Exploring the Relationship between G-Quadruplex Nucleic Acids and Plants: From Plant G-Quadruplex Function to Phytochemical G4 Ligands with Pharmaceutic Potential

G-quadruplex (G4) oligonucleotides are higher-order DNA and RNA secondary structures of enormous relevance due to their implication in several biological processes and pathological states in different organisms. Strategies aiming at modulating human G4 structures and their interrelated functions are first-line approaches in modern research aiming at finding new potential anticancer treatments or G4-based aptamers for various biomedical and biotechnological applications. Plants offer a cornucopia of phytocompounds that, in many cases, are effective in binding and modulating the thermal stability of G4s and, on the other hand, contain almost unexplored G4 motifs in their genome that could inspire new biotechnological strategies. Herein, we describe some G4 structures found in plants, summarizing the existing knowledge of their functions and biological role. Moreover, we review some of the most promising G4 ligands isolated from vegetal sources and report on the known relationships between such phytochemicals and G4-mediated biological processes that make them potential leads in the pharmaceutical sector

Weighted gene co-expression network analysis of nitrogen (N)-responsive genes and the putative role of G-quadruplexes in N use efficiency (NUE) in rice

Rice is an important target to improve crop nitrogen (N) use efficiency (NUE), and the identification and shortlisting of the candidate genes are still in progress. We analyzed data from 16 published N-responsive transcriptomes/microarrays to identify, eight datasets that contained the maximum number of 3020 common genes, referred to as N-responsive genes. These include different classes of transcription factors, transporters, miRNA targets, kinases and events of post-translational modifications. A Weighted gene co-expression network analysis (WGCNA) with all the 3020 N-responsive genes revealed 15 co-expression modules and their annotated biological roles. Protein-protein interaction network analysis of the main module revealed the hub genes and their functional annotation revealed their involvement in the ubiquitin process. Further, the occurrences of G-quadruplex sequences were examined, which are known to play important roles in epigenetic regulation but are hitherto unknown in N-response/NUE. Out of the 3020 N-responsive genes studied, 2298 contained G-quadruplex sequences. We compared these N-responsive genes containing G-quadruplex sequences with the 3601 genes we previously identified as NUE-related (for being both N-responsive and yield-associated). This analysis revealed 389 (17%) NUE-related genes containing G-quadruplex sequences. These genes may be involved in the epigenetic regulation of NUE, while the rest of the 83% (1811) genes may regulate NUE through genetic mechanisms and/or other epigenetic means besides G-quadruplexes. A few potentially important genes/processes identified as associated with NUE were experimentally validated in a pair of rice genotypes contrasting for NUE. The results from the WGCNA and G4 sequence analysis of N-responsive genes helped identify and shortlist six genes as candidates to improve NUE. Further, the hitherto unavailable segregation of genetic and epigenetic gene targets could aid in informed interventions through genetic and epigenetic means of crop improvement

Recent Advances in Genetics and Breeding of Major Staple Food Crops

To meet the global food demand of an increasing population, food production has to be increased by 60% by 2050. The main production constraints, such as climate change, biotic stresses, abiotic stresses, soil nutrition deficiency problems, problematic soils, etc., have to be addressed on an urgent basis. More than 50% of human calories are from three major cereals: rice, wheat, and maize. The harnessing of genetic diversity by novel allele mining assisted by recent advances in biotechnological and bioinformatics tools will enhance the utilization of the hidden treasures in the gene bank. Technological advances in plant breeding will provide some solutions for the biofortification, stress resistance, yield potential, and quality improvement in staple crops. The elucidation of the genetic, physiological, and molecular basis of useful traits and the improvement of the improved donors containing multiple traits are key activities for variety development. High-throughput genotyping systems assisted by bioinformatics and data science provide efficient and easy tools for geneticists and breeders. Recently, new breeding techniques applied in some food crops have become game-changers in the global food crop market. With this background, we invited 18 eminent researchers working on food crops from across the world to contribute their high-quality original research manuscripts. The research studies covered modern food crop genetics and breeding

Centromeres under pressure: Evolutionary innovation in conflict with conserved function

Centromeres are essential genetic elements that enable spindle microtubule attachment for chromosome segregation during mitosis and meiosis. While this function is preserved across species, centromeres display an array of dynamic features, including: (1) rapidly evolving DNA; (2) wide evolutionary diversity in size, shape and organization; (3) evidence of mutational processes to generate homogenized repetitive arrays that characterize centromeres in several species; (4) tolerance to changes in position, as in the case of neocentromeres; and (5) intrinsic fragility derived by sequence composition and secondary DNA structures. Centromere drive underlies rapid centromere DNA evolution due to the “selfish” pursuit to bias meiotic transmission and promote the propagation of stronger centromeres. Yet, the origins of other dynamic features of centromeres remain unclear. Here, we review our current understanding of centromere evolution and plasticity. We also detail the mutagenic processes proposed to shape the divergent genetic nature of centromeres. Changes to centromeres are not simply evolutionary relics, but ongoing shifts that on one side promote centromere flexibility, but on the other can undermine centromere integrity and function with potential pathological implications such as genome instability

RNA G-quadruplexes and polyamines effects on translation: data analysis and development of software for ribosome pause prediction

Ribosome profiling (Ribo-seq) technique provides a global snapshot of actively translating ribosomes by capturing the ribosome protected mRNA fragments. Ribo-seq technique can be implemented to study various translation aspects such as differential gene expression at the translation level, ribosomal frameshifting, decoding rate, ribosome pausing etc. The process of translation can be affected locally by the sequence of translated mRNA, e.g. by specific secondary structures as well as globally by cellular conditions, e.g. concentrations of metabolites such as polyamines. G-quadruplexes is a type of RNA secondary structures that has been implicated in mRNA translation. These G-quadruplex structures have been suggested to play roles in capdependent, cap-independent and repeat associated non-AUG (RAN) translation. Gquadruplex structures are also known to stimulate ribosomal frameshifting and ribosome pausing. In my work, I explored the effect of G-quadruplex on translation by utilizing the publicly available ribosome-profiling datasets to find the relationship between G-quadruplex structures and ribosome footprint density. This analysis revealed the presence of very low ribosome footprint density in the vicinity of the G-quadruplex structures. Further I have analysed the effect of the pharmacological stabilisation of the G-quadruplex on translation using PhenDC3 drug. The treatment of PhenDC3 drug was found to have no significant effect on the translational level. This is described in chapter 2. The presence of ribosome pauses in the upstream of G-quadruplex regions and the lack of an existing tool to identify ribosome pause sites motivated me to develop a tool for the prediction of the ribosome pauses using ribosome profiling data. This lead to the development of PausePred tool which has been discussed in chapter 3. The PausePred tool can be used to infer ribosome pauses which are scored based on their magnitude relative to the background density within the surrounding area. In addition to the score, PausePred provides the coordinates of the pause location, the footprint density at the pause site and the flanking nucleotide sequence. Further I utilized PausePred to analyse ribosome pausing using ribosome profiling data obtained under varying concentrations of polyamines (described in chapter 4). I showed that in cells with higher polyamine levels, proline-proline or proline-glycine amino acids are enriched in the E and P-sites of paused ribosomes

Plant Proteomic Research 4.0

As an important tool of systems biology, proteomics has enabled a deep understanding of different plant processes and functions. Complemented with genomic data, computational tools, and improved sample preparation strategies, proteomics has an unprecedented opportunity to characterize plant proteoforms in high spatial and temporal resolution. This special issue of Plant Proteomic Research 4.0 captures the recent advancements in proteomics and addresses the current challenges of plant stress response and resilience in the ever-changing climate. It contains 12 articles, including three reviews and nine original research articles. The three reviews deal with pollen phosphoproteomics, starch biosynthesis-related proteins and posttranslational modifications (PTMs) in rice developing seeds, and PTMs of waxy proteins in rice grain. The nine research articles include three related to temperature, two on water stress, two on salt stress, one on fungal pathogen, and the last one on field-grown potato apoplast proteome. The articles reflect the current frontiers of plant proteomics, focusing on themes of environmental stresses, proteoforms/PTMs, crop species, and new development in data-independent acquisition mass spectrometry. They provide readers insights into current technologies, their utility in understanding plant growth and resilience, as well as directions of proteomics in the frontiers of systems biology and synthetic biology

Isolation and characterization of potato homologues of Arabidopsis thaliana genes operating in defense signal transduction

An increasing number of pathogen-defense related genes are being identified and characterized in Arabidopsis thaliana. So far, it is not known whether and which structural and functional homologues of these Arabidopsis genes have any role in natural variation of resistance to pathogens in crops. Using sequence database mining and PCR-based approaches, potato (Solanum tuberosum L.) gene fragments with high sequence similarity to 16 Arabidopsis defense signal transduction genes were obtained, sequenced and genetically positioned on potato molecular maps. Of 16 novel loci, five were positional candidates for known potato pathogen resistance QTL. One of the candidate loci, StAOS2 co-localizing with QTL for resistance to P. infestans and E. carotovora on linkage group XI, was further characterized in more detail. StAOS2 encodes a gene for allene oxide synthase, a cytochrome P450-enzyme, acting upstream in the jasmonic acid biosynthesis pathway. A metabolic block at the level of AOS completely abolishes JA production, which affects plant development (e.g. sterile pollen production) and various abiotic and biotic stress responses (e.g. P. infestans resistance in tomato, E. carotovora resistance in Arabidopsis). The chloroplastic localization of StAOS2-GFP was confirmed by confocal microscopy and functionality of the potato protein was proven by complementation of the male-sterile Arabidopsis aos mutant. StAOS2-RNAi transgenic lines in potato were generated in order to test role of StAOS2 in P. infestans resistance. The measurements of endogenous OPDA and JA in the silenced lines after wounding treatment revealed drastic decrease in the levels of above mentioned compounds (up to 25 folds less than in wild type plants). In addition, natural variation of StAOS2 locus was characterized. Sequencing of the locus across 38 potato chromosomes revealed high polymorphism. Thirteen distinct alleles were found, and four of them showed highly significant (P=0.000, R2=14%) linkage to P. infestans and E. carotovora QTL. Five alleles of StAOS2 were cloned. Sequence analyses revealed a substantial polymorphism on amino acid level, including non-conservative substitutions and an insertion/deletion within the cytochrome P450 domain. Currently, an ongoing quantitative complementation of the Ataos mutant with the five different StAOS2 alleles fused to the native AtAOS promoter, followed by OPDA and JA levels measurements in the transgenic lines, will possibly provide direct evidence for StAOS2 being the first plant resistance QTL identified

Physiological, agronomic and genomic evaluation of drought stress tolerance in potato (Solanum tuberosum L.)

156 p.La patata (Solanum tuberosum L.) es uno de los cultivos más importantes a nivel mundial y contribuye de forma importante al suministro mundial de alimentos, produciéndose más de 370 millones de toneladas. El cultivo tiene, asimismo, una gran relevancia en la seguridad alimentaria, reducción de la pobreza y mejora de la nutrición humana. Debido a los efectos del cambio climático, las condiciones tienden a ser cada vez más extremas, siendo la disponibilidad de agua uno de los factores limitantes en la producción del cultivo, por lo que es importante poder seleccionar variedades que puedan mantener el rendimiento y hacer frente a las consecuencias del cambio climático. La mejora tradicional de patata es un proceso largo, costoso y con problemas como la naturaleza heterocigota y tetraploide. Durante las últimas décadas se han desarrollado tecnologías de secuenciación de nueva generación (NGS) que cada vez son más asequibles y permiten a los mejoradores identificar alelos específicos beneficiosos que son responsables de las variaciones deseables en los cultivos. Esta tesis intenta abordar el problema del efecto del estrés hídrico como consecuencia del cambio climático en el cultivo de la patata desde distintos puntos de vista.Por un lado, se evaluó la respuesta diferencial de parámetros fisiológicos y de producción en seis variedades de patata bajo condiciones de déficit hídrico. Para ello se llevó a cabo un ensayo deinvernadero con las variedades Ágata, Agria, Kennebec, Monalisa, Santé y Zorba. Se tomaron todas las medidas en la hoja más joven completamente expandida y en 4 tiempos distintos: antes del estrés (T0), a los 17 días (T1) y a los 24 días (T2) de retirarlas el riego y 5 días después de regarlas nuevamente (R1). Los parámetros fisiológicos evaluados fueron el contenido y la fluorescencia de la clorofila, contenido relativo de agua (RWC), apertura estomática, conductividad electrolítica y potencial hídrico. Tras el periodo de sequía se cortó la parte aérea de la mitad de las plantas para obtener la biomasa. Se estimaron los parámetros de producción como como número y peso medio de tubérculos, producción, materia seca y almidón. Los parámetros más afectados por el déficit hídrico fueron la apertura estomática y el potencial hídrico. La selección indirecta basada en parámetros asociados al estrés hídrico puede ser una herramienta útil en programas de selección en patata para la identificación de variedades más tolerantes.Por otro lado, con el objetivo de comprender los mecanismos de respuesta al estrés hídrico a nivel de transcriptoma, se llevó a cabo un ensayo en el que se utilizaron las variedades Agria y Zorba para ver la diferencia de expresión de genes entre plantas control y plantas sometidas a estrés hídrico. Para ello, se secuenciaron mediante RNAseq, obteniendo alrededor de 50 millones de transcritos para cada variedad. Al comparar los transcritos significativos obtenidos de las plantas control con las de sequía de la variedad Agria, se vio que 931 genes estaban regulados ¿up¿, es decir, aumentaba su expresión, y 2077 genes estaban regulados ¿down¿, disminuyendo su expresión bajo condiciones de estrés. Cuando se compararon ambos tratamientos en las plantas de la variedad Zorba, se encontraron 735 genes regulados ¿up¿ y 923 genes regulados ¿down¿. Además, se analizaron los transcritos con el fin de averiguar sus funciones y destacar las vías metabólicas potencialmente relacionadas con la tolerancia a la sequía. Para ello se hizo una clasificación de ontología de genes (GO), las cuales describen la función molecular de los productos génicos, su rol en los procesos biológicos y su localización en componentes celulares. En este ensayo se ha visto que las principales ontologías a las que pertenecen los transcritos obtenidos pertenecen a componentes celulares, y más en concreto relacionados con la membrana y la pared celular. Estos resultados proporcionan una información valiosa relacionada con los mecanismos moleculares de tolerancia a estrés hídrico.Por último, se evaluó un panel de 144 genotipos de patata durante dos años para observar la variación biológica de varios rasgos fisiológicos (contenido de clorofila, fluorescencia de clorofila, conductancia estomática, NDVI, área y perímetro foliar) y agronómicos (rendimiento, peso y número de tubérculos, contenido de almidón, materia seca y azúcares reductores). Estas mismas variedades se genotiparon mediante el chip ¿GGP V3 Potato array¿, obteniendo un total de 31190 marcadores SNP que tras el filtrado se quedaron en 18259. Se determinó la subestructura de la población utilizando el software STRUCTURE 2.3.4 y el análisis de mapeo asociativo se realizó utilizando el paquete estadístico GwasPoly. Se detectaron SNPs asociados tanto con los rasgos fenotípicos como con los agronómicos en varios cromosomas y se asociaron con funciones genéticas conocidas. Estos resultados podrían ser útiles para diseñar marcadores y utilizarlos en futuros programas de selección asistida relacionados con la tolerancia a la sequía en patata.Neike

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers