Functional conservation of the plant EMBRYONIC FLOWER2 gene between bamboo and Arabidopsis.

A novel EMBRYONGIC FLOWER2 gene, DlEMF2, was isolated from bamboo, Dendrocalamus latiflorus. It is 2518 bp in length encoding a protein of 629 amino acids. It was expressed in all organs and at a higher level in shoot tissue than in inflorescences. Transgenic Arabidopsis plants expressing antisense DlEMF2 displayed a spectrum of early-flowering phenotypes. Quantitative real-time PCR experiments indicated that the antisense transgene DlEMF2 reduced endogenous AtEMF2 transcript levels leading to the flowering time reduction. Besides, transforming the bamboo DlEMF2 gene into Arabidopsis emf2-1 mutant could rescue phenotype in vegetative and reproductive developmental stages, which reveals that EMF2 has functional conservation between monocots and eudicots.

A high-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation.

The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD-TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF-DNA and TF-protein interactions: the CHE-CCA1 promoter interaction by Y1H and NPR1-TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.

Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes.

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.