LEUNIG and SEUSS co-repressors regulate miR172 expression in Arabidopsis flowers.

Central to the ABCE model of flower development is the antagonistic interaction between class A and class C genes. The molecular mechanisms underlying the A-C antagonism are not completely understood. In Arabidopsis thaliana, miR172 is expressed in the inner floral whorls where it downregulates the class A gene APETALA 2 (AP2). However, what controls this predominantly inner whorl-specific expression of miR172 is not known. We show that the LEUNIG (LUG) and SEUSS (SEU) co-repressors repress miR172 expression in the outer whorls of A. thaliana flowers. The recruitment of LUG/SEU to the miR172 promoters is dependent on AP2, suggesting that AP2 represses the expression of its cognate microRNA. Our study provides new insights into the molecular mechanisms underlying the A-C antagonism and shed light on the transcriptional regulation of miR172 during flower development.

Floral-dip transformation of Arabidopsis thaliana to examine pTSO2::beta-glucuronidase reporter gene expression.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, beta-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

The Arabidopsis floral homeotic proteins APETALA3 and PISTILLATA negatively regulate the BANQUO genes implicated in light signaling.

The Arabidopsis thaliana MADS box transcription factors APETALA3 (AP3) and PISTILLATA (PI) heterodimerize and are required to specify petal identity, yet many details of how this regulatory process is effected are unclear. We have identified three related genes, BHLH136/BANQUO1 (BNQ1), BHLH134/BANQUO2 (BNQ2), and BHLH161/BANQUO3 (BNQ3), as being directly and negatively regulated by AP3 and PI in petals. BNQ1, BNQ2, and BNQ3 encode products belonging to a family of atypical non-DNA binding basic helix-loop-helix (bHLH) proteins that heterodimerize with and negatively regulate bHLH transcription factors. We show that bnq3 mutants have pale-green sepals and carpels and decreased chlorophyll levels, suggesting that BNQ3 has a role in regulating light responses. The ap3 bnq3 double mutant displays pale second-whorl organs, supporting the hypothesis that BNQ3 is downstream of AP3. Consistent with a role in light response, we show that the BNQ gene products regulate the function of HFR1 (for LONG HYPOCOTYL IN FAR-RED1), which encodes a bHLH protein that regulates photomorphogenesis through modulating phytochrome and cryptochrome signaling. The BNQ genes also are required for appropriate regulation of flowering time. Our results suggest that petal identity is specified in part through downregulation of BNQ-dependent photomorphogenic and developmental signaling pathways.

Regulatory mechanisms for floral homeotic gene expression.

Proper regulation of floral homeotic gene (or ABCE gene) expression ensures the development of floral organs in the correct number, type, and precise spatial arrangement. This review summarizes recent advances on the regulation of floral homeotic genes, highlighting the variety and the complexity of the regulatory mechanisms involved. As flower development is one of the most well characterized developmental processes in higher plants, it facilitates the discovery of novel regulatory mechanisms. To date, mechanisms for the regulation of floral homeotic genes range from transcription to post-transcription, from activators to repressors, and from microRNA- to ubiquitin-mediated post-transcriptional regulation. Region-specific activation of floral homeotic genes is dependent on the integration of a flower-specific activity provided by LEAFY (LFY) and a region- and stage-specific activating function provided by one of the LFY cofactors. Two types of regulatory loops, the feed-forward and the feedback loop, provide properly timed gene activation and subsequent maintenance and refinement in proper spatial and temporal domains of ABCE genes. Two different microRNA/target modules may have been independently recruited in different plant species to regulate C gene expression. Additionally, competition among different MADS box proteins for common interacting partners may represent a mechanism in whorl boundary demarcation. Future work using systems approaches and the development of non-model plants will provide integrated views on floral homeotic gene regulation and insights into the evolution of morphological diversity in flowering plants.

Two GATA transcription factors are downstream effectors of floral homeotic gene action in Arabidopsis.

Floral organogenesis is dependent on the combinatorial action of MADS-box transcription factors, which in turn control the expression of suites of genes required for growth, patterning, and differentiation. In Arabidopsis (Arabidopsis thaliana), the specification of petal and stamen identity depends on the action of two MADS-box gene products, APETALA3 (AP3) and PISTILLATA (PI). In a screen for genes whose expression was altered in response to the induction of AP3 activity, we identified GNC (GATA, nitrate-inducible, carbon-metabolism-involved) as being negatively regulated by AP3 and PI. The GNC gene encodes a member of the Arabidopsis GATA transcription factor family and has been implicated in the regulation of chlorophyll biosynthesis as well as carbon and nitrogen metabolism. In addition, we found that the GNC paralog, GNL (GNC-like), is also negatively regulated by AP3 and PI. Using chromatin immunoprecipitation, we showed that promoter sequences of both GNC and GNL are bound by PI protein, suggesting a direct regulatory interaction. Analyses of single and double gnc and gnl mutants indicated that the two genes share redundant roles in promoting chlorophyll biosynthesis, suggesting that in repressing GNC and GNL, AP3/PI have roles in negatively regulating this biosynthetic pathway in flowers. In addition, coexpression analyses of genes regulated by AP3, PI, GNC, and GNL indicate a complex regulatory interplay between these transcription factors in regulating a variety of light and nutrient responsive genes. Together, these results provide new insights into the transcriptional cascades controlling the specification of floral organ identities.