Strigolactone induces D14-dependent large-scale changes in gene expression requiring SWI/SNF chromatin remodellers.

Strigolactones (SL) function as plant hormones in control of multiple aspects of plant development, mostly via the regulation of gene expression. Immediate early-gene regulation by SL remains unexplored due to difficulty in dissecting early from late gene expression responses to SL. We used synthetic SL, rac-GR24 treatment of protoplasts and RNA-seq to explore early SL-induced changes in gene expression over time (5-180 minutes) and discovered rapid, dynamic and SL receptor D14-dependent regulation of gene expression in response to rac-GR24. Importantly, we discovered a significant dependence of SL signalling on chromatin remodelling processes, as the induction of a key SL-induced transcription factor BRANCHED1 requires the SWI/SNF chromatin remodelling ATPase SPLAYED (SYD) and leads to upregulation of a homologue SWI/SNF ATPase BRAHMA. ATAC-seq profiling of genome-wide changes in chromatin accessibility in response to rac-GR24 identified large-scale changes, with over 1400 differentially accessible regions. These changes in chromatin accessibility often precede transcriptional changes and are likely to harbour SL cis-regulatory elements. Importantly, we discovered that this early and extensive modification of the chromatin landscape also requires SYD. This study, therefore, provides evidence that SL signalling requires regulation of chromatin accessibility, and it identifies genomic locations harbouring likely SL cis-regulatory sequences.

Genome-wide identification of accessible chromatin regions by ATAC-seq upon induction of the transcription factor bZIP11 in Arabidopsis.

Basic leucine zipper 11 (bZIP11) is a transcription factor that is activated under low energy conditions in plants and plays a crucial role in enabling plants to adapt to starvation situations. Although previous results indicate that bZIP11 regulates chromatin accessibility based on evidence obtained from single genomic loci, to what extent this transcription factor regulates the chromatin landscape at the whole genome level remains unknown. Here we addressed this by performing an ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) on Arabidopsis thaliana (Arabidopsis) leaf protoplasts to obtain a profile of chromatin patterning in response upon bZIP11 induction. We identified, on average, 10,000 differentially accessible regions upon bZIP11 induction, corresponding to over 8,420 different genes out of the 25,000 genes present in the Arabidopsis genome. Our study provides a resource for understanding how bZIP11 regulates the genome at the chromatin level and provides an example of the impact of a single transcription factor on a whole plant genome.

Advancing tree genomics to future proof next generation orchard production.

The challenges facing tree orchard production in the coming years will be largely driven by changes in the climate affecting the sustainability of farming practices in specific geographical regions. Identifying key traits that enable tree crops to modify their growth to varying environmental conditions and taking advantage of new crop improvement opportunities and technologies will ensure the tree crop industry remains viable and profitable into the future. In this review article we 1) outline climate and sustainability challenges relevant to horticultural tree crop industries, 2) describe key tree crop traits targeted for improvement in agroecosystem productivity and resilience to environmental change, and 3) discuss existing and emerging genomic technologies that provide opportunities for industries to future proof the next generation of orchards.

Horticultural innovation by viral-induced gene regulation of carotenogenesis.

Multipartite viral vectors provide a simple, inexpensive and effective biotechnological tool to transiently manipulate (i.e. reduce or increase) gene expression in planta and characterise the function of genetic traits. The development of virus-induced gene regulation (VIGR) systems usually involve the targeted silencing or overexpression of genes involved in pigment biosynthesis or degradation in plastids, thereby providing rapid visual assessment of success in establishing RNA- or DNA-based VIGR systems in planta. Carotenoids pigments provide plant tissues with an array of yellow, orange, and pinkish-red colours. VIGR-induced transient manipulation of carotenoid-related gene expression has advanced our understanding of carotenoid biosynthesis, regulation, accumulation and degradation, as well as plastid signalling processes. In this review, we describe mechanisms of VIGR, the importance of carotenoids as visual markers of technology development, and knowledge gained through manipulating carotenogenesis in model plants as well as horticultural crops not always amenable to transgenic approaches. We outline how VIGR can be utilised in plants to fast-track the characterisation of gene function(s), accelerate fruit tree breeding programs, edit genomes, and biofortify plant products enriched in carotenoid micronutrients for horticultural innovation.

Epigenome guided crop improvement: current progress and future opportunities.

Epigenomics encompasses a broad field of study, including the investigation of chromatin states, chromatin modifications and their impact on gene regulation; as well as the phenomena of epigenetic inheritance. The epigenome is a multi-modal layer of information superimposed on DNA sequences, instructing their usage in gene expression. As such, it is an emerging focus of efforts to improve crop performance. Broadly, this might be divided into avenues that leverage chromatin information to better annotate and decode plant genomes, and into complementary strategies that aim to identify and select for heritable epialleles that control crop traits independent of underlying genotype. In this review, we focus on the first approach, which we term 'epigenome guided' improvement. This encompasses the use of chromatin profiles to enhance our understanding of the composition and structure of complex crop genomes. We discuss the current progress and future prospects towards integrating this epigenomic information into crop improvement strategies; in particular for CRISPR/Cas9 gene editing and precision genome engineering. We also highlight some specific opportunities and challenges for grain and horticultural crops.

Pre-introduction introgression contributes to parallel differentiation and contrasting hybridization outcomes between invasive and native marine mussels.

Non-native species experience novel selection pressures in introduced environments and may interbreed with native lineages. Species introductions therefore provide opportunities to investigate repeated patterns of adaptation and introgression across replicated contact zones. Here, we investigate genetic parallelism between multiple introduced populations of the invasive marine mussel, Mytilus galloprovincialis, in the absence (South Africa and California) and presence of hybridization with a native congener (Mytilus planulatus in Batemans Bay and Sydney Harbour, Australia). Repeatability in post-introduction differentiation from native-range populations varied between genetically distinct Atlantic and Mediterranean lineages, with Atlantic-derived introductions displaying high differentiation (maxFST  > 0.4) and parallelism at outlier loci. Identification of long noncoding RNA transcripts (lncRNA) additionally allowed us to clarify that parallel responses are largely limited to protein-coding loci, with lncRNAs likely evolving under evolutionary constraints. Comparisons of independent hybrid zones revealed differential introgression most strongly in Batemans Bay, with an excess of M. galloprovincialis ancestry and resistance to introgression at loci differentiating parental lineages (M. planulatus and Atlantic M. galloprovincialis). Additionally, contigs putatively introgressed with divergent alleles from a closely related species, Mytilus edulis, showed stronger introgression asymmetries compared with genome-wide trends and also diverged in parallel in both Atlantic-derived introductions. These results suggest that divergent demographic histories experienced by introduced lineages, including pre-introduction introgression, influence contemporary admixture dynamics. Our findings build on previous investigations reporting contributions of historical introgression to intrinsic reproductive architectures shared between marine lineages and illustrate that interspecific introgression history can shape differentiation between colonizing populations and their hybridization with native congeners.

Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation.

5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones.

An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant.

All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

De novo transcriptome assembly and annotation for gene discovery in avocado, macadamia and mango.

Avocado (Persea americana Mill.), macadamia (Macadamia integrifolia L.) and mango (Mangifera indica L.) are important subtropical tree species grown for their edible fruits and nuts. Despite their commercial and nutritional importance, the genomic information for these species is largely lacking. Here we report the generation of avocado, macadamia and mango transcriptome assemblies from pooled leaf, stem, bud, root, floral and fruit/nut tissue. Using normalized cDNA libraries, we generated comprehensive RNA-Seq datasets from which we assembled 63420, 78871 and 82198 unigenes of avocado, macadamia and mango, respectively using a combination of de novo transcriptome assembly and redundancy reduction. These unigenes were functionally annotated using Basic Local Alignment Search Tool (BLAST) to query the Universal Protein Resource Knowledgebase (UniProtKB). A workflow encompassing RNA extraction, library preparation, transcriptome assembly, redundancy reduction, assembly validation and annotation is provided. This study provides avocado, macadamia and mango transcriptome and annotation data, which is valuable for gene discovery and gene expression profiling experiments as well as ongoing and future genome annotation and marker development applications.

SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling.

Chromatin modification has gained increased attention for its role in the regulation of plant responses to environmental changes, but the specific mechanisms and molecular players remain elusive. Here, we show that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) mediates genome-wide changes in H3K36 methylation at specific genomic loci functionally relevant to nitrate treatments. Moreover, we show that the specific H3K36 methyltransferase encoded by SDG8 is required for canonical RNA processing, and that RNA isoform switching is more prominent in the sdg8-5 deletion mutant than in the wild type. To demonstrate that SDG8-mediated regulation of RNA isoform expression is functionally relevant, we examined a putative regulatory gene, CONSTANS, CO-like, and TOC1 101 (CCT101), whose nitrogen-responsive isoform-specific RNA expression is mediated by SDG8. We show by functional expression in shoot cells that the different RNA isoforms of CCT101 encode distinct regulatory proteins with different effects on genome-wide transcription. We conclude that SDG8 is involved in plant responses to environmental nitrogen supply, affecting multiple gene regulatory processes including genome-wide histone modification, transcriptional regulation, and RNA processing, and thereby mediating developmental and metabolic processes related to nitrogen use.

A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing.

BACKGROUND: Woody tropical plants contain high levels of complex organic compounds that inhibit the chemical procedures needed to extract RNA or DNA, thus compromising downstream applications such as RNA sequencing and analysis of gene expression. To overcome this issue, researchers must use extraction protocols using CTAB/PVP buffer instead of commercially available DNA/RNA extraction kits. However, these protocols are time-consuming, use toxic chemicals like phenol and chloroform, and can only be used to process a small number of samples at a time. To overcome these issues, we developed a new CTAB/PVP based protocol for RNA or DNA extraction that eliminates the traditional phenol/chloroform step. Furthermore, the protocol was developed for 96-well plates to speed up processing. RESULTS: Our new protocol enabled us to successfully extract RNA from macadamia, avocado, and mango tissues that are traditionally difficult to work with. This RNA was then successfully used to synthesise cDNA for real-time quantitative PCR and to generate good quality RNA-Seq libraries. Our protocol can be easily converted for rapid DNA extraction from different tropical and sub-tropical tree species. CONCLUSION: This method enables safer and faster DNA and RNA extraction from recalcitrant species, thus facilitating future work on tropical trees.

Juvenility and Vegetative Phase Transition in Tropical/Subtropical Tree Crops.

In plants, juvenile to adult phase transition is regulated by the sequential activity of two microRNAs: miR156 and miR172. A decline in miR156 and increase in miR172 abundance is associated with phase transition. There is very limited information on phase transition in economically important horticultural tree crops, which have a significantly long vegetative phase affecting fruit bearing. Here, we profiled various molecular cues known to be involved in phase transition and flowering, including the microRNAs miR156 and miR172, in three horticultural tree crops: avocado (Persea americana), mango (Mangifera indica), and macadamia (Macadamia integrifolia). We observed that miR156 expression decreases as these trees age and can potentially be used as a juvenility marker. Consistent with findings in annual plants, we also observed conserved regulation of the miR156-SPL3/4/5 regulatory module in these genetically distant tree crops, suggesting that this pathway may play a highly conserved role in vegetative identity. Meanwhile, the abundance of miR172 and its target AP2-like genes as well as the accumulation level of SPL9 transcripts were not related with plant age in these crops except in avocado where miR172 expression increased steadily. Finally, we demonstrate that various floral genes, including AP1 and SOC1 were upregulated in the reproductive phase and can be used as potential markers for the reproductive phase transition. Overall, this study provides an insight into the molecular associations of juvenility and phase transition in horticultural trees where crop breeding and improvement are encumbered by long juvenile phases.

De Novo Plant Transcriptome Assembly and Annotation Using Illumina RNA-Seq Reads.

The ability to identify and quantify transcribed sequences from a multitude of organisms using high-throughput RNA sequencing has revolutionized our understanding of genetics and plant biology. However, a number of computational tools used in these analyses still require a reference genome sequence, something that is seldom available for non-model organisms. Computational tools employing de Bruijn graphs to reconstruct full-length transcripts from short sequence reads allow for de novo transcriptome assembly. Here we provide detailed methods for generating and annotating de novo transcriptome assembly from plant RNA-seq data.

Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants.

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other's transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.

Sex Determination in Ceratopteris richardii Is Accompanied by Transcriptome Changes That Drive Epigenetic Reprogramming of the Young Gametophyte.

The fern Ceratopteris richardii is an important model for studies of sex determination and gamete differentiation in homosporous plants. Here we use RNA-seq to de novo assemble a transcriptome and identify genes differentially expressed in young gametophytes as their sex is determined by the presence or absence of the male-inducing pheromone called antheridiogen. Of the 1,163 consensus differentially expressed genes identified, the vast majority (1,030) are up-regulated in gametophytes treated with antheridiogen. GO term enrichment analyses of these DEGs reveals that a large number of genes involved in epigenetic reprogramming of the gametophyte genome are up-regulated by the pheromone. Additional hormone response and development genes are also up-regulated by the pheromone. This C. richardii gametophyte transcriptome and gene expression dataset will prove useful for studies focusing on sex determination and differentiation in plants.

Long non-coding regulatory RNAs in sponges and insights into the origin of animal multicellularity.

How animals evolved from a single-celled ancestor over 700 million years ago is poorly understood. Recent transcriptomic and chromatin analyses in the sponge Amphimedon queenslandica, a morphologically-simple representative of one of the oldest animal phyletic lineages, have shed light on what innovations in the genome and its regulation underlie the emergence of animal multicellularity. Comparisons of the regulatory genome of this sponge with those of more complex bilaterian model species and even simpler unicellular relatives have revealed that fundamental changes in genome regulatory complexity accompanied the evolution of animal multicellularity. Here, we review and discuss the results of these recent investigations by specifically focusing on the contribution of long non-coding RNAs to the evolution of the animal regulatory genome.

Sponge Long Non-Coding RNAs Are Expressed in Specific Cell Types and Conserved Networks.

Although developmental regulation by long non-coding RNAs (lncRNAs) appears to be a widespread feature amongst animals, the origin and level of evolutionary conservation of this mode of regulation remain unclear. We have previously demonstrated that the sponge Amphimedon queenslandica-a morphologically-simple animal-developmentally expresses an array of lncRNAs in manner akin to more complex bilaterians (insects + vertebrates). Here, we first show that Amphimedon lncRNAs are expressed in specific cell types in larvae, juveniles and adults. Thus, as in bilaterians, sponge developmental regulation involves the dynamic, cell type- and context-specific regulation of specific lncRNAs. Second, by comparing gene co-expression networks between Amphimedon queenslandica and Sycon ciliatum-a distantly-related calcisponge-we identify several putative co-expression modules that appear to be shared in sponges; these network-embedded sponge lncRNAs have no discernable sequence similarity. Together, these results suggest sponge lncRNAs are developmentally regulated and operate in conserved gene regulatory networks, as appears to be the case in more complex bilaterians.

Convergent microevolution of Cryptococcus neoformans hypervirulence in the laboratory and the clinic.

Reference strains are a key component of laboratory research, providing a common background allowing for comparisons across a community of researchers. However, laboratory passage of these strains has been shown to lead to reduced fitness and the attenuation of virulence in some species. In this study we show the opposite in the fungal pathogen Cryptococcus neoformans, with analysis of a collection of type strain H99 subcultures revealing that the most commonly used laboratory subcultures belong to a mutant lineage of the type strain that is hypervirulent. The pleiotropic mutant phenotypes in this H99L (for "Laboratory") lineage are the result of a deletion in the gene encoding the SAGA Associated Factor Sgf29, a mutation that is also present in the widely-used H99L-derived KN99a/α congenic pair. At a molecular level, loss of this gene results in a reduction in histone H3K9 acetylation. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating not only the first example of hypervirulence in clinical C. neoformans samples, but also parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.

Landscape of histone modifications in a sponge reveals the origin of animal cis-regulatory complexity.

Combinatorial patterns of histone modifications regulate developmental and cell type-specific gene expression and underpin animal complexity, but it is unclear when this regulatory system evolved. By analysing histone modifications in a morphologically-simple, early branching animal, the sponge Amphimedonqueenslandica, we show that the regulatory landscape used by complex bilaterians was already in place at the dawn of animal multicellularity. This includes distal enhancers, repressive chromatin and transcriptional units marked by H3K4me3 that vary with levels of developmental regulation. Strikingly, Amphimedon enhancers are enriched in metazoan-specific microsyntenic units, suggesting that their genomic location is extremely ancient and likely to place constraints on the evolution of surrounding genes. These results suggest that the regulatory foundation for spatiotemporal gene expression evolved prior to the divergence of sponges and eumetazoans, and was necessary for the evolution of animal multicellularity.