Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, napB. Comparisons of the 5'-upstream region of napB to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non-expressing tissue.Seed storage proteins serve as a nitrogen and sulfur source for the developing plant seedling during the period of early germination. Storage proteins from cultivated plant species also constitute a major animal and human protein source. Since storage proteins are only expressed in embryonic and/ or endosperm tissues during seed development, they are presently in the focus for studies on developmentally regulated gene expression in plants. Napin is one of the dominating storage proteins of Brassica napus (rape) and variant forms are found among the Brassicaceae, e. g. Raphanus sativus (radish) [l], Sinapis alba (white mustard) [2], and Arabidopsis thaliana [3]. In addition, homologous storage proteins have been characterised in Bertholletia exelsa (brazil nut) [4], Hellianthus annus (sun flower) [5], Triticum aestivum (wheat) [6], and Ricinus communis (castor bean) [7]. The presence of a related protein in the spores of a fern (Matteucia struthiopteris) has also been reported [8, 91. Napin is encoded by a gene family [lo, 111 and several isoforms of napin exist [12]. The sequences of three napin genes, napA [lo], pGNA [ll], and BngNAPl [13], have so far been published. Radke et al. [14] have shown that 300 bp of the 5' flanking sequence in front of the translation initiation codon are sufficient to give a developmentally faithful expression of a chimeric gene construct in transgenic B. napus plants. However, it has not been ascertained whether all sequence motives regulating the napin gene expression are included in this segment of the upstream region.With the underlying assumption that regulatory promoter elements will prove to be conserved between the different Correspondence to
G. 1991. Distinct sequence elements in a napin promoter interact in vitro with DNA-binding proteins from Brassica napus. -Physiol. Ptant. 82: 205-212.Nuclear extracts obtained from developing seeds of oilseed rape, Brassica napus cv. Svalov Karat K2O516, were shown to contain severai distinct DNA-binding proteins as evidenced by gel retardation experiments. Four of the proteins were capable of interacting in vitro with oligonucleotide probes containing sequences reiated to motives in a napin gene promoter atsd its upstream region. Another protein interacted with an A/T-rich repeated sequence present at the 3' end of the gene. The proteins appear to be present also in leaf nuclei and do not show any deteaable variations that correlate with napin expression during seed development. Thus, analogous with many transcription factors, the DNA-binding proteins that we have identified are present in both expressing and non-expressing cells.
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.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
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