ETHYLENE-INSENSITIVE5 Encodes a 5\u27→3\u27 Exoribonuclease Required for Regulation of the EIN3-Targeting F-Box Proteins EDF1⁄2

Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence of ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F-box proteins, EBF1 and EBF2. Here we report the identification of ETHYLENE-INSENSITIVE5 as the 5′→3′ exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response

Diversity in the Architecture of ATLs, a Family of Plant Ubiquitin-Ligases, Leads to Recognition and Targeting of Substrates in Different Cellular Environments

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20–28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress

The ATXN2 Orthologs CID3 and CID4, Act Redundantly to In-Fluence Developmental Pathways throughout the Life Cycle of Arabidopsis thaliana

RNA-binding proteins (RBPs) are key elements involved in post-transcriptional regulation. Ataxin-2 (ATXN2) is an evolutionarily conserved RBP protein, whose function has been studied in several model organisms, from Saccharomyces cerevisiae to the Homo sapiens. ATXN2 interacts with poly(A) binding proteins (PABP) and binds to specific sequences at the 3′UTR of target mRNAs to stabilize them. CTC-Interacting Domain3 (CID3) and CID4 are two ATXN2 orthologs present in plant genomes whose function is unknown. In the present study, phenotypical and transcriptome profiling were used to examine the role of CID3 and CID4 in Arabidopsis thaliana. We found that they act redundantly to influence pathways throughout the life cycle. cid3cid4 double mutant showed a delay in flowering time and a reduced rosette size. Transcriptome profiling revealed that key factors that promote floral transition and floral meristem identity were downregulated in cid3cid4 whereas the flowering repressor FLOWERING LOCUS C (FLC) was upregulated. Expression of key factors in the photoperiodic regulation of flowering and circadian clock pathways, were also altered in cid3cid4, as well as the expression of several transcription factors and miRNAs encoding genes involved in leaf growth dynamics. These findings reveal that ATXN2 orthologs may have a role in developmental pathways throughout the life cycle of plants

Overall domain comparison between ATL and Rabring7/BCA2/BTL RING-H2 ubiquitin-ligases.

<p>A schematic representation of canonical ATL and Rabring7/BCA2/BTL E3 ligases indicating the position of the two relevant domains on each class: hydrophobic and RING-H2 in ATLs and BZF and RING-H2 in Rabring7/BCA2/BTLs. A sequence LOGO comparison between the two RING-H2 domains is shown; LOGOs were generated from the collected Rabring7/BCA2/BTLs sequences and from a previous analysis of ATLs. The numbers indicate the residues involved in zinc ligation. The arrowhead indicates an absent amino acid residue in Rabring7/BCA2/BTLs and broken lines indicate conserved residues between the two RING-H2 domains. An alignment of representative proteins displaying the BZF and the RING-H2 regions of Rabring7/BCA2/BTLs is displayed below. ClustalX was used for sequence alignment and a default color code was applied. The numbers indicate the residues involved in zinc ligation in the RING-H2 domain and letters the conserved cysteines in BZF.</p

Expansion and Diversification of BTL Ring-H2 Ubiquitin Ligases in Angiosperms: Putative Rabring7/BCA2 Orthologs

<div><p>RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for <i>B</i>ZF A<i>TL</i>s. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to <i>ATL</i>s, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes.</p> </div

Distribution of BTLs from embryophites in six groups.

<p>Heat map representation of the number of BTLs from the 29 species in each one of the six groups by a gray scale. The species tree displayed to the left is adapted from the National Center of Biotechnology Information (NCBI) taxonomy server (<a href="http://www.ncbi.nlm.nih.gov/taxonomy" target="_blank"><u>http://www.ncbi.nlm.nih.gov/Taxonomy</u></a>). The total number of genes in each group is shown at the bottom (the catalog of the 396 BTLs in 6 groups in displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s009" target="_blank">Table S4</a>). The species tree is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone-0072729-g002" target="_blank">Figure 2</a>. Novel sequence LOGOs mapped to regions III and V on each group are displayed below the heat map; the occurrence for each of these LOGOs is more than 5%.</p

Number of retrieved Rabring7/BCA2/BTLs in eukaryotes.

<p>The phylogenetic relationship between thirty-three viridiplantae, forty-one animal, eighteen fungal, and fifteen protist genomes is displayed in a circle. Relationships were adapted from the National Center of Biotechnology Information (NCBI) taxonomy server (<a href="http://www.ncbi.nlm.nih.gov/taxonomy" target="_blank">http://www.ncbi.nlm.nih.gov/Taxonomy</a>). The color code for major group f organisms is shown at the bottom. The species abbreviations are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s006" target="_blank">Table S1</a> and the genes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s007" target="_blank">Tables S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s009" target="_blank">S4</a>.</p

Protein–protein interaction domains in BTLs.

<p>(A) The upper diagram depicts the modular organization of ath|BTL4 based on sequence LOGOs. The lower diagram is a schematic representation of four clones encompassing different regions of ath|BTL4 that were used for the yeast two-hybrid screening and assays. Twelve clones depicted below the clone BTL(III) within a gray circle were retrieved from a yeast two-hybrid screening using the BTL4(III-IV–V). The segment in BTL4 that mediates the interaction with all of them was further mapped to region III by a yeast two-hybrid assay; the description of the interactors is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s010" target="_blank">Table S5</a>. (B) Interaction between BZF and ubiquitin. Representative plates showing yeast two-hybrid interactions between BTL4(BZF) and four <i>A. thaliana</i> ubiquitin clones. The left panel shows the template of the plates. The BTL4 BZF-containing fragment was ligated into the DNA-binding domain of pGBKT7, and the ubiquitin clones are in pGAD10, which is a pGADT7-related vector. The yeast strain AH109 was cotransformed with pGBKT7 and pGADT7 derivatives, selecting for transformants on SC lacking Trp and Leu. Two representative transformants were then streaked onto SC medium lacking Trp and Leu (SC) and onto SC medium with 7 mM 3-AT and lacking Trp, Leu, His, and Ade. The plates were incubated at 30^oC for four days; growth is observed as dense streaks of yeast over background. The interaction with pGBKT7 was included as a negative control (depicted as a gray sector in the template plate).</p

Sequence LOGOs mapped to Rabring7/BCA2/BTLs regions.

<p>Below the canonical Rabring7/BCA2/BTLs diagram, geometric figures represent the five modules and the sequence LOGOs mapped to each region for major groups of organisms. Species are arranged into two groups: animals, fungi and protists in one group, and viridiplantae in the other group. The sum of logos in each group is indicated, and the total number of logos is shown at the bottom. The prominent sequence LOGOs for the two zinc fingers is displayed above the diagram; the distance between the pairs of cysteines in 97% of Rabring7/BCA2/BTLs ranges between 11 and 19 amino acids. The catalog of the 73 sequence LOGOs is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s008" target="_blank">Table S3</a>.</p

Phylogeny of 99 Rabring7/BCA2 proteins from animals, fungi and protists.

<p>The tree was generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone-0072729-g003" target="_blank">Figure 3</a>; the tree obtained by concatenating domains is displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone.0072729.s001" target="_blank">Figure S1</a>. The color code on the branches for groups of organisms is that same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072729#pone-0072729-g002" target="_blank">Figure 2</a>.</p