Non-B DNA in plant genomes: prediction, mapping, and emerging roles.

Regulating gene expression in plant development and environmental responses is vital for mitigating the effects of climate change on crop growth and productivity. The eukaryotic genome largely shows the canonical B-DNA structure that is organized into nucleosomes with histone modifications shaping the epigenome. Nuclear proteins and RNA interactions influence chromatin conformations and dynamically modulate gene activity. Non-B DNA conformations and their transitions introduce novel aspects to gene expression modulation, particularly in response to environmental shifts. We explore the current understanding of non-B DNA structures in plant genomes, their interplay with epigenomics and gene expression, and advances in methods for their mapping and characterization. The exploration of so far uncharacterized non-B DNA structures remains an intriguing area in plant chromatin research and offers insights into their potential role in gene regulation.

Epigenetic control during root development and symbiosis.

The roots of plants play multiple functions that are essential for growth and development, including anchoring to the soil as well as 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, for example, the root nodule symbiosis (RNS) 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 the RNS 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 highlight how DNA methylation and histone post-translational modifications, as well as chromatin remodeling factors and long noncoding 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.

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.

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.

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.

The Arabidopsis APOLO and human UPAT sequence-unrelated long noncoding RNAs can modulate DNA and histone methylation machineries in plants.

BACKGROUND: RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. RESULTS: Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. CONCLUSIONS: Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

ChronoRoot: High-throughput phenotyping by deep segmentation networks reveals novel temporal parameters of plant root system architecture.

BACKGROUND: Deep learning methods have outperformed previous techniques in most computer vision tasks, including image-based plant phenotyping. However, massive data collection of root traits and the development of associated artificial intelligence approaches have been hampered by the inaccessibility of the rhizosphere. Here we present ChronoRoot, a system that combines 3D-printed open-hardware with deep segmentation networks for high temporal resolution phenotyping of plant roots in agarized medium. RESULTS: We developed a novel deep learning-based root extraction method that leverages the latest advances in convolutional neural networks for image segmentation and incorporates temporal consistency into the root system architecture reconstruction process. Automatic extraction of phenotypic parameters from sequences of images allowed a comprehensive characterization of the root system growth dynamics. Furthermore, novel time-associated parameters emerged from the analysis of spectral features derived from temporal signals. CONCLUSIONS: Our work shows that the combination of machine intelligence methods and a 3D-printed device expands the possibilities of root high-throughput phenotyping for genetics and natural variation studies, as well as the screening of clock-related mutants, revealing novel root traits.

The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold.

Plant long noncoding RNAs (lncRNAs) have emerged as important regulators of chromatin dynamics, impacting on transcriptional programs leading to different developmental outputs. The lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO) directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation, leading to transcriptional shifts. Here, we show that APOLO recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) and controls RHD6 transcriptional activity, leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4. Furthermore, we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter. WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion. Collectively, our results indicate that a novel ribonucleoprotein complex with APOLO and WRKY42 forms a regulatory hub to activate RHD6 by shaping its epigenetic environment and integrate signals governing RH growth and development.

Insights into long non-coding RNA regulation of anthocyanin carrot root pigmentation.

Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.

Long noncoding RNAs shape transcription in plants.

The advent of novel high-throughput sequencing techniques has revealed that eukaryotic genomes are massively transcribed although only a small fraction of RNAs exhibits protein-coding capacity. In the last years, long noncoding RNAs (lncRNAs) have emerged as regulators of eukaryotic gene expression in a wide range of molecular mechanisms. Plant lncRNAs can be transcribed by alternative RNA polymerases, acting directly as long transcripts or can be processed into active small RNAs. Several lncRNAs have been recently shown to interact with chromatin, DNA or nuclear proteins to condition the epigenetic environment of target genes or modulate the activity of transcriptional complexes. In this review, we will summarize the recent discoveries about the actions of plant lncRNAs in the regulation of gene expression at the transcriptional level.

Evolution of the Small Family of Alternative Splicing Modulators Nuclear Speckle RNA-binding Proteins in Plants.

RNA-Binding Protein 1 (RBP1) was first identified as a protein partner of the long noncoding RNA (lncRNA) ENOD40 in Medicagotruncatula, involved in symbiotic nodule development. RBP1 is localized in nuclear speckles and can be relocalized to the cytoplasm by the interaction with ENOD40. The two closest homologs to RBP1 in Arabidopsis thaliana were called Nuclear Speckle RNA-binding proteins (NSRs) and characterized as alternative splicing modulators of specific mRNAs. They can recognize in vivo the lncRNA ALTERNATIVE SPLICING COMPETITOR (ASCO) among other lncRNAs, regulating lateral root formation. Here, we performed a phylogenetic analysis of NSR/RBP proteins tracking the roots of the family to the Embryophytes. Strikingly, eudicots faced a reductive trend of NSR/RBP proteins in comparison with other groups of flowering plants. In Medicagotruncatula and Lotus japonicus, their expression profile during nodulation and in specific regions of the symbiotic nodule was compared to that of the lncRNA ENOD40, as well as to changes in alternative splicing. This hinted at distinct and specific roles of each member during nodulation, likely modulating the population of alternatively spliced transcripts. Our results establish the basis to guide future exploration of NSR/RBP function in alternative splicing regulation in different developmental contexts along the plant lineage.

The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth.

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2 Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development.

TMV induces RNA decay pathways to modulate gene silencing and disease symptoms.

RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms.

The chloroplastic DEVH-box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing.

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH-box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron-containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2-silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing-regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell-cell communication in plants.

Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula.

Translation of mRNAs is a key regulatory step that contributes to the coordination and modulation of eukaryotic gene expression during development or adaptation to the environment. mRNA stability or translatability can be regulated by the action of small regulatory RNAs (sRNAs), which control diverse biological processes. Under low nitrogen conditions, leguminous plants associate with soil bacteria and develop a new organ specialized in nitrogen fixation: the nodule. To gain insight into the translational regulation of mRNAs during nodule formation, the association of mRNAs and sRNAs to polysomes was characterized in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Quantitative comparison of steady-state and polysomal mRNAs for 15 genes involved in nodulation identified a group of transcripts with slight or no change in total cellular abundance that were significantly upregulated at the level of association with polysomes in response to rhizobia. This group included mRNAs encoding receptors like kinases required either for nodule organogenesis, bacterial infection or both, and transcripts encoding GRAS and NF-Y transcription factors (TFs). Quantitative analysis of sRNAs in total and polysomal RNA samples revealed that mature microRNAs (miRNAs) were associated with the translational machinery, notably, miR169 and miR172, which target the NF-YA/HAP2 and AP2 TFs, respectively. Upon inoculation, levels of miR169 pronouncedly decreased in polysomal complexes, concomitant with the increased accumulation of the NF-YA/HAP2 protein. These results indicate that both mRNAs and miRNAs are subject to differential recruitment to polysomes, and expose the importance of selective mRNA translation during root nodule symbiosis.

The LOB-like transcription factor Mt LBD1 controls Medicago truncatula root architecture under salt stress.

Lateral root (LR) formation and emergence are influenced by the environment and determines the architecture of the root system in the soil. Whereas auxins appear as the main hormone controlling LR initiation, patterning and emergence, abscisic acid (ABA) is the key hormone mediating the effect of the environment on root architecture. Hormone signaling act through transcription factors (TFs) and the Medicago truncatula LOB-like TF LBD1 was shown to be auxin-inducible but repressed by the HD-Zip I TF MtHB1 in response to salt stress and ABA during LR formation. Here, we demonstrate that the constitutive expression of Mt LBD1 in Medicago roots alters their global architecture when the plant is subjected to salt stress. Hence, LBD1 may control the final form of the root system in the soil environment.

Environmental regulation of lateral root emergence in Medicago truncatula requires the HD-Zip I transcription factor HB1.

The adaptation of root architecture to environmental constraints is a major agricultural trait, notably in legumes, the third main crop worldwide. This root developmental plasticity depends on the formation of lateral roots (LRs) emerging from primary roots. In the model legume Medicago truncatula, the HD-Zip I transcription factor HB1 is expressed in primary and lateral root meristems and induced by salt stress. Constitutive expression of HB1 in M. truncatula roots alters their architecture, whereas hb1 TILLING mutants showed increased lateral root emergence. Electrophoretic mobility shift assay, promoter mutagenesis, and chromatin immunoprecipitation-PCR assays revealed that HB1 directly recognizes a CAATAATTG cis-element present in the promoter of a LOB-like (for Lateral Organ Boundaries) gene, LBD1, transcriptionally regulated by auxin. Expression of these genes in response to abscisic acid and auxin and their behavior in hb1 mutants revealed an HB1-mediated repression of LBD1 acting during LR emergence. M. truncatula HB1 regulates an adaptive developmental response to minimize the root surface exposed to adverse environmental stresses.

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula.

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.

Cross-talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb-4 transcription factor.

Hahb-4 is a member of the Helianthusannuus (sunflower) subfamily I of HD-Zip proteins that is transcriptionally regulated by water availability and abscisic acid. Transgenic Arabidopsis thaliana plants overexpressing this transcription factor (TF) exhibit a characteristic phenotype that includes a strong tolerance to water stress. Here we show that this TF is a new component of ethylene signalling pathways, and that it induces a marked delay in senescence. Plants overexpressing Hahb-4 are less sensitive to external ethylene, enter the senescence pathway later and do not show the typical triple response. Furthermore, transgenic plants expressing this gene under the control of its own inducible promoter showed an inverse correlation between ethylene sensitivity and Hahb-4 levels. Potential targets of Hahb-4 were identified by comparing the transcriptome of Hahb-4-transformed and wild-type plants using microarrays and quantitative RT-PCR. Expression of this TF has a major repressive effect on genes related to ethylene synthesis, such as ACO and SAM, and on genes related to ethylene signalling, such as ERF2 and ERF5. Expression studies in sunflower indicate that Hahb-4 transcript levels are elevated in mature/senescent leaves. Expression of Hahb-4 is induced by ethylene, concomitantly with several genes homologous to the targets identified in the transcriptome analysis (HA-ACOa and HA-ACOb). Transient transformation of sunflower leaves demonstrated the action of Hahb-4 in the regulation of ethylene-related genes. We propose that Hahb-4 is involved in a novel conserved mechanism related to ethylene-mediated senescence that functions to improve desiccation tolerance.