PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis
Christian Breton 1,3,, Annie Chaboud 1 Elisabeth Matthys-Rochon 1, Elizabeth E.M. Bates 1, J. Mark Cock 1, Hillel Fromm 2 and Christian Dumas 1 1E.N.S. Lyon, Reconnaissance Cellulaire et Amdlioration des Plantes-CNRS-INRA, 46 all~e d'Italie, 69364 Lyon Cedex 07, France," 2Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel," * Present address: LN.R.A. Orldans, Centre de Recherches ForestiOres, 45160 Ardon, France (*author for correspondence)
Received 6 April 1994; accepted in revised form 18 October 1994
One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 x 105 recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant em- bryos and represents a breakthrough to isolate genes involved in embryo differentiation.
Introduction
Based on morphological changes and physiologi- cal data, plant embryogenesis has been divided into three phases of development that are respec- tively embryo differentiation, embryo maturation and seed dehydration [15]. 3500 genes are esti- mated to participate in this particular develop- mental program [20]. However, most molecular studies to date have focused on seed maturation and dehydration for which greater amounts of material are available [15]. Few reports are re-
lated to the early differentiation phase which is crucial for the elaboration of the body structure of the embryo and the acquisition of bilateral symmetry [ 6 for review]. This lack of information is mainly explained by the fact that the early em- bryos are included within the tissues of the de- veloping seeds and not easily accessible. Never- theless, lethal mutants which abort during early embryogenesis have been isolated in Arabidopsis thaliana and Zea mays revealing the existence of single genes that play key roles in embryo pattern formation [5, 8, 20]. In Zea mays, male and re-
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X77396 and X77397.
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male gametes can also be isolated and fused in
vitro providing a new approach to study early fertilization events and perhaps, in the future, early embryogenesis in plants [9, 21]. Indeed, maize plants have been regenerated in our labo- ratory from fertilized embryo sacs isolated while the embryos were at the one or two-cell stages [24], and microdissected young zygotic embryos were considered as a material of choice for gene expression studies and cDNA library construc- tion.
In this report, we describe the construction of a maize cDNA library obtained from 100 zygotic embryos microdissected at the transition stage. Due to the small size of these embryos and the time-consuming dissections required to isolate them, PCR amplification of whole cDNA popu- lations has been used to increase the amount of cDNA available before cloning. Previous reports have shown that cDNA populations could be am- plified from a few animal cells or viruses repre- senting minute amounts of RNA [13, 29]. This type of approach has been used to construct an amplified cDNA library from distal parts of 7.5 days post-coitum mouse embryos and to clone two glucose transporter cDNAs preferentially ex- pressed at this stage [281. In plants, the obtention of PCR-generated cDNA libraries has been de- scribed [7, 16, 19] but, to our knowledge, no clones isolated from them have yet been charac- terized. Here, the quality of the early embryo li- brary we have obtained was confirmed by the cloning and sequencing of two maize calmodulin cDNAs. Calmodulin is an ubiquitous protein known to be involved in cell response via calcium [22] and has been shown to be expressed during early carrot somatic embryogenesis [30]. Its ex- pression was studied throughout maize zygotic embryogenesis at the mRNA, amplified cDNA and protein levels.
Construction of the early embryos eDNA library
Zea mays L. (inbred line A188) were grown in a phytotronic growth chamber as described [24]. Embryos were obtained from in vivo pollinated
ears and microdissected in sterile conditions under a stereomicroscope [24]. Emphasis was put on the earliest stages of embryogenesis and, due to developmental gradients existing along the ears, they were individually selected according to their morphological stages [1, 5] (Fig. 1). Batches of 100 embryos at the transition stage (Fig. 1A) or 50 coleoptilar embryos (Fig. 1B) were used for poly(A) + RNA extraction. The transition stage corresponds to the 'globular' stage defined for dicotyledonous plants; the embryos are still char- acterized by the round shape of the embryo proper (Fig. 1A) and correspond to rapidly differentiat- ing structures that are on the verge of producing apical meristems and switching from axial to plane symmetry (Fig. 1B). Their viability was checked by the fluorochromatic reaction [ 17] and in vitro regeneration [24]. After microdissection the embryos were immediately frozen in liquid nitrogen and stored at -80 °C.
The non-selective amplification of cDNA populations was adapted from [29]. Poly(A) + RNAs were directly extracted from frozen tissues in 100 mM Tris-HC1 pH 8,500 mM LiC1, 10 mM EDTA, 1~o SDS, 5 mM DTT using magnetic oligodT Dynabeads (Dynal, Norway) according to the manufacturer's indications except that no EDTA was added during the washing step. Mag- netic beads (0.4 mg) were added to the early em-
Fig. 1. Early stages of maize embryogenesis (bar = 500/~m). A. Embryo at the transition stage microdissected between 5 and 7 days after pollination (dap); ep, embryo proper; s, sus- pensor. B. Typical triangular shaped embryos at the coleop- tilar stage (Col) were isolated between 8 and 10 dap. The scutellum (sc) and the shoot meristem (sin) were clearly vis- ible at this stage, s, suspensor. For the later stages of devel- opment only the number of dap was taken into account.
bryo extracts and the mRNAs were recovered in 50 #l of DEPC-treated water. The mRNAs were directly used for cDNA synthesis in two separate reactions. Poly(A) + RNAs obtained from samples available in non-limiting quantity such as older embryos, unfertilized ovules, 5 mm root tips, leaves, seedlings (5 days after germination) were also analyzed. For these samples 0.1 #g of poly(A) + RNA was routinely used for cDNA synthesis. First-strand cDNAs were synthetized with M-MLV reverse transcriptase (Gibco-BRL). The cDNAs were then oligo dC-tailed at their 3'termini with terminal transferase and the m R N A templates were digested by RNase H (Boehringer). All of the early embryo cDNAs were ethanol precipitated prior to amplification whereas only one tenth of the cDNAs corre- sponding to ca. 20 ng of poly(A) + RNAs ob- tained from large samples were used. Non- selective amplification reactions were performed in 100 #1 of 10 mM Tris-HC1 pH 8.3, 50 mM KC1, 1.5 mM MgC12, 200 #M of each dNTP, 2.5 U of Taq polymerase (Perkin-Elmer) and 1/~M of each non-selective primers 5 ' -GGGGCTC- GAG(T)16-3' and 5 ' -GGGGAATTC(G)I I -3 ' complementary to the original poly(A) tail and to the oligo dC tail. For cloning purposes, these primers contained Xho I and Eco RI restriction sites at their 5' ends, respectively. Each sample was subjected to two successive amplifications. The first amplification was 94 ° C for 5 min, 50 ° C for 2 min, 72 ° C for 5 min, 94 ° C for 1 min, 40 ° C for 1 min, 72 °C for 5 min, followed by ten cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 5 min. The primary amplification products were then divided into 24 #1 aliquots and ream- plified for 5 more cycles in new amplification mix- tures using the last thermal conditions. When am- plified cDNAs were to be analyzed by Southern blot, 10 #1 aliquots of primary products were reamplified by a second amplification of 25 cycles. After agarose gel electrophoresis and ethidium bromide staining, the amplified cDNAs appeared as smears ranging from 0.3 to 3 kb with a major- ity of fragments between 0.7 and 1 kb (data not shown). Preliminary results obtained with the Rab 17 marker gene expressed only during late
107
embryogenesis have shown that the NS-PCR method adapted here was sensitive and reliable enough to be applied for cloning purposes (un- published results). Amplified cDNA populations were then used to study the expression of specific genes and to construct PCR-generated cDNA li- braries.
Amplified cDNAs obtained from 100 transi- tion stage embryos were used for the construction of the early embryos library. Final amplification products were pooled, ethanol precipitated and separated on a 1.25~o Sea-plaque agarose gel (FMC Bio Products). Fragments of 1 to 4 kb (45 ng) were recovered from the gels by the 'squeeze-freeze' technique using Spin-X columns (Costar), digested with Eco RI and Xho I and li- gated to 2ZAP predigested arms (Uni-ZAP XR cloning kit, Stratagene, USA). Ligation products were packaged with the Gigapack II Gold system (Stratagene). The primary library contained a total of 2.5 x 105 phages. 95~o of these phages were recombinants as estimated by color selec- tion with IPTG/X-gal using Escherichia coli XL 1- Blue as host. This gives the same number of re- combinants as generally observed for cDNA libraries obtained by conventional methods. The phages contained inserts whose sizes varied from 0.4 to 2 kb with a majority in the range of 0.6 to 0.9 kb as visualized from PCR amplifications with T3 and T7 primers of 15 randomly selected re- combinant plaques (data not shown). This library was amplified to a final titer of 3.0 x 10 7 pfu/#l and used for screening experiments.
Characterization of two maize calmodulin cDNAs
In carrot somatic embryogenesis, calmodulin ex- pression has been described in induced cells and in early stages of embryo differentiation [30]. Calmodulin is also ubiquitous, well conserved, and known to play important roles in cell activ- ity and cell response to various signals via cal- cium regulation [22]. Calmodulin-binding pro- teins have also been characterized in plants [2, 12]. A calmodulin-binding kinase exhibiting strong homology with the mammalian type II
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CaM kinase was found to be expressed in apple cells [32] showing that calmodulin targets and downstream regulation systems may also be conserved between species. Therefore, we con- sidered calmodulin genes as good candidates to test the quality and the representativeness of the library.
A petunia calmodulin clone was used to isolate homologous maize cDNAs expressed at the tran- sition stage. Insert DNA of calmodulin petunia clone petCaM81 (GenBank accession number M80836) was labelled with c~32p-dCTP by ran- dom priming (Boehringer). Ca. 10 6 phage were plated and plaque lifts performed on Hybond-N membranes (Amersham) [27]. Filters were hy- bridized for 16 h at 42 °C in 5 x SSPE, 50~o (v/v) formamide, 2 x Denhardt's solution, 0.5~o SDS and 100 #g/ml denatured, sheared herring sperm DNA. The final wash was performed in 1 x SSC, 0.1~o SDS at 55 °C for 30 min. Sev- enteen positive phages were observed. Four of these phages, which gave the strongest signal in the second screening step were purified in a third step. PCR amplification of their inserts with T3 and T7 primers revealed two different insert sizes. One plasmid of each category was recovered by in vivo excision (Uni-ZAP XR cloning kit, Strat- agene). The inserts were fully sequenced on both strands using the Sequenase version 2.0 kit (US Biochemical) and sequence analysis performed with LaserGene software (DNASTAR).
Two clones ZMCaM1 and ZMCaM2 (Gen- Bank accession numbers X77396 and X77397 respectively) were characterized. Comparison of their nucleotide sequences showed that at least two calmodulin genes were expressed in early maize embryos. At the DNA level, ZMCaM1 and ZMCaM2 showed an overall homology of 59~ o but most of the differences were located within the 5' and 3' non-coding regions that are 44 and 24~o homologous, respectively. The sequences of both non-selective primers were found at each end of ZMCaM1 indicating that this clone cor- responds to a full-length cDNA of 919 bp. In contrast, the ZMCaM2 sequence was partial and the absence of the 5' non-selective primer is ex- plained by an Eco RI site within the 5' untrans-
lated region of the cDNA. The two 450 bp cod- ing sequences were 89~ homologous at the DNA level. They were predicted to start at the first encountered ATG codon based on the alignment with other calmodulin genes and on the DNA context of their ATG codons which match 100 ~o the maize G/AC/AC/GATGGC/AG consensus initiation context [23 ]. An important bias in favor of G or C in the third position of the codons was noticed in the coding regions of ZMCaM1 and ZMCaM2 (84~o and 78~o, respectively). This agrees with the particular codon usage described for maize [ 10, 31 ] indicating that the two cDNAs cloned are genuine maize clones and not con- taminating sequences amplified during the NS- PCR.
The estimated molecular masses of ZMCAM 1 and ZMCAM2 proteins are 16 792 and 16832Da. Their 149 amino acid deduced se- quences differ for five residues at the positions 8, 10, 105, 106 and 140 and are 78 to 99~o homolo- gous with more than 20 other plant and animal calmodulins found in the databases (Fig. 2). ZM- CAM1 and ZMCAM2 were also, over a short stretch of 45 amino acids available, 44 and 82~o homologous to TCH2 and TCH3 that have been shown to be touch-induced calmodulin-related proteins in Arabidopsis thaliana [3]. Although we do not exclude the occurrence of nucleotide changes due to PCR, both the high level of ho- mology and the nature of the differences noticed between ZMCAM1 and ZMCAM2 suggest that they may be rare. Indeed, 41 out of 49 nucleotide changes between the two coding regions affect the third base of the codons and are silent. The con- served leucine found at the position 86 in both cDNAs is also encoded by two very distinct codons (AGG and CGT for ZMCaM1 and ZM- CAM2, respectively) for which the two nucleotide changes are probably not due to the Taq poly- merase. Finally, 4 of the 5 amino acids differing between the two maize clones correspond to ho- mologous substitutions that are found in other calmodulins (Fig. 2). Only the threonine located at position 10 in ZMCAM1 appears to be novel and may either be specific to ZMCAM1 or re- lated to PCR amplification errors.
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Fig. 2. Comparison of ZMCAM1 and ZMCAM2 proteins with other calmodulin and calmodulin-related proteins. ZMCAM1 and ZMCAM2 have been aligned to 23 other plant and animal calmodulins. Only the most representative sequences are shown here. Residues that match the consensus sequence (majority) are boxed. The Ca 2 ÷ binding domains are indicated by dashed lines. The threonine specific to ZMCAM 1 is shown by a star. PETCAM81, PETCAM72, OSCAM2 and ZMCALM accession numbers are M80836, M80832, Z12828 and X74490. Arabidopsis thaliana ACAM1-6 and TCH1-3 sequences were obtained from Gawienowski et al. [ 14] and Braam and Davis [3]. Expression studies and sequence homology have shown that TCH 1 corresponds to ACAM-1 and is not specifically touch-induced whereas TCH2 and TCH3 are calmodulin-related touch-induced genes [3].
Finally, the calmodulin gene family in maize was investigated by Southern hybridization ofge- nomic DNA using the full length sequence of ZMCaMI as a probe (Fig. 3). Due to the high degree of conservation of calmodulin genes this probe is taken to reveal the different members of the gene family. Multiple bands were observed for each digestion analyzed suggesting the presence of a small calmodulin gene family in the A188 maize genome (Fig 3). This result agrees with previous studies concerning other plants and hu- mans [ 11, 14].
Calmodulin expression during maize embryogen- esis
Calmodulin mRNAs expression was investigated during embryo development and in different maize tissues using the full sequence of ZMCaM1 as a probe (Fig. 4). Indeed, no differences of ex- pression were noticed between ZMCaM1 and ZMCaM2 when gene-specific probes correspond- ing to the 3' untranslated regions of each gene were used, nor when RT-PCR experiments with gene-specific primers were performed (data not
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Fig. 3. Southern blot of maize A188 genomic DNA hybrid- ized to ZMCaM1. Maize A188 genomic DNA was extracted from the leaves of young (10-15 days) plants according to Rogers and Bendich [26]. 10 #g of DNA were digested with Barn HI (B), Eco RI (E) and Hind III (H), separated on a 0.8% agarose gel and transferred to Hybond N + (Amer- sham) using an alkaline capillary transfer method [27]. ZM- CaMI insert DNA was labelled by random priming (Boe- bringer). Hybridization of the filter was at 42 °C for 16 h in 6 x SSPE, 50~ (v/v) formamide, 5 x Denhardt's solution, 0.1% S DS, and 200 #g/ml denatured, sheared herring sperm DNA [27]. The final wash was in 0.1 x SSC, 0.1% SDS at 65 ° C. The sizes of molecular weight markers are indicated in bp on the left.
shown). For northern blots 0.5 #g of poly(A) + RNA were electrophoresed under denaturing conditions through 1.5~o agarose gels [27] and transferred to Hybond-Nfp membranes (Amer- sham) (Fig. 4A). Filters were hybridized for 16 h at42 °Cin 5 x SSPE, 50~o (v/v) formamide, 2 x Denhardt's solution, 0.5~o SDS and 100/~g/ml denatured, sheared herring sperm DNA. The final wash was in 0.5 x SSC, 0.1~o SDS at 55 °C. Calmodulin mRNAs were detected as a single band showing different levels of expression in
Fig. 4. Expression of calmodulin mRNAs during maize em- bryogenesis. A. Calmodulin mRNAs directly analyzed by northern blot. B. Calmodulin cDNAs revealed after NS-PCR by Southern blot. The full-length sequence of ZMCaM1 was used as a probe. Samples analyzed are pollen (Pol), isolated ovules (Ov), embryos at the transition stage (T), embryos differentiating from the transition to the coleoptilar stage (TC), embryos at the coleoptilar stage (Col), embryos of 10, 20, 30, 40 dap (10, 20, 30, 40 respectively), 5 days after germination seedlings (Ger), root tips (Root) and leaves (Leaf). The early embryo stages that could only be analyzed by NS-PCR are specified between dashed lines. Water treated in the same manner as mRNA samples was used to check for nucleic acid contamination during the NS-PCR procedure (H20).
every tissue analyzed (Fig. 4A). Their estimated size was 1000 nucleotides which corresponds to the fulMength sequence of ZMCaMI. Expression was stronger in ovules and root tips than in pol- len, seedlings and leaves. During embryo devel- opment, calmodulin mRNAs were most abun- dant between 10 and 30 days after pollination (dap), decreased 40 dap and were almost unde- tectable in seedlings. Earlier stages of embryo de- velopment were studied by Southern blot analy- sis of NS-PCR amplified cDNAs (Fig. 4B). NS- PCR products were assayed, separated through 1.2 ~o agarose gels and transferred onto Hybond- Nfp membranes [27]. Hybridization, washing conditions and film exposure were the same as those for northern analysis. A band of 1 kb was revealed among the populations of amplified cDNA. Calmodulin cDNAs were detected as
early as the transition stage. The intensity of the signal increased during the differentiation from transition to coleoptilar stage and decreased thereafter. Although the NS-PCR analysis is not quantitative, the most striking features of the ex- pression pattern after NS-PCR are similar to those observed directly at the mRNA level (e.g. high expression in root, ovules and young em- bryos). On the other hand, stronger signals were observed after NS-PCR in the case of samples presenting initially low amounts of calmodulin mRNAs (leaves and pollen). This could reflect a preferential amplification of small DNA frag- ments during the NS-PCR procedure. In general, calmodulin mRNAs were more abundant in tis- sues corresponding to small developing struc- tures. Indeed, calcium activated calmodulin was clearly localised in the differentiating proembryo- genic masses and root meristems of developing carrot somatic embryos [30]. In potato, calmodu- lin mRNAs levels were also shown to be high in stolon tips and five times lower in differentiated tubers, suggesting that calmodulin may be in- volved in signal transduction during tuberization [18]. During maize embryogenesis, calmodulin mRNA levels were high in the early stages and decreased later upon embryo maturation (Fig. 4). This decrease can be explained by the relative increase within the mRNA populations of spe- cific RNAs coding for storage or LEA (late embryogenesis-abundant) proteins expressed during late embryogenesis and/or to a reduced metabolism when abscisic acid linked activity takes over.
Finally, calmodulin proteins were detected by western blotting (Fig. 5) using a mouse mono- clonal antibody CAM72-17-28 raised against the recombinant petunia calmodulin obtained from the clonepetCaM72 (GenBank accession number M80832; H. Fromm, unpublished results). Total proteins were extracted, assayed, separated by SDS-PAGE [4] and transferred onto BA 83 cel- lulose nitrate membranes (Schleicher & Schuell) using a semi-dry blotting apparatus. They were fixed with 0.2 ~o glutaraldehyde in 25 mM potas- sium phosphate pH 7 and the calmodulin- antibody complexes were revealed by chemilumi-
111
Fig. 5. Expression of calmodulin proteins during maize em- bryogenesis. Calmodulin proteins were revealed with a mono- clonal antibody directed against a recombinant calmodulin from petunia. Samples are: 50 ng of purified spinach calm- odulin (Sigma P5779) (Spi) and 60 #g of total proteins ex- tracted from pollen (Pol), ovules (Ov), 20, 30, 40 dap embryos (20, 30, 40 respectively), 5 days after germination seedlings (Ger), root tips (Root) and leaves (Leaf). Molecular masses in kDa are indicated on the left.
nescence (ECL kit, Amersham) using an anti- mouse Ig conjugated to horseradish peroxidase (Fig. 5). This antibody recognized a set of three major bands of 16 to 19 kDa in the purified spin- ach calmodulin sample used as positive control, and in the maize extracts analyzed (bands A, B and C). Band B corresponded to the strongest signal for most of the tissue analyzed whereas band A and C were strongly represented in seed- lings and pollen respectively. One minor band (C') appeared during embryo maturation 30 and 40 dap. This band could correspond to a specific isoform, a post-translationally modified protein or to an embryo-specific conformer since calm- odulin is known to have a flexible structure. The overall amounts of calmodulin did not vary sig- nificantly between the different samples and dur- ing embryo development. Indeed, biochemical analysis had previously shown that the levels of calmodulin were constant throughout in vitro car- rot cell growth curve and somatic embryo devel- opment [25].
Concluding remarks
In this report, we have adapted a non-selective cDNA amplification method [29] to construct a PCR-generated cDNA library from maize zygotic embryos microdissected at the transition stage. The library that we have obtained contains
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2.3 x 10 s recombinant clones representing genes or gene fragments expressed at this crucial stage of embryo differentiation. Late specific genes cod- ing for storage, RAB or LEA proteins, are not yet expressed at the transition stage, thus increasing the complexity of the sequences present in the library. To our knowledge, this library is the first available for this type of embryos. It represents an interesting reservoir of genes expressed at this stage and a complementary approach to genetic analysis focusing on single mutated genes [5, 8, 20]. Two calmodulin cDNAs have been charac- terized and calmodulin expression was confirmed at the mRNA, amplified cDNA and protein lev- els throughout embryogenesis. The expression pattern corresponded to that expected according to the general role of this protein in cell metabo- lism and to the results described for carrot so- matic embryogenesis [22, 30]. Sequence analysis has allowed us to confirm the maize origin of both genes cloned. They also suggest that punctual PCR-linked nucleotide changes may be rare. In- deed, if their occurrence is not excluded, they did not prevent us from generating gene-specific probes or RT-PCR primers that can be used to diagnose the expression of each gene cloned at any stages of maize development (data not shown). In animals, two glucose transporter cDNAs have been cloned from an early mouse embryos PCR-generated library [28]. To our knowledge, ZMCaM1 and ZMCaM2 represent the first cDNAs whose sequences were charac- terized from such libraries in plants. In addition, we have recently isolated a clone encoding the characteristic DNA binding domain of a heat shock transcription factor ('HSF') and that is ex- pressed during embryogenesis at very low level compared to calmodulin (Gagliardi and Breton, unpublished results). In consequence, we assume that this library represents an interesting tool to search for new genes specifically expressed dur- ing early embryogenesis.
Acknowledgements
This work was supported by grants from the In- stitut National de Recherche Agronomique
(France) to C.B. and the Human Frontier Sci- ence Program Organization (Japan) to C.D. and E.E.M.B.H.F. was supported by a grant from the Ministry of Science and Technology (Israel) and the Gesellschaft Far Biotechnologische Fors- chung (Germany). The authors would like to thank R. Blanc for growing the plants, Dr R. Mol for his help in dissecting the embryos and G. Green for typing the manuscript.
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