PvAlf, an embryo-specific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters.

Mutations in Vp1 and ABl3 genes of maize and Arabidopsis lead to drastic reductions in the synthesis of a subset of maturation-specific products including seed storage proteins. Gene Phaseolus vulgaris ABl3-like factor (PvAlf), whose protein product is similar to the ABl3 and Vp1 proteins, has been cloned. Here, it is shown that PvAlf positively regulates phaseolin and phytohemagglutinin (PHA-L) promoters in particle bombardment assays. PvAlf mRNA expression is embryo-specific and temporally complex. PvAlf mRNA abundance is highest during two periods (9-14 and 22-35 days after flowering) that precede the onsets of seed maturation and seed abscission, respectively. Protein fusions with the DNA-binding domain of the yeast transcriptional activator GAL4 demonstrated that the N-terminal 243 amino acids of PvAlf function as a strong transcriptional activation domain in yeast (Saccharomyces cerevisiae) and plant cells. This domain consists of a central cluster rich in serine, threonine and proline (STP cluster) flanked by two negatively charged regions containing bulky hydrophobic residues similar to acidic activation domains of Vp1, the herpes simplex virus virion protein VP16 and transcription factors GCN4 and HAP4 from yeast. Together with the Vp1 proteins of maize and rice and ABl3, PvAlf constitutes a class (Vp1/ABl3-like factors or VAlfs) of regulatory factors that are pivotal for the promotion of seed maturation and dormancy in angiosperms.

The developmentally regulated bZIP factor ROM1 modulates transcription from lectin and storage protein genes in bean embryos.

Expression of the MAT genes DLEC2 and PHS beta, coding for phytohemagglutinin L-subunit and beta-phaseolin, respectively, is coordinately induced in bean embryos at the onset of seed maturation and regulated at the level of transcription. Previously, it was reported that expression of DLEC2 and PHS beta genes is positively regulated by the acidic transcription activator PvALF. This article describes the regulator of MAT1 (ROM1), a basic/leucine-zipper protein that binds to hybrid G box/C box motifs found on the DLEC2 and PHS beta seed enhancers. ROM1 repressed DLEC2- and PHS beta-driven expression and antagonized transactivation of either promoter by PvALF, in a dosage-dependent manner. By contrast, a PvALF/ROM1 hybrid protein activated transcription. Both repression by ROM1 and activation by the hybrid protein required the presence of intact G-box/C-box motifs. The abundance of ROM1 mRNA in embryos was highest during the cotyledon stage and decreased rapidly at the beginning of maturation, as phytohemagglutinin and phaseolin mRNAs were induced. The results demonstrate that ROM1 is a DNA-binding site-dependent repressor and point to its possible role as a negative modulator of PvALF-activated transcription of MAT genes.

Conserved RY-repeats mediate transactivation of seed-specific promoters by the developmental regulator PvALF.

Transcription of genes DLEC2 and PHSbeta is specifically and coordinately regulated during maturation of Phaseolus embryos. Over-expression of the seed- specific factor PvALF in cotyledon cells results in transactivation of either promoter. PvALF is related to the VP1 protein of maize, which transactivates gene expression via G-boxes, Sph elements and AT-rich sequences. We used deletions and base substitutions in the DLEC2 and PHSbeta promoters to demonstrate that several conserved RY-repeats were necessary for PvALF induction of both genes. A comprehensive mutational and transactivation analysis was used to define functionally the sequence of the DLEC2 repeat RY3 asG/CCATGCxxG/C. We also found that an interaction between RY3 and the 3'-flanking tetranucleotide CCAC increased PvALF transactivation. A preferred spacing and phasing requirement for the RY3 and CCAC motifs suggested the possibility of interactions between cellular factors that recognize either element. The high conservation of Sph-RY motifs in seed-specific promoters from monocots and dicots indicates that organ and temporal specification by factors similar to VP1 and PvALF is common among seed plants.

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.

The regulator of MAT2 (ROM2) protein binds to early maturation promoters and represses PvALF-activated transcription.

The regulation of maturation (MAT)- and late embryogenesis (LEA)-specific gene expression in dicots involves factors related to ABI3, a seed-specific component of the abscisic acid signal transduction pathways from Arabidopsis. In French bean (Phaseolus vulgaris), the ABI3-like factor, PvALF, activates transcription from MAT promoters of phytohemagglutinin (DLEC2) and beta-phaseolin (PHS beta) genes. We describe the regulator of MAT2 (ROM2) as a basic leucine zipper (bZIP) DNA binding protein that recognizes motifs with symmetric (ACGT) and asymmetric (ACCT) core elements present in both MAT promoters. ROM2 antagonizes trans-activation of the DLEC2 promoter by PvALF in transient expression assays. Repression was abolished by mutations that prevented binding of ROM2 to the DLEC2 seed enhancer region. Moreover, a hybrid protein composed of a PvALF activation domain and the DNA binding and dimerization domain of ROM2 activated gene expression, indicating that ROM2 recognizes the DLEC2 enhancer in vivo; consequently, ROM2 functions as a DNA binding site-dependent repressor. Supershift analysis of nuclear proteins, using a ROM2-specific antibody, revealed an increase in ROM2 DNA binding activity during seed desiccation. A corresponding increase in ROM2 mRNA coincided with the period when DLEC2 mRNA levels declined in embryos. These results demonstrate developmental regulation of the ROM2 repressor and point to a role for this factor in silencing DLEC2 transcription during late embryogenesis.

Developmental modulation of tubulin protein and mRNA levels during somatic embryogenesis in cultured carrot cells.

The number of cortical microtubules (MTs) increases considerably as cultured carrot (Daucus carota L.) cells initiate and progress through somatic embryogenesis. The basis for this increase in MT number was investigated. A radioimmune assay was used to show that tubulin-protein per cell first decreased as the undifferentiated cells initiated embryonic development, but subsequently increased approximately fivefold between the globular and torpedo/plantlet stages. The increase during the torpedo/plantlet stage was correlated with the increase in cell size that occurred during the latter stages of embryogenesis. The cellular levels of tubulin mRNA were determined by Northern blot analysis, using labeled probes derived from soybean α- and ß-tubulin genomic sequences, cloned in the vectors pSP64 and pSP65. This analysis demonstrated that the levels of tubulin-gene transcripts varied with the tubulin-protein levels. Cell-free translation of polyadenylated RNA, followed by immunoprecipitation with an anti-tubulin antiserum, established that these transcripts represented functional tubulin mRNA. These results indicate that MT formation in early embryogenesis is controlled by factors other than the availability of tubulin, but that MT formation later in embryogenesis is coordinated with concomitant changes in tubulin-gene transcription and in the size of the total tubulin-heterodimer pool.

Light Regulation of beta-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.).

The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of beta-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)(+)RNA with a probe derived from the coding region of a previously characterized soybean beta-tubulin gene. (MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of beta-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of beta-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of beta-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.

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.

Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter.

Mutations affecting spatial and temporal regulation of a beta-phaseolin gene encoding the major storage protein of bean (Phaseolus vulgaris) were analyzed by stable and transient transformation approaches. The results substantiate the value of transient assays for rapid determination of the functionality of cis-acting sequences and the importance of stable transformation to identify tissue-specific determinants. Spatial information is specified primarily by two upstream activating sequences (UAS). UAS1 (-295 to -109) was sufficient for seed-specific expression from both homologous and heterologous (CaMV 35S) promoters. In situ localization of GUS expression in tobacco embryos demonstrated that UAS1 activity was restricted to the cotyledons and shoot meristem. A second positive domain, UAS2 (-468 to -391), extended gene activity to the hypocotyl. Temporal control of GUS expression was found to involve two negative regulatory sequences, NRS1 (-391 to -295) and NRS2 (-518 to -418), as well as the positive domain UAS1. The deletion of either negative element caused premature onset of GUS expression. These findings indicate combinatorial interactions between multiple sequence motifs specifying spatial information, and provide the first example of the involvement of negative elements in the temporal control of gene expression in higher plants.

Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene.

A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.

Expression, glycosylation and secretion of phaseolin in a baculovirus system.

In this report, we describe the efficient expression and glycosylation, in insect cells, of ß-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/10(6) 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 (G¼C) 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.

Opportunities for bioactive compounds in transgenic plants.

A variety of bioactive compounds have now been introduced into plants through recombinant DNA techniques. Early examples included genes encoding proteins conferring herbicide tolerance and insect or virus resistance. More recently, pharmacologically useful compounds such as enkephalin and immunoglobulin have been produced in transgenic plants. Modification of existing compounds to provide better nutritional value or improved functional properties is exemplified in the case of seed storage proteins. The value of RNAs as bioactive compounds for suppression of undesirable products and viral infection has now been demonstrated in plants. The developmentally regulated expression of novel bioactive compounds in defined tissues, and their targeting to specific subcellular locations, is becoming of ever increasing economic and sociological importance as knowledge of the molecular mechanisms involved accumulates.

The isolation, characterization and sequence of two divergent ß-tubulin genes from soybean (Glycine max L.).

Two divergent ß-tubulin genes (designated Sß-1 and Sß-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii ß-tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different ß-tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of ß-tubulin genes (thus far undetected) exist in the soybean genome. The Sß-1 and Sß-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode ß-tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with ß-tubulins from several organisms showed that they are most homologous to Chlamydomonas ß-tubulin (85-87%), with lesser degrees of homology to ß-tubulins of vertebrate species (79-83%), Trypanosoma brucei (80-81%) and Saccharomyces cerevisiae (66-68%). The amino acid sequences of Sß-1 and Sß-2 are as divergent from each other as they are from the Chlamydomonas ß-tubulin. The amino acids at the diverged positions in Sß-2 are nearly all conservative substitutions while in Sß-1, 18 of the 69 substitutions were non-conservative. Both soybean ß-tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas ß-tubulin genes. Codon usage in the two soybean ß-tubulins is remarkably similar (D (2)=0.87), but differs from codon usage in other soybean genes.