The SvFUL2 transcription factor is required for inflorescence determinacy and timely flowering in Setaria viridis.

Inflorescence architecture in cereal crops directly impacts yield potential through regulation of seed number and harvesting ability. Extensive architectural diversity found in inflorescences of grass species is due to spatial and temporal activity and determinacy of meristems, which control the number and arrangement of branches and flowers, and underlie plasticity. Timing of the floral transition is also intimately associated with inflorescence development and architecture, yet little is known about the intersecting pathways and how they are rewired during development. Here, we show that a single mutation in a gene encoding an AP1/FUL-like MADS-box transcription factor significantly delays flowering time and disrupts multiple levels of meristem determinacy in panicles of the C4 model panicoid grass, Setaria viridis. Previous reports of AP1/FUL-like genes in cereals have revealed extensive functional redundancy, and in panicoid grasses, no associated inflorescence phenotypes have been described. In S. viridis, perturbation of SvFul2, both through chemical mutagenesis and gene editing, converted a normally determinate inflorescence habit to an indeterminate one, and also repressed determinacy in axillary branch and floral meristems. Our analysis of gene networks connected to disruption of SvFul2 identified regulatory hubs at the intersection of floral transition and inflorescence determinacy, providing insights into the optimization of cereal crop architecture.

Tuberomics: a molecular profiling for the adaption of edible fungi (Tuber magnatum Pico) to different natural environments.

BACKGROUND: Truffles are symbiotic fungi that develop underground in association with plant roots, forming ectomycorrhizae. They are primarily known for the organoleptic qualities of their hypogeous fruiting bodies. Primarily, Tuber magnatum Pico is a greatly appreciated truffle species mainly distributed in Italy and Balkans. Its price and features are mostly depending on its geographical origin. However, the genetic variation within T. magnatum has been only partially investigated as well as its adaptation to several environments. RESULTS: Here, we applied an integrated omic strategy to T. magnatum fruiting bodies collected during several seasons from three different areas located in the North, Center and South of Italy, with the aim to distinguish them according to molecular and biochemical traits and to verify the impact of several environments on these properties. With the proteomic approach based on two-dimensional electrophoresis (2-DE) followed by mass spectrometry, we were able to identify proteins specifically linked to the sample origin. We further associated the proteomic results to an RNA-seq profiling, which confirmed the possibility to differentiate samples according to their source and provided a basis for the detailed analysis of genes involved in sulfur metabolism. Finally, geographical specificities were associated with the set of volatile compounds produced by the fruiting bodies, as quantitatively and qualitatively determined through proton transfer reaction-mass spectrometry (PTR-MS) and gas-chromatography-mass spectrometry (GC-MS). In particular, a partial least squares-discriminant analysis (PLS-DA) model built from the latter data was able to return high confidence predictions of sample source. CONCLUSIONS: Results provide a characterization of white fruiting bodies by a wide range of different molecules, suggesting the role for specific compounds in the responses and adaptation to distinct environments.

Comparative transcriptome analysis of two citrus germplasms with contrasting susceptibility to Phytophthora nicotianae provides new insights into tolerance mechanisms.

KEY MESSAGE: Host perception of Phytophthora nicotianae switching to necrotrophy is fundamental for disease tolerance of citrus. It involves an HR-like response, strengthening of the cell wall structure and hormonal signaling. Stem rot caused by P. nicotianae is a worldwide disease of several important crops, including citrus. Given the growing awareness of chemical fungicides drawbacks, genetic improvement of citrus rootstocks remains the best alternative. However, the molecular basis underlying the successful response of resistant and/or tolerant genotypes remains poorly understood. Therefore, we performed a transcriptomic analysis to examine the differential defense response to P. nicotianae of two germplasms-tolerant sour orange (SO, Citrus aurantium) and susceptible Madam Vinous (MV, C. sinensis)-in both the biotrophic and necrotrophic phases of host-pathogen interaction. Our results revealed the necrotrophic phase as a decisive turning point, since it included stronger modulation of a number of genes implicated in pathogen perception, signal transduction, HR-like response, transcriptional reprogramming, hormone signaling, and cell wall modifications. In particular, the pathogen perception category reflected the ability of SO to perceive the pathogen even after its switch to necrotrophy, and thus to cope successfully with the infection, while MV failed. The concomitant changes in genes involved in the remaining functional categories seemed to prevent pathogen spread. This investigation provided further understanding of the successful defense mechanisms of C. aurantium against P. nicotianae, which might be exploited in post-genomic strategies to develop resistant Citrus genotypes.

Identification and characterization of durum wheat microRNAs in leaf and root tissues.

MicroRNAs are a class of post-transcriptional regulators of plant developmental and physiological processes and responses to environmental stresses. Here, we present the study regarding the annotation and characterization of MIR genes conducted in durum wheat. We characterized the miRNAome of leaf and root tissues at tillering stage under two environmental conditions: irrigated with 100% (control) and 55% of evapotranspiration (early water stress). In total, 90 microRNAs were identified, of which 32 were classified as putative novel and species-specific miRNAs. In addition, seven microRNA homeologous groups were identified in each of the two genomes of the tetraploid durum wheat. Differential expression analysis highlighted a total of 45 microRNAs significantly differentially regulated in the pairwise comparisons leaf versus root. The miRNA families, miR530, miR395, miR393, miR5168, miR396 and miR166, miR171, miR319, and miR167, were the most expressed in leaves in comparison to roots. Putative microRNA targets were predicted for both five and three prime sequences derived from the stem-loop of the MIR gene. Gene ontology analysis showed significant overrepresented gene categories in microRNA targets belonging to transcription factors, phenylpropanoids, oxydases, and lipid binding-protein. This work represents one of the first genome wide characterization of MIR genes in durum wheat, identifying leaf and root tissue-specific microRNAs. This genomic identification of microRNAs together with the analysis of their expression profiles is a well-accepted starting point leading to a better comprehension of the role of MIR genes in the genus Triticum.

microRNAs differentially modulated in response to heat and drought stress in durum wheat cultivars with contrasting water use efficiency.

Plant stress response is a complex molecular process based on transcriptional and posttranscriptional regulation of many stress-related genes. microRNAs are the best-studied class of small RNAs known to play key regulatory roles in plant response to stress, besides being involved in plant development and organogenesis. We analyzed the leaf miRNAome of two durum wheat cultivars (Cappelli and Ofanto) characterized by a contrasting water use efficiency, exposed to heat stress, and mild and severe drought stress. On the whole, we identified 98 miRNA highly similar to previously known miRNAs and grouped in 47 MIR families, as well as 85 novel candidate miRNA, putatively wheat specific. A total of 80 known and novel miRNA precursors were found differentially expressed between the two cultivars or modulated by stress and many of them showed a cultivar-specific expression profile. Interestingly, most in silico predicted targets of the miRNAs coming from the differentially expressed precursors have been experimentally linked in other species to mechanisms controlling stomatal movement, a finding in agreement with previous results showing that Cappelli has a lower stomatal conductance than Ofanto. Selected miRNAs were validated through a standardized and reliable stem-loop qRT-PCR procedure.

Genomic skimming for identification of medium/highly abundant transposable elements in Arundo donax and Arundo plinii.

Transposable elements (TEs) are the most abundant genetic material for almost all eukaryotic genomes. Their effects on the host genomes range from an extensive size variation to the regulation of gene expression, altering gene function and creating new genes. Because of TEs pivotal contribute to the host genome structure and regulation, their identification and characterization provide a wealth of useful data for gaining an in-depth understanding of host genome functioning. The giant reed (Arundo donax) is a perennial rhizomatous C3 grass, octadecaploid, with an estimated nuclear genome size of 2744 Mbp. It is a promising feedstock for second-generation biofuels and biomethane production. To identify and characterize the most repetitive TEs in the genomes of A. donax and its ancestral A. plinii species, we carried out low-coverage whole genome shotgun sequencing for both species. Using a de novo repeat identification approach, 33,041 and 28,237 non-redundant repetitive sequences were identified and characterized in A. donax and A. plinii genomes, representing 37.55 and 31.68% of each genome, respectively. Comparative phylogenetic analyses, including the major TE classes identified in A. donax and A. plinii, together with rice and maize TE paralogs, were carried out to understand the evolutionary relationship of the most abundant TE classes. Highly conserved copies of RIRE1-like Ty1-Copia elements were discovered in two Arundo spp. in which they represented nearly 3% of each genomic sequence. We identified and characterized the medium/highly repetitive TEs in two unexplored polyploid genomes, thus generating useful information for the study of the genomic structure, composition, and functioning of these two non-model species. We provided a valuable resource that could be exploited in any effort aimed at sequencing and assembling these two genomes.

Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome.

BACKGROUND: Eragrostis tef is an allotetraploid (2n = 4 × = 40) annual, C4 grass with an estimated nuclear genome size of 730 Mbp. It is widely grown in Ethiopia, where it provides basic nutrition for more than half of the population. Although a draft assembly of the E. tef genome was made available in 2014, characterization of the repetitive portion of the E. tef genome has not been a subject of a detailed analysis. Repetitive sequences constitute most of the DNA in eukaryotic genomes. Transposable elements are usually the most abundant repetitive component in plant genomes. They contribute to genome size variation, cause mutations, can result in chromosomal rearrangements, and influence gene regulation. An extensive and in depth characterization of the repetitive component is essential in understanding the evolution and function of the genome. RESULTS: Using new paired-end sequence data and a de novo repeat identification strategy, we identified the most repetitive elements in the E. tef genome. Putative repeat sequences were annotated based on similarity to known repeat groups in other grasses. Altogether we identified 1,389 medium/highly repetitive sequences that collectively represent about 27% of the teff genome. Phylogenetic analyses of the most important classes of TEs were carried out in a comparative framework including paralog elements from rice and maize. Finally, an abundant tandem repeat accounting for more than 4% of the whole genome was identified and partially characterized. CONCLUSIONS: Analyzing a large sample of randomly sheared reads we obtained a library of the repetitive sequences of E. tef. The approach we used was designed to avoid underestimation of repeat contribution; such underestimation is characteristic of whole genome assembly projects. The data collected represent a valuable resource for further analysis of the genome of this important orphan crop.

miRVine: a microRNA expression atlas of grapevine based on small RNA sequencing.

BACKGROUND: miRNAs are the most abundant class of small non-coding RNAs, and they are involved in post-transcriptional regulations, playing a crucial role in the refinement of genetic programming during plant development. Here we present a comprehensive picture of miRNA regulation in Vitis vinifera L. plant during its complete life cycle. Furthering our knowledge about the post-transcriptional regulation of plant development is fundamental to understand the biology of such an important crop. RESULTS: We analyzed 70 small RNA libraries, prepared from berries, inflorescences, tendrils, buds, carpels, stamens and other samples at different developmental stages. One-hundred and ten known and 175 novel miRNAs have been identified and a wide grapevine expression atlas has been described. The distribution of miRNA abundance reveals that 22 novel miRNAs are specific to stamen, and two of them are, interestingly, involved in ethylene biosynthesis, while only few miRNAs are highly specific to other organs. Thirty-eight miRNAs are present in all our samples, suggesting a role in key regulatory circuit. On the basis of miRNAs abundance and distribution across samples and on the estimated correlation, we suggest that miRNA expression define organ identity. We performed target prediction analysis and focused on miRNA expression analysis in berries and inflorescence during their development, providing an initial functional description of the identified miRNAs. CONCLUSIONS: Our findings represent a very extensive miRNA expression atlas in grapevine, allowing the definition of how the spatio-temporal distribution of miRNAs defines organ identity. We describe miRNAs abundance in specific tissues not previously described in grapevine and contribute to future targeted functional analyses. Finally, we present a deep characterization of miRNA involvement in berry and inflorescence development, suggesting a role for miRNA-driven hormonal regulation.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells.

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

Boundary domain genes were recruited to suppress bract growth and promote branching in maize.

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

Publisher Correction: Long noncoding RNAs in the model species Brachypodium distachyon.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The regulatory landscape of early maize inflorescence development.

BACKGROUND: The functional genome of agronomically important plant species remains largely unexplored, yet presents a virtually untapped resource for targeted crop improvement. Functional elements of regulatory DNA revealed through profiles of chromatin accessibility can be harnessed for fine-tuning gene expression to optimal phenotypes in specific environments. RESULT: Here, we investigate the non-coding regulatory space in the maize (Zea mays) genome during early reproductive development of pollen- and grain-bearing inflorescences. Using an assay for differential sensitivity of chromatin to micrococcal nuclease (MNase) digestion, we profile accessible chromatin and nucleosome occupancy in these largely undifferentiated tissues and classify at least 1.6% of the genome as accessible, with the majority of MNase hypersensitive sites marking proximal promoters, but also 3' ends of maize genes. This approach maps regulatory elements to footprint-level resolution. Integration of complementary transcriptome profiles and transcription factor occupancy data are used to annotate regulatory factors, such as combinatorial transcription factor binding motifs and long non-coding RNAs, that potentially contribute to organogenesis, including tissue-specific regulation between male and female inflorescence structures. Finally, genome-wide association studies for inflorescence architecture traits based solely on functional regions delineated by MNase hypersensitivity reveals new SNP-trait associations in known regulators of inflorescence development as well as new candidates. CONCLUSIONS: These analyses provide a comprehensive look into the cis-regulatory landscape during inflorescence differentiation in a major cereal crop, which ultimately shapes architecture and influences yield potential.

Transcriptional and Posttranscriptional Regulation of Drought Stress Treatments in Brachypodium Leaves.

Plant sensing drought stress conditions activate complex molecular networks leading to a rapid reprogramming of plant physiology and metabolism, in order to survive in suboptimal conditions.Here, we describe a standardized in vivo soil drought assay to investigate the effects of drought stress on leaf growth. Since it is now clear that stress responses can be specific to developmental stages and cell types, we describe a procedure to dissect the leaf in three distinct areas in order to study transcriptional and posttranscriptional gene regulation on both organ and cellular levels. Noncoding RNAs, both small RNAs and long noncoding RNAs, are emerging to be deeply involved in abiotic stress responses, acting as molecular switches, interconnecting different response pathways. Here, we illustrate the methodology that has been used to identify miRNAs involved in drought response and to analyze the modulation of expression of their putative targets, in order to gain a complete picture of transcriptional and posttranscriptional regulation driven by noncoding RNAs.

Brachypodium distachyon Long Noncoding RNAs: Genome-Wide Identification and Expression Analysis.

Recent advances in high throughput sequencing technology have revealed a pervasive and complex transcriptional activity of all eukaryotic genomes and have allowed the identification and characterization of several classes of noncoding RNAs (ncRNAs) with key roles in various biological processes. Among ncRNAs, long ncRNAs (lncRNAs) are transcripts typically longer than 200 nucleotides whose members tend to be expressed at low levels, show a lack of phylogenetic conservation and exhibit tissue-specific, cell-specific, or stress-responsive expression profiles.Although a large set of lncRNAs has been identified both in animal and plant systems, the regulatory roles of lncRNAs are only beginning to be recognized and the molecular basis of lncRNA mediated gene regulation remains largely unexplored, particularly in plants.Here, we describe an efficient methodology to identify long noncoding RNAs using next-generation sequencing data.

Effects of different nitrogen fertilizers on two wheat cultivars: An integrated approach.

Investigation of cultivated plant physiology grown under low energy input plays an important role to indicate their fitness to the new environmental conditions. The durum-wheat cultivars Creso and Dylan were tested to evaluate the growth, production, and proteomic and transcriptomic profiles of the crop under different synthetic and organic nitrogen fertilization regimes. In this work, a two-dimensional gel electrophoresis (2-DE) approach combined with liquid chromatography-mass spectrometry (LC-MS) was used to investigate the protein changes induced by the use of different nitrogen sources (hydrolysate of proteins 1 and 2, rhizovit, synthesis, leather) on wheat plants. Proteomic studies were integrated with qPCR analysis of genes related to glutamine synthetase/glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) and tricarboxylic acid (TCA) metabolic pathways because most relevant for nitrogen-dependent plants growth. The proteomic analysis lead to the isolation of 23 spots that were able to distinguish the analyzed samples. These spots yielded the identification of 60 proteins involved in photosynthesis, glycolysis, and nitrogen metabolism. As an example, the quinone oxidoreductase-like protein and probable glutathione S-transferase GSTU proteins were identified in two spots that represents the most statistically significant ones in Dylan samples. Transcript analysis indicated that related genes exhibited different expression trends; the heat map also revealed the different behaviors of the hydrolysates of the proteins 1 and 2 nitrogen sources. The effects of nitrogenous fertilizers at the proteomic and agronomic levels revealed that plants fertilized with synthesis or rhizovit gave the best results concerning yield, whereas rhizovit and protein hydrolysates were most effective for proteins content in the grain (% of dry weight). Therefore, all parameters measured in this study indicated that different kinds of nitrogen fertilization used have a relevant impact on plant growth and production.

Long noncoding RNAs in the model species Brachypodium distachyon.

Eukaryotic genomes are pervasively transcribed and only a small portion of the transcribed sequences belongs to protein coding genes. High-throughput sequencing technology contributed to consolidate this perspective, allowing the identification of numerous noncoding RNAs with key roles in biological processes. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt with limited phylogenetic conservation, expressed at low levels and characterized by tissue/organ specific expression profiles. Although a large set of lncRNAs has been identified, the functional roles of lncRNAs are only beginning to be recognized and the molecular mechanism of lncRNA-mediated gene regulation remains largely unexplored, particularly in plants where their annotation and characterization are still incomplete. Using public and proprietary poly-(A)+ RNA-seq data as well as a collection of full length ESTs from several organs, developmental stages and stress conditions in three Brachypodium distachyon inbred lines, we describe the identification and the main features of thousands lncRNAs. Here we provide a genome-wide characterization of lncRNAs, highlighting their intraspecies conservation and describing their expression patterns among several organs/tissues and stress conditions. This work represents a fundamental resource to deepen our knowledge on long noncoding RNAs in C3 cereals, allowing the Brachypodium community to exploit these results in future research programs.

Transcriptome analysis of Phoenix canariensis Chabaud in response to Rhynchophorus ferrugineus Olivier attacks.

UNLABELLED: Red Palm Weevil (RPW, Rhynchophorus ferrugineus Olivier) threatens most palm species worldwide. Until now, no studies have analyzed the gene regulatory networks of Phoenix canariensis (Chabaud) in response to RPW attacks. The aim of this study was to fill this knowledge gap. Providing this basic knowledge is very important to improve its management. RESULTS: A deep transcriptome analysis was performed on fully expanded leaves of healthy non-infested trees and attacked trees at two symptom stages (middle and late infestation). A total of 54 genes were significantly regulated during middle stage. Pathway enrichment analysis showed that phenylpropanoid-related pathways were induced at this stage. More than 3300 genes were affected during late stage of attacks. Higher transcript abundances were observed for lipid fatty acid metabolism (fatty acid and glycerolipids), tryptophan metabolism, phenylpropanoid metabolism. Key RPW-modulated genes involved in innate response mediated by hormone crosstalk were observed belonging to auxin, jasmonate and salicylic acid (SA) pathways. Among transcription factors, some WRKYs were clearly induced. qRT-PCR validation confirmed the upregulation of key genes chosen as validation of transcriptomic analysis. CONCLUSION: A subset of these genes may be further analyzed in future studies to confirm their specificity to be induced by RPW infestations.

Draft Whole-Genome Sequence of the Biocontrol Agent Trichoderma harzianum T6776.

Trichoderma harzianum T6776 is a promising beneficial isolate whose effects consist of growth promotion, positive response of photosynthetic activity, hormonal signaling, and carbon partitioning in tomato, coupled with biocontrol of plant pathogens. Here, we present the first genome assembly of T6776, providing a useful platform for the scientific community.

Molecular and physiological analysis of growth-limiting drought stress in Brachypodium distachyon leaves.

The drought-tolerant grass Brachypodium distachyon is an emerging model species for temperate grasses and cereal crops. To explore the usefulness of this species for drought studies, a reproducible in vivo drought assay was developed. Spontaneous soil drying led to a 45% reduction in leaf size, and this was mostly due to a decrease in cell expansion, whereas cell division remained largely unaffected by drought. To investigate the molecular basis of the observed leaf growth reduction, the third Brachypodium leaf was dissected in three zones, namely proliferation, expansion, and mature zones, and subjected to transcriptome analysis, based on a whole-genome tiling array. This approach allowed us to highlight that transcriptome profiles of different developmental leaf zones respond differently to drought. Several genes and functional processes involved in drought tolerance were identified. The transcriptome data suggest an increased energy availability in the proliferation zones, along with an up-regulation of sterol synthesis that may influence membrane fluidity. This information may be used to improve the tolerance of temperate cereals to drought, which is undoubtedly one of the major environmental challenges faced by agriculture today and in the near future.

Addressing the role of microRNAs in reprogramming leaf growth during drought stress in Brachypodium distachyon.

Plant responses to drought are regulated by complex genetic and epigenetic networks leading to rapid reprogramming of plant growth. miRNAs have been widely indicated as key players in the regulation of growth and development. The role of miRNAs in drought response was investigated in young leaves of Brachypodium distachyon, a drought-tolerant monocot model species. Adopting an in vivo drought assay, shown to cause a dramatic reduction in leaf size, mostly due to reduced cell expansion, small RNA libraries were produced from proliferating and expanding leaf cells. Next-generation sequencing data were analyzed using an in-house bioinformatics pipeline allowing the identification of 66 annotated miRNA genes and 122 new high confidence predictions greatly expanding the number of known Brachypodium miRNAs. In addition, we identified four TAS3 loci and a large number of siRNA-producing loci that show characteristics suggesting that they may represent young miRNA genes. Most miRNAs showed a high expression level, consistent with their involvement in early leaf development and cell identity. Proliferating and expanding leaf cells respond differently to drought treatment and differential expression analyses suggest novel evidence for an miRNA regulatory network controlling cell division in both normal and stressed conditions and demonstrate that drought triggers a genetic reprogramming of leaf growth in which miRNAs are deeply involved.