Plant Science, 69 (1990) 239--247 239 Elsevier Scientific Publishers Ireland Ltd.

COMPARATIVE ANALYSIS OF TRANSLATABLE mRNA POPULATIONS IN ZYGOTIC AND POLLEN-DERIVED EMBRYOS OF BARLEY {HORDEUM VULGARE L.)

PATRICIA HIGGINS* and DIANNA J. BOWLES**

Department of Biochemistry, University of Leeds, Leeds, LS~ 9JT /U.K.]

(Received October 10th, 1989) (Revision received January 15th, 1990) {Accepted March 15th, 1990)

Zygotic embryos of barley have been compared to those formed in anther culture. Polysomal RNA has been isolated from these two classes of embryo at different stages of development and translated in vitro, uS-Tranalation products have been analysed by 2-D gel electrophoresis. Comparison of the profiles indicates that substantial differences exist in the pattern of gene expression in zygotic and pollen-derived embryos. Many translation products characteristic of the pollen-derived embryos are also present in callus.

Key words: embryogenesis; anther culture; embryo-specific; barley; zygotic embryo; polien~ierived embryo

Introduction

The life cycle of higher plants can be divided into two distinct phases each of which is charac- terised by a precise sequence of developmental events. The dominant sporophytic generation begins with fertilisation and produces the diploid zygote which will continue through embryogenesis and eventually develop into a mature plant. In contrast, during the gamet~ phytic stage of development haploid gametes are formed which will subsequently be involved in fertilisation to produce a diploid zygote.

Under particular experimental conditions, cells of the angiosperm sporophyte and gametc~ phyte can be induced to behave as a zygote and mimic the developmental process which leads to the formation of embryc~like structures [1]. This phenomonon was first clearly described by Steward et al. [2] in cell suspension cultures of

*Present address: Department of Biology, University of Massachusetts, Boston. MA 02143, U.S.A. **To whom correspondence should be addressed. Abbreviations: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline (10 mm phosphate buffer, pH 7.4, 145 mm sodium chloride); TCA, trichloroacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid

carrot and to date this is one of the most comprehensively studied systems with respect to the biochemistry and molecular biology of embryogenesis (reviewed in Nomura and Komamine [3]). In contrast to many dicot spe- cies, there are few monocots which will rou- tinely produce embryo structures when placed in liquid culture, one notable exception being Dactylis giomerata [4]. Embyrogenic structures of monocots formed in vitro generally comprise a somatic embryo structure on the surface of proliferating callus [5]. However under defined conditions it is also possible to produce embryo structures directly from pollen. In barley, these embryos have no obvious callus tissue and show close morphological similarity to their zygotic counterparts [6]. Such structures formed from pollen will be referred to as pollen~lerived embryos.

The aim of the present study was to use anther culture of barley as a model system to identify the biochemical events occurring during development of monocot embryos in vitro. In order to elucidate changes associated with embryo development and maturation, zygotic embryos were harvested and analysed to provide reference points to which the in

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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vitro derived material could be compared. As a comparison for the material formed in culture, proliferating callus tissue of barley was also analysed. Tissues were compared on the basis of polypeptide composition and translatable mRNA populations at selected time points dur- ing a developmental time-course.

Methods

Hordeum vulgate var. Igri was prepared for anther culture as described by Hunter [7]. Pre- treated anthers were placed on the surface of a nutrient medium at a density of 2 per ml of medium. The medium was modified from the callus induction medium of Foroughi-Wehr et al. [8] and contained no auxin, 5.0 mM glutam- ine, 0.18 mM maltose and was solidified with either 0.8°/0 (w/v) sea plaque agarose or 200/0 (w/ v) Ficoll 400. Cultures were incubated in the dark at 25 °C for 14 days, thereafter under con- tinuous illumination also at 25°C. Embryos were identified in the cultures after two weeks and continued to develop asynchronously over a period of approximately 5 weeks. All embryos were harvested manually and their size meas- ured us ingan eyepiece gratic~d~, in a binocular microscope.

Zygotic embryos were dissected from devel- oping grains of field grown material at known days after anthesis and measured under a binocular microscope.

Callus cultures were established from imma- ture embryos of H. vulgare dissected 14 days after the onset of flowering. The grains were surface sterilised in 10% (v/v) Domestos (Lever Bros., U.K.) for 15 rain and rinsed thoroughly in sterile distilled water. Embryos were dissected from the grains aseptically under a binocular microscope and placed on a callus medium with the scutellum exposed and the radicle and plu- mule embedded in the medium. The callus medium was based on Murashige and Skoog [9] and included 30/0 (w/v) sucrose, 5 rag/1 2,4-D and 0.7% (w/v} Bactoagar. Cell proliferation from the scutellum of these cultured embryos was sub-cultured every 4 weeks to a fresh medium of similar composition. All callus cultures were

maintained at 25°C under continous illumina- tion and had been maintained over several years before use in the experiments described.

All material was fast frozen in liquid N 2 then stored at - 70 °C until used.

Isolation of RNA and translation in vitro Polysomal RNA was extracted from embryo

and callus material as described by Dalkin and Bowles [10]. Approximately 200 mg of embryo tissues and 1 g of all other tissue was required for each preparation to yield at least 250 ~g of polysomal RNA. The isolated RNA was trans- lated in vitro using the mRNA-dependent rabbit reticutocyte cell-free system and [85S]methionine. Rabbit reticulocyte lysate was obtained from Dr. T. Hunt, University of Cam- bridge and was prepared according to the method of Pelham and Jackson [11]. Transla- tions were performed as described by Dalkin and Bowles [10]; in general between 1 and 5 ~g of RNA was used per 11 ~d reaction.

Proteins synthesised in vitro from cell-free translation systems were prepared for SDS- PAGE and two dimensional electrophoresis as described by Dalkin and Bowles [10]. The first dimension isoelectric separation p roduced a linear gradient between pH 4.2 and 8.5. All sec- ond dimension preparations were carried out using 12o/0 (w/v) polyacrylamide slab gels.

Preparation of protein samples for electropho- resis

Plant tissue was homogenised in cold PBS and centrifuged for 3 rain at 12 000 × g. Ali- quots of the supernatant were removed for pro- tein estimation as described by Sedmak and Grossberg [12] the remainder was then precipi- tated by cotd 60% (w/v) TCA to a final concen- tration of 10% (w/v). After 30 rain on ice the precipitate was collected by centrifugation at 12 000 × g for 3 rain. The pellet was washed twice with cold 900/0 (v/v) acetone and resus- pended in SDS-PAGE sample buffer. Samples were solubilised, heated at 95°C for 3 rain and centrifuged for 3 rain at 12 000 × g prior to loading. Samples not used immediately were frozen in liquid nitrogen and stored at - 2 0 °C.

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SDS-PAGE was performed as described in Bowles et al. [13].

Results

Selection of the embryo material Embryo material was classified on the basis

of scuteUum length. Three time points were selected during a course of zygotic embryo development each of which could be identified by the characteristics of the developing grain as described by Rogers and Quatrano [14]: (1) describes embryos less than 1.5 mm in length (stage II), (2) describes embryos 2.6--3.0 mm in length (stages III-- IV) and (3) describes mature embryos dissected from desiccating grains (stage V). Pollen-derived embryos were harvested directly from plates at regular inter- vals following the onset of culture. Only that material in which an obvious embryo axis and scutellum could be identified was harvested; residual anther walls and unrecognisable struc- tures were discarded. Many pollen-derived embryos germinated precociously on the tissue

culture plates, these were also discarded. The development of embryo structures and their subsequent germination was not synchronous amongst the pollen grains of any one batch of anthers; each culture showed embryos at all stages of development (Fig. 1).

One-dimensional gel analysis of embryo and callus products

Polypeptide profiles of zygotic, pollen derived embryos and callus are shown in Fig. 2A. Most of the abundant polypeptides detected in embryo extracts (lanes 1 and 2) were also identified in callus (lane 3). Further- more, comparisons between the samples revealed no major quantitative changes in the abundance of particular polypeptide species, one exception being that of approximate M r 100 kDa. This band was identified in both embryo samples but not in callus.

Similarly, one-dimensional SDS-PAGE anal- ysis of in vitro translation products revealed that most of the abundant polypeptides syn- thesised in vitro could be detected in both classes of embryo (lanes 1 and 2) and callus (lane 3). However, quantitative differences in the abundance of several polypeptide species were identified, particularly those of M r 54, 35, 32 and 21 kDa indicated by the open arrows. Two other polypeptides of M 83 and 73 kDa were visualised in both pollen-derived embryos and callus but were not detected in zygotic embryo samples (closed arrows).

Fig. 1. Anther cultures of Hordeum vulgate. Structures at various stages of development are obvious in each culture plate. Each embryo s t ructure comprises a scutellum (s) and axis (a)

Developmental changes in zygotic and pollen- derived embryos

Changes in the abundance of translatable mRNA species were followed at different stages of embryo development by 2-D gel analysis of in vitro translation products. Results from zygotic embryos are shown in Fig. 3 and indicate that few changes are detectable between embryos of 1.5 mm and more mature stages. Some differences in the relative abun- dance of particular products were observed however, and these have been highlighted. For example, two polypeptides, (boxes 1 and 2) of M 40 and 33 kDa were detected at low levels in

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the translation profiles of immature embryos, whilst in more mature structures they were present as fairly abundant products.

Similar analyses of two different size classes of pollen-derived embryos is shown in Fig. 4. Again, the overall pattern was very similar for the two stages, but some differences in the rela- tive abundance of particular translation prod- ucts were identified and these have been highlighted. Two polypeptides, (boxes 3 and 4) with M r 27 and 17 kDa were present at higher levels in translations of mRNA from embryos

1.5 ram, relative to larger structures. In con- trast, polypeptides (boxes 5 and 6) of M r 17 and 15 kDa increased in abundance in the translation profiles of more mature structures.

Comparision of products from zygotic embryos, pollen-derived embryos and call~s

In vitro translation products of mRNA from zygotic embryos and pollen derived embryos were compared with one another and with those from callus. The results are shown in Fig. 5. Due to the complexity of the patterns, refer-

A B

kD 1 2 3 1 2 3 kD

06-

29.

21_

14_

Fig. 2. Polypeptide profiles of embryos and callus. Cellular protein extracts were prepared from embryos and callus and polypeptides were separated using I0" 15% w/w polyaerylam/de gradient gels and visual/sod with Coomassie Blue (A). Poly- soma] RNA was extracted from simillr samples, translated in vitro and synthos/sod polypeptides were separated as before prior to fluorography (B). ~ s represen~t extracts frbm ~/J zygotic embryos, (2) pollen derived embryos and C3) callus.

243

2

3

Fig. 4. Translation profiles of pollen-der/ved embryos. Fluorograpbs of in vitro translation products of pollen derived embryos separated using two-dimensionai PAGE. (1) Embryos < 1.5 mm in length; (2) embryos 1.5-3.1 mm in length.

Fig. 3. Translation profiles of zygotic embryos. Fluorographs of in vitro translation products of zygotic embryos separated using two-dimensionai PAGE. (I) Embryos < 1.5 mm in length; (2) embryos 1.5-3.1 mm in length; (3) embryos from mature grains.

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Table I. ~S-Trauslation products from mRNA prepared from zygotic embryos, pollen-derived embryos and callus, analysed by two-dimensional gel eleetrophoresis. Boxes refer to those designated in Fig. 5 (nd, not detected; +, det~ted; + +, detected at high levels)

Box M pl Zygotic Pollen-derived Callus (kDa) embryos embryos

A 46 6 .2 - -6 .6 nd + + 44 5 .5 - -5 .9 + + + +

B 4 4 . 5 - - 4 5 6 . 8 - - 6 . 9 + + + + + 37.5--38 7.1--7.3 + + + +

C 29 7.5 + + + + 2 5 - 25.5 7 . 3 - 7.9 + + + +

D

E

20 6.6--6.9 nd + + +

30 6.3 + ++ + + 30 7.0 + + nd nd 25 6.2 + nd nd

ence is made to constellations of polypeptides found within defined regions of the gel (boxes A - E) rather than to single species.

Although many translation products were similar in the profiles of zygotic and pollen- derived embryos, substantial differences did exist. For example, products highlighted in boxes B, C and E were abundant in profiles of zygotic embryos, but were either not detected or at substantially reduced levels in profiles of petten~terived embryos, Similarly, those high- lighted in boxes A and D were either more abundant or specific to the pollen~ierived embryos. Full details of the M, and p! of these products are given in Table I.

It was also of interest, however, to compare the two classes of embryo with callus. The cal- lus profile of in vitro translation products is shown in the third section of Fig. 5. As expected, the callus profile was quite different to that of zygotic embryos. But unexpectedly, many of the polypeptide constellations identi- fied as characteristic of pollen-derived rather than zygotic embryos, were detected also in callus. Details of these findings are given in Table I.

D i ~ l ~ o n

In recent years, considerable effort has been directed towards analysing the development of zygotic embryos from both monocot and dicot species in terms of the differential expression of particular polypeptides or mRNAs. Such studies have been described for many species, including wheat [14,15], maize [16~ soybean [17] and cotton [18,19] and the developmental events involved have been extensively reviewed (e.g., Refs. 20 and 21). Attempts have also been made to characterise similar events in culture~lerived material. To-dato most attention has focused on somatic embry- ogenesis in carrot suspension~ultured cells [22 - 2 7 ] . There have also been recent studies on pea [:~], rice [29land or©hard.ass [4].

Analysis of an extended developmental time- course from initial cell division through matura- tion to desiccation of the embryo has not been possible in this study of embryogenesis in bar- ley anther culture, since many of the embryos formed in culture precociously germinate as soon as organ primordia differentiate. The pre- sent data have revealed that although both pol-

2

3

Fig. 5. Translation profiles of embryos and callus. Fluorographs of in vitro translation products of embryos and callus separated using two-dimensional PAGE. (1) Zyg- otic embryos 1.5--3.1 ram; (2) pollen-derived embryos 1.5-- 3.1 ram; (3) callus.

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len~lerived and zygotic embryos are similar to each other in terms of gross morphology and polypeptide profiles, differences do exist in the relative abundance of particular mRNA tran- scripts in the two classes of embryo. Previous reports in the l i terature describing comparisons between embryo material p r o duced in vitro and that produced in a develop- ing seed have described changes in constituent polypeptides, but no studies on translatable mRNAs have been reported previously. Chen and Luthe [29] compared polypeptides of zyg- otic embryos and embryogenic cultures of rice and identified several products common to both tissues. Using immunological methods, Crouch [30] measured levels of the 12S storage protein characteristic of zygotic embryos of Brassica napus through a time-couse of both pollen- derived and zygotic embryo development. Although this protein was detected in cultured embryos, its pat tern of expression was altered in terms of absolute levels and the time of appearance as compared to the in vivo situa- tion.

Many of the differences between zygotic and pollen-derived embryos reported in this study are quantitative changes in the abundance of particular in vitro translation products. Since many of the products of pollen~lerived embryos are present at similar levels to those of callus, it seems likely that the level of expression of these mRNAs is markedly affected by the culture environment. Using cell suspensions of D. glomerata, Hahne et al. [4] demonstrated great similarity in the in vitro translation prod- ucts of embryogenic and non-embryogenic cul- tures, however they also identified several products unique to each particular system. Unfortunately, they did not analyse translation products of mRNA from zygotic embryos for comparison.

Although frequently cited as a source of undifferentiated cells, several studies have shown that callus derived from scutellar tissue of wheat and barley embryos contains meris- tematic zones which can give rise to root and shoot meristems [31]. Coppens and Gillis [32] further demonstrated that isozyme activity

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characteristic of developed plant organs can be detected in barley callus in the absence of any morphological changes in the tissue. In view of this, it would be incorrect to consider callus as a source of undifferentiated non-embryogenic culture material.

The formation of an embryo structure is an interaction between both the genetic pro- gramme of the cell and the environmental stimuli perceived by the cells during develop- ment. In contrast to the very ordered growth pattern deseribed for barley embryos in vivo [33], development in culture is plastic and sensitive to the original properties of the explant as well as the culture conditions. Given these differences, it is perhaps surprising that pollen-derived embryos resemble zygotic embryos both morphologically and develop- mentally. However, this study has shown that although this resemblance exists, overall patterns of gene expression are different in the two classes of embryo. The study provides a basis for further analysis of the temporal and spatial regulation of 'embryo-specific genes' in structures, formed in vivo and those derived in culture in Vitro.

Ao-lmowledgements

Dr. Rob Lyne of Shell Research Ltd., Sitting- bourne is thanked for many useful discussions. The research was supported as an SERC Co- Operative Studentship to P. Higgins, with Shell Research Ltd.

References

1 V. Raghavan, Biochemistry of somatic embryogenesis, in: D.A. Evans, W.R. Sharp, P.V. Ammirato and Y. Yamada (Eds.), Handbook of Plant Cell Culture, Vol. 1, Macmillan Publishing Co., 1984, pp. 654- 671.

2 F.C. Steward, M.O. Mapes and K. Mears, Growth and organized development of cultured cells. 2. Organiza- tion in cultures grown from freely suspended cells. Am. J. Bet., 45 (1958) 705-- 708.

3 K. Nomura and A. Komamine, Molecular mechanisms of somatic embryogenesis. Oxford Surveys, Plant Mol. Cell BioL, 3 (1986) 456 - 466.

4 G. Hahne, J.E. Meyer and H. Lorz, Embryogenic and callus-specific proteins in somatic embryogenasls of

the grass Dactylis glomerata L. Plant Sci., 55 (1988) 267 -- 279.

5 S.W.J. Bright and M.G.K. Jones (Eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff/Junk, Utrecht, 1985.

6 R.L. Lyne, R.L Bennett and C.P. Hunter, Embryoid and plant production from cultured barley anthers, in: L.A. Withers and P.G. Anderson (Eds.), Plant Tissue Culture and its Agricultural Applications, Butter- worth, London, 1984, pp. 405--411.

7 C.P. Hunter, The effect of anther orientation on the production of mierospore derived embryoids and plants of Hordeum tmlgare cv Sabarlis. Plant Cell Rep., 4 (1985) 267-- 268.

8 B. Foroughi-Wehr, G. Mix, H. Gaul and H.M. Wilson, Plant production from cultured anthers of Hordeum vulgate L. Z. Pflanzenzuchtg., 77 (1976) 198- 204.

9 T. Murashige and F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cul- tures. Physiol. Plant., 15 (1962) 474--497.

10 K. Dalkin and DJ. Bewles, Local and systemic changes in gene expression induced in tomato plants by wound- ing and by elicitor treatment. Planta, 179 (1989) 367 -- 375.

11 H.R.B. Pelham and R.J. Jackson, An efficient mRNA- dependent translation system from reticulocyte lys- ates. Eur. J. Biochem., 67 (1976) 247- 255.

12 J.J. Sedmak and G.E. Grossberg, A rapid sensitive and versatile assay for protein using Coomaasie Brilliant Blue G250. Anal. Biochem., 79 (1977} 544--552.

13 D.J. Bewles, S.E. Marcus, D.J. Pappin, J.B.C. Findlay, E. Eliopoulous, P.R. Marcus and J. Burgess, Post- translational processing of eoncanavalin A precursors in jaekbean cotyledons. J. Cell Biol., 102 (1986) 1284-- 1297.

14 S.O. Rogers and R.S. Quatrano. Morphological staging of wheat caryopsis development. Am. J. Bot., 70 (1988) 308-311.

15 J.D. Wiliiamson and R.S. Quatrano, ABA regulation of two classes of embryo spocifie sequences in mature wheat embryos. Plant Physiol., 86 (1988) 208- 215.

16 D. Sanchez-Martinez, P. Puig Domenach and M. Pages, Regulation of gene expression in developing Zea mays embryos. Plant Physiol., 82 (1986) 543 -- 549.

17 R.B. Goldberg, G. Hosehek, S.H. Tam, G.S. Ditta and R.W. Beidenbaeh, Abundance, diversity and regula- tion of mRNA sequence sets in soybean embryogene- sis. Dev. Biol., 83 (1981) 201 --207.

18 G.A. Galau, N. Bijaisuradat and D.W. Hughes, Accu- mulation kinetics of cotton late embryogenesis abun- dant mRNAs and storage protein mRNAs: coordinate regulation during embryogenosis and the role of abs- cisie acid. Dev. Bio1.,:~23 (1987) 192--212.

19 D.W. Hughes and G.A. Galan, Temporally modular gene expression during development. Genes and Development, 3 (1989) 358- 369.

20 M.L. Crouch, Regulation of gene expression during

seed development in flowering plants, in: L.W. Brow- der (Ed.), Developmental Biology: A Comprehensive Survey, Academic Press, New York, 1987, pp. 367- 404.

21 R.B. Goldberg, Plants: novel developmental process. Science, 240 (1988) 1460--67.

22 C. Sengupta and V. Raghavan, Somatic embryogenesis in carrot cell suspension. I. Pattern of protein and nucleic acid synthesis. J. Exp. Bot., 31 (1980) 247-258.

23 S.R. Sung and R. Okimoto, Embryonic proteins in somatic embryos of carrot. Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 3683--3687.

24 S.R. Sung and R. Okimoto, Coordinate gene expression during somatic embryogenesis in carrot. Proc. Natl. Acad. Sci. U.S.A., 80 (1933) 2661 - 2665.

25 J.H. Choi and Z.R. Sung, Two dimensional gel analysis of carrot somatic embryonic proteins. Plant Mol. Biol. Rep., 2 (1984) 19--25.

26 S.C. de Vries, H. Booij, P. Meyerink, G. Huisman, H.D. Wilde, T.L. Thomas and A. Van Kammen, Aquisition of embryogenic potential in carrot cell-suspension cul- tures. Planta, 176 (1988) 196--204.

27 H.D. Wilde, W.S. Nelson, H. Booij, S.C. de Vries and

247

T.L. Thomas, Gene expression programmes in embry- ogenic and non embryogenic carrot cultures. Planta, 176 (1988) 205--211.

28 S. Stirn and H.J. Jaeobson, Marker proteins for embry- ogenic differentiation pattern in pea callus. Plant Cell Rep., 6 (1987) 50 - 54.

29 L.J. Chen and D.S. Luthe, Analysis of proteins from embryogenic and non-embryogenic rice (Oryza sativa L. ) calli. Plant Sci., 48 (1987) 181 -- 188.

30 M.L. Crouch, Non-zygotic embryos of Brassica napus L. contain embryo specific storage proteins. Planta, 156 (1982) 520--524.

31 R. Luhrs and H. Lorz, Plant regeneration in vitro from embryogenic cultures of spring- and winter-type bar- ley (Hordeum vulgate L.) varieties. Theor. Appl. Genet., 75 (1987) 16-25.

32 L. Coppens and E. Gillis, Isozyme electrofocussing as a biochemical marker system of embryogenesis and organogenesis in callus tissues of Hordeum ~'ulgare. J. Plant Physiol., 127 (1986) 153-158.

33 K. Norstog, Early development of the barley embryo: fine structure. Am. J Bot., 59 (1972) 123-- 132.