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IN DOUGLAS-FIR (PSEUDOTSUGA MENZIESII [MIRB.] FRANCO)
by
Malinee Chatthai
B. Sc., Chulalongkom University, 1990
A Dissertation Submitted in Partial Fulfillment o f the Requirements for the Degree o f DOCTOR OF PHILOSOPHY
in the Department o f Biochemistry and Microbiology We accept this dissertation as conforming to the required standard
Dr. Santosh M ^sfar^up^rvisor (Department o f Biochemistry and Microbiology)
________________
ental M ember (Department ot Biochemistry and Microbiology)
___________________________________________
Dr. Williarn W. Kay, D epartm entajÆ em ber (Department o f Biochemistry and
Microbiology) / /
Dr. Paul J. Rdmahiuk, Departmental Member (Department o f Biochemistry and Microbiology)
Dr. Nigel J. Pfivingston, Outside Member (Department o f Biology)
r. John E. Carlson, External E
Dr. John E. Carlson, External Examiner (School o f Forest Resources, The Pennsylvania State University)
©Malinee Chatthai, 1999 University o f Victoria
All rights reserved. This dissertation may not be reproduced in whole or in part, by photocopying or other means, without the permission o f the author.
Il
Supervisor: Dr. Santosh Misra
ABSTRACT
As a direct approach to investigate the molecular basis o f embryogenesis in Douglas-fir {Pseiidotsuga menziesii [Mirb.] Franco), a cDNA library made from poly(A)^ RNA o f developing seeds was differentially screened for clones representing transcripts abundant in the developing seeds but absent in mature seeds. O f a number o f clones isolated, two groups were selected for further sequence and gene expression analysis.
A group o f four cDNA clones (PM2S1, PM2S2, PM2S3 and PM2S4) shared a significant nucleotide and deduced amino acid sequence similarity with each other and with gymnosperm 2S seed storage protein cDNAs. The deduced amino acid sequences had low similarity with angiosperm 2S storage proteins but contained all conserved cysteine residues in an arrangement suggestive o f a structural similarity between the 2S seed storage proteins from gymnosperms and angiosperms. Northern blot analysis revealed PM2S mRNAs were present specifically in seeds and temporally during seed development. However, the relatively low abundance o f PM2S3 mRNAs and the decline o f PM2S2 mRNAs in megagametophyte which occurred before that o f the other mRNAs suggested that their expression was regulated differentially. The accumulation o f PM2S transcripts in megagametophyte started during the early embryogenesis and reached a peak before that in zygotic embryos. PM2S mRNAs were present in Douglas-fir somatic embryos at the same developmental stages as those in zygotic embryos, and ABA and osmoticum stress were necessary for the expression o f PM2S genes in somatic embryos. Southern blot analysis o f genomic DNA suggested that the Douglas-fir 2S seed protein genes consisted o f at least two sub-families each including several gene members. A gene designated g P m 2 S l was isolated and sequenced. A comparison o f the upstream sequence o f gP m 2Sl with the promoters o f known 2S storage protein genes did not reveal significant sequence similarity except the presence o f RY-repeated element (GCATGC),
and the frequent occurrence o f ACGT-containing motifs and E-box motifs (CANNTG). The 1.2-kb g P m lS l promoter was fused to a P-glucuronidase (uidA) reporter gene and transformed into developing Douglas-fir seeds using particle bombardment and into tobacco via Agrobacterium tumefaciens. Histochemical analysis showed that the promoter was active in both systems and the gene expression was confined to endosperm and embryos o f transgenic tobacco, indicating a common seed-specific regulatory mechanism between angiosperms and conifers.
Another cDNA clone, PM2.1, hybridized to a 0.5 kb transcript and was predicted to encode a metallothionein (MT)-like protein. Alignment o f the PM2.1 predicted amino acid sequence with other plant MT-like gene products revealed a general paucity o f Cys and Cys-Xaa-Cys sequences and the presence o f serine residues within the conserved Cys-Xaa-Cys motifs in the C-terminal domain. Phylogenetic analysis showed that PM2.1 grouped with class 1/type 3 MT-like genes. The PM 2.1 was expressed in somatic and zygotic embryos, in megagametophyte, as well as in hormone- and metal- treated seeds and seedlings. The PM2.1 transcripts were detected in the needles o f 10- week-old seedlings, but not the root tissue or mature pollen. The expression o f the PM 2.1 gene in embryos was dependent upon ABA and osmoticum and was differentially modulated by metals, suggesting that the PM2.1 gene product m ay play a role in the control o f microelement availability during Douglas-fir seed development and germination. Southern blot analysis o f genomic DNA suggested that the PM2.1 was encoded by a multigene family. Three genomic clones were isolated and one o f these clones (gPmMTa) was cloned and sequenced. The sequence analysis o f its 5'-flanking region identified a number o f putative regulatory elements such as ACGT-containing motifs, metal-responsive element (TGCGCC) and ethylene-responsive elements (ATTTCAAA) which may be responsible for gene transcription. DNase I-footprinting experiments with nuclear extracts isolated from Douglas-fir megagametophyte identified two protein-protected sites, a 31-bp sequence locating in the -176/-146 region that
IV
contained two ACGT-core motifs, and a 12-bp sequence, 5-TGCCACGGAAGG-3', o f unknown function. To identify promoter regions responsible for the regulation o f
gPmMTa gene expression, a series o f deletions in the 0.9-kb fragment o f the gPmMTa
promoter was fused to the uidA reporter gene and the chimeric gene constructs were assayed in Douglas-fir and transgenic tobacco. Transient expression assays in megagametophyte and zygotic embryos indicated that the sequence lying between -190 and +88 o f gPmM Ta was sufficient to drive the expression o f the reporter gene and the 225-bp fragment (-677 to -453) contained sequences necessary for high level expression. The gPmMTa promoter was not active in the seeds o f transgenic tobacco.
Dr. SantosinMism, Supervisor (Department o f Biochemistry and Microbiology)
ental Member (Department o f Biochemistry and Microbiology)
Dr. William W. Kay, Departmepç(rM ember (Department o f Biochemistry and Microbiology)
Dr. Paul Jf./Romaniuk, Departmental Member (Department o f Biochemistry and Microbiol
Dr. Nigel J. Livingston, Outside Member (Department o f Biology)
Dr. John E. Carls^bn, External Examiner (School o f Forest Resources, The Pennsylvania State University)
VI TABLE OF CONTENT ABSTRACT... ü TABLE OF CO NTEN TS... vi LIST OF T A B L E S... xi LIST OF F IG U R E S ... xii
LIST OF A BBREVIATIONS... xiv
ACKNOWLEDGEMENTS... xvi
CHAPTER 1 : INTRODUCTION... I CHAPTER 2: LITERATURE R E V IE W S ... 4
1. Molecular biology of em bryogenesis... 4
1.1. Seed development... 4
1.2. Gene expression during embryogenesis... 5
1.2.1. Early embryogenesis... 5
1.2.2. Seed maturation and desiccation... 1
2. Seed storage proteins... 9
2.1. Structure of 2S seed storage proteins... 10
2.2. Synthesis and deposition of 2S seed storage proteins... 12
3. Storage protein gene fam ily... 15
3.1. Storage protein gene expression... 16
4. Transcriptional regulation of 2S seed storage protein gene expression... 17
4.1. cjs-acting elem ents... 17
4.2. fran5-acting factors... 20
5. Post-transcriptional and translational regulation... 23
6. Factors that influence the developmental expression of seed storage protein genes . . . . 24
6.1. Effects o f ABA on seed developmental gene ex p ressio n ... 25
6.2. Regulation of gene expression by A B A... 26
6.3. Down-regulation of storage protein synthesis by desiccation... 28
7. Metallothionein proteins... 29
7.1. Structure of metallothioneins... 29
7.2. Synthesis and functions of metallothioneins... 30
7.3. Molecular characterization and regulation of metallothionein g e n e s ... 31
7.4. Mammalian metallothionein functions in p la n ts... 32
8. Metallothionein-like genes in plants... 33
8.1. Identification o f genes encoding plant metallothionein-like proteins... 33
8.3. Modulated expression and potential roles of MT-like proteins in p la n ts... 38
8.4. Regulation of plant metallothionein gene expression... 41
9. Conifer embryogenesis... 42
9.1. Molecular events and gene expression during conifer embryogenesis... 44
10. Somatic embryogenesis in conifers... 46
10.1. Conifer somatic embryo developm ent... 47
10.2. Developmental gene expression during somatic embryogenesis... 47
10.2.1. Initiation of embryogénie cultures... 48
10.2.2. Developing somatic embryos... 50
10.3. Effects of phytohormones and osmoticum on conifer somatic embryo development and gene expression... 51
CHAPTER 3: MATERIALS AND M ETHODS... 54
1. Plant materials: 1.1. Developing seeds... 54
1.2. Somatic embryos... 55
1.3. Seed germination and treatment conditions... 57
2. Vectors, bacterial strains and culture condition... 58
3. Material, reagents and m e d ia ... 58
4. RNA techniques: 4.1. Total RNA extraction... 59
4.2. Poly(A)* RNA preparation... 59
4.3. Northern hybridization... 60
5. DNA techniques: 5.1. DNA preparation 5.1.1. Plasmid DNA preparation... 61
5.1.2. Bacteriophage DNA preparation... 61
5.1.3. Douglas-fir genomic DNA preparation... 62
5.1.4. Oligonucleotide preparation... 63
5.1.5. DNA fragment purification... 64
5.2. Radioactive lebelling o f DNA fragment 5.2.1. First-single stranded cDNA probe... 64
5.2.2. Random-primer labeling... 65
5.2.3. 5'-end lebeling of oligonucleotide primer... 66
5.2.4. Single-stranded 5'-end labeled DNA fragment... 66
5.3. Construction of Douglas-fir embryogenesis cDNA library... 66
5.4. Differential screening o f the Douglas-fir cDNA library... 68
vin
5.6. Screening of the Douglas-fir genomic library... 69
5.7. Southern hybridization... 70
6. Molecular cloning: 6.1. fn vivo phage excision... 70
6.2. Preparation of E. call competent c e ll s ... 71
6.2.1. Hanahan's m e th o d ... 71
6.2.2. CaCl: s to c k ... 72
6.3. DNA restriction digestion and dephosphorylation... 72
6.4. DNA ligation... 73
6.5. Transformation of E. c o ll... 73
6.6. Unidirectional deletion of plasmid DNA... 74
7. DNA sequence analysis 7.1. Dideoxy chain termination method... 75
7.2. Nucleotide modification cleavage method for G sequencing reaction... 76
7.3. Computer analysis... 76
8. Primer extension analysis... 76
9. DNA-protein interaction analysis 9.1. Nuclei isolation... 77
9.2. Preparation of nuclear extract... 78
9.3. DNase I-footprinting assay... 78
10. Plant transformation 10.1. Tissue preparation... 79
10.2. Construction of GUS expression vectors... 79
10.3. Particle bombardment... 80
10.4. Tobacco transformation... 81
10.5. Histochemical assay for GUS transient expression... 82
11. Protein purification and detection 11.1. Purification of 28 seed storage proteins 11.1.1. Protein extraction... 82
11.1.2. Gel filtration... 83
11.1.3. High-performance liquid chromatography... 83
11.1.4. Reduction and alkylation o f proteins... 83
11.2. Determination of protein concentration... 84
11.3. Tricine/sodium dodecyl sulphate polyacrylamide gel electrophoresis... 84
11.4. Two-dimesional isoelectric focusing/SDS polyacrylamide gel electrophoresis . . 85
CHAPTER 4: RESULTS I. Cloning of embryogenesis-assoclated cDNAs of Douglas-fir... 86
1. Isolation of embryogenesis-specific cDNAs... 86
n . Molecular characterizatiou of a 2S seed storage protein g e n e ... 89
1. Identification and characterization o f cDNAs encoding 2S seed storage protein precursors... 89
2. Sequence comparison of a Douglas-fir 28 seed storage protein with other similar proteins from gymnosperms and angiosperms... 92
3. Genomic Southern blot analysis of PM2S c D N A s... 99
4. Cloning of 2S seed storage protein genes in D ouglas-fir... 99
5. Characterization of the Douglas-fir g e n e ... 100
6. Developmental expression of PM2S mRNAs during Douglas-fir zygotic embryogenesis... 106
7. Developmental expression of PM2S mRNAs during Douglas-fir somatic embryogenesis... 107
8. Effects of ABA and osmoticum on PM2S mRNA accumulation... 108
9. Analysis of the gPm2Sl/uidA chimeric gene activity in Douglas-fir and transgenic tobacco... 108
10. Characterization of 2S seed storage proteins from Douglas-fir seeds... 116
m . Molecular analysis of a metaUothionein-like g e n e ...123
1. Sequence analysis of the PM2.1 cDNA clone... 123
2. Sequence comparison of PM2.1 with metallothionein-like gene products from gymnosperms and angiosperms... 123
3. Genomic Southern blot analysis o f PM2.1 c D N A ... 128
4. Cloning of a Douglas-fir metallothionein-like g e n e ...129
5. Characterization of the Douglas-fir gPmMTa gene... 129
6. Developmental expression of PM2.1 mRNA during Douglas-fir zygotic embryogenesis... 138
7. Tissue-specific accumulation of PM2.1 mRNA...138
8. Developmental expression of PM2.1 mRNA during Douglas-fir somatic embryogenesis... 139
9. Effects of ABA and osmoticum on PM2.1 mRNA accumulation in somatic em bryos...139
10. Effects of ABA and metal ions on PM2.1 mRNA accumulation...140
11. Analysis o f the gPmMTa/tiidA chimeric gene activity in Douglas-fir and transgenic tobacco...149
CHAPTER 5: DISCUSSION... 159
1. Embryogenesis-related genes in Douglas-fir...159
2. M o le c u la r a n a ly sis o f a 2 S s e e d sto r a g e p ro tein g e n e f a m i l y ... 159
2.1. Structures o f PM2S cDNAs and their translated products...159
2.2. Douglas-fir 2S seed storage protein gene fam ily...164
2.3. Douglas-fir 2S storage protein genes and their differential expression...166
2.4. Developmental expression o f PM2S mRNAs during embryogenesis... 167
2.5. Influence of ABA and osmoticum on PM2S gene expression...169
2.6. Transcriptional regulation of 2S seed storage protein gene expression...171
3. Molecular analysis of a metallothionein-like protein gene family... 173
3.1. Structure of PM2.1 cDNA and its translated p ro d u c t... 173
3.2. Developmental expression of PM2.1 mRNA during embryogenesis... 174
3.3. Effects of ABA and osmoticum on PM2.1 gene expression... 176
3.4. Effects of metal ions on PM2.1 gene expression...177
3.5. Transcriptional regulation of metallothionein-like gene expression...178
REFER EN C ES... 183
APPENDIX 1. Alignment of translated amino acid sequences of 28 seed storage protein precursors...206
APPENDIX 2. Alignment of coding regions of 28 seed storage protein cDNAs from conifers... 208
APPENDIX 3. Alignment of 3'-untranslated regions o f 28 seed storage protein cDNAs from co n ifers...210
LIST OF TABLES
Table 1. Metallothionein-like gene products identified in plants and their expression
patterns... 35 Table 2. Collection dates and stages of development in zygotic embryos of
Douglas-fir ... 55 Table 3. Synthetic oligonucleotides used in this s tu d y ... 63 Table 4. Embryogenesis-specific cDNA clones isolated from a Douglas-fir embryo
genesis cDNA lib rary ... 87 Table 5. Isolated embryogenesis-specific cDNAs used in this study... 88 Table6. Percentage similarities of PM2S1, PM2S2, PM2S3 and PM2S4 cDNAs. . . . 92 Table 7. Transient expression assays for gPmMTa/uidA chimeric genes in Douglas-fir. 156
X ll
L IST O F n C U R E S
Figure 1. Gene expression during seed development and germination... 6
Figure 2. Structures and processing of 2S seed storage proteins... II Figure 3. Comparison of the amino acid sequence deduced from cDNAs and genes encoding metallothionein-like proteins in p la n ts... 36
Figure 4. Four stages of Douglas-fir somatic embryo development... 56
Figure 5. Steps in differential screening of Douglas-fir embryogenesis cDNA library. . 67
Figure 6. The nucleotide and deduced amino acid sequences of PM 2S1, PM2S2, PM2S3 and PM2S4 cDNAs... 90
Figure 7. Hydropathy plot of PM2S1, PM2S2, andPM2S3 translated products... 93
Figure 8. Comparison of the amino acid sequences deduced from PM2S cDNA clones with similar gene products from angiosperms... 95
Figure 9. Phylogenetic tree of 2S seed storage protein precursors... 97
Figure 10. Phylogenetic tree of coding regions and 3'-untranslated regions of 2S seed storage protein genes from conifers... 98
Figure 11. Southern blot analysis of Douglas-fir genomic DNA... 101
Figure 12. Analysis of a 2S seed storage protein genomic clone... 102
Figure 13. Partial nucleotide and deduced amino acid sequences o f the Douglas-fir g P rn iS / gene... 103
Figure 14. Mapping of the transcriptional initiation site of the gPm lSl gene... 105
Figure 15. PM2S expression in Douglas-fir developing seeds... 109
Figure 16. PM2S expression during Douglas-fir seed germination... 110
Figure 17. PM2S expression in developing somatic embryos of Douglas-fir... I l l Figure 18. Effects of ABA and PEG on PM2S expression in somatic embryos... 112
Figure 19. Transient expression analysis of the gPm lSl promoter in megagametophyte, and zygotic embryos of Douglas-fir... 114
Figure 20. Histochemical localization of GUS activity in transgenic tobacco seeds expressing gPm2Sl/uidA chimeric g e n e ... 115
Figure 21. Gel filtration chromatography of saline extracts from Douglas-fir seeds . . . . 119
Figure 22. Reversed-phase high-performance chromatography of 16-kDa proteins . . . . 120
Figure 23. Reversed-phase HPLC of the reduced 16-kDa proteins of Douglas-fir 121 Figure 24. Accumulation profile of salt soluble proteins during seed development and germination of Douglas-fir... 122
Figure 25. The cDNA sequences of Douglas-fir metallothionein-like c D N A s... 124
Figure 26. Hydropathy plot o f the PM2.1 translated product... 125
Figure 27. Comparison of the amino acid sequences deduced from the PM2.1 cDNA with MT-like gene products from other organisms... 126
Figure 28. Phylogenetic tree of MT-like gene products... 127 Figure 29. Genomic Southern hybridization with the PM2.1 cDNA p r o b e ... 131 Figure 30. Analysis of metallothionein-like genomic clones... 132 Figure 31. Partial restriction maps of kPMMTa, IPMMTb and IPMMTc clo n es 134 Figure 32. Structure of the Douglas-fir gPrnAfTa g e n e ... 135 Figure 33. Mapping of the transcriptional initiation site of the gfmMTb g e n e ... 137 Figure 34. PM2.1 expression in Douglas-fir developing seeds... 142 Figure 35. PM2.1 expression during seed germination and in other organs of Douglas-fir 143 Figure 36. PM2.1 expression in developing somatic embryos of Douglas-fir... 144 Figure 37. Effects of ABA and PEG on PM2.1 expression in Douglas-fir somatic
em bryos... 145 Figure 38. Effects of ABA and metal ions on PM2.1 expression in stratified seeds . . . . 146 Figure 39. Effects of ABA and metal ions on PM2.1 expression in seedlings... 147 Figure 40. Accumulation of PM2.1 transcripts in response to various metal ion
treatments in stratified seed s... 148 Figure 41. Transient expression analysis of the gPmMTa promoter in megagameto
phyte, zygotic embryos and somatic embryos of Douglas-fir... 152 Figure 42. Transient expression analysis of deletion constructs of the gPmMTa promoter 154 Figure 43. Histochemical localization of GUS activity in transgenic tobacco seeds
expressing the gP/nA/ra/«/Vi4 chimeric g e n e ... 157 Figure 44. DNAse I-footprint analysis o f the -280/+25 promoter fragment... 158 Figure 45. Prediction for vacuolar processing sites of a Douglas-fir 2S storage protein. . 163
L IS T O F A B B R EV IA TIO N S XIV A B A Am ino acid: ATP ATPF bases bp BSA cpm C T .\B CTPF DEPC deoxynucleotides dideoxynucleotides DMSO D N A D T I EDTA GUS h HEPES HPLC lEF IPF IPTG kb kDa LB M Pg pi mg mfn A bscisic acid A : alanine M : m ethionine C : cysteine N : asparagine
D : aspatic acid P : proline
E : glutamic acid Q : glutamine
F : phenylalanine R : arginine G : glycine S : serine H : histidine I : threonine I : isoleucine V : valine K : lysine W : tryptophan L : leucine Y : tyrosine A denosine 5 ' triphosphate Amino-terminal propeptide A : Adenine C : Cytosine G : Guanine I : Thymine Base pairs
Bovine serum albumin Counts per minute
Hexacetyltrimethylam monium bromide Carboxy-terminal propeptide
Diethyl pyrocarbonate
dATP : deoxyadenosine-5 -triphosphate
dCTP : d eoxycytidine-5 -triphosphate dGTP : deoxyguanosine-5 -triphosphate dTTP : deoxythym idine-5'-triphosphate ddATP : dideoxyadenosine-5-triphosphate ddCTP : dideoxycytidine-5 -triphosphate ddGTP : dideoxyguanosine-5-triphosphate
ddTTP : dideoxythym idine-5 -triphosphate
Dimethyl sulfoxide D eoxyribonucleic acid Dithiothreitol
Ethylenediamine tetraacetic acid P-glucuronidase
Hour(s)
Y -2-hydroxyethylpiperazine-A -2-ethanesulfonic acid High-performance liqiud chromatography
Isoelectric focusing Internal propeptide Isopropylthiogalactoside kilobase pairs kilodaltons Luria-Bertani medium
(1% NaCI, 1% Bacto-tryptone, 0.5% yeast extracL 10 mM M gS 0 4 , 0.2% maltose) Molar microgram microliter milligram minute(s)
mM MOPS MT MW ng C D ORF PAGE PEG pfu Pg pi pmol PMSF PSV R N A m RNA rRNA RNAase rpm S SDS SDS-PAGE SE SM SSC TAE THE TCA TE TEMED Tricine Ir is X -gal X-gluc m illimolar 3-[jV-morpholino]propanesulfonic acid M etallothionein M olecular w eight nanogram Optical density Open reading frame
Polyacrylam ide gel electrophoresis Polyethylene glycol
Plaque form ing units picogram
Isoelectric point picom olar
Phenylm ethylsulfonyl fluoride protein storage vacuoles R ibonucleic acid
M essenger ribonucleic acid Ribosomal ribonulceic acid Ribonuc lease
revolutions per minute Svedberg sedim entation unit Sodium dodecyl sulfate
Sodium dodecyl sulfate-polyacrylam ide gel electrophoresis Standard error (standard deviation o f the mean)
Suspension medium
(0.1 M NaCI. 8 mM MgSO^.VHiO. 50 mM Tris-HCl pH 7.5, 0.01% gelatin)
Sodium chloride-sodium citrate buffer
(IX SSC ; 0.15 M sodium chloride, 0.015 M sodium citrate) Tris-acetate EDTA buffer
( I X TAE : 0 .0 4 M Tris-acetate. 0.002 M disodium -ED TA . pH 8.0) Tris-borate EDTA buffer
( IX TBE : 0.089 M Tris. 0.089 M boric acid, 0.008 M disodium- EDTA. pH 8.0) Trichloroacetic acid Tris-EDTA buffer (I X TE : 10 mM Tris-HCl pH 8.0. 1 mM ED TA) N.N.N'.N’- tetramethylethylenediamine iV-tris-[hydroxymethyl]-methylglycine Tris-[hydroxym ethyl]-aminom ethane
5-brom o-4-chloro-3-indolyl-P-D -galactopyranoside 5-brom o-4-chloro-3-indolyl-P-D -glucuronidase
XVI
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation for the patient guidance and consistent encouragement o f my supervisor. Dr. Santosh Misra. Her friendship and generosity have been invaluable and inspired me throughout the progress o f my study.
I would like to thank the members o f my supervisory committee Dr. Juan Ausio for his helpful advice on the biochemical studies and purification o f seed storage proteins, Dr. William W. Kay, Dr. Paul J. Romaniuk and Dr. Nigel J. Livingston for their constructive suggestions and guidance throughout my study and on the dissertation, and Dr. Zamir K. Punja for his kind acceptance and input as an external examiner. Thanks are extended to Dr. Pramod K. Gupta for providing Douglas-fir somatic embryos, Canadian Pacific Forest Products for access to developing Douglas-fir seeds and the Center for Forest Biology, University o f Victoria, for the use o f growth chambers and facilities. I gratefully acknowledged the Natural Science and Engineering Research Council o f Canada, the Royal Thai Government, and the Office o f Education Affairs (Thai Embassy, Washington, DC) for financial support through scholarships and grants for this research.
I would like to thank all my colleagues in Dr. Misra's laboratory: Ben Forward, Karia Kaukinen, Luba Osuska, Milan Osusky, Anna-Mary Schmidt, Ivan Stefanov, Tim Tranbarger and Bill Yu for their friendship and valuable discussion. I would also like to thank host families including the Bunyans, Deardens, Kopes, Mortimers and Nesbitts, the Thai commimity and many friends, too numerous to mention individually, who made my stay in Victoria enjoyable. I give my appreciation to my loving sisters, Ladawan and M ullika Chatthai, my dear friend Anotai Suksangpanomrung and family members for their moral support through rough times.
Most importantly, I owe a debt o f gratitude to m y mother and father for their encouragement, confidence and unfailing affection.
Pseudotsuga menziesii [Mirb.] Franco, commonly known as Douglas-fir, is a
member o f the Pinaceae family widely distributed in British Columbia and the Pacific Northwest. Its superior growth characteristics and high quality lumber make it the most important economic conifer species o f this region (Silen, 1978). As a result, there is increasing demand for Douglas-fir seeds for use in reforestation.
A major priority o f the reforestation program is the improvement o f conifer seed quality and productivity. Forestry practices and conventional breeding have been commonly used to maintain seed quality and production o f genetically superior Douglas- fir trees (Silen, 1978). Nonetheless, the achievement has been slow because o f the long reproductive cycle (Allen and Owens, 1972) and unpredictable seed crops due to failure during reproductive processes, including lack o f cone bud development, lack o f pollination, premature embryo abortion and seed immaturity (Owens et al., 1991).
In vitro clonal propagation by somatic embryogenesis has provided new prospects
for rapid conifer propagation and the delivery o f superior material to the commercial forest (Attree and Fowke, 1993). With recent advances in gene transfer and genetic manipulation techniques in conifers (Ellis, 1995; Levée et al., 1997; Bommineni et al., 1998), genes responsible for elite traits can be incorporated in the conifer genome and transformed conifers subsequently recovered via somatic embryogenesis. This will significantly shorten the time span for introducing genetically engineered conifers and the clone selection process.
The development o f somatic embryos has been reported in several coniferous species (Tautorus et al., 1991) including Douglas-fir (Durzan and Gupta, 1987). Much effort has been devoted to studying the effects o f media composition and plant growth regulators on somatic embryo cultures and determining the optimal conditions which
yield somatic embryos whose development is analogous to their zygotic counterparts (Gupta and Pullman, 1991; Gupta et al., 1995). In most conifers, somatic embryos undergo the sequence o f development morphologically similar to those o f zygotic embryos. Ultrastructural and histochemical studies have demonstrated that somatic embryos develop storage proteins, starch granules and lipid bodies in a pattern similar to that o f zygotic embryos (M isra and Green, 1990; Owens et al., 1993). Developmental expression o f seed storage proteins and their transcripts are also comparable in somatic and zygotic embryos (Flinn et al., 1991b; 1993; Hakman, 1993a; Leal et al., 1995; Misra et al., 1993). The optimal concentration o f polyethylene glycol (PEG) in combination with increasing concentration o f the phytohormone abscisic acid (ABA) in the maturation medium promotes higher accumulation o f both storage proteins and their transcripts (Misra et al., 1993; Leal et al., 1995). The developmental changes and the effect o f these exogenous factors on conifer somatic embryos confirm the potential to use somatic embryos to facilitate the study o f molecular mechanisms o f gene regulation in conifers.
Embryogenesis in conifers can be divided into three phases: the morphogenetic or cell-division phase, the maturation or cell-expansion phase, and the desiccation phase. Each o f the stages is characterized by specific morphological, physiological, biochemical and molecular changes (Misra, 1994, 1995). The identification o f genes expressed during conifer embryogenesis is a fundamental step towards an understanding o f the molecular events o f conifer embryo development. Characterization o f their expression and the function o f their encoded products will provide extensive information on the biochemical processes occurring during embryogenesis.
In conifers, only a limited number o f the embryogenesis-specific molecular markers have been described (reviewed in Misra, 1994). Previously, the isolation o f a cDNA encoding an 1 IS legumin-like storage protein from a Douglas-fir expression cDNA library with polyclonal antibodies against crystalloid proteins was reported (Leal and Misra, 1993b). However, the immunoscreening technique was limited to genes
developing embryos and screen for genes differentially expressed at specific times and/or within specific tissues during embryogenesis.
Using Douglas-fir as a model system, this investigation was initiated with the isolation and molecular cloning o f embryogenesis-specific cDNAs. Two sets o f embryogenesis-abundant mRNAs, corresponding to 2S seed storage proteins and metallothionein-like proteins, were used as molecular markers to explore factors and regulatory mechanism(s) involved in the developmental expression during zygotic and somatic embryogenesis.
The following objectives were examined in this study:
(1) To isolate and identify transcripts and corresponding genes whose expression is associated with Douglas-fir embryogenesis
(2) To examine the mRNA accumulation patterns o f these genes during seed development and somatic embryogenesis
(3) To study the effects o f ABA, osmoticum and environmental factors on gene expression in somatic embryos, and
(4) To characterize and identify gene promoter region(s) responsible for controlling the transcription o f embryogenesis-specific genes.
CHAPTER 2 LITERATURE REVIEWS
1. Molecular biology of embryogenesis
1.1. Seed development and embryogenesis
Seed development represents a unique transition stage in the life cycle o f higher plants, involving embryo developmental events and physiological adaptation processes that occur within the seed to ensure progeny survival. In angiosperms, seeds are products o f double fertilization, in which one o f the two sperm nuclei fertilizes the haploid egg cell giving rise to a diploid zygote, and the other sperm nucleus fertilizes the polar nuclei in the central cell giving rise to triploid endosperm (Reiser and Fischer, 1993). In some dicotyledonous plants, the endosperm degenerates during seed development, and embryonic cotyledons become dominant (Lopes and Larkins, 1993). As the embryo and the endosperm develop, the ovule enlarges into a seed. Maternal tissues o f inner and outer integument surrounding an embryo sac form a seed coat.
Embryogenesis describes the subsequent period o f development, during which the zygote undergoes a complex series o f morphological and cellular changes resulting in the formation o f a developmentally arrested mature embryo comprised o f an embryo axis with shoot and root apices and cotyledon(s). The process o f embryogenesis can be roughly divided into three phases: early embryogenesis, maturation and late embryogenesis. Early embryogenesis is the phase during w hich cell division and morphological pattern formation take place, yielding an embryo with shoot and root apices, incipient cotyledons and provascular tissue (West and Harada, 1993). During maturation, the embryo enlarges as a result o f cell expansion and the concomitant accumulation o f storage reserve molecules including storage proteins, lipids and carbohydrates (Higgins, 1984). These macromolecules accumulate predominantly in the endosperm or cotyledons, and subsequently serve as nutrient sources for the developing
period o f developmental arrest (Kermode, 1990). This final phase inhibits precocious germination and ensures seed dormancy. The mature embryo remains dormant until it encounters conditions appropriate for germination. Generally, once the seed is hydrated, germination commences as the radical emerges, followed by the mobilization o f the storage reserves (Bewley, 1997).
1.2. Gene expression during embryogenesis
Gene expression during seed development, maturation and germination has been examined in several angiosperm species and distinct sets o f developmentally regulated genes have been identified (Figure 1, Bewley and Marcus, 1990; Goldberg et al., 1989; Thomas, 1993). Most o f the transcripts persist throughout embryogenesis, and are present in mature plants. These include the "housekeeping" genes encoding proteins such as enzymes and structural proteins. Specific sets o f genes are expressed during each o f the three phases o f embryogenesis, although expression patterns characteristic o f one stage or another often overlap. For example, synthesis o f seed storage proteins occurs primarily during maturation but overlaps with both early- and late- embryogenesis. It is believed that developmental stage-specific genes encode proteins whose functions are relevant to the key events o f each embryogenesis phase. The genes in each set may respond to distinct regulatory signals (Schmidt et al., 1994).
1.2.1. EARLY EMBRYOGENESIS
In early embryogenesis, the fundamental events are the establishment o f a bipolar embryo following a series o f asymmetric and transverse cell divisions, and the formation o f embryogénie tissue and organ system (W est and Harada, 1993). Very little is known about the genes expressed during early zygotic embryogenesis due to difficulties in
F igu re 1. Schem atic representation o f m RNA sets during seed developm ent. Shaded areas denote the timing o f appearance and relative abundance o f m R N A populations (after Goldberg et al., 1989) c o N S T m i n v E EMBRYO-SPECIFIC EARLY EMBRYOGENESiS SEED PROTEIN LATE EMBRYOGE.N ES IS
LATE EMBRYOCENESIS/EARI Y GERMINATION
GERMINATION SPECIFIC___
J____ I
CC7TYLEDON &^RLY MID- LATE VIAOIRE 12b Uh STAGE M AfLRATinN VWTLRAnON M ArURAnON SEED
POST-EM BRYOGENESIS G ER M IN A TIO N
accessing the small zygotic embryos. Recently a PCR-based method, mRNA differential display (Liang and Pardee, 1992), was employed to isolate early embryogenesis-specific enolase cDNA from rice zygotic embryos (Hsing et al., 1995). An alternative is to characterize genes that are expressed in early stage somatic embryos. Several genes have been identified from carrot somatic embryo cultures (Zimmerman, 1993), such as those encoding extracellular proteins (EPs). EP2 was identified as encoding a lipid transfer protein. The expression o f the carrot EP2 gene was observed as early as 60-celled globular zygotic embryos, as well as in preglobular somatic embryos, showing restriction to cells o f the protoderm (Sterk et al., 1991). EP3 encodes an acidic endochitinase that affects protoderm development o f somatic embryos (de Jong et al., 1993). Another secreted enzyme, a cationic peroxidase, can restore globular embryo development in carrot cultures in which somatic embryogenesis is impaired by tunicamycin (Cordewener et al., 1991). Use o f these genes as molecular markers for developmental events in early embryogenesis has been suggested (Zimmerman, 1993).
determination o f the apical-basal organization (de Jong et al., 1993; Dodeman et al., 1997). For example, the gnom mutant o f Arabidopsis thaliana exhibits a ball-shaped embryo lacking both apical (cotyledon) and basal (root) domains (Mayer et al., 1993). Rather than imdergoing an asymmetric cleavage, the gnom zygote produces nearly equal sized daughter cells, and subsequent cell divisions are also abnormal. It has been suggested that GNOM gene activity affects the two most important determinants o f plant morphogenesis, the correct position o f the cell division plane and the controlled directional cell expansion (Lloyd, 1991). Mutants defective in the pattern formation have been reported in monopteros seedlings that lack basal structures (Berleth and Jurgens, 1993), m d shoot-meristemless (stm) and gurke mutants whose embryos lack shoot apical meristem (Barton and Poethig, 1993; Torress-Ruiz et al., 1996), indicating the role o f corresponding genes in the normal organization o f the basal and apical regions, respectively. The am pl and p in l mutants are examples o f mutants perturbed in the balance o f phytohormones, auxins and cytokinins, causing abnormalities in the formation o f cotyledons. These mutants highlight the role o f growth regulators as signaling molecules during zygotic embryogenesis (Dodeman et al., 1997). Presently, the nature o f these gene products is unknown.
1.2.2. SEED MATURATION AND DESICCATION
The stages o f maturation and desiccation are coincident with increased levels o f several mRNA species, particularly those encoding storage proteins and late embryogenesis abimdant (LEA) proteins, respectively. Storage proteins are utilized as food reserves for germinating seeds, and the LEA proteins are thought to participate in desiccation tolerance (Bewley and Marcus, 1990). W hereas storage protein mRNAs are normally expressed after embryo formation has been completed, LEA protein mRNAs
are expressed at the mid- to late-maturation stages. The expression o f these genes is highly regulated, both spatially and temporally during embryogenesis, and as a consequence, there have been extensive studies on the structure and expression o f these genes from numerous plant species (reviewed in Bewley and Marcus, 1990; Gatehouse and Shirsat, 1993; Thomas, 1993; Wobus et al., 1995).
Mutations that reduce seed dormancy and cause precocious germination have been identified in a number o f plant species. Many o f these mutants are deficient in the synthesis o f abscisic acid (ABA), a phytohormone that is responsible for seed dormancy and inhibits seed germination when supplied exogenously. In Arabidopsis, ABA- deficient (aba) mutants are impaired in the ABA biosynthesis pathway, leading to reduced levels o f ABA (Rock and Zeevaart, 1991). The aba mutation results in reduced accumulation o f various LEA mRNAs, with slight or no effect on the accumulation o f storage proteins. ABA-insensitive (abi2) mutants in Arabidopsis and viviparous (vpl) mutants in maize are distinct from the aba mutants in that they contain normal levels o f endogenous ABA, but that does not prevent the seeds from precocious germination (K oom neef et al., 1989; Robichaud and Sussex, 1986). Moreover, their phenotype is not reversed by an exogenous supply o f ABA. Seeds o f these mutants possess reduced levels o f embryogenesis-specific mRNAs, including storage protein and LEA mRNAs (Nambara et al., 1992; Pal va and Kriz, 1994), indicating that the ABI3 and V Pl proteins play a central role in modulating the ABA response and regulating gene expression during embryogenesis. M olecular cloning o f the A B B and VPl genes has revealed that these genes encode transcription activator proteins (McCarty et al., 1991; Giraudat et al., 1992). Leafy cotyledon ( le d ) and Jusca mutants in Arabidopsis exhibit unusual cotyledons with greenish leaf-like structures, characterized by trichrome, stomata and mesophyll cell differentiation (Meinke et al., 1994; Keith et al., 1994). Like the abi3 mutant, l e d and Jiisca3 mutants do not establish dormancy correctly and lack protein and lipid storage organelles. However, in contrast to the abi3 mutant, exogenous ABA in
2. Seed storage proteins
An important feature o f seed development is the synthesis and accumulation o f protein reserves (Goldberg et al., 1989). Seed storage proteins represent a major group o f seed proteins in both angiosperms and gymnosperms (Higgins, 1984; Misra, 1994). Traditionally, seed storage proteins are classified as albumins, globulins, glutelins or prolamins based on their solubility in water, salt solution, acid or alkali, or aqueous alcohol, respectively (Higgins, 1984). The major types o f seed storage proteins vary with plant species; for example, globulins are major storage proteins in legumes whereas prolamins predominate in cereals. Each type o f seed storage proteins can be divided further based on their molecular mass and sedimentation coefficient. For instance, in legumes two globulin types, 7S (vicilins) and 1 IS (legumins), constitute much o f the seed protein content, whereas the low molecular weight (28) fraction makes up less than 10% o f the total seed protein (Croy et al., 1984; Higgin et al., 1986). In seeds o f non- leguminous plants, in addition to the 78 and 118 proteins, the 28 storage proteins appear to make up a large percentage o f total seed proteins (30-60%) (Youle and Huang, 1981). The 28 storage proteins o f latter plants have been the focus o f investigation because o f their higher content o f essential sulfur-containing amino acids, i.e. cysteine and methionine.
The 28 proteins are the main storage proteins in a wide variety o f plants (Youle and Huang, 1981). They are classified as albumins or globulins depending upon their solubility. 8ome 28 storage proteins are structurally related to prolamins o f cereals (8hewry et al., 1995), the a-am ylase/trypsin inhibitors (Garcia-Olmedo et al., 1985; Richardson, 1991), and sweet protein mabinlins (Nirasawa et al., 1993). Like other seed storage proteins, the 28 storage proteins are rich in glutamate/glutamine and aspartate/asparagine, supporting their role as a carbon and nitrogen source for the
10
growing seedlings. Due to their high cysteine content, these proteins also represent a major sulfur storage reserve in seeds (Ampe et al., 1986). Some 28 storage proteins, such as napins from canola (Ericson et al., 1986; Laroche-Raynal and Delseny, 1986; Monsalve et al., 1994) and arabidins form Arabidopsis (Krebbers et al., 1988) appear to have only a storage function. However, some others have been shown to exert biological activities. For example, 28 albumins from canola and radish exhibit antifungal properties
in vitro (Terras et al., 1992, 1993). The 28 albumins from oriental mustard (Monsalve et
al., 1993), rice (Adachi et al., 1993) and Brazil nut (Nordlee et al., 1996) are known to cause allergic reactions in hypersensitive individuals.
2.1. Structure of 28 seed storage proteins
In most angiosperms, the 28 storage proteins are heterodimers composed o f a small (3-5 kDa) and a large (8-12 kOa) polypeptide linked by disulfide bridges (Altenbach et al., 1987; Ericsson et al., 1986; Irwin et al., 1990; Krebbers et al., 1988). An exception is sunflower 28 albumin, which is a single polypeptide chain folded into a compact structure by intramolecular disulfide bridges (Kortt et al., 1991; Thoyts et al., 1996). In addition, a number o f cDNAs encoding low molecular weight proteins with sequence similarity to the 28 storage proteins have been identified in seeds o f monocots (Adachi et al., 1993; Alvarez et al., 1995; Cammue et al., 1995; Gautier et al., 1994; 8horrosh et al., 1992). The encoded proteins range from 19-kDa globulins (8horrosh et al. 1992), allergenic proteins (Alvarez et al., 1995), non-specific lipid transfer proteins (Vignols et al., 1994), to puroindolines (Gautier et al., 1994). Despite the differences in their structures, all 28 storage proteins show a common pattern o f eight cysteine residues (...C1...C2.../...C3C4...C5XC6...C7...C8..). It is believed that their conserved cysteine
residues play a crucial role in stabilizing the protein structure via the formation o f inter- and intra-molecular disulfide bonds (8hewry et al., 1995). The disulfide map o f 28 proteins determined by amino acid sequencing o f HPLC-purified cysteine-containing
F igu re 2. Schem atic structures (A ) and processing (B) o f 2S seed storage proteins. Conserved cysteine residues are indicated with stars (♦), and those forming disulfide bridges are linked with lines. Signal peptides are shown as black boxes, propeptides as shaded boxes, and mature proteins as w hite boxes. The abbreviations SS and LS are small and large subunits, respectively. The diagrams are based on data o f D e Castro et al. (1987), Gaylor et al. (1 9 9 0 ), Irwin et al. (1 9 9 0 ), Kortt et al. (1991), Hara-Nishimura et al. (1993b), Nirasawa et al. (1 9 9 3 ), R ico et al. ( 1996), Shewry et al. (1995) and Müntz (1998).
proteins plant species
I— r A l . I l ^ ° ° " I I H
J i
| SS ,IS , , K ° ° H « « « « « « « « « 1 I — 1 1 napm, 2S albumin mabinlin Brassica napus Arabidopsis thaliana Bertholletia excelsa Cucurbila pepo Capparis masaikaiconglutin ô Lupinus angustifolium
1 JTT * $ « « «« I T T> C O O H 2S albumin (SFA8) Helianthus annuus B
1— n r
l~ C O O H « « «« «« 1 — 1 SS m i LS 1 -C O O H 1 1 A Ik m m mm mm m m1 1 1 SS mm LS B -C O O H 1 1 A Ik • « «• «« 1 1--- 1 L_|_k_ 1 1 ---1 1 non-specific lipid transfer protein Triticum aestivum 2ea mavnapin, Brassica napus
2S albumin Cucurbila pepo
signal peptidase
protein disulfide isomerase?
LS ~ 1 -C O O H
rn
T i
■ SS w f-nha S S J - N H : LS i -C O O H J Î N H s - 1 ^ LS ^ I-C O O H I CCX)H-I S S ^ NHivacuolar processing enzyme aspartic endopeptidase unknown processing enzymes'?
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peptides (Nirasawa et al., 1993; Egorov et al., 1996), and two-dimensional NMR spectroscopy (Gincel et al., 1994; Rico et al., 1996) is shown in Figure 2A.
2.2. Synthesis and deposition of 2S seed storage proteins
Extensive research with angiosperms has led to a detailed understanding o f synthesis, processing and deposition o f 2S seed storage proteins (Shewry et al., 1995; Müntz, 1996; 1998). 28 storage proteins are synthesized as precursor proteins which undergo co-translational and post-translational proteolytic processing steps including removal o f the signal peptide, amino-terminal (ATPF), internal (ITPF) and carboxy- terminal (CTPF) propeptides (Figure 2B; Chrispeels, 1991; Shewry et al., 1995). Concomitant to their biosynthesis, storage proteins are translocated into the rough endoplasmic reticulum (RER) lumen and transported into protein storage vacuoles, which are then transformed into protein bodies (Müntz, 1996; 1998).
The signal sequences o f storage proteins display characteristics o f transported proteins, such as length (20-30 amino acids), hydrophobicity, and the presence o f an amino acid with a small uncharged side chain at the C-terminus o f the sequence (von Heijne, 1992). A survey o f signal peptides in several seed storage proteins has further suggested the sequences MA(N)KL and MKTFLIFALL as consensus signal peptide sequences for dicot and monocot storage proteins, respectively (Roy and Mandel, 1996). The function o f signal sequences in plant storage proteins is to facilitate the translocation o f storage proteins into the lumen o f the ER where they are cleaved and the proteins are further modified (Vitale and Chrispeels, 1992; Müntz, 1998). In the RER, storage proteins are subjected to modifications such as glycosylation, disulfide bridge formation, chaparone-aided folding and oligomerization (Chrispeels, 1991). In the case o f pro2S albumins, disulfide bridging is the only modification contributing to the tertiary structure o f the proteins.
protein storage vacuoles (PSV) where they are processed proteolytically by vacuolar processing enzymes to be converted to the mature proteins (Müntz, 1998). Their routes to the PSV may be different, depending on the type o f storage protein and plant species. Seed storage proteins such as 1 IS-legumin and 7S-vicilin in pea cotyledons (Hohl et al., 1996) and rice glutelin (Li et al., 1993) are transported to the PSV via the Golgi apparatus network, the process mediated by Golgi-derived vesicles. In contrast, transport o f prol IS globulin and pro2S albumin in maturing seeds o f pumpkin and castor bean (Hara- Nishimura et al., 1998) and prolamin in rice (Li et al., 1993) and in wheat (Levanony et al., 1992) is independent o f the Golgi apparatus network. Using immunocytochemical analysis, Hara-Nishimura et al. (1998) demonstrated that pro2S albumins form electron- dense aggregates surrounded by an electron-translucent layer within the RER. The protein aggregates leave the RER as electron-dense cores o f precursor-accumulating (PAC) vesicles which eventually are translocated to the PSV (Hara-Nishimura et al.,
1993b).
Comparison o f canola pronap ins with Arabidopsis pro2S albumins shows that the propeptide sequences are more conserved than the mature chains (Krebbers et al., 1988). However, deletion o f major portions o f the propeptides, or even entire propeptides, did not inhibit the transport or processing o f napin (Murén et al., 1995) o r Arabidopsis 2S albumin (D'Hondt et al., 1993b) in transgenic tobacco seeds. In addition, the removal or modification o f the propeptides had no effect on the folding and architecture o f the 2S storage proteins (Murén et al., 1996). The 2S albumins from Brazil nut (BE2S) show sequence similarity o f approximately 20% in both the processed and mature regions (Krebbers et al., 1988; Gander et al., 1991). Based on these observations, the role o f propeptides in the targeting o f 2S storage proteins as well as a common vacuolar targeting mechanism is questionable.
Chimeric polypeptides composed o f proBE2S fragments fused to a yeast invertase reporter enzyme were used to study targeting o f pro2S albumin in Brazil nut (Saalbach et
14
al., 1996). A 20-residue C-terminal fragment containing tetrapeptide lAGF was sufficient to target the yeast enzyme into vacuoles o f transgenic tobacco embryos. Deletion o f this C-terminal tetrapeptide was shown to prevent vacuolar targeting o f pro2S albumin in transgenic plants, indicating that this propeptide acted as a targeting signal to the PSV (Saalbach et al., 1996). The C-terminal targeting sequence was shown to interact with an 80-kDa protein, a receptor for vacuolar proteins transported from Golgi- derived vesicles to the PSV (Kirch et al., 1996).
In developing pumpkin seeds, deposition o f pro2S albumin into the PSV is mediated by RER-derived PAC vesicles (Hara-Nishimura et al., 1998). Recently, Shimada et al. (1999) demonstrated that a 72-kDa protein found in the membranes o f pumpkin PAC vesicles bound to two peptide domains, a RRE sequence and a NLPS sequence located near the amino- and carboxy-termini o f the large subunit, respectively, o f the pro2S albumin. Mutation o f these domains abolished the binding abilities to the receptor but only the mutation o f the NLPS sequence prevented the maturation o f the pumpkin 2S albumin in transgenic Arabidopsis seeds. Hayashi et al. (1999) demonstrated that a fusion protein consisting o f the 79-amino acid portion (containing the RRE sequence but lacking the NLPS sequence) o f the pumpkin 2S albumin and a phosphinothricin acetyltransferase was deposited in the PAC-like vesicles in transgenic
Arabidopsis. Together, these authors proposed that for pumpkin pro2S albumin the RRE
sequence is involved in the formation o f PAC vesicles, whereas the NLPS sequence is responsible for the intracellular transport to the PSV, and both processes are mediated by interaction with the 72-kDa sorting receptor.
The pro2S storage proteins undergo additional processing in the PSV. Immunocytochemical analysis and cell fractionation experiments o f developing plant seeds located mature 2S albumins in the matrix o f the PSV (De Clercq et al., 1990; Hara- Nishimura et al., 1993a; Krebbers et al., 1988). Cell fractionation o f pulse-chase labeled pumpkin cotyledons revealed that the pro2S albumin accumulated transiently in the
vacuoles (Hara-Nishimura et al., 1993a). The processing o f the 2S storage proteins appears to involve several processing enzymes (D'Hondt et al., 1993a; Murén et al.,
1995). A vacuolar protein with a molecular mass o f 37 kDa, designated a vacuolar processing enzyme isolated from castor bean seeds, was shown to convert pro2S albumin into heterodimer mature 2S albumin in vitro (Hara-Nishimura et al., 1991) by cleaving a peptide bond at the C-terminus o f an asparagine residue (Hara-Nishimura et al., 1993b). Two asparagine residues, located in the amino-terminal and the internal propeptides, are conserved in pro2S albumins o f various plants (Godinho da Silva et al., 1996; Gonzalez de la Pena et al., 1996; Irwin et al., 1990; Krebbers et al., 1988; Murén et al., 1996). D'Hondt et al. (1993a) reported that an aspartic endopeptidase from rapeseed cleaved a synthetic peptide containing the internal propeptide o f Arabidopsis pro2S albumin. Recent studies by Hiraiwa et al. ( 1997) confirmed the previous result, however, argued that the aspartic endopeptidase alone was unable to convert Arabidopsis pro2S albumins into the mature forms. These authors proposed that the aspartic endopeptidase may play a role in trimming the C-terminal propeptides from the subunits that are produced by the action o f the vacuolar processing enzyme.
The steps involved in processing o f the C-terminal propeptide o f pro2S albumin are not well characterized. A comparison o f the DNA-derived amino acid sequences with the corresponding mature 2S albumins highlighted a conserved aromatic amino acid, such as phenylalanine (F), tyrosine (Y) o r tryptophan (W), as the N-terminal amino acid o f the CTPF. In the 2S albumin precursors, these aromatic amino acids are preceded by serine or glycine. It is likely that an additional enzyme cleaves the N-terminal side o f F, Y or W (Gehrig et al., 1996).
3. Storage protein gene family
cDNAs and genes encoding 28 storage proteins have been characterized in several plants \no\\\d\n% Arabidopsis (Krebbers et al., 1988; Guerche et al., 1990), canola
16
(Baszcynski and Fallis, 1990; Josefsson et al., 1987; Scofield and Crouch, 1987), radish (Raynal et al., 1991), Brazil nut (Gander et al., 1991), sunflower (Allen et al., 1987), and rice (Adachi et al., 1993). In all these plants, the 2S storage proteins are encoded by multigene families with copy numbers in the range o f 4-20. Except for the BE2S1 and
BE2S2 genes from Brazil nut and the HaG5 gene from sunflower, which contain a single
intron, most 28 storage protein genes show a simple intronless gene structure.
3.1. Storage protein gene expression
The expression o f storage protein genes is restricted both spatially, to the tissues o f the embryo and/or the endosperm, and temporally to maturation and late embryogenesis o f seed development (Goldberg et al., 1989). Thus, these genes represent an interesting model for studying the mechanisms o f tissue- and development-specific regulation o f gene expression in higher plants. For the past two decades, the regulation o f seed storage protein gene expression has been described comprehensively for 118 legumin and 78 vicilin genes in legumes, as well as prolamin and glutelin genes in monocots (Higgin et al, 1984; Goldberg et al., 1989; Morton et al., 1995; Gatehouse and 8hirsat, 1993; 8hirsat, 1990). Herein, information accumulated mainly from studies on the regulation o f 28 storage protein gene expression will be emphasized.
During seed development, 28 storage proteins are differentially expressed in various tissues. Guerche et al. (1990) showed that the expression o f the four genes encoding 28 albumins from Arabidopsis differed in the cell types in which they were expressed. In situ hybridization showed that at282, at283 and at284 mRNAs were present throughout the embryo, whereas a t2 8 1 mRNA was only expressed at significant levels in the embryo axis. In canola, in situ hybridization was used to display the patterns o f napin gene induction during seed developm ent (Fernandez et al., 1991). The napin (pN2) mRNA initially accumulated in the cortex o f the axis during late heart stage, in the outer layers o f the cotyledons during torpedo stage, and in the inner layers o f the
cotyledons during cotyledonary stage. The storage protein transcripts were not detected in root and shoot meristem in early embryos, but appeared in the shoot meristem as the embryos entered maturation and started desiccation. The physiological significance o f this phenomena is not known; however, it may reflect gene induction by diffusible signal molecule(s) or differences in cell maturation (Fernandez et al., 1991).
4. Transcriptional regulation of 2S seed storage protein gene expression
The strict spatial and temporal expression o f seed storage protein genes is believed to be controlled primarily at the transcriptional level (Goldberg et al., 1989; Stâlberg et al., 1993). The increase in mRNA accumulation occurs simultaneously with an increase in transcriptional activity (DeLisle and Crouch, 1989). Similarly, the peak and decline in mRNA levels as seed development proceeds is also paralleled by a peak and decline in transcription (Gatehouse et al., 1986). A number o f studies have identified regulatory promoter elements associated with the expression level, developmental stage, and tissue-specific gene expression o f 28 storage protein genes (De Clercq et al., 1990; Grossi de Sa et al., 1994; Radke et al., 1988; Stâlberg et al., 1993). The concerted action o f sequence-specific transcription factors that interact with cw-regulatory elements residing in promoter regions is a primary component o f the transcriptional regulation o f 2S albumin gene expression (Vincentz et al., 1997).
4.1. cû-acting elements
Several approaches, including (1) comparison o f promoter sequences to search for conserved motifs, (2) analysis o f gene fusion constructs in transgenic plants, and (3) protein binding assays, have been used to identify regulatory elements.
Comparisons o f the 5'-fianking regions o f known 2S albumin genes from the same or related plant species have revealed a number o f conserved sequences. M ost o f the conserved sequences localize at similar distances from the putative TATA box. An
18
example o f such conserved sequences is the sequence, 5-TACATA-3', which is repeatedly shown in the 200-bp region upstream from the transcriptional start site (Scofield and Crouch, 1987). Also in the proximal region, multiple copies o f the G-box (5'-CACGTG-3') and the RY repeat (5'-CATGCA-3') are present (Josefsson et al., 1987; Dasgupta et al., 1993). The G-box and related sequences are found in the 5' region o f many seed-specific genes and are involved in conferring the spatial and temporal control o f gene expression via interaction with transcription factors (Guilfoyle, 1997). The CATGCA m otif frequently found in other seed protein gene promoters has been shown to be essential for seed-specific expression, as well as in ABA-regulated gene expression (Morton et al., 1995). One copy o f the CATGCA m otif occurs near the TATA box and overlaps with the octomer sequence ATGCAAAT, known from many other eukaryotic promoters (Ericson et al., 1991). Transcription o f 1 IS legumin genes depends on regulatory interactions between proximal and distal CATGCA motifs (Chamberland et al., 1992). Similarly, the frequent occurrence and the conserved spacing pattern o f the TGCA (or ACGT) palindromes are a common feature o f genes coding for 2S albumins (Gander et al., 1991).
The functional analysis o f 5' flanking regions o f genes for c/5-acting sequences can be complemented by identifying sequences that interact with DNA-binding proteins. Analyses o f the upstream region o f a napin gene, napA, by gel retardation assays have revealed several o f the conserved elements that bind seed nuclear proteins from canola. The sequences include the TAG AC AT m otif (Ericson et al., 1991) and the napA -152/ -120 (B-box) region (Gustavsson et al., 1991) in which two conserved sequences, 5'- CACTTGTCACT-3' and 5'-TTCCAACACCTAA-3', are present. DNAse-I footprinting assays revealed that the regions close to or containing either ACGT or CCAC motifs in the Brazil nut BE2S1 gene promoter specifically bind seed nuclear extracts (Grossi de Sa et al., 1994).
responsible for storage protein gene expression. Radke et ai. (1988) reported that the 300-bp sequence upstream from the translation initiation codon o f a napin gene is sufficient to give an embryo-specific and developmentally regulated expression o f a chimeric gene construct in transgenic canola. Likewise, the 1.8-kb promoter o f
Arabidopsis a t lS l gene directed seed-specific expression in heterologous plants such as
transgenic tobacco and canola (De Clercq et al., 1990). Deletion analyses o f the napA promoter showed that the -1527+44 sequence was sufficient to direct seed-specific and developmental expression o f a reporter gene {uidA) encoding P-glucuronidase (GUS), whereas addition o f the sequences further upstream to -309 directed strong expression in the seeds o f both transgenic tobacco and canola (Stâlberg et al., 1993; 1996). A further deletion to the position -144 completely abolished the GUS activity (Ellerstrom et al., 1996; Stâlberg et al.; 1996). These authors, while not excluding the significance o f other regulatory motifs, concluded that four AT-rich sequences present in the -3097-211 region, and the E-box (5'-CACTTG-3') disrupted in the -144 construct were crucial for quantitative and correct expression o f the napin genes. Similar results were obtained in studies on the Brazil nut 2S albumin gene regulation. Deletion experiments demonstrated that the -2677-210 region is responsible for quantitative expression (Grossi de Sa et al.,
1994), and the -1777+56 region is suffrcient to confer seed-specific and temporal expression o f a reporter gene (Vincentz et al., 1997). Further deletion to position -88 abolished the GUS activity. However, the individual c/s-acting elements have not been defined.
Evidence is accumulating for the role o f specific c/5-acting sequences in determining the spatial expression o f 2S albumin genes. For example, Ellerstrom et al. (1996) showed that, when the [-1527+44]«ap^-w/dL4 chimeric construct was used in tobacco transformation, transgenic plants retained GUS activity in both the embryo and endosperm. However, internal deletion o f the region -1337-121 from this construct caused a decreased GUS activity in the embryo and an increased activity in endosperm
