R-Loop Mediated trans Action of the APOLO Long Noncoding RNA.

In eukaryotes, three-dimensional genome organization is critical for transcriptional regulation of gene expression. Long noncoding RNAs (lncRNAs) can modulate chromatin conformation of spatially related genomic locations within the nucleus. Here, we show that the lncRNA APOLO (AUXIN-REGULATED PROMOTER LOOP) recognizes multiple distant independent loci in the Arabidopsis thaliana genome. We found that APOLO targets are not spatially associated in the nucleus and that APOLO recognizes its targets by short sequence complementarity and the formation of DNA-RNA duplexes (R-loops). The invasion of APOLO to the target DNA decoys the plant Polycomb Repressive Complex 1 component LHP1, modulating local chromatin 3D conformation. APOLO lncRNA coordinates the expression of distal unrelated auxin-responsive genes during lateral root development in Arabidopsis. Hence, R-loop formation and chromatin protein decoy mediate trans action of lncRNAs on distant loci. VIDEO ABSTRACT.

The plant noncoding transcriptome: a versatile environmental sensor.

Plant noncoding RNA transcripts have gained increasing attention in recent years due to growing evidence that they can regulate developmental plasticity. In this review article, we comprehensively analyze the relationship between noncoding RNA transcripts in plants and their response to environmental cues. We first provide an overview of the various noncoding transcript types, including long and small RNAs, and how the environment modulates their performance. We then highlight the importance of noncoding RNA secondary structure for their molecular and biological functions. Finally, we discuss recent studies that have unveiled the functional significance of specific long noncoding transcripts and their molecular partners within ribonucleoprotein complexes during development and in response to biotic and abiotic stress. Overall, this review sheds light on the fascinating and complex relationship between dynamic noncoding transcription and plant environmental responses, and highlights the need for further research to uncover the underlying molecular mechanisms and exploit the potential of noncoding transcripts for crop resilience in the context of global warming.

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controls shade avoidance syndrome in Arabidopsis.

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. In response to far-red (FR) light, expression of APOLO anti-correlates with that of its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. Accumulation of APOLO transcription fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves and in a late response to far-red light treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of auxin synthesis gene YUCCA2, and auxin efflux genes PID and WAG2. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant-environment interactions may therefore become a new tool for sustainable agriculture.

An atlas of the tomato epigenome reveals that KRYPTONITE shapes TAD-like boundaries through the control of H3K9ac distribution.

In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.

sincFold: end-to-end learning of short- and long-range interactions in RNA secondary structure.

MOTIVATION: Coding and noncoding RNA molecules participate in many important biological processes. Noncoding RNAs fold into well-defined secondary structures to exert their functions. However, the computational prediction of the secondary structure from a raw RNA sequence is a long-standing unsolved problem, which after decades of almost unchanged performance has now re-emerged due to deep learning. Traditional RNA secondary structure prediction algorithms have been mostly based on thermodynamic models and dynamic programming for free energy minimization. More recently deep learning methods have shown competitive performance compared with the classical ones, but there is still a wide margin for improvement. RESULTS: In this work we present sincFold, an end-to-end deep learning approach, that predicts the nucleotides contact matrix using only the RNA sequence as input. The model is based on 1D and 2D residual neural networks that can learn short- and long-range interaction patterns. We show that structures can be accurately predicted with minimal physical assumptions. Extensive experiments were conducted on several benchmark datasets, considering sequence homology and cross-family validation. sincFold was compared with classical methods and recent deep learning models, showing that it can outperform the state-of-the-art methods.

Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

LncRNA DANA1 promotes drought tolerance and histone deacetylation of drought responsive genes in Arabidopsis.

Although many long noncoding RNAs have been discovered in plants, little is known about their biological function and mode of action. Here we show that the drought-induced long intergenic noncoding RNA DANA1 interacts with the L1p/L10e family member protein DANA1-INTERACTING PROTEIN 1 (DIP1) in the cell nucleus of Arabidopsis, and both DANA1 and DIP1 promote plant drought resistance. DANA1 and DIP1 increase histone deacetylase HDA9 binding to the CYP707A1 and CYP707A2 loci. DIP1 further interacts with PWWP3, a member of the PEAT complex that associates with HDA9 and has histone deacetylase activity. Mutation of DANA1 enhances CYP707A1 and CYP707A2 acetylation and expression resulting in impaired drought tolerance, in agreement with dip1 and pwwp3 mutant phenotypes. Our results demonstrate that DANA1 is a positive regulator of drought response and that DANA1 works jointly with the novel chromatin-related factor DIP1 on epigenetic reprogramming of the plant transcriptome during the response to drought.

Genetic-epigenetic interplay in the determination of plant 3D genome organization.

The 3D chromatin organization plays a major role in the control of gene expression. However, our comprehension of the governing principles behind nuclear organization remains incomplete. Particularly, the spatial segregation of loci with similar repressive transcriptional states in plants poses a significant yet poorly understood puzzle. In this study, employing a combination of genetics and advanced 3D genomics approaches, we demonstrated that a redistribution of facultative heterochromatin marks in regions usually occupied by constitutive heterochromatin marks disrupts the 3D genome compartmentalisation. This disturbance, in turn, triggers novel chromatin interactions between genic and transposable element (TE) regions. Interestingly, our results imply that epigenetic features, constrained by genetic factors, intricately mold the landscape of 3D genome organisation. This study sheds light on the profound genetic-epigenetic interplay that underlies the regulation of gene expression within the intricate framework of the 3D genome. Our findings highlight the complexity of the relationships between genetic determinants and epigenetic features in shaping the dynamic configuration of the 3D genome.

Epigenetic control during root development and symbiosis.

The roots of plants play multiples functions that are essential for growth and development, including anchoring to the soil and water and nutrient acquisition. These underground organs exhibit the plasticity to modify their root system architecture in response to environmental cues allowing adaptation to change in water and nutrient availability. In addition, roots enter in mutualistic interactions with soil microorganisms, e.g. the root nodule symbiosis established between a limited group of plants and nitrogen fixing soil bacteria and the arbuscular mycorrhiza symbiosis involving most land plants and fungi of the Glomeromycetes phylum. In the past 20 years, genetic approaches allowed the identification and functional characterization of genes required for the specific programs of root development, root nodule and arbuscular mycorrhiza symbioses. These genetic studies provided evidence that the program of root nodule symbiosis recruited components of the arbuscular mycorrhiza symbiosis and the root developmental programs. The execution of these programs is strongly influenced by epigenetic changes -DNA methylation and histone post-translational modifications- that alter chromatin conformation modifying the expression of key genes. In this review, we summarize recent advances that highlighted how DNA methylation and histone post-translational modifications, as well as chromatin remodeling factors and long non-coding RNAs, shape the root system architecture and allow the successful establishment of both root nodule and arbuscular mycorrhiza symbioses. We anticipate that the analysis of dynamic epigenetic changes and chromatin 3D structure in specific single-cells or tissue types of root organs will illuminate our understanding of how root developmental and symbiotic programs are orchestrated, opening exciting questions and new perspectives to modulate agronomical and ecological traits linked to nutrient acquisition.

Polycomb-dependent differential chromatin compartmentalization determines gene coregulation in Arabidopsis.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

A lateral organ boundaries domain transcription factor acts downstream of the auxin response factor 2 to control nodulation and root architecture in Medicago truncatula.

Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans-Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes. Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP-PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2. Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program.

Transcription factor NAC1 activates expression of peptidase-encoding AtCEPs in roots to limit root hair growth.

Plant genomes encode a unique group of papain-type Cysteine EndoPeptidases (CysEPs) containing a KDEL endoplasmic reticulum (ER) retention signal (KDEL-CysEPs or CEPs). CEPs process the cell-wall scaffolding EXTENSIN (EXT) proteins that regulate de novo cell-wall formation and cell expansion. Since CEPs cleave EXTs and EXT-related proteins, acting as cell-wall-weakening agents, they may play a role in cell elongation. The Arabidopsis (Arabidopsis thaliana) genome encodes 3 CEPs (AtCPE1-AtCEP3). Here, we report that the genes encoding these 3 Arabidopsis CEPs are highly expressed in root-hair (RH) cell files. Single mutants have no evident abnormal RH phenotype, but atcep1-3 atcep3-2 and atcep1-3 atcep2-2 double mutants have longer RHs than wild-type (Wt) plants, suggesting that expression of AtCEPs in root trichoblasts restrains polar elongation of the RH. We provide evidence that the transcription factor NAC1 (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) activates AtCEPs expression in roots to limit RH growth. Chromatin immunoprecipitation indicates that NAC1 binds to the promoter of AtCEP1, AtCEP2, and, to a lower extent, AtCEP3 and may directly regulate their expression. Inducible NAC1 overexpression increases AtCEP1 and AtCEP2 transcript levels in roots and leads to reduced RH growth while the loss of function nac1-2 mutation reduces AtCEP1-AtCEP3 gene expression and enhances RH growth. Likewise, expression of a dominant chimeric NAC1-SRDX repressor construct leads to increased RH length. Finally, we show that RH cell walls in the atcep1-3 atcep3-2 double mutant have reduced levels of EXT deposition, suggesting that the defects in RH elongation are linked to alterations in EXT processing and accumulation. Our results support the involvement of AtCEPs in controlling RH polar growth through EXT processing and insolubilization at the cell wall.

TCP15 interacts with GOLDEN2-LIKE 1 to control cotyledon opening in Arabidopsis.

After germination, exposure to light promotes the opening and expansion of the cotyledons and the development of the photosynthetic apparatus in a process called de-etiolation. This process is crucial for seedling establishment and photoautotrophic growth. TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors are important developmental regulators of plant responses to internal and external signals that are grouped into two main classes. In this study, we identified GOLDEN2-LIKE 1 (GLK1), a key transcriptional regulator of photomorphogenesis, as a protein partner of class I TCPs during light-induced cotyledon opening and expansion in Arabidopsis. The class I TCP TCP15 and GLK1 are mutually required for cotyledon opening and the induction of SAUR and EXPANSIN genes, involved in cell expansion. TCP15 also participates in the expression of photosynthesis-associated genes regulated by GLK1, like LHCB1.4 and LHCB2.2. Furthermore, GLK1 and TCP15 bind to the same promoter regions of different target genes containing either GLK or TCP binding motifs and binding of TCP15 is affected in a GLK1-deficient background, suggesting that a complex between TCP15 and GLK1 participates in the induction of these genes. We postulate that GLK1 helps to recruit TCP15 for the modulation of cell expansion genes in cotyledons and that the functional interaction between these transcription factors may serve to coordinate the expression of cell expansion genes with that of genes involved in the development of the photosynthetic apparatus.

The long intergenic noncoding RNA ARES modulates root architecture in Arabidopsis.

Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression in plants. They have been linked to a wide range of molecular mechanisms, including epigenetics, miRNA activity, RNA processing and translation, and protein localization or stability. In Arabidopsis, characterized lncRNAs have been implicated in several physiological contexts, including plant development and the response to the environment. Here we searched for lncRNA loci located nearby key genes involved in root development and identified the lncRNA ARES (AUXIN REGULATOR ELEMENT DOWNSTREAM SOLITARYROOT) downstream of the lateral root master gene IAA14/SOLITARYROOT (SLR). Although ARES and IAA14 are co-regulated during development, the knockdown and knockout of ARES did not affect IAA14 expression. However, in response to exogenous auxin, ARES knockdown impairs the induction of its other neighboring gene encoding the transcription factor NF-YB3. Furthermore, knockdown/out of ARES results in a root developmental phenotype in control conditions. Accordingly, a transcriptomic analysis revealed that a subset of ARF7-dependent genes is deregulated. Altogether, our results hint at the lncRNA ARES as a novel regulator of the auxin response governing lateral root development, likely by modulating gene expression in trans.

Differential chromatin binding preference is the result of the neo-functionalization of the TB1 clade of TCP transcription factors in grasses.

The understanding of neo-functionalization of plant transcription factors (TFs) after gene duplication has been extensively focused on changes in protein-protein interactions, the expression pattern of TFs, or the variation of cis-elements bound by TFs. Yet, the main molecular role of a TF, that is, its specific chromatin binding for the direct regulation of target gene expression, continues to be mostly overlooked. Here, we studied the TB1 clade of the TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTORS (TCP) TF family within the grasses (Poaceae). We identified an Asp/Gly amino acid replacement within the TCP domain, originated within a paralog TIG1 clade exclusive for grasses. The heterologous expression of Zea mays TB1 and its two paralogs BAD1 and TIG1 in Arabidopsis mutant plants lacking the TB1 ortholog BRC1 revealed distinct functions in plant development. Notably, the Gly acquired in the TIG1 clade does not impair TF homodimerization and heterodimerization, while it modulates chromatin binding preferences. We found that in vivo TF recognition of target promoters depends on this Asp/Gly mutation and directly impacts downstream gene expression and subsequent plant development. These results provided new insights into how natural selection fine-tunes gene expression regulation after duplication of TFs to define plant architecture.

Plant long non-coding RNAs: biologically relevant and mechanistically intriguing.

Long non-coding RNAs (lncRNAs) are a group of RNAs greater than 200 nucleotides in length exhibiting low or no coding potential that are involved in diverse biological functions through their molecular interaction with proteins, DNA, or other RNAs. With the emergence of advanced high-throughput RNA sequencing technologies, tens of thousands of novel long non-coding RNAs have been identified in plant transcriptomes in the last decade. More importantly, functional studies revealed that several lncRNAs play key regulatory roles in plant development and stress responses. In this review, we focus on summarizing recent progress uncovering regulatory roles and mechanisms of lncRNAs during the plant life cycle, and briefly discuss the possible biotechnological applications of lncRNAs for plant breeding.

Exogenous RNAs: promising tools for the second green revolution.

The impending need for increasing amounts of food for the world population poses enormous challenges to agriculture. Moreover, global warming has exacerbated abiotic and biotic stresses, accelerating the emergence of new pests and pathogens which threatens crop productivity. Therefore, the scientific community urgently needs to develop innovative solutions for sustainable agriculture, notably replacing synthetic pesticides by active and highly specific biomolecules for pest control. In this context, RNA-based technologies emerge as an outstanding genetically modified organism-free approach offering versatile solutions to boost productivity while conserving and harnessing the wide variety of local landraces. Here we review recent advances in the field, including RNA synthesis approaches and the development of the nanotechnology required for RNA stabilization and delivery, and we discuss the potential of RNA as the key molecule for versatile applications in the second green revolution.

Trichoderma root colonization in maize triggers epigenetic changes in genes related to the jasmonic and salicylic acid pathways that prime defenses against Colletotrichum graminicola leaf infection.

Beneficial interactions between plant roots and Trichoderma species lead to both local and systemic enhancements of the plant immune system through a mechanism known as priming of defenses. Previously, we have reported a number of genes and proteins that are differentially regulated in distant tissues of maize plants following inoculation with Trichoderma atroviride. To further investigate the mechanisms involved in the systemic activation of plant responses, here we have further evaluated the regulatory aspects of a selected group of genes when priming is triggered in maize plants. Time-course experiments from the beginning of the interaction between T. atroviride and maize roots followed by leaf infection with Colletotrichum graminicola allowed us to identify a gene set regulated by priming in the leaf tissue. In the same experiment, phytohormone measurements revealed a decrease in jasmonic acid concentration while salicylic acid increased at 2 d and 6 d post-inoculation. In addition, chromatin structure and modification assays showed that chromatin was more open in the primed state compared with unprimed control conditions, and this allowed for quicker gene activation in response to pathogen attack. Overall, the results allowed us to gain insights on the interplay between the phytohormones and epigenetic regulatory events in the systemic and long-lasting regulation of maize plant defenses following Trichoderma inoculation.

Novel SARS-CoV-2 encoded small RNAs in the passage to humans.

MOTIVATION: The Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) has recently emerged as the responsible for the pandemic outbreak of the coronavirus disease 2019. This virus is closely related to coronaviruses infecting bats and Malayan pangolins, species suspected to be an intermediate host in the passage to humans. Several genomic mutations affecting viral proteins have been identified, contributing to the understanding of the recent animal-to-human transmission. However, the capacity of SARS-CoV-2 to encode functional putative microRNAs (miRNAs) remains largely unexplored. RESULTS: We have used deep learning to discover 12 candidate stem-loop structures hidden in the viral protein-coding genome. Among the precursors, the expression of eight mature miRNAs-like sequences was confirmed in small RNA-seq data from SARS-CoV-2 infected human cells. Predicted miRNAs are likely to target a subset of human genes of which 109 are transcriptionally deregulated upon infection. Remarkably, 28 of those genes potentially targeted by SARS-CoV-2 miRNAs are down-regulated in infected human cells. Interestingly, most of them have been related to respiratory diseases and viral infection, including several afflictions previously associated with SARS-CoV-1 and SARS-CoV-2. The comparison of SARS-CoV-2 pre-miRNA sequences with those from bat and pangolin coronaviruses suggests that single nucleotide mutations could have helped its progenitors jumping inter-species boundaries, allowing the gain of novel mature miRNAs targeting human mRNAs. Our results suggest that the recent acquisition of novel miRNAs-like sequences in the SARS-CoV-2 genome may have contributed to modulate the transcriptional reprograming of the new host upon infection. AVAILABILITY AND IMPLEMENTATION: https://github.com/sinc-lab/sarscov2-mirna-discovery. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cytochrome c and the transcription factor ABI4 establish a molecular link between mitochondria and ABA-dependent seed germination.

During germination, seed reserves are mobilised to sustain the metabolic and energetic demands of plant growth. Mitochondrial respiration is presumably required to drive germination in several species, but only recently its role in this process has begun to be elucidated. Using Arabidopsis thaliana lines with changes in the levels of the respiratory chain component cytochrome c (CYTc), we investigated the role of this protein in germination and its relationship with hormonal pathways. Cytochrome c deficiency causes delayed seed germination, which correlates with decreased cyanide-sensitive respiration and ATP production at the onset of germination. In addition, CYTc affects the sensitivity of germination to abscisic acid (ABA), which negatively regulates the expression of CYTC-2, one of two CYTc-encoding genes in Arabidopsis. CYTC-2 acts downstream of the transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), which binds to a region of the CYTC-2 promoter required for repression by ABA and regulates its expression. The results show that CYTc is a main player during seed germination through its role in respiratory metabolism and energy production. In addition, the direct regulation of CYTC-2 by ABI4 and its effect on ABA-responsive germination establishes a link between mitochondrial and hormonal functions during this process.