A recombineering-based gene tagging system for Arabidopsis.

One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.

TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.

Plants have evolved a tremendous ability to respond to environmental changes by adapting their growth and development. The interaction between hormonal and developmental signals is a critical mechanism in the generation of this enormous plasticity. A good example is the response to the hormone ethylene that depends on tissue type, developmental stage, and environmental conditions. By characterizing the Arabidopsis wei8 mutant, we have found that a small family of genes mediates tissue-specific responses to ethylene. Biochemical studies revealed that WEI8 encodes a long-anticipated tryptophan aminotransferase, TAA1, in the essential, yet genetically uncharacterized, indole-3-pyruvic acid (IPA) branch of the auxin biosynthetic pathway. Analysis of TAA1 and its paralogues revealed a link between local auxin production, tissue-specific ethylene effects, and organ development. Thus, the IPA route of auxin production is key to generating robust auxin gradients in response to environmental and developmental cues.

Molecular mechanisms of ethylene signaling in Arabidopsis.

Ethylene is a gaseous plant hormone involved in several important physiological processes throughout a plant's life cycle. Decades of scientific research devoted to deciphering how plants are able to sense and respond to this key molecule have culminated in the establishment of one of the best characterized signal transduction pathways in plants. The ethylene signaling pathway starts with the perception of this gaseous hormone by a family of membrane-anchored receptors followed by a Raf-like kinase CTR1 that is physically associated with the receptors and actively inhibits downstream components of the pathway. A major gap is represented by the mysterious plant protein EIN2 that genetically works downstream of CTR1 and upstream of the key transcription factor EIN3. Transcriptional regulation by EIN3 and EIN3-family members has emerged as a key aspect of ethylene responses. The major components of this transcriptional cascade have been characterized and the involvement of post-transcriptional control by ubiquitination has been determined. Nevertheless, many aspects of this pathway still remain unknown. Recent genomic studies aiming to provide a more comprehensive view of modulation of gene expression have further emphasized the ample role of ethylene in a myriad of cellular processes and particularly in its crosstalk with other important plant hormones. This review aims to serve as a guide to the main scientific discoveries that have shaped the field of ethylene biology in the recent years.