The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.Polycomb group (PcG) proteins from complexes in vivo at regulatory sites, the Polycomb response elements (PREs), which mediate the silencing of neighboring genes. Transposons containing PREs generate new binding sites for PcG proteins on polytene chromosomes, indicating that PREs are the physical targets for PcG complex formation. Chromatin cross-linking experiments have also shown that PcG proteins are bound to and in the vicinity of known PRE sites (28,35,36). In these experiments, PcG proteins are found cross-linked over a few kilobases centered over fragments with known PRE activity, suggesting the possibility that a silencing complex initiated at a PRE involves at least a few kilobases either because of a cooperative spreading of the complex or because the PRE is in fact not a site but a region containing multiple sequences that interact with PcG proteins. None of the well-characterized PcG proteins can be shown to bind to DNA in vitro. One simple explanation for the fact that PcG complex formation appears to be specific for the PRE might be that it depends on other hitherto-unknown PcG proteins. One such candidate, the product of the pleiohomeotic (pho) gene, has recently been identified (3). However, it has not been shown yet that PHO interacts with other PcG proteins, and the presumptive consensus sequence for the binding of PHO is not always present in DNA fragments that have PRE activity, suggesting that other DNA-binding proteins might be involved. Other possibilities are suggested by the properties of PcG proteins and of PREs. Several of the PcG proteins can interact with one another, and experiments with Pc protein targeted to LexA or GAL4 binding sites indicate that a single PcG protein can recruit a silencing complex (26; S. Poux, D. McCabe, and V. Pirrotta, submitted for publication). Different genomic PcG sites show different degrees of dependence on different members of the PcG genes, both in the strength of the signal generated by immunostaining and the effect of different PcG mutations on the silencing of the accompanying genes. The silencing ability of a given PRE is strongly dependent on the genomic context in which it is introduced and on homologous pairing or the physical proximity to other PRE sequences in the nucleus (5, 10, 17, 34). All these observations suggest that the formation of a PcG c...

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