c o m p o u n d s which effect the DNA more immediately. These studies indeed show that BrdU is a p o t e n t inducer o f pisatin in peas as well as an enhancer o f p h e n y l a l a n i n e ammonia-lyase activity. In comparison with actinomycin D, the effect of BrdU is delayed by the time n e e d e d for incorporation of the BrdU into DNA. Also, studies with metabolic inhibitors show that both DNA and RNA synthesis are needed before the inducing effect o f BrdU is expressed. Evidence for the incorporation of BrdU into pea DNA and some structural changes in the nuclei of BrdU-incorporated plant cells are also described. Methods

Plant material and induction Immature Alaska pea (l~'sum sativum) pods at less than 2 cm in length were used in all induction experiments. The pea pods were harvested, the floral parts removed and the pods were split exposing the sterile endocarp cells to treatment. The split pods were placed in a Petri dish with the endocarp exposed and

280 the treatment solutions were applied to these pod halves with a Pasteur pipet. The Petri dishes were covered and stored in a humid atmosphere for the required induction time (~>24 h). Some pods were also treated while still attached to the plants in the greenhouse as a control on the integrity of excised pods. On some plants the blossoms were removed and the pods were partly split with a spatula and the pod endocarp injected with a b o u t 10 pl of treatment solution using an Eppendorf pipet.

Assays for pisatin and L-phenylalanine ammonia-lyase Pisatin was extracted from the pods with hexane [6]. The extract was quantitated on the basis of absorbance at 309 nm following purification by thin-layer chromatography. The assays are expressed as pg pisatin/g pods. PAL was assayed as described previously [7]. The assays are expressed in nM cinnamic acid/g pods. 5-Bromo-2'-deoxyuridine, actinomycin D and cordycepin were purchased from Sigma Chemical Co., St. Louis, MO, and L-['4C]phenyl alanine from New England Nuclear, Boston, MA.

Transmission electron microscopy Pea pods were treated with BrdU while still attached to the plant. Treatment was with 5 or 10 ~l/pod of BrdU (1--10 mg/ml) for 24 or 48 h. After incubation, pods were washed and diced by clean cross-sectional cuts with a razor blade. Freshly cut slices (approx. i mm ~) from the center o f the pod were dropped directly into a buffered fixing solution (3.5% glutaraldehyde, 4% formaldehyde in 0.2 M cacodylate, pH 7.2) prepared according to Karnofsky [15]. After 2 h at 4°C the pieces were washed in 0.2 M cacodylate buffer, postfixed in 2% OsO4 for 1.5 h and washed successively in 0.2 M cacodylate buffer and distilled water. The pieces were transferred to 15% ethanol and then to a 30% ethanol containing 1% uranyl acetate for 3 h. The pieces were dehydrated via a graded ethanol series and then subjected to an ascending series of solutions containing propylene oxide and Spurr's resin. After polymerization the tissue pieces were sectioned on a Porter-Blum Mt-2B ultramicrotome equipped with a diamond knife. Sections were examined with a Hitachi HY-125 E transmission electron microscope. Nuclear spreads. Fresh slices of endocarp tissue from BrdU-treated and control pea pods were placed on a nylon mesh screen positioned in the upper portion of 15 ml centrifuge tube containing a sucrose (0.1 M) solution buffered with 0.01 M Tris, pH 8. Centrifugation (5000 rev./min, 15 s, Sorvall SS34 rotor) released and finally pelleted the nuclei at the b o t t o m of the tube. The nuclei were gently dispersed in 200 ml o f swelling solution (0.001 M EDTA, pH 9, 15 min). The swelling solution was then made 1% formalin. Aliquots (50 ~l) of the dispersed nuclear suspension were added to a centrifuge tube (1 ml, 1 × 0.5 cm, lucite positioned in the b o t t o m of a 60 ml cellulose nitrate tube) containing 0.1 M sucrose in 1.0% formalin. The dispersed nuclei were then centrifuged {5000 rev.]min, 15 s, SW 25 Spinco L2) and pelleted on to a carbon-coated 300 mesh grid. The grid was stained with an ethanol solution of phosphotungstic acid prior to viewing with the Hitachi transmission electron microscopy.


Scan n ing electron microscopy Freeze-fracture, freeze-dry preparations. BrdU-treated and control p o d halves were directly immersed in liquid freon and then transferred to liquid nitrogen as described previously [9]. The frozen pods were transferred to a D e l m a r / S t u m p f / R o t h cryosorption p u m p for lyophilization. Both arms of the apparatus were cooled by liquid nitrogen. After a minimum of 3 days, the samples were removed for gold coating and viewed in an ETEC auto-scanning electron microscope. Critical-point drying technique. BrdU-treated and control tissue were also prepared for scanning electron microscopy using the critical-point drying technique [16]. In this technique samples were first treated as for transmission electron microscopy using Karnofsky's fixative, OSO4, and ethanol dehydration (see above). From the 100% ethanol the tissues were transferred in series to freon TF (15--100%) and put into the critical-point drying b o m b (saturated with Freon 13, tc = 28.9 at 561 lb/inch 2) and bled for 20 min. The tissues were then m o u n t e d and sputtered with gold for viewing in the ETEC auto-scanning electron microscope. Sedimentation profile of thymidine-labeled nuclear components. The m e t h o d of McGrath and Williams [17] as modified by Ide et al. [18] was utilized as a physical parameter in addition to the electron microscope analysis of the chromatin in pea p o d endocarp nuclei. Intact plant cells could n o t be lysed directly on prepared gradients because of the rigid cell walls. It was important, however, to obtain minimally sheared chromatin for layering over 5--20% sucrose gradients and to minimize the high activity o f endogenous nucleases. The difficulties were overcome by developing speed and precision in the procedures, using the two halves of the same p o d for treatment and control respectively, including an additional gentle homogenizing step in the preparation and increasing the levels of EDTA and NaC1 in the gradients. The procedure used was as follows: Treated and control halves o f pea pods after incubation were washed thoroughly, thin (approx. six cell layer) slices of endocarp surface were put into 1 ml of 1% sucrose, 0.05 M Tris, pH 8.0, and homogenized for 15 s at 45 V (Virtis). This step released the nuclei. The homogenate was filtered through a nylon screen and overlayed on 4 ml o f 3% sucrose (in 0.05 M Tris, pH 8.0) and gently centrifuged in an SW 50L Spinco rotor at 5000 rev./min for 2 min. The pellet containing mainly nuclei was resuspended in 0.01 M Tris buffer and homogenized in a smooth wall homogenizer (six strokes) and overlayed on a 5--20% sucrose gradient which included a gradient o f 0.05--0.01 M EDTA in 1.0 M NaC1 and 0.01 M Tris at pH 7.4. Procedures from cell disruption to this point were performed rapidly at 4°C. The gradients were centrifuged in an SW 5 0 L Spinco rotor for 2 h at 36 000 rev./min. Fractions of four drops each were collected onto Millipore filters washed with 5% trichloroacetic acid and counted in Triton X-100 scintillation fluid in a Packard Tri-Carb 2405 Counter. The counts are arbitrarily expressed as a percentage o f total counts for that gradient.

DNA extraction and CsCl density gradients DNA was extracted directly from pea pods by grinding in 1% SDS buffered with 0.01 M Tris, 0.01 M EDTA and 0.15 M NaCI (pH 8) at 0--4°C. The solu-


tion was filtered and deproteinized with water saturated with freshly distilled phenol at neutral pH. The aqueous phase was purified by washing with ether and dialysed in 0.01 M Tris. RNA was hydrolyzed with RNAase A and T] RNAase. DNA was also extracted from chromatin by sedimenting from 4 M CsC1 [19]. CsC1 density gradients were formed in an SW 50L rotor run at 40 000 rev./min for 3 days in a Spinco L2 centrifuge run at 25°C. Micrococcus luteus DNA was used as a marker. Thermal denaturation Thermal denaturation was studied optically using a Gilford sample chamber attached to a Beckman DU spectrophotometer. Temperature was controlled either manually or with a Gilford Thermo-programmer 2527. Results

Fig. 1 shows the changes in the level of PAL activity resulting from treatments with BrdU (1 mg/ml per g pods) and actinomycin D (10 mg/ml per g pods) as a function of time after treatment. It is seen that within a few hours actinomycin D (a pisatin inducer described previously [ 2 0 ] ) rapidly induces a high level o f PAL activity but that this level rapidly drops. The enhancement of PAL activity by BrdU however, is somewhat delayed. A large increase in PAL

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HOURS Fig. 1. C o m p a r i s o n o f e f f e c t s o f B r d U a n d a c t i n o m y c i n D t r e a t m e n t s in e n h a n c i n g P A L a c t i v i t y in d e t a c h e d s p l i t p o d s . i , a c t i n o m y c i n D a t 1 0 /~g/ml p e r g p o d s ( T h i s t y p i c a l d a t u m i n c l u d e d f o r c o m p a r a tive p u r p o s e s w a s d e r i v e d f r o m a d i f f e r e n t l o t o f p e a p o d s ) ; A, B r d U a t 1 r a g / n i l p e r g p o d s ; e, c o n t r o l . A s s a y t i m e s are s h o w n a f t e r i n i t i a t i o n o f t r e a t m e n t ; r o o m t e m p e r a t u r e a n d in t h e d a r k .


activity occurred at a b o u t 12--16 h, reached a maximum at a b o u t 24--32 h and finally decreased gradually 40--60 h after treatment. These experiments show that enhancement of PAL activity in BrdU-treated pods is delayed b y a b o u t the time needed for dividing cells to incorporate the BrdU. No such delay is observed in actinomycin D-treated pods. The action of actinomycin D appears more direct than that of BrdU apparently due to the need for the latter, which is a precursor of DNA, to be incorporated. Pisatin production in BrdU-treated detached pods (BrdU, 1 mg/ml per g pods) as a function of time is shown in Fig. 2. The upper curve shows that after a delay of about 20 h BrdU-treated pods produced up to 300 ~g pisatin per g pods. At about 60 h the rate of pisatin accumulation began to level off. The initial delay in pisatin production in BrdU-treated pods is attributed to the time required for a sufficient number of cells to go through a cell cycle and concomitantly incorporate BrdU. No delay was observed with DNA-interacting inducers such as actinomycin D and intercalating dyes [20]. The concentration dependence of BrdU induction of pisatin is shown in Fig. 3. Pisatin production was measured at 48 h after the initial BrdU treatment and is shown for BrdU concentrations up to 2 mg/ml per g pods. The production of pisatin is seen to level o f f somewhat at concentrations o f BrdU of i mg/g pods or greater. A comparison of the pisatin yield is shown at 24 h and 72 h after an initial treatment with BrdU at 1 mg/ml per g pods. This experiment was performed on detached split pods in the dark and shows that the optimal concentration o f BrdU in pisatin production was greater than 2 mg/ml of BrdU per g pods. Fig. 4 shows an experiment with a batch of pods which were treated with BrdU (1 mg/ml per g pods} in the presence of cordycepin (0.5 mg/ml per g pods). A BrdU control experiment at 1 mg/ml per g pods is included. Cordy-

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HOURS F i g . 2. T i m e c o u r s e o f pisatin p r o d u c t i o n in (o) B r d U - t r e a t e d (1 m g / m . l p e r g p o d s ) w a t e r c o n t r o l . T r e a t m e n t s w e r e o n d e t a c h e d split p o d s at 2 5 - - - 2 8 ° C , and in the dark.

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Fig. 3. Pisatin p r o d u c t i o n as a f u n c t i o n o f B r d U c o n c e n t r a t i o n in d e t a c h e d split p o d s . T r e a t m e n t s w e r e for 2 4 ( o ) , 4 8 ( ~ ) , a n d 7 2 (o) h ; t = 2 5 - - 2 8 ° C , and in t h e dark.

cepin is a potent inhibitor of the addition o f the polyadenylate moieties in m R N A and is reported to completely block RNA synthesis in radish seedlings at 0.2 mg/ml [ 2 1 - - 2 3 ] . It also inhibits the induction of mouse leukovirus production by 5-iodo-2'-deoxyuridine, an analogue o f BrdU [24]. In this experiment cordycepin inhibits the BrdU induction of pisatin to the levels of water controls (lower curve}. The upper curve shows the data obtained with BrdU alone and these are qualitatively similar to that obtained in the previous experiment. These data suggest that the addition of polyadenylate groups to m R N A is necessary in the induction o f pisatin by BrdU. In Fig. 5 we see the effect of hydroxyurea on the enhancement o f PAL activ-



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278 Biochirnica et Biophysiea Acta, 5 6 3 ( 1 9 7 9 ) 2 7 8 - - 2 9 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press BBA 9...
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