Several zein genes have been cloned and sequenced and the comparison of their 5' flanking regions showed a common feature: the presence of a distal and a proximal dual promoter with respect to the ATG codon, indicated as P1 and P2, respectively. Analysis of the expression in transgenic Petunia hybrida of chimeric constructs containing the different promoter regions (P1, P2 or P1+P2), upstream of the GUS coding region, revealed GUS activity not only in the endosperm of transformed seeds, thereby retaining their original tissue specificity, but also in anthers, particularly in the tapetal cell layers (Quattrocchio et al., Plant Mol. Biol. 15:81-93, 1990). This observation prompted the authors to investigate in more detail the situation in maize inflorescences, where Northern blot analysis revealed the presence of a 1.9 kb mRNA, indicating a preferential transcription from the distal promoter P1 (Quattrocchio et al., ibid.).
In order to detect even low levels of zein gene expression in tissues other than endosperm, total RNA was extracted from male inflorescences of the line A69Y at different developmental stages, reverse transcribed using oligo-dT primer and McMuLV reverse transcriptase, and amplified by PCR using primers corresponding to consensus sequences derived from a comparative analysis of the 5' and 3' regions of about 10 different structural zein genes. Before reverse transcription total RNA was treated with RNase-free DNase, to remove any possible contaminant trace of genomic DNA present in the RNA sample. Subsequent Southern blot analysis, using as probes a mixture of sequences coding for different light zein (ZL) chains, showed the presence of amplification products, confirming that transcripts for this zein class are present in developing tassels. The same analysis on transcripts corresponding to the heavy zein (ZH) chains is being performed at present.
To assess for the presence of zein polypeptides, Western blot analysis was carried out with anti-zein sera on protein extracts obtained from developing male inflorescences. Our results indicate that polypeptides belonging to both classes, ZH and ZL, are synthesized in these organs, even if in much smaller amounts than in endosperm. Their presence has also been confirmed by immunogold detection on anther sections containing pollen grains at different developmental stages. Zein polypeptides were localized in the ER of the inner layer of the tapetum.
In order to determine if the expression of zein genes in male inflorescences is regulated in a manner similar to or different from endosperm, the first step was to investigate whether the regulatory gene opaque2 is also expressed in this tissue. This was done by RT-PCR on total RNA, previously treated with RNase-free DNase, using two sequences internal to the O2 coding region as primers. Amplification products were analyzed by Southern blot with the entire coding sequence as probe. The expected length of the amplified sequences is 680 bp for products obtained from mRNA molecules and 1027 bp for those derived from genomic DNA, as three introns are present in this region. Only the expected smaller fragment was amplified when DNase-treated RNA samples were used, thereby indicating that not only structural zein genes, but also the regulatory O2 gene, are expressed in male inflorescences.
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