Catálogo de publicaciones - revistas

<jats:title>Abstract</jats:title><jats:p>In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. <jats:italic>IPI1/emb175/PPR103</jats:italic> is a nuclear gene encoding a PLS-type PPR protein essential for survival in <jats:italic>Arabidopsis thaliana</jats:italic> and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and <jats:italic>Nicotiana</jats:italic> IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in <jats:italic>N. benthamiana</jats:italic> to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the <jats:italic>Nicotiana</jats:italic> and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of <jats:italic>NbIPI1</jats:italic> led to defects in chloroplast ribosomal RNA processing and changes to stability of <jats:italic>rpl16</jats:italic> transcripts, revealing conserved function with its maize ortholog. <jats:italic>NbIPI1</jats:italic>-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in <jats:italic>N. benthamiana</jats:italic> chloroplasts<jats:italic>.</jats:italic></jats:p>

<jats:title>Abstract</jats:title><jats:p>The dynamic interaction of RNA-binding proteins (RBPs) with their target RNAs contributes to the diversity of ribonucleoprotein (RNP) complexes that are involved in a myriad of biological processes. Identifying the RNP components at high resolution and defining their interactions are key to understanding their regulation and function. Expressing fusions between an RBP of interest and an RNA editing enzyme can result in nucleobase changes in target RNAs, representing a recent addition to experimental approaches for profiling RBP/RNA interactions. Here, we have used the MS2 protein/RNA interaction to test four RNA editing proteins for their suitability to detect target RNAs of RBPs <jats:italic>in planta</jats:italic>. We have established a transient test system for fast and simple quantification of editing events and identified the hyperactive version of the catalytic domain of an adenosine deaminase (hADARcd) as the most suitable editing enzyme. Examining fusions between homologs of polypyrimidine tract binding proteins (PTBs) from <jats:italic>Arabidopsis thaliana</jats:italic> and hADARcd allowed determining target RNAs with high sensitivity and specificity. Moreover, almost complete editing of a splicing intermediate provided insight into the order of splicing reactions and PTB dependency of this particular splicing event. Addition of sequences for nuclear localisation of the fusion protein increased the editing efficiency, highlighting this approach’s potential to identify RBP targets in a compartment-specific manner. Our studies have established the editing-based analysis of interactions between RBPs and their RNA targets in a fast and straightforward assay, offering a new system to study the intricate composition and functions of plant RNPs in vivo.</jats:p>

<jats:title>Abstract</jats:title><jats:p>Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.</jats:p>

<jats:title>Abstract</jats:title><jats:p>Pyruvate kinase (Pyk<jats:italic>,</jats:italic> EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. <jats:italic>Synechocystis</jats:italic> sp. PCC 6803 possesses two pyk genes (encoded <jats:italic>pyk1</jats:italic>, sll0587 and <jats:italic>pyk2</jats:italic>, sll1275). A previous study suggested that <jats:italic>pyk2</jats:italic> and not <jats:italic>pyk1</jats:italic> is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed <jats:italic>Synechocystis</jats:italic> Pyk2 (hereafter, <jats:italic>Sy</jats:italic>Pyk2). The optimum pH and temperature of <jats:italic>Sy</jats:italic>Pyk2 were 7.0 and 55 °C, respectively, and the <jats:italic>K</jats:italic><jats:sub>m</jats:sub> values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. <jats:italic>Sy</jats:italic>Pyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that <jats:italic>Sy</jats:italic>Pyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. <jats:italic>Sy</jats:italic>Pyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.</jats:p>

<jats:title>Abstract</jats:title><jats:p>The plant-specific homeodomain-leucine zipper I subfamily is involved in the regulation of various biological processes, particularly growth, development and stress response. In the present study, we characterized four <jats:italic>BnaHB6</jats:italic> homologues from <jats:italic>Brassica napus</jats:italic>. All BnaHB6 proteins have transcriptional activation activity. Structural and functional data indicate the complex role of <jats:italic>BnaHB6</jats:italic> genes in regulating biological processes, with some functions conserved and others diverged. Transcriptional analyzes revealed that they are induced in a similar manner in different tissues but show different expression patterns in response to stress and circadian rhythm. Only the <jats:italic>BnaA09HB6</jats:italic> and <jats:italic>BnaC08HB6</jats:italic> genes are expressed under dehydration and salt stress, and in darkness. The partial transcriptional overlap of <jats:italic>BnaHB6</jats:italic>s with the evolutionarily related genes <jats:italic>BnaHB5</jats:italic> and <jats:italic>BnaHB16</jats:italic> was also observed. Transgenic <jats:italic>Arabidopsis thaliana</jats:italic> plants expressing a single pro<jats:italic>BnaHB6</jats:italic>::GUS partially confirmed the expression results. Bioinformatic analysis allowed the identification of TF-binding sites in the <jats:italic>BnaHB6</jats:italic> promoters that may control their expression under stress and circadian rhythm. ChIP-qPCR analysis revealed that <jats:italic>BnaA09HB6</jats:italic> and <jats:italic>BnaC08HB6</jats:italic> bind directly to the promoters of the target genes <jats:italic>BnaABF4</jats:italic> and <jats:italic>BnaDREB2A</jats:italic>. Comparison of their expression patterns in the WT plants and the <jats:italic>bnac08hb6</jats:italic> mutant showed that <jats:italic>BnaC08HB6</jats:italic> positively regulates the expression of the <jats:italic>BnaABF4</jats:italic> and <jats:italic>BnaDREB2A</jats:italic> genes under dehydration and salt stress. We conclude that four <jats:italic>BnaHB6</jats:italic> homologues have distinct functions in response to stress despite high sequence similarity, possibly indicating different binding preferences with <jats:italic>BnaABF4</jats:italic> and <jats:italic>BnaDREB2A</jats:italic>. We hypothesize that <jats:italic>BnaC08HB6</jats:italic> and <jats:italic>BnaA09HB6</jats:italic> function in a complex regulatory network under stress.</jats:p>