Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A–GUS fusion genes using tobacco protoplasts revealed novel types of positive cis/-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis/-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.     

Immunoelectrophoretic analysis of seed proteins from Pisum sativum L.

Summary Soluble proteins of pea seed were investigated by quantitative immunological methods. Vicilin, legumin, pea seed lectin (PEA), 26 albumins and a globulin (B₁) were detected and observed during seed development, germination and under different extraction and fractionation procedures. Vicilin and legumin were found to be immunologically distinctly different. Legumin was found to be comprised of two similar proteins, Legumin species I and II. Vicilin, but no legumin, was detected in the embryonic axis.Three albumins, B₁ and PEA were found to be synthesized after the onset of legumin synthesis.Among the pea lines investigated, one line exhibited distinct differences with respect to the albumins and PEA.Some observations indicate that PEA might interact with other seed proteins of pea.     

Analysis of regulatory elements involved in stress-induced and organ-specific expression of tobacco acidic and basic β-1,3-glucanase genes

Infection of tobacco by tobacco mosaic virus (TMV) induces coordinate expression of genes encoding acidic and basic -1,3-glucanase isoforms. These genes are differentially expressed in response to other treatments. Salicylate treatment induces acidic glucanase mRNA to a higher level than basic glucanase mRNA. Ethylene treatment and wounding strongly induce the basic glucanase genes but have little effect on genes encoding the acidic isoforms. Furthermore, the basic glucanase genes are constitutively expressed in roots and lower leaves of healthy plants, whereas the acidic glucanase genes are not. In order to investigate how these expression patterns are established, we fused promoter regions of an acidic and a basic glucanase gene to the -glucuronidase (GUS) reporter gene and examined expression of these constructs in transgenic tobacco plants.A fragment of 1750 bp and two 5-truncated fragments of 650 bp and 300 bp of the acidic glucanase promoter were tested for induction of GUS gene expression after salicylate treatment and TMV infection. Upstream sequences of 1750 bp and 650 bp were sufficient for induction of the reporter gene by salicylate treatment and TMV infection, but the activity of the 300 bp fragment was strongly reduced. The results suggest that the 1750 bp upstream sequence of the acidic glucanase gene contains multiple regulatory elements.For the basic glucanase promoter it is shown that 1476 bp of upstream sequences were able to drive expression in response to TMV infection and ethylene treatment, but no response was found to incision wounding. Furthermore, high GUS activity was found in lower leaves and roots of healthy transgenic plants, carrying the 1476 bp basic glucanase promoter/GUS construct. When the promoter was truncated up to position –446 all activity was lost, indicating that the region between –1476 and –446 of the basic glucanase promoter is necessary for organ-specific and developmentally regulated expression as well as for induced expression in response to infection and other stress treatments.     

Pea legumin overexpressed in wheat endosperm assembles into an ordered paracrystalline matrix

Legumin, a major component of pea seed storage vacuoles, is synthesized by a number of paralogous genes. The polypeptides are cleaved posttranslationally and can form mixed hexamers. This heterogeneity hampers structural studies, based on the production of hexamer crystals in vitro. To study a single type of homogenous legumin we produced pea legumin A in transgenic wheat (Triticum aestivum) endosperm where prolamins are predominant and only small amounts of globulins accumulate in separate inclusions. We demonstrated that the legumin precursor was cleaved posttranslationally and we confirmed assembly into 11S hexamers. Legumin was deposited within specific regions of the inclusion bodies. Angular legumin crystals extended from the inclusion bodies into the vacuole, correlating with the high legumin content. This suggests that the high-level production of a single type of legumin polypeptide resulted in the spontaneous formation of crystals in vivo. The use of a heterologous cereal system such as wheat endosperm to produce, isolate, and recrystallize homogenous 11S legume globulins offers exciting possibilities for structural analysis and characterization of these important seed storage proteins.     

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco

Summary Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumintype genes of several legume species. Constructs containing-833 and-1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions-97 and-549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions-549 and-1203 bp appear to function as enhancer-like elements, to increase expression.     

Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene

A 2.4 kb fragment containing the 5'-flanking region and the 5'-noncoding sequence of the Vicia faba legumin gene LeB4 mediates high level seed-specific expression in transgenic tobacco plants. Deleted derivatives of this legumin upstream sequence were fused to the npt-II reporter gene to determine the tissue-specific activity of the chimeric constructs in stably transformed tobacco plants. The results indicate the presence of positive regulatory, enhancer-like cis elements within 566 bp of the upstream sequence. Most importantly, however, these elements are only fully functional in conjunction with the core motif CATGCATG of the legumin box around position -95, since destruction of the motif by a 6 bp deletion in an otherwise intact 2.4 kb upstream sequence drastically reduces expression in seeds. At the same time, low level expression in leaves is observed. The occurrence of similar CATGCATG consensus cis elements with alternating purine and pyrimidine base pairs in front of several other plant genes suggests a functional role of the motif in a wider range of plant promoters.     

Two genes encoding ''minor'' legumin polypeptides in pea (Pisum sativum L.). Characterization and complete sequence of the LegJ gene.

A genomic clone from pea (Pisum sativum L.) contains all of one gene encoding a 'minor' (B-type) legumin polypeptide, and most of a second very similar gene. The two genes, designated LegJ and LegK, are arranged in tandem, separated by approx. 6 kb. A complete sequence of gene LegJ and its flanking sequences is given, with as much of the sequence of gene LegK as is present on the genomic clone. Hybridization of 3' flanking sequence probes to seed mRNA, and sequence comparisons with cDNA species, suggested that gene LegJ, and probably gene LegK, was expressed. The partial amino acid sequences of 'minor' legumin alpha- and beta-polypeptides were used to confirm the identity of these genes. The transciption start in gene LegJ was mapped. The 5' flanking sequence of gene LegJ contains a sequence conserved in legumin genes from pea and other species, which is likely to have functional significance in control of gene expression. Sequence comparisons with legumin genes and cDNA species from Vicia faba and soya bean show that separation of legumin genes into A- and B-type subfamilies occurred before separation of the Viciae and Glycinae tribes.     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.