Milo Aukerman

Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes

MicroRNAs (miRNAs) are ∼21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.

Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally

A T-DNA insertion in the Arabidopsis thaliana DEK1 gene, encoding a calpain-like cysteine proteinase with a predicted membrane anchor, causes unorganized embryo development displaying irregular mitotic divisions in the embryo proper and suspensor. Embryo development is arrested at the globular stage, and the embryo proper lacks a defined protoderm. In the endosperm, the aleurone-like peripheral cell layer is partly or completely lacking. The Arabidopsis DEK1 wild-type transcript is expressed evenly throughout the endosperm and the embryo in developing seed as determined using in situ hybridization. The conclusion that the observed phenotype is caused by a T-DNA insertion in the Arabidopsis DEK1 gene is confirmed by complementation with the Arabidopisis DEK1 genomic sequence, as well as analysis of a second T-DNA insertion allele. Over-expression of the Arabidopsis DEK1 gene coding sequence under the control of the 35S promoter causes a number of developmental phenotypes, including a global lack of trichomes, leaves exhibiting improper dorsiventral symmetry and aberrant cell organization in flowers. We interpret the data to suggest a role for DEK1 in providing cells with positional clues for an appropriate developmental context within plant tissues.

Artificial trans-acting siRNAs confer consistent and effective gene silencing.

Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing, and artificial microRNAs take advantage of endogenous posttranscriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs in Arabidopsis (Arabidopsis thaliana). Replacing the endogenous small interfering RNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with an miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of small interfering RNAs from the TAS1c locus.