Steven F. Fabijanski

A gene showing sequence similarity to pectin esterase is specifically expressed in developing pollen of Brassica napus. Sequences in its 5' flanking region are conserved in other pollen-specific promoters.

Differential screening of a Brassica napus genomic library led to the isolation of the clone named Bp 19 containing a gene which is highly expressed during microspore development. The accumulation of Bp 19 mRNA starts in uninucleate microspores, increases during development reaching a peak in the late stages but declines considerably in mature pollen. The nucleotide sequence of the entire coding region and of extended portions of the 5′ and 3′ flanking regions was determined. Several homologous cDNA clones were also isolated and sequenced. The Bp 19 gene contains a single intron of 137 bp and gives origin to a mRNA of ca. 1.9 kb which codes for a polypeptide of 584 amino acids. Bp 19 protein has an estimated molecular weight of 63 kilodaltons and has a highly hydrophobic amino terminal region which shows features of a signal peptide. The carboxy half of the Bp 19 protein, starting at amino acid 269, has striking sequence similarity to the pectin esterases of tomato and of the plant pathogen Erwinia chrysanthemi. Four short domains are extremely well conserved in all the three proteins and therefore could represent catalytic sites responsible for enzyme activity. Comparison of the 5′ flanking region of the Bp 19 gene with the sequence of other pollen-specific promoters revealed the presence of several conserved regions. These short promoter sequences could correspond to regulatory elements responsible for pollen-specific gene expression.

CHARACTERIZATION OF A POLLEN-SPECIFIC GENE FAMILY FROM BRASSICA-NAPUS WHICH IS ACTIVATED DURING EARLY MICROSPORE DEVELOPMENT

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5′ region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprising gene 1 and gene 2 from Agrobacterium tumefaciens whereby gene 1 was controlled by the seed-specific phaseolin promoter modified to contain a binding site for the Escherichia coli TET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination. Plants containing the SL construct were crossed with plants containing the tet R gene to derive plant lines where the expression of the SL construct is repressed. Plant lines that contained both constructs allowed normal seed formation and germination, whereas seeds in which the SL construct was separated from the R gene through segregation did not germinate. The requirements of such a method to efficiently control the flow of novel traits among sexually compatible plants are discussed.

Antisense RNA inhibition of β-glucuronidase gene expression in transgenic tobacco plants

Antisense RNA was used to specifically inhibit the expression of a GUS gene introduced in a transgenic plant. A tobacco transformant containing a single intact copy of the GUS gene and showing relatively high constitutive levels of GUS activity (GUS+) was re-transformed with an Agrobacterium Ti-derived binary vector containing an antisense version of this reporter gene. The sense and antisense GUS genes were each under the regulation of the CaMV 35S promoter. Re-transformed plants contained 1–5 copies of the antisense construct and all showed a greater than 90% reduction in GUS activity relative to the original GUS+ plant. This reduction in GUS activity correlated closely with the levels of GUS enzyme and steady state GUS mRNA observed in these plants. The relatively low levels of sense and antisense GUS transcripts found in the re-transformed plants may indicate a rapid degradation of the RNA:RNA duplex in the cell.

Evidence for translational control of storage protein biosynthesis during embryogenesis ofAvena sativa L. (oat endosperm).

Oat polysomes direct the synthesisin vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A+ RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased amount of the other products synthesizedin vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments using oat poly A+ mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is partially responsible for the levels of globulin in the mature grain.

Antigenic homologies between oat and wheat globulins

Total globulins of oat and wheat were compared by SDS‐electophoresis immunoblotting and in vitro protein synthesis experiments. Numerous homologies were found. The same 4 protein subgroups, with respect to molecular mass, were found in the two cereals. Immunoblotting with antibodies for total oat globulins allowed the detection of homologous proteins in each wheat protein group, especially in the 60‐kDa group, which corresponds to the major 12 S oat globulin. The same antibodies also reacted with in vitro synthesized proteins of the two cereals. However, compared to mature proteins, only a limited number of polypeptides react, suggesting that wheat globulins, like oat globulins, undergo post‐translational processes.

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs.

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.

Characterization of genes expressed during the development ofBrassica napus pollen

SummaryThe study of the formation of pollen in plants has been the focus of extensive morphologic and cytologic observations. This complex developmental process requires the coordinated activity of both gametophytic and sporophytic tissues. The events that occur during microspore development represent a carefully orchestrated program of physiologic, biochemical, and genetic activities. Genes expressed specifically in pollen or in sporophytic tissues that support pollen development have only recently been identified and desribed. In the present paper we describe several genes expressed during pollen development in the important oil seed speciesBrassica napus (oil seed rape/canola). The characterization of three gene families expressed during microspore development is reviewed which provides a basis for comparison with other genes expressed during pollen maturation. The, potential value of these genes for the development of novel plant breeding strategies and hybrid seed production is discussed.

Heat Shock Response of Brassica oleracea L. (Broccoli)

Summary The response of broccoli (Brassica oleracea var. Italica) to rapid changes in temperature has been examined by labelling proteins in vivo. Time course analysis using both one and two-dimensional gel electrophoresis reveals that rapid changes in protein synthesis occur when leaf material is subjected to temperature stress. One-dimensional gel analysis demonstrates at least 14 polypeptides are preferentially synthesized at 37 °C when compared to 20 °C. Two-dimensional gel analysis of these in vivo labelled proteins demonstrate that many of these 14 proteins induced at 37 °C are resolved into more components which are isoelectric variants of the same molecular weight protein. There are upwards of 30 proteins induced at 37 °C visible on two-dimensional gels, and based on comparison with heat-induced proteins from other plants, we have identified these proteins in broccoli as heat shock proteins.