Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.

BACKGROUND: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. RESULTS: We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. CONCLUSIONS: Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility

Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions

MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant

MiR172-APETALA2-like genes integrate vernalization and plant age to control flowering time in wheat

Plants possess regulatory mechanisms that allow them to flower under conditions that maximize reproductive success. Selection of natural variants affecting those mechanisms has been critical in agriculture to modulate the flowering response of crops to specific environments and to increase yield. In the temperate cereals, wheat and barley, the photoperiod and vernalization pathways explain most of the natural variation in flowering time. However, other pathways also participate in fine-tuning the flowering response. In this work, we integrate the conserved microRNA miR172 and its targets APETALA2-like (AP2L) genes into the temperate grass flowering network involving VERNALIZATION 1 (VRN1), VRN2 and FLOWERING LOCUS T 1 (FT1 = VRN3) genes. Using mutants, transgenics and different growing conditions, we show that miR172 promotes flowering in wheat, while its target genes AP2L1 (TaTOE1) and AP2L5 (Q) act as flowering repressors. Moreover, we reveal that the miR172-AP2L pathway regulates FT1 expression in the leaves, and that this regulation is independent of VRN2 and VRN1. In addition, we show that the miR172-AP2L module and flowering are both controlled by plant age through miR156 in spring cultivars. However, in winter cultivars, flowering and the regulation of AP2L1 expression are decoupled from miR156 downregulation with age, and induction of VRN1 by vernalization is required to repress AP2L1 in the leaves and promote flowering. Interestingly, the levels of miR172 and both AP2L genes modulate the flowering response to different vernalization treatments in winter cultivars. In summary, our results show that conserved and grass specific gene networks interact to modulate the flowering response, and that natural or induced mutations in AP2L genes are useful tools for fine-tuning wheat flowering time in a changing environment

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei

BackgroundThe genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.ResultsIn this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.ConclusionsWe have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei

Abstract Background The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification. Results In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels. Conclusions We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops

An efficient transformation method for genome editing of elite bread wheat cultivars

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far

Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize