Planta

Planta (1990)182:58-63

9 Springer-Verlag1990

Specific phosphoproteins in the initial period of tobacco pollen embryogenesis M a s a h a r u Kyo i and Hiroshi H a r a d a 2

1 Department of Bioresource Science, Facultyof Agriculture, KagawaUniversity,Kagawa761-07, and 2 Institute of BiologicalSciences,Tsukuba University,Ibaraki 305, Japan Received 22 January; accepted 15 March 1990 Abstract. Several phosphoproteins specifically correlated

with the induction of embryogenic cells were detected in immature pollen grains of Nicotiana tabacum L. By regulating the concentration of glutamine in the medium the developmental pathways of immature pollen grains isolated at the mid-bicellular stage could be controlled, resulting in the formation of either mature pollen grains or embryogenic cells. Different phosphoproteins, designated as a~i and as e-i, respectively, were detected when the pollen grains either became embryogenic cells in glutamine-free medium, or when they were allowed to mature in glutamine-containing medium. The formation of embryogenic cells was suppressed by adding glutamine or cytokinin to the glutamine-free medium, nor did it occur with pollen grains at younger or older stages, and in these cases the phosphoproteins a-d were detectable only partially or faintly. The phosphoproteins a-d and e-i thus may be one of the factors necessary to direct the developmental pathway of immature tobacco pollen grains to embryogenic cells and to mature pollen grains, respectively. Key words: Embryogenesis (in pollen) - Glutamine and embryogenesis - Nicotiana (pollen embryogenesis) -

Phosphoprotein - Pollen embryogenesis (phosphoproteins)

Introduction

Embryogenesis of pollen grains is an example of the expression of totipotency in higher plants, and is also of considerable interest for plant-breeding work. However, this totipotency has not been demonstrated in all plant species and regeneration frequency varies depending on the plant species, making a general application of pollen embryogenesis to breeding work using new genetic and cell technology methods difficult. From the viewpoint of both applied and basic studies, it is therefore important to characterize the properties of pollen

cells having embryogenic capacity, and to understand the mechanism of transformation to an embryogenic cell. Previous microscopic observations indicated some cytological changes in the early period of pollen embryogenesis. In Nicotiana tabacum, degradation of cytoplasm and ribosomes, development of lysosomes, and structural change of mitochondria and plastids were observed before the first embryogenic cell division (Dunwell and Sunderland 1974). In Datura innoxia, accumulation of compounds with high electron density on the tonoplast was reported (Dunwell and Sunderland 1976). Some biochemical changes in embryogenic pollen were also observed, such as decrease in total contents of protein and RNA in N. tabacum (Bhojwani et al. 1973), polyamine accumulation in D. innoxia (Villanueva et al. 1985) and vigorous synthesis of polyadenylated RNA (poly(A+) RNA) in Hyoscyamus niger (Raghavan 1979). However, the induction mechanism of transformation from pollen grain to embryogenic cell is still unknown, one reason probably being that the previous methods for inducing pollen embryogenesis, i.e. mainly anther culture, are not suitable for biochemical analysis since in-vitro-cultured anthers consist of a heterogeneous cell population, including anther-wall cells, maturing pollen at different stages, dead pollen, and a small number of embryogenic cells. Recently, we have developed a new method for culture of Nicotiana rustica and N. tabacum pollen which allows carrying out biochemical investigations of pollen embryogenesis using pure culture of pollen (Kyo and Harada 1985, 1986). Using a highly homogeneous population of immature pollen at the mid-bicellular stage and regulating certain culture conditions we could direct the developmental pathway of the pollen toward either gametogenesis or embryogenesis. When cultured in basal medium with glutamine, most pollen grains underwent gametogenic development, i.e. formed mature pollen grains capable of germination and pollen-tube growth at a high frequency when transferred to another medium, suitable for germination. In contrast, when first cul-

M. Kyo and H. Harada : Specific phosphoproteins in embryogenic pollen of tobacco t u r e d in m e d i u m w i t h o u t g l u t a m i n e m o s t p o l l e n u n d e r w e n t self-digestion o f their c y t o p l a s m a n d did n o t u n d e r go m a t u r a t i o n . I f these starved pollen grains were transferred to m e d i u m with sucrose a n d g l u t a m i n e they u n d e r w e n t cell division which seemed to be the start o f embryogenesis. Evidently, the i m m a t u r e pollen grains h a d been t r a n s f o r m e d d u r i n g the g l u t a m i n e s t a r v a t i o n to e m b r y o g e n i c cells. As a first step for u n d e r s t a n d i n g the i n d u c t i o n m e c h a n i s m o f this t r a n s f o r m a t i o n , we att e m p t e d to discover specific b i o c h e m i c a l changes in midbicellular pollen starved o f g l u t a m i n e in vitro. We ass u m e d t h a t such p u t a t i v e b i o c h e m i c a l m a r k e r s s h o u l d be f o u n d in h i g h - m o l e c u l a r substances, especially in proteins. However, we could n o t detect a n y p r o t e i n s specific for e m b r y o g e n i c cells u s i n g t w o - d i m e n s i o n a l gel electrophoresis followed by silver s t a i n i n g or f l u o r o g r a p h y (Kyo 1987). Based o n the general idea that p h o s p h o r y l a tion o f certain p r o t e i n s plays a n i m p o r t a n t role in the p e r c e p t i o n o f e x o g e n o u s factors or in the expression o f v a r i o u s cell f u n c t i o n s at least in a n i m a l s a n d m i c r o o r g a n i s m s (e.g. C h o e n 1982; R o n s o n et al. 1987), we have n o w c o n d u c t e d a search for differences in p h o s p h o p r o reins. I n this report, we show t h a t the a p p e a r a n c e o f certain p h o s p h o p r o t e i n s coincides with the t r a n s f o r m a tion f r o m i m m a t u r e p o l l e n to e m b r y o g e n i c cells, while other p h o s p h o p r o t e i n s seem to be characteristic for dev e l o p m e n t to m a t u r e pollen.

M a t e r i a l and methods

Plant and pollen material. Plants of Nicotiana tabacum L. cv. Samsun (seeds from Japan Tobacco Co., Iwata Experimental Station) were grown under natural light in a greenhouse. The frequency of cell division in pollen of N. tabaeum had been found to be low when pollen was taken from plants just after they began to flower or when the temperature in the greenhouse was higher than 30~ C. Therefore, pollen was obtained from flower buds on plants which had started to flower one month before conducting the experiments, during November to February. Selection of flower buds, isolation of pollen, Percoll fractionation, culture, transfer from a medium to another, and observations were conducted in accordance with the methods described in Kyo and Harada (1986), with minor modifications in the size of culture (5 ml medium/5-cm-diameter Petri dish, 2-104 pollen grains.ml-1) and in medium components. In the experiment shown in Table 2 and Fig. 5, pollen at various developmental stages was prepared in a manner similar to that described in Kyo and Harada (1986) except for the length of flower buds and the concentration of Percoll for the fractionation. Each lot of pollen obtained from flower buds 10-12, 15-17, 21-23, 25-27 and 30-35 mm long was fractionated by two-step Percoll centrifugation with different density gradients (in the sequence given, 0/40, 40/50, 50/60, 60/70 and 70/80%). The pollen remaining at each interface, representing the main fraction of each pollen lot, is referred to as Fractions I-V. Culture media. The components of the various media used are listed in Table 1. Media B' and B" are identical with medium B in Kyo and Harada (1986) except that the concentration of phosphate was decreased to 0.1 and 0 mM, respectively, for reasons described below. Media B' and B" were used for the starvation treatment of pollen described in the Introduction and medium B' with glutamine at a concentration of 1 or 3 mM was used for inducing normal development (maturation). In these media with lower phosphate concentration pollen maturation and formation of embryogenic cells were found not to be inhibited. Media C and D were the

59

Table 1. Composition of the culture medium used. Figures = concentration in mM Component

KCI MgSO4 CaC12 KH2PO 4 H3BO3 Glutamine Sucrose Mannitol pH a

Medium B'

B"

C

D

20 1 1 0.1 0 0 0 300 6.8

20 1 1 0 0 0 0 300 _ b

20 1 1 1 0 3 1 300 6.8

0 0 0 0 2 0 300 0 6.8

a Adjusted with KOH b Not adjusted same as described in Kyo and Harada (1986), and were used for inducing cell division after the starvation treatment and pollen germination following maturation, respectively. Experiments with growth substances Abscisic acid was purchased from Sigma Chemical Co., St. Louis, Mo., USA, and the other phytohormones, N6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid and c~-naphthaleneacetic acid were from Nacalai Tesque, Kyoto, Japan. Each phytohormone was first solubilized in dimethyl sulfoxide (DMSO) at a concentration of 0.1 M and was diluted with medium B" first to 10 - 4 M ; the 10 - 4 M phytohormone in medium B" was further diluted by medium B" with 0.1% (v/v) DMSO to 10-SM and 10 -6 M. These solutions were sterilized by filtration and further dilutions (10 -7 M, 10 -8 M)made with 0.1% DMSO. Feeding labeled compounds to pollen grains. Pollen was cultured in medium with 0.37 MBq. ml- 1 [a2p]orthophosphate (32pi) (carrier-free; Japan Atomic Energy Research Institute, Tohkaimura, Ibaraki) for various durations indicated in Results and discussion. In order to promote the uptake of 3zPi the concentration of phosphate in the media was adjusted to 0 or 0.1 mM, lower than that in the medium B previously designed by Kyo and Harada (1986). Sample preparation and two-dimensional gel electrophoresis. Pollen which had been fed with labeled compounds was collected and rinsed twice with 0.3 M mannitol containing 20 mM KCI by centrifugation (150.g, 1 min). The pellets (pollen grains) were stored in microcentrifuge tubes (1.5 ml size) at - 8 0 ~ until use. They were homogenized in 100 ~tl icecold 20% trichloroacetic acid with a glass-Teflon homogenizer (1 ml size). The homogenates were put into microcentrifuge tubes and kept on ice for I h. After centrifugation (12000.g, 30 min) the pellets were rinsed once with icecold acetone and then three times with icecold ether by centrifugation (12000.g, 3 x 15 min), and were dried on ice to remove the ether. Then the pellets were resuspended in 20 txl of 9 M urea containing 5% 2-mercaptoethanol and 2% Nonidet P-40 (Sigma Chemical Co., St. Louis, Mo., USA). After centrifugation (12000-g, 5 rain), the supernatant was used for miniaturized O'Farrell's two-dimensional gel electrophoresis (O'Farrell etal. 1977; Mikawa etal. 1981). The protein in the supernatant was separated by nonequilibrium pH-gradient electrophoresis (NEPHGE) for the first dimension and sodium dodecyl sulfate-polyacrylamide (12.5%) gel electrophoresis (SDS-PAGE) for the second. After electrophoresis, the gel was soaked for 10 rain in 10% trichloroacetic acid, followed by three change of 50% methanol overnight, and was then dried and exposed to Kodak X-Omat K film (Eastman-Kodak Co., Rochester, N.Y., USA) for 3-5 d.

60

M. Kyo and H. Harada: Specific phosphoproteins in embryogenic pollen of tobacco

Fig. 1. Changes in the electrophoretic pattern of phosphoproteins of N. tabacum pollen during dedifferentiation and maturation. Mid-bicellular pollen was cultured in medium B' with addition of 32pi (0.37 MBq-m1-1) and glutamine at 0 or 1 mM. Pollen was harvested after 12, 24, and 36 h of culture. For easier comparison of the autoradiograms, 2-105 ( - g l u t a m i n e ) and 105 (+glutamine) pollen grains were used for two-dimensional gel electrophoresis

Results and discussion

Embryogenic cells formed in pollen cultured in vitro possessed a degenerated cytoplasm and lacked cell-division capacity; mature pollen grains possessed a normally developed cytoplasm and were capable of germination (Kyo and Harada 1986). Hereafter, we refer to the transformation from the immature pollen grain to the embryogenic cell as pollen dedifferentiation and to differentiation from immature to mature pollen as pollen maturation. In preliminary experiments we observed that the ATP level and the amount of synthesis rate of total protein in cultured pollen rapidly decreased in the dedifferentiation process. These cytological and biochemical changes in the dedifferentiating pollen are in agreement with the microscopic observations of Dunwell and Sunderland (1974) and Bhojwani et al. (1973). The degenerative changes by self-digestion may be one of the factors necessary for dedifferentiation. However, a similar change was also observed in the non-embryogenic cells which could be induced by starvation from pollen older or younger than the mid-bicellular stage (Kyo and Harada 1986). To detect other biochemical changes specific to dedifferentiating pollen, we examined the protein-phosphorylation pattern of cultured pollen. The pattern of total phosphorylated proteins of pollen cultured in medium B' or that with glutamine was analyzed by two-dimensional gel electrophoresis and autoradiography (Fig. 1). In medium B' a group of spots a-d in the acidic side (left half) of the chromatogram were intensified after 24 h of culture (spot b consists of three or four individual spots located closely together), while another spot, e, became lighter with time. Spot d is faint in the figures shown in this paper except for Fig. 5 but its appearance was reproducible. In pattern cultured on medium B' with

l mM glutamine, the intensification of spots a-d on the chromatograms was not observed, while spots a' and g-i in the basic side (right half) of the chromatogram appeared heavier. The density of spots e and f underwent hardly any change with time. Spots a and a' have closely adjacent positions but were distinguishable judging from other results (e.g., Fig. 5). Figure 2 shows the relationship between the duration of the starvation period (i.e. with no glutamine) and the frequency of pollen dedifferentiation. After the first culture period in medium B' for various periods, the pollen was transferred to and cultured in medium C for three weeks. The frequency of cell division on medium C can be considered as an index of pollen dedifferen-

6O 5o 40

,,z, 30

~ 2o "

10 0

9

I

11

I

0 24 48 72 120 192 FIRST CULTURE PERIOD ( h )

Fig. 2. Changes in the frequency of dedifferentiated pollen of N. tabacum with different durations of the starvation period. Mid-bicelo lular pollen was cultured in medium B' for various time periods and then transferred to and cultured in medium C for three weeks. Cell division was observed as described in Kyo and Harada (1986)

M. Kyo and H. Harada: Specific phosphoproteins in embryogenic pollen of tobacco

61

A601

GERM

CELL DIVlS Z

5oi

IJJ

Fig. 4A-C. Effect of glutamine on the electrophoretic pattern of phosphoproteins in N. tabacum pollen. Mid-bicellular pollen was cultured in medium B' with addition of 32Pi (0.37 MBq. ml-1) and glutamine at 0 mM (A), 0.3 mM (B) and 3.0 mM (C) for 36 h. Protein prepared from 105 pollen grains was used for each run

0ul 4 oi

0

~ 0

0.01

0.03

0.i

0.3

1

3

GLUTAMINE (mM) Fig. 3. Effect of gtutamine on dedifferentiation and maturation of tobacco pollen grains. Mid-bicellular pollen was cultured in medium B' with addition of glutamine at various concentrations for 48 h and aliquots were transferred to media C and D

tiation during the starvation period (first culture). As can be seen in Fig. 2, the frequency of dedifferentiated pollen increased rapidly after 24 h and attained a maximum within 48 h. This increase in dedifferentiation coincided with intensification of spots a-d, on the chromatograms or was somewhat delayed in comparison with the latter. The decrease in the frequency of dedifferentiated pollen after 192 h was probably a result of irreversible degeneration during too long a starvation period. Figure 3 shows the frequency of pollen maturation and dedifferentiation induced in medium B' containing glutamine at various concentrations. Mid-bicellular pollen was first cultured in medium B' with glutamine (03 mM) for 48 h and aliquots were transferred to medium D or C. The frequencies of germination and cell division were observed after 10 h and three weeks, respectively. These frequencies can be regarded as indices of pollen dedifferentiation and maturation, respectively. With increasing glutamine concentration in the medium, above 0.01 mM, increasing numbers of pollen grains matured and decreasing numbers dedifferentiated; at 1 mM and above dedifferentiation was hardly observed. The protein-phosphorylation patterns of pollen cultured in some of these media are shown in Fig. 4. The total amount o f 32p incorporated into the proteins varied depending on the glutamine concentration in the medium, but differences in the pattern were also apparent. In the presence of glutamine in the medium at 0.3 mM or higher, five spots (e-i) became distinctively apparent on the chromatograms, their density increasing with the glutamine concentration. In the absence of glutamine or at

0.3 mM glutamine, other spots (a~d) were observed (d is not clear in Fig. 4) whose intensity was inversely proportional to the glutamine concentration. All of the above results indicate that spots a-d are strongly correlated with glutamine starvation and pollen dedifferentiation, while spots e-i are correlated with pollen maturation. We examined the relationship between the frequency of pollen dedifferentiation and the appearance of spots a~t in pollen grains at various developmental stages cultured in the absence of glutamine (Table 2, Fig. 5). Pollen grains at various stages were obtained as described in Material and methods and are designated as Fractions I-V. Fraction I consisted of unicellular pollen grains and a small number of mitotic ones. Fractions II, III and IV mainly consisted of early, mid and late bicellular pollen grains which were described in our previous paper (Kyo and Harada 1986); however, in Fraction IV considerable amounts of mid-bicellular pollen grains were observed. Fraction V consisted of the pollen showing higher density and more accumulation of starch grains than in the late bicellular pollen. To determine the frequency of pollen dedifferentiation, an aliquot of each fraction was cultured in medium B" for 72 h (Fractions I-IV) or 48 h (Fraction V), and was then transferred to medium C for three weeks. In Fractions I, II and V, no cell division was observed while in Fractions III and IV, it was observed at a frequency of 72 and 20%, respectively (Table 2). Therefore, no pollen in Fractions I, II and V, and 72 and 20% pollen in Fractions III and IV, respectively, had undergone dedifferentiation during the starvation period. In Fraction V, a large number of pollen grains showed the mature form at high frequency within 24 h even in the absence of glutamine; then the pollen grains began to burst and die. If these matureform pollen grains were transferred before bursting to medium D (Table 1), approx. 20% of them produced pollen tubes (data not shown). The pollen in Fraction V seemed to contain considerable amounts of nutrients to complete maturation. Four aliquots of the pollen at each development stage (I-V) were cultured in medium B" with 32pi, harvested after 6, 24, 48 and 72 h, and the protein-phosphorylation patterns were examined (Fig. 5). In the chromatograms of Fraction-I and -II pollen, spots a and c were found

62

M. Kyo and H. Harada : Specific phosphoproteins in embryogenic pollen of tobacco

Table 2. Intensity of spots a-i and a' and the frequencies of dedifferentiated pollen in Fractions I-V of tobacco pollen Fraction Spots in autoradiogram a

a'

b

c

d

e

f

g

h

i

I

+

-

-

+

-

-

_+

-

-

-

II III IV V

++ +++ ++ -

• ++ ++

+++ ++ -

++ +++ ++ +

++ • -

+ + + ++

+ + + +

+ + ++

• ++ +++

• + +

Dedifferentiated pollen (%) 0

(0-

4)"

0 (0- 4) 72(81 62) 20(13-29) 0 (0- 4)

Figures in 0 = 95% confidence intervals for binominal distribution + + + : Very dense, + + : dense, + : detectable, +_ : faint, - : not detectable

a f t e r 24 h o f culture, while s p o t s b a n d d were n o t detecta b l e in either o f these two f r a c t i o n s w i t h i n 72 h. In F r a c tion III, s p o t s a - d h a d the g r e a t e s t d e n s i t y a n d in F r a c tion IV, t h e y a p p e a r e d still quite dense after 24 h. In F r a c t i o n V, s p o t s ~ d were f a i n t o r n o t d e t e c t a b l e b u t s p o t s g - i were q u i t e d e n s e a t 24 h. T h e e l e c t r o p h o r e t i c

p a t t e r n o f p h o s p h o p r o t e i n o f this p o l l e n was similar to t h a t o f m a t u r e p o l l e n which h a d been i n d u c e d f r o m the m i d - b i c e l l u l a r stage in m e d i u m c o n t a i n i n g g l u t a m i n e (see Fig. 2). As m e n t i o n e d before, s p o t a' is definitely different f r o m s p o t a, t h o u g h the two are p r e s e n t in a d j a c e n t positions. T h e intensity o f s p o t s a - i a n d a' after 48 h ( F r a c t i o n s I IV) or 24 h ( F r a c t i o n V) o f the s t a r v a t i o n p e r i o d a n d the f r e q u e n c y o f d e d i f f e r e n t i a t i o n in each f r a c t i o n are s u m m a r i z e d in Table 2. These results s u p p o r t the a s s u m p t i o n t h a t the a p p e a r a n c e o f s p o t s a ~ t , especially o f b, is r e l a t e d to p o l l e n d e d i f f e r e n t i a t i o n r a t h e r t h a n to g l u t a m i n e s t a r v a t i o n , a n d t h a t the intensification o f spots a' a n d e - i , especially g - i , is a s s o c i a t e d with p o l l e n m a t u r a t i o n . A d d i t i o n o f N 6 - b e n z y l a d e n i n e (BA) to the m e d i u m d u r i n g the s t a r v a t i o n p e r i o d was i n h i b i t o r y to p o l l e n d e d i f f e r e n t i a t i o n w h e r e a s = - n a p h t h a l e n e a c e t i c acid, 2,4d i c h l o r o p h e n o x y a c e t i c acid, gibberellin A3 a n d abscisic acid h a d no such effect (Fig. 6). A n e x a m i n a t i o n o f the

5 (3

20,

~

~

-oo

A

A

]

x,

I

.

!

-8 CONCENTRATION

Fig. 5. Changes in the electrophoretic patterns of phosphoproteins prepared from N . t a b a c u m pollen at various developmental stages, during the starvation period

B

-7

-6

( log M )

Fig. 6. Effect of phytohormones on the dedifferentiation of tobacco pollen. Mid-bicellular pollen was cultured in medium B" with various phytohormones and 0.001% (v/v) DMSO for 48 h and then transferred to and cultured in medium C for three weeks. A B A = abscisic acid; B A =N6-benzylaminopurine (benzyladenine), 2 , 4 D = 2,4-dichlorophenoxyacetic acid, G A 3 = gibberellic acid, N A A = ~-naphthaleneactic acid

M. Kyo and H. Harada: Specific phosphoproteins in embryogenic pollen of tobacco

63

Fig. 7 A-D. Effect of phytohormones on the electrophoretic pattern of phosphoproteins. Mid-bicellular pollen was cultured in medium B" with various concentrations of benzyladenine (BA), 32pi

(0.37MBq.m1-1) and 0.001% DMSO for 48 h. A Control; B 10 - S M B A ; C 1 0 7 M B A ; D 1 0 - 6 M B A

effect of different concentrations of BA on the intensity o f spots a - d is shown in Fig. 7. As the concentration o f BA was increased, the intensity of spots a - d was reduced. The other growth substances at 10 -6 M had no such effects. These results again indicate that the density o f spots a~:t is closely related to pollen dedifferentiation. None the phytohormones used here had any effect on normal development from mid-bicellular pollen to mature pollen in vitro at a concentration of 10-6 M (data not shown). I n c o n c l u s i o n , a comparison by two-dimensional gel electrophoresis and autoradiography of the patterns of phosphorylated proteins in cultured grains o f N i c o t i a n a t a b a c u m pollen which were in the process of dedifferentiation or o f maturation has now shown that several phosphoproteins (a-d) appeared in chromatograms of dedifferentiating pollen grains and several others (g-i) appeared in those o f maturing grains. The close coincidence of the appearance of phosphoproteins a - d with pollen dedifferentiation under different circumstances, namely, with the time course (Figs. 1, 2) and depending on the concentration of glutamine in the medium (Figs. 3, 4), the developmental stage of the pollen (Table 2, Fig. 5) and the suppression of the dedifferentiation process by BA in the medium (Figs. 6, 7), indicates that the appearance of these phosphoproteins may be one of the essential factors for the onset of pollen embryogenesis. According to preliminary results, phosphoproteins were not detectable after the beginning of cell division in medium C, indicating that their function in the process of pollen embryogenesis is transient. So far, we have not examined whether or not the appearance of these phosphoproteins is dependent on the expression of protein-kinase activity and-or on de-novo synthesis of the corresponding proteins. Studies on the localization of these phosphoproteins in the cell are also needed.

the preparation of the paper. This work was supported by a Grantin-Aid for special project research from the Ministry of Education, Science and Culture of Japan.

The authors thank Dr. V.S. Jaiswal (Botany Department, Banaras Hindu University, Varanasi, India) for his valuable suggestion in

References

Bhojwani, S.S., Dunwell, J.M., Sunderland, N. (1973) Nucleic acid and protein contents of embryogenic tobacco pollen. J. Exp. Bot. 24, 863-871 Choen, P. (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296, 613619 Dunwell, J.M., Sunderland, N. (1974) Pollen ultrastructure in anther cultures of Nicotiana tabacum. II. Changes associated with embryogenesis. J. Exp. Bot. 25, 363-373 Dunwell, J.M., Sunderland, N. (1976) Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division. J. Cell Sci. 22, 493-501 Kyo, M. (1987) Physiological and biochemical studies on pollen embryogenesis of Nicotiana. Dissertation, Tsukuba University, Japan Kyo, M., Harada, H. (1985) Studies on conditions for cell division and embryogenesis in isolated pollen culture of Nicotiana rustica. Plant Physiol. 79, 90-94 Kyo, M., Harada, H. (1986) Control of the developmental pathway of tobacco pollen in vitro. Planta 168, 427432 Mikawa, T., Takeda, S., Shimizu, T., Kitaura, T. (1981) Gene expression of myofibrillar proteins in single muscle fibers of adult chicken: micro two dimensional gel electrophoretic analysis. J. Biochem. 89, 1951-1962 O'Farrell, P.Z., Goodman, H.M., O'Farrell, P.H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142 Raghavan, V. (1979) An autoradiographic study of RNA synthesis during pollen embryogenesis in Hyoscyamus niger (henbane). Am. J. Bot. 66, 36-39 Ronson, C.W., Nixon, B.T., Ausubel, F.M. (1987) Conservative domains in bacterial regulatory proteins that respond to environmental stimuli. Cell 49, 579-581 Villanueva, V.R., Mathivet, V., Sangwan, R.J. (1985) RNA, protein and polyamines during gametophytic and androgenic development of pollen in Nicotiana tabacum and Datura innoxia. Plant Growth Regul. 3, 293-307