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Identification and characterization of homeobox genes involved in soybean{Glycine max L.) embryo development
Ma, Hongchang, Ph.D.
The Ohio State University, 1994
UMI 300 N. ZeebRd. Ann Arbor, MI 48106 IDENTIFICATION AND CHARACTERIZATION OF HOMEOBOX GENES
INVOLVED IN SOYBEAN (GLYCINEMAXL.) EMBRYO DEVELOPMENT
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Hongchang Ma, B.S., M.S.
+ + * * +
The Ohio State University
1994
Dissertation Committee: Approved by
J.G. Streeter
J.J. Finer
L.M. Lagrimini / ] V. S — ° X J Advisor K.A. Campbell Department of Agronomy ACKNOWLEDGMENTS
I express my sincere appreciation to Dr. John J. Finer and Dr. John G. Streeter for their guidance, encouragement, and consistent support throughout my research. My gratitude goes to other members of my advisory committee, Dr. Mark Lagrimini for his invaluable suggestions in my research and for his help in analyzing some of my research data, Dr. Kimberly A. Campbell for her valuable comments on my manuscripts and providing laboratory space and equipment. I am especially grateful to Dr. Michael D.
McMullen, who first suggested isolation of homeobox genes from soybean.
The technical assistance of Barbara L. Norris, Mark W. Jones, Brenda S. Schult, and Kevin Simcox is gratefully acknowledged. I also thank Thomas G. Lanker, Seppo O.
Salminen, Dimple R. Abernathy, and Marie E. Semko for their technical and instrument supports.
I thank to my fellows, Masood Z. Hadi and Tai-Sheng Cheng for sharing exciting scientific information with me and making my graduate studies more enjoyable and fruitful. I also wish to thank Dr. Sarah Hake for providing theK nl cDNA clone, Dr. David
P. Jackson and Dr. Sarah Hake for sharing their unpublished information and for their
technique assistance in in situ hybridization. The supply of soybean ubiquitin gene from
Dr. Desh-Pal Verma is also appreciated.
To my wife, Huiling, I offer sincere thanks for her understanding, support, and encouragement throughout this journey. To my daughter, Li, 1 thank for her willingness to endure my frequent absences in her normal life. VITA
April 14, 1955 ...... Bom in Dongguang, Hebei, P.R. China
March, 1982 ...... B.S., Hebei Agricultural University, Baoding, Hebei, P.R. China
1982-1987 ...... Research Scientist, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang, Hebei, P.R. China
December, 1989 ...... M.S., Kansas State University, Manhattan, Kansas
March, 1990-Present ...... Graduate Research Associate, Ohio Agricultural Research and Development Center/The Ohio State University, Wooster, Ohio
PUBLICATIONS
1. Ma H, McMullen MD, Finer JJ: Identification of a homeobox-containing gene with enhanced expression during soybean (Glycine max L.) somatic embryo development. Plant Mol. Biol. 24: 465-473 (1994).
2. Ma H, McMullen MD, Finer JJ: A pyruvate kinase cDNA from soybean somatic embryos. Plant Physiol. 102: 1345 (1993). 3. Ma H, Liang GH, Wassom CE: Effects of growth regulators and genotypes on callus and embryoid induction from maize anther culture. Plant Breeding 106: 47-52 (1991).
4. Ma H, Wassom CE, Liang GH: Direct generation of maize haploids via anther culture. Cytologia 56: 103-106(1991).
5. Gao K, Ma H, Liu A: Study on population size of hand-pollinated plants in seed renewal of maize cultivars. ACTA Agronomica Sinica 17: 314-319 (1991) [in Chinese].
FIELDS OF STUDY
Major Field: Agronomy
Studies in Tissue Culture and Cell Biology, Dr. John J. Finer
Studies in Plant Genetics, Dr. Kimberly A. Campbell
Studies in Plant Physiology, Dr. John G. Streeter
Studies in Molecular Biology, Dr. Michael D. McMullen Dr. Mark Lagrimini TABLE OF CONTENTS
ACKNOWLEDGMENTS ...... ii
VITA ...... iv
LIST OF FIGURES ...... viii
INTRODUCTION ...... 1
CHAPTER PAGE
I. ISOLATION AND CHARACTERIZATION OF A PYRUVATE KINASE cDNA FROM SOYBEAN ...... 21
Abstract ...... 21 Introduction ...... 22 Materials and Methods ...... 28 Results and Discussion ...... 31
II. IDENTIFICATION OF HOMEOBOX-CONTAINING GENES FROM SOMATIC EMBRYOS AND CHARACTERIZATION OF THE FIRST SOYBEAN HOMEOBOX-CONTAINING GENE ...... 48
Abstract ...... 48 Introduction ...... 49 Materials and Methods ...... 60 Results and Discussion ...... 62 CHAPTER PAGE
III. TEMPORAL AND SPATIAL EXPRESSIONS OF SBH1 DURING PLANT DEVELOPMENT ...... 73
Abstract ...... 73 Introduction ...... 75 Materials and Methods ...... 79 Results ...... 83 Discussion ...... 87
GENERAL DISCUSSION ...... 99
LIST OF REFERENCES ...... 102
APPENDIX ...... 119
LIST OF ABBREVIATIONS ...... 146 LIST OF FIGURES
FIGURE PAGE
1.1. The development of soybean somatic embryos...... 38
1.2. DNA dot hybridizations of selected clones with other clones and themselves ...... 39
1.3. RNA dot blots showing the expression patterns of the selected clones during soybean somatic embryogenesis...... 40
1.4. RNA dot blots to detect the effects of auxin on expression of the selected clones ...... 41
1.5. Nucleic acid sequence of Sbpk and deduced amino-acid sequence of SBPK...... 42-43
1.6. Alignment of the deduced amino acid sequence of soybean pyruvate kinase [S] with that of potato [P] and tobacco [T] pyruvate kinases...... 44
1.7. Southern hybridization analysis of soybean DNA using Sbpk cDNA as a probe ...... 45
1.8. Expression ofSbpk in somatic embryos developed under anaerobic or aerobic conditions ...... 46
1.9. Expression ofSbpk in soybean somatic embryos developed in liquid medium containing different concentrations of sucrose ...... 47
2.1. Nucleotide acid sequence of Sbhl and deduced amino-acid sequence of SBH1 ...... 67-68
2.2. The comparison between the predicted SBH1 protein (top line) and KN1 (bottom line) ...... 69 FIGURE PAGE
2.3. The amino acid sequence of SBH1 homeodomain aligned with several homeodomains from other organisms...... 70
2.4. Southern hybridization analysis of soybean DNA using Sbhl cDNA as a probe ...... 71
2.5. Hybridization of the selected clones with the whole cDNA {Sbhl cDNA), the homeobox region{Sbhl hox), and the S’-region {Sbhl 5’) o f Sbhl ...... 72
3.1. Northern hybridization analysis ofSbhl transcript ...... 92
3.2. In situ hybridization ofSbhl RNA probes to soybean somatic embryos at different developmental stages ...... 93
3.3. In situ hybridization ofSbhl RNA probes to soybean zygotic embryos at different developmental stages ...... 95
3.4. In situ hybridization showing the localization ofSbhl mRNA in different organs and tissues of soybean plants ...... 97
ix INTRODUCTION
Plant embryogenesis is the process of embryo initiation and development. This process starts at zygote formation, ends with the production of a mature seed and marks the beginning of the sporophytic generation of the life cycle of plants (Goldberget al .,
1988). Plant embryogenesis consists of three overlapping stages. The early stage is characterized by cell division and morphogenesis. During this stage, the embryo becomes defined and the shoot and root apices are formed. The next stage is the embryo maturation stage and is characterized by the accumulation of storage proteins, carbohydrates and lipids. During the final stage of embryogenesis, the embryo prepares for developmental arrest, desiccates, and enters dormancy (Goldberg et al ., 1989, West and Harada, 1993). Accompanying embryogenesis in monocotyledonous species, the nonembryogenic triploid endosperm develops and carbohydrates, lipids, and proteins are accumulated. In dicotyledonous plants, the cotyledons are the actual storage tissues.
These storage tissues provide important sources of food for human and animals as well as nutrient sources for plants during the germination process. Since embryogenesis represents the beginning of the life cycle in higher plants and the end products are essential for human existence, this subject has attracted a great deal of interest from many scientists in different disciplines. In this introduction, the discussion of plant embryogenesis will concentrate on dicots and focus on the information obtained from histological, genetic and molecular biological studies.
1 Histological Studies on Plant Embryogenesis
Morphological and anatomical changes during the early stages of plant embryogenesis have been described in detail (Mansfield and Briarty, 1990, Johansen,
1950). The early stages of dicot embryogenesis start at zygote formation and continue until the cotyledons are formed. Early embryogenesis is characterized by 1) pattern formation during which the precise spatial organization of embryonic organs is established and 2) tissue differentiation during which the cellular diversity within an embryo is generated (Lindsey and Topping, 1993).
After fertilization, the first division of the zygote is transverse and asymmetric, giving rise to a small apical cell and a large basal cell. Through a series of divisions, the apical cell gives rise to the embryo proper. The embryo passes through the following classical stages of development: four-celled embryo, octant proembryo, globular-, heart-, and cotyledon-shaped embryo. The basal cell undergoes a series of divisions and forms the hypophysis and suspensor. The derivatives of the hypophysis will form the root cap and part of the root initial. The suspensor at the base of the embryo, is believed to aid embryo growth at the early stages by archoring the embryo in the nutrient-rich endosperm
(West and Harada, 1993) and perhaps by conducting nutrients and growth factors from maternal tissue to the embryo (Yeung and Sussex, 1979, Yeung and Meinke, 1993,
Lindsey and Topping, 1993). The suspensor degenerates late in embryogenesis as its role is minimal in the developed seed.
Tissue differentiation begins prior to the formation of the globular staged embryo.
The octant embryo divides periclinally resulting in the first histologically detectable tissue, the protoderm, which is the precursor of the epidermis. A dramatic morphological change occurs during the transition from globular to heart stage. The shift of embryo symmetry from radial at the globular stage to bilateral at the heart stage indicates the 3 emergence of two embryonic organs, the cotyledons and the axis. The procambium,
which is the precursor of the vascular tissue, and the ground tissue are intiated at this transition stage. Therefore, by the time that the heart-staged embryos are formed, pattern
formation and tissue differentiation have been initiated. At the end of the cotyledon stage, the embryonic organs and tissues are evident and the embryo contains a well-developed polar axis and primary tissues. The polar axis of an embryo consists of an apical domain with cotyledons, the shoot apex, and the upper hypocotyl, a central domain including mainly the hypocotyl, and a basal domain with the root initial and root cap. The three fundamental tissues of the plant including the epidermis, ground tissue and vascular tissue are also well-defined (West and Harada, 1993, Lindsey and Topping, 1993).
Although embryo development at the early stages is very critical and the morphological patterns have been extensively studied using histological and anatomical methods, the mechanics underlying morphogenesis are not yet known. To better understand the mechanisms underlying plant embryogenesis, genetic and molecular biological approaches are currently being utilized.
Genetic Studies on Plant Embryogenesis
Some clues to the basis of embryogenesis can be obtained by the use of mutants.
For clonal analysis, single meristematic cells are genetically marked and the cell division pattern is analyzed by observing the distribution of the resulting sectors (Poethig, 1989).
Clonal analysis has provided extensive information on the origin and developmental fate of cells within the embryonic shoot apex of maize (Poethiget al ., 1986). During embryogenesis, the number of cells in the embryonic shoot meristem increases. The fate of cells becomes progressively restricted as an embryo develops. Cells toward the periphery of the meristem give rise to the lower part of the plant while central cells form 4 more distal parts. The restriction of cell fate progresses from the periphery towards the
center of the meristem. However, the fate of a cell is still relatively flexible and cell
lineages play a minor role in plant development (Jegla and Sussex, 1989). Clonal analysis
has not been widely used to study cell lineages during early stages of embryogenesis for
the lack of cell-autonomous genetic markers that can be readily scored throughout
embryo development and the difficulty in precisely marking specific cells at certain stage
of the development (Christianson, 1986, Meinke, 1991b).
Mutants can also be used to identify genes with important developmental
functions. A large number of mutants have been generated from Arabidopsis and maize
by X-irradiation, chemical mutagenesis, transposon tagging, and T-DNA insertional
mutagenesis (Meinke, 1991a, 1991b, Jilrgens et al ., 1991, Clark and Sheridan, 1991,
Dolfini and Sparvoli, 1988, Errampalli et a l 1991). Meinke (1991b) placed most of the
embryonic mutants into the following four classes: 1) defective in essential housekeeping
genes such as the biotin auxotroph in Arabidopsis (Haughn and Somerville, 1988), 2)
defective in early stages of cell division and morphogenesis such as embryonic emb ( )
mutants of Arabidopsis (Meinke, 1991a), defective-kernel (dek), and germless (germ )
mutants of maize (Sheridan, 1988, Clark and Sheridan, 1991), 3) defective in accumulation of pigments and storage materials such as those with abnormal endosperm development in maize (Coe et al ., 1988), 4) defective in preparation for dormancy and germination including viviparous mutant of maize (McCartyet al., 1989) and hormone mutants ofArabidopsis (Finkelstein et al., 1988, Lincolnet al., 1990).
In order to obtain pattern formation mutants, Jilrgenset al. (1991) selected a
number o f Arabidopsis embryo mutants with relatively normal germination but abnormal
seedling development. Four classes of pattern mutants were identified. The first class of
mutants lacked specific regions of the mature embryo, the second had duplications of 5
embryogenic structures, the third class was characterized by multiplication of cotyledons,
and in the fourth one, the cotyledons were transformed into shoots. One of the most
interesting pattern mutant, gnom, was studied in detail by Mayer et al . (1991). Gnom
belongs to the first class of mutants described above in which the root is absent. By tracing gnom back to the zygote, it was found that the first cell division was abnormal with a nearly symmetric rather than a normal asymmetric division. Moreover, the position as well as the plane of the first division ingnom was abnormal. The lack of root formation may result from the failure ofgnom a zygote to form a normal basal cell that should, in turn, give rise to the hypophysis, the precursor of part of the root initial. Mayer et al. (1991) also observed that in some gnom mutants, after fertilization of the egg cell, expansion of the zygote was suppressed. As a result, the partitioning of the zygote into a cytoplasm-rich apical cell and a vacuolated basal cell was affected. Therefore, the wild type of thegnom gene regulates important events, the position and plane of cell division and the direction of cell expansion during plant embryogenesis. Another type of pattern mutants is doppelwurzel in which the apical portion of the embryo was deleted and replaced with a mirror image of the basal portion. Therefore, a seedling with two polar root systems is produced indoppelwurzel mutants. Other mutants have been identified from embryonic lethal and defectives of Arabidopsis (Meinke, 1991b, 1992) with interesting morphogenic phenotypes such as fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, split hypocotyls, embryos with altered symmetry, embryos with leafy cotyledons, and so on.
Using a chemical mutagenic strategy, Mayeret al. (1991) identified mutants with abnormal tissue and organ organization in Arabidopsis seedlings. Genetic complementation studies indicated that those aberrant phenotypes were determined by 6
nine genes which can be divided into 3 classes based on the effect of the genes on
morphological organization. The first class contains apical-basal pattern mutants,
characterized by organ deletion which does not affect primary tissues in the seedling.
Four genes were identified for these pattern deletions. Mutant alleles of the gurke gene
affected the apical part of the seedling and caused cotyledon and shoot meristem
deletions in the strongest phenotype. Mutants of thefackel gene eliminated the hypocotyl
in the central part of axis resulting direct attachment of cotyledons to the root.
Monopteros mutations caused basal deletions characterized by the absence of both the
hypocotyl and the root in seedlings.Gnom mutants have terminal deletions in which the
root is deleted and cotyledon is reduced or eliminated resulting in a cone- or ball-shaped
seedling. Pairing of complementary mutant phenotypes indicated a combinational model: gurk and gnom specify apical, fackel and monopteros specify central, and gnom and
monopteros basal. The second class is the radial pattern mutation, characterized by
abnormal development and organization of plant embryonic tissues including the epidermis, ground tissue, and vascular tissue. Mutations in two genes (biolie and keule) caused defects in epidermis formation. However, mutants affecting the other two tissues, vascular and ground tissues, have not been identified. The third class of mutants is characterized by abnormal seedling shape with major organs and tissues remaining normal. Three genes were identified for this class. They are fass, mutants with stout, short, and compressed seedlings; knopf with small, round, and pale seedlings; and minckey, with thick disc-shaped cotyledons.
The mutant phenotypes in the seedlings were correlated with abnormalities in cellular patterning in the heart, globular and even pre-globular stages of the embryos. In gurk embryos, the apical surface was rounded and the cotyledon primordia were missing.
Fackel embryos were broader than normal because of the absence of hypocotyl resulting 7
in root and cotyledon primordia conjugation.Gnom embryos looked round indicating the
lack of the terminal parts of axis, and momopteros embryos were triangle-shaped
suggesting the absence of the basal parts of the embryos. These correlations are consistent
with the concept that the organization of organs and tissues in the seedling is determined
at the early stages of embryo development (Lindsey and Topping, 1993, Mayeret al.,
1991). Further analysis of all of these mutants will provide dramatic insights in the genetic control of plant embryogenesis.
Maize provides a model for genetic studies of monocot embryogenesis. Compared with dicot species, embryogenesis in monocots is characterized by more pronounced asymmetric cell divisions and advanced development of the embryo and endosperm in the seed (Lindsey and Topping, 1993). Sheridan and Thorstenson (1986) identified three defective kernel (dek) mutants in maize with uniform blockage at specific developmental stages. The development of ptd*-N 30 embryos was retarded at the transition stage and the embryos failed to form shoot and root meristems.Ep*-1418 reached the early coleoptilar stage with a well formed root meristem but lacked leaf primordia and the shoot meristem. Bno*-747B embryos grew normally to the early coleoptilar stage but became irregularly shaped masses by kernel maturity. These three genes are most likely essential for embryo development.
Two mutants, dek22 and dek23, with developmental blockage prior to the formation of leaf primordia were identified (Clark and Sheridan, 1986). The dek22 embryos stopped development at the transition stage indicating that the function of the wild type gene is essential prior to the coleoptilar stage of embryogenesis. Indek23 embryos, the formation of the shoot apex is selectively blocked suggesting that the wild- type gene is involved in shoot formation. Thedek22 and dek23 loci are located on different chromosomes. With the transposable element, Mutator, 51 embryo-specific 8 (emb) mutants were identified by Clark and Sheridan (1991). Using B-A translocations,
25 mutations were located on six of eight chromosome arms. These mutants produced morphologically abnormal embryos with normal endosperm indicating that they function in morphogenesis and not as housekeeping genes involved in general cellular growth and metabolism.
Recent genetic studies on rice embryogenesis suggest the presence of a hierarchical regulation for organ formation (Kitanoet al., 1993). With the analysis of different phenotypes of mutants, four regulatory processes,organ determination, organ position, and embryo size, were suggested to operate the formation of shoot and root during rice embryogenesis. In the organ determination group, some mutants do not form organs and some developed roots but no shoots. Kitanoet al. (1993) suggested that organ determination is controlled at two levels, global control represented by mutants without any organ, and local control identified by the mutants lacking a specific organ. In the organ position group, one type of mutant contained shoots and roots that were displaced apically. In the other type, only one organ, either the shoot or root, was displaced. They suggested that the positioning of the embryonic organs is regulated at two levels: displacement of total organ system by the global control and displacement of individual organ by the local control. In the embryo size group, two types of mutants were identified and two levels of regulations were also suggested. In one type of mutant, the embryo size was affected by the sizes of all organs and in the other type, embryo size is affected only by the size of a specific organ. From these mutant analyses, they proposed that these three regulatory processes take place before shoot and root morphogenesis. They act independently or cooperatively to affect the morphogenesis of embryonic organs and hence, the embryo. Two types of genes participate these processes. One provides a basic 9
framework for the whole body and the other functions more locally or restricted to
compartment.
In addition to Arabidopsis, maize, and rice, embryonic mutants have also been
identified from other plant species such as barley (Bosnes et al., 1987) and pea
(Bhattachayyaet al., 1990). It is relatively easy to generate a large number of mutants
with any mutagenesis approach. The high frequency of embryonic mutants indicates that
a large number of genes are active and involved in this process. The numbers of genes
essential for embryogenesis were estimated to be between 500 (Meinke, 1991b) and 4000
(JUrgens et al., 1991) in Arabidopsis. Among these, about 40 in total may be sufficient to
control pattern formation (Mayeret al., 1991).
Genetic studies provide some insights in plant embryo development. In addition,
many genes involved in this process have been identified. Further studies of these
mutants depends on the isolation of defective and wild type genes (Meinke, 1991b). Since
most mutants were produced using chemical mutagenesis, it is quite difficult to isolate
the respective genes. Some promising approaches being employed to isolate these genes
are transposon tagging, T-DNA insertional mutagenesis, and chromosome walking from
adjacent restriction fragment length polymorphism and random amplified primer DNA markers. However, these approaches do have their limitations. Approximately half of the embryogenic mutants identified are not tagged: most contain duplications and rearrangements of T-DNA that complicate molecular analysis. Transposon tagging is restricted to few species such as maize and chromosome walking is presently tedious and time-consuming (Meinke, 1991b). To understand the molecular mechanisms underlying embryogenesis, other approaches are required. 10 Molecular Biological Studies on Plant Embryogenesis
The morphological changes that occur during plant embryogenesis are a result of
biochemical and molecular events characterized by the selective expression of numerous
genes in precise cell types at defined developmental times (Sheridan and Neuffer, 1982).
During embryogenesis, many events occur simultaneously or in proper sequence.
Hierarchies of gene interactions exist in which genes are coordinately regulated to
complete particular genetic programs (Sheridan, 1988). Pattern formation requires the
function of the so-called patterning genes. The expression of these genes establishes planes of cell division by regulating the expression of cell division genes. Once patterning is defined, cell differentiation proceeds by the expression of cell type-specific genes which control cell expansion and cell differentiation (Lindsey and Topping, 1993).
Many genes are expressed during plant embryogenesis. It is estimated that at least
20,000 genes are expressed throughout seed development (Goldberg et al ., 1989). Most of these genes are expressed both during and after embryogenesis and only a small number of genes are specific to embryos or present at much higher level in embryos than in other parts of plants. In order to understand the molecular events that control plant embryo development, it is essential to identify the genes that are specifically expressed at different stages or in specific tissues.
By studying gene expression in developing embryos of maize, Sdnchez-Martinez et al . (1986) identified three sets of polypeptides that are specifically synthesized either in young embryos, in mature embryos, or during seed germination. These polypeptide sets provided a molecular framework for maize embryo development. Hughes and Galau
(1989) performed a detailed temporal analysis of gene expression during cotton embryogenesis and identified key modules regulating cell differentiation from the early stages through desiccation of the mature embryos. From late cotyledon-staged embryos of 11 cotton, Galauet al. (1991) identified five mRNAs which were expressed only during the maturation stage, twelve lea mRNAs expressed at the postabscission stage, and three mRNA which were limited to early germination.
Seed proteins are highly regulated during embryo development. Although the function of some seed proteins are not yet known, some storage proteins serve as nutrient sources for the seedling during germination. These proteins provide an attractive model for molecular studies because of their highly specific expression, their importance as food sources for human and animal consumptions, abundance of the mRNA during the maturation stage of embryogenesis, and the ease in obtaining large quantity of materials for study. Studies of storage protein genes have concentrated on gene expression patterns.
Several conclusions have been drawn from these studies (Goldberg et al., 1989). First, the expression of storage protein genes is regulated temporally during embryogenesis and the genes are mostly expressed at high levels during mid-late maturation stages of seed development. Second, storage proteins are specifically present in plant embryos and are found at low levels in other plant organs. Third, storage protein genes are expressed spatially within embryonic organs or within specific cells and not in the surrounding nonembryonic seed tissues. Finally, the specific expression pattern of these genes is conserved among different plant species suggesting that the DNA sequences and protein factors controlling their expression are highly conserved.
The expression of storage protein genes during embryogenesis is regulated by both transcriptional and posttranscriptional processes (Goldberget al., 1981, Baker et al.,
1988, Harada et al., 1989, Walling et al., 1986). The transcription of a gene is controlled by both cis-acting elements and trans-acting factors. The cis-acting elements that are usually located in the flanking region of that gene include the promoter, enhancers, and other activation sequences (Kuhlemeier et al., 1987). Usually, the proximal promoter 12 region specifies seed expression and the more distal regions function to modify and enhance the basic expression pattern (Thomas, 1993), Trans-acting factors include transcription factors and other DNA binding proteins facilitating or inhibiting the binding and action of the RNA polymerase and controlling the rate of transcription. The regulation of gene expression at the transcriptional level is facilitated by the interaction between cis-acting elements and trans-acting factors. The temporal and spatial expression of a gene is determined by the presence of trans-acting factors at specific time and in certain tissues interacting with the unique cis-acting sequence of the regulated gene
(Schibler et al., 1987, Bogue et al., 1990). The identification of regulatory genes involved in transcriptional activation and analysis of the promoter and enhancer sequences of the regulated genes involved in development will contribute substantially to our understanding of this process (Chandlee, 1991).
Since the identification of cis-acting elements conferring tissue- and development- specific expression is important in understanding the regulation of gene expression, much research has concentrated in this area and many cis-acting elements have been identified.
The soybean lectin gene is expressed at much higher level in mid-maturation staged embryos than in other plant organs. Through an assay performed in transgenic tobacco plants, developmental and quantitive regulatory regions important for lectin gene expression were identified (Goldberg et al., 1989). The 77 bp (+1 to -77) 5' region is required for the temporal expression of the gene and another region, about 1 kb upstream of the lectin gene, is required for the correct quantitive expression in the embryos. This quantitive region raises lectin mRNA levels more than 1000-fold during seed development. Several conclusions were drawn from soybean lectin gene expression studies. Sequences required for correct spatial and temporal expression of seed protein genes were located in the 5'-flanking regions, distinct domains within the 5’-region appear 13 to play different roles, and the positions of these elements are specific for each gene
(Goldberg et al., 1989). By the analysis of 5*-flanking sequence, a 170 bp DNA sequence
(-257 to -78) containing essential elements was found for the seed specific and temporal expression of the a' subunit of P-conglycinin gene (Chenet a l 1989). Lessard et al.
(1993) reported that the proximal promoter region (-140 to +13) was sufficient for seed- specific expression of the a ’ subunit of P-conglycinin in transgenic tobacco. This region contains a binding site for the soybean embryo factor SEF4, a vicilin box GCCACCTC,
ACGT motif, and an E-box (CANNTG). In maize, a TGTAAAG sequence in zein gene functioned as an activation sequence (Thompson, 1990). In pea, the promoter sequence between -97 and -549 was responsible for promoter activity, seed specificity, and temporal regulation ofLeg A gene (Shirsat, 1989). The CATGCATG (RY) motif is widely distributed among dicot and monocot genes and comprises a part of the 28-bp legumin box found in legumin protein genes (Dickinson et al ., 1988). Deletions of this motif in the soybean gtycinin promoter region resulted in a decrease of GUS expression using a glycinin promoter-GUS expression vector in transgenic tobacco (Lelievreet al.,
1992).
Trans-acting factors have been isolated and identified from a number of plant species. Allen et al. (1989) identified two trans-acting factors from immature soybean seeds. Soybean embryo factor 3 (SEF3) bound exclusively to the -183 to -134 region, the previously reported seed-specific enhancer. SEF3 binding activity correlated with the accumulation pattern of the a' subunit of p-conglycinin and SEF3 accumulated only in nuclear extracts of soybean seed developed at the middle and late maturation stages. In com, an endosperm DNA binding protein was found to interact with a 15 bp DNA motif present at the 5' region of all zein storage protein genes (Maieret al ., 1987). The Opaque-
2 (02) is another trans-acting factor regulating zein gene expression in maize. Schmidtet 14 al. (1992) showed that the 02 protein is a basic leucine zipper protein (BZip) and recognizes the TCCACGTAGA sequence only present in the promoter of 22-kD zein gene. Another maize BZip protein, 02 heterodimerizing protein 1 (OHP1), was isolated by Pysh et al. (1993). OHP1 bound to the 02 target sequence in vitro as a homodimer or as a heterodimer with 02. The function of OHP1 may be to form a heterodimer with 02 to regulate the expression of 22-kD zein gene.
Molecular studies have provided further insights into the basic biology of plant embryogenesis through gene isolation and regulation. However, this study is only limited to genes expressed during mid to late stages of embryo development. The isolation of genes, involved in early events such as pattern formation and tissue differentiation, has not been accomplished and molecular and biochemical analyses of early embryogenesis are limited (Zimmerman, 1990, West and Harada, 1993). As Thomas (1993) reported, few genes specifically expressed in the early staged embryos have been isolated. The main reason for this is that early staged embryos are very small and embedded by both maternal tissues and endosperm. In addition, it is very difficult to get enough materials for biochemical and molecular studies (Goldberg et al., 1988, 1989, Galau, 1991,
Thomas, 1993). To overcome this difficulty, somatic embryogenesis has been used as an alternative for early embryogenesis studies. Some recent studies have provided significant progress in understanding the molecular and cellular events taking place during early embryo development (Chasan, 1993, De Jonget a l 1993, Zimmerman,
1993, Thomas, 1993).
Somatic Embryogenesis Provides a Model for Plant Embryogenesis
Somatic embryogenesis is a process, where somatic plant cells form embryos and then whole plants. This pathway for plant regeneration can be utilized as a potential 15
model for studying early events during plant embryogenesis (Zimmerman, 1993).
Knowledge of embryogenesis is also important for use in somatic hybridization and DNA
transformation studies (Dudits et al., 1991).
Somatic embryogenesis has been used as an alternative for the early
embryogenesis studies for a few reasons. Somatic and zygotic embryos are
morphologically similar and display some common gene expression patterns. It is
relatively easy to obtain large quantity of synchronized somatic embryos at discrete
developmental stages. This makes study of the early embryo development possible.
Moreover, the two most critical events for morphogenesis, asymmetric cell division and
controlled cell expansion, are important mechanisms for both somatic and zygotic
embryogenesis systems indicating that the same basic cellular mechanisms are used
although the start points in both systems are quite different (De Jonget al ., 1993). This
similarity is especially evident at the early stages where globular-, heart-, and cotyledon-
staged embryos are formed. The organization of the plant tissues such as the epidermis, vascular tissue and ground tissue in the somatic embryo is similar to that observed in zygotic embryo (Prez-Grau and Goldberg, 1989).
Gene expression studies showed that the some genes active within the cotyledons of a zygotic embryo are also active in the analogous cells of somatic embryos (Crouchet al., 1984, Franz et al., 1989). The soybean trypsin inhibitor gene, KTi3, was expressed in the same spatial and temporal pattern in both somatic and zygotic embryos (Perez-Grau and Goldberg, 1989). The KTi3 mRNA was observed within the axis but not within the vascular tissue in both systems. Shoemaker et al. (1987) found that the pattern of some protein synthesis, processing and accumulation in zygotic embryos of cotton was broadly similar to those in somatic embryos. Sterket al. (1991) reported that the extracellular protein 2 (EP2) gene of carrot was expressed in the periphery, i.e. the protoderm cells of 16 globular and heart staged somatic embryos. As embryos developed, the expression of EP2 gene became localized. By the seedling stage, the EP2 mRNA was restricted to the shoot apex. The expression of EP2 in zygotic embryos of carrot was similar to that in the somatic embryos both temporally and spatially. The pattern of gradual restriction of EP2 gene expression toward the shoot apex was strikingly similar too. Usingin situ hybridization, Wurtele et al. (1993) showed that the EMB-1 mRNA, isolated from carrot as an embryo abundant mRNA, began to accumulate uniformly and at low levels at the globular stage in both zygotic and somatic embryos. As somatic and zygotic embryos developed to the heart stage, the EMB-1 mRNA accumulated to higher levels and predominantly in the peripheral regions i.e. the regions forming the cotyledon, protoderm, and root. In the torpedo stage of both somatic and zygotic embryos, the EMB-1 mRNA accumulated to high levels in the shoot and root meristems
The similarities between zygotic and somatic embryos, based on both gene expression and morphology, indicate that the specific expression of some genes and the major morphological events during this process are controlled by the developmental program of the embryos and not by the maternal or endosperm factors (Chasan, 1992,
Wurtele et a l 1993, Zimmerman, 1993). This shows that information obtained from somatic embryogenesis can be applied to zygotic embryogenesis.
Carrot (Daucus carota) has been used as a model system for somatic embryogenesis studies in many laboratories. Normura and Komamine (1985) demonstrated the presence of "type I" cells in suspension culture at high cell density and high concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). The "type I" cells are capable of developing into proembryogenic masses after exposure to low level of 2,4-D.
After transfer of cells to a 2,4-D-free medium, the proembryonic masses develop to 17 globular and subsequently, heart, torpedo, and cotyledon staged embryos closely resembling to the zygotic embryo development.
Using carrot somatic embryos as a model, numerous genes have been isolated.
Zimmerman (1993) summarized the characteristics of 21 genes cloned from carrot somatic embryos. Although the function of most of them are not known, someLea are genes. Two extracellular protein genes, EP2 and EP3, have attracted the interest of some researchers for their potential functions in carrot somatic embryogenesis. Some of these genes from carrot somatic embryos are also expressed in the zygotic embryos such as
DC59, EMB-1, ECP31, ECP40, and EP2. Most of these genes are preferentially expressed in somatic embryos and are not detected or are detected at much lower levels in nonembryogenic cells. This indicates that these genes can be used as molecular markers for embryo development and they may play important roles in carrot somatic embryogenesis. Further studies of these genes will help us to understand both somatic and zygotic embryogenesis.
By studying the effects of conditioned medium on somatic embryogenesis of carrot, Fisenberg et al. (1984) found that increased polyamine levels were required for cellular differentiation and development. Inhibition of polyamine biosynthesis by a- difluromethylarginine (DFMA) inhibited somatic embryogenesis in carrot cell cultures.
Promotion of polyamine synthesis by a-difluromethylomithine (DFMO) allowed the development of somatic embryos even in the presence of auxin (Robie and Minocha
1989). De Vries et al. (1988) found that the excreted polypeptides were active components for the acquisition of embryogenic potential of carrot suspension culture.
Three types of extracellular proteins associated with embryogenesis have recently been isolated and partially characterized. They are cationic peroxidases (Cordewener et al.,
1991), chitinases (De Jong et al., 1992), and lipid transfer proteins (Sterk et al., 1991). 18
Using tunicamycin, the potential function of the cationic peroxidase was analyzed
(Cordewener et a l, 1991). Tunicamycin is a fungal antibiotic that prevents N- glycosylation of proteins and is found to inhibit somatic embryo development at the preglobular stage with an abnormal expansion of small embryogenic cells. The inhibitory effect of tunicamycin and the abnormal cell expansion can be overcome by adding cationic peroxidase to culture medium. This indicates that the cationic peroxidase functions to restrict the cell size perhaps, by catalyzing the formation of oxygen radicals which cross-link cell wall proteins and polysaccharides. Therefore, this restriction is an important prerequisite for somatic embryogenesis (Van Engelen and De Vries, 1992). De
Jong et al. (1992) purified another secreted extracellular protein, EP3, which was shown to be an acidic endochitinase. The function of this protein was recognized by its effect on the rescue of t s ll embryos. T slI is a temperature sensitive mutant. At the restrictive temperature, the embryos oft s ll do not develop beyond the globular stage. The major feature of arrested t s l l embryos is the lack of proper protoderm formation. The addition of EP3 to the medium allows completion of somatic embryo development at restrictive temperature and restores the embryo protoderm. The chitinase activity may utilize cell wall components as substrates to modify cell wall strength in order to regulate cell size and plane of cell division (De Jong et a l, 1992). EP2, a lipid transfer protein, is only synthesized by embryogenic cells and somatic embryos. It is present in the celt wall of carrot somatic embryos, and expressed in the protoderm of the embryos. Based on the extracellular location, the expression pattern, and the lipid transfer function of this class of protein, Sterk et al. (1991) proposed that EP2 transfers cutin monomers through the extracellular matrix to the site of cutin synthesis. The cutin layer around the developing embryos is suggested to, perhaps, regulate water uptake and hence, cell size. Although these extracellular proteins have been shown to be very important for carrot somatic 19 embryo development, their functions in other somatic embryogenesis systems and in zygotic embryogenesis have not yet been determined. It will be interesting to determine their functions in other systems. If they are really important in carrot somatic embryogenesis, they must also play similar roles in somatic and zygotic embryogenesis of other plant species.
Although somatic embryogenesis can provide a reliable alternative for plant embryogenesis studies, this approach is limited to only a few systems in which somatic embryos can be readily obtained. Studies of embryogenesis in other plant species are restricted by the difficulty in obtaining suitable materials. An embryogenic suspension culture of soybean was obtained by Finer and Nagasawa (1988). The development pattern of this culture is similar to that reported in carrot. Soybean embryogenic tissue proliferates in the presence of 2,4-D. Upon transferring the tissue to a growth regulator- free medium, the embryos develop through the globular, heart, and cotyledonary stages.
The development is fairly synchronous and this system is therefore suitable for embryogenesis studies. In my dissertation research, this culture material was used and our objectives were to isolate genes expressed during the early stages of both somatic and zygotic embryogenesis and to gain some insight to their potential functions in plant embryogenesis.
The expression of most genes is tightly regulated during embryogenesis. In order to isolate genes expressed at the early stages, differential screening technique was employed. A cDNA clone encoding pyruvate kinase was identified from the cDNA library constructed from early staged somatic embryos. In the first chapter of this dissertation, the isolation and characterization of this pyruvate kinase cDNA will be described. 20
Homeobox genes have been shown to play important roles in various
developmental processes of a wide range of organisms including plants, especially in
Drosophila embryogenesis. We believe that the homeobox genes are present in soybean
embryos and play important roles in soybean embryogenesis. Using heterologous probing
with Knotted-l cDNA, a homeobox gene from maize, the first soybean homeobox-
containing gene (S b h l ) was identified. Probing with Sbhl, several different homeobox-
containing cDNA clones were subsequently isolated. In chapter II, the identification of these homeobox-containing genes and characterization of the first homeobox-containing
gene (Sbhl) will be discussed.
In order to understand to potential function of theSbhl involved in plant development, expression studies were performed using northern and in situ hybridizations. The techniques involved in and the results obtained from these experiments will be described in the third chapter of this dissertation. CH A PTER I
Isolation and Characterization of a Pyruvate Kinase cDNA from Soybean
ABSTRACT
This work describes the isolation and characterization of genes expressed during the early stages of soybean somatic embryogenesis. Differential screening was utilized to recover cDNAs representing mRNAs of early staged somatic embryos. Four cDNA clones were identified with higher expression at early stages. One of these 4 clones was used for more extensive studies. Sequencing analysis showed that this cDNA clone encodes pyruvate kinase. This pyruvate kinase cDNA is one of a few pyruvate kinase genes identified from plant species and the first one from soybean (Glycine max L.).
The soybean pyruvate kinase cDNA (Sbpk) is 1795 bp long excluding the polyadenylation tail and contains an open reading frame of 1533 nucleotides, a 61- nucleotide 5' and a 198-nucleotide 3' untranslated region. The predicted protein consists of 511 amino acids with a translation start codon AUG at position 62 and a stop codon at nucleotide 1595, This protein is rich in leucine, alanine, and valine with a molecular mass of about 5.5 kD. Comparison of the deduced protein with data base proteins revealed extensive homology with many nonplant and plant pyruvate kinases. The SBPK has
89.4% identity with potato pyruvate kinase, 32.5% with PKpa, the a-subunit of castor bean pyruvate kinase, and 77.9% with tobacco pyruvate kinase.
21 22
Southern hybridization analysis indicated that a single copy of pyruvate kinase
gene is present in the soybean genome. Although more work is required to clarify the
expression pattern ofSbpk, preliminary studies showed that the expression ofSbpk was
not induced by auxin (2,4-D) or under anaerobic and osmotic stress conditions. Since we
were more interested in genes controlling morphogenesis than functioning as
"housekeeping", studies on Sbpk were not further pursued. Therefore, the potential
function of this gene in plant embryogenesis remains unknown. However, since early
staged somatic embryos consist of active dividing cells and pyruvate kinase is a key enzyme in energy production, the higher expression of this gene at the early stages of soybean somatic embryogenesis may be critical in providing an energy source for active cell division.
INTRODUCTION
Like zygotic embryogenesis, somatic embryo development is accompanied by distinct morphological changes which result from alterations in expression of different sets of genes. In order to study embryogenesis, it is necessary to isolate genes expressed specifically during this process and then to understand their functions. Several approaches have been developed for the isolation of genes expressed during embryogenesis.
Differential screening is based on a comparison of gene expression in different materials.
Heterologous probing is to use the genes cloned from other developmental points as probes. Genetic mutants also provides another way to facilitate gene isolation (De Jonget al., 1992, Zimmerman, 1993). Thus far, the differential screening is the most commonly used technique for gene isolation in embryo development.
Carrot somatic embryogenesis is one of the most comprehensively studied systems in term of biochemistry and molecular biology. Somatic embryos of carrot are 23 induced from unorganized cells (Nomura and Komanine, 1985). The dramatic transition
from unorganized cell growth to organized embryo development, the active RNA
synthesis (Fujimura and Komamine, 1980) and the protein profile changes at this
transition state suggest that a substantial reprogramming of gene expression exists
(Zimmerman, 1993). Choi and Sung (1984) showed that 10% of approximately 100
prevalent proteins were embryo-specific. They then identified two embryo-specific and
two callus-specific proteins by comparing the protein profiles between carrot somatic embryos and calli. Kiyosueet al. (1991) purified an embryogenic cell protein, ECP31
from carrot. This protein was present in large amount in embryogenic cells and early staged embryos but at very low level in nonembryogenic cells and root hairs. ECP31 preferentially accumulated in the peripheral cells of embryogenic cell clusters, where the proembryogenic mass originated. The amount, the location, and the time of its accumulation seemed to correlate with competence of cells to form somatic embryos. In carrot culture, several biochemical differences were observed between the calli and the embryos (Sung and Okimoto, 1981). Callus tissues produced callus-specific proteins and conditioning factors that were necessary for the synthesis of callus-specific proteins. In parallel, embryos produced embryo-specific proteins. During the transition from calli to the globular embryos, the callus-specific proteins disappeared with the appearance of embryo-specific proteins. Based on protein pattern changes, Sung and Okimoto (1983) proposed a "switch" model: callus-specific protein synthesis is turned on while embryo- specific protein synthesis is turned off in carrot callus culture. On the other hand, when embryo-specific protein synthesis turns on in embryos, callus-specific protein synthesis is turned off.
Similar patterns of protein alteration have been observed in other systems. In orchard grass (Dactylis glomera) tissue culture, embryos and undifferentiated cells can be 24
clearly distinguished from each other by their protein patterns. With three independent
detection techniques: silver-staining, in vivo labeling, and in vitro translation, Hahne et
al. (1988) identified 32 embryo-specific proteins from somatic embryos and 18 callus-
specific proteins from unorganized cells ofDactylis. In Cichorium, somatic embryos are
easily induced directly from cells of freshly cultured leaves. Using this system, Hilbertet
al. (1992) compared the protein profiles of the induced leaves with that of non-induced
leaves and two embryo-related proteins were identified. They were detected only after
embryogenic induction and remained present during embryogenesis. These proteins were
not detected either in uninduced leaves or in leaves of an non-responsive line. The
accumulation of these two proteins before any morphological changes suggested that their synthesis was associated with the induction of embryogenesis rather than being a consequence of embryogenesis. In rice Oryza( saliva) tissue culture, there are two types
of calli: embryogenic and nonembryogenic. In embryogenic calli, somatic embryogenesis
is easily induced while cells in a nonembryogenic calli grow in an unorganized manner.
Chen and Luthe (1987) analyzed the protein patterns of nonembryogenic calli, embryogenic calli, and rice zygotic embryos. They found some qualitative and quantitative differences between proteins in the three different types of materials. Most of
polypeptides present in zygotic embryos were also present in embryogenic calli but not in
nonembryogenic calli indicating that they were embryogenic proteins. Stim and Jacobsen
(1987) identified several proteins which were specifically expressed in embryogenic
callus of pea.
Based on differential protein synthesis, somatic embryogenesis may be obtained
by switching off the callus-specific genes and switching on the embryo-specific genes. A
strategy comparing differences in gene expression between somatic embryos and callus
cells has been employed. This strategy should identify genes associated with embryonic 25 organization and morphogenesis. A common way to identify genes involved in embryogenesis is to construct a cDNA library using embryogenic materials and then screen for genes expressed preferentially in these materials. The nonembryogenic callus can provide as a control for genes involved in the house-keeping and cell growth. With differential screening of this cDNA library against poly(A+)RNA from callus cells, the house-keeping genes could be eliminated and the genes expressed specifically in embryos can be recognized (Choi et al ., 1987). With differential screening of a carrot cDNA library constructed from cells cultured on 2,4-D-free medium with cDNA probes reverse transcribed from po!y(A+)RNAs isolated from carrot cultures with and without 2,4-D, 27 clones were identified which were expressed preferentially in (-) 2,4-D culture (Wurtele et al., 1993). The elongation factor-la cDNA clone was also identified from carrot with differential screening of globular embryos and unorganized callus cells of carrot
(Kawahara et al., 1992). Using this strategy, many other cDNA clones expressed preferentially in carrot somatic embryos have also been isolated such as DC3 and DCS
(Wilde et al., 1988), DC 8 (Borkird et al., 1988), and EMB-1 (Ulrich et al., 1990).
Meristematic cells are often used for differential screening in order to eliminate genes common to rapidly dividing cells. When a barley embryogenesis cDNA library was screened against meristematic tissues from leaf bases of young seedlings, Smith et al.
(1992) identified a cDNA clone, pZE40, which was expressed at high level in specific tissues of barley embryos.
Differential screening, based on gene expression differences between embryos and calli, has some drawbacks. This technique is designed with the bias that genes important for embryo development should not be expressed at all or at much lower level in callus cells. However, the expression of some isolated genes showed that many genes expressed in somatic embryos were expressed in calli before embryo induction. It is very 26
likely that differential screening by comparing embryo cDNA with callus cDNA will eliminate some genes important for embryo development (Zimmerman, 1993). Higgins and Bowles (1990) compared in vitro translation products of poly(A+)RNAs from nonembryogenic culture and somatic and zygotic embryos of barley. Since there were no significant differences in protein patterns either quantitatively or qualitatively, they concluded that callus is not good source for the differential screening when interests are to clone embryo-specific genes.
To avoid the loss of important genes for embryo development, another approach has been employed in which the differential screening is based on preferential expression of genes during different stages of embryo development. In vitro translation of isolated polyadenylated RNAs from proembryogenic masses and torpedo embryos of carrot revealed some differences in gene expression between these two stages (Wilde et al.,
1988). With in vivo labeling and in vitro translation, Sdnchez-Martinez et al. (1986) identified two sets of polypeptides from maize embryos. In the embryogenic set, the polypeptides were synthesized in young and mature embryos. In the maturation sets, the polypeptides were present in mature but not in young embryos. In order to identify genes induced at globular staged embryos of carrot, differential screening was used and 50 different clones were isolated (Zimmerman, 1993). Using this technique, other clones such as DC1.2, DC2.26, DC2.15, DC7.1, DC9.1, DC10.1, and DC3.1 have been isolated by comparing different staged embryos and calli (Aleith and Richer, 1990).
Somatic embryo development of soybean culture is similar to that of carrot.
Embryos in the suspension culture containing 2,4-D keep proliferating and remain at globular or preglobular stages. About one month after transfer to the development medium, they develop to the cotyledon stage (Figure 1.1). This dramatic morphological alteration indicates significant gene expression changes between these two stages. We 27
thought that by differential screening cDNA from globular staged embryos with that from
cotyledon embryos, the genes expressed during the early stages of embryogenesis could
be isolated. As a result of a differential screening of a cDNA library constructed from
poly(A+)RNA of globular embryos, a cDNA clone was isolated which hybridized to a
cDNA probe from globular embryos but not to that from cotyledon embryos. Sequence
analysis revealed that this cDNA clone encodes pyruvate kinase.
Pyruvate kinase is one of the key regulatory enzymes controlling glycolysis in plants and animals. It catalyses the irreversible formation of pyruvate and ATP by the following reaction: phosphoeno/pyruvate + ADP—>pyruvate + ATP. The enzyme from nonplant species has been purified in a wide range of species and studied more extensively. Most nonplant pyruvate kinases are homotetramers with a native mass of
190-250 kD. Mammalian pyruvate kinases occur in distinct tissue-specific isozymes which are modulated by mechanisms including allosteric regulation and reversible protein-kinase-mediated phosphorylation. In plants, pyruvate kinases can be either hetero- or homotetramers depending on the sources of the isozymes (Plaxton, 1989). Only a few of pyruvate kinase enzymes have been purified or partially purified (Plaxton, 1988) and little is known about their regulatory mechanisms and physical properties (Plaxton,
1989). Pyruvate kinase genes have only recently been cloned from a few plant species such as Ricinus communis (Blakeley et al., 1991), Solanum tuberosum (Blakeley et a!.,
1990), and Nicotiana tabacum (Gottlob-McHugh et al., 1994). The isolation of soybean pyruvate kinase cDNA from our research not only added one more gene to the short list of plant pyruvate kinase genes but also may be important to plant physiologists and plant biochemists who study the regulation and function of pyruvate kinase in glycolysis and plant development. 28 MATERIALS AND METHODS
Plant Materials
Embryogenic suspension cultures were initiated from soybean(Glycine max [L.]
Men.) cv Fayette and maintained according to Finer and Nagasawa (1988). Suspensions were subcultured monthly in a liquid soybean proliferating medium (SBP6) which consists of MS salts, B5 vitamin, 6% sucrose, 23 pM 2,4-dichlorophenoxyacetic acid
(2,4-D) and 5 mM asparagine. For embryo development, embryogenic tissues were transferred to a hormone-free solid MS3M medium (MS salts, B5 vitamins and 3% maltose) at 23°C (Finer and McMullen, 1991). Developing embryos were harvested 0, 3,
7, 14, and 28 days following transfer of tissue to the embryo development medium
(Figure 1.1).
RNA Isolation
Total RNAs were extracted from various staged soybean somatic embryos from proliferating embryogenic suspension culture (day 0) to cotyledon stage (day 28) (Figure
1.1). In brief, fresh tissues were collected, ground in liquid nitrogen and then extracted using guanidinum thiocyanate (Davis et al., 1986) [Appendix 1]. After centrifugation, the supernatants were pelleted through a CsCl gradient. The pellets were resuspended in
NETS (NaCl, £DTA, Iris, and SDS) buffer and then phenol/chloroform purified. The total RNAs in the supernatants were precipitated with ethanol and finally resuspended in sterile H 2 O. For poly(A+)RNA isolation, the total RNAs were fractionated by one passage through an oligo (dT)-cellulose column according to Aviv and Leder (1972)
[Appendix 2]. cDNA Library Constructions
cDNA libraries were constructed in XgtlO according to the protocols in the cDNA
Synthesis System Plus and cDNA Cloning System-XgtlO Kits (Amersham, Arlington 29
Heights, Illinois) with some modifications. The first strand cDNAs were synthesized using poly(A+)RNAs isolated from different development staged embryos as templates with reverse transcriptase and oligo (dT) primers. The poly(A+)RNA templates were nicked with RNaseH and the nicked poly(A+)RNAs were used as primers for the synthesis of second strand of cDNA by T4 DNA polymerase.
For cDNA cloning, £coRI adaptors were ligated to the double stranded cDNAs .
After removing the unreacted adaptors by chromatography through a column packed with
Sephacryl S-200 (Pharmacia Biotech, Piscataway, NJ), the adapted cDNAs were phosphorylated and then ligated with XgtlO arms. The XgtlO cDNA recombinants were packaged in vitro into phage particles. cDNA Library Screening
The early staged (day 0) cDNA library was used to infect the E coli NM514 host strain and then plated on LB agarose medium in 100 mm Petri dishes at density of 500 pfu/plate. Three replica filters were prepared from each plate based on Benton and Davis
(1977) [Appendix 3]. After denaturation and neutralization of phage DNA, the filters were baked under vacuum for 2 hours at 80°C. Prehybridizations were performed in solution A (3x SSC) for one hour, solution B (3x SSC, 2x Denhardts) for two hours, and then solution C (3x SSC, 2x Denhardts, 10 pg ml-* poly[A]-[dT]i2, and 50 pg ml-^ boiled salmon sperm DNA) for 4-6 hours at 65°C. Hybridizations were performed for 2 days using the prehybridization solution C plus 0.1% SDS and cDNA probes. For differential screening, cDNA probes were synthesized from poly(A+)RNAs of different staged embryos [Appendix 4]. Each of the three replica filters was used for hybridization with different probes. One filter was hybridized with cDNA probe synthesized from early staged (day 0) embryos, one with cDNA probe from late staged (day 28) embryos, and the other with cDNA probe from soybean leaves. After hybridizations, filters were 30 washed with 3x SSC plus 0.1% SDS, and then O.lx SSC and 1% SDS at 65°C. After
autoradiography, plaques hybridizing to the day 0 cDNA probe but not to the others were
further purified by three rounds of plaque purification.
Subctoning and Sequencing
Phage were propagated in liquid culture and purified using CsCl gradients
[Appendix 5]. Phage DNAs were isolated [Appendix 5] and digested with EcoKl to release intact cDNAs. The fragments were purified using Geneclean (BIO 101, La Jolla,
CA) and subcloned into pUCl 19 in both orientations. Single strand DNAs were produced
(Vieira and Messing, 1987) [Appendix 6], Ordered deletions were created according to
IBI Cyclone I Kit. Single stranded DNAs from deletions were used for sequencing with dideoxy chain termination method (Sambrook et al., 1989). Sequence data was analyzed using IntellGenetics (Mountain View, CA) sequence analysis programs.
Southern Hybridization Analysis
Genomic DNA was isolated from leaf tissue of soybean cv."Fayette” according to
Saghai-Maroofet al. (1984) [Appendix 7]. Genomic DNA was digested with restriction enzymes, electrophoresed, and transferred onto nitrocellulose membrane according to
Sambrook et al. (1989). The membranes were prehybridized six hours in 5x SSC, 5x
Denhardts, 0.1% SDS, 50 mM Tris (pH 8.0), 10 mM EDTA, and 100 pg ml-1 denatured salmon sperm DNA and then hybridized for two days with random primer labeled
(Feinberg and Vogelstein, 1983) [Appendix 8] Sbpk, a soybean pyruvate kinase cDNA in the above solution plus 10% dextran sulfate. Membranes were washed either at low stringency (lx SSC and 0.1% SDS at 65°C) or at high stringency (O.lx SSC and 0.1%
SDS at 65°C) and then exposed using Kodak film with intensifying screens for 2-3 days. 31
Northern Hybridization Analysis
Ten jig of total RNAs were used for gel electrophoresis and northern
hybridization analysis as described by Thomas (1983) [Appendix 9]. Following
hybridization with random primer labeled (Feinberg and Vogelstein, 1983) Sbpk cDNA,
membranes were washed at high stringency (O.lx SSC and 0.1% SDS at 65°C) and then exposed using Kodak film with intensifying screens for 2-3 days.
DNA and RNA Dot Blots
DNA dot blots [Appendix 10] were performed on nitrocellulose filters treated with lOx SSC and then dried at room temperature for 1 hour. Phage DNAs were boiled for ten minutes, alkaline denatured at room temperature for 20 minutes, and then neutralized with HC1. The DNAs were precipitated and resuspended in H 2 O and spotted on nitrocellulose filter. After baking at 80°C for 2 hours, the filters were used for prehybridization and hybridization essentially the same as described for Southern hybridization analysis.
RNA dot blots (Thomas, 1983) [Appendix 11] were performed with nitrocellulose filters soaked in 20x SSC. Total RNAs were denatured with glyoxal at 55°C for one hour and then spotted onto the filter. After baking at 80°C for two hours, the filters were briefly boiled in 20 mM Tris-HCl (pH 8.0) to remove glyoxal. Prehybridization and hybridization were performed essentially the same as described for northern hybridization analysis.
RESULTS AND DISCUSSION
Construction o f cDNA Libraries
Different developmental staged somatic embryos of soybean were collected for isolations of total RNAs and then poly(A+)RNAs. Five pg of poly(A+)RNAs from each 32
staged embryos were used for cDNA synthesis. Different amounts of cDNA (from 8 ng to
110 ng) were used for ligation with 1.0 ng of A,gt 10 arms. Afterin vitro packaging, five
cDNA libraries were obtained. cDNA library constructed from embryos 0 day in
development (proliferating embryos) is referred as day 0 cDNA library and same for
other cDNA libraries. Day 0 cDNA library had a titration of 3 x 10^ plaque forming units
(pfu), day 3 cDNA library had 4 x 10^ pfu, day 7 cDNA library had 9 x 10^ pfu, day 14
cDNA library had 2 x 10^ pfu, and day 28 cDNA library had 4x10^ pfu.
DifferentiaI Screening o f the Day 0 cDISA Library
Since we were more interested in isolating genes expressed at early stages of embryo development, the day 0 cDNA library was used for differential screening. Filters obtained from this library were hybridized with cDNA probes prepared from poly(A+)RNA from day 0 soybean embryos, cotyledon embryos and soybean leaves.
Filters prepared from XgtlO phage were used as controls to show hybridization background. After screening about 15,000 plaques, several clones were identified which showed stronger hybridization signals to day 0 cDNA probe than to cotyledon embryo and leaf cDNA probes. They were designated as Al, A2, A4, A5, A8, A 10, and All.
Characterization o f the Isolated Clones
To determine if some of these clones were similar to each other, DNA dot hybridizations were performed (Figure 1.2). When selected clones were hybridized with other isolated clones as well as themselves, the cross-hybridization was only detected between the A8 and A10 clones. The same intensity of hybridization signals was observed when A8 and A10 clones were probed with each other as well as with themselves indicating that they were the same clone. The rest of the isolated clones were different from each other according to the hybridization patterns. 33
In order to confirm their differential expression during embryogenesis, RNA dot
blots were performed (Figure 1.3). Three types of expression patterns were identified.
The first one was represented by Al clone which was constitutively expressed during
embryo development and also expressed in soybean leaf. The second pattern showed
expression at much higher levels in the early staged (0, 3 days) embryos than in the late
staged embryos and soybean leaf such as A2, A5, A8, A10, and Al 1. A4 clone was
expressed at same levels at all stages of embryo development but at very low level in
soybean leaf. Since we were more interested in genes expressed during early stages of
embryo development, the second class of clones (A2, A5, A 10, and All) was used for
further study. The same expression patterns of A8 and A 10 further indicated that they
were same and, therefore, A8 clone was excluded.
A2, A5, A 10, and Al 1 clones were expressed at higher levels in the proliferating
embryos than in cotyledon embryos. One of the possibilities was that they could be auxin
inducible genes since the proliferating embryos were cultured in the medium containing
2,4-D while the embry os developed in the absence of 2,4-D. To test for this possibility,
following experiment was performed based on Hagen et ah (1984). After three days of
germination, hypocotyls were excised from soybean seeds, precultured four hours in
hormone-free SBP6 medium (HFSBP6), and then treated as follows;
Treatment 1 (Tl): no further treatments.
Treatment 2 (T2): transferred to HFSPB6 for another 2 hours
Treatment 3 (T3): transferred to HFSBP6 + 2,4-D (5 mg 1"1) for 2 hours
Treatment 4 (T4): transferred to HFSBP6 for another 4 hours
Treatment 5 (T5): transferred to HFSBP6 + 2,4-D (5 mg 1-1) for 4 hours
After these treatments, the hypocotyls were collected and used for total RNA isolation as described before. RNA dot blots (Figure 1.4) showed that (1) A2 clone was expressed at 34
the same level in hypocotyls cultured in media with and without 2,4-D and expressed at a
much lower level in hypocotyls than in somatic embryos. These results indicated that A2
clone might be embryo-specific and non-auxin inducible; (2) A5 clone was not auxin
inducible but was not embryo-specific since hybridization signals were nearly the same in
hypocotyl RNA dot blot as in embryo RNA dot blot; and (3) A 10 and Al 1 clones were
expressed at higher levels in the presence of 2,4-D than in the absence of 2,4-D.
Therefore, they might be auxin inducible genes. Since the A2 clone did not appear to be
auxin inducible and seemed to be embryo-specific, it was chosen for further studies.
Sequence Analysis o f A2 cDNA and Deduced Protein
Nucleotide and deduced amino acid sequences of clone A2 are shown in Figure
1.5. Excluding the polyadenylation tail, the nucleotide sequence of this cDNA is 1795 bp
long and contains an open reading frame of 1533 nucleotides, a 61-nucleotide 5' and a
198-nucleotide 3' untranslated region. In the 3' untranslated region, there is a putative
polyadenylation signal, AAATAA, upstream of the polyadenylation sequence. The
predicted protein consists of 511 amino acids with a translation start codon AUG at
position 62 and a stop codon UAA at nucleotide 1595. The predicted protein is rich in
leucine, alanine, and valine with a molecular mass of about 5.5 kD. It is similar to the
subunits of PKc homotetramer (Plaxtonet al., 1989) and the smaller subunits of PKp heterohexamer (Blakeley et al ., 1990) of caster bean.
Comparison of the deduced protein of the A2 clone with data base proteins revealed extensive homology with many nonplant (data not shown) and plant pyruvate kinases (Figure 1.6). The soybean pyruvate kinase has 89.4% identity with potato pyruvate kinase (Blakeley et al., 1990), 32.5% identity with PKpcc, the a-subunit of castor bean pyruvate kinase (Blakeley et al., 1991), and 77.9% identity with tobacco pyruvate kinase (Gottlob-McHugh et al., 1994). The high homology at the amino acid 35 sequence indicated that A2 clone encodes pyruvate kinase. The A2 clone is hereafter referred as soybean pyruvate kinase cDNA (Sbpk).
Sbpk Is a Member o f Small Gene Family
When leaf DNA of soybean was digested with several restriction enzymes and hybridized with Sbpk cDNA, few fragments were detected (Figure 1.7) after membranes were washed at either low (left panel) and high (right panel) stringencies. This indicates that Sbpk is present in the soybean genome at low copy. Two bands were revealed when genomic DNA was digested with Hindlll. Because one Hindlll site is present within the cDNA, there is probably a single gene for this pyruvate kinase in soybean which is the same as the PKpa in caster bean, Ricinus communis (Blakeley et al., 1990).
Expression Analysis o f Sbpk
Since pyruvate kinase is involved in regulation of glycolysis, its function in somatic embryogenesis may be "housekeeping". However, its higher expression during early embryo development was intriguing. This expression pattern could be the result of several factors. Auxin induction may be excluded by the results from the previous experiment. Another two possibilities for the expression pattern could be anaerobic or osmotic induction. Since early staged embryos were maintained in liquid culture, oxygen deficiency could stimulate expression of Sbpk because many enzymes involved in glycolysis are induced by anaerobic stress (Kennedyet al ., 1991, Russell et al ., 1989). As the early staged embryos were also maintained in medium containing high concentration of sugar, osmotic stress may give higher expression ofSbpk.
To test if the enhanced expression of Sbpk is anaerobically induced, the proliferating somatic embryos were placed onto solid development medium for ten days.
Some of them were then put into liquid development medium for four days. Total RNAs were isolated and used for an RNA dot blot. RNA dot blot hybridization (Figure 1.8) 36 analysis showed that Sbpk was expressed at same level in solid medium as well as in liquid medium indicating that the enhanced expression of Sbpk during early stage of somatic embryo development was not due to anaerobic induction.
To find out if the increased expression was induced by osmotic treatment, soybean somatic embryos were permitted to develop in liquid medium containing either high or low concentrations of sucrose. The embryos were collected after different days of development and used for total RNA isolations. RNA dot blot and northern hybridization analysis were performed. No significant differences in Sbpk expression were detected when embryos developed at either higher or lower concentrations of sucrose by both
RNA dot blot and northern hybridization analysis (Figure 1.9). This indicates that enhanced expression of Sbpk in the early staged embryos was not induced by the presence of a high concentration of sugar. However, RNA dot blot and northern hybridization analysis did not show enhanced expression ofSbpk during the early stage of embryo development since the hybridization signals were uniform at all stages. This is contradictory to what we observed before. Although no further experiment was performed to clarify this point, we still believe thatSbpk was expressed at a higher level during early stages of embryogenesis because of the nature of differential screening and the evidence from the previous RNA dot hybridization analysis (Figure 1.3). The controversy inSbpk expression may be the result of old tissue culture material and poor development of embryos in liquid culture condition.
The expression of Sbpk responding to auxin treatment is also contradictory to a recent report by Sussex (personal communication) who showed that a radish pyruvate kinase gene was one of many auxin inducible genes. Auxin influences diverse growth processes such as cell elongation and cell division. The active cell division during embryogenesis requires ample input of energy generated by catalytic reactions driven by 37 enzymes such as pyruvate kinase. Therefore, it is reasonable to hypothesize that the expression of Sbpk is associated with cell division and may be auxin inducible. The insensitivity of Sbpk expression to auxin in our experiments may be the result of improper treatments. We used 2,4-D at concentration of 23 pM which is normally used in our embryo proliferating culture but was much lower than that used by Hagenet al.
(1984). This level of 2,4-D may be not sufficient to induce expression ofSbpk in hypocotyls.
The enhanced expression of many auxin inducible genes was transient (Hagen et al., 1984). Induced translation products were first observed 15 minutes after auxin application, with maximum expression 2-4 hours following auxin treatment. The expression level usually decreased 24 hours after auxin application. Our auxin treatments were performed for 2 and 4 hours. The expression ofSbpk may be induced to the highest level prior to these time points. Therefore, more time points should be applied if further experiments are performed.
Pyruvate kinase is an important enzyme regulating glycolysis and it could play a critical role in plant embryogenesis. However, since we were more interested in genes controlling morphogenesis rather than functioning as "housekeeping" genes, we did not pursue studies on Sbpk. Therefore, the potential function of soybean pyruvate kinase in plant embryogenesis remains unknown. Figure 1.1. The development of soybean somatic embryos. A: A clump of embryogenic soybean tissue from proliferation medium (referred as 0 day in the text), B: Three days after transfer to development medium, C: After 7 days, D: After 14 days, E: After 28 days. The letter C indicates cotyledons, H indicates hypocoty!. PROBES themselves. Figure 1.2. DNA dot hybridizations o f selected clones with other clones and and clones other with clones selected f o hybridizations dot DNA 1.2. Figure A10 A11 CLONES 39 40
0d 3d 7d 14d 28d L Od 3d 76 14d 28d L
Figure 1.3. RNA dot blots showing the expression patterns of the selected clones during soybean somatic embryogenesis. Total RNAs from different development staged (first row above the filters) somatic embryos and leaves of soybean were hybridized with the selected clones (indicated on the blots). On each filter, different amounts of RNAs were loaded (left column). 41
T1 T2 T3 T4 T5 Od T1 T2 T3 T4 T5 Od
10 mq
5pg
1 M 9
10 M9
5 Mg
1 Mg
Figure 1,4. RNA dot blots to detect the effects of auxin on expression of the selected clones. The clones used as probes for hybridizations were shown on each blot. Different amounts (left column) of total RNAs from different treatments (above the blots) were spotted on nitrocellulose filters. The treatments are T1: the excised hypocotols were precultured in HFSBP6 for 4 hours, T2: precultured in HFSBP6 for 6 hours, T3: T1 + 2,4-D SBP6 for 2 hours, T4: precultured in HFSBP6 for 8 hours, and T5: T1 + 2,4-DSBP6 for 4 hours. Figure 1.5. Nucleic acid sequence o f Sbpk and deduced amino-acid sequence of SBPK. Putative polyadenylation signal is underlined. The translation stop codon is marked with *.
42 43
TGAGACTGAGGTTGGGTTTGGAAGGCAAGGGCTTGTGTTGTCACAATCCAAGAGAGAAGTAATOGCGAAC 7 0 M A N 3 ATAGACATCGAAGGGATCTTGAAGCAGCAGCAGCCTTATGATGGGCGCGTTCCGAAGACGAAGATAGTG 13 9 XDXBGXLKQQQFYDGRVPRTKXV 26 TGTACTTTGGGCCCTGCTTCTCGATCCGTAGAAATGACCGAGAAGCTTCTGAGGGCAGGGATGAACGTT 208 CTIiGPASRSVRMTBXLLRAGMHV 49 GCTCGTTTCAATTTCTCTCATGGCACCCACGACTATCACCAGGAAACCCTCAACAATCTCAAGACTGCC 2 77 ARFNFSHGTHDYHQ8TLNMLKTA 72 ATGCACAACACTGGCATACTCTGTGCCGTCATGCTTGACACTAAGGGACCTGAGATTCGGACTGGTTTT 34 6 MHNTGILCAVMLDTKGPBIRTGF 95 CTGAAAGATGGAAAACCTATTCAACTTAAAGAAGGGCAAGAAGTCACCATAACTACTGATTATGACATT 415 LKDGXFXQLKEGQBVTXTTDYDX 118 AAGGGGGATCCGGAGATGATATCCATGAGTTACAAGAAGCTGCCTGTCCACTTGAAGCCTGGAAATACC 4 84 KGDPBHI SMSYKXIiPVHLKPGNT 141 GACGGGACGATTACTCTCACTGTCTTGTCTTGTGACCCTGATGCTATACTGTGCTCTGGTACTGTTAGA 5 5 3 I LCSDGTITLTVLSCDPDAGTVR 164 TGTCGTTGTGAAAACACTGCAACGTTGGGTGAGAGAAAAAATGTTAACCTTCCTGGTGTTGTGGTGGAT 62 2 CRCBNTATLGERKMVNLPGVVVD 187 CTACCCACACTTACTGAGAAGGATAAGGAAGACATTCTTGGATGGGGTGTACCCAACAAGATTGACATG 6 91 LFTLTBKDKBDXLGWGVPNKXDM 210 ATTGCTCTTTCATTTGTTCGTAAAGGCTCGGATCTTGTTAATGTCCGCAAGGTTCTGGGGCCACATGCA 76 0 XALSFVRKOSDIiVHVRRVLQPHA 233 AAGAATATTCAGTTGATGTCAAAGGTTGAGAATCAGGAGGGAGTCCTGAATTTTGACGAAATCCTGCGG B 2 9 KNXQLMSKVBHQBGVLNFDEX LR 256 GAGACTGATGCATTCATGGTGGCACGTGGTGATC'TTGGAATGGAGATCCCAGTAGAAAAGATTTTCCTG 8 98 ETDAFMVARGDLGKB IPVBKI FL 279 GCACAGAAGATGATGATATACAAGTGTAATCTTGTTGGGAAGCCAGTGGTGACTGCTACCCAAATGCTT 967 AQKMMXYKCNLVGKPVVTATQMX. 302 GAATCAATGATAAAGTCTCCCAGGCCAACCCGAGCTGAAGCAACTGATGTAGCTAATGCAGTTCTTGAT 1036 BSMXKSFRFTRAEATDVANAVLD 325 GGAACAGATTGTGTGATGCTTAGTGGTGAAAGTGCTGCTGGGGCATACCCAGAACTTGCTGTGAAAATC 1105 GTDCVHLSGBSAAGAYPBLAVKX 348 ATGG CTCGCATTTG CATTGAAG CAGAAT CAT CCCTTGACTATGGTGC CAT CTTCAAAGAGATGATAAGG 1174 MARX CXBAB SSLDYGAX FKBHXR 371 T CTACCC CATTGC CTATGAGTCCATTGGAG AGCCTTGCAT CAT CTG CTGTCCGCACAGCAAACAAGG C C 124 3 STPLPMS PLB3LA3 SAVRTAHKA 394 AAAGCAAAACTCATTGTTGTGCTGACACGTGGCGGGTCTACAGCCAAGTTAGTTGCCAAGTATAGGCCA 1312 KAKLXVVLTRGGSTAKLVARYRP 417 GCGGTTCCAATATTGTCGGTGGTGGTTCCAGTGTTGAGCACGGACTCATTTG ATTGG AC CTGCAGTGAT 13 81 A V P ILSVVVPVLETDSFDWTCSD 440 GAGACGCCAGCAAGGCACAGCCTGATATACAGGGGCTTGATTCCTATACTGGGCGAGGGATCTGCAAAG 14 50 BTPARHSLIYRGLXPILGEGSAK 463 GCTACCGATGCAGAATCCACAGAGGTCATTCTCGAAGCTGCTCTTAAGTCCGCAACAGAAAGGGCCCTT 1519 ATDABSTBVXLBAALKSATBRAL 486 TGTAAGC CTGGTG ATG CAGTTG TTGCCCTG CAT CGTATTGGAGCTGCCTCTGT CATAAAGATCTGCAT A 158 6 CKFGDAVVALHRXGAASVXKXCX 509 GT CAAATAATGCATGCTCAACT CAACGTGCCGCCAACTAATGCAGGGTT ATGTTTTG C CTTTTATTTCT 16 57 V K * 511 TTCTTTCTGCTGTTTATCCGTATCATAAAATGGAGGATTTAGTTACTAGCTGTAGATCGAGTGTTTTTT 172 6 GGTATACTCTGTTTATATTGCGGAGTACTTCTAAATCAGATTATCATGGAAATAATACTCTTACCTTTT 17 95
Figure 1.5 44
[S] MANIDIBQI IiKQQQP YDORVPKTK IVCTLOPASR SVEKTBKLLRAOMNVARFNFSHQTKDYHQBT 6 5 [ P ] ------D-A—M-DIt -N 1 ------S — -T-P-L ------B ...... 6 4 [T] -- -BHHHHOVNFCTVX ...... F-I ...... S ...... 6 2
[S] LHNIJCTAMHNTGXLCAVMEiDTKOFBIRTGFLKDGKFIQLKEQQBVTITTO YDIKQDP KHISHS YK 13 0 [ P ] - D I - - Q - - Q ...... T ...... I-VS ...... MB...... 1 2 9 [TJ ID--RQ--BS ...... A--V Q I--S S BS--C 127
[S] KLP VHLKPSNTILC S DQTITLTVT.S CDFDAaTVRCRCBMTATLQBRKNVNLFGVWDZiPTLTBKD 195 [P] --V-D...... A ...... S ...... S ...... 294 [T] --AEDV--QSV A--Q--P ...... KBN-LD ...... V ...... I D - - 2 9 2
[S] KKDILOWOVPHKIDMIALSFVRKOSDLVNVRKVLOPHAKNlQLMSKVBNQBaVIiNFDBILRBTDA 26 0 [ P ] K N ...... A ...... R ...... I ...... S 2 5 9 IT ] - D N ------H ------B ------L--E ------L ------A ------D- -LNS-- 257
[S] FMVARGDLGHBIFVSKIFIJkQlOOlIYKCMLVGKFVVTATQMLRSKIKSFRPTRARATDVANAVU} 32 5 [ P ] ...... A--A ...... 3 2 4 [T].... I ...... V ...... IQ ...... 3 2 2
[ S ] aTDCVNLSaBSAAOAYPBLAVKIMARICIBABSSLOYOAIFKBMIRSTPLPMSPLBSLASSAVRT 3 9 0 I P ] ------3 ------NB ------C ------3 8 9 [T]--- T ------D ------OT--K ------T I- -PDV--RIMflNA-V ------3 8 7
[S] AHKAKAKLIWliTRGQSTAKLVAXYRFAVPLSIL3WVPVTD3FDIfTCSDBTPARHSLIYRQLIP 455 [ P ] ------R ...... T ...... SI ...... V ...... 4 5 4 [T ] - -3 A - - L V ...... OH ...... BIK— ...... F ------V - 4 5 2
[Si DAWALHRiaAASVIKICIVKILGEGSAKATDASSTEVILRAALKSATERALCKFG 511 [ P ] ------S ------VT-O ------S ------V - - 5 1 0 IT ] - S ------V-T ------VT--V-HA ------R - S B 8 ------EA-DF--QH-KTKG ------5 0 8
Figure 1.6. Alignment of the deduced amino acid sequence of soybean pyruvate kinase [S] with that o f potato [P] (Blakeleyet al., 1990) and tobacco [T] (Gottlob-McHugh et al., 1994) pyruvate kinases. Amino acid residues identical to soybean pyruvate kinase are marked by 45
Figure 1.7. Southern hybridization analysis of soybean DNA usingSbpk cDNA as a probe. Ten pg of soybean leaf DNA were digested with flg/II(Bg), BstE II (Bs), Clal (C), EcoRV (E), and HindU\ (H). The membranes were washed at low (left, lxSSC, 65°C) and high (right, O.lxSSC, 65°C) strigencies. 46
Figure 1.8. Expression o fSbpk in somatic embryos developed under anaerobic or aerobic conditions. Total RNAs were isolated from somatic embryos of soybean proliferated in the suspension culture (A), developed on solid development medium for 10 days (B), and then on the same medium for another 3 days (C), developed on solid development medium for 10 days and then in liquid medium for 3 days (D), Od and 7d mean Oday and 7days following transfer of tissues to the development medium. 47
Figure 1.9. Expression ofSbpk in soybean somatic embryos developed in liquid medium containing different concentrations of sucrose. Total RNAs were isolated from somatic embryos proliferated in SBP6 medium (A), 7 days in die liquid development medium with 3% sucrose (B), 7 days in the medium with 6% sucrose (C), 14 days in the medium with 3 % sucrose (D), 14 days in the medium with 6% sucrose (E), 30 days in the medium with 3% maltose (F). G: Another preparation of 0 day total RNA. Left panel is a RNA dot blot and right panel is a northern blot with 10 pg of total RNAs loaded on each lane. C H A PTER II
Identification of Homeobox-Containing Genes from Somatic Embryos and
Characterization of the First Soybean Homeobox-Containing Gene
ABSTRACT
Homeotic genes are key 'switches' that control developmental processes.
Homeotic genes containing the consensus 'homeobox' domain have been identified from a number of organisms including Drosophila melanogaster, Caenorhabditis elegans, Homo sapiens, and Zea mays. Although homeobox-containing genes have been demonstrated to be important in embryo development of some insects, amphibians, and mammals, there are no reports of their involvement in plant embryogenesis. Through my dissertation research, I identified several cDNA clones for homeobox-containing genes expressed in somatic embryos of soybean. These are the first homeobox-containing genes isolated from plant embryos and the first ones from soybean species.
The first soybean homeobox-containing gene S( b h l ) was isolated from a cDNA library constructed from poly(A+)RNAs of soybean somatic proliferating embryos using maize Knotted-1 (Knl) cDNA as a heterologous probe. Subsequently, several homeobox- containing cDNA clones were isolated from the same cDNA library using different regions ofSbhl as probes. The hybridization patterns and signal intensities indicated that at least 2 different clones were identified.
48 49
Extensive studies were performed on the first homeobox gene. The Sbhl cDNA clone is 1515 bp long and encodes a predicted protein consisting of 379 amino acids. The homeobox region ofSbhl consists of 192 nucleotides and encodes a 64 amino-acid homeodomain. Within the homeodomain, the amino acid sequence of a helix-tum-helix structure, and invariant and conserved residues were identified. The deduced SBH1 protein shares a high amino acid identity with KN1 protein (47.0% overall and 87.5% for the homeodomain). The homologies of SBH1 with other plant and nonplant homeodomains are only limited to the invariant and conserved residues. Southern hybridization analysis indicated that Sbhl is a member of a small gene family.
The identification of these genes has proved the presence of homeobox-containing genes in plant embryos and provided an entry point for the isolation of other homeobox- containing genes from embryos of other plant species. The expression of these genes in the early staged embryos indicates that homeobox-containing genes may act independently and/or coordinately in regulating plant embryo development.
INTRODUCTION
Higher plants and animals display fundamental differences in their development
(Goldberg, 1988, Chasan and Walbot, 1993). In animals, the entire body plan and organ systems are established during embryogenesis. Postembryonic development is the elaboration of the structures, and as organs grow, they receive new cells from stem cells.
By contrast, most major ontogenetic events in higher plants occur postembryonically and all postembryonic structures of plants arise from meristems which are formed during early embryogenesis. These meristematic plant cells are analogous to animal stem cells and serve as sources of cells throughout plant development. However, in contrast to animal stems, plant meristems lead to the formations of complex organ systems. 50
Therefore, embryo development in plants, unlike that in animals, does not result in the production of an organism that resembles a mature plant. Another difference between plants and animals is the origin of gametes. In animals, the gametes are originated from a germ line that consists of specific cells formed during early embryogenesis while the plant gametes arise from cells of sexual organs of the flower. These cells are not present in plant embryos, thus, plants do not have a specialized germ line. During animal embryogenesis, some maternal molecules within an egg cell may define asymmetry of the early embryo. However, no maternal materials have been described that are necessary for plant embryogenesis so far. In fact, the totipotence of many plant somatic cells suggests that plant embryo development may be independent of maternal factors (Zimmerman,
1993, Goldberg, 1988). In animals, morphogenesis is accomplished by controlled cell migration and differential cell assembly while the presence of the plant cell wall restricts cell migration and therefore, plant morphological patterns are rigidly defined by a combination of asymmetric cell divisions, division in distinct planes, different rates of division and differential growth after cell division.
Although development program differences exist between plants and animals, development in both systems involves pattern formation and tissue specification. This implies that some basic mechanisms of developmental control may be shared by both animals and plants. This speculation is supported by several lines of evidence. Plant genomes are as large and complex as those of animals (Goldberg, 1988) and both plant and animal genes contain introns and exons (Heidecker and Messing, 1986, Goldberg,
1988). Gene expression programs that control plant development are similar to those controlling animal development. Expression programs are regulated by the interaction of
DNA with cis-acting elements and trans-acting factors. In both animals and plants, cis- acting elements include promoters and enhancers while trans-acting factors consist of 51 transcription factors and other regulatory proteins. The structures of DNA-binding domains in regulatory proteins previously described in animals such as helix-loop-helix, leucine-zipper, and zinc fingers have also been found in plants (Ludwig and Wessler,
1990, Weisshaar et al., 1991, Lam et al., 1990). The structural similarities of these DNA- binding domains suggest that they may have originated before plants and animals diverged and that eukaryotes use a limited number of regulatory factors to achieve various purposes (Katagiri and Chua, 1992). Regulatory factors found in other organisms can regulate plant gene expression suggesting that basic regulatory mechanisms are conserved in plants and other organisms (Ma et al., 1988, Hilsonet al., 1990, Gasch et al., 1990, Schena et al., 1991). Another characteristic common to both kingdoms is the presence of homeotic mutations and homeobox genes. Homeobox genes are present in the animal, fungal, and plant kingdoms and the homeodomain proteins encoded by the homeobox genes may be used to determine the developmental fate of cells in many different organisms (Vollbrecht et al., 1993, Chasan, 1992). This further indicates that the fundamental regulatory mechanisms that control development may be shared among organisms (Carabelli et al., 1993, Sessa et al., 1993, Gehring, 1992).
Homeosis and homeotic mutants have been studied more extensively in
Drosophila melanogaster than any organism. In Drosophila, homeotic genes specify identity and spatial arrangement of the body segments. Homeotic mutations cause developmental abnormalities in which one structure of the body is transformed into the homologous structure of another body part resulting in an organism with altered architecture (Gehring and Hiromi, 1986, Gehring, 1987). In Drosophila, two clusters of homeotic genes, bithorax complex and Antennapedia complex, have been the focus of most of the developmental studies. In the Antennapedia (Antp) mutant, the antennae on the head of the fly are transformed into a pair of second legs (Kaufmanet al., 1980). In 52 the mutant of Ubx, a gene in the bithorax complex, the third thoracic segment becomes
transformed into a second thoracic segment with a second pair of wings (Lewis, 1978).
Since the transformation of single structure requires the concerted action of numerous
genes, these homeotic genes can be considered master control genes that regulate other
genes and certain developmental pathways (Scott et al., 1989).
When two homeotic genes, Antp and Ubx were cloned from Drosophila
(McGinnis et al., 1984, Scott and Weiner, 1984), sequence analysis revealed a highly
conserved DNA sequence termed "homeobox". The homeobox is approximately 180 bp
long and encodes a polypeptide segment called homeodomain, which is about 60 amino
acids long. At the DNA sequence level, Ubx and Antp share 74% identity. The Antp and
Ubx homeodomains are identical at 54 out of 61 amino acids (88%). The greater
conservation of protein sequences than that of the nucleotide sequences suggested that the
protein sequence were selected and maintained during evolution (Scott et al., 1989).
Since each of the genes in the two complexes is expressed in a certain region of the
Drosophila embryo and the genes have similar functions, the homeobox-containing genes in Drosophila are believed to have evolved from an ancestral gene by duplication and divergence (Lewis, 1978). Because of their importance in controlling development, intensive studies have concentrated on identification and characterization of more homeobox genes as well as the identification and isolation of genes that activate and are activated by the homeobox genes.
The high conservation of nucleotide sequences of the homeobox has allowed the efficient and rapid isolation of homeobox-containing genes from diverse sources. Since the first homeobox genes were discovered in Drosophila in 1984 (McGinnis et al., 1984,
Scott and Weiner, 1984), more than 300 homeobox genes have been found in a wide range of organisms including yeast, frogs, mice, human, and plants (Komberg, 1993, 53 Chasan, 1992, Gehring, 1992). In most cases, they were identified by cross-hybridization
(Scott et al., 1989) and using degenerate probes (Burglin et al., 1989, Ruberti et al., 1991,
Feng and Kung, 1994). In other cases, homeobox genes were identified after genes were cloned by other methods such as isolation ofAntp and Dfd by chromosome walking
(Gehring, 1992).
Homeodomains are usually located near the C-terminus of the much larger homeodomain proteins. By comparison of many homeodomains, Scottet al. (1989) revealed some striking similarities at the primary sequence level. Four amino acid residues are conserved in all non-yeast homeodomains. These invariant residues are tryptophan, phenylalanine, asparagine, and arginine at the position 49, 50, 52, and 54 from N-terminal of the homeodomain respectively. Another eight residues are highly conserved. Conservation of these 12 amino acid residues is one of the characteristics of most homeodomains. The predicted secondary structure of homeodomains serves as another distinguishing feature. Homeodomains confer a helix-tum-helix motif. The presence of this motif was first suggested on the basis of sequence homologies between
Drosophila homeodomain proteins and some prokaryotic regulatory proteins (Laughon and Scott, 1984, Shepherd et al., 1984). This information was later confirmed by nuclear magnetic resonance (NMR) spectroscopy (Qianet al., 1989). Helix I is located on the most N-terminal of the helix-tum-helix motif. It is proposed to be involved in protein- protein contact to allow formation of protein dimers. Helix II lies over the recognition helix (helix III) and contacts the phosphate groups of the DNA. This helix is believed to stabilize the interaction between helix III and the target DNA. The contacts with phosphate groups may be important for sequence-specific DNA binding. Helix III fits directly into the major groove of the DNA, makes extensive contacts with the bases, and is thought to play the major role in sequence recognition. The N-terminal arm of the 54 homeodomain in DNA binding has been shown in recent genetic studies to be very important. The functional specificity of two very similar homeodomain resides in the N- terminal segments (Furukubo-Tokunaga et al., 1993) and a structural analysis showed that a mutation of six residues in the N-terminal arm caused 10-fold reduction of the
DNA binding (Qian et al., 1994). The interaction between the homeodomain and target
DNA has been investigated using genetic studies (Hanes and Brent, 1991, Hanes and
Brent, 1989, Treisman et al., 1989), DNA binding assays (Desplan et al., 1988) and co crystallization analysis (Wolbergeret al., 1991, Kissinger et al., 1990). In vitro binding studies revealed that different homeodomains exert only minor distinctions in their preference for different binding sites (Hory et al., 1988, Dessain et al., 1992). The function specificity of homeodomain proteins may be enhanced either by sequences outside of homeodomains (Ekker et al., 1991) or by the interaction between homeodomain proteins and other DNA binding factors (Keleheret al., 1988, Goutte and
Johnson, 1994).
Homeodomain proteins play important roles in pattern formation and tissue differentiation. In Drosophila, more than 30 homeobox-containing genes have been found and most of them are known to regulate developmental processes (Schier and Gehring,
1993). Bicoid (bed) is a maternal gene that is crucial for the development of head and thorax and the gradient of bed protein seems to determine anterior-posterior polarity of the embryo (Driever and Ntlsslein-Volhard, 1989). Antp is a homeotic gene that controls antenna differentiation during Drosophila embryo development (Gehring, 1987). Fushi tarazu iftz) is a segmentation gene. An embryo homozygous forftz a mutation develops with half the normal number of body segments (Scott and Weiner, 1984). Some homeobox-containing genes found in C. elegans have been shown to regulate cell specification. The mec-3 gene controls differentiation of touch cells (Way and Chalfie, 55
1988) and mab-5 controls the development of an array of cells that form structures
characteristic of the posterior of the worm (Costaet al., 1988). In mutants lacking mec-3
activity, cells have normal patterns of division but have no characteristics of the touch
cells because of abnormal differentiation. Without mab-5 function, the posterior cells
develop in anterior patterns. In yeast, some homeobox genes such asMATo.2 and MATslX
control mating type cell differentiation.
Some homeobox genes control processes other than development.P H 02 in yeast
is a transcription activator of an acid phosphatase and is apparently involved in regulating
cell physiology rather than cell fate (Burglin, 1988).Zen, a homeobox gene required for
differentiation of the dorsal structure ofDrosophila embryo (Doyleet al ., 1989), was
found to repress the expressions of both DNA polymerase a and proliferating cell nuclear antigen genes, and inhibits DNA replication and therefore, cell proliferation (Hirose et al., 1994). It seems that homeobox genes may not be restricted to development and cell differentiation control.
Homeodomain proteins generally function as transcriptional factors regulating the expression of target genes. Their effects on transcription can be either positive or negative. The transcriptional function has been shown directly and indirectly. The first clue to homeodomain protein function came from comparison of their protein sequences with the proteins encoded by the yeast mating-type lociMATo.2 and MdTal (Shepherd et al., 1984) and some prokaryotic DNA binding proteins such as bacteriophageX repressor and bacterial trp repressor (Laughon and Scoot, 1984). SinceMAT cl2 and the prokaryotic
DNA binding proteins were shown to regulate transcription (Pabo and Sauer, 1984), the homologies between them suggested that homeodomain proteins may have the same function. Immunolocalization studies indicate that homeodomain proteins accumulate in the nuclei (Scott et al., 1989, Kessel et al., 1987), This subceilular location is consistent 56 with their roles as transcription factors. NMR spectroscopy has demonstrated the helix- tum-helix structure of the homeodomain (Qianet al ., 1989, Ottig et al., 1990). The crystal structure of homeodomain-DNA complexes suggested interactions between helices of homeodomain and target DNA (Wolbergeret al., 1991, Kissinger et al., 1990).
About 30% of amino acids within the homeodomain are basic indicating a possible association between the homeodomain and DNA (Scottet al., 1989). Additional evidence came from in vitro DNA binding studies (Desplan et al., 1988) which have shown that some isolated homeodomain proteins bind to specific DNA sequences and the sequence
TAAT is often included in the binding sites. More direct evidence is that homeodomain proteins can regulate transcriptionin vitro (Biggin and Tjian, 1989) and in vivo (Krasnow et al., 1989). When the Ubx gene was introduced into Drosophila culture cells along with potential target promoters linked to a reporter gene, the Ubx protein repressed expression of the reporter gene linked toAntp promoter and activated the reporter gene linked to Ubx gene promoter (Krasnowet al., 1989). Studies on ftz protein demonstrated that ftz protein directly interacts with ftz autoregulatory enhancers and enhances the expression of its own gene via an autocatalytic positive-feedback loop (Schier and Gehring, 1992). This suggested that high-specificity, high affinity homeodomain-DNA interaction and multiple interactions with other auxiliary factors contribute to the functional specificity of homeodomain proteins (Schier and Gehring, 1993). Some gene products were first identified as transcription factors that bound to cis-acting elements of target genes and later were found to contain homeodomains such as Pit-1 in rat (Ingrahamet al., 1988) and
Oct-2 in human (Ko et al., 1988).
Analysis of the regulatory pathway of homeodomain protein synthesis and characterization of the downstream molecules regulated by the homeodomain proteins is also critical in understanding development (Edelman and Jones, 1993). Research on 57
expression of Drosophila and vertebrate homeobox genes showed that these genes
autoregulate and cross-regulate each other (Bienz and Tremml, 1988, Kuziora and
McGinnis, 1988, Zappavigna et al ., 1991). Growth factors (e.g. retinoic acid) can activate or repress the expression of homeobox genes (Simeoneet al., 1990).
In vertebrates, downstream target genes for the homeodomain proteins have been identified (Edelman and Jones, 1993). They are cell adhesion molecules (CAM) and substrate adhesion molecules (SAM) as well as various growth factors. These molecules affect morphogenetic movements and formation of cell collectives which are fundamental in vertebrate development. Cotransfection of a homeobox gene with a reporter gene driven by a CAM promoter showed that the homeobox gene controlled CAM promoter activity (Jones et at ., 1992a, 1993). A 47-bp DNA segment in this CAM promoter contains two potential homeodomain binding sites which are sufficient for promoter modulation by certain homeodomain proteins. Mutations in these homeodomain binding sites abolished these effects (Jones et al., 1992b). Furthermore, Jones et al. (1993) showed that homeodomain proteins bind to the target site in a DNA sequence-specific manners. For example, a homeodomain encoded by the Hox C6 gene (Joneset al., 1993) was bound to a specific TAAT containing sequence CCTAATTATTAA in one CAM promoter. On the basis of these results, a morphoregulatory control loop was hypothesized (Edelman and Jones, 1993). In vertebrates, cells at a particular place in the embryos adopt shapes, movement, and proliferation patterns based on location-dependent integration of three kinds of signaling components, growth factors, homeodomain proteins, and CAMs and SAMs. During development, the activities of CAM and SAM are regulated by homeodomain proteins in precise patterns. Homeodomain proteins may affect responses to appropriate growth factors as well. The activities of CAM and SAM 58
bring cells together which, in turn, alter the subsequent expression of a different mix of
adhesion molecules, growth factors and homeobox genes in a new place.
Since the isolation ofknotted-1 (K n l), the first homeobox gene from plants (Hake et a l, 1989), more than a dozen different homeobox genes have been isolated from maize
(Vollbrecht et a l, 1991, Bellman and Wer, 1992), Homeobox genes have also identified from other plant species such asArabidopsis (Schindler et al., 1993, Carabelli et al.,
1993, Schena and Davis, 1992, Ruberti et al., 1991, Mattsson et a l, 1992), rice
(Matsuoka et a l, 1993), tomato (Vollbrechtet al., 1993), and tobacco (Feng and Kung,
1994). On the basis of the structural characteristics, these isolated plant homeodomain proteins can be divided into three groups. The first group consists of most maize homeodomain proteins (Vollbrechtet al., 1993) and is characterized by the presence of the ELK (glutamic acid, leucine, and lysine) region, adjacent to the amino side of the homeodomain. Although the function of this region is unclear, the ELK domain could form a amphipathic a helix and facilitate protein-protein interactions. The second group is represented by most Arabidopsis homeodomain proteins (Carabelliet a l, 1993, Schena and Davis, 1992, Ruberti et al. , 1991, Mattsson et a l, 1992). This group is characterized by the presence of leucine zipper motif located immediately at the C-terminal side of homeodomain. Leucine zipper motifs are found in some regulatory proteins of both plants and animals and are involved in dimerization which is associated with recognition of
DNA (Landschultz et a l, 1988, Sessa et a l, 1993). The isolation and structure analysis of a new Arabidopsis homeobox gene, HAT3.1 (Schindler et a l, 1993) and comparison with a maize homeobox gene, Zmhoxla (Bellman and Wer, 1992), has revealed yet another group of homeodomain proteins in plants. This group is characterized by the presence of a new protein motif called PHD-fmger (plant homeodomain-finger). This motif is located 59 at the N-terminal side of homeodomain proteins and may play an important role in protein-protein and protein-DNA interactions (Schindleret al., 1993).
The functions of all homeobox genes isolated from plants are unknown.
Mutations in K nl result in a "knotted" phenotype. Based on this mutant phenotype and by comparison with homeobox genes in animals, this gene is believed to serve as a control point in cell specification (Chasan, 1992, Hakeet al., 1992, Vollbrecht et al, 1991). Knl may direct cells to stay or become meristematic. IfK nl is ectopically expressed in cells of a differentiated tissue, these cells wilt undergo meristematic growth relative to neighboring celts resulting in knot formation. Overexpression ofK nl in transgenic tobacco provides additional clues to the function of this homeobox gene (Sinhaet al.,
1993). K nl -transformed tobacco exhibited various phenotypes. Plants with low level of
KN1 showed puckered leaves whereas those with high level of KN1 formed ectopic shoots on the leaf surface. The homeodomain protein encoded by Zmhoxla recognizes sequences close to the transcription start site ofShrunken promoter and may mediate the transcription feedback control of the Shrunken gene (Bellman and Wer, 1992). The expression pattern and the in vitro binding assay of homeobox gene HAT3.1 indicates that it may be involved in Arabidopsis root development (Schindler et al., 1993).
Although homeobox genes have been shown to play important roles in embryo development in many organisms, especially inDrosophila, and homeobox genes have been isolated from plants, there are no reports of their involvement in plant embryogenesis. Considering the conservation of homeodomain proteins in many organisms and the general functions they play inDrosophila embryogenesis, homeobox genes should play important roles in plant embryogenesis.
Using K nl cDNA as a heterologous probe, we isolated a homeobox-containing gene (Sbhl) from soybean somatic embryos. It is the first homeobox cDNA isolated from 60 plant embryos and the first one from soybean. Several different soybean homeobox cDNAs were subsequently isolated using Sbhl as a probe. The isolation of these homeobox cDNAs has proved the expression of homeobox genes in plant embryos and provided an entry point for the isolation of other homeobox genes from embryos of other plant species. Further studies on these homeobox genes may provide important information on the molecular mechanisms underlying plant embryo development.
MATERIALS AND METHODS cDNA Library Screening
The cDNA library constructed from early staged somatic embryos of soybean was used to screen for homeobox-containing genes.E coli NM514 was infected with phage and plated on 100 mm plates at density of 500 pfu/plate. Three replica filters were prepared from each plate (Benton and Davis, 1977) [Appendix 3], After baking under vacuum for 2 hours at 80°C, the filters were prehybridized in 5x SSC, 5x Denhardts,
0.1% SDS, and 100 pg ml'* denatured salmon sperm DNA at 55°C for 6 hours.
Hybridization was performed for 2 days using the same solution as the prehybridization plus probe. The first soybean homeobox-containing gene(Sbhl) was identified using K nl cDNA (Vollbrecht et al,, 1991, kindly provided by S. Hake, USDA-ARS, Albany, CA) as a probe prepared by random priming (Feinberg and Vogelstein, 1983) [Appendix 4].
Replica filters were washed with either 4x, 2x, lx SSC plus 0.1% SDS at 55°C. After autoradiography, plaques hybridizing to theK nl cDNA probe were further purified by two rounds of plaque purification. Other homeobox-containing genes were identified using the whole cDNA, the 5’-region, and the homeobox region ofSbhl as probes. The conditions used to isolate these clones were essentially the same as those used forSbhl isolation. 61 Subcioning and Sequencing
The phage DNA containing Sbhl was digested with HindlU and Bglll to release
the intact cDNA fragment. The fragment was purified using Geneclean (BIO 101, La
Jolla, CA) and subcloned into pBIuscript ICS- and SK- in both orientations. Ordered
deletions were created using exonuclease 111 as described by Sambrook et al. (1989).
Single strand DNAs were produced (Vieira and Messing, 1987) [Appendix 6] and used
for sequencing with the dideoxy chain termination method (Sambrook et al., 1989).
Sequence data was analyzed using InteMGenetics (Mountain View, CA) sequence
analysis programs and NCBI BLAST E-Mail Server.
The phage DNAs from other homeobox-containing clones were digested with
either fcoRI orSmal. The released cDNA fragments were subcloned into pBluescript
SK+.
Southern Hybridization Analysis
Genomic DNA was isolated from leaf tissue of soybean cv."Fayette" according to
Saghai-Maroof et al. (1984) [Appendix 7]. DNAs were digested with appropriate
restriction enzymes, electrophoresed, and transferred onto GeneScreenPlus membranes
using the "dry blot" method (Kempter et al., 1991) [Appendix 12], The membranes were
prehybridized six hours in 5x SSC, 5x Denhardts, 0.1% SDS, 50 mM Tris (pH 8.0), 10
mM EDTA, and 100 pg ml"^ denatured salmon sperm DNA and then hybridized for 2 days with random primer labelled (Feinberg and Vogelstein, 1983) [Appendix 8] Sbhl cDNA in the above solution plus 10% dextran sulfate. Membranes were washed either at
low stringency (lx SSC, 0.1% SDS, 65°C) or at high stringency (O.lx SSC, 0.1% SDS,
65°C). For visualization of hybridization bands, the membranes were exposed to Kodak film with intensifying screens for 2-3 days. 62 RESULTS AND DISCUSSION
Isolation o f Sbhl, the First Soybean Homeobox-Containing Gene
Approximately 6,000 plaques from the proliferating soybean somatic embryo cDNA library were screened using the maize K nl cDNA (Vollbrecht et al., 1991) as a probe. Ten plaques initially appeared to hybridize to K nl cDNA. From these 10 clones, one clone, which gave strong signals on three replica filters washed at different stringencies (4x, 2x, lx SSC, with 0.1% SDS) was selected for further characterization.
We designated the cDNA clone Sbhl for soybean homeobox-containing gene 1. The other nine original clones were discarded due to the lack of strong hybridization in subsequent screening with K nl cDNA.
Structures o f Sbhl Clone and Deduced Protein
The DNA sequence of the Sbhl cDNA is 1515 base pairs long (Figure 2.1). The predicted SBH1 protein is 379 amino acids long with a translation start codon AUG at position 156 and stop codon UAG at nucleotide 1293. The overall amino acid identity between SBH1 and KN1 is 47.0% (87.5% identity with KN1 homeodomain) (Figure 2.2).
The SBH1 homeodomain, located near the carboxyl terminus, is 64 amino acids long and contains the four invariant residues in the recognition helix (helix III) and six of the eight highly conserved residues or similar substitutions (Figure 2.3). Although a high sequence similarity was observed between the homeodomain of SBH1 and homeodomains of three maize proteins (KN1, ZMHI, ZMH2), the homology between the homeodomain of SBH1 and other homeodomain proteins was limited to the most conserved residues (Figure 2.3).
The ELK region found in some plant homeodomain proteins (Vollbrechtet a l,
1993) is also present in the SBH1 protein (Figure 2.2). The "invariant" glutamic acid is replaced by an aspartic acid and the periodicity of the hydrophobic residues is conserved.
The PYP (Proline, Tyrosine, Proline) sequence between helices 1 and 2, conserved in all 63 five maize homeodomain proteins (Bellmann and Werr, 1992) is also present in SBH1 protein (Figure 2.3). An acidic region, characteristic of transcription activating domains
(Ptashne, 1988), is present at the amino side of the SBH1 homeodomain (Figure 2.1).
This region includes 110 amino acids with a net charge of -15. A leucine zipper region that is found in certain homeobox-containing genes fromArabidopsis thaliana (Mattsson et al., 1992, Ruberti et al., 1991, Schena and Davis, 1992) is not present in either SBH1 or KN1 proteins.
Although high homology is shown between SBH1 and KN1 proteins, several unique features are present in Sbhl cDNA and SBH1 protein. There is a longer leader sequence prior to the translation start site of the predicted SBH1 protein (Figure 2.1).
Within this leader sequence, there is a small ORF (Open Reading Frame) from position
108-141 which potentially encodes the peptide MCVCVCVCVCC. Comparison of this sequence with the Protein Database did not reveal similarity with any other leader peptides. We have not yet determined whether this leader peptide is present in vivo. If it is present, it may be involved in translational control similar to other leader peptides of some regulatory proteins such as the three ORFs inOpaque-2 of maize (Lomeret al.,
1993) and the four short ORFs in GCN-4 of yeast (Hinnebusch, 1988).
Although most eukaryotic proteins initiate at the first AUG, about 5-10% deviation exists from the "first-AUG rule" (Kozak, 1987, Kozak, 1989). In the case of
SBH1, the predicted protein starts at the second AUG instead of the first (Figure 2.1).
When comparing the sequence context of both the first AUG (CAGUGAAUGU1 and the second AUG fAAAGCUAUGG) with the consensus sequence for initiation in higher eukaryotes GCCfA/GlCCAUGG. the purine in position -3 and G in position +4 are present in the second AUG context indicating that the second AUG is more favorable for protein initiation. 64 Sbhl Is a Member o f a Small Gene Family
When DNA of soybean was digested with several restriction enzymes, the recognition sequences of which were not present in theS b h l cDNA sequence, and hybridized with Sbhl cDNA, 2-3 fragments were clearly detected (Figure 2.4). The size and intensity of the fragments suggest that more than one copy is present in the soybean genome. This is supported by the isolation of other homeobox-containing genes from the same cDNA library using the homeobox region fromSbhl as a probe (see next section).
At least 2 clones are different from Sbhl based on restriction patterns and hybridization patterns.
Identification o f other Hameobox-Containing Genes
Another screening of the proliferating soybean somatic embryo cDNA library was performed using the whole sequence, the homeobox region, and the 5’ region ofSbhl cDNA as probes. After screening of approximately 25,000 plaques, twelve additional clones were isolated. After subsequent purifications, seven clones were selected for further study. In order to determine if these clones are different fromSbhl and from each other, the phage DNAs were initially digested with £coRI orSmal to release the intact cDNAs. The released cDNAs were subcloned into pBluescript SK+. The phage DNAs and the plasmid DNAs were digested with appropriate restriction enzymes and used for
Southern hybridization analysis (Figure 2.5). Southern hybridization analysis showed that the Cl clone hybridized to the homeobox region but not to the 5'-region ofSbh l. This indicates that Cl contains a homeobox region and is distinct from Sbhl. No signal was detected when Cl was hybridized with the whole cDNA sequence of Sbh l. This membrane was washed at high stringency (65°C) while the membranes that were used for hybridization with the homeobox- and the 5'-region ofSb h l were washed at lower stringency (55°C). Since the size of Cl is similar to that ofSbhl and a much weaker 65
hybridization signal was observed when Cl clone was hybridized to Sbhl compared to
itself (Figure 2.5B lane 1 vs lane 10), we concluded that Cl clone is a new member of the
soybean homeobox gene family. When Cl clone was used as a probe to hybridize to other
clones, no cross-hybridizations were detected after a high stringency wash, except for a
weak hybridization signal with C5 (data not shown). This indicated that Cl clone is
different from the others homeobox clones.
Hybridization of C2 clone with the wholeSbhl cDNA gave a weaker signal
compared to the homeobox region ofS b h l> indicating that C2 has a higher homology
with Sbhl within but not outside of the homeobox region. It may represent another
member of the homeobox gene family. Since C2 is only about 600 bp, it may be a
truncated clone.
Hybridization of C5 with the whole Sbhl cDNA and the homeobox region of
Sbhl gave similar signal intensities to that ofSbhl hybridized to itself. Since it is smaller
than Sbhl cDNA (about 800 bp), C5 clone may be a truncated Sbhl clone.
When C6 and C9 clones were probed with the Sbhl whole cDNA, the 5' region,
and the homeobox, the hybridization signals were similar to that ofSbhl hybridized to
itself indicating that C6 and C9 may be either full length clones of Sbhl or have very high
homologies withSbhl. C6 and C9 did not show hybridization with Sbhl homeobox
region (Figure 2.5B lane 7 and 8). This is because the small £coRI fragments of the 3'-
region including the homeobox region of C6 and C9 were not subcloned into pBluescript
SK+. Subsequent hybridization of C6 and C9 phage DNAs with Sbhl homeobox region
showed strong hybridization signals (data not shown). C6 and C9 are similar in size
(about 1.9 kb), and display similar hybridization patterns and signal intensity when hybridized with the three probes. Therefore, C6 and C9 may represent the same clone. 66
When C6 was hybridized with other clones (data not shown), no cross-hybridizations were detected between C6, C l, and C2 indicating C6 is different from Cl and C2.
CIO has the same size and shows the same restriction pattern and hybridization signal intensity as Sbhl. Therefore, CIO is probably another copy ofS b h l. C4 did not hybridize with either probe in this Southern hybridization analyses and may be a false positive clone from filter hybridization.
In summary, at least 3 members of homeobox-containing genes were isolated from soybean somatic embryos. The isolation of these clones will provide an entry point for the isolation of regulatory genes involved in plant development. The presence of these clones in early staged somatic embryos indicates that they may act coordinately and/or independently to control soybean embryogenesis and plant development. Further studies on these clones will lead us to a better understanding of the molecular mechanisms underlying plant embryogenesis. Figure 2.1. Nucleotide acid sequence of Sbhl and deduced amino-acid sequence of SBHl. The homeobox and homeodomain are boxed. Putative polyadenylation signals are underlined. The suggested acidic region is noted by double underline. The nucleotide acid sequence which encodes a possible short leader peptide is bolded.
67 AATAAGAGAATTGTGTGTCGTGTTTGTTTTTGTTTGGTTTGTTGTAAGGTTAGCTAGTG 59 AGTATTCTACACAAGGGTGGTGGTAGGGCAAAAAGGATAAGACAGTGAATOTOTOTGTOT 119 G TGT OT GTGT Q TOT QT OTT QTTOACAAG CAAAAG CTAT GGAGGG TGG TAGTAG TAG CT CT 17 9 MEGGSSSS 8 AAT GG CACTT CTTAT CTG TTGG CTTTTGGAGAAAA CAACAGTGG TGGG CTATG C C CAA T G 2 3 9 NGTSYLLAFGENNSGGLCPM 28 ACGATGAT G C CTTTGG TGA CTT C C CAT CA CG CT GGT CATCAT C CAATAAAT CCTAG TAAT 2 99 TMMPLVTSHHAGHHP INPSN 48 AATAATAATGTAAACACAAACTGTCTCTTCATTCCCAACTGCAGTAACAGTACTGGAACT 3 59 NNNVNTNCLFI PNCSNSTGT 6 8 CCTTCTATCATGCTCCACAATAATCACAACAACAACAAAACTGATGATGATGATAACAAC 419 PSIMLHNMHNNNKTDDDDNN 8 8 AACAACACTGGGTTAGGGTACTATTTCATGGAGAGTGACCACCACCACCATCACCACGGC 479 NNTGLGYYFMESDHHHHHHG 108 AAGAACAACAACAATGGAAGCTCCTCCTCCTCCTCCTCTTCTGCTGTCAAGGCCAAGATC 53 9 NNNNNG9SSSSSSSAVKAK I 128 ATGGCTCATCCTCACTATCACCGTCTCTTGGCAGCTTACGTCAATTGTCAGAAGGTTGGG 59 9 MAHPHYHRLLAAYVNCQKVG 148 GCCCCGCCTGAAGTGGTGGCAAGGTTAGAAGAAGCATGTGCTTCTGCAGCGACAATGGCT 6 5 9 A P P EVVARLEEA C___ A--S— A . A T M A 168 GG TGG TGATG CAGCAG CTCX3AT C AAG CT G CAT AGG TGAAGAT C CAG CTTTG GAT CAG T T C 7 1 9 GGPAAAGSS._CIGEDPALDOF 188 ATGGAGGCTTACTGTGAGATGCTCACAAAGTATGAGCAAGAACTCTCCAAACCCTTAAAG 77 9 M___E A Y C EM LTKY E Q E L S K P LK 208 GAAGCCATGCTCTTCCTTCAAAGGATCGAGTGCCAGTTCAAAAATCTTACAATTTCTTCC 83 9 EAMLFLQR I.ECQFKHLT ISS 228 TCCGACTTTGCTAGCAATGAGGGTGGTGATAGGAATGGATCGTCTGAAGAGGATGTTGAT 8 99 SPFASHEGgPRNGSBEEDVD 2 4 8 CT A CACAACAT GAT AG AT C CC CAGG CAGAGGACAGGGATTTAAAGGGT CAG CTT’TT G CG C 9 59 L HHMIDPOAEDRD L K G Q L L R 268 AAGTATAGCGGATACTTGGGCAGTCTGAAGCAAGAATTC ATGAAGAAGAGGAAGAAAGGA 1 0 1 9 KYSGYLGSLKQEF 4 K K R K K G 288 AAGCTACCTAAAGAAGCAAGGCAACAATTACTTGAATGGTGGAACAGACATTACAAATGG 1 0 7 9 KLPKEARQQLLEWWNRHYKW 308 CCTTACCCATCCGAATCCCAGAAGCTGGCTCTTGCAGAGTCGACAGGTCTGGATCAGAAG 1 1 3 9 PYPSESQKLALAESTGL D Q K 328 CAAATCAACAACTGGTTTATTAATCAAAGGAAACGGCACTGGAAGCCTTC7 OAGGACATG 1199 QXHNHF.IHQRKRHW KPS E D M 348 CAGTTTGTGGTGATGGATCCAAGCCATCCACACTATTACATGGATAATGTTCTAGGCAAT 1259 QFVVMDPSHPHYYMDNVLGN 3 6 8 CCATTTC CCATGGATCTTTCCCATCCCATGCTCTAGAAAATTATCCCTCGTTTGTGGGCT 1319 PFPMDESHPML* 3 7 9 GCTGATAATAGATTCATAAACTCGTGCTGTCACTTATTAAAACCTTACAATTATTAATAT 1379 TAATTAATATGCATTCTAAGAAATCCTAGATTGCTATACTATAATATAGTACGCAGGTGT 1439 A T C C CTTG CTAG CTTTTTAGA CGGT C CTTTGT□TGGATCATCTAGTTGAAGGAGTTATGA 14 99 ATAAATAAAATTCCAT 1515
Figure 2.1 69
MEGGSSSSNGTSYLLA.FGENNSGGLCPMTMMPLVTSHHAGHHPINPSNNNNV1ITNCLFIPNCSNSTGTPS II I II III I I I I I I MEEITQHFG-- -VGASSHGHGHGQHHHHHHHHHPWASSLSAW APLPPQPPSAGLP-
IMLHNNHNNNKTDDDDNNNNTGLG YYFME S DHHHHHHGNNNJJNGS S S S S S SSAVKAKIMAHPHYH II I III INI I INI INI - -LTLN TVAATGN-SGGSGNPVLQLANGGGLDDACVKAKEPSSSSPYAGDVEAIRAKIISHPHYY
RLLAAYVNCQKVGAPPEWARL- EEACASAA- - - TMAGGDAAAGSSCIGEDPALDQFMEAYCEMLTKYEQ I I I I I I 111 I I I I III I I I I II III I IIIMIII III I S L LT AYL E CNKVGAP PEVS ARLTEIAQ EVEARQRTALGGLAAA------TEPELDQFMEAYHEMLVKFRE
ELSKPLKEAMLFLQRIECQFKNLTIS S SDFASNEGGDRNGS SEED ------VDLHNMIDPQAED ii ii 111 i i i i 111 i mill mi i ELTRPLQEAMEFMRRVESQLNSLS IS GRSLRNILS SGSSEEDQEGSGGETELPEVDAH ------GVD
RDLKGQLLRKYSGYLGSLKQEFMKKRKKGKLPKEARQQLLEWWNRHYKWPYPSESQKLALAESTGLDQKQ ii ii m m him ii miimmm 11 imimi 11 m m m 11 OELKHHLLKKY S GYLS S IiKQ ELS KKKKKG KL PKEAROOL LS WWDOHYKWP Y PS ETOKVALAE S TGLDLKO
INNWFINQRKRHWKPSEDMQFWMDPSH- - PHYYMDNVLGNPFPMDLSHPML Mmm iimmi i ii i m i i i INNWFINQRKRHWKP S EEMHHI jMMDG YHTTNAFYMD ------GHFINDGGLYRL
Figure 2.2. The comparison between the predicted SBH1 protein (top line) and K.N1 (bottom line). The residues identical to those of SB HI are indicated by hyphens and the ELK region is underlined. The conserved and hydrophobic residues in the ELK region are in bold type. 70
HflliX I______H elix _ II_tum Helix III S b h l MKKRKKGKLPKEARQQLLEWWNRHYKWPYPSESQKLALAESTGLDQKQINNWFINQRKRHWKPS G l y c i n e Knl S -- K ...... S --D Q ------T--V ------L ------Z e a Zmhl LR--RA------GDTTSI-KQ--QE-S------T-DD-AK-V-E------QL------N-HNN Z e a Zmh2 LR--R A ------GDTA5T-KA--QA-S------T-ED-AR-VQE - QL N-HNN Z ea Z m hoxla NSTARKG H FG PVINQKLHEHFKTQ R -V -E S EL- -TFR-V-K- -ETR-HSARVA- Z e a Zmhoxlb NI-DRKGHFGPVISQKLHEHFKTQ R-L-ES EL--TFH-V-R--E-R-HFARLA- Z e a Athbl QLPE-KRRLTTE-VHLLEKSFETENKLEPER-TQ--KKL--QPR-VAV--Q-R-A-WKTKQ Arabidopsis Athb2 DNSR-KLRLSKD-SAILEETFKDHSTLNPK--Q ------SQL--RAR-VEV--Q-R-A-TKLKQ A rabidopsis Athb3 MLGE-KKRLNLE-VRALEKSFELGNKLEPER-MQ--KAL--QPR--AT - -Q-R-A-WKTKQ A rabidopsis HAT4 NSR-KLRLSKD-SAILEETFKDHSTLNPK--Q-- -KQL--RAR-VEV--Q-R-A-TKLKQ A rabidopsis HAT5 LPE-KRRLTTE-VHLLEKSFETENKLEPER-TQ--KKL--QPR-VAV--Q-R-A-WKTKQ A rabldopsis PR1 AR - - RRNFN-Q-TEI -N-YFYS- LSN...... EA-EE- - KKC- ITVS-VS G-K-I-YK-NI H omo PBX2 AR- -FRNFS-Q-TEV-N-YFYS-LSN------EA-EE--KKC-ITVS-VS G-K-I-YK-NI H omo MATPI MTTVR- QCS- CTKPH-MR- LLL- - DN------N- EFYD-SAATG- TRTQLRN- - S - R- R Saccharomyces MATO 2 T-PYRGHRFT--NVRI-ES-FAKNIEN-- LDTKGLEN-MKN-S-SRI- -K --VS-R-RKEKTIT Saccharonsyces CEH-5 P--PRTDNAD-QLEK-E-SF-...TSG-L-G-TRAK L--SDN-VKV--Q-R-TKQK- ID Caenorhabditis Antp ER--GRQTYTRYQTLE-EKEF.. , HFNR-LTRRRRIEl-HALC-TER- -KI--Q -R- MKWK-EN D r o s o p h i l a Enl ED- -PRTAFTA-QL-R-KAEF, . . QANR-ITEQRRQT--QELS-NES--KI--Q-K-AKIK-AT H u s
t ■ « • ■ * ♦ ♦ ♦ ■ • Consensus R Q L F LI/VWF N R K K
Figure 2.3. The amino acid sequence of SBH1 homeodomain aligned with several homeodomains from other organisms. The residues identical to those of SBH1 are indicated by hyphens. The helix-tum-helix structure is positioned according to the motif of KN1. The 4 invariant residues in the recognition helix are marked with an asterisk (*) and the eight highly conserved residues are marked with a period (■). 71
B* Bt C E H k b
t ill!
Figure 2.4. Southern hybridization analysis of soybean DNA using Sbhl cDNA as a probe. Ten |ig soybean leaf DNA were digested with Bgl II (Bg), Bst Eli (Bs), Cla I (C), EcoRV (E), and Hind III (H). The membrane was washed at high stringency (O.lx SSC, 65°C). 72
Sbhl cDNA Sbhl Hox Sbhl 5’
o _c Or o _c: OOOOOOOWOOOOOUOOOCOOOOOOOOOOCO
Figure 2.5. Hybridization of the selected clones with the whole cDNA(Sbhl cDNA), the homeobox region(Sbhl Hox), and the 5’-region (Sbhl 5r) o fSbhl. Phage DNAs were used for the first gel, plasmid DNAs for the second and third gels. fcoRJ was used for all digestions except Smal for C 10 and Sbhl on the 3rd gel. The first membrane was washed at 65°C and 2nd and 3rd at 55°C with O.lx SSC and 0.1% SDS. CHAPTER III
Temporal and Spatial ExpressionsS bhof l during Plant Development
ABSTRACT
In order to elucidate the potential function ofSbhl in plant development,
especially in embryogenesis, its expression patterns were analyzed using northern andin situ hybridizations. Northern hybridization analysis indicates that the expression ofSbhl
is developmentally regulated. The transcript of Sbhl was present in the early staged
somatic embryos, increased as embryos developed, and then decreased by the time that embryos developed to the cotyledon stage. The highest expression occurred at the globular-heart transition stage during which the root and shoot meristems and provascular tissues are formed. Sbhl was also expressed in an organ-specific manner. Sbhl was expressed at low levels in the young stem and hypocotyl while noSbhl mRNA was detectable in leaf, root, and nonembryogenic cultures of soybean.
In situ hybridization showed that Sbhl was expressed in a tissue-specific manner during both zygotic and somatic embryo development.Sbhl was expressed in all stages of both somatic and zygotic embryos but in some what differential locations.Sbhl mRNA was evenly distributed in globular-staged embryos. At the heart-shaped stage, the transcript ofSbhl was present at higher levels in the shoot meristem and procambium, the region forming vascular tissues. In the cotyledon staged embryos, the expression ofSbhl was restricted to the shoot meristem and provascular tissues. Sbhl transcript was not
73 74
detectable in the root meristem, cotyledons, ground tissue, and epidermis of the embryos
developed beyond the globular stage. No Sbhl mRNA was found in primary leaves and
cells within well-developed vascular tissue of soybean zygotic embryos. Hybridization
signal was not observed in the suspensor. The Sbhl mRNA was also not present in
nonembryogenic tissues of soybean seed such as the seed coat and endosperm, indicating
that Sbhl was expressed specifically in embryogenic tissues during soybean embryo
development.
In situ hybridization analysis also showed thatSbhl was expressed in the shoot
apical meristem and axillary buds of soybean plants. The Sbhl mRNA distribution pattern and intensity within these regions were very simitar to that observed in globular staged zygotic embryos.Sbhl mRNA was present uniformly throughout the shoot apical meristem. The expression levels of Sbhl decreased sharply in the ground tissue just
below the apical meristem. Sbhl mRNA was also present at high levels in the newly formed procambium located just below the shoot apical meristem. As the intemode elongated, the levels of Sbhl expression in the provascular tissue decreased. In mature stems, Sbhl was expressed in strands along the vascular tissue. Sbhl was not expressed in mature stem containing well-differentiated vascular tissue. Sbhl transcript was also detected in the basal tissue of the flower. There were no signals detectable in young leaf primordia, older leaves and in the root apical meristem. These results indicate that Sbhl is only expressed in cells remaining at an undifferentiated state and not after the fates of cells are determined. The absence of Sbhl mRNA in the root apical meristem indicates that the expression of Sbhl distinguishes the root and shoot apical meristems that give rise to two morphologically different parts of the plants.
In summary, Sbhl was expressed in somatic and zygotic embryos and in the shoot apical meristem in similar patterns. Sbhl transcript is specifically localized in some 75 undifferentiated regions and not within cells with determined fates. Therefore, Sbhl may function in keeping cells in an undifferentiated state. The presence of Sbhl transcript in rapidly dividing cells of the embryos but not in nonembryogenic calli that consist of rapid and unorganized dividing cells indicates that its product does not act purely as a cell division factor and that Sbhl may be involved in either programmed cell division or cell differentiation or both. The similar expression patterns ofSbhl in both zygotic and somatic embryos of soybean indicates that it plays an important role in plant embryogenesis and that plant somatic embryogenesis can be used as a model for embryogenesis studies.
INTRODUCTION
Homeobox genes were first discovered in the fruit fly {Drosophila melanogaster).
In Drosophila, homeobox genes are expressed in spatial and temporal patterns during development, especially, in an anteroposterior order, with maximal expression in the segments that they specify (Gehring and Hiromi, 1986). The order ofDrosophila homeobox genes along the chromosome corresponds to their sites of expression along the body of the fly. In some higher eukaryotic organisms such as mice and human, homeobox genes have also been found to be arranged in clusters along the chromosome. As in
Drosophila, there is a correlation between the location of gene expression in an organism and their order along the chromosome (Holland and Hogan, 1987). Vertebrate homeobox genes are expressed in a co-linear fashion; that is, during development, each gene is sequentially expressed in an order consistent with its position within the cluster on the chromosome. The gene expressed earliest is located at the 3'-end of the cluster and gene expression progresses toward the 5'-rcgion of the cluster as the organism develops. Genes expressed earlier yield more posterior structures, and later expressing genes yield more 76 anterior structures (Graham et ai., 1989). In many cases, the distinct spatial and temporal
expression patterns of homeobox genes are consistent with their roles in pattern formation
in higher eukaryotes (Scottet ai., 1989). The regional expression of these genes is correlated with tissue function and physiology (Edelman and Jones, 1993).
During the past few years, more than a dozen different homeobox genes have been isolated from several plant species. This has been facilitated through genetic studies and homologies with known homeobox genes. In the genetic studies, the biological functions of the isolated genes can be analyzed by the phenotype of homeobox gene mutants. The elucidation of the function of isolated genes through heterologous probing stilt remains a challenging task. A high overall amino acid sequence homology with known regulatory proteins usually indicates related function. However, in most cases, the homology is restricted to certain regions such as the DNA-binding domain of homeodomain proteins. It is difficult to elucidate the specific function of the whole protein based on partial sequence homologies. Knockout- and over-expression of the target genes as well as site directed mutagenesis can be used to study the function of the isolated genes. Studies on the expression patterns provide valuable information and this is a good first step in studying gene function in plant development (Katagiri and Chua,
1993, Chasan, 1992).
In plant species, expression studies have been used in characterization of genes involved in plant development (Wilkson and Nieto, 1993). However, there are no reports on the location and arrangement of homeobox genes along the chromosome (Langdale,
1994). Most homeobox genes are expressed developmentally and spatially in tissues that they specify. In order to understand the function of a gene in development, it is important to analyze the expression pattern of the gene. 77
Molecular cloning of homeobox genes makes it possible to study their spatial and
temporal expression during plant development. Northern hybridization analysis allows
the detection of general temporal and organ-specific patterns of gene expression.In situ
hybridization has become an essential toot for studying the regulation of gene expression
in an organism or tissues. It permits the localization of specific mRNA transcripts at the
cellular level and provides sensitive detection of mRNAs that are present in a smalt
population of cells (Langdale, 1994, Sassoon and Rosenthal, 1993). For example, a weak
signal in a northern hybridization analysis could result from either a larger number of
cells transcribing at low levels or a small number of cells within a population transcribing
high mRNA levels (Sassoon and Rosenthal, 1993). Since many developmental genes
such as homeobox genes are expressed either in a minority of cells in complex tissues or
for only a brief period during development, in situ technique provides an effective tool
for expression studies of homeobox-containing genes.
In situ hybridization has been used commonly in animal systems and has been
applied to plants only in recent years (McKhann and Hirsch, 1993). In plants,in situ
hybridization has been used to analyze gene expression patterns involved during
flowering and seed development. Floricaula {Jlo) is a gene controlling the transition of
inflorescence to the floral meristem inAntirrhinum majus. The expression ofJlo in the wild type flower is restricted to sepal, petal, and carpel primordia and no signal is detected in stamen primordium (Coen et al., 1990). Flo is also expressed in sequential manner with the earliest expression in bract primordia followed by sepal, petal, and carpel primordia. This expression order corresponds to the initiation sequence of these organs. Therefore, the expression ofJlo has implications for the function of this gene in flower development. The expression ofJlo in certain primordia may be required to 78
activate genes necessary for their normal development. The absence offto expression in
stamen primordia may be necessary for normal stamen development (Coen, 1991).
With in situ hybridization analysis, the expression patterns of four different homeobox genes in maize were compared (Jackson et al., 1994). K nl was expressed in the shoot apical meristem, developing stem, and certain provascular cells associated with developing lateral vein traces in the stem. The expression pattern of KNOX8 (Knotted related homeobox &) is similar to that ofK nl but with a weaker signal, and no signal is detected in cells associated with provascular traces. The localization of RSI (Rough
Sheath 1) and KNOX3 (Knotted related homeobox 2) mRNAs is more restricted to the vegetative shoot apex and small groups of cells near the base of each leaf. RSI labels provascular cells in the stem. Detailed localization ofK nl, RSI and KNOX3 expression revealed that K nl mRNA was expressed throughout the shoot apical meristem with the exception of two groups of cells that are believed to be the site of the leaf preprimordium
(Pg). RSI and KNOX3 were expressed in the cells located between Pg and the site of insertion of P] (the youngest leaf primordium), corresponding to the incipient intemode and axillary bud. Therefore, through gene expression analysis, Jackson et al. (1994) concluded that the absence of K nl expression marks the presumed position of the incipient leaf, and RSI and KNOX3 expression indicates the location of the incipient intemode and axillary bud. The expression patterns of these three genes predict the sites of initiation of leaf, stem, and axillary bud primordia that give rise to the segmentation unit known as the phytomer of the maize shoot.
Many genes associated with plant embryogenesis have been cloned and their expression patterns have been extensively studied. Most of these studies have concentrated on genes expressed at high levels or expressed at later stages of embryogenesis. The important events of embryo development at the early stages, such as 79 pattern formation and tissue differentiation, have attracted the interest of many molecular and developmental biologists. However, the difficulty in obtaining large amounts of early staged embryos has limited expression studies using northern hybridization analysis. In this case, in situ hybridization can be used as a powerful method for studies of gene expression during the early stages of embryo development.
In order to elucidate the potential function ofSbhl in plant development, especially in embryogenesis, the expression pattern was analyzed using northern andin situ hybridizations. Northern hybridization analysis indicates that the expression ofSbhl is developmentally regulated with the highest expression occurring at the embryo transition stage. During this stage, the meristematic regions such as root and shoot meristems and provascular tissue are formed. In situ hybridization showed that Sbhl is expressed in a tissue-specific manner during plant development. It is expressed in meristematic celts such as the shoot apical meristem and provascular tissues. The similar expression patterns ofSbhl in both zygotic and somatic embryos of soybean indicate that it plays an important role in plant embryogenesis and that plant somatic embryogenesis can be used as a model for embryogenesis studies. The expression pattern obtained from both northern and in situ hybridizations together with its homology with theK nl gene suggests that Sbhl may be involved in determining the cells to remain in a meristematic and differentiation-competent state.
MATERIALS AND METHODS
Northern Hybridization Analysis
Total RNAs and poly(A+)RNAs were isolated as described in Chapter I from soybean somatic embryos developed to different stages (0, 3, 7, 28 days after development). Total RNAs were also extracted from soybean nonembryogenic cultures 80 and other plant tissues such as leaf, stem, and root. One pg of poly(A+)RNAs or 10 pg of total RNAs were glyoxalated and then used for gel electrophoresis and northern hybridization analysis as described by Thomas (1983) [Appendix 9] except a modified
1'dry blot" procedure (Kempter et a!., 1991) [Appendix 12] was used for RNA transfer.
The modified "dry blot" was performed as follows: After electrophoresis, the gel was laid on a single sheet of Whatman 3M paper saturated with 10 mM phosphate buffer (pH 6.5).
A GeneScreenPlus membrane (Du Pont) was moistened with 10 mM phosphate buffer and then placed on the top of gel. The membrane was covered with one sheet of Whatman
3M paper soaked in 10 mM phosphate buffer followed by four dry sheets of Whatman
3M paper and a 5 cm stack of paper towels. A 0.5 to 1 kg weight was placed on top of the paper towels and the transfer proceeded for 1 -2 hours. After the dry blot, the membranes were prehybridized for six hours in 3x SSC, 5x Denhardts, 0.1% SDS, 50 pg ml"l yeast tRNA (Sigma), and 50 pg m H denatured salmon sperm DNA. Transferred DNAs were then hybridized for 2 days with random primer labeled (Feinberg and Vogelstein, 1983)
[Appendix 8] Sbhl cDNA in the above solution. Following hybridization, membranes were washed at high stringency (O.lx SSC, 0.1% SDS, 65°C) and exposed using Kodak
XAR film with intensifying screens for 2-3 days.
In Situ Hybridizations
In situ hybridization was performed essentially as described by Jackson (1991 and personal communication) except that embedding, sectioning, and preparation of poly-L- lysine coated slides were following McKhann and Hirsch's protocols (1993). The procedure is described briefly as follows:
Plant Materials
Embryogenic cultures were initiated from soybean(Glycine max [L.] Merr.) cv
Chapman. For embryo development, the embryogenic tissues were transferred to a 81 hormone-free MS6M (MS salts, B5 vitamins, and 6% maltose) medium and incubated at
28°C (Finer and Nagasawa, 1988). Soybean somatic embryos were collected at various developmental stages, from globular to cotyledon. Soybean zygotic embryos were collected from Chapman plants grown in the greenhouse at different times after flowering. Soybean leaves, stems, and flowers were excised from young plants grown in the greenhouse. Shoot apical meristems and roots were taken from seeds one week after germination in Petri dishes.
Tissue Fixation. Dehydration. Wax Infiltration, and Sectioning
As soon as plant materials were collected, they were fixed in 4% paraformaldehyde in PBS (phosphate buffered saline) at 4°C overnight [Appendix 13].
Dehydration was performed using an ethanol series at 4°C with 90 minutes for each step.
Before infiltration, tissues were treated with Histoclear (National Diagnostics). The infiltration was performed in a 60°C oven with tissues immersed in melted TissuePrep
(Fisher Scientific) for 3 days. Fresh melted TissuePrep was changed twice every day, once in the morning and once in the evening. After infiltration, tissues were embedded in wax and then sectioned with a microtome to 10 pm [Appendix 14]. The sections were affixed to poly-L-lysine coated glass slides [Appendix 15].
Preparation of RNA Probes
Sbhl cDNA was subcloned into the expression vector, pBluescript SK+. The plasmid DNA was digested with appropriate restriction enzymes so that transcription would yield an intact RNA probe lacking plasmid sequences. For the sense RNA probe synthesis, SpeI was used and for the antisense RNA probe synthesis, EcoRV was used
[Appendix 16]. After purification with phenol/chloroform, the linearized plasmid DNA was suspended in TE (Tris, EDTA) buffer. Digoxygenin-labeled probes were synthesized by in vitro transcription according to manufactor’s recommendation (Boehringer 82
Mannheim). The antisense RNA probe was synthesized with T3 RNA polymerase. The sense RNA probe was synthesized with T7 RNA polymerase. After digestion of DNA templates with RNase-free DNase, the RNA probes were alkaline hydrolyzed to a final size of 150-200 bp to facilitate probe penetration.
Prehvbridization. Hybridization, and Posthvbridization Washing
Before hybridization, sections were dewaxed with Histoclear and then rehydrated through an ethanol series [Appendix 17]. Pronase (Sigma) was used to digest cellular proteins, thus rendering the target RNA more accessible to the RNA probe. Acetic anhydride was used to block the positive charge on both slides and sections, thus preventing nonspecific binding of probe. The sections were dehydrated through an ethanol series. Hybridization was performed at 55°C overnight in solution containing 0.3
M NaCl, 10 mM Tris-HCl (pH 6.8), 10 mM phosphate buffer (pH 6.8), 5 mM EDTA,
50% formamide, 10% dextran sulphate, lx Denhardts, 1 mg m l'l yeast tRNA (Sigma) and an RNA probe [Appendix 18]. The RNA probe was used at a concentration of 0.5 ng/jil/kb complexity. After hybridization, sections were washed in 0.2x SSC at 55°C and then treated with RNaseA to remove probe that had not annealed to the target RNA
[Appendix 19].
Immunological Detection
The sections were incubated with 0.5% Boehringer block in buffer A (100 mM
Tris-HCl, pH 7.5 and 150 mM NaCl) and then with buffer B (buffer A plus 1% BSA and
0.3% Triton X-100) [Appendix 19]. An alkaline phosphatase-coupled antidigoxygenin antibody (Boehringer Mannheim) with dilution of 1:1250 in buffer B was used to incubate slides for 2 hours. After washing with buffer B and then buffer C (100 mM Tris-
HCl, pH 9.5, 50 mM MgCl2, 100 mM NaCl), the substrates, nitro blue tetrazolium and 5- bromo-4-chloro-3-indolyl-phosphate, of alkaline phosphatase were included in the color 83
reaction. After 2-3 days of incubation at room temperature in the dark, the sections were
washed with TE, dehydrated through ethanol series, rinsed in Histoclear, and mounted
onto glass slides with Permount (Fisher Scientific). The sections were observed with a
light microscope.
RESULTS
Temporal and Organ-Specific Expression o f Sbhl
Soybean embryogenic suspension cultures proliferate as clumps of globular
embryos (Figure 1.1 A). After transfer to the hormone-free solid development medium,
globular embryos developed to the heart, torpedo, and cotyledon stages (Figure 1.1).
Hybridization of the Sbhl cDNA to poly(A+)RNAs isolated from soybean somatic
embryos at different stages of development detected two transcripts (Figure 3.1 A). The
larger transcript (about 1.9 kb) did not vary in intensity up to day 14 and might represent
unprocessed Sbhl transcript or transcript from a different homeobox-containing gene.
The smaller transcript (about 1.6 kb) of similar size to the Sbhl cDNA was present during the embryo proliferation stage, increased during early somatic embryo development (7 days after transfer to hormone-free medium), and decreased thereafter (14 days post- transfer). Both the larger and smaller transcripts were present at very low levels at 28 day post transfer to the development medium. Additional northern hybridization analysis using poly(A+)RNAs from another soybean embryogenic suspension culture line
indicated a similar expression pattern although the decrease in Sbhl expression at the 28 day time point was not as pronounced (data not shown).Sbhl transcript was not detected
in nonembryogenic soybean cultures, roots, and leaves but occurred at a low level in stems (Figure 3.IB) and hypocotyl (data not shown). When the same membranes were used for hybridization with soybean ubiquitin as an internal control, the intensity of 84 hybridization signal was uniform among all the lanes (Figure 3.1, low panels) indicating the signal detected by Sbhl represents Sbhl mRNA abundance at different stages of somatic embryo development and in different soybean organs.
Spatial and Temporal Expression o f Sbhl during Soybean Embryogenesis
In order to study the spatial expression ofSbhl, soybean somatic embryos at different developmental stages were collected for in situ hybridization analysis using digoxygenin-labeled riboprobes of Sbhl cDNA. The blue color shown in median longitudinal sections of somatic embryos developed from globular through the late cotyledon stage represents the hybridization signal and indicates expression of the Sbhl gene (Figure 3.2). Sbhl mRNA accumulation began early in embryo development. With in situ hybridization, we detected high expression at the globular stage, which was the earliest stage used in this study. Therefore, the start ofSbhl expression is unknown. Sbhl was expressed in all staged somatic embryos from globular to the late cotyledon stage but showed differential localization. Sbhl mRNA was evenly distributed in globular-staged embryos (Figure 3.2A). At the elongation and heart-shaped stages, the transcript ofSbhl showed higher expression levels in the shoot meristem and procambium (Figure 3.2B,C).
In embryos at the cotyledon stage, the expression ofSbhl was restricted to some meristematic regions such as the shoot meristem and provascular tissues (Figure 3.2D).
Sbhl transcript was not detected in cotyledons and ground tissue of somatic embryos
(Figure 3.2C,D). No signals were detected when sense Sbhl probe (control) was used in the hybridization (Figure 3.2E) indicating that the signal detected with antisense probe was not due to background.
If Sbhl is important in controlling embryogenesis as some of the homeobox genes in Drosophila, it should be expressed in zygotic embryos of soybean as well. In order to determine this possibility, different staged zygotic embryos were collected forin situ 85 hybridization analysis. The expression ofSbhl in zygotic embryos was very similar to that observed in somatic embryos both spatially and temporally, but was more pronounced (Figure 3.3). Sbhl transcript was present throughout all stages of embryo development from globular to late cotyledon stage. Similar to somatic embryos,Sbhl mRNA showed no preferential localization in globular embryos (Figure 3.3A, B). As the embryos developed to heart (Figure 3.3C) and early cotyledon (Figure 3.3D) stages, the preferential localization ofSbhl transcript became more evident with high expression levels in the regions forming the shoot meristem and vascular tissues. No signal was detected in the cotyledons and root meristems. By the time that the embryos developed to the cotyledon (Figure 3.3E) and late cotyledon (Figure 3.3F) stages,Sbhl mRNA showed more specific localization with expression limited to the shoot meristem and procambium. Again, no Sbhl mRNA was found in the root meristem, cotyledons, ground tissue, epidermis, young primary leaves or cells within well-developed vascular tissue, when the zygotic embryos developed beyond the globular stage. Hybridization signal of
Sbhl was never observed in the suspensor at the various stages of embryo development.
The Sbhl mRNA was not present in nonembryogenic tissues of soybean seed such as the seed coat and endosperm, indicating that Sbhl was expressed specifically in embryogenic tissues during soybean embryo development (Figure 3.3A). No signals were detected when sense Sbhl probe (control) was used in the hybridization (data not shown) again indicating that the signal observed using antisense probe represents the presence of Sbhl mRNA in vivo. The specific expression ofS b h l in provascular tissues was clearly shown by the hybridization pattern in a hypocotyl cross-section (Figure 3.3G).
Spatial Expression o f Sbhl during Plant Development
All of the vegetative organs of a plant arise from postembryogenic cell divisions and differentiation in the root and shoot apical meristems that are established during 86
embryogenesis. To determine if Sbhl is expressed postembryogenically, different plant
organs were collected to determine the organ- and tissue-specific expression ofSbhl
within an organ (Figure 3.4). The expression ofSbhl is shown in median longitudinal
sections throughout the shoot of a one week old soybean seedling.In situ hybridization
analysis showed that Sbhl was expressed in the shoot apical meristem (Figure 3.4A, B)
and the axillary bud (Figure 3.4D,E). The pattern and intensity of Sbhl expression in the
shoot apical meristem and axillary bud were very similar to that observed in the globular
staged zygotic embryos.Sbhl mRNA was present uniformly throughout the shoot apical
meristem and expression levels decreased sharply in the ground tissue just below the
shoot apical meristem.Sbhl expression was restricted to the meristem region and there
was no signal detectable in young leaf primordia and in older leaves (Figure 3.4B). Sbhl
mRNA was present at high levels in the newly formed procambium located just below
the shoot apical meristem (Figure 3.4B,C). As the intemode elongated, the level ofSbhl
expression in the provascular tissue decreased and Sbhl was expressed in strands along
vascular tissue (Figure 3.4C). In more mature stems with well-differentiated vascular tissue, Sbhl was not expressed (Figure 3.4A). These results indicate that Sbhl is only expressed in cells remaining in an undifferentiated state, and after the fates of cells are determined, Sbhl expression is minimal. Sbhl transcript was also detectable in the basal tissue of the flower (Figure 3.4F). No signals were detected in the root apical meristem and the leaf (data not shown). The results of these tissue-specific expressions are consistent with those obtained from northern hybridization analysis. Using both techniques, the Sbhl transcript was detected at low levels in soybean stems but not in
leaves and roots. 87 DISCUSSION
The Specific Expression of Sbhl Indicates its function in Soybean Development
We detected high levels of Sbhl expression in soybean somatic embryos
developed from 7 to 14 days following transfer to a hormone-free medium. In soybean
somatic embryogenesis, this corresponds to embryo elongation and globular-heart
transition stage (Figure 1.1). This stage is critical in development during which the cells
giving rise to cotyledons and provascular tissue are determined (Meinke, 1991). In situ
hybridization enabled us to detect the specific localization ofSbhl expression. Sbhl was
expressed evenly in globular embryos in which all cells were rapidly dividing and cells
had not undergone differentiation except for protoderm cells. In heart staged embryos, the cells forming cotyledons and vascular tissue have been determined. The Sbhl transcript
was present in cells that remained in an undifferentiated state such as cells in the shoot
meristem and procambium. The cells in differentiated tissues and organs such as cotyledons and well-developed vascular tissue did not express Sbhl. This expression pattern became more evident as embryos developed further.
The shoot apical meristem typically forms a dome-shaped structure and is the origin of the entire above-ground portion of the plant. The shoot apical meristem functions to proliferate cells and to initiate new tissues and organs (Medford, 1992). Like an embryo that gives rise to embryogenic tissue and organs through organized divisions and differentiation, the shoot apical meristem gives rise to vegetative organs such as leaves and the stem. However, at least two similarities exist between the shoot meristem and the young embryo. One is that they consist of rapidly dividing cells. The other is that the cells undergo organized and preprogrammed division and differentiation. In this respect, many of the genes expressed during embryogenesis would be expected to be transcriptionally active in other meristematic tissues (Lindsey and Topping, 1993). 88 Therefore, it is not surprising to see thatS b h l , the gene isolated from embryos was also expressed in the shoot apical meristem. Surprisingly, Sbhl was expressed in the shoot apical meristem in similar pattern to that in an embryo. Sbhl mRNA was present in rapidly dividing and undifferentiated cells. When the fates of cells had been determined to form a specific organ and a tissue such as leaf primordia and mature vascular tissue, there was no expression ofSbhl. Sbhl transcript decreased sharply in the region just below the shoot apical meristem creating a boundary corresponding to the rib zone that is believed to form a border between the shoot apical meristem and fully differentiated cells
(Medford, 1992). Sbhl was expressed in the procambium in young stems. Procambial cells stay in a state of rapid division and eventually give rise to vascular tissue after differentiation.
The root apical meristem is also composed of rapidly and organized dividing cells. However, S b h l transcript was not detected in soybean root apical meristems either embryogenic or vegetative. The root apical meristem is initiated at the pole opposite of the shoot apical meristem during embryogenesis. The root meristem gives rise to the underground parts of a plant that are morphologically totally different from the shoot.
This difference must be controlled by the action of different set of genes since in some mutants of Arabidopsis, the shoot apical meristem is disrupted without affecting the organization of the root apical meristem (Mayeret a l 1991, Medford, 1992). Since many homeobox genes controlling pattern formation and tissue differentiation are expressed in specific domain of an organism,Sbhl may be one of these genes that make the distinction between shoot and root apical meristems. As proposed by Medford (1992) that meristematic genes are these expressed in regions of active cell division such as shoot and root apical meristems and cambium. Some of these genes may be expressed in all meristematic regions and some may be specific for each type of apical meristems.S bh l 89
was expressed in shoot but not in root apical meristems and it may belong to the type of
genes with expression specific to the shoot apical meristem.
Sbhl Was Expressed in both Somatic and Zygotic Embryos in Same Patterns
The similarity in development between somatic and zygotic embryos has been shown by many morphological and gene expression studies. It is generally believed but not universally accepted that somatic embryogenesis can be used as a model for plant embryogenesis studies. Recently, the similarity between these two systems has been supported by detailed gene expression studies using in situ hybridization. Most of thesein situs have been performed on genes with abundant products in embryos and genes constitutively expressed during embryo development. My dissertation research onSbhl expression indicates that the genes involved in pattern formation and cell specification are also expressed similarly during both somatic and zygotic embryogenesis.
Sbhl mRNA was present in zygotic embryos at a much higher level than in somatic embryos at all corresponding developmental stages. This phenomenon might result from the slower development of somatic embryos. Zygotic embryos develop much faster than somatic embryos. Under normal field or greenhouse conditions, the zygote divides very rapidly and a globular embryo is formed within 5 days after flowering. It usually takes about 1 *3 days for the globular embryos to develop to the heart stage, about another week to get to cotyledon stage (Lersten and Carlson, 1987). However, about 4 weeks are required for the globular embryo to develop to the cotyledon stage during somatic embryogenesis. The embryogenic culture used for thisin situ hybridization has been maintained for about four years. Some somatic embryos developed slowly while others did not develop at all. Therefore, it is understandable that Sbhl was expressed at lower level in somatic embryos consisting of cells with less active divisions and slower differentiation. 90
Comparison o f Gene Expression between Sbhl and KnI
Sbhl cDNA clone was isolated using K nl cDNA as a heterologous probe under low hybridization and washing stringencies. The sequence analysis shows 47.0% and
87.5% identities in amino acid sequence at the whole protein and within the homeodomain respectively. The high sequence identity between homeobox genes from soybean, a dicot species, and maize, a monocot species indicates fhat the plant homeobox genes may be conserved during evolution. This conclusion is also reflected by similarities in gene expression patterns between Sbhl and K nl. K nl was expressed in zygotic embryos, apical meristems of vegetative shoots and undifferentiated cells within the developing vascular tissue but not in leaves and roots (Smith et al ., 1992, Jackson, personal communication). The spatial expression ofKnl in the shoot apical meristem has been described in detail by Jackson et al. (1993). Similar to the expression ofSbhl in soybean shoot apical meristem,Knl was expressed in maize apical meristem except for regions forming leaf primordia. These similarities indicate the Sbhl is a homologue of
K nl in soybean and may function similarly toKnl in maize development.
Some differences in gene expression have been observed between Sbhl and K nl.
Although K nl mRNA has been detected in shoot meristem and procambium of zygotic embryos developed 14 days after pollination, it has not been detected in globular embryos
(Hake, personal communication). AlsoSbhl transcript was present in the LI cells of soybean shoot apical meristem while theKnl transcript was not detectable in this layer of maize shoot apical meristem (Jacksonet al., 1993). Whether these differences are true reflection of the differences between monocot species and dicot species or due to the technique used for in situ hybridization is not known.
In summary, Sbhl was expressed in somatic and zygotic embryos and in the shoot apical meristem in a similar pattern. The location of its expression is specifically in some 91 meristematic and undifferentiated regions and not in cells with determined fates indicating that Sbhl may function as keeping cells stay in an undifferentiated state. The presence of Sbhl transcript in rapidly dividing cells does not indicate that it is a cell division factor since the Sbhl transcript was not detected in rapidly dividing soybean nonembryo genic calli (Figure 3.1). The presence ofSbhl in organized and not in unorganized dividing cells may implicate that Sbhl may be involved in either programmed cell division or cell differentiation or both since these two phenomena were absent in nonembryogenic culture of soybean.
Based on the homologies between Sbhl and K nl both at sequence level and in expression patterns, together with the specific pattern ofSbhl expression as described above, we suggest that the Sbhl may function as a transcription factor directing cells to remain in an undifferentiated state, and programming cell division. Efforts are currently underway to better understand the function of SBH1 protein by plant transformation with sense and antisense constructions ofS b h l. The sense construction should cause over- and/or ectopic-expression ofSbhl resulting in some phenotypic alterations such as knotted genes in maize. The antisense construction should reduce the endogenousSbhl expression and may result in the arrest of embryo development. With a more thorough understanding of the functions of the Sbhl and other isolated homeobox genes and their interactions, the molecular mechanism underlying plant embryogenesis will be better understood. 92
0 3 7 14 28 . t L S R N
Figure 3.1. Northern hybridization analysis ofSbhl transcript. A) One pg of poly(A+)RNA from soybean somatic embryos collected 0, 3, 7, and 28 days following transfer to the development medium was loaded per lane. B) Ten pg of total RNA from leaf (L), stem (S), root (R), and nonembryogenic callus (N) were loaded per lane. The RNAs on each membrane were first hybridized with Sbhl cDNA (upper panels) and then rehybridized with soybean ubiquitin cDNA (low panels). Figure 3.2. In situ hybridization ofSbhl RNA probes to soybean somatic embryos at different developmental stages. The longitudinal sections of somatic embryos at globular (A), pre-heart (B), heart (C), and cotyledon (D) stages were hybridized to digoxygenin labeled anti-mRNA probe of S b h l. A section from a cotyledon staged embryo was hybridized to the sense-RNA probe of Sbhl to show hybridization background (E). The pictures were taken using a light microscope under bright field. The blue color represents hybridization signal. Abbreviation: Co-cotyledon, Pc-procambium, SM-shoot meristem.
93 Figure 3.2 Figure 3.3. In situ hybridization ofSbhl RNA probes to soybean zygotic embryos at different developmental stages. The longitudinal sections of zygotic embryos collected at different times after flowering were used to hybridize to digoxygenin labeled anti-mRNA probe of Sbhl. Different stages of embryos were obtained which are globular (A, B), heart (C), early cotyledon (D), cotyledon (E), and late cotyledon (F). A hypocotyl transverse section of a cotyledon embryo (F) was used to show vascular localization ofSbhl expression (G). Abbreviation: Co-cotyledon, En-endosperm, Pc-procambium, PL-primary leaf, SC-seed coat, SM-shoot meristem, Sp-suspensor, VB-vascular bundle.
95 Figure 3.3 Figure 3.4. In situ hybridization showing the localization ofSbhl mRNA in different organs and tissues of soybean plants. The longitudinal sections of the vegetative shoot apical meristem (A,B), axillary bud (D,E) and flower (F), and a cross-section of the shoot apical meristem (C) were hybridized to digoxygenin labeled anti-mRNA probe of S b h l. Abbreviation: AB-axillary bud, LP-leaf primordia, Pc-procambium, SAM-shoot apical meristem, VB-vascular bundle.
97 Figure 3.4 GENERAL DISCUSSION
Through my dissertation research, a pyruvate kinase gene and several homeobox-
containing genes were isolated from soybean somatic embryos. They represent two
classes of genes, one class acts primarily as a house-keeping and the other has a
regulatory function.
The pyruvate kinase gene encodes an important regulatory enzyme involved in
regulation of glycolysis during which energy is generated for various processes. The
rapid-dividing cells in young embryos are metabolically active. Genes encoding enzymes
involved in respiration, cell division and cell wall synthesis are expected to be actively
transcribed in these cells (Lindsey and Topping, 1993). Even though its function in plant
embiyogenesis is unknown, it is reasonable to believe that the higher expression of
pyruvate kinase gene at early stage of embryogenesis could be critical for the rapid cell
divisions.
Homeobox genes belong to another class of genes that are involved in pattern
formation and/or cell specification. The homeobox genes we isolated represent the first
homeobox genes discovered in plant embryos and from soybean. The expression ofSbhl
was spatially and temporally regulated during embryo development. It was expressed at
the highest level at the globular-heart transition stage, during which the cells forming root
and shoot meristems, vascular tissues, and cotyledons are determined. Sbhl was also expressed spatially in some meristematic regions including the procambium. Although
Sbhl was isolated from soybean embryos, its expression was not limited to the embryos.
The expression pattern ofSbhl in the shoot apical meristem is strikingly similar to that in
99 100
embryos. Because Sbhl was expressed in both embryos and shoot apical meristem that
consist of cells with some common characteristics such as organized cell division and
programmed cell differentiation, Sbhl expression may be critical for these functions. The
function ofSbhl product as a cell proliferating factor has been excluded since it was not
expressed in nonembryogenic soybean calli that consist of rapidly dividing and
unorganized cells. Sbhl was not expressed in the root apical meristem indicating that its
expression may mark the distinction between root and shoot apical meristems.
Furthermore, Sbhl was expressed specifically in undifferentiated but organized dividing
cells in the shoot apical meristem. After celts are committed to a certain pathway such as
cells in leaf primordia and differentiated cells just located below the shoot apical
meristem, Sbhl is no longer expressed. These expression patterns together with high
homology withKnl indicate that Sbhl may be involved in keeping cells in an
undifferentiated state.
Southern hybridization analysis and the presence of several homeobox genes in an
early staged embryo cDNA library indicate that several homeobox genes are expressed
during soybean embryogenesis. They may act coordinately and/or independently to
control embryogenesis. Similar expression patterns ofSbhl in both somatic and zygotic
embryogenesis provide further evidence that somatic embryogenesis can be used as a
model for plant embryogenesis studies. The isolation ofSbhl by a distant heterologous
probe indicates that plant homeobox genes are conserved during evolution. The isolation
o f Sbhl will provide an entry point for isolating other regulatory genes in plant
development and for understanding the molecular mechanism underlying plant
embryogenesis and other developmental processes.
It has been estimated that about 2x10^ diverse genes are expressed during plant embryogenesis (Goldberg et al., 1989). It will take some time before to clone all these 101 genes and understand their functions and hierarchical interactions. Isolation and characterization of master control genes such as homeobox genes should be high priority.
Homeobox genes encode transcription factors that act as important components of the signal transduction pathway. These genes can be considered the start point of a series of genes that result in biochemical and morphological changes during plant embryogenesis.
It will be important to identify the signals activating the homeobox genes and to determine how the homeodomain proteins control their target genes. With the isolation of the upstream genes controlling the homeobox genes, we can get closer to the beginning of signal transduction. By isolating downstream genes regulated by homeobox genes, the structural genes can be obtained.
Assessing the role of identified genes expressed early in plant embryogenesis is still challenging. Antisense techniques are an attractive approach in which the expression of endogenous genes are reduced by the introduced antisense gene. Overexpression and ectopic expression of the target genes provide yet other approaches. Abnormal phenotypes resulting from the modifying expression of homeobox genes will provide valuable information about their function. With the isolation and characterization of more genes involved in embryogenesis, we will gain a better understanding of the mechanisms underlying plant embryo development. LIST OF REFERENCES
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Protocols and Solutions
1. Extraction of Plant TotalRNA (Davis eta!., 1986) (1) Grind 1-2 gram of fresh or frozen tissues in liquid nitrogen, transfer to another mortar, add 10 ml GIT buffer, and grind more. (2) Transfer the ground tissues to a cortex tube, spin at 8,000 rpm, 20°C for 10 minutes, and filter supernatant through cheesecloth to another tube. (3) Layer the solution on 3 ml 5.7 M CsCI mix in a SW 40.1 tube, fill the tube to the top with GIT buffer, and spin at 37,000 rpm, 20°C for 24 hours. Protein will remain in aqueous guanidine, DNA bands in CsCI solution, and RNA pellets to the bottom of the tube. (4) Discard most of supernatant with broad mouth pipette, drain the rest, and swab around tube side with Kim wipes to remove any remaining buffer. (5) Resuspend pellet in 4 ml GIT buffer, layer the buffer on 1 ml CsCI mix in a SW 50.1 tube, and fill the tube with GIT buffer. Spin at 45,000 rpm, 20°C overnight and collect pellet as step 4. (6) Resuspend pellet in 0.5 ml lx NETS, transfer to a microfuge tube, and then add 0.5 ml NETS saturated phenol/chloroform. Mix well for 15 minutes and spin for 10 minutes. Transfer upper aqueous phase to a new tube, extract again with 0.5 ml phenol/chloroform for 10 minutes, and spin 10 minutes. (7) Remove the upper aqueous phase to another microfuge tube, add 0.1 volume of 3 M sodium acetate (pH 6.0) and 2 volume of 95% cold ethanol, mix well, and store
119 120 at -20°C overnight.
( 8) Spin 10 minutes at 4°C, discard supernatant, wash with 70% ethanol, and spin
again. Resuspend the pellet in 500 ml sterile H 2 O and keep in -20°C or -70°C freezer.
(9) Determine RNA quantity with OD2 6 O reading using 4 pi sample mixed with 996
pi water and using 1 ml H 2 O as a blank. Reading x 40 x 250 = RNA pg/ml or Reading x 10 = RNA pg/pl Wear gloves all the time and change frequently. Glassware should be baked at 220°C overnight. Guanidine Isothiocyanate (GIT) buffer
1 0 0 ml final concentration 47.3 g guanidine isothiocyanate 4 M 0.84 ml 3 M sodium acetate (pH 6,0) 25 mM
volume to 100 ml with DEPC-H2 O
sterilize through 0 .2 2 pm filter add 1.0 g N-lauroylsarcosine 1% add 0.84 ml (3-mercaptoethanol (in fume hood) keep at room temperature Cesium Chloride (CsCI) M ix 50 ml final concentration 48.0 g CsCI 5.7 M 0.42 ml 3 M sodium acetate (pH 6.0) 25 mM volume to 50 ml with DEPC-H2 O
sterilize through 0 .2 2 pm filter I x N E T S 500 ml final concentration 10 ml 5 M NaCl 100 mM 10 ml 1 M Tris (pH 7.5) 20 mM 1 ml 0.5 M EDTA 1 mM 25 ml 10% SDS 0.5% volume to 500 ml with DEPC-H2 O Do not autoclave 121 2. Poly(A+)RNA Isolation(Aviv and Leder, 1972)
(1) Suspend 0.2 g (get 0.5 ml volume) of oligo (dT)-cellulose in washing buffer.
(2) Wash with 3 ml of washing buffer.
(3) Wash with H2 O until pH<8.0.
(4) Wash with 5 ml loading buffer A to equilibrate the column.
(5) Heat the RNA solution (500 ml) to 65 °C for 5 minutes, add equal volume of
prewarmed (65°C) loading buffer A, mix, and let cool to room temperature for 2
minutes.
(6) Apply to the column and collect the eluate. When all RNA solution has entered
the column, add 0.5 ml loading buffer B.
(7) Heat eluate to 65°C for 5 minutes and cool to room temperature for 2 minutes.
(8) Reapply eluate to the column followed by washing the column with 5 ml of
loading buffer B.
(9) Add 1,5 ml of elution buffer and collect 4 drop fractions.
(10) Check the fractions by placing 1.0 pi of each on the EtBr TE agarose plates and
observing under UV light after 20 minutes.
(11) Pool the positive fractions, add 0.1 volume of 3 M sodium acetate (pH 6.0) and
2.5 volume of cold 95% ethanol, and precipitate at -70°C overnight.
(12) Spin for 20 minutes at 4°C, wash with 70% ethanol, and spin again.
(13) After drying pellet, suspended in H 2 O, and keep in -70°C freezer.
Usually poly(A+)RNA is 1-2% of total RNA 122 Loading Buffer A
1 0 0 ml final concentration 4.0 ml 1 M Tris (pH 7.4) 40 mM 2 0 ml 5 M NaCl 1 M 0 .2 ml 0.5 M EDTA 1 mM 75 ml d e p c -h 2o mix and autoclave 25 minutes add 1 ml 10%SDS 0.1% Precipitate is easily formed at room temperature. If so, keep in 65°C bath until dissolved.
Loading Buffer B 100 m l final concentration 2.0 ml 1 M Tris (pH 7.4) 20 mM 2.0 ml 5 M NaCl 0.1 M 0.2 ml 0.5 M EDTA 1 mM 95 ml DEPC-H20 mix and autoclave 25 minutes add 1 ml 10%SDS 0 . 1%
Elution Buffer
1 0 0 ml final concentration 1.0 ml 1 M Tris (pH 7.4) 10 mM 0.2 ml 0.5 M EDTA 1 mM 98.3 ml DEPC-H20 mix and autoclave 25 minutes add 0.5 ml 10% SDS 0.05%
Washing Buffer 50 ml final concentration 1.0 ml 5 N NaOH 0.1 M 0.5 ml 0.5 M EDTA 5.0 mM 48.5 ml DEPC-H20 Do not autoclave 123 3. Preparation of Replica Filters (Benton and Davis, 1977) (1) Culture host cells in LB medium containing 0.4% maltose overnight and add equal
volume of 10 mM MgS0 4 - (2) Add desirable amounts of phage to 300 |il of host cells and incubate at 37°C for 15 minutes. (3) Add 3 ml top agarose kept at 55°C, disperse on the plate evenly, cool for 5 minutes, and incubate at 37°C overnight. (4) Place plates at 4°C for at least two hours before filter lift. (5) Mark plates asymmetrically by punching three holes through medium.
(6) Carefully lay filters onto plates. Be sure: a) to center the filter on the plate, b) to avoid trapping air bubbles, c) as soon as filter touch the plate never remove again. (7) Mark the filter corresponding to the markers on the plates.
Leave filter on the plate, the 1s* lift for 1 minute, 2 nd lift for 2 minutes, 3r<* lift for 3 minutes.
(8) Carefully remove the filters with blunt forceps and avoid ripping top agarose. (9) Denature phage DNA (plaque side up) in denaturing solution for 20 second with agitation. (10) Dip filter (plaque side up) in Tris 2x SSCP for 20 second with agitation. (11) Air dry filter on paper tower (plaque side up). (12) Bake the filter in vacuum oven at 80°C for 2 hours. (13) Store the filters at room temperature before hybridization. Denature Solution
1 0 0 0 ml final concentration 87.7 g NaCl 1.5 M 4.0 g NaOH 0.1 M Tris 2x SSCP 1 0 0 0 ml final concentration 24.2 g Tris 0.2 M add 500 ml H 2 O, adjust pH to 7.5 with HC1, and then add 100 ml 2Ox SSC 2x SSC 3.0 g K 2 H P 0 4 13 mM 1.77 g KH 2 PO 4 13 mM autoclave for 30 minutes 124 4. cDNA Labeling
(1) Add components in following order and quantity. X Ml h 2o 5 Ml lOx RT buffer 5 Ml 1 Ox cold labeling nucleotides 2 Ml Actinomycin D (0.5 mg/ml) 0.5 pi 1 M DTT 2 Ml RNasein (25 U/ml) 1 Ml oligo dT X Ml (3 pg) poly(A+)RNA 5 Ml 32p-dCTP 2 Ml AMV reverse transcriptase
50 pi total
(2) Incubate at 37°C for 2 hours.
(3) Stop the reaction by adding 4 pi 0.25 M EDTA.
(4) Add 25 pi of 150 mM NaOH, incubate at 65°C for 1 hour, and then put on ice.
(5) Add 25 pi 1 M Tris (pH 8.0) and 5 p ll M HC1.
(6) Fraction probe with nick column as random primer labeling (Appendix 8). lOx Cold Labeling Nucleotides
1 0 0 pi final concentration 10 pi lOOmMdATP lOmM 10 pi lOOmMdGTP 10 mM 10 pi lOOmMdTTP 10 mM 1 pi lOOmMdCTP 1 mM 69 pi ddH20 lOxRT B u ffer 0.5 M Tris-HCl (pH 8.3)
0 .1 M MgCl2 0.7 M KC1 1 mg/m I BSA (enzyme grade) 125 5. Isolation of Phage and Phage DNA(Finer, personal communication)
(1) Culture NM514 in LB medium containing 0.4% maltose overnight.
(2) Spin down 100 pi cells for 2 minutes and resuspend in 100 pi of TM buffer.
(3) Add agar plug of 1 fresh plaque and incubate at 37°C for 10 minutes.
(4) Transfer into 25 ml CY medium and incubate at 37°C with vigorous shaking (lysis
in 7-8 hours, check every hour after 4 hours of culture).
(5) Add 0.63 g NaCl and 50 pi chloroform to each lysate and shake at 37°C for 5
minutes.
(6) Spin at 10,000 rpm for 10 minutes, transfer the supernatant to a new flask
containing 2.5 g PEG 6000, add DNase and RNase at concentration of lpg/ml,
and shake gently at 37°C until PEG dissolved.
(7) Chill on ice from 30 minutes to overnight.
(8) Spin at 10,000 rpm, 4°C for 10 minutes and discard the supernatant.
(9) Dissolve the pellet in 1 ml 1 M NaCl (in TM) and spin in microfuge at maximum
speed for 10 minutes.
(10) Layer the supernatant onto CsCI gradients
Gradient: top 1 ml (1 pt 62.5% CsCI: 2 pts TM)
1 ml (1 pt 62.5% CsCI : 1 pt TM)
1 ml (2 pts 62.5% CsCI: 1 pt TM)
bottom 0.5 ml (62.5% CsCI)
(11) Spin at 40,000 rpm for 35 minutes and collect phage band with syringe 20 G
needle (phage should remain a sharp band at interface between 2 n^ and 3rt^
gradient steps from bottom).
(12) Add H 2 O to final volume of 2 ml.
(13) Add 40 pi 0.5 M EDTA and heat 65°C for 10 minutes. 126 (14) Add 133 til of 3 M sodium acetate (pH 5.0) and 4 ml of 95% cold ethanol and
keep at -20°C for 1 hour.
(15) Spin at 10,000 rpm, 20°C for 30 minutes and suspend the pellet in 500 pi TE.
(16) Extract with phenol, phenol/chloroform, and then with chloroform.
(17) Precipitate with ethanol and wash with 70% ethanol twice.
(18) Suspend pellet in H 2 O.
TM B uffer
500 ml final concentration
0.605 g Tris 10 mM
1.23 g M g S 0 4 10 mM
volume to 500 ml and autoclave 20 minutes
62.5% CsCI in TM
50 g CsCI
volume to 50 ml with TM
C Y m ed iu m
10 g Casein hydrolysate
5 g Yeast extract (Difco)
3 g NaCl
2 g KC1
3 g Tris base
2 g MgCl2
volume to 1000 ml, adjust pH to 7.0 and then autoclave 20 minutes 127
6. Production of Single-Stranded DNA(Vieira and Messing, 1987)
(1) Inoculate 5 ml 2x YT medium containing 75 pg/ml ampicillin with a single
colony, culture at 37°C for 1-2 hours with gentle shaking, and then add 15 pi K07
helper phage.
(2) Grow shaking at 37°C for 1-2 hours and add kanamycin to final concentration of
70 pg/ml.
(3) Grow at 37°C with vigorous shaking until saturation (16-24 hours).
(4) Spin 1.5 ml of cells for 5 minutes in microfuge.
(5) Transfer 1 ml of supernatant to another tube and add 150 pi of solution containing
20% PEG (6000)/2.5 M NaCl. Precipitate on ice for 15 minutes.
(6) Spin for 5 minutes in microfuge, drain and respin, and clean pellet with pipette tip
(pellet should be visible).
(7) Resuspend the pellet in 400 pi of 0.3 M sodium acetate (pH 6.0)/l .0 M EDTA by
vortexing vigorously.
(8) Extract with one volume of saturated phenol/chloroform.
(9) Spin, transfer the top phase to a tube, and add 1 ml of 95% cold ethanol. Spin for 5
minutes (-20°C precipitation may help low yields).
(10) Wash with 70% ethanol and dry in vacuum.
(11) Resuspend the pellet in 25 pi T 1/10 E.
2xYT 500 ml Bacto trypotone 16 g Bacto yeast extract 1 0 g NaCl 5 g volume to 500 ml, adjust pH to 7.5 and autoclave for 30 minutes 128 7. DNA Extraction (Saghai-Maroofet al., 1984) (1) Grind 0.3-0.4 g tissue into powder immediately before DNA extraction.
(2) Transfer the ground tissue to a 15 ml tube, add 8 ml CTAB extraction buffer, and quickly invert tube to mix. (3) Incubate 30 to 60 minutes at 65°C and mix occasionally by tapping (hold cap tightly). (4) Cool 10 minutes on bench. (5) Add 4.5 ml of 24:1 chloroformroctanol mixture and mix gently for 5 minutes while holding caps down.
(6) Spin for 10 minutes at 2000 rpm in table top centrifuge. (7) Draw off supernatant into clean snap top tube with 10 ml pipette (can be dumped
carefully leaving pelleted chloroform and crud under the interface) and add 6 ml of cold isopropanol, and mix gently.
(8) Hook DNA with a glass hook, transfer to 5 ml tube containing 2 ml of 76% ethanol, 0.2 M sodium acetate for 20 minutes (DNA can be held at this step if needed). (9) Transfer hooked DNA to 1 ml of 76% ethanol, 10 mM ammonium acetate in 5 ml tube, and dip for 5 second. (10) Transfer to 1.5 ml microfuge tube containing TE. DNA should slide off the hook easily and soaking will facilitate removal from the hook. (11) Place on rocker in cold room overnight. (12) Spin tubes for 10 minutes in microfuge and transfer to clean tube leaving crud behind. CTAB Extraction Buffer 500 ml final concentration 10 g Hexadecyltrimethylammonium bromide 2% 50 ml 1 M Tris-HCl (pH 8.0) 100 mM 40.9 g NaCl 1.4 M 20 ml 0.5 M EDTA 20 mM 430 ml H 2 O Dissolve ingredients and add Ji-mercaptoethanol 1% 8. Random Primer Labeling (Feinberg and Vogelstein, 1983)
(1)Add x pl(50ng) DNA x pi H 2 O
10 til total
(2) Boil 5 minutes and chill on ice.
(3) Add 11.5 (j.1 LS mix 1.0 til BSA (10 pg/pl)
2.0 Ml 3 2 P-dCTP 0.5 til (5 units/pl) Klenow enzyme
(4) Incubate at room temperature for 2 hours and stop reaction by adding 5 pi 0.5 M
EDTA.
(5) Flush column twice with TE.
(6) After stop dripping, add random primer reaction mix.
(7) Immediately add 400 pi TE and discard this fraction.
(8) Add another 400 pi TE and collect this fraction.
(9) Boil 5 minutes and place on ice.
L S M ix 500 pi nucleotide mixture 252 pi hexanucleotide mixture 250 pi 2 M Hepes (pH 6.0) 118 pi H20
1120 pi total
Nucleotide Mixture T M B u ffe r 1 pi lOOmMdATP 250 pi 1 M Tris (pH 8.0)
1 pi lOOmMdGTP 25 pi 1 M MgCl2 1 pi lOOmMdTTP 3.5 pi BME 997 pi TM buffer 721.5 pi H20 130 9. Northern Hybridization(Thomas, 1983)
(1) Denature RNA x pi (10 pg) total RNA or (1.0 pg) poly(A+)RNA 4.2 pi 4 M glyoxal 2.5 pi 100 mM phosphate buffer (pH 6.5) x pi DEPC-H 2 O
25 pi total Incubate at 50°C for 1 hour, cool on ice, and spin (keep on ice until load). (2) Prepare 1.2% agarose gel in 10 mM phosphate buffer (pH 6.5). The concentration of gel depends on the size of RNA species. (3) Add 4.0 pi RNA loading buffer to each sample. (4) Load samples and run at 90 volts until bromophenol blue migrate to 3/4 place of the get length using recirculation device after 10 minutes of running. The running buffer is 10 mM phosphate buffer(pH 6.5) buffer. (5) Cut the marker lane out, de-glyoxalate in 10 volumes 50 mM NaOH, shake gently for 30 minutes at room temperature, neutralize in 20 volume of 50 mM phosphate buffer (pH 6.5) 3 times, and at last time include EtBr at 1.0 pg/ml.
(6) Transfer: essentially same as Southern transfer, transfer 12 hours, air dry, and bake at 80°C for 2 hours. (7) Place the membrane in 200 ml of 20 mM Tris-HCl (pH 8.0) at 100°C and then allow to cool to room temperature.
Phosphate Buffer (pH 6.5) 100 mM
1 0 0 0 ml final concentration NaH2P04(FW 138) 5.38 g 39 mM
N a2H P0 4 (FW 268) 16.35 g 61 mM
volume to 1000 ml with DEPC-H2 O and autoclave 25 minutes Loading buffer 5 ml Glycerol 2.5 ml 100 mM phosphate buffer (pH 6.5) 0.5 ml
d e p c -h 2o 2 .0 ml Bromo-phenol blue a bit 10. DNA Dot Blot
(1) Wet nitrocellulose filter in lOx SSC.
(2) Air dry filters for 1 hour. (3) Prepare DNA samples.
(a) Place DNA (10 ng/dot) into a microfuge tube with a final volume of 50 pi in 20
mM Tris (pH 7.6) and 1 mM EDTA.
(b) Heat in a boiling water for 10 minutes (prevent cap open).
(c) Add 50 pi of 1 M NaOH and incubate at room temperature for 20 minutes.
(d) Neutralize by sequential addition of 50 pi of 1 M Tris (pH 8.0), 50 pi of I N HC1,
and then 20 pg of carrier tRNA (yeast tRNA).
(e) Add 2.5 volume of 95% cold ethanol and place at -70°C for 30 minutes. Spin 10
minutes at 4°C.
(f) Discard supernatant and wash twice with 95% cold ethanol.
(g) Dry the pellet under vacuum.
(h) Redissolve the pellet in 20 pi of water and place on ice.
(4) Spot 2.0 pi aliqots of each sample onto nitrocellulose filter each time. After drying
spot 2 pi more, repeat until getting to the desirable amounts.
(5) Air dry and then bake at 80°C for 2 hours. 132 11. RNA Dot Blots(Thomas, 1983)
(1) Prepare RNA samples:
x pi (1-10 pg/dot) total RNA
1.5 pi (30 pg) yeast tRNA
x pi DEPC-H 2 O
10 pi total
(2) Add 10 pi of denaturation solution to each sample and incubate at 50°C for 1
hour.
(3) Prepare nitrocellulose filters by wetting them in DEPC-H2 O and then in 20x SSC
for 10 minutes and drying at 65°C for 1 hour.
(4) Make desirable RNA dilutions with 0.1% SDS and spot 2 pi on the nitrocellulose
filters each time. Add 2 pi more after drying and repeat until getting to desirable
amounts.
(5) Air dry and then bake the filters at 80°C for 2 hours.
(6) Place the filters in 200 ml of 20 mM Tris (pH 8.0) at 100°C and allow cool to
room temperature.
Denaturation Solution
34 pi 4 M de-ionized glyoxal
20 pi 100 mM sodium phosphate buffer (pH 6.5)
46 pi DEPC-H 2 O
20 m M Tris (pH 8.0)
4.0 ml 1.0 M Tris (pH 8.0)
196 ml DEPC-H20 133 12. Dry Blot DNA Transfer(Kempter el al ., 1991) (1) After electrophoresis, depurinate gels in two volumes of 0.25 N HC1 until the bromophenol blue dye marker turns yellow.
(2) Rinse gels with ddH 2 0 and denature gets in two volumes of 0.4 M NaOH (2X15 minutes).
(3) Rinse gels with ddH 2 0 and equilibrate gels in two volumes of 0,25 M Tris- Acetate, 0,1 M NaCl (pH 8,0) for 15 minutes.
(4) Rinse gels with ddH 2 0 and equilibrate gels in two volumes of 0.025 M Tris- Acetate, 0.1 M NaCl (pH 8.0) for 15 minutes (or until the pH of the solution is
about 8.0 ).
(5) After spreading out a sheet of plastic wrap, the gel is laid on a single sheet of Whatman 3M paper soaked with 0.025 M Tris-Acetate, 0.1 M NaCl (pH 8.0).
(6) Wet a membrane in 0.025 M Tris-Acetate, 0.1 M NaCl (pH 8.0) and place on top of the gel. Roll the membrane with a pipet to make sure no air bubbles are present. Cover the membrane with one sheet of Whatman 3M paper soaked in 0.025 M Tris-Acetate, 0.1 M NaCl (pH 8.0), roll out the bubbles. Complete the stack with 4 dry sheets of Whatman 3M paper and a stack of paper towels. Add a 0.5 to 1 kg weight. (7) Blot for 1 hour or longer and then disassemble the stack. 134 025 N HCI 2L 2L 1L 0.5L Cone. HCI (ml) 64.5 43.1 21.5 10.7
ddH 2 0 (ml) 2936 1957 978 439
0.4 M NaOH 2L 2L 1L
NaOH 48 g 32 g 16
025 M Tris-Acetate, 0.1 M NaCl (pH 8.0)
1L 2L 2L Trizma base (g) 30.3 60.6 90.9
Glacial acetic acid (ml) 8.5 17.0 25.5
Dilute with ddH 2 0 (ml) 991 1983 2974
Add NaCl 5.3 g for 900 ml buffer
0.025 M Tris-Acetate, 0.1 M NaCl (pH 8.0)
Dilute 100 ml of 0.25 M Tris-Acetate (pH 8.0) to 900 ml of ddH2 0 and add 5.8 g
NaCl. 135 13. Tissue Fixation, Dehydration, and Infiltration(Jackson, 1991)
D ay 1
(1) Collect fresh tissues and cut if they are too large.
(2) Place tissues in fixative on ice (if tissues keep floating, use vacuum infiltration),
renew fixative, and leave at 4°C overnight with gentle shaking.
D ay 2
(3) Pour off fixative, replace with ice-cold 0.85% NaCl, and leave at 4°C for 30
minutes with gentle shaking.
(4) Dehydrate tissues through ethanol series, 50%, 70%, 85%, each containing 0,85%
NaCl, 95% and 100% containing no NaCl, at 4°C, 90 minutes for each step.
(5) Renew 100% ethanol and leave at 4°C overnight.
D ay 3
(6) Renew 100% ethanol and leave for 2 hours at room temperature. Replace with
50% ethanol:50% Histoclear and leave for 1 hour.
(7) Repeat three times with 100% Histoclear, at the last change, add TissuePrep chips
(Fisher Scientific) to about half the volume of the Histoclear, and then leave
overnight.
D ay 4
(8) Incubate at 40-50°C until chips dissolve.
(9) Replace with melted TissuePrep and leave in 60°C oven overnight.
D ay 5 -7
(10) Renew melted TissuePrep each morning and evening. On day 7, the tissues are
ready for embedding. Use a large excess of each solution. TissuePrep should be freshly melted before use and should not be heated above 60°C as this destroys synthetic polymers which are added to wax to aid sectioning. 136 4% Formaldehyde Fixative Solution
2 0 0 ml final concatenation
20 ml lOx PBS buffer lx
180 ml DEPC-H20
Adjust pH to 11 with NaOH (1 pellet)
Heat to 60°C
8 g paraformaldehyde (Sigma) 4%
Stir until dissolved (1-2 minutes)
Cool on ice and then adjust pH to 7.0 with H2 SO4
Make fresh just before use.
Formaldehyde vapor is toxic, work in a fume hood.
Do not use HCI to adjust pH as the combination of HCI and formaldehyde releases a
powerful carcinogen.
I Ox PBS IPhosphate Buffered Saline) 1000 ml final concentration 76 g NaCl (FW 58.44) 1.3 M
18.76 g Na 2 HP0 4 (FW 268.07) 70 mM
4.14 g NaH 2 P 0 4 (FW 137.99) 30 mM
Volume to 1000 ml with DEPC-H20 and then autoclave. 137 14. Embedding and Sectioning(McKhann and Hirsch, 1993)
(1) Pour melted TissuePrep and infiltrated tissues into a mould.
(2) Place the mould on a warming table and orient the tissues with wanned needles or
a forceps under a dissection microscope.
(3) Carefully remove to a clod plate, add more melted wax, and allow to cool
completely.
(4) Store at 4°C until use (blocks can be stored at 4°C for several months).
(5) Carefully cut block, leave 2 mm of wax around the plant material, and remove
excess wax.
(6) Mount a knife and the trimmed block on a microtome.
(7) Section with a rotary microtome at 7-10 pm by moving the wheel with steady and
even stroke.
(8) Spread the ribbon of sections on paper.
(9) Examine the sections under a dissection microscope to determine which sections
are to be affixed on the slide.
(10) Affix the sections to the poly-L-lysine coated slides.
(a) Place several drops of DEPC-H2 O on the slide.
(b) Place the section on the flooded area, align and spread them with needles.
(c) Place the slide on a hotplate (40-50°C) for a few minutes until any wrinkles in the
ribbon are removed (ribbon may be stretched slightly with needles).
(d) Remove excess H 2 O with "Kimwipes".
(e) Dry slides on the hotplate overnight in a dust-free area.
(11) Keep the slides in a slide box at 4°C until use (stable for at least 2 months). 138 15. Preparation of Poly-L-Lysine Coated Slides(McKhann and Hirsch, 1993)
(1) Soak glass slides in concentrated H 2 SO4 with 2% NOCHROMIX (Godax, New
York) overnight.
(2) Rinse several times with distilled water.
(3) Drain the water and bake the slides overnight at 200°C.
(4) Cool them gradually.
(5) Immerse slides in 100 pg ml~l poly-L-lysine (mol. wt. 150-300,000, Sigma)
solution in 10 mM Tris (pH 8.0) for 30 minutes at room temperature.
(6) Air dry overnight in a clean area.
(7) Store the slides in a slide box at 4°C (stable for several months).
Poly-L-Lysine Solution (109 pg m t1)
25Qml
2.5 ml 1 M Tris-HCl, pH 8.0 (RNase-free)
247.5 ml DEPC-H 2 O
25 mg poly-L-lysine (mol. wt. 150-300,000)
Store at 4°C and can be reused within 2 months. 139 16. Preparation oRNA f probes(Jackson, 1991, Hake and Jackson, per. comm.) Prepare DNA Templates (1) Subclone SbhJ cDNA (or other DNA fragments) into pBluescript SK+. (2) Linearize plasmid DNA (20 pg) with a proper restriction enzyme (excess enzyme and longer incubation time to get complete digestion). (3) Purify DNA twice with phenol/chloroform and then with chloroform. (4) Precipitate at -20°C overnight or at -80°C for 2 hours. (5) Spin, wash with 70% ethanol, and air dry. (6) Resuspend DNA at concentration of 0.5 pg/pl in TE and store at -20°C. Prepare DIG-RNA Probes
(1) Add the components to a microfuge tube in following order;
8 pi DEPC-H 2 O 2 pi (1 pg) linearized plasmid DNA 2 pi lOx DIG RNA labeling mixture (Boehringer Mannheim Biochemica) 4 pi 5x transcription buffer (Promega) 1 pi 25 u/pl RNasin (Promega) 2 pi lOOmMDTT 1 pi 20 u/pl RNA polymerase (T7 or T3, Promega) (2) Mix gently and incubate 1 hour at 37°C. (3) Add another 1 pi T7 (or T3) and incubate for another 1 hour. (4) Remove 1 pi for gel analysis (to check the size and yield of the probe). (5) Add 5 pi yeast tRNA (20 mg mH), 5 pi lu/pl RQ1 DNase (Promega), and then
70 pi DEPC-H 2 O. Incubate at 37°C for 10 minutes.
(6) Precipitate RNA with 10 pi 4 M LiCl (or 3 M sodium acetate) and 300 pi 100%
ethanol. (7) Keep at -80°C for 30 minutes or at -20°C for 4 hours.
(8) Spin 20 minutes at 4°C, wash with 70% ethanol, and air dry. 140 Hydrolyze RNA Probes
(1) Resuspend RNA in 50 pi DEPC-H 2 O, add 50 pi carbonate buffer, and incubate at
60°C for required time (hydrolyze to the final size of 100-200 base long according to the equation: t = Li-Lf/k x Li x Lf, in which t = time in min, k = 0.11 kb/min, the rate constant, Li = initial length in kb, Lf = final length in kb).
(2) Neutralize the reaction by adding equal volume of hydrolysis-neutralization buffer.
(3) Precipitate RNA with 600 pi 100% ethanol and leave at -80°C for 2 hours or at -20°C overnight.
(4) Spin 30 minutes at 4°C, wash pellet with 70% ethanol, and air dry.
(5) Resuspend pellet in 100 pi DEPC-H 2 O by warming to 37°C for 10 minutes with frequent vertexing.
(6) Store at -80°C until use. Carbonate Buffer
10 ml final concentration 127.2 mg Na 2 CC>3 (FW 106) 120 mM
67.2 mg NaHCC >3 (FW 84.01) 80 mM 10 ml DEPC-H20 DEPC-treat, autoclave 15 minutes, and store at 4°C (stable for 2 weeks) Hydrolysis-Neutralization Buffer
1 0 ml final concentration 0.7 ml 3 M sodium acetate (pH 6.0) 200 mM 0 . 1 ml glacial acetic acid 1% 9.2 ml DEPC-H20
Store at 4°C (stable for 6 months) 4M LiC l 50 ml 8.478 g LiCl (FW 42.39)
50 ml DEPC-H2 O DEPC-treat, autoclave, and store at 4°C 141 17. Prchybridization Treatments(Jackson, 1991)
Place slides in a stain dish or on a rack and go through following treatments.
Treatments lim e
( 1) Dewax with Histoclear 1 0 min
Histoclear 1 0 min
(2) Rehydration: 100% ethanol 1 min
1 0 0 % ethanol 30 sec
95% ethanol 30 sec
85% ethanol, 0.85% NaCl 30 sec
70% ethanol, 0.85% NaCl 30 sec
50% ethanol, 0.85% NaCl 30 sec
30% ethanol, 0.85% NaCl 30 sec
0.85% NaCl 2 min
(3) Pronase treatment: wash with lx PBS 2 min
pronase (0.1 mg/ml) 15 min
0.2% glycine in lx PBS 2 min
(4) Fixation: wash with lx PBS 2 min
4% formaldehyde (in PBS)(®) 10 min
(5) Acetylation: wash with lx PBS 2 min
Acetic anhydride wash with 1 x PBS 2 min (6) Dehydration: same as step (2) but in reverseorder. (a) Make fresh, as described in Appendix 13. (b) Acetic anhydride is unstable in water. Stir rapidly as acetic anhydride is added. Dip the slide rack a couple of times and then stir gently. Make fresh buffer each time. 142 Pronase Stock 5 ml final concentration 125 mg pronase E (Sigma) 25 mg/ml 4.94 ml DEPC-H20 50 pi 1 M Tris-HCl (pH 7.5) 10 mM 10 pi 5 M NaCl 10 mM Divide 400 pi in each microfuge tube, incubate at 37°C for 3 hours, and store at -20°C. Use 400 pi (one tube) per 100 ml pronase E buffer for the pronase treatment. Pronase E Buffer 1 0 0 ml final concentration 5 ml 1 M Tris (pH 7.5) 50 mM 1 ml 0.5 M EDTA 5 mM 94 ml DEPC-H20 I Ox Glycine buffer 2 0 0 ml final concentration 4 g glycine 2% 180ml DEPC-H20 20 ml lOx PBS 1 x DEPC-treat and autoclave 0.25% Acetic Anhydride in 0.1 M Triethanofamine-HCl 240 ml final concentration 4.45 g triethanolamine (FW 185.7, Sigma) 0.1 M 240 ml DEPC-H20 Adjust pH to 8.0 by adding 5 pellets of NaOH After dissolving and just before use, add 1.2 ml acetic anhydride with vigorous stirring. 143 18. In Situ Hybridization(Jackson, 1991) (1) Calculate the amount of probe needed for hybridization to get the final concentration of 0.5 ng/pl/kb probe complexity. For example, with 100 pi probe mix applied on each slide and probe is 0 .2 kb long, the amount of probe is0 .5 x 1 0 0 x 0 .2 = 1 0 ng probe/slide (always make some extra). (2) Denature probe (5x concentrated in 50% formamide) at 80 °C for 2 minutes, cool on ice, and then spin briefly. (3) Prepare hybridization buffer 1.0 ml 1.25x conc. Final conc. of 1 x hybridization buffer ______hybridization buffer 70.0 pi 5.36 M NaCl 0.3 M 12.5 pi 1 M Tris (pH 6.8) lOmM 125.0 pi 100 mM phosphate buffer (pH6 .8) 10 mM 12.5 pi 0.5 M EDTA 5 mM 500.0 pi formamide (ultrapure RNase-free, BRL) 50% 250.0 pi 50% dextran sulfate 10% 12.5 pi 1 OOx Denhardts lx 25.0 pi 20 mg/ml yeast tRNA 1 mg/ml (4) Mix 1 volume of 5x conc. probe with 4 volume of 1.25x conc. hybridization buffer. (5) Mark with pencil for probe used on each slide. (6) Apply 100 pi probe mix to each slide, spread in a line along the center of the section, carefully cover the slide with a baked coverslip avoiding bubbles. (7) Place the slides in a box containing tissues soaked in 2x SSC and 50% formamide. (8) Keep the sealed box in water bath set at 55°C overnight. 144 19. I n S itu Washes and Immunological Detection(Hake and Jackson, per. comm.) W ashing (1) Allow the coverslips to come off slides in 0.2x SSC. (2) Wash the slides in 0.2x SSC at 55°C for 1 hour and repeat. (3) Rinse 5 minutes in NTE and repeat. (4) Incubate slides at 37°C in NTE containing 20 pg ml"* RNase A for 30 minutes. (5) Rinse 5 minutes in NTE and repeat. (6) Wash the slides in 0.2x SSC at 55°C for 1 hour. Immunological Detection (1) Rinse in 1 x PBS. (2) Incubate in 0.5% Boehringer block (dissolved in buffer A at 60°C for 1 hour) for 45 minutes with gentle shaking. (3) Incubate in buffer B for 45 minutes. (4) Dilute antibody in 1:1250 in buffer B (7.0 pi antibody + 8.75 ml buffer B). (5) Add 100 pi of diluted antibody to each slide, place coverslip, put slides in a box, and incubate 2 hours at room temperature. (6) Wash slides with buffer B at room temperature for four times, 20 minutes each with gentle shaking. (7) Wash 5 minutes in buffer C and repeat. (8) Prepare color reaction buffer by adding 22 pi NBT to 10 ml buffer C, mixing, and then adding 16 pi BCIP (make fresh and avoid light). (9) Add 100 pi color reaction buffer on each slide, put coverslip, and place slides in a box containing tissues soaked with buffer C. (10) Incubate in dark for 2-3 days. (11) Rinse slides twice in TE, dehydrate through ethanol series, rinse in Histoclear, ;> drain briefly, and mount with Permount (Fisher Scientific). (12) After drying, observe under light microscope with bright field. NTE 600 ml final concentration 60.0 ml 5 M NaCl 0.5 M 6.0 ml 1 M Tris (pH 8.0) 10 mM 1.2 ml 0.5 M EDTA 1 mM 532.8 ml distilled H 2 O R N a se A 5 ml final concentration 50 mg RNase A (Sigma) 10 mg/ml 50 pi 1 M Tris (pH 7.5) 10 mM 15 pi 5 M NaCl 15 mM 4.9 ml distilled H 2 O Do not boil, dispense into aliquots, and store at -20° B u ffe r A 800 ml final concentration 80 ml I M Tris (pH 7.5) 100 mM 24 ml 5 M NaCl 150 mM 696 ml distilled H 2 O B u ffe r B 6QQ ml final concentration 598 ml buffer A 6 g BSA (Boehringer Mannheim) 1 % 1.8 ml Triton X-100 0.3% B u ffe r C 230 ml final concentration 23 ml 1 M Tris (pH 9.5) 100 mM 4.6 ml 5 M NaCl 100 mM 11 .5 ml 1 M MgCI2 50 mM 191 ml distilled H 2 O LIST OF ABBREVIATION 2,4-D 2,4-dichlorophenoxyacetic acid ADP adenosine diphosphate ATP adenosine triphosphate BCIP 5-bromo-4-chloro-3-indolyl-phosphate bp base pair BSA bovine serum albumin CAM cell adhesion molecule cDNA complementary DNA DEPC diethylpyrocarbonate dig digoxygenin DTT dithiotheritol EDTA ethylene diamine tetraacetate ELK glutamic acid, leucine, lysine EtBr Ethidium bromide GIT guanidine isothiocyanate GUS P-glucuronidase HFSBP6 hormone-free SBP6 medium kb kilobase kD kilodalton mRNA messenger RNA MS3M MS salts, B5 vitamin, 3% maltose NBT nitro blue tetrazolium NETS NaCl, EDTA, Tris, SDS 146 147 NMR nuclear magnetic resonance NTE NaCl, Tris, EDTA ORF open reading frame PBS phosphate buffered saline PEG polyethylene glycol pfu plaque forming unit Pkc cytosolic pyruvate kinase pkpa a-subunit of plastid pyruvate kinase Pkp plastid pyruvate kinase PYP proline, tyrosine, proline rpm revolution per minute RT reverse transcription SAM substrate adhesion molecule SBP6 soybean proliferating medium containing6% sucrose SDS sodium dodecylsulfate SSC sodium chloride, sodium citrate TE Tris, EDTA Tris Tris base