Correct glycosylation,Golgi-processing,and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5 upstream and 1600 bp 3 downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571–3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.Abbreviations Asn asparagine - Endo H endoglycosidase H - HPLC high-performance liquid chromatography - IgG immunoglobulin G - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecylsulfate - TFMS trifluoromethanesulfonic acid     

Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco

The storage protein phaseolin accumulates during seed development in protein bodies in cotyledons of the common bean Phaseolus vulgaris. Hall et al. (In L Van Vloten-Doting, TC Hall, eds, Molecular Form and Function of the Plant Genome, 1985 Plenum Press, In press) recently reported the expression of a gene coding for phaseolin and the accumulation of phaseolin protein in developing seeds of tobacco plants regenerated from transformed callus cells. The protein did not accumulate in other organs of the plants. Mature seeds from normal and transformed tobacco plants were obtained and the subcellular distribution of phaseolin in the seeds was examined using both light and electron microscopic immunocytochemical methods. Phaseolin was found in six of seven transformed tobacco embryos examined, but was present in only one endosperm of five. When present, phaseolin was located exclusively in the protein bodies of the embryonic and endospermic cells. Furthermore, phaseolin was restricted solely to the amorphous matrix of the protein bodies and was excluded from the globoid and proteinaceous crystalloid components of these organelles. The subcellular location of phaseolin in seeds from transformed tobacco plants is similar to that seen in mature seeds of the common bean indicating that in the transformed cells the protein is targeted to the right subcellular compartment.     

A Single-Nucleotide Polymorphism in an Endo-1,4-β-Glucanase Gene Controls Seed Coat Permeability in Soybean

Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of β-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-β-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of β-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of β-1,4-glucan derivatives such as xyloglucan and/or β-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.     

Chromosomal integration is required for spatial regulation of expression from the β-phaseolin promoter

The stringency of spatial expression of phaseolin, the major storage protein of bean (Phaseolus vulgaris) seeds, has been rigorously evaluated using stable and transient transformation techniques. Transgenic tobacco plants known to be homozygous for the β-glucuronidase (gus) reporter sequence under the regulation of various lengths of the β-phaseolin gene (phas) promoter were shown to express gus only in developing seed tissues. No expression was detected in calli initiated from stems, leaves and immature seeds, showing that expression was not leaky in undifferentiated tissues. Control plants and cultures containing gus fused to the CaMV 35S promoter actively expressed gus under identical conditions. It was not possible to induce expression in phas/gus calli with ABA, GA or jasmonic acid. Treatment of the cultures with 5-azacytidine did not result in expression, excluding methyletlon as the major factor regulating the phas promoter. However, strong gus expression was detected in seed of plants regenerated from these callus cultures, confirming that neither gene rearrangements nor deletion were responsible for the lack of activity seen in tissues other than the developing seed. In contrast to the above observations, strong transient expression of gus was detected in tobacco, bean and soybean leaves following introduction of the phas/gus fusion constructs via biolistic approaches and in electroporated bean leaf and hypocotyl protoplasts. These experiments show unequi-vocally that the phas promoter is under rigorous spatial control when integrated into the genome, but lacks spatial control when present as extrachromosomal naked DNA. A putative model explaining these differences is presented.     

In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.     

Mung bean (<Emphasis Type="Italic">Vigna radiata</Emphasis>) cultivars mediated oviposition preference and development of <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis> (Coleoptera: Chrysomelidae: Bruchinae)

The Azuki bean weevil, Callosobruchus chinensis (L.), is a destructive pest of stored mung bean [Vigna radiata (L.) Wilczek] as well as other leguminous seeds. The development of resistant seeds to manage this pest is of current great interest to plant breeders. In this study, we investigated the oviposition preference and development of C. chinensis on two susceptible mung bean cultivars (Seonhwa and Gyeongseon) and one previously reported resistant cultivar (Jangan), compared to the susceptible cowpea (Vigna unguiculata L.), cultivar (Yeonbun) using both multiple-choice and no-choice tests. In addition, the development of C. chinensis was also examined at four constant temperatures (20, 25, 30, and 35 °C). Both tests found cowpea to be the most suitable seed for oviposition. Total developmental time from oviposition to adult emergence ranged from 27.01 to 38.2 days, being shortest on cowpea and longest on the mung bean, cv. Jangan. However, no successful development of C. chinensis larvae on mung bean, cv. Jangan, occurred at any temperature. The highest rate of adult emergence and the longest adult longevity both occurred on cowpea and certain mung bean cultivars (Seonhwa and Gyeongseon), with the dramatic exception of cv. Jangan. These results suggest that the higher preference and performance of C. chinensis on cowpea (3.3 egg/seed) and least on mung bean, cv. Jangan (0.4 egg/seed). This information may facilitate the exploration of resistant genetic materials and chemicals associated with seeds for successful breeding. Further studies should examine the chemicals associated with mung bean cultivars and its resistant mechanism to develop a control method against bruchines.     

Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean

Incubation of protoplasts with polyethylene glycol (PEG) and plasmid DNA containing the coding region for aminoglycoside phosphotransferase gene (NPT II) proved to be a simple transformation method for Nicotiana tabacum and Vigna aconitifolia, a drought-tolerant grain legume. In both plant species examined, the plant cultivar was an important factor, which clearly influenced transformation rates. The use of different expression signals derived from gene VI of the cauliflower mosaic virus (plasmid pABD1) or from the nopaline synthase gene of Agrobacterium tumefaciens (plasmid pLGV neo2103) also resulted in different frequencies of stably transformed colonies. Plants could be regenerated from kanamycin-resistant line of tobacco and moth bean.     

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [³H]glucosamine or [³H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M_r 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M_r 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [³H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.     

Effect of Canavanine from Alfalfa Seeds on the Population Biology of Bacillus cereus

Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685–691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B. cereus, Bacillus thuringiensis, and Vibrio cholerae. The B. cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B. cereus.     

Transcriptional Activities of a Winged Bean Kunitz Chymotrypsin Inhibitor Gene Promoter in Stable and Transient Expression Systems

Sequential deletions of the promoter region of the WCI-3b gene, which encodes the major chymotrypsin inhibitor of winged bean, were constructed and their expression was analyzed in transgenic tobacco plants and in bombarded winged bean seeds. In transgenic tobacco plants, a critical promoter region which is important for high levels of expression in seeds was identified, but deletion of this region had essentially no effect when bombarded into winged bean seeds.     

Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana

Reports from a number of laboratories describe the presence of a family of proteins (the major intrinsic protein family) in a variety of organisms. These proteins are postulated to form channels that function in metabolite transport. In plants, this family is represented by the product of NOD26, a nodulation gene in soybean that encodes a protein of the peribacteroid membrane, and tonoplast intrinsic protein (TIP), an abundant protein in the tonoplast of protein storage vacuoles of bean seeds (KD Johnson, H Höfte, MJ Chrispeels [1990] Plant Cell 2: 525-532). Other homologs that are induced by water stress in pea and in Arabidopsis thaliana and that are expressed in the roots of tobacco have been reported, but the location of the proteins they encode is not known. We now report the presence and derived amino acid sequences of two different TIP proteins in A. thaliana. α-TIP is a seed-specific protein that has 68% amino acid sequence identity with bean seed TIP; γ-TIP is expressed in the entire vegetative body of A. thaliana and has 58% amino acid identity with bean seed TIP. Both proteins are associated with the tonoplast. Comparisons of the derived amino acid sequences of the seven known plant proteins in the major intrinsic protein family show that genes with similar expression patterns (e.g. water stress-induced or seed specific) are more closely related to each other than the three A. thaliana homologs are related. We propose that the nonoverlapping gene expression patterns reported here, and the evolutionary relationships indicated by the phylogenetic tree, suggest a functional specialization of these proteins.     

Crystallization of glycosylated and nonglycosylated phytohemagglutinin-L

In the seeds of legume plants a class of sugar-binding proteins can be found, generally called legume lectins. In this paper we present the crystallization of phytohemagglutinin-L (PHA-L), a glycosylated lectin from the seeds of the common bean (Phaseolus vulgaris). Single PHA-L crystals were grown by vapor diffusion, using PEG as precipitant. The protein crystallizes in the monoclinic space group C2, and diffracts to a resolution of 2.7 Å. The unit cell parameters are a = 106.3 Å, b = 121.2 Å, c = 90.8 Å, and β = 93.7°. The asymmetric unit probably contains one PHA-L tetramer. Crystals of a recombinant nonglycosylated form of PHA-L, grown under identical conditions, and crystals of the native PHA-L, grown in the presence of isopropanol, did not survive the mounting process.     

Protein markers and seed size variation in common bean segregating populations

Selection and random genetic drift are the two main forces affecting allele frequencies in common bean breeding programs. Therefore, knowledge on allele frequency changes attributable to these forces is of fundamental importance for breeders. The changes in frequencies of alleles of biochemical markers were examined in F₂ to F₇ populations derived from crosses between cultivated Mesoamerican and Andean common bean accessions (Phaseolus vulgaris L.). Biochemical markers included the seed proteins phaseolin, lectin and other seed polypeptides, and six isozymes. The Schaffer’s test detected a high significant linear trend of the 63% of the polymorphic loci studied, meaning that directional selection was acting on those loci. Associations between seed size traits, phaseolin seed-storage protein and isozyme markers were detected based on the comparisons of the progeny genotypic means. In the interracial populations the intermediate form PhaH/T, b6, and Rbcs ⁹⁸ alleles had a positive effect on seed size. In the inter-gene pool populations, a higher transmission of Mesoamerican alleles in all loci was showed, although the Andean alleles Pha^T, Skdh ¹⁰⁰ , Rbcs ⁹⁸ , and Diap ¹⁰⁰ showed positive effects on seed weight. Our results suggest that phaseolin and other seed proteins markers are linked to loci affecting seed size. These markers have good potential for improving the results of the selection and should be considered as a strategy for germplasm enhancement and to avoid the reduced performance of the inter-gene pool populations.