Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

Expression of DNA binding proteins in carrot somatic embryos that specifically interact with a cis regulatory element of the French bean phaseolin gene

Nuclear proteins from bean (Phaseolus vulgarus) embryos bind specifically to a 55 bp DNA sequence located upstream of the seed storage protein gene phaseolin. This sequence is capable of elevating gene expression in transgenic tobacco plants by as much as 150-fold when fused to a chimeric -glucuronidase reporter gene. Results presented in this paper demonstrate that nuclear extracts from carrot embryos bind to a phaseolin DNA sequence that includes a phaseolin activator sequence. This specific DNA binding activity is modulated during somatic embryogenesis. Two separable protein species react specifically with the labeled phaseolin DNA fragment (58.0 and 51.7 dDa). These results suggest that the cis- and trans-acting elements controlling gene expression have been highly conserved during evolution.Abbreviations bp base pairs - CAMV cauliflower mosaic virus - GUS -glucuronidase - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A modified storage protein is synthesized,processed, and degraded in the seeds of transgenic plants

In vitro mutagenesis was used to supplement the sulfur amino acid codon content of a gene encoding -phaseolin, a Phaseolus vulgaris storage protein. The number of methionine codons in the phaseolin gene was increased from three to nine by insertion of a 45 base pair (bp) synthetic duplex. Either modified or normal phaseolin genes were integrated into the genome of tobacco plants through Agrobacterium tumefaciens-mediated transformation. Although similar levels of phaseolin RNA are detected in seeds of plants transformed with either the normal or modified (himet) gene, the quantity of himet protein is consistently much lower than normal -phaseolin. Himet phaseolin is expressed in a temporal- and organ-specific fashion, and is N-glycosylated and assembled into trimers in the manner of normal phaseolin. After germination, both types of phaseolin are hydrolyzed, but the himet protein is more quickly degraded. Electron microscopic immunocytochemical observations of developing seeds indicate that the himet protein is primarily localized in the endoplasmic reticulum (ER) and in Golgi apparatus secretion vesicles. Himet phaseolin is absent from protein storage vacuoles, termed protein bodies, where normal phaseolin is deposited in transgenic tobacco. We interpret the immunocytochemical data to indicate that himet phasolin is transported through the ER and Golgi apparatus and is then degraded in Golgi secretion vesicles or the protein bodies.Mention of trademark, proprietary product, or vendor does not constitute a guarantee of warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Publication No. 70 from Agrigenetics Advanced Science Company.     

Bean arcelin

Summary SDS-PAGE of seed proteins from the seeds of a nondomesticated bean of Mexican origin (Phaseolus vulgaris L., PI 325690) revealed the presence of a novel 38 kd protein which appeared to be neither an altered phaseolin nor a lectin fraction. The protein was named arcelin, after Arcelia, the town in the state of Guerrero near which PI 325690 had been collected. The pure line, UW 325, was derived by self fertilization of the plant from a single arcelin-containing seed of PI 325690. Despite a low percentage seed phaseolin (14.6%), seed phenotype, seed germination, plant growth, pollen fertility, and percentage seed protein of UW 325 were normal. Analyses of F₂ and F₃ seeds from a single F₁ plant of the cross SanilacXPI 325690-3 revealed that arcelin expression was inherited as a single gene and that presence was dominant to absence of arcelin. The mean percentage phaseolin in the seeds of homozygous dominant Arc/Arc F₃ families (14.0%) was significantly lower than that of the homozygous recessive arc/arc seeds (44.7%). The distribution of percentage phaseolin values for seeds within segregating families was bimodal and nonoverlapping. Without exception, seeds containing arcelin (Arc+phenotype) contained a lower percentage phaseolin than seeds lacking arcelin (Arc-phenotype). Although arcelin presence was associated with low percentage phaseolin, the Arc/Arc and Arc/arc genotypes were similar for seed weight and percentage total seed protein.     

Enhanced available methionine concentration associated with higher phaseolin levels in common bean seeds

Summary The relationship between available methionine concentration and the levels of phaseolin — the major seed storage proteins of the common bean — was studied using three groups of genetic materials: First, the F₂ progenies of interspecific crosses between P. vulgaris cultivars and aP. coccineus subsp. coccineus line (cv. Mexican Red Runner) having no detectable phaseolin; second, the F₂ progenies and segregating F₃ families of crosses between cultivated P. vulgaris lines and a Mexican wild bean accession (PI 325690-3) carrying a gene producing a reduction in phaseolin content; third, two inbred backcross populations: SanilacxBush Blue Lake 240 (population 2) and Sanilacx15R 148 (population 6). Total seed N levels were determined by micro-Kjeldahl, phaseolin levels by rocket immunoelectrophoresis and available methionine levels by the Streptococcus zymogenes bioassay. Our results indicate that in all the genetic materials studied, with the exception of population 6, higher phaseolin levels lead to increased available methionine concentration. Although phaseolin has a low methionine concentration, it is actually a major source of available methionine in common bean seeds, because it represents a large part of total seed nitrogen and because limited differences exist between the methionine concentrations of the different protein fractions. This contrasts with the situation in cereals such as maize, barley and sorghum, where increased levels of the major limiting amino acid (lysine) can be achieved through a decrease in the amounts of the main seed storage protein fraction (prolamines). In population 6, no relationship was observed between available methionine and phaseolin content. Other factors, such as additional methionine-rich polypeptides or the presence of tannins, might obscure the positive relationship between phaseolin and available methionine content in population 6.     

Apical and lateral shoot apex-specific expression is conferred by promoter of the seed storage protein β-phaseolin gene

A previous analysis with deletion mutants of the native -phaseolin gene demonstrated that removal of a negative element 5 upstream of–107 permitted phaseolin expression in stem cortex and secondary root (Burowet al., 1992). Here we employed the -glucuronidase (GUS) reporter gene to visualize, by histochemical staining, the cell type-specificity of phaseolin expression in stem and root, and to understand further the spatial control of the -phaseolin gene. The 782 bp 5 upstream promoter and its deletion mutants were fused to the GUS gene, and these chimaeric genes were used to transform tobacco. Histochemical staining for GUS activity demonstrated that phaseolin promoters truncated downstream of –227 conferred cell-type specific expression in internal/external phloem and protoxylem of mature stem. Surprisingly, GUS staining was prominent in both apical and lateral shoot apices of plants that contain the full-length –782 promoter and mutant promoters deleted up to –64. GUS expression was extended to all cell types of shoot tips, including epidermis, cortex, vasculature, procambium and pith. Expression in vasculature of petioles was limited to plants with promoters truncated to –106 and –64. The current results are in agreement with our previous findings with the native phaseolin gene: that the major positive element (–295/–228) is sufficient for seed-specific late-temporal expression of the phaseolin gene. We conclude that the 5 upstream sequence of the -phaseolin gene directs spatially- and temporally-controlled gene expression in developing seeds during the reproductive phase, but also confers expression in shoot apices during the vegetative phase of plant development.     

Expression,glycosylation and secretion of phaseolin in a baculovirus system

In this report, we describe the efficient expression and glycosylation, in insect cells, of -phaseolin polypeptides (M _r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M _r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/10⁶ cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.     

Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope himet (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.Abbreviations ER endoplasmic reticulum - BiP binding protein - IgG immunoglobulin G - M_r relative molecular mass The mention of vendor or product does not imply that they are endorsed or recommended by the US Department of Agriculture over vendors of similar products not mentionedThis work was supported by a grant from the National Science Foundation (Cell Biology) to M.J. Chrispeels and a fellowship from the Ministry of Education and Science, Spain-Fullbright Program to J.J. Pueyo. We thank H. Pelham for a gift of the constructs containing c-myc-SEKDEL and cmyc-FEHDEL and for a gift of anti-HDEL monoclonal antibodies. The original HiMet phaseolin construct was made by L. Hoffman and the phaseolin-HDEL or KDEL and HiMet-HDEL or KDEL constructs were made by D. Hunt as part of his doctoral research.     

Linkage between isozyme markers and a locus affecting seed size in Phaseolus vulgaris L.

Summary Backcross and F₂ progenies were produced between two bean genotypes, XR-235 and Calima, which differ in seed weight by a factor of two. The small-seeded XR-235 was used as the pistillate and recurrent parent. These genotypes showed polymorphisms at nine isozyme loci and at the phaseolin locus. Seed size parameters (weight, length, width, and thickness) were determined for each BC₁ and F₂ individual, i.e., for seeds harvested from XR-235 after pollination with F₁ and from the F₁ after selfing, respectively. A combination of starch gel electrophoresis and enzyme activity staining was used to determine the genotype of each BC₁ and F₂ individual at the segregating loci. SDS-PAGE and Coomassie blue staining were used to determine geno-type at the phaseolin locus. Tests for independent assortment using two-way contingency and maximum likelihood tables revealed three linkage pairs: Aco-1 — 20 cM — Dia-1; Adh-1 — 2 cM — Got-2; and Est-2 — 11 cM — Pha. Statistical comparisons were made between the means of genotype classes at each segregating locus for all seed size parameters. The results from two independently obtained BC₁s and the F₂ consistently indicated that the Adh-1-Got-2 segment was linked to a locus that affected seed size and overcame maternal control over seed size. This locus has been designated Ssz-1. This gene exhibited additive gene action and accounted for 30–50% of the seed size difference between the parents.Florida Agricultural Experiment Station, Journal Series No. R00696     

Correlated changes in steady-state levels of Balbiani ring mRNAs and secretory polypeptides in salivary glands of Chironomus tentans

Balbiani rings (BRs) on polytenized chromosomes in Chironomid salivary glands contain members of a homologous multigene family that encodes a family (the sp-I family) of high M _r secretory polypeptides. Each of these BR genes is comprised largely of tandemly duplicated core repeat sequences consisting of related constant (C) regions and intergenically divergent subrepeat (SR) regions. A set of oligodeoxyribonucleotide probes were synthesized that correspond to the transcribed strand of the SR region of BR1, BR2, BR2/, and BR6 core repeats. Under a defined set of conditions, it was possible to show that each oligonucleotide probe hybridized exclusively to its cognate repeat type without hybridization to other repeat types in cloned DNA templates. These BR probes were then used in dotblot hybridization experiments to simultaneously follow alterations in the steady-state level of BR mRNAs in response to prolonged exposure of larvae to galactose. The results indicated that the relative amounts of these four BR mRNAs may change in a noncoordinate manner. These BR probes were also used in experiments to compare simultaneously the salivary gland content of sp-I components and specific BR mRNAs in larvae that exhibited naturally occurring or induced alterations in BR gene expression. A correlation was found which suggested that sp-Ia is encoded in a gene comprised of BR1 repeats, sp-Ib is encoded by BR2 repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2 repeats.     

Pisum lipoxygenase genes

Summary The copy number, genomic arrangement and linkage relationships of two classes of lipoxygenase gene have been investigated in Pisum(pea) lines. Each of the two classes contained two to three members in P. sativum lines. RFLPs associated with genomic fragments containing the 5 sequences of one gene class permitted its correlation in genetical analyses with a lipoxygenase locus on linkage group 4, which was previously identified through polypeptide variation. Genetical analyses of RFLPs associated with other fragments identified by low- and medium-stringency hybridization to lipoxygenase cDNAs indicate the existence of other unlinked lipoxygenase gene loci.     

Immunosystematical comparison ofPhaseolus coccineus seed proteins with those of other legumes

An immunosystematical research was conducted on the seed proteins of 32 species ofFabaceae. By comparing all immunelectrophoretic patterns with the self reaction of a reference system (Phaseolus coccineus) all proteins detected have been identified and their distribution within the family has been analyzed. Half of the proteins identified inP. coccineus gave positive cross reactions with proteins present in all other species. Among this group are protein I and phaseolin. This result supports the homologization between phaseolin and vicilin. Among the other proteins, three are irregularly distributed throughout the family, and only three are restricted to a few taxa. This last group includes phytohaemagglutinin, which does not present any cross reaction outside the tribePhaseoleae.     

An ultrahigh-sulphur keratin gene of the human hair cuticle is located at 11q13 and cross-hybridizes with sequences at 11p15

A human hair cuticle ultrahigh-sulphur keratin (UHSK) gene (KRN1) has been mapped by Southern analysis of a somatic cell hybrid panel and by in situ hybridization. A probe containing the coding region of this gene mapped to 11pter->11q21 using the hybrid cell panel and on in situ hybridization mapped to two regions on chromosome 11: the distal part of 11p15, most likely 11p15.5, and the distal part of 11q13, most likely 11q13.5. A probe from the 3 non-coding region of KRN1 mapped to 11q13.5 indicating that this was the map location of the cloned gene. The sequence of 11p15.5 is termed KRN1-like (KRN1L). The results reveal that the cuticle UHSK gene family is clustered in the human genome. Present address: The Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3, 9DU, United Kingdom.     

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from -carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an -helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core -barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressedin vivo.     

Molecular gene mapping of human aldolase A (ALDOA) gene to chromosome 16

Summary Mapping of human aldolase A (ALDOA) gene was performed by molecular hybridization techniques using a panel of human-mouse cell hybrids and sorted fractions of human metaphase chromosomes besides in situ hybridization. For the purpose, three kinds of DNA probes derived from the coding region (probe-1), the 3 noncoding region (probe-2), and the coding and 3 noncoding regions (probe-3) of human aldolase A cDNA clone, pHAAL116-3, were selectively employed. The results of RNA and DNA blot analyses indicated that the human ALDOA gene is located on chromosome 16. The in situ hybridization experiment also indicated that the ALDOA gene was localized to 16q22–q24.     

Extensive modifications for methionine enhancement in the β-barrels do not alter the structural stability of the bean seed storage protein phaseolin

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. AnEscherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded inE. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61°C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the-barrel and loop structures, with extremely small effects on protein stability.     

No evidence for sequences structurally related to the RB1 gene in the human genome

Summary The retinoblastoma (RB1) gene is a ubiquitously expressed gene encoding a cell-cycle control protein. Inactivation of this gene plays a crucial role in the development of retinoblastoma, osteosarcoma, and other tumors. In a search for structurally related gene sequences we identified a 5.5-kb BamHI fragment strongly cross-hybridizing with the 5 end of the RB1 cDNA. Molecular cloning, in situ hybridization, restriction mapping, and sequence analysis identified this DNA segment as the 28S rRNA gene. The absence of other cross-hybridizing sequences suggests that the RB1 gene is not part of a structurally related gene family.     

Towards an integrated linkage map of common bean 2. Development of an RFLP-based linkage map

Summary A restriction fragment length polymorphism (RFLP)-based linkage map for common bean (Phaseolus vulgaris L.) covering 827 centiMorgans (cM) was developed based on a F₂ mapping population derived from a cross between BAT93 and Jalo EEP558. The parental genotypes were chosen because they exhibited differences in evolutionary origin, allozymes, phaseolin type, and for several agronomic traits. The segregation of 152 markers was analyzed, including 115 RFLP loci, 7 isozyme loci, 8 random amplified polymorphic DNA (RAPD) marker loci, and 19 loci corresponding to 15 clones of known genes, 1 virus resistance gene, 1 flower color gene, and 1 seed color pattern gene. Using MAPMAKER and LINKAGE-1, we were able to assign 143 markers to 15 linkage groups, whereas 9 markers remained unassigned. The average interval between markers was 6.5 cM; only one interval was larger than 30 cM. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios (121 or 31) and mapped into four clusters. Probes of known genes belonged to three categories: seed proteins, pathogen response genes, and Rhizobium response genes. Within each category, sequences homologous to the various probes were unlinked. The I gene for bean common mosaic virus resistance is the first disease resistance gene to be located on the common bean genetic linkage map.     

Selection for increased percentage phaseolin in common bean

Summary Two selection methods were compared to determine which was more efficient for increasing percentage phaseolin in the common bean (Phaseolus vulgaris L.). A base population consisting of families segregating for six seed protein alleles (Phas ^S , Phas ^C , Phas ^T , phas ^-, lec^-, and Arcl ⁺), all of which have measurable effects on percentage phaseolin, was subjected to either three cycles of S₁ family recurrent selection for increased percentage phaseolin (PPS), or one cycle of selection for combinations of the protein alleles (PAS) known to have positive effects on phaseolin accumulation. One cycle of PAS resulted in an increase in percentage phaseolin that was equivalent to three cycles of PPS. Selection under both methods produced increases in several correlated traits including percentage total protein, phaseolin as a percent of total protein, mg protein/seed, and mg phaseolin/seed. The amount of nonphaseolin protein per seed decreased, while seed yield was unaffected by either selection procedure. By selecting for favorable seed protein alleles identified by electrophoresis, it was possible to rapidly increase percentage phaseolin without the need for field evaluation.     

Maturation and germination of Phaseolus vulgaris embryonic axes in culture

In this study of embryo development in Phaseolus vulgaris L., we found that immature embryonic axes placed in culture show a growth lag before germinating. The length of this lag phase varies according to axis age at excision, but is not affected by transfer to fresh medium, alteration of sucrose concentration between 0.5 and 2%, or whether the culture medium is liquid or agar-solidified. The lag phase was shortened by both actinomycin D and cordycepin treatment, and by treatment with 10^-5 to 10^-6 M benzyladenine. The effect of abscisic acid (ABA) varied with concentration: below a certain level, it had no effect on the lag phase, but above that level it inhibited, germination. This threshold concentration was 10^-7 M for 20-d-old axes but increased to 10^-5 M by the time the axes were 32 to 34 d old. To determine whether the axes were continuing their embryonic development during the lag phase, we tested them for desiccation-tolerance and for synthesis of phaseolin, a seed storage protein which is specific for embryos of P. vulgaris. The ability to germinate after rapid desiccation was acquired by axes at 26 d past anthesis; when axes younger than this were placed in culture, they developed desiccation-tolerance during the lag phase of growth, indicating that they were continuing embryonic maturation. Phaseolin was present in isolated axes, although at lower levels than in cotyledons. It accumulated during axis development in parallel with total protein, staying at about 1% of total protein content. When isolated immature axes were pulsed with ³H-or ¹⁴C-amino acids, they incorporated label into phaseolin, shown by precipitation with anti-phaseolin antibody. Isolated axes from mature seeds, however, did not synthesize detectable amounts of phaseolin. Immature axes cultured in vitro for a period of one to several days continued synthesizing phaseolin until the day prior to visible germination. Treatment of cultured axes with ABA increased the amount of precursor amino acids incorporated into protein, but had a small or no effect on the relative proportion of phaseolin synthesized. We conclude that P. vulgaris axes in culture continue to develop embryonically for a period of time which seems to be under intrinisc control by the axis. This contrasts with precocious germanation, a pattern of embryo behavior seen in many other species. When such embryos are excised from seeds while immature and placed in culture, they switch promptly from embryo development into germination. If ABA or water stress is responsible for preventing precocious germination, it may be that a high level of ABA is maintained or synthesized internally by embryonic axes of Phaseolus, while in other embryos the maternal environment supplies ABA and/or causes water stress.Abbreviations ABA Abscisic acid - BA benzyladenine