Phaseolin Gene Research Articles

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Nucleotide diversity based on phaseolin and iron reductase genes in common bean accessions of different geographical origins

Discriminating genotypes within plant collections is imperative, and DNA sequence approaches for detecting single nucleotide polymorphisms (SNPs) have proved essential in any modern analysis of germplasm. By sequencing the α-Phs and PvFRO1 genes that, respectively, encode phaseolin and an iron reductase, we prospected for SNPs in exonic and intronic regions of both genes in a sample of 31 accessions of Phaseolus vulgaris from Mesoamerican and Andean gene pools, and one accession of Phaseolus lunatus, chosen as an outgroup. Sequence alignment showed 95 SNPs in α-Phs and 83 in PvFRO1, but diversity along the nucleotide sequences was not evenly distributed in both genes. Accessions from the same gene pool showed greater similarity than those from different gene pools, and the cluster patterns obtained in this study were consistent with the hierarchical organization into two P. vulgaris gene pools. The polymorphisms detected in the α-Phs gene allowed better discrimination among the accessions within each cluster than the PvFRO1 polymorphisms. Furthermore, some variations within exons changes amino acids in both predicted protein sequences. In an unprecedented result, the phaseolin-predicted amino acid variation allowed most of the accessions to be typified.

Gene Networks and Chromatin and Transcriptional Regulation of the Phaseolin Promoter in Arabidopsis

The complete lack of seed storage protein expression in vegetative tissues and robust expression during embryogenesis makes seed development an ideal system to study tissue-specific expression of genes. The promoter for the Phaseolin (phas) gene, which encodes the major seed storage protein in bean (Phaseolus vulgaris), is activated in two sequential steps: Phaseolus vulgaris ABI3-like factor (Pv-ALF)-dependent potentiation and abscisic acid-mediated activation. In this study, a heterologous in vivo Pv-ALF/phas-GUS (for β-glucuronidase) expression system in transgenic Arabidopsis thaliana leaves was used in conjunction with the powerful RNA-Seq approach to capture transcriptional landscapes of phas promoter expression. Remarkably, expression of over 1300 genes from 11 functional categories coincided with changes in the transcriptional status of the phas promoter. Gene network analysis of induced genes and artificial microRNA-mediated loss-of-function genetic assays identified transcriptional regulators RINGLET 2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5) as being essential for phas transcription. Pv-ALF binding to the RLT2 and AIL5 promoter regions was confirmed by electrophoretic mobility shift assay. RLT2 and AIL5 knockdown lines displayed reduced expression of several endogenous seed genes, suggesting that these factors are involved in activation of endogenous Arabidopsis seed storage gene expression. Overall, the identification of these key factors involved in phas activation provides important insight into the two-step transcriptional regulation of seed-specific gene expression.

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (Phaseolus vulgaris L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome.

Chromatin structure and phaseolin gene regulation.

Chromatin structure, the organized packaging of DNA with histones in the nucleus, is now seen as a dynamic fabric that changes with development. Here, we use studies on the phaseolin (phas) gene that encodes a seed protein to show how chromatin structure interacts with the transcription machinery to accomplish rigorous spatial regulation of expression. In leaf and other vegetative tissues, a nucleosome is rotationally and translationally positioned over an ensemble of three phased TATA boxes, denying access to TBP. Current interest focuses on the mechanisms by which this architecture is remodeled during embryogenesis. The transcription factor PvALF is intrinsically involved, as are other non-histone proteins and abscisic acid. These concepts, and the possible modular nature of phas expression, are summarized together with speculations concerning the re-establishment of the nucleosome over the phas promoter during terminal stages of embryogenesis.

PHASEOLIN RFLP IN BEAN (PHASEOLUS VULGARIS) CULTIVARS

A 0.5 kbp phaseolin gene fragment was used as a probe to study phaseolin restriction fragment length polymorphism (RFLP) in P. vulgaris cultivars. Results obtained from plants displaying type S and type T phaseolin suggest a genomic organization more variable than that previously described. This variability could be exploited to aid gene identification as well as to improve germ plasm bank organization. The same probe was useful in detecting phaseolin linked RFLP in other leguminosae.

Beta-Phaseolin gene activation is a two-step process: PvALF- facilitated chromatin modification followed by abscisic acid-mediated gene activation.

We have shown previously that a rotationally and translationally positioned nucleosome is responsible for the absence of transcriptional expression from the phaseolin (phas) gene promoter in leaf tissue and that the repressive chromatin structure is disrupted on transcriptional activation during embryogenesis. To investigate how the chromatin structure is modified, we ectopically expressed PvALF, a putative seed-specific phas activator, in leaf tissue of a tobacco line transgenic for a chimeric phas/uidA construct. DNase I footprinting in vivo revealed that the ectopic expression of PvALF resulted in remodeling of the chromatin architecture over the TATA region of the phas promoter but did not lead to transcriptional activation in the absence of abscisic acid (ABA). Treatment of the transgenic tobacco leaves with ABA in the absence of PvALF neither alleviated the repressive chromatin architecture nor activated transcription. However, in the presence of PvALF, high levels of beta-glucuronidase expression were obtained on exposure of leaves to ABA. These results reveal that expression from the phas promoter involves at least two discrete steps: chromatin potentiation by PvALF followed by ABA-mediated transcriptional activation.

Participation of chromatin in the regulation of phaseolin gene expression

Summary It has long been recognized that multiple transcription factors contribute to the overall regulation of gene expression. Since it is inconceivable that a specific transcription factor is assigned to each proteinencoding gene, it has been suggested that several factors interact in various combinations (Goldberg, 1988; Thomas, 1993). However, it is not clear if such combinations are established by the relative linear positions of the corresponding cis-element, by the order in which binding sites are occupied (providing alternative surfaces for protein-protein interactions), or by both of these parameters, possibly in association with other influences. One of these influences is undoubtedly chromatin. While this has previously considered being only a repressive structure, the recent finding that several transcriptional regulators can acetylate histones is rapidly leading to the conclusion that modification of histones, and thus chromatin, is a vital step in activation of the transcriptional machinery (Wade and Wolffe, 1997). Evidence for replacement of the repressive nucleosome structure by TFIID during transcription activation has been postulated by Van Holde and Zlatanova (1996) and may well be involved in phas promoter activation. Discerning actual steps by which the repressive nucleosome structure is modified or displaced will provide new insight to plant gene regulation, and it is tempting to think that histone acetylation will be part of the key. Once the chromatin structure is relaxed, transcription factors can gain access to the promoter, and a careful evaluation of changes in cis-element occupancy during embryogenesis using the sensitive DMS footprinting and LMPCR analysis technique will give important clues to the types of proteins involved. Another future opportunity for these approaches will be to explore events that lead to the cessation of expression as the embryo matures.

Induction of a beta-phaseolin promoter by exogenous abscisic acid in tobacco: developmental regulation and modulation by external sucrose and Ca2+ ions.

Phaseolin genes are induced by unidentified factors at the onset of seed maturation in embryos of both Phaseolus and tobacco. We show that in tobacco, expression of a beta-phaseolin promoter-GUS (PHSbeta-uidA) mRNA and the corresponding GUS activity, could be induced by abscisic acid (ABA). The effect paralleled an increase in the amount of endogenous 12S globulin (Glb12S) mRNA. In contrast, ABA repressed the expression of isocitrate lyase (ll9) mRNA. The responses of PHSbeta-uidA and Glb12S to ABA declined markedly between 11 and 13 DAF, indicating that they are developmentally regulated. We also show evidence that the ABA response of PHSbeta-uidA can be modulated by the external concentrations of sucrose and Ca2+ ion. These compounds inhibited the response if added to the medium separately, in the concentration ranges of 80-200 mM for sucrose and 0.76-20 mM for CaCl2. However, the presence of both sucrose and CaCl2 restored the ABA response to 20-40% of the maximum value measured in sucrose- and CaCl2-free media. These results suggest that ABA induction of beta-phaseolin gene expression is modulated by developmental signals and by the external supply of sucrose and calcium to the embryos.

Pulsed-field gel electrophoresis analysis of the phaseolin locus region inPhaseolus vulgaris

Phaseolin is the major seed storage protein of common bean (Phaseolus vulgaris L.). It is encoded by a small multigene family of 6-9 genes that are clustered in a single complex locus (Phs). We have constructed a long-range restriction map of the phaseolin genomic region, including the Phs locus and two flanking marker loci, D1861 and Bng060. Using a combination of high molecular weight DNA isolation, one- and two-dimensional pulsed-field gel electrophoresis of single and double restriction digests followed by Southern hybridization, and PCR analysis of individual fragments, we found that: (i) the maximum size of the Phs locus is 190 kb, (ii) the Phs locus may have increased in size during the evolution of P. vulgaris, (iii) the genomic region marked by D1861-Phs-Bng060 spans 5 cM, which corresponds to a maximum of 1.9 Mb, and (iv) the Phs locus could be oriented with respect to the two adjacent markers. Further progress in determining the gene arrangement in the Phs locus will require cloning and analysis of large DNA fragments containing phaseolin genes via BAC libraries. Key words : multigene family, physical distance, genome mapping, seed protein.

A novel basic region/helix-loop-helix protein binds to a G-box motif CACGTG of the bean seed storage protein β- phaseolin gene

Expression of the bean seed storage protein β-phaseolin is under strict developmental control primarily at the level of transcription. A G-box motif CACGTG has been shown to be a major positive cis-acting element of the β- phaseolin gene and to be recognized by a DNA binding protein from bean seed nuclei. To understand the molecular nature of the G-box-binding protein, we screened a cDNA expression library from immature bean seed and identified a positive clone based on binding activity of the proteins expressed in Escherichia coli to an oligodeoxyribonucleotide probe. DNA sequence analysis showed that the cDNA for the phaseolin G-box-binding protein (PG1) encodes a basic region/helix-loop-helix (bHLH) domain near the carboxyl terminus. Sequence comparison with other known plant bHLH proteins such as maize R B and Antirrhinum DEL suggests that PG1 represents a new member of bHLH protein family in plants. Electrophoresis mobility-shift and DNase I footprinting assays demonstrated that PG1 protein binds preferentially to the G-box (CA CG TG) and not to other two phaseolin E-box motifs (CA CC TG) and CA TA TG). Expression of PG1 RNA was detectable in all stages of seed development as well as inflower, root, and leaf. We discuss the implications of the bHLH protein for transcriptional regulation of the β- phaseolin gene.

Cell Ablation Reveals That Expression from the Phaseolin Promoter Is Confined to Embryogenesis and Microsporogenesis.

Most previous studies of the [beta]-phaseolin (phas) gene, which encodes the major storage protein in bean (Phaseolus vulgaris L.), have shown its expression to be rigorously confined to the developing seed, both in bean and transgenic tobacco (Nicotiana tabacum L. cv Xanthi) plants. To confirm unequivocally the lack of phas expression in vegetative tissues, we placed the diphtheria toxin A-chain (DT-A) coding region under the control of [beta]-phaseolin promoter sequences. Tobacco plants transgenic for phas/DT-A were phenotypically normal until flowering, when they produced anthers that were externally normal but contained no viable pollen. Microscopic examination of immature anthers revealed a normal tapetum, but the pollen mother cells did not undergo meiosis and subsequently degenerated, resulting in male-sterile plants. This demonstration of phas expression during microsporogenesis was corroborated by the expression of [beta]-glucuronidase in pollen of plants transformed with comparable phas/uidA constructs. Although these findings suggested that similarities in phas expression may exist between seed and pollen maturation, no phas activity could be detected in bean pollen. After fertilization of the DT-A-transformed plants with pollen from wild-type tobacco, 50% of the resulting embryos aborted at the heart stage, defining this as the earliest time for phas expression during embryogenesis.

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm.

The seed storage protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice seed storage protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

Detection of rRNA and phaseolin genes on polytene chromosomes of Phaseolus coccineus by fluorescence in situ hybridization after pepsin pretreatment

This is the first report of fluorescence in situ hybridization (FISH) on plant polytene chromosomes. Different protease pretreatments have been tested to improve fluorescence in situ hybridization FISH on polytene chromosomes of a plant, Phaseolus coccineus, with the aim to enable the detection of low-copy genes. The structural preservation of the chromosomes and the distinctness of the FISH signals were comparatively analysed with a probe for the ribosomal RNA genes after digestion with pepsin and trypsin. The pepsin pretreatment resulted in a general loosening of chromatin with good conservation of chromosome morphology and an increased number and density of signal points. The six nucleolus organizers exhibited significant differences in condensation. The pretreatment with pepsin enabled the detection of the low-copy genes encoding the seed storage protein phaseolin.

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 β-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 β-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 β-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 β-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.

Constitutive expression of the ?-phaseolin gene in different tissues of transgenic alfalfa does not ensure phaseolin accumulation in non-seed tissue

Phaseolin is a glycoprotein that constitutes the major storage protein in bean seeds. The phaseolin gene promoters function in a seed-specific manner. In an attempt to understand if events following transcription of the gene also contribute to the seed-specific accumulation of the phaseolin protein, we studied the effect of substituting the constitutive CaMV-35S promoter for the beta-phaseolin gene promoter on expression of the phaseolin gene in different plant organs. A chimeric gene consisting of the 35S promoter, the coding sequence of the beta-phaseolin gene (all five introns and six exons) and the 3'-flanking region of the beta-phaseolin gene, was introduced into alfalfa via Agrobacterium tumefaciens. While all organs examined shared high levels of phaseolin transcripts, the only organ that showed significant accumulation of the phaseolin protein were the mature seeds. Co-migration of the major immunoreactive polypeptides from the non-seed organs with the authentic beta-phaseolin polypeptides on SDS-PAGE indicates that the protein in non-seed organs undergoes correct post-translational processing and modification, but are more unstable in a non-seed environment.

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene 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 beta-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 (beta wti-) and mutant phaseolin glycoforms (beta dgly1, beta dgly2 and beta dgly1,2) 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 beta dgly1 and beta dgly2 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 beta dgly1,2 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.

Expression of Bean Storage Protein Minigene in Tobacco Seeds: Introns are not Required for Seed Specific Expression

Summary A native and mini (intronless) phaseolin gene were inserted into a mutant derivative of Agrobacterium tumefaciens plasmid pTi15955, in which the oncogenic tms2 and tmr loci were deleted. This pTi vector is referred to as a rooty-shooty mutant, pTiRS 2 014. The vector pTiRS 2 014 was used for the transfer and integration of phaseolin genes into the genome of tobacco followed by the regeneration of phenotypically normal tobacco plants. The presence of phaseolin polypeptides in R 1 and R 2 seeds was confirmed by immunological methods. These results demonstrate that native and mini-phaseolin genes are integrated stably into the respective tobacco plant lines and that the absence of the phaseolin introns did not affect its developmental expression.

Localization of phaseolin genes in the polytene chromosomes ofPhaseolus coccineus (Leguminosae)

The location of the genes for the major storage protein, phaseolin, was determined in the polytene chromosomes of the embryo-suspensor ofPhaseolus coccineus, cv. Hammond's Dwarf Scarlett, byin situ hybridization with a35S-labelled cloned probe. The genes were adjoined to chromosome pair no. 3. The distribution of the label indicates a puffed state of the sites during embryogenesis of the legume studied. The location of the phaseolin genes was certified by hybridization of a ribosomal DNA probe to the polytene chromosomes, because their position is well known.