The proper number and distribution of stomata are essential for the efficient exchange of gases between the atmosphere and the aerial parts of plants. We show that the density and development of stomatal complexes on the epidermis of Arabidopsis thaliana leaves depend, in part, on the microRNA-mediated regulation of Agamous-like16 ( AGL16 ), which is a member of the MADS box protein family. AGL16 mRNA is targeted for sequence-specific degradation by miR824, a recently evolved microRNA conserved in the Brassicaceae and encoded at a single genetic locus. Primary stomatal complexes can give rise to higher-order complexes derived from satellite meristemoids. Expression of a miR824-resistant AGL16 mRNA, but not the wild-type AGL16 mRNA, in transgenic plants increased the incidence of stomata in higher-order complexes. By contrast, reduced expression of AGL16 mRNA in the agl16-1 deficiency mutant and in transgenic lines overexpressing miR824 decreased the incidence of stomata in higher-order complexes. These findings and the nonoverlapping patterns of AGL16 mRNA and miR824 localization led us to propose that the miR824/AGL16 pathway functions in the satellite meristemoid lineage of stomatal development.

The tobacco class I chitinase gene CHN48 driven by CaMV 35S RNA expression signals introduced into Nicotiana sylvestris can inactivate its own expression as well as expression of homologous host genes at the mRNA level. Comparison of the silencing of chitinase and β‐1,3‐glucanase transgenes with the same expression signals and breeding experiments with transgenes inserted in different chromosomes showed that chitinase‐gene silencing depends on transcribed sequence hornology and trans‐gene dose. The CHN48 transgene silenced host class I but not class II chitinases suggesting that greater than about 60% identity of sequences encoding the catalytic domain is required for silencing. Although steady‐state mRNA contents were greatly reduced in silent plants, the levels of nascent chitinase transcripts detected in nuclear run‐on experiments were the same in nuclei from high‐expressing and silent plants. This strongly suggests that chitinase‐gene silencing is a post‐transcriptional phenomenon. Silencing and resetting to high‐level expression of chitinase genes is developmentally regulated. Silencing occurs stochastically in 25–100% of seedlings at the 6–10 leaf stage of development and is preceded by a marked, transient increase in transgene‐encoded chitinase. Resetting of silent genes to the high‐expressing state is a non‐stochastic event that occurs in developing seeds 8–11 days after pollination. Experiments with lateral buds and plants regenerated from cultured leaf disks show that once established, competence for silencing can persist in dormant, actively growing, and de novo established shoot meristems. Some plants show a variegated pattern of silencing. Analyses of these patterns indicate that chitinase‐gene silencing is not a clonal event. Thus, it is likely that communication between cells rather than the lineage of the cells is important in stabilizing the silent state. Possible mechanisms for developmentally regulated post‐transcriptional silencing are discussed.

Agroinfiltration--the infiltration of Agrobacterium tumefaciens into intact plant levels--provides a rapid and simple way of screening large numbers of transgene constructs for silencing in response to a resident transgene. Transgenic Nicotiana sylvestris plants homozygous for the tobacco class I chitinase A gene CHN48 under the control of the cauliflower mosaic virus 35S RNA promoter (P35S) show a high incidence of postranscriptional gene silencing. We forced suspensions of A. tumefaciens, carrying P35S-CHN48 in a binary Tiplasmid vector, into wild-type and transgenic N, sylvestris leaves with a blunt-tipped plastic syringe. The infiltrated CHN48 transgene was expressed in leaves transformed with the vector alone, but not in CHN48-transformed leaves showing the silent phenotype. In contrast, expression of a chimeric P35S-E. coli beta-glucuronidase gene (uidA) infiltrated into leaves was not affected by the presence of the CHN48 transgene stably integrated in the host genome. These results show that extra copies of CHN48 are silenced by resident, silent copies of the same gene and confirm that CHN48 silencing is not the result of promoter interactions. The results also suggest that silencing of the additional CHN48 copies does not require their integration into chromosomes.

Genomic imprinting is exclusive to mammals and seed plants and refers to parent-of-origin-dependent, differential transcription. As previously shown in mammals, studies in Arabidopsis have implicated DNA methylation as an important hallmark of imprinting. The current model suggests that maternally expressed imprinted genes, such as MEDEA (MEA), are activated by the DNA glycosylase DEMETER (DME), which removes DNA methylation established by the DNA methyltransferase MET1. We report the systematic functional dissection of the MEA cis-regulatory region, resulting in the identification of a 200-bp fragment that is necessary and sufficient to mediate MEA activation and imprinted expression, thus containing the imprinting control region (ICR). Notably, imprinted MEA expression mediated by this ICR is independent of DME and MET1, consistent with the lack of any significant DNA methylation in this region. This is the first example of an ICR without differential DNA methylation, suggesting that factors other than DME and MET1 are required for imprinting at the MEA locus.

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