Genome-wide analysis of RNA-chromatin interactions in lizards as a mean for functional lncRNA identification.

BACKGROUND: Long non-coding RNAs (lncRNAs) are defined as transcribed molecules longer than 200 nucleotides with little to no protein-coding potential. LncRNAs can regulate gene expression of nearby genes (cis-acting) or genes located on other chromosomes (trans-acting). Several methodologies have been developed to capture lncRNAs associated with chromatin at a genome-wide level. Analysis of RNA-DNA contacts can be combined with epigenetic and RNA-seq data to define potential lncRNAs involved in the regulation of gene expression. RESULTS: We performed Chromatin Associated RNA sequencing (ChAR-seq) in Anolis carolinensis to obtain the genome-wide map of the associations that RNA molecules have with chromatin. We analyzed the frequency of DNA contacts for different classes of RNAs and were able to define cis- and trans-acting lncRNAs. We integrated the ChAR-seq map of RNA-DNA contacts with epigenetic data for the acetylation of lysine 16 on histone H4 (H4K16ac), a mark connected to actively transcribed chromatin in lizards. We successfully identified three trans-acting lncRNAs significantly associated with the H4K16ac signal, which are likely involved in the regulation of gene expression in A. carolinensis. CONCLUSIONS: We show that the ChAR-seq method is a powerful tool to explore the RNA-DNA map of interactions. Moreover, in combination with epigenetic data, ChAR-seq can be applied in non-model species to establish potential roles for predicted lncRNAs that lack functional annotations.

Transient Posttranscriptional Gene Silencing in Medicago truncatula: Virus-Induced Gene Silencing (VIGS).

Successful application of virus-induced gene silencing for functional genomics requires a virus vector that can initiate a systemic infection of the host plant. Agroinoculation of the pea early browning virus vectors pCAPE1 and pCAPE2 can establish infection in several genotypes of Medicago truncatula and can reduce target gene RNA levels to an extent that allows investigation of gene function.

The AP2/ERF Transcription Factor DRNL Modulates Gynoecium Development and Affects Its Response to Cytokinin.

The gynoecium is the female reproductive system in flowering plants. It is a complex structure formed by different tissues, some that are essential for reproduction and others that facilitate the fertilization process and nurture and protect the developing seeds. The coordinated development of these different tissues during the formation of the gynoecium is important for reproductive success. Both hormones and genetic regulators guide the development of the different tissues. Auxin and cytokinin in particular have been found to play important roles in this process. On the other hand, the AP2/ERF2 transcription factor BOL/DRNL/ESR2/SOB is expressed at very early stages of aerial organ formation and has been proposed to be a marker for organ founder cells. In this work, we found that this gene is also expressed at later stages during gynoecium development, particularly at the lateral regions (the region related to the valves of the ovary). The loss of DRNL function affects gynoecium development. Some of the mutant phenotypes present similarities to those observed in plants treated with exogenous cytokinins, and AHP6 has been previously proposed to be a target of DRNL. Therefore, we explored the response of drnl-2 developing gynoecia to cytokinins, and found that the loss of DRNL function affects the response of the gynoecium to exogenously applied cytokinins in a developmental-stage-dependent manner. In summary, this gene participates during gynoecium development, possibly through the dynamic modulation of cytokinin homeostasis and response.

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

Two euAGAMOUS genes control C-function in Medicago truncatula.

C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.

Functional specialization of duplicated AP3-like genes in Medicago truncatula.

The B-class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3-like genes of a legume species. To obtain insight into the extent to which B-class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non-overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3-like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.