Evolutionary Footprints Reveal Insights into Plant MicroRNA Biogenesis.

MicroRNAs (miRNAs) are endogenous small RNAs that recognize target sequences by base complementarity and play a role in the regulation of target gene expression. They are processed from longer precursor molecules that harbor a fold-back structure. Plant miRNA precursors are quite variable in size and shape, and are recognized by the processing machinery in different ways. However, ancient miRNAs and their binding sites in target genes are conserved during evolution. Here, we designed a strategy to systematically analyze MIRNAs from different species generating a graphical representation of the conservation of the primary sequence and secondary structure. We found that plant MIRNAs have evolutionary footprints that go beyond the small RNA sequence itself, yet their location along the precursor depends on the specific MIRNA We show that these conserved regions correspond to structural determinants recognized during the biogenesis of plant miRNAs. Furthermore, we found that the members of the miR166 family have unusual conservation patterns and demonstrated that the recognition of these precursors in vivo differs from other known miRNAs. Our results describe a link between the evolutionary conservation of plant MIRNAs and the mechanisms underlying the biogenesis of these small RNAs and show that the MIRNA pattern of conservation can be used to infer the mode of miRNA biogenesis.

SUMOylation Inhibition Mediated by Disruption of SUMO E1-E2 Interactions Confers Plant Susceptibility to Necrotrophic Fungal Pathogens.

Protein modification by SUMO modulates essential biological processes in eukaryotes. SUMOylation is facilitated by sequential action of the E1-activating, E2-conjugating, and E3-ligase enzymes. In plants, SUMO regulates plant development and stress responses, which are key determinants in agricultural productivity. To generate additional tools for advancing our knowledge about the SUMO biology, we have developed a strategy for inhibiting in vivo SUMO conjugation based on disruption of SUMO E1-E2 interactions through expression of E1 SAE2UFDCt domain. Targeted mutagenesis and phylogenetic analyses revealed that this inhibition involves a short motif in SAE2UFDCt highly divergent across kingdoms. Transgenic plants expressing the SAE2UFDCt domain displayed dose-dependent inhibition of SUMO conjugation, and have revealed the existence of a post-transcriptional mechanism that regulates SUMO E2 conjugating enzyme levels. Interestingly, these transgenic plants displayed increased susceptibility to necrotrophic fungal infections by Botrytis cinerea and Plectosphaerella cucumerina. Early after fungal inoculation, host SUMO conjugation was post-transcriptionally downregulated, suggesting that targeting SUMOylation machinery could constitute a novel mechanism for fungal pathogenicity. These findings support the role of SUMOylation as a mechanism involved in plant protection from environmental stresses. In addition, the strategy for inhibiting SUMO conjugation in vivo described in this study might be applicable in important crop plants and other non-plant organisms regardless of their genetic complexity.

A Simplified and Rapid Method for the Isolation and Transfection of Arabidopsis Leaf Mesophyll Protoplasts for Large-Scale Applications.

Arabidopsis leaf mesophyll protoplasts constitute an important and versatile tool for conducting cell-based experiments to analyze the functions of distinct signaling pathways and cellular machineries using proteomic, biochemical, cellular, genetic, and genomic approaches. Thus, the methods for protoplast isolation and transfection have been gradually improved to achieve efficient expression of genes of interest. Although many well-established protocols have been extensively tested, their successful application is sometimes limited to researchers with a high degree of skill and experience in protoplasts handling. Here we present a detailed method for the isolation and transfection of Arabidopsis mesophyll protoplasts, in which many of the time-consuming and critical steps present in the current protocols have been simplified. The method described is fast, simple, and leads to high yields of competent protoplasts allowing large-scale applications.

Analysis of Protein-Lipid Interactions Using Purified C2 Domains.

C2 domains (C2s) are regulatory protein modules identified in eukaryotic proteins targeted to cell membranes. C2s were initially characterized as independently folded Ca(2+)-dependent phospholipids binding domains; however, later studies have shown that C2s have evolutionarily diverged into Ca(2+)-dependent and Ca(2+)-independent forms. These forms interact and regulate their affinity to diverse lipid species using different binding mechanisms. In this protocol we describe a biochemical approach to produce, purify, and solubilize functional C2 domains bound to GST for the identification of their putative Ca(2+)-dependent and Ca(2+)-independent lipid-binding partners.

Structural determinants of Arabidopsis thaliana Hyponastic leaves 1 function in vivo.

MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity.

Reprint of: construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates.

MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems.

Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates.

MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems.

Multiple RNA recognition patterns during microRNA biogenesis in plants.

MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.

Processing of plant microRNA precursors.

MicroRNAs are endogenous small RNAs known to be key regulators of gene expression in animals and plants. They are defined by their specific biogenesis which involves the precise excision from an imperfect fold-back precursor. These precursors contain structural determinants required for their correct processing. Still, there are significant differences in the biogenesis and activities of plant and animal microRNAs. This review summarizes diverse aspects of precursor processing in plants, contrasting them to their animal counterparts.

Plasma membrane repair in plants.

Resealing is the membrane-repair process that enables cells to survive disruption, preventing the loss of irreplaceable cell types and eliminating the cost of replacing injured cells. Given that failure in the resealing process in animal cells causes diverse types of muscular dystrophy, plasma membrane repair has been extensively studied in these systems. Animal proteins with Ca(2+)-binding domains such as synaptotagmins and dysferlin mediate Ca(2+)-dependent exocytosis to repair plasma membranes after mechanical damage. Until recently, no components or proof for membrane repair mechanisms have been discovered in plants. However, Arabidopsis SYT1 is now the first plant synaptotagmin demonstrated to participate in Ca(2+)-dependent repair of membranes. This suggests a conservation of membrane repair mechanisms between animal and plant cells.

Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

The Arabidopsis tetratricopeptide repeat-containing protein TTL1 is required for osmotic stress responses and abscisic acid sensitivity.

Mutations in the Arabidopsis (Arabidopsis thaliana) TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 1 (TTL1) cause reduced tolerance to NaCl and osmotic stress that is characterized by reduced root elongation, disorganization of the root meristem, and impaired osmotic responses during germination and seedling development. Expression analyses of genes involved in abscisic acid (ABA) biosynthesis and catabolism suggest that TTL1 is not involved in the regulation of ABA levels but is required for ABA-regulated responses. TTL1 regulates the transcript levels of several dehydration-responsive genes, such as the transcription factor DREB2A, and genes encoding dehydration response proteins, such as ERD1 (early response to dehydration 1), ERD3, and COR15a. The TTL1 gene encodes a novel plant protein with tetratricopeptide repeats and a region with homology to thioredoxin proteins. Based on homology searches, there are four TTL members in the Arabidopsis genome with similar intron-exon structure and conserved amino acid domains. Proteins containing tetratricopeptide repeat motifs act as scaffold-forming multiprotein complexes and are emerging as essential elements for plant hormonal responses (such as gibberellin responses and ethylene biosynthesis). In this report, we identify TTL1 as a positive regulator of ABA signaling during germination and seedling development under stress.

TPR Proteins in Plant Hormone Signaling.

There is a large number of proteins in nature containing Tetratrico Peptide Repeats (TPRs). TPR motifs are defined as a protein-protein interaction module involved in regulation of different cellular functions. We have recently identified TTL1 as a protein containing TPR motifs required for abscisic acid responses and osmotic stress tolerance. In recent years several of these proteins have been found to be essential for responses to other hormones such ethylene, cytokinin, gibberelling and auxin in Arabidopsis. Thus, proteins containing TPRs are emerging as essential determinants for signal transduction pathways mediated by most plant hormones.