Plant Cell Reports
Plant Cell Reports (1987) 6:70-73
© Springer-Verlag 1987
Nitro-blue tetrazolium: A specific stain for photosynthetic activity in protoplasts* Dominique Robertson ** and Elizabeth D. Earle Department of Plant Breeding, Cornell University, Ithaca, NY 14853, USA Received September 2, 1986 / Revised version received November 19, 1986 - Communicated by O. Gamborg
ABSTRACT
A qualitative assay for the d e t e c t i o n of photosynthetic a c t i v i t y in p r o t o p l a s t s is described. Leaf p r o t o p l a s t s from atrazine resistant and susceptible biotypes of Brassica napus suspended in m e d i u m with 0.01% nitro-blue t e t r a z o l i u m and i0 to i00 ~M atrazine showed clear d i f f e r e n c e s in staining. Staining was detectable with 0.001% fluorescein-labelled t e t r a z o l i u m which, unlike n i t r o - b l u e tetrazolium, did not cause collapse of the protoplasts. ABBREVIATIONS
atrazine:2-chloro-4-(ethylamino)-6(isopropyl-amino)s-triazine DCPIP:
dichlorophenolindophenol
f l u o r e s c e i n - l a b e l l e d tetrazolium: 2,5-bis-(4-nitrop h e n y l ) - 3 - ( 5 - f l u o r e s c e i n y l ) - 2 H - t e t r a z o l i u m chloride Hepes:N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid Mes:2-(N-morpholino)ethanesulfonic
acid
n i t r o - b l u e tetrazolium: 2,2',5,5'-tetraphenyl-(3,3' - d i m e t h o x y - 4 , 4 ' - b i p h e n y l e n e ) - d i t e t r a z o l i u m chloride PS:0.6M sorbitol + 10mM NaHC03 + 50mM Hepes, pH 7.6 S M : 0 . 5 M sorbitol + 10mM Mes, pH 5.8 INTRODUCTION
The reduction of n i t r o - b l u e t e t r a z o l i u m to the insoluble diformazan salt occurs rapidly in p h o t o s y n t h e t i c tissues and results in a change in color of chloroplasts from green to blue-black. The use of this dye to localize p h o t o s y n t h e t i c a c t i v i t y in chloroplasts of leaf tissue was first d e s c r i b e d by Dyar (1953). Downton and Pyliotis (1970) used tetranitro-blue tetrazolium chloride staining of hand-cut sections from S o r g h u m leaves to d e m o n s t r a t e loss of p h o t o s y s t e m II a c t i v i t y d u r i n g o n t o g e n y of bundle sheath chloroplasts. Although the mature bundle sheath chloroplasts show photosystem I activity, they do not reduce the stain (Downton et al. 1970). Vaughn and Outlaw (1983) used t h i o c a r b a m y l nitro-blue t e t r a z o l i u m as a stain for e l e c t r o n m i c r o s c o p y to d e m o n s t r a t e p h o t o s y s t e m II a c t i v i t y in guard cell chloroplasts. A d d i t i o n of DCMU, w h i c h blocks p h o t o s y s t e m II e l e c t r o n transport, prevented reduction of this stain in guard cell chloroplasts.
Stains have some a d v a n t a g e s over q u a n t i t a t i v e methods, such as rate of oxygen evolution, as assays for p h o t o s y n t h e t i c activity. Staining requires a small amount of tissue and is economical and easy to perform. This paper d e s c r i b e s a procedure for t~e use of n i t r o - b l u e tetrazolium and fluoresceinlabelled t e t r a z o l i u m as stains to detect p h o t o s y n t h e tic a c t i v i t y in leaf protoplasts. Atrazine, like DCMU, blocks photosystem II electron transport (Moreland 1980) and prevents reduction of the tetrazolium stains. The threshold of atrazine i n h i b i t i o n of staining in protoplasts from a t r a z i n e sensitive and resistant b i o t y p e s of B r a s s i c a napus is reported. MATERIALS
AND
METHODS
Protoplasts were isolated from atrazine sensitive Brassica o l e r a c e a var botrytis (Green Comet broccoli, Harris and Moran Seed Co., Rochester, NY) and from atrazine resistant and sensitive b i o t y p e s of B r a s s i c a napus cv Tower. The a t r a z i n e resistance o r i g i n a t e d in b i r d s - r a p e (B. campestris) and was transferred into the c u l t i v a t e d oilseed crop, B. napus, by repeated b a c k c r o s s i n g (Beversdorf et al. 1980). Fully expanded leaves from plants grown at 23 to 26°C, 65% RH with a 12 h p h o t o p e r i o d of i00 ~E/m2/s light intensity were used for p r o t o p l a s t isolation. In some e x p e r i m e n t s leaves were kept in the dark for 24 h before i s o l a t i n g protoplasts. The d e t a i l e d procedure for p r o t o p l a s t isolation has been d e s c r i b e d (Robertson and Earle 1986). Leaves from B. o l e r a c e a were sterilized for one min in 70% ethanol and 4 min in 10% Clorox and then rinsed 3 times w i t h sterile water. Brassica napus leaves were s t e r i l i z e d for 2 min in 70% ethanol. A f t e r scoring into 1 m m strips, 0.3 g of leaves was p l a c e d abaxial side down in 4 ml of an enzyme solution c o n s i s t i n g of 0.5% C e l l u l y s i n (Calbiochem B e h r i n g Corp.), 0.25% M a c e r o z y m e (Yakult P h a r m a c e u t i c a l Industry Co.), 0.1% D r i s e l a s e (Kyowa Hakko Kogy Co.), 0.44 M sorbitol, 24 m M CaCI2, 4 m M Mes (pH 5.6) and 1 m M KH2PO 4. Incubation o c c u r r e d at 50 rpm for 16 h in darkness. Leaf tissue was gently agitated to release protoplasts, and the solution was f i l t e r e d t h r o u g h a 167 ~ m mesh and c e n t r i f u g e d 5 min at 55g~ The p r o t o p l a s t pellet was r e s u s p e n d e d in 3 ml 0.5 M sucrose w i t h 5 m M Mes (pH 5.8); 0.5 ml SCMP (0.5 M sorbitol, i0 m M CaCI2, 5 m M Mes, and 1 m M KH2P04, pH 5.8) was layered on top. After c e n t r i f u g a t i o n at 55g for 5 min intact p r o t e p l a s t s floated to the s u c r o s e / SCMP interface. This band was t r a n s f e r r e d to a n o t h e r tube, m i x e d with 3 ml SCMP and recentrifuged. The
* This work has been submitted by D. R. in partial fulfillment of the requirement for the Ph.D. degree ** Present address: Department of Zoology, Duke University, Durham, NC 27706, USA Offprint requests to: E.D. Earle
7! protoplast pellet was resuspended in m e d i u m and either stored at 4 ° C in the dark or used immediately. The p h o t o s y n t h e s i s m e d i u m (PS medium) c o n s i s t e d of 0.6 M sorbitol, i0 m M NaHC03, and 50 m M Hepes, pH 7.6 (KOH) (Hampp and Zeigler 1980). A 0.1% (w/v) stock solution of n i t r o - b l u e t e t r a z o l i u m (Sigma) was p r e p a r e d in PS medium, f i l t e r e d and stored at 4°C in the dark. Fluorescein-labelled tetrazolium (Polysciences) was p r e p a r e d as a 0.1% (w/v) stock solution in m e t h a n o l and stored at -20°C in tMe dark. A 50 m M stock solution of a t r a z i n e (gift f r o m C i b a Geigy) was p r e p a r e d in 95% e t h a n o l and stored at -20°C. Media were sterilized by autoclaving. A t r a z i n e was added to a u t o c l a v e d m e d i a d i r e c t l y f r o m the stock solution. P r o t o p l a s t s (approx. 105/ml) were p l a t e d in 0.09 ml aliquots in 96 well multiwells (Falcon). N i t r o - b l u e t e t r a z o l i u m was added to a c o n c e n t r a t i o n of 0.01% and the plates i n c u b a t e d at 150 ~ E / m 2 / s for 30 min. The light source c o n s i s t e d of a c o m b i n a t i o n of G r o - L u x and cool white f l u o r e s c e n t lights. The percentage of stained protoplasts was determined u s i n g an i n v e r t e d m i c r o s c o p e . The same p r o c e d u r e was used for staining with fluorescein-labelled t e t r a z o l i u m e x c e p t that the stain was d i l u t e d I:i0 w i t h PS m e d i u m i m m e d i a t e l y before use. Fluorescein fluorescence was detected using a Zeiss epifluorescenee microscope with the filter c o m b i n a t i o n B P 4 5 0 - 4 9 0 (excitati0n filter) and LF 520 (barrier filter) w i t h an FT 510 b e a m splitter. RESULTS
AND
DISCUSSION
Nitro-blue tetrazolium s t a i n i n g in p r o t o p l a s t s s u s p e n d e d in PS m e d i u m was d e t e c t a b l e a f t e r 1-2 min e x p o s u r e to light (Fig. I). After 30 min light, the c h l o r o p l a s t s turned c o m p l e t e l y b l u e - b l a c k and stained protoplasts were easily identified at lower magnifications (Fig. 2). The proportion of protoplasts with stained chloroplasts varied in d i f f e r e n t treatments, but m o s t p r o t o p l a s t s c o n t a i n e d either stained or u n s t a i n e d chloroplasts. Protoplast collapse o c c u r r e d c o n c o m i t a n t l y w i t h r e d u c t i o n of the stain (Fig. 2); p r o t o p l a s t s e x p o s e d to the same light i n t e n s i t y in the a b s e n c e of stain were not c o l l a p s e d (Fig. 3). There was no s t a i n i n g w h e n p r o t o p l a s t s
were incubated in PS medium with nitro-blue t e t r a z o l i u m in the dark. Protoplasts i n c u b a t e d in nutrient medium (Schenck and R o b b e l e n 1982) with t e t r a z o l i u m in the d a r k showed l i g h t - b r o w n s t a i n i n g after 16 to 24 h. This was c l e a r l y d i s t i n g u i s h a b l e from p h o t o c h e m i c a l r e d u c t i o n of the stain. The o p t i m u m c o n c e n t r a t i o n o f stain was 0.01%. At lower c o n c e n t r a t i o n s the p e r c e n t a g e of stained protoplasts was reduced, and staining was less intense. Protoplasts exposed to nitro-blue t e t r a z o l i u m i m m e d i a t e l y a f t e r i s o l a t i o n showed 70 to 80% staining. If p r o t o p l a s t s were stored for 1 d in S C M P in the d a r k at 25°C, the p e r c e n t a g e of s t a i n e d protoplasts i n c r e a s e d to 95-99%. Protoplasts from leaves kept in the dark for one d before p r o t o p ! a s t isolation had 95% staining even when assayed immediately after enzyme treatment. Other investigators (Morris and Thain 1980; S e r v a i t e s and 0gren 1980; Yazawa et al. 1983) have n o t e d i m p r o v e d rates of p h o t o s y n t h e s i s in leaf cells and p r o t o p l a s t s when the level of starch was decreased by p r e t r e a t m e n t of leaf tissue in the dark. Protoplasts incubated in PS m e d i u m (with or w i t h o u t stain) c o l l a p s e d w i t h i n 24 h. In an e f f o r t to define media suitable for long t e r m i n c u b a t i o n of photosynthetically active protoplasts, the e f f e c t s of various medium components on nitro-blue t e t r a z o l i u m r e d u c t i o n and p r o t o p l a s t s t a b i l i t y w e r e c o m p a r e d (Table i). A d d i t i o n of i0 m M c a l c i u m to PS m e d i u m r e s u l t e d in the a p p e a r a n c e of e x t r a c e l l u l a r crystals (probably CaCO3) , and staining was d e t e c t a b l e o n l y in those p r o t o p l a s t s a d j a c e n t to the crystals. An e q u i v a l e n t percentage of p r o t o p l a s t s (95-100%) stained in PS m e d i u m and in SM medium; however staining in SM, which lacked added bicarbonate, was not as intense as in PS medium. A f t e r 24 h in media w i t h o u t calcium, 95-100% of the protoplasts c o l l a p s e d even w i t h o u t the a d d i t i o n of n i t r o - b l u e tetrazolium. A d d i t i o n of c a l c i u m to the sorb-Mes solution p r e v e n t e d c o l l a p s e but r e d u c e d the p e r c e n t a g e of stained p r o t o p l a s t s to 90% at 1 m M and 41% at i0 m M calcium. When b i c a r b o n a t e (i0 mM) was added to the sorb-Mes solutions, the p e r c e n t a g e of stained p r o t o p l a s t s i n c r e a s e d to 95-100% w i t h 5 m M calcium and 74% with i0 m M calcium. Protoplasts remained intact after several days in media
F i g u r e s 1-3: N i t r o - b l u e t e t r a z o l i u m s t a i n i n g in leaf p r o t o p l a s t s from B. oleracea. Protoplasts were incubated at 4°C in the dark in S C M P for 24 h and then r e s u s p e n d e d in PS m e d i u m w i t h or w i t h o u t 0.01% n i t r o - b l u e t e t r a z o l i u m i m m e d i a t e l y before e x p o s u r e to light, i. P r o t o p l a s t in PS m e d i u m w i t h t e t r a z o l i u m after 5 min e x p o s u r e to light. A r r o w points to c h l o r o p l a s t with dark area of r e d u c e d stain in its center. M o s t of the chloroplasts showed some staining. (bar=10~m) 2. P r o t o p l a s t s in PS m e d i u m w i t h t e t r a z o l i u m after 30 m i n e x p o s u r e to light. In the c o l l a p s e d p r o t o p l a s t s all of the c h l o r o p l a s t s were stained and looked black. In the other p r o t o p l a s t s m o s t of the c h l o r o p l a s t s w e r e green. (bar=20~m) 3. P r o t o p l a s t s in PS m e d i u m w i t h o u t n i t r o - b l u e t e t r a z o l i u m after 30 min e x p o s u r e to light. Intact p r o t o p l a s t s c o n t a i n e d green c h l o r o p l a s t s . ( b a r = 2 0 D m )
72 containing 5 to i0 m M calcium. In summary, the o p t i m a l m e d i u m for long t e r m p h o t o s y n t h e t i c studies with protoplasts included 1 to 5 mM calcium in a d d i t i o n to b i c a r b o n a t e and osmoticum. A pH value of 5.8, w h i c h is close to that of m o s t p r o t o p l a s t culture media, did not significantly reduce t e t r a z o l i u m staining. O t h e r i n v e s t i g a t o r s have shown that, in contrast to i s o l a t e d chloroplasts which require a pH between 7 and 8, p h o t o s y n t h e s i s in p r o t o p l a s t s shows a w i d e t o l e r a n c e to pH (Kaiser and Heber 1983; Morris and Thain 1980). Table i. E f f e c t of D i f f e r e n t M e d i u m Components on Staining and Membrane Stability in Leaf Protoplasts from Brassica oleracea P r o t o p l a s t s were i n c u b a t e d in SCMP in the d a r k 48 h, resuspended in the specified medium with 0.01% n i t r o - b l u e tetrazolium, and e x p o s e d to light for 30 min. The percentage of stained protoplasts was determined from counts of 300-500 protoplasts. P r o t o p l a s t collapse was d e t e r m i n e d after 24 h dark i n c u b a t i o n in m e d i u m w i t h o u t stain.
Medium
Calcium (mM)
pH
NaHCO 3 (mM)
Staining (%)
Collapse after 24 h
PS PS SM SM SM SM SM SM SM
0 l0 0 0.5 1 5 i0 5 i0
7.6 7.6 5.8 5.8 5.8 5.8 5.8 5.8 5.8
10 i0 0 0 0 0 0 10 i0
95-100 20 a 95-100 95-100 90 65 41 95-100 74 a
yes no yes yes no no no no no
a extracellular
crystals
present
in m e d i u m
Table 2 compares the effects of a t r a z i n e on the r e d u c t i o n of n i t r o - b l u e t e t r a z o l i u m by p r o t o p l a s t s f r o m atrazine sensitive and r e s i s t a n t b i o t y p e s of B. napus. Atrazine inhibited s t a i n i n g in p r o t o p l a s t s from the sensitive biotype with a threshold of b e t w e e n 1 and i0 pM, but had no effect on p r o t o p l a s t s from the r e s i s t a n t b i o t y p e at c o n c e n t r a t i o n s up to i00 pM atrazine. The c o n t r a s t between r e s i s t a n t and Susceptible biotype protoplasts in i0 to i00 p M atrazine was easily distinguished without a microscope. E x p e r i m e n t s using p r o t o p l a s t s isolated from a t r a z i n e r e s i s t a n t and s e n s i t i v e b i o t y p e s of B. campestris gave i d e n t i c a l results.
Table 2. Effect of Atrazine on the Reduction of Nitro-blue Tetrazolium by Leaf Protoplasts from Atrazine Sensitive and Resistant B i o t y p e s of B. napus P r o t o p l a s t s were i n c u b a t e d in SCMP in the d a r k for 24 h and resuspended in PS m e d i u m with v a r y i n g concentrations of atrazine and 0.01% nitro-blue tetrazolium. The p e r c e n t a g e of stained p r o t o p l a s t s after 30 m i n e x p o s u r e to light was d e t e r m i n e d f r o m counts of 300-500 protoplasts.
Protoplast atrazine atrazine
Source
sensitive resistant
0
Atrazine 1
80% 92%
73% 93%
C o n c e n t r a t i o n (pM) 5 i0 50 I00 8% 82%
0% 86%
0% 86%
0% 80%
The threshold reported here for atrazine i n h i b i t i o n of t e t r a z o l i u m s t a i n i n g of p r o t o p l a s t s is n e a r the c o n c e n t r a t i o n that is r e p o r t e d to c o m p l e t e l y i n h i b i t e l e c t r o n t r a n s p o r t in i s o l a t e d chloroplasts. Darr et al. (1981) studied the effect of a t r a z i n e on the rate of e l e c t r o n t r a n s p o r t (measured as DCPIP reduction) in isolated chloroplasts of atrazine sensitive and r e s i s t a n t b i o t y p e s of B. campestris. Electron transport rates in c h l o r o p l a s t s f r o m the resistant b i o t y p e were 60% of the control in the presence of i00 ~M atrazine. Atrazine inhibited e l e c t r o n t r a n s p o r t in the sensitive b i o t y p e 50% at 0.i to 0.3 pM. At 1 ~M the rate of e l e c t r o n t r a n s p o r t was about 25% of the control, w h i c h m a y be enough to cause r e d u c t i o n of n i t r o - b l u e tetrazolium. The use of protoplasts to select for p h o t o s y n t h e t i c m u t a n t s is a d v a n t a g e o u s b e c a u s e large p o p u l a t i o n s of single cells are easily o b t a i n e d and the risk of o b t a i n i n g c h i m e r i c m u t a n t s is d e c r e a s e d (Cseplo and Maliga 1984). N i t r o - b l u e t e t r a z o l i u m at 0.01% was toxic to p h o t o s y n t h e t i c a l l y active cells and m a y t h e r e f o r e be useful as a m e t h o d for i s o l a t i n g mutants with defective photosystem II electron transport. For the selection of herbicide resistant mutants, a n o n d e s t r u c t i v e screen for p h o t o s y n t h e t i c activity would be desirable. Figs. 4 to 6 show p r o t o p l a s t s that have been stained w i t h f l u o r e s c e i n l a b e l l e d t e t r a z o l i u m blue. After 30 min i n c u b a t i o n in the light, aliquots were removed to a hem0cytometer for microscopic examination. Fluorescein fluorescence was d e t e c t a b l e only after a l~g time of from 3 to i0 min e x p o s u r e to the
F i g u r e s 4-6: Fluorescein-labelled t e t r a z o l i u m s t a i n i n g in leaf p r o t o p l a s t s from B. o l e r a c e a (bar=20 pm). 4. F l u o r e s c e n c e of stained p r o t o p l a s t s after i0 min e x p o s u r e to 450-490 n m light. M o v e m e n t of the p r o t o p l a s t s d u r i n g the exposure o b s c u r e d the fact that the f l u o r e s c e n c e came from the chloroplasts. 5. Bright field exposure of p r o t o p l a s t s in Fig. 4. Protoplasts show no signs of collapse. 6. F l u o r e s c e n c e of stained p r o t o p l a s t s is clearly a s s o c i a t e d w i t h the chloroplasts.
73 e x c i t a t i o n light. The r e a s o n for the lag time is not clear but it may be caused by quenching from chlorophyll. Protoplasts containing brightly f l u o r e s c e n t c h l o r o p l a s t s (Fig. 4, 6) r e m a i n e d i n t a c t (Fig. 5). To d e t e r m i n e p r o t o p l a s t v i a b i l i t y a f t e r s t a i n i n g with fluorescein-labelled tetrazolium, leaf protoplasts from B. o l e r a c e a w e r e i n c u b a t e d in PS m e d i u m with 0.001% f l u o r e s c e i n - l i b e l l e d tetrazolium for 30 min in the light, resuspended in n u t r i e n t m e d i u m B (Pelletier et al. 1983) at a c o n c e n t r a t i o n of 5 x 104/ml and p l a c e d in the d a r k for 2 d. A l i q u o t s of p r o t o p l a s t s r e m o v e d after 1 and 2 d still showed bright fluorescein fluorescence upon illumination, and the lag time before d e t e c t i o n of fluorescence was reduced. After 2 d, p r o t o p l a s t s w e r e p l a c e d in a culture r o o m i l l u m i n a t e d w i t h 80 ~ E / m 2 / s for 16 h/d. A f t e r 5 d about 50% of these protoplasts showed e v i d e n c e of wall f o r m a t i o n (loss of spherical shape), and about 5% showed first divisions. At this point c h l o r o p h y l l fluorescence was much reduced in both stained and unstained protoplast cultures, and it was difficult to d i s t i n g u i s h between y e l l o w f l u o r e s c e i n fluorescence and a slightly g r e e n e r a u t o f l u o r e s c e n c e seen in b o t h stained and u n s t a i n e d p r o t o p l a s t cultures. Although it is not known w h e t h e r p r o t o p l a s t s that d i v i d e d were in fact stained with fluorescein-labelled tetrazolium, at least some of the stained p r o t o p l a s t s were capable of wall formation. If c h l o r o p l a s t s that stain are still capable of division, fluorescein-labelled t e t r a z o l i u m m a y be v a l u a b l e as a m a r k e r for p h o t o s y n t h e t i c a l l y active cells. Methods for nondestructively labelling photosynthetically active cells are c u r r e n t l y not available. The results r e p o r t e d here suggest that fluorescein-labelled tetrazolium, or some modification of this stain, may provide such a method. In c o m b i n a t i o n w i t h a f l u o r e s c e n c e - a c t i v a t e d cell sorter, this stain m a y be used to select for herbicide resistant protoplasts. Because nitro-blue tetrazolium is toxic to a c t i v e l y p h o t o s y n t h e s i z i n g cells, it can be used as a m e t h o d for s e l e c t i n g cells with impaired photosystem II activity. Both tetrazolium stains may be useful in experiments
involving manipulations with herbicide resistance genes by p r o v i d i n g a m a r k e r for their e x p r e s s i o n at the c e l l u l a r level (Robertson et al. 1985). ACKNOWLEDGEMENTS
Seed from atrazine resistant and sensitive b i o t y p e s of B. napus and B. c a m p e s t r i s was k i n d l y provided by Dr. W. Beversdorf, Allelix Inc., Mississauga, Ontario. We thank Dr. T. G. Owens, Cornell university, for the suggestion to use tetrazolium. This w o r k was s u p p o r t e d by grants no. 8 2 - C R C R - I - 1 0 2 6 f r o m U S D A and D C B - 8 2 0 7 7 0 1 f r o m NSF. REFERENCES
Beversdorf WD, Weiss-Lerman J, Erickson LR, Souza-Machado V (1980) Can J G e n e t Cytol 22: 167-172 C s e p l o A, M a l i g a P (1984) Mol Gen Genet 1 9 6 : 4 0 7 - 4 1 2 Darr S, Souza M a c h a d o V, A r n t z e n CJ (1981) B i o c h i m B i o p h y s Acta 6 3 4 : 2 1 9 - 2 2 8 Downton WJS, Pyliotis NA (1970) Can J Bot 49:179-180 Downton WJS, Berry JA, Tregunna EB (1970) Z Pflanzenphysiol 63:194-198 Dyar MT (1953) A m J Bet 4 0 : 2 0 - 2 5 Hampp H, Zeigler E (1980) Planta 1 4 7 : 4 8 5 - 4 9 4 K a i s e r G, Heber U (1983) Planta 1 5 7 : 4 6 2 - 4 7 0 M o r e l a n d DE (1980) Ann R e v Plant Physiol 3 1 : 5 9 7 - 6 3 8 M o r r i s P, Thain JF (1980) J Exp Bot 3 1 : 8 3 - 9 5 P e l l e t i e r G, P r i m a r d C, V e d e l F, C h e t r i t P, R e m y R, Rouselle P, R e n a r d M (1983) Mol Gen G e n e t 191:244-252 Robertson D, Earle ED (1986) Plant Cell R e p o r t s 5:61-64 R o b e r t s o n D, Earle ED, M u t s c h l e r M A (1985) E u c a r p i a Cruciferae Newsletter 10:88-89 S c h e n c k HR, R o b b e l e n G (1982) Z P f l a n z e n z u c h t 89: 278-288 S e r v a i t e s JC, Ogren WL (1980) M e t h o d s in E n z y m o l 69:642-648 Vaughn KC, Outlaw WH (1983) Plant Physiol 71:420-424 Y a z a w a M, M u t o S, M i y a c h i S (1983) Plant and Cell Physiol 2 4 : 1 3 4 3 - 1 3 4 7
Plant Cell Reports (1987) 6:70-73
© Springer-Verlag 1987
Nitro-blue tetrazolium: A specific stain for photosynthetic activity in protoplasts* Dominique Robertson ** and Elizabeth D. Earle Department of Plant Breeding, Cornell University, Ithaca, NY 14853, USA Received September 2, 1986 / Revised version received November 19, 1986 - Communicated by O. Gamborg
ABSTRACT
A qualitative assay for the d e t e c t i o n of photosynthetic a c t i v i t y in p r o t o p l a s t s is described. Leaf p r o t o p l a s t s from atrazine resistant and susceptible biotypes of Brassica napus suspended in m e d i u m with 0.01% nitro-blue t e t r a z o l i u m and i0 to i00 ~M atrazine showed clear d i f f e r e n c e s in staining. Staining was detectable with 0.001% fluorescein-labelled t e t r a z o l i u m which, unlike n i t r o - b l u e tetrazolium, did not cause collapse of the protoplasts. ABBREVIATIONS
atrazine:2-chloro-4-(ethylamino)-6(isopropyl-amino)s-triazine DCPIP:
dichlorophenolindophenol
f l u o r e s c e i n - l a b e l l e d tetrazolium: 2,5-bis-(4-nitrop h e n y l ) - 3 - ( 5 - f l u o r e s c e i n y l ) - 2 H - t e t r a z o l i u m chloride Hepes:N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid Mes:2-(N-morpholino)ethanesulfonic
acid
n i t r o - b l u e tetrazolium: 2,2',5,5'-tetraphenyl-(3,3' - d i m e t h o x y - 4 , 4 ' - b i p h e n y l e n e ) - d i t e t r a z o l i u m chloride PS:0.6M sorbitol + 10mM NaHC03 + 50mM Hepes, pH 7.6 S M : 0 . 5 M sorbitol + 10mM Mes, pH 5.8 INTRODUCTION
The reduction of n i t r o - b l u e t e t r a z o l i u m to the insoluble diformazan salt occurs rapidly in p h o t o s y n t h e t i c tissues and results in a change in color of chloroplasts from green to blue-black. The use of this dye to localize p h o t o s y n t h e t i c a c t i v i t y in chloroplasts of leaf tissue was first d e s c r i b e d by Dyar (1953). Downton and Pyliotis (1970) used tetranitro-blue tetrazolium chloride staining of hand-cut sections from S o r g h u m leaves to d e m o n s t r a t e loss of p h o t o s y s t e m II a c t i v i t y d u r i n g o n t o g e n y of bundle sheath chloroplasts. Although the mature bundle sheath chloroplasts show photosystem I activity, they do not reduce the stain (Downton et al. 1970). Vaughn and Outlaw (1983) used t h i o c a r b a m y l nitro-blue t e t r a z o l i u m as a stain for e l e c t r o n m i c r o s c o p y to d e m o n s t r a t e p h o t o s y s t e m II a c t i v i t y in guard cell chloroplasts. A d d i t i o n of DCMU, w h i c h blocks p h o t o s y s t e m II e l e c t r o n transport, prevented reduction of this stain in guard cell chloroplasts.
Stains have some a d v a n t a g e s over q u a n t i t a t i v e methods, such as rate of oxygen evolution, as assays for p h o t o s y n t h e t i c activity. Staining requires a small amount of tissue and is economical and easy to perform. This paper d e s c r i b e s a procedure for t~e use of n i t r o - b l u e tetrazolium and fluoresceinlabelled t e t r a z o l i u m as stains to detect p h o t o s y n t h e tic a c t i v i t y in leaf protoplasts. Atrazine, like DCMU, blocks photosystem II electron transport (Moreland 1980) and prevents reduction of the tetrazolium stains. The threshold of atrazine i n h i b i t i o n of staining in protoplasts from a t r a z i n e sensitive and resistant b i o t y p e s of B r a s s i c a napus is reported. MATERIALS
AND
METHODS
Protoplasts were isolated from atrazine sensitive Brassica o l e r a c e a var botrytis (Green Comet broccoli, Harris and Moran Seed Co., Rochester, NY) and from atrazine resistant and sensitive b i o t y p e s of B r a s s i c a napus cv Tower. The a t r a z i n e resistance o r i g i n a t e d in b i r d s - r a p e (B. campestris) and was transferred into the c u l t i v a t e d oilseed crop, B. napus, by repeated b a c k c r o s s i n g (Beversdorf et al. 1980). Fully expanded leaves from plants grown at 23 to 26°C, 65% RH with a 12 h p h o t o p e r i o d of i00 ~E/m2/s light intensity were used for p r o t o p l a s t isolation. In some e x p e r i m e n t s leaves were kept in the dark for 24 h before i s o l a t i n g protoplasts. The d e t a i l e d procedure for p r o t o p l a s t isolation has been d e s c r i b e d (Robertson and Earle 1986). Leaves from B. o l e r a c e a were sterilized for one min in 70% ethanol and 4 min in 10% Clorox and then rinsed 3 times w i t h sterile water. Brassica napus leaves were s t e r i l i z e d for 2 min in 70% ethanol. A f t e r scoring into 1 m m strips, 0.3 g of leaves was p l a c e d abaxial side down in 4 ml of an enzyme solution c o n s i s t i n g of 0.5% C e l l u l y s i n (Calbiochem B e h r i n g Corp.), 0.25% M a c e r o z y m e (Yakult P h a r m a c e u t i c a l Industry Co.), 0.1% D r i s e l a s e (Kyowa Hakko Kogy Co.), 0.44 M sorbitol, 24 m M CaCI2, 4 m M Mes (pH 5.6) and 1 m M KH2PO 4. Incubation o c c u r r e d at 50 rpm for 16 h in darkness. Leaf tissue was gently agitated to release protoplasts, and the solution was f i l t e r e d t h r o u g h a 167 ~ m mesh and c e n t r i f u g e d 5 min at 55g~ The p r o t o p l a s t pellet was r e s u s p e n d e d in 3 ml 0.5 M sucrose w i t h 5 m M Mes (pH 5.8); 0.5 ml SCMP (0.5 M sorbitol, i0 m M CaCI2, 5 m M Mes, and 1 m M KH2P04, pH 5.8) was layered on top. After c e n t r i f u g a t i o n at 55g for 5 min intact p r o t e p l a s t s floated to the s u c r o s e / SCMP interface. This band was t r a n s f e r r e d to a n o t h e r tube, m i x e d with 3 ml SCMP and recentrifuged. The
* This work has been submitted by D. R. in partial fulfillment of the requirement for the Ph.D. degree ** Present address: Department of Zoology, Duke University, Durham, NC 27706, USA Offprint requests to: E.D. Earle
7! protoplast pellet was resuspended in m e d i u m and either stored at 4 ° C in the dark or used immediately. The p h o t o s y n t h e s i s m e d i u m (PS medium) c o n s i s t e d of 0.6 M sorbitol, i0 m M NaHC03, and 50 m M Hepes, pH 7.6 (KOH) (Hampp and Zeigler 1980). A 0.1% (w/v) stock solution of n i t r o - b l u e t e t r a z o l i u m (Sigma) was p r e p a r e d in PS medium, f i l t e r e d and stored at 4°C in the dark. Fluorescein-labelled tetrazolium (Polysciences) was p r e p a r e d as a 0.1% (w/v) stock solution in m e t h a n o l and stored at -20°C in tMe dark. A 50 m M stock solution of a t r a z i n e (gift f r o m C i b a Geigy) was p r e p a r e d in 95% e t h a n o l and stored at -20°C. Media were sterilized by autoclaving. A t r a z i n e was added to a u t o c l a v e d m e d i a d i r e c t l y f r o m the stock solution. P r o t o p l a s t s (approx. 105/ml) were p l a t e d in 0.09 ml aliquots in 96 well multiwells (Falcon). N i t r o - b l u e t e t r a z o l i u m was added to a c o n c e n t r a t i o n of 0.01% and the plates i n c u b a t e d at 150 ~ E / m 2 / s for 30 min. The light source c o n s i s t e d of a c o m b i n a t i o n of G r o - L u x and cool white f l u o r e s c e n t lights. The percentage of stained protoplasts was determined u s i n g an i n v e r t e d m i c r o s c o p e . The same p r o c e d u r e was used for staining with fluorescein-labelled t e t r a z o l i u m e x c e p t that the stain was d i l u t e d I:i0 w i t h PS m e d i u m i m m e d i a t e l y before use. Fluorescein fluorescence was detected using a Zeiss epifluorescenee microscope with the filter c o m b i n a t i o n B P 4 5 0 - 4 9 0 (excitati0n filter) and LF 520 (barrier filter) w i t h an FT 510 b e a m splitter. RESULTS
AND
DISCUSSION
Nitro-blue tetrazolium s t a i n i n g in p r o t o p l a s t s s u s p e n d e d in PS m e d i u m was d e t e c t a b l e a f t e r 1-2 min e x p o s u r e to light (Fig. I). After 30 min light, the c h l o r o p l a s t s turned c o m p l e t e l y b l u e - b l a c k and stained protoplasts were easily identified at lower magnifications (Fig. 2). The proportion of protoplasts with stained chloroplasts varied in d i f f e r e n t treatments, but m o s t p r o t o p l a s t s c o n t a i n e d either stained or u n s t a i n e d chloroplasts. Protoplast collapse o c c u r r e d c o n c o m i t a n t l y w i t h r e d u c t i o n of the stain (Fig. 2); p r o t o p l a s t s e x p o s e d to the same light i n t e n s i t y in the a b s e n c e of stain were not c o l l a p s e d (Fig. 3). There was no s t a i n i n g w h e n p r o t o p l a s t s
were incubated in PS medium with nitro-blue t e t r a z o l i u m in the dark. Protoplasts i n c u b a t e d in nutrient medium (Schenck and R o b b e l e n 1982) with t e t r a z o l i u m in the d a r k showed l i g h t - b r o w n s t a i n i n g after 16 to 24 h. This was c l e a r l y d i s t i n g u i s h a b l e from p h o t o c h e m i c a l r e d u c t i o n of the stain. The o p t i m u m c o n c e n t r a t i o n o f stain was 0.01%. At lower c o n c e n t r a t i o n s the p e r c e n t a g e of stained protoplasts was reduced, and staining was less intense. Protoplasts exposed to nitro-blue t e t r a z o l i u m i m m e d i a t e l y a f t e r i s o l a t i o n showed 70 to 80% staining. If p r o t o p l a s t s were stored for 1 d in S C M P in the d a r k at 25°C, the p e r c e n t a g e of s t a i n e d protoplasts i n c r e a s e d to 95-99%. Protoplasts from leaves kept in the dark for one d before p r o t o p ! a s t isolation had 95% staining even when assayed immediately after enzyme treatment. Other investigators (Morris and Thain 1980; S e r v a i t e s and 0gren 1980; Yazawa et al. 1983) have n o t e d i m p r o v e d rates of p h o t o s y n t h e s i s in leaf cells and p r o t o p l a s t s when the level of starch was decreased by p r e t r e a t m e n t of leaf tissue in the dark. Protoplasts incubated in PS m e d i u m (with or w i t h o u t stain) c o l l a p s e d w i t h i n 24 h. In an e f f o r t to define media suitable for long t e r m i n c u b a t i o n of photosynthetically active protoplasts, the e f f e c t s of various medium components on nitro-blue t e t r a z o l i u m r e d u c t i o n and p r o t o p l a s t s t a b i l i t y w e r e c o m p a r e d (Table i). A d d i t i o n of i0 m M c a l c i u m to PS m e d i u m r e s u l t e d in the a p p e a r a n c e of e x t r a c e l l u l a r crystals (probably CaCO3) , and staining was d e t e c t a b l e o n l y in those p r o t o p l a s t s a d j a c e n t to the crystals. An e q u i v a l e n t percentage of p r o t o p l a s t s (95-100%) stained in PS m e d i u m and in SM medium; however staining in SM, which lacked added bicarbonate, was not as intense as in PS medium. A f t e r 24 h in media w i t h o u t calcium, 95-100% of the protoplasts c o l l a p s e d even w i t h o u t the a d d i t i o n of n i t r o - b l u e tetrazolium. A d d i t i o n of c a l c i u m to the sorb-Mes solution p r e v e n t e d c o l l a p s e but r e d u c e d the p e r c e n t a g e of stained p r o t o p l a s t s to 90% at 1 m M and 41% at i0 m M calcium. When b i c a r b o n a t e (i0 mM) was added to the sorb-Mes solutions, the p e r c e n t a g e of stained p r o t o p l a s t s i n c r e a s e d to 95-100% w i t h 5 m M calcium and 74% with i0 m M calcium. Protoplasts remained intact after several days in media
F i g u r e s 1-3: N i t r o - b l u e t e t r a z o l i u m s t a i n i n g in leaf p r o t o p l a s t s from B. oleracea. Protoplasts were incubated at 4°C in the dark in S C M P for 24 h and then r e s u s p e n d e d in PS m e d i u m w i t h or w i t h o u t 0.01% n i t r o - b l u e t e t r a z o l i u m i m m e d i a t e l y before e x p o s u r e to light, i. P r o t o p l a s t in PS m e d i u m w i t h t e t r a z o l i u m after 5 min e x p o s u r e to light. A r r o w points to c h l o r o p l a s t with dark area of r e d u c e d stain in its center. M o s t of the chloroplasts showed some staining. (bar=10~m) 2. P r o t o p l a s t s in PS m e d i u m w i t h t e t r a z o l i u m after 30 m i n e x p o s u r e to light. In the c o l l a p s e d p r o t o p l a s t s all of the c h l o r o p l a s t s were stained and looked black. In the other p r o t o p l a s t s m o s t of the c h l o r o p l a s t s w e r e green. (bar=20~m) 3. P r o t o p l a s t s in PS m e d i u m w i t h o u t n i t r o - b l u e t e t r a z o l i u m after 30 min e x p o s u r e to light. Intact p r o t o p l a s t s c o n t a i n e d green c h l o r o p l a s t s . ( b a r = 2 0 D m )
72 containing 5 to i0 m M calcium. In summary, the o p t i m a l m e d i u m for long t e r m p h o t o s y n t h e t i c studies with protoplasts included 1 to 5 mM calcium in a d d i t i o n to b i c a r b o n a t e and osmoticum. A pH value of 5.8, w h i c h is close to that of m o s t p r o t o p l a s t culture media, did not significantly reduce t e t r a z o l i u m staining. O t h e r i n v e s t i g a t o r s have shown that, in contrast to i s o l a t e d chloroplasts which require a pH between 7 and 8, p h o t o s y n t h e s i s in p r o t o p l a s t s shows a w i d e t o l e r a n c e to pH (Kaiser and Heber 1983; Morris and Thain 1980). Table i. E f f e c t of D i f f e r e n t M e d i u m Components on Staining and Membrane Stability in Leaf Protoplasts from Brassica oleracea P r o t o p l a s t s were i n c u b a t e d in SCMP in the d a r k 48 h, resuspended in the specified medium with 0.01% n i t r o - b l u e tetrazolium, and e x p o s e d to light for 30 min. The percentage of stained protoplasts was determined from counts of 300-500 protoplasts. P r o t o p l a s t collapse was d e t e r m i n e d after 24 h dark i n c u b a t i o n in m e d i u m w i t h o u t stain.
Medium
Calcium (mM)
pH
NaHCO 3 (mM)
Staining (%)
Collapse after 24 h
PS PS SM SM SM SM SM SM SM
0 l0 0 0.5 1 5 i0 5 i0
7.6 7.6 5.8 5.8 5.8 5.8 5.8 5.8 5.8
10 i0 0 0 0 0 0 10 i0
95-100 20 a 95-100 95-100 90 65 41 95-100 74 a
yes no yes yes no no no no no
a extracellular
crystals
present
in m e d i u m
Table 2 compares the effects of a t r a z i n e on the r e d u c t i o n of n i t r o - b l u e t e t r a z o l i u m by p r o t o p l a s t s f r o m atrazine sensitive and r e s i s t a n t b i o t y p e s of B. napus. Atrazine inhibited s t a i n i n g in p r o t o p l a s t s from the sensitive biotype with a threshold of b e t w e e n 1 and i0 pM, but had no effect on p r o t o p l a s t s from the r e s i s t a n t b i o t y p e at c o n c e n t r a t i o n s up to i00 pM atrazine. The c o n t r a s t between r e s i s t a n t and Susceptible biotype protoplasts in i0 to i00 p M atrazine was easily distinguished without a microscope. E x p e r i m e n t s using p r o t o p l a s t s isolated from a t r a z i n e r e s i s t a n t and s e n s i t i v e b i o t y p e s of B. campestris gave i d e n t i c a l results.
Table 2. Effect of Atrazine on the Reduction of Nitro-blue Tetrazolium by Leaf Protoplasts from Atrazine Sensitive and Resistant B i o t y p e s of B. napus P r o t o p l a s t s were i n c u b a t e d in SCMP in the d a r k for 24 h and resuspended in PS m e d i u m with v a r y i n g concentrations of atrazine and 0.01% nitro-blue tetrazolium. The p e r c e n t a g e of stained p r o t o p l a s t s after 30 m i n e x p o s u r e to light was d e t e r m i n e d f r o m counts of 300-500 protoplasts.
Protoplast atrazine atrazine
Source
sensitive resistant
0
Atrazine 1
80% 92%
73% 93%
C o n c e n t r a t i o n (pM) 5 i0 50 I00 8% 82%
0% 86%
0% 86%
0% 80%
The threshold reported here for atrazine i n h i b i t i o n of t e t r a z o l i u m s t a i n i n g of p r o t o p l a s t s is n e a r the c o n c e n t r a t i o n that is r e p o r t e d to c o m p l e t e l y i n h i b i t e l e c t r o n t r a n s p o r t in i s o l a t e d chloroplasts. Darr et al. (1981) studied the effect of a t r a z i n e on the rate of e l e c t r o n t r a n s p o r t (measured as DCPIP reduction) in isolated chloroplasts of atrazine sensitive and r e s i s t a n t b i o t y p e s of B. campestris. Electron transport rates in c h l o r o p l a s t s f r o m the resistant b i o t y p e were 60% of the control in the presence of i00 ~M atrazine. Atrazine inhibited e l e c t r o n t r a n s p o r t in the sensitive b i o t y p e 50% at 0.i to 0.3 pM. At 1 ~M the rate of e l e c t r o n t r a n s p o r t was about 25% of the control, w h i c h m a y be enough to cause r e d u c t i o n of n i t r o - b l u e tetrazolium. The use of protoplasts to select for p h o t o s y n t h e t i c m u t a n t s is a d v a n t a g e o u s b e c a u s e large p o p u l a t i o n s of single cells are easily o b t a i n e d and the risk of o b t a i n i n g c h i m e r i c m u t a n t s is d e c r e a s e d (Cseplo and Maliga 1984). N i t r o - b l u e t e t r a z o l i u m at 0.01% was toxic to p h o t o s y n t h e t i c a l l y active cells and m a y t h e r e f o r e be useful as a m e t h o d for i s o l a t i n g mutants with defective photosystem II electron transport. For the selection of herbicide resistant mutants, a n o n d e s t r u c t i v e screen for p h o t o s y n t h e t i c activity would be desirable. Figs. 4 to 6 show p r o t o p l a s t s that have been stained w i t h f l u o r e s c e i n l a b e l l e d t e t r a z o l i u m blue. After 30 min i n c u b a t i o n in the light, aliquots were removed to a hem0cytometer for microscopic examination. Fluorescein fluorescence was d e t e c t a b l e only after a l~g time of from 3 to i0 min e x p o s u r e to the
F i g u r e s 4-6: Fluorescein-labelled t e t r a z o l i u m s t a i n i n g in leaf p r o t o p l a s t s from B. o l e r a c e a (bar=20 pm). 4. F l u o r e s c e n c e of stained p r o t o p l a s t s after i0 min e x p o s u r e to 450-490 n m light. M o v e m e n t of the p r o t o p l a s t s d u r i n g the exposure o b s c u r e d the fact that the f l u o r e s c e n c e came from the chloroplasts. 5. Bright field exposure of p r o t o p l a s t s in Fig. 4. Protoplasts show no signs of collapse. 6. F l u o r e s c e n c e of stained p r o t o p l a s t s is clearly a s s o c i a t e d w i t h the chloroplasts.
73 e x c i t a t i o n light. The r e a s o n for the lag time is not clear but it may be caused by quenching from chlorophyll. Protoplasts containing brightly f l u o r e s c e n t c h l o r o p l a s t s (Fig. 4, 6) r e m a i n e d i n t a c t (Fig. 5). To d e t e r m i n e p r o t o p l a s t v i a b i l i t y a f t e r s t a i n i n g with fluorescein-labelled tetrazolium, leaf protoplasts from B. o l e r a c e a w e r e i n c u b a t e d in PS m e d i u m with 0.001% f l u o r e s c e i n - l i b e l l e d tetrazolium for 30 min in the light, resuspended in n u t r i e n t m e d i u m B (Pelletier et al. 1983) at a c o n c e n t r a t i o n of 5 x 104/ml and p l a c e d in the d a r k for 2 d. A l i q u o t s of p r o t o p l a s t s r e m o v e d after 1 and 2 d still showed bright fluorescein fluorescence upon illumination, and the lag time before d e t e c t i o n of fluorescence was reduced. After 2 d, p r o t o p l a s t s w e r e p l a c e d in a culture r o o m i l l u m i n a t e d w i t h 80 ~ E / m 2 / s for 16 h/d. A f t e r 5 d about 50% of these protoplasts showed e v i d e n c e of wall f o r m a t i o n (loss of spherical shape), and about 5% showed first divisions. At this point c h l o r o p h y l l fluorescence was much reduced in both stained and unstained protoplast cultures, and it was difficult to d i s t i n g u i s h between y e l l o w f l u o r e s c e i n fluorescence and a slightly g r e e n e r a u t o f l u o r e s c e n c e seen in b o t h stained and u n s t a i n e d p r o t o p l a s t cultures. Although it is not known w h e t h e r p r o t o p l a s t s that d i v i d e d were in fact stained with fluorescein-labelled tetrazolium, at least some of the stained p r o t o p l a s t s were capable of wall formation. If c h l o r o p l a s t s that stain are still capable of division, fluorescein-labelled t e t r a z o l i u m m a y be v a l u a b l e as a m a r k e r for p h o t o s y n t h e t i c a l l y active cells. Methods for nondestructively labelling photosynthetically active cells are c u r r e n t l y not available. The results r e p o r t e d here suggest that fluorescein-labelled tetrazolium, or some modification of this stain, may provide such a method. In c o m b i n a t i o n w i t h a f l u o r e s c e n c e - a c t i v a t e d cell sorter, this stain m a y be used to select for herbicide resistant protoplasts. Because nitro-blue tetrazolium is toxic to a c t i v e l y p h o t o s y n t h e s i z i n g cells, it can be used as a m e t h o d for s e l e c t i n g cells with impaired photosystem II activity. Both tetrazolium stains may be useful in experiments
involving manipulations with herbicide resistance genes by p r o v i d i n g a m a r k e r for their e x p r e s s i o n at the c e l l u l a r level (Robertson et al. 1985). ACKNOWLEDGEMENTS
Seed from atrazine resistant and sensitive b i o t y p e s of B. napus and B. c a m p e s t r i s was k i n d l y provided by Dr. W. Beversdorf, Allelix Inc., Mississauga, Ontario. We thank Dr. T. G. Owens, Cornell university, for the suggestion to use tetrazolium. This w o r k was s u p p o r t e d by grants no. 8 2 - C R C R - I - 1 0 2 6 f r o m U S D A and D C B - 8 2 0 7 7 0 1 f r o m NSF. REFERENCES
Beversdorf WD, Weiss-Lerman J, Erickson LR, Souza-Machado V (1980) Can J G e n e t Cytol 22: 167-172 C s e p l o A, M a l i g a P (1984) Mol Gen Genet 1 9 6 : 4 0 7 - 4 1 2 Darr S, Souza M a c h a d o V, A r n t z e n CJ (1981) B i o c h i m B i o p h y s Acta 6 3 4 : 2 1 9 - 2 2 8 Downton WJS, Pyliotis NA (1970) Can J Bot 49:179-180 Downton WJS, Berry JA, Tregunna EB (1970) Z Pflanzenphysiol 63:194-198 Dyar MT (1953) A m J Bet 4 0 : 2 0 - 2 5 Hampp H, Zeigler E (1980) Planta 1 4 7 : 4 8 5 - 4 9 4 K a i s e r G, Heber U (1983) Planta 1 5 7 : 4 6 2 - 4 7 0 M o r e l a n d DE (1980) Ann R e v Plant Physiol 3 1 : 5 9 7 - 6 3 8 M o r r i s P, Thain JF (1980) J Exp Bot 3 1 : 8 3 - 9 5 P e l l e t i e r G, P r i m a r d C, V e d e l F, C h e t r i t P, R e m y R, Rouselle P, R e n a r d M (1983) Mol Gen G e n e t 191:244-252 Robertson D, Earle ED (1986) Plant Cell R e p o r t s 5:61-64 R o b e r t s o n D, Earle ED, M u t s c h l e r M A (1985) E u c a r p i a Cruciferae Newsletter 10:88-89 S c h e n c k HR, R o b b e l e n G (1982) Z P f l a n z e n z u c h t 89: 278-288 S e r v a i t e s JC, Ogren WL (1980) M e t h o d s in E n z y m o l 69:642-648 Vaughn KC, Outlaw WH (1983) Plant Physiol 71:420-424 Y a z a w a M, M u t o S, M i y a c h i S (1983) Plant and Cell Physiol 2 4 : 1 3 4 3 - 1 3 4 7
