--Silvana Faccio Dolfini, Michela Landoni, Chiara Tonelli, L.Bernard¹ and A.Viotti¹

¹C.N.R. - Istituto Biosintesi Vegetali

The cellular heterogeneity of the endosperm may be related to a differential regulation of gene expression during the development of this tissue. To elucidate this aspect, the spatial expression during the seed formation of genes encoding zeins and glutelins was investigated by in situ hybridization on wildtype lines (W64A and A69Y) and genotypes carrying mutations at loci affecting zein synthesis (o2, o7, fl2 and pro1-1). Preliminarily, the lines, obtained from various sources and investigated during different harvesting periods, were characterized for their DNA restriction patterns.

The localization of storage-protein gene transcripts was visualized in serial sections of developing seeds using anti-mRNA probes transcribed from sequences representative of genes encoding heavy- (M1) and light-chain (E19 and M6) zeins and glutelins (G1) (ViottiA et al., EMBO J. 4:1103, 1985; Prat et al., Gene 52:41, 1987). In normal endosperms the zein and glutelin mRNAs are expressed in all cells, except for the aleurone layer. However, each mRNA type accumulates at a different level in the various endosperm regions, allowing recognition within the tissue of specific territories of expression for each storage-protein mRNA. The genes of the heavy-chain zeins (M1) show a higher expression in the apical region, while light-chain zein genes (E19 and M6) are more abundant on the abgerminal side. The glutelins show a greater amount of transcripts mainly in the germinal region (Fig. 1).

Figure 1. Accumulation of zein and glutelin mRNAs during W64A seed development.

In general, this transcript distribution clearly follows a decreasing gradient from the outer layers of the endosperm to the inner, contrasting the hypothesis that DNA amplification occurring in the central cells is correlated to a higher level of zein synthesis (Kowles and Phillips, Int. Rev. Cytol. 112:97, 1988). These spatial expression patterns, established early for each gene type, are maintained during the subsequent period of seed maturation.

The various probes were also hybridized to sections obtained from different mutants: W64A o2, Rossman o2, W22 o7, W64A fl2 and W22 pro1-1. Each mutant reveals, in comparison with the wildtype, a specific pattern of expression, showing different and profound reductions in the accumulation of zeins and glutelins, in agreement with the hypothesis of a "regional" control of zein gene transcription and in accord with results of Northern analyses. As a consequence, the molecular constitution of protein bodies, as regards the relative proportion of heavy- and light-chain zeins, may be different in the various regions of the endosperm.

The size and abundance in the different genotypes of the transcript of the trans-acting regulatory gene opaque-2 (O2) was determined by Northern blotting and its cellular localization was analyzed by in situ hybridization, using as probe the O2 cDNA (Schmidt et al., P.N.A.S. 87:46, 1990).

Results show that opaque-2 transcript is expressed and localized differently in normal and in various opaque-2 endosperms. In fact, a transcript is present over nucleus and cytoplasm in W64A and A69Y inbred lines, as well as in o7 and fl2 mutants (Fig. 2d). The various o2 mutations analyzed, however, proved to be molecularly different, since a strong hybridization signal is present almost exclusively over nuclei in W64A o2 (Fig. 2a-c), while only a few grains are visible over nucleus and cytoplasm of Rossman o2 and A69Y o2. These data are supported by Northern results. In particular, the recovery of the transcript of W64A o2, having a molecular weight greater than the normal, suggests the absence of a post-transcriptional modification (possibly intron splicing) and correlates with its nuclear localization.

Figure 2. Hybridization of O2 anti-mRNA probe to (a-c) W64A opaque-2 endosperm at 15 DAP (a x30, b x200, c x400) and (d) W64A endosperm at 15 DAP.

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