developmental gene expression in conifer embryogenesis and germination. iii. analysis of crystalloid...

Plant Science, 88 (1993) 25-37 25 Elsevier Scientific Publishers Ireland Ltd.

Developmental gene expression in conifer embryogenesis and germination. III. Analysis of crystalloid protein mRNAs and

desiccation protein mRNAs in the developing embryo and megagametophyte of white spruce (Picea glauca (Moench) Voss)

Isabel Leal and San tosh Misra

Department of Biochemistry and Microbiology, University of Victoria, Victoria, B. C V8 W 3P6 (Canada)

(Received June 1 lth, 1992; revision received August 14th, 1992; accepted September 14th, 1992)

Messenger RNAs were extracted from developing embryonic axes and megagametophytes of white spruce. Products of in vitro translation were immunoprecipitated using crystalloid protein antiserum and analysed by SDS-PAGE. Results showed that the crystalloid mRNAs directed synthesis of 55-60-, 21- and 18-kDa polypeptides. A comparison of immunoprecipitated polypeptides synthesized in vitro with those synthesized in vivo revealed that the crystalloids were synthesized as high molecular weight precursors. A eDNA library of poly(A)+RNA was constructed and a crystalloid eDNA clone WS2 was isolated. The eDNA probe hybridized to a transcript of 1.8 kb, as revealed by RNA gel blot analysis of seed tissues. Northern blot analysis of RNA extracted from various developmental stages showed that crystalloid transcripts were present at maximal levels in embryonic axes at the club-shaped stage. In megagametophytes, the crystalloid transcripts accumulated to high levels as early as the embryonal mass stage. The crystalloid transcripts were not detected in mature seeds and in seedling tissues. We also employed a heterologous eDNA probe for a desiccation protein of radish (pSB6), to examine the expression of corresponding sequences in conifers. This probe cross hybridized to an RNA class of - 600 nucleotides the accumulation of which began a few days prior to the desiccation phase and reached a maximum in the dry seeds. The pattern of temporal expression, as well as the sequence similarities as judged by the cross-hybridization, suggests that the 'Lea' proteins of conifers and angiosperms have a common ancestral origin.

Key words: embryogenesis; Picea glauca; crystalloid transcripts; desiccation (Lea) transcripts; crystalloid protein cDNA; nucleic acid hybridization

Introduction

Maturation drying is the terminal phase of seed development in most plants, leading to a state of metabolic quiescence [ 1 ]. Subsequent hydration of mature dry seed leads to its germination. These processes are generally considered to involve a switch from ~i pattern of embryogenesis to one characteristic of germination and subsequent seedling development [2,3]. Gene expression during seed development, maturation and germination has been characterized in a number of angiosperm

Correspondence to: Santosh Misra, Dcpartraent of Biochem. istry and Microbiology, University of Victoria, Victoria, B.C. VSW 3P6, Canada.

species and distinct subsets of developmentally regulated genes which respond to distinct regulatory signals have been identified [4] (see Refs. 2, 5 and 6 for review). For example, a major metabolic event during seed development is the expression of storage protein genes. In contrast there is little or no such activity in germinated seeds; rather, there is production of enzymes responsible for the mobilization and utilization of storage reserves. Studies of angiosperms have shown that storage protein gene expression is regulated mainly at the transcriptional level (see Refs. 5 and 6). Re- cent evidence tends to point to a link between changes in the level of ABA and osmoticum to the control of embryo maturation and deposition of storage reserves in angiosperm species [5-7].

0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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During late embryogensis another set of genes, Lea (late embryogenesis abundant) is prefer- entially expressed [4,8-11]. The products of some of these genes may be responsible for the development of desiccation tolerance in the embryo (see Refs. 7,12). Under experimental conditions, the level of these maturation-associated proteins can be manipulated using ABA or osmoticum during imbibition as well as by premature drying of seeds (see Ref. 7 for review). Similar proteins have yet to be characterized in conifers although a number of studies have shown that ABA [13-15] and more recently ABA and osmoticum [16,17] are required for proper development, differentiation and desiccation of conifer embryos in vitro. In general, in contrast to the extensive work done on angiosperms to date, the biochemical and molecular controls operative in conifer embryogenesis remain largely unknown.

We showed earlier that crystalloids are the major storage proteins of white spruce embryonic axes and the haploid megagametophytes [18]. SDS-PAGE and immunoblot analysis revealed that synthesis of these proteins is developmentally regulated with maximal synthesis occurring between mid-to-late-cotyledonary stages [19]. As a prelude to further studies involving the effect of ABA and osmoticum on embryo-specific gene regulation in conifers, we report in this paper the temporal changes in crystalloid mRNAs and a class of desiccation mRNA in white spruce embryonic axes and megagametophytes. The crystalloid mRNAs were examined by: (a) immunoprecipitation of in vitro translated mRNA products with crystalloid-specific antiserum, followed by SDS-PAGE analysis, (b) isolation of a homologous crystalloid cDNA clone from a white spruce cDNA library and Northern blot analysis of crystalloid transcripts during the developmental stages. We have also examined the steady-state levels of desiccation protein transcripts, using a heterologous cDNA probe (p8B6) for a radish desiccation protein [9].

Our results showed that the crystalloid cDNA clone of white spruce WS2 hybridized to 1.8 kb transcripts in RNA from developing seeds; and that its expression was regulated in a develop-

mental-and tissue-specific manner. Unlike the trend seen in angiosperms the crystalloid transcripts in megagametophytic tissue were detected as early as the embryonal mass stage and around the club-shaped stage in embryonic axes, much before the completion of cell division and differentiation of the embryo was achieved. The crystalloid transcripts were not detected either in the late maturation stage, in mature seeds or in seedling tissues. The desiccation probe from radish hybridized to a class of transcripts, approximately 600 nucleotides long. The accumulation of these transcripts coincided with the onset of desiccation phase in white spruce embryonic axes, reaching a maximum in fully mature desiccated embryos, which is indicative of their potential role in maturation of conifer embryos.

Materials and Methods

Plant material Immature cones of white spruce (Picea glauca

(Moench) Voss) were collected from a clonal seed orchard of grafted 15-year-old clones, at the Kalamalka Research Station, B.C., Ministry of Forests. The cones were wind pollinated and cross pollinated with mixed pollen (supplemental mass pollination), covered with bags and allowed to mature. Collections were made biweekly beginning June 6, 1989 and were continued throughout sum- mer 1989 (until September 12). Collections were repeated in 1990, beginning May 22.

The freshly harvested cones were packed in coolers and sent by Air-Express to the University of Victoria. Developing seeds were removed from cones, their seed coats removed and separated megagametophytes and embryonic axes were frozen in liquid nitrogen and stored at -80°C until further use. All samples were from the same mater- nal clones. The collection dates correspond to the following developmental stages: June 6, embryonal mass stage; June 20, club-shaped; July 5, early- cotyledonary stage; July 17, mid-cotyledonary stage; August 2, late cotyledonary stage; August 29, full size embryo; mature seeds of white spruce seed lot #8782 were obtained from the Surrey Seed Centre, B.C. Ministry of Forests.

RNA isolation Total RNA was extracted from white spruce

embryos or megagametophytes at eight different stages of development by a modification of the procedure of Verwoerd et al. [20]. After grinding 100 mg of frozen seed tissue, 0.5 ml of hot extraction buffer (phenol-100 mM LiCI, 100 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS (1:1), at 80°C) and 0.25 ml of chloroform-isoamyl alcohol (24:1) were added and vortexed. The aqueous phase was removed by centrifugation and mixed with one volume of 4 M LiC1. RNAs were allowed to precipitate overnight at 4°C and collected by centrifugation. The pellets were then dissolved in 0.25 ml of water, 0.1 volume of 3 M sodium acetate (pH 5.2) and the RNAs were reprecipitated with 2 vols. of cold 95% ethanol. Yields of between 30-50/~g of total RNA were routinely obtained from 100 mg of seed tissue.

In vitro translation Cell-free translation of 10/zg of total RNA as a

source of mRNA was carried out in a rabbit reticulocyte lysate translation system (BRL) in the presence of either [35S]methionine or [3H]leucine. To measure incorporation of radioactivity into polypeptides, 2 #1 of the mixture was spotted onto Whatman paper filters. Proteins were pre- cipitated in cold TCA and the filters were dried. Protein samples with 20 000 counts/min were dissolved in sample buffer (65 mM Tris (pH 6.8), 2% (w/v) SDS, 10% (w/v) glycerol and 5% (v/v)/3- mercaptoethanol. Electrophoresis was carried out in one dimension on 12% SDS polyacrylamide gels, which were then subjected to fluorography and autoradiography.

Radioactive labeling of proteins in vivo Megagametophyte and embryonic tissue from

different stages of development were dissected from immature seeds of white spruce and placed on 35S-labelled methionine and 3H-labelled leucine at room temperature for 4 h in Eppendorf tubes covered with aluminum foil. The labeling solution contained 1000/~Ci/ml of [3H]leucine for both megagametophytes and embryos and 10 000 #Ci/ml for [35S]methionine. After the incubation

27

period, the labelled tissue was rinsed with sterile distilled water and stored at -80°C until protein extraction. Fractionation of proteins into soluble matrix and insoluble crystalloids was carried out according to Misra and Green [19].

Immunoprecipitation The in vitro translated products and the in vivo

labelled extracted proteins were first precleared as described in Maniatis et al. [21] by pretreating them with preimmune serum drawn from the same rabbit that was immunized against the 57-kDa white spruce crystalloid protein complex [19]. After the preclearing treatment, the lysate samples were immunoprecipitated with white spruce crystalloid protein antiserum essentially as described by the procedure of Howard and Buckley [22]. After boiling 50 000 counts/min of each lysate sample for 2 min in 0.1 ml of immunoprecipitation buffer (1% SDS, 190 mM NaCI, 60 mM Tris-HCl pH 7.4, 6 mM EDTA), each sample was diluted with 4 vols. of 1.25% Triton X- 100, 190 mM NaC1, 60 mM Tris-HC1, pH 7.4, containing 0.1 mM PMSF and 1 mg BSA and 20 ml of a 10% (w/v) Staphylococcus aureus cell solution. After 1 h incubation at 4°C, the samples were centrifuged for 15 min and the supernatants were transferred to new microfuge tubes. To each sample, 2/zl of neat anti-white spruce crystalloid protein complex antiserum was added and immune complexes were allowed to form overnight. The immune complexes were then collected by centrifugation with a 10% w/v S. aureus cell solution (previously incubated for 15 min in 30 mg/ml BSA in phosphate-buffered saline). The immune complexes were then washed, dissolved in sample buffer (62.5 mM Tris (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol and 0.1 M DTT) and subjected to electrophoresis on a SDS 12% polyacrylamide gel. Gels were then subjected to fluorography and autoradiography.

Construction of a white spruce embryonic cDNA library

Messenger RNAs used for cDNA cloning were isolated from total RNA of developing megagametophytes over one cycle of oligo (dT) column

28

using prepacked oligo (dT) cellulose spun columns (Pharmacia). After ethanol precipitation, the poly (A) ÷ RNA was dissolved in sterile water. From 500/zg of total RNA, 15/zg of poly(A) ÷ RNA was obtained, of which 5 /~g were used to construct double stranded eDNA by the method of Gubler and Hoffman [23], as described in Pharmacia eDNA synthesis manual. Approximately 100 ng of the EcoRI-ended cDNA were ligated with 1 #g of

ZAP II arms (Stratagene), assembled into phage particles in vitro by using the Gigapack system (Stratagene) and the resultant recombinant phage was used to infect the E. coli host XL1 Blue. A total of 3 x 106 recombinant clones were obtained and the library was stored at 4°C.

Screening the cDNA library by nucleic acid hybridization

A total of 10 × 104 phage clones were transferred to nitrocellulose filters as described by Man- iatis et al. [21] and screened using a crystalloid full length eDNA clone of Douglas fir (DF1) which was previously isolated from a Douglas fir embryonic eDNA library by immunoscreening [24]. The Douglas fir eDNA fragment was radioactively labelled by random priming (BRL) and hybridized in 50% Formamide, 5x SSC, 0.2 M Tris pH 7.6, 1X Denhardt solution, 10% Dextran sulphate, 0.1% SDS and 0.1 mg/ml sheared calf thymus DNA for 18 h at 42°C. The filters were then washed twice for 15 min with 2x SSC, 0.1% SDS at room temperature and then twice for 15 min with 0.2x SSC, 0.1% S.D.S at 55°C. A positive cDNA clone (WS2) was excised from X ZAP II as a recombinant pBluescript SK(-) plasmid according to the manufacturer's protocol. The insert from recombinant pBluescript SK(-) plasmid was purified on an 0.8% low melting point agarose and EcoRI digestion.

Dot-blot hybridization Total denatured RNA (5 /~g) from each

developmental stage was blotted on Zeta-probe membrane (Bio-Rad) by microfiltration. The eDNA probes WS2 and p8B6 were labelled by random priming (BRL) with 5'-[c~-32p]dCTP according to the manufacturer's protocols to a specific activity of approximately 1 x 108 counts/

min//xg. The 32p-labelled recombinant probe was hybridized to the RNA blot according to the method of Church and Gilbert [25]. The membrane was prehybridized for 5 rnin and then hybridized for 12 h at 42°C in 50% formamide, 0.25 M NaHPO4 (pH 7.2), 0.25 M NaC1, 7% (w/v) SDS, 1 mM EDTA. The membrane was then washed with 2x SSC, 0.1% SDS, followed by 0.5x SSC, 0.1% SDS at room temperature for 15 min, respectively and then twice for 15 rain with 0.1 x SSC, 0.1% SDS at 65°C. The filters were exposed to X-ray films overnight.

Northern blot analysis Total denatured RNA (10 /~g) were electro-

phoresed on a 1% (w/v) agarose-formaldehyde gel according to the method of Fourney et al. [26]. After electrophoresis, formaldehyde was removed from the gel by three washings with 10x SSC at room temperature and the RNA was transferred overnight to a Zeta-probe membrane by capillary flow of 10x SSC. Hybridization and washes were performed as described for dot-blot hybridization. The filters were exposed to X-ray film overnight for Figs. 4A and 4B and 3 days for Fig. 5A.

Results

In vitro protein synthesis and immunoprecipitation Total RNA was isolated from eight different

stages of zygotic embryogenesis from seeds collected on: (1) June 6, (2) June 22, (3) July 5, (4) July 17, (5) August 2, (6) Aug 15, (7) Aug 29 and (8) dry mature seeds. Fertilization is believed to have taken place around June 3 (J.N. Owens, pers. commun.) and on July 5 it was possible to separate the megagametophyte from the embryonic axis.

The mRNA from all these different stages of embryonic development was analyzed by using the rabbit reticulocyte in vitro translation assay and [35S]methionine, followed by SDS-PAGE analysis and autoradiography. The results showed that the mRNAs from different stages code for a variety of polypeptides (Fig. 1A-B), however, polypeptides of 60, 55, 21 and 18 kDa were predominant. These abundant mRNAs accumulated during the early and middle stages of zygotic embryogenesis. In megagametophytes the prevalent mRNAs were

A

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1 2 3 Fig. 1. Fluorographs of proteins synthesized in vitro using total RNA extracted from: (A) megagametophytes at the following developmental stages: May 19 (1); June 6 (2); June 22 (3); July 5 (4); July 17 (5); Aug 2 (6); Mature seed (7). (B) embryos at the following developmental stages: July 5 (I); July 17 (2); Aug 2 (3). Translation was carried out in a rabbit reticulocyte lysate, in presence of [35S]methionine and analysed by SDS-PAGE and fluorography.

present on June 6 and 22, July 5 and 17 (Fig. 1A, lanes 2, 3, 4 and 5, respectively) and in embryos on July 5 and 17 (Fig. 1B, lanes 1 and 2, respectively). They then decline dramatically at later stages of embryogenesis, e.g. in megagametophytes, August 2 0ane 6), mature seeds 0ane 7) and in embryos, August 2 0ane 3).

29

The products of in vitro translation were immunoprecipitated with a polyclonal antiserum raised against purified non-reduced crystalloid 57- kDa protein complex [19]. SDS-PAGE analysis of immunoprecipitated polypeptides indicated that some of the abundant mRNA species observed during the course of embryogenesis corresponded to white spruce crystalloid proteins. All four polypeptides were immunoprecipitated from the mixture and appear to correspond to the major translation products of approximate molecular weights of 60, 55, 21 and 18 kDa. These results in- dicate that the level of white spruce crystalloid mRNAs was high during the early stages of embryogenesis for both megagametophytes (Fig. 2A: June 6, lane 1; June 22, lane 2; July 5, lane 3; July 17, lane 4) and for embryos (Fig. 2B: July 5, lane 2; July 17, lane 3). However, the crystalloid mRNAs were not present at later stages of maturity for either megagametophytes (Fig. 2A: Aug 2, lane 5; Aug 15, lane 6; and Aug 29, lane 7) or embryos (Fig. 2B: Aug 2, lane 4; Aug 15, lane 5; and Aug 29, lane 6). Embryonic axes (Fig. 2B, lane 7) and megagametophytes (Fig. 2A, lane 8) from mature seeds also lacked detectable mRNAs for the crystalloid storage proteins.

In vivo protein labeling and immunoprecipitation To compare synthesis of crystalloid polypep-

tides in vitro with those synthesized in vivo, megagametophytes from two different stages of development, June 22 and July 5 were incubated in [3H]leucine containing medium. The products were immunopreciptated with the antiserum raised against the 57-kDa crystalloid complex and the immunoprecipitated polypeptides were analysed by SDS-PAGE gel electrophoresis and autoradiography. In parallel, mRNA was isolated from immature megagametophytes (June 22) and assayed for its activity to direct protein synthesis in a rabbit reticulocyte lysate system in the presence of [3H]leucine. The polypeptides were immunoprecipitated using crystalloid antibodies and analysed as described above. Figure 3 shows that the major classes of immunoprecipitated polypeptides synthesized in vitro were 55-60 kDa and 18-22 kDa with the former being the most prevalent species (Fig. 3, lane 1). The 55-60-kDa immunoprecipit-

30

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1 2 3 4 5 6 7 Fig. 2. Immunoprecipitation of total translation products from: (A) megagametophytes at the following developmental stages: June 6 (1); June 22 (2); July 5 (3); July 17 (4); Aug 2 (5); Aug 15 (6); Aug 29 (7); mature seed (8) and control (9). (B) embryos at the following developmental stages: Control (1); July 5 (2); July 17 (3); Aug 2 (4); Aug 15 (5); Aug 29 (6) and mature seed (7) with anti white spruce crystalloid protein complex antibodies.

ated polypeptides were no longer present in the in vivo pattern (Fig. 3, matrix fraction: July 5, lane 3 and crystalloid fraction: June 22, lane 4 and July 5, lane 5). In June 22 samples (matrix fraction, lane 2) faint bands that co-migrated with the 55-60- kDa polypeptides were observed. It is possible that the 60- and 55-kDa precursor polypeptides are processed to give rise to the 42-45- and 8-kDa polypeptides in the matrix fraction (lanes 2 and 3) and the 35- and 20-kDa polypeptides of the

crystalloid fraction (lanes 4 and 5) observed in the immunoprecipitation of in vivo products.

Isolation of crystalloid cDNA clone and analysis of temporal expression

In order to investigate the developmental regulation of crystalloid mRNAs, we constructed a cDNA library from an early-maturation-stage mRNA of megagametophytes of white spruce (June 22). In vitro translation studies indicated

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31

1 2 3 4 5 Immunoprecipitation of: in vitro translation products from megagametophytes, June 22 (1) and in vivo labelled products

from megagametophytes, matrix fraction: June 22 (2), July 5 (3), and crystailoid fraction: June 22 (4), July 5 (5). The polypeptides synthesized in vitro and in vivo were labelled with [3H]leucine.

that at this stage crystalloid storage protein mRNAs were abundant. The library was screened by hybridization with a Douglas fir crystalloid full length cDNA clone (DF1) previously isolated and characterized in our laboratory [24]. Several positive clones were identified and one designated WS2 was used for the studies described here. Analysis of the insert from this clone on a 1% agarose gel indicated that it was approximately 0.7 kb in size.

(i) Northern blot analysis. Northern hybridization was carried out in order to show that the selected cDNA WS2 clone represented a distinct embryo message, and to determine the size of complementary mRNAs. The hybridizations were performed by using an equal amount of total RNA from eight developmental stages, mature seeds, leaves and roots of 2-week-old seedlings and the cDNA WS2 clone as a probe. The WS2 clone hybridized to 1.8-kb transcripts in RNA extracted

from seed tissues and exhibited a pattern of dif- ferential gene expression during development. The complementary mRNAs began accumulating at an early developmental stage in megagametophytes (Fig. 4A: lanes 1 and 2) and in embryonic axes (Fig. 4B: lane 1) remained at high levels during mid-stages of development (Fig. 4A: lanes 3 and 4 for megagametophytes and Fig. 4B: lane 2 for embryos), but were no longer present during late stages of development (Fig. 4A: lanes 5, 6 and 7 for megagametophytes and Fig. 4B: lanes 3, 4 and 5, for embryos), mature seeds (Fig. 4A: lane 8, megagametophytes, and Fig. 4B: lane 6, embryos), in leaves (Fig. 4B: lane 7) and in roots (Fig. 4B: lane 8) of 2-week-old seedlings.

(ii) Dot-blot hybridization. In order to quantitate levels of crystalloid mRNA accumulation, dot-blot experiments were performed by reacting total RNA from megagametophytes, embryos and dry seeds with 32p-labeled cDNA clone WS2 and

32

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measur ing radioactive incorpora t ion by scintilla- t ion counting. According to the quant i ta t ive da ta obta ined (Fig. 4C), the m R N A s for crystalloid storage protein were most prevalent during early

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(iii) Cross-hybridization of radish desiccation cDNA and temporal expression changes in white spruce. The desiccation cDNA clone p8B6 from radish [9] was used in Northern blot and dot-blot analysis with total RNA from embryos of white spruce at specific developmental stages. Figure 5A shows that the white spruce mRNAs that cross- hybridized with this clone were about 600 nucleotides in size and began accumulating during the late stages of development (Aug 2, lane 3; Aug 15, lane 4; Aug 29, lane 5; and mature seeds, lane 6), but were not detected at early stages of development corresponding to July 5 (lane 1) and July 17 (lane 2). This period corresponds to the beginning of the desiccation phase and at this stage mRNAs for crystalloid storage proteins were not expressed at detectable levels. In order to quantitate the relative extent of hybridization of the desiccation cDNA clone p8B6 from radish with white spruce, total RNA from megagametophytes, embryos and dry seeds obtained from the same eight developmental stages as shown in Fig. 4C was dot-blotted and hybridized with this clone. In contrast to the quantitative results obtained for the crystalloid storage protein cDNA clone WS2, Fig. 5B shows that the desiccation transcripts began accumulating to increased levels during the late developmental stages (Aug 2 and Aug 15), reaching a maximum during the last developmental stage (Aug 29), and were stored at high levels in mature dry seeds.

Discussion

We report in this paper the isolation of a crystalloid cDNA probe of white spruce and its use for the study of temporal changes in crystalloid transcripts in embryonic axes and megagametophytes. We also report, for the first time, developmental changes in desiccation (Lea) mRNAs in white spruce zygotic embryos using a heterologous desiccation eDNA probe from radish.

A eDNA library was constructed from poly(A) ÷ mRNA of immature megagametophytes and was then screened using a crystaUoid eDNA of Douglas fir. The isolated eDNA clone WS2 represented a developmentally regulated gene whose expression was induced during early embryogenesis. This gene was not expressed in

root or leaf tissues of young seedlings. Our results showed that the 1.8-kb transcripts hybridizing to this eDNA probe began accumulating soon after fertilization in the megagametophytic tissue and accumulated to high levels as early as the embryonal mass stage. In the embryonic axes, crystalloid transcripts reached a maximal level at the club-shaped stage when embryonic axes were easily dissected from the megagametophytic tissue. These data support the results of in vitro translation and immunoprecipitation described here. In each organ, the maximal levels of crystalloid transcript accumulation preceded the period of rapid accumulation of crystalloid storage proteins as reported earlier by Misra and Green [19]. This latter study showed that the most dramatic changes in storage protein accumulation occurred between the stages of mid-embryo to the beginning of late embryogenesis. In the past few years, a number of laboratories have examined storage protein accumulation in zygotic [18,19,27,28] as well as somatic embryos of various conifers [29-31]. However, characterization of embryo- specific gene expression at the transcriptional and post-transcriptional level is rather limited [32]. Whitmore and Kriebel [32] studied the expression of a gene transiently expressed around the time of fertilization by in vitro translation of the mRNAs extracted from ovules and developing embryos of Pinus strobus. The temporal expression of crystalloid storage protein described in our study differs from the 23-kDa protein gene described in Pinus strobus. Also, unlike the storage protein gene expression described in our study, the expression of the 23-kDa protein gene was limited to the seed coat and nucellus tissue.

Developmental changes in the seed storage protein mRNAs have previously been described for a variety of angiosperms (see Ref. 5 for review). In general, storage protein genes are expressed abun- dantly during the cell expansion phase which oc- curs midway through embryo development, after cell division has ceased and tissues have differen- tiated. In contrast to these results, in white spruce megagametophytes the maximum expression of crystalloid genes was achieved during the embryonal mass stage, before the formation of meri- stematic regions of the embryo. Thus, our study suggests there are differences in the temporal

regulation of storage protein gene expression between angiosperms and conifers. This may reflect basic cellular differences in embryo development between these two diverse groups [33,341.

Immunoprecipitation of crystalloids synthesized in vitro from white spruce mRNAs showed the ex- istence of several major precursors of crystalloid storage proteins. These precursors may be processed to give rise to the mature subunits of crystalloid complex present in in vivo immunoprecipitated products. Similar subunits have been reported previously by Western-blot (immunoblot) analysis of extant proteins [19].

The 11-12 S globulin (legumin) storage proteins of angiosperms have been well characterized [35]. The legumins from several angiosperms are synthesized as 60-kDa precursors that are cleaved to yield 40- and 20-kDa polypeptides [10]. The 55-60-kDa precursor polypeptides of white spruce may correspond to these high-molecular weight legumin precursors and the 1.8-kb crystalloid transcript of white spruce may correspond to the 60-kDa precursor. In fact, the amino acid sequence derived from a full length (1.7 kb) crystalloid storage protein cDNA clone of Douglas fir (DF1) shares 34% identity with the legumin precursor of cotton (Leal and Misra, manuscript in preparation). The deduced crystalloid precursor protein sequence of Douglas fir contains a signal peptide with domain A and B separated by a highly conserved cleavage site, as observed in a range of legumin precursor cDNAs [10l.

Another set of genes, late embryogenesis abundant (Lea) genes which appear to be involved in desiccation and dormancy, have also been reported in a variety of angiosperms (see Ref. 7 for review). To examine the temporal pattern of Lea gene expression in developing seeds of white spruce, a desiccation cDNA clone of radish p8B6 [9] was employed. The p8B6 cDNA clone is homologous to the group 1 Lea proteins, D19 of cotton [36l and Em protein of wheat [37]. In Nor- them analysis of white spruce RNA from developing embryos, the p8B6 probe hybridized to a 600-nucleotide mRNA class which began accumulating just prior to the onset of desiccation (late- cotyledonary) phase, reached maximal level in the fully mature embryo and was stored in dry seeds.

35

The time course of synthesis of these mRNAs was very different from that of the crystalloid mRNAs, whose expression occurred at a very high level in early and mid-embryogeny. The temporal pattern of accumulation of desiccation (Lea) transcripts in white spruce is comparable to the developmental pattern reported in radish [91 and in other angiosperms [4,7,11]. The cross-hybridization of radish desiccation eDNA to corresponding sequences in white spruce RNA suggests that there are sequence similarities between the Lea genes of angiosperm and gymnosperms, and these genes share a common ancestral origin.

In conclusion, our data show temporal changes in two distinct gene sets, namely: crystalloids and desiccation (Lea) genes during white spruce zygotic embryogenesis. Using the WS2 crystalloid eDNA of white spruce and p8B6 desiccation cDNA of radish as probes it may be possible to determine how these genes respond to ABA, osmoticum and desiccation in conifer zygotic and somatic embryogenesis. We are presently using the p8B6 probe to isolate homologous cDNA clones of white spruce desiccation proteins. Our long- term goal is to isolate the genes encoding seed- specific proteins and to dissect the cis- and trans-

acting regulatory factors affecting gene regulation in haploid megagametophytic tissue and in the diploid embryos of conifers.

Acknowledgements

This work was supported by an operating grant to Santosh Misra from the Natural Sciences and Engineering Research Council of Canada. Isabel Leal is the recipient of an NSERC postgraduate fellowship and the Science Council of British Col- umbia G.R.E.A.T. award. We wish to thank Dr. Michel Delseny for the gift of desiccation clone p8B6.

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