A link between mRNA turnover and RNA interference in Arabidopsis.

In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in Arabidopsis was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5'-3' exonucleolysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.

Transcriptional activation of APETALA1 by LEAFY.

Plants produce new appendages reiteratively from groups of stem cells called shoot apical meristems. LEAFY (LFY) and APETALA1 (AP1) are pivotal for the switch to the reproductive phase, where instead of leaves the shoot apical meristem produces flowers. Use of steroid-inducible activation of LFY demonstrated that early expression of AP1 is a result of transcriptional induction by LFY. This AP1 induction is independent of protein synthesis and occurs specifically in the tissues and at the developmental stage in which floral fate is assumed. Later expression of AP1 appears to be only indirectly affected by LFY.

Temperature-sensitive splicing in the floral homeotic mutant apetala3-1.

The floral homeotic gene APETALA3 (AP3) is required for stamen and petal development in Arabidopsis. The previously described ap3-1 allele is temperature sensitive and carries a missense mutation near a 5' splice site. The missense mutation lies within a domain of the AP3 protein that is thought to be important for protein-protein interactions, which suggests that temperature sensitivity of ap3-1 could reflect an unstable interaction with cofactors. Here, we show instead that the ap3-1 mutation causes a temperature-dependent splicing defect and that temperature sensitivity is not a property of the protein products of ap3-1 but of RNA processing, possibly because of unstable base pairing between the transcript and small nuclear RNAs. The unexpected defect of the ap3-1 mutant offers unique opportunities for genetic and molecular studies of splice site recognition in plants.

A homolog of NO APICAL MERISTEM is an immediate target of the floral homeotic genes APETALA3/PISTILLATA.

To understand how homeotic genes affect morphogenesis and differentiation, their target genes must be identified. In Arabidopsis flowers, the homeotic protein heterodimer APETALA3/PISTILLATA is necessary for petal and stamen formation. Here, AP3/PI function was put under posttranslational control to analyze its immediate effect on the floral mRNA population, with indirect effects blocked by cycloheximide. Using differential display, a target gene of AP3/PI was identified (NAP:NAC-LIKE, ACTIVATED BY AP3/PI), which is homologous to genes required for meristem establishment and separation of floral organs. The expression pattern of NAP and the phenotypes caused by its misexpression suggest that it functions in the transition between growth by cell division and cell expansion in stamens and petals.

Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter.

The promoter of the bean PAL2 gene (encoding phenylalanine ammonia-lyase; EC 4.3.1.5) is a model for studies of tissue-restricted gene expression in plants. Petal epidermis is one of the tissues in which this promoter is activated in tobacco. Previous work suggested that a major factor establishing the pattern of PAL2 expression in tobacco petals is the tissue distribution of a protein closely related to Myb305, which is a Myb-like transcriptional activator from snapdragon. In the present work, we show that Myb305 expression in tobacco leaves causes ectopic activation of the PAL2 promoter. To achieve Myb305 expression in planta, a viral expression vector was used. This approach combines the utility of transient assays with the possibility of direct biochemical detection of the introduced factor and may have wider application for studying the function of plant transcription factors.

Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco.

Genes encoding phenylalanine ammonia-lyase (PAL) are expressed in a complex pattern during plant development and in response to light, pathogen ingress, mechanical damage and other stresses. Analysis of the promoter of the bean PAL2 gene in transgenic tobacco has shown that some regions responsible for developmental expression are functionally compensatory. The minimum sequence containing all cis sequences necessary for developmental patterns of expression is within -254 bp of the transcription start site. Footprinting and electrophoretic mobility shift assay studies of this region revealed potential cis sequences which coincided with the functional domains defined by small deletions and promoter fusions. Mutations in these potential cis sequences in the context of the minimal -254 bp promoter altered tissue-specific expression patterns, confirming the importance of these sequences for expression in vivo. A functional model for the promoter is presented which predicts that three AC-elements, which are possible Myb protein binding sites, together with a G-box, interact to direct the complex patterns of tissue-specific expression observed.

A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes.

Synthesis of flavonoid pigments in flowers requires the co-ordinated expression of genes encoding enzymes in th phenylpropanoid biosynthetic pathway. Some cis-elements involved in the transcriptional control of these genes have been defined. We report binding of petal-specific activities from tobacco and Antirrhinum majus (snapdragon) to an element conserved in promoters of phenylpropanoid biosynthetic genes and implicated in expression in flowers. These binding activities were inhibited by antibodies raised against Myb305, a flower-specific Myb protein previously cloned from Antirrhinum by sequence homology. Myb305 bound to the same element and formed a DNA-protein complex with the same mobility as the Antirrhinum petal protein in electrophoretic mobility shift experiments. Myb305 activated expression from its binding site in yeast and in tobacco protoplasts. In protoplasts, activation also required a G-box-like element, suggesting co-operation with other elements and factors. The results strongly suggest a role for Myb305-related proteins in the activation of phenylpropanoid biosynthetic genes in flowers. This is consistent with the genetically demonstrated role of plant Myb proteins in the regulation of genes involved in flavonoid synthesis.