Staffan Sjödahl

Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families

A gene encoding a subunit of the 12S storage globulin, cruciferin, in Brassica napus (oilseed rape) has been isolated and characterized. The gene consists of about 2200 bp including three short intervening sequences. Primer extension analysis showed that the major transcription start site is located 30 bp 5′ of the predicted ATG start codon. This gene belongs to one of three different major families encoding cruciferin subunits. By use of gene-family-specific probes and Southern blotting analysis the number of genes of the three different cruciferin subtypes in B. napus was estimated.

Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions

To define sequences in the cruciferin gene cru1 promoter of importance for expression, tobacco (Nicotina tabacum L.) plants were transformed with constructs in which the cru1 promoter, in front of the intact cru1 structural gene, was truncated at −1216, −974, −736, −515, −306, −46 and −17 bp relative to the cap-site. Cru1 expression in tobacco seeds was studied by Northern analysis, Western analysis and in-situ hybridizations. Comparisons of the Northern analysis of RNA from tobacco seeds harvested at 18 d after pollination with the Western analysis of protein from mature seeds showed that the regions between −974 to −736 and −306 to −46 were important for the expression of cru1 at an early developmental stage, whereas the regions −736 to −515 and −515 to −306 were important for expression throughout embryogenesis. By investigating the mRNA levels in transgenic seeds at different stages of development, indications were obtained that the two latter regions exerted their effects during the later stages. The in-situ hybridization showed that cru1 mRNA was distributed in parenchyma cells throughout the embryo in seeds expressing constructs −974 and −736. Constructs −515 and −306 showed an expression restricted to the axis or axis and parts of the cotyledons. Sequence comparisons of the cru1 promoter with other storage-protein gene promoters, identified several motifs implicated in gene regulation. Gel retardation assays with synthetic oligonucleotides showed that a region present in both cru1 and BnC1 promoters, a CANNTG motif, an SEF3 motif, an abscisic-acid-responsive element and an RY-like motif interacted specifically in vitro with DNA-binding proteins present in nuclear extracts from seeds of Brassica napus L. harvested 40 d after pollination.

Cruciferin gene families are expressed coordinately but with tissue-specific differences during Brassica napus seed development

The major storage protein in seeds of Brassica napus, the 12S globulin cruciferin, is composed of three different groups of subunits; cru1, cru2/3 and cru4. By using gene family-specific probes, we have investigated the accumulation, rate of synthesis and spatial distribution of transcripts corresponding to the different groups of cruciferin subunits in developing seeds. Cruciferin transcripts derived from different gene families accumulate coordinately to comparable amounts during seed development. The corresponding gene families are, however, transcribed at different rates. Investigation of the spatial distribution of transcripts corresponding to each group of cruciferin subunits in the developing seed by in situ hybridization, revealed that mRNAs of all three types accumulate in both axis and cotyledons. Transcripts derived from cru1 and cru4 gene families show a similar cell specificity and accumulate in a similar spatial manner during seed development. In contrast, mRNAs corresponding to the cru2/3 gene family are expressed with a partly different cell specificity and show a slightly different pattern of accumulation in the axis and cotyledons, with a delayed accumulation in epidermal cells. In the cotyledons, the initial accumulation of this type of cruciferin mRNAs is also distinguished from the two other types. The differences in cell specificity are seen in the root cap and in provascular cells, where mRNAs belonging to the cru2/3 family are absent.

Purification and Characterization of a Microsomal Phospholipase A2 from Developing Elm Seeds

The phospholipases A2 (PLA2s) hydrolyse specifically the sn-2-fatty acyl ester bond of phosphoglycerides (Waite, 1987). PLA2s in animal systems are involved in many important processes, such as signal transduction, eicosanoid synthesis and inflammation. The available information about PLA2 from plant tissues is, however, very limited.