Exploring the potential of endophyte-plant interactions for improving crop sustainable yields in a changing climate.

Climate change poses a major threat to global food security, significantly reducing crop yields as cause of abiotic stresses, and for boosting the spread of new and old pathogens and pests. Sustainable crop management as a route to mitigation poses the challenge of recruiting an array of solutions and tools for the new aims. Among these, the deployment of positive interactions between the micro-biotic components of agroecosystems and plants can play a highly significant role, as part of the agro-ecological revolution. Endophytic microorganisms have emerged as a promising solution to tackle this challenge. Among these, Arbuscular Mycorrhizal Fungi (AMF) and endophytic bacteria and fungi have demonstrated their potential to alleviate abiotic stresses such as drought and heat stress, as well as the impacts of biotic stresses. They can enhance crop yields in a sustainable way also by other mechanisms, such as improving the nutrient uptake, or by direct effects on plant physiology. In this review we summarize and update on the main types of endophytes, we highlight several studies that demonstrate their efficacy in improving sustainable yields and explore possible avenues for implementing crop-microbiota interactions. The mechanisms underlying these interactions are highly complex and require a comprehensive understanding. For this reason, omic technologies such as genomics, transcriptomics, proteomics, and metabolomics have been employed to unravel, by a higher level of information, the complex network of interactions between plants and microorganisms. Therefore, we also discuss the various omic approaches and techniques that have been used so far to study plant-endophyte interactions.

Physiological Responses to Salt Stress at the Seedling Stage in Wild (Oryza rufipogon Griff.) and Cultivated (Oryza sativa L.) Rice.

Domesticated rice Oryza sativa L. is a major staple food worldwide, and the cereal most sensitive to salinity. It originated from the wild ancestor Oryza rufipogon Griff., which was reported to possess superior salinity tolerance. Here, we examined the morpho-physiological responses to salinity stress (80 mM NaCl for 7 days) in seedlings of an O. rufipogon accession and two Italian O. sativa genotypes, Baldo (mildly tolerant) and Vialone Nano (sensitive). Under salt treatment, O. rufipogon showed the highest percentage of plants with no to moderate stress symptoms, displaying an unchanged shoot/root biomass ratio, the highest Na+ accumulation in roots, the lowest root and leaf Na+/K+ ratio, and highest leaf relative water content, leading to a better preservation of the plant architecture, ion homeostasis, and water status. Moreover, O. rufipogon preserved the overall leaf carbon to nitrogen balance and photosynthetic apparatus integrity. Conversely, Vialone Nano showed the lowest percentage of plants surviving after treatment, and displayed a higher reduction in the growth of shoots rather than roots, with leaves compromised in water and ionic balance, negatively affecting the photosynthetic performance (lowest performance index by JIP-test) and apparatus integrity. Baldo showed intermediate salt tolerance. Being O. rufipogon interfertile with O. sativa, it resulted a good candidate for pre-breeding towards salt-tolerant lines.

Durum wheat genome highlights past domestication signatures and future improvement targets.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

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.

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.

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.

The Influence of Genotype and Environment on Small RNA Profiles in Grapevine Berry.

Understanding the molecular mechanisms involved in the interaction between the genetic composition and the environment is crucial for modern viticulture. We approached this issue by focusing on the small RNA transcriptome in grapevine berries of the two varieties Cabernet Sauvignon and Sangiovese, growing in adjacent vineyards in three different environments. Four different developmental stages were studied and a total of 48 libraries of small RNAs were produced and sequenced. Using a proximity-based pipeline, we determined the general landscape of small RNAs accumulation in grapevine berries. We also investigated the presence of known and novel miRNAs and analyzed their accumulation profile. The results showed that the distribution of small RNA-producing loci is variable between the two cultivars, and that the level of variation depends on the vineyard. Differently, the profile of miRNA accumulation mainly depends on the developmental stage. The vineyard in Riccione maximizes the differences between the varieties, promoting the production of more than 1000 specific small RNA loci and modulating their expression depending on the cultivar and the maturation stage. In total, 89 known vvi-miRNAs and 33 novel vvi-miRNA candidates were identified in our samples, many of them showing the accumulation profile modulated by at least one of the factors studied. The in silico prediction of miRNA targets suggests their involvement in berry development and in secondary metabolites accumulation such as anthocyanins and polyphenols.

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.

Post-transcriptional and post-translational regulations of drought and heat response in plants: a spider's web of mechanisms.

Drought and heat tolerance are complex quantitative traits. Moreover, the adaptive significance of some stress-related traits is more related to plant survival than to agronomic performance. A web of regulatory mechanisms fine-tunes the expression of stress-related traits and integrates both environmental and developmental signals. Both post-transcriptional and post-translational modifications contribute substantially to this network with a pivotal regulatory function of the transcriptional changes related to cellular and plant stress response. Alternative splicing and RNA-mediated silencing control the amount of specific transcripts, while ubiquitin and SUMO modify activity, sub-cellular localization and half-life of proteins. Interactions across these modification mechanisms ensure temporally and spatially appropriate patterns of downstream-gene expression. For key molecular components of these regulatory mechanisms, natural genetic diversity exists among genotypes with different behavior in terms of stress tolerance, with effects upon the expression of adaptive morphological and/or physiological target traits.

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution.

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

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.

On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development.

The ABC model of flower development explains how three classes of homeotic genes confer identity to the four types of floral organs. In Arabidopsis thaliana, APETALA2 (AP2) and AGAMOUS (AG) represent A- and C-class genes that act in an antagonistic fashion to specify perianth and reproductive organs, respectively. An apparent paradox was the finding that AP2 mRNA is supposedly uniformly distributed throughout young floral primordia. Although miR172 has a role in preventing AP2 protein accumulation, miR172 was reported to disappear from the periphery only several days after AG activation in the center of the flower. Here, we resolve the enigmatic behavior of AP2 and its negative regulator miR172 through careful expression analyses. We find that AP2 mRNA accumulates predominantly in the outer floral whorls, as expected for an A-class homeotic gene. Its pattern overlaps only transiently with that of miR172, which we find to be restricted to the center of young floral primordia from early stages on. MiR172 also accumulates in the shoot meristem upon floral induction, compatible with its known role in regulating AP2-related genes with a role in flowering. Furthermore, we show that AP2 can cause striking organ proliferation defects that are not limited to the center of the floral meristem, where its antagonist AG is required for terminating stem cell proliferation. Moreover, AP2 never expands uniformly into the center of ag mutant flowers, while miR172 is largely unaffected by loss of AG activity. We present a model in which the decision whether stamens or petals develop is based on the balance between AP2 and AG activities, rather than the two being mutually exclusive.

Correction: High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera.

UNLABELLED: The version of this article published in BMC Genomics 2009, 10:558, contains data in Table 1 which are now known to be unreliable, and an illustration, in Figure 1, of unusual miRNA processing events predicted by these unreliable data. In this full-length correction, new data replace those found to be unreliable, leading to a more straightforward interpretation without altering the principle conclusions of the study. Table 1 and associated methods have been corrected, Figure 1 deleted, supplementary file 1 added, and modifications made to the sections "Deep sequencing of small RNAs from grapevine leaf tissue" and "Microarray analysis of miRNA expression". The editors and authors regret the inconvenience caused to readers by premature publication of the original paper. BACKGROUND: MicroRNAs are short (~21 base) single stranded RNAs that, in plants, are generally coded by specific genes and cleaved specifically from hairpin precursors. MicroRNAs are critical for the regulation of multiple developmental, stress related and other physiological processes in plants. The recent annotation of the genome of the grapevine (Vitis vinifera L.) allowed the identification of many putative conserved microRNA precursors, grouped into multiple gene families. RESULTS: Here we use oligonucleotide arrays to provide the first indication that many of these microRNAs show differential expression patterns between tissues and during the maturation of fruit in the grapevine. Furthermore we demonstrate that whole transcriptome sequencing and deep-sequencing of small RNA fractions can be used both to identify which microRNA precursors are expressed in different tissues and to estimate genomic coordinates and patterns of splicing and alternative splicing for many primary miRNA transcripts. CONCLUSIONS: Our results show that many microRNAs are differentially expressed in different tissues and during fruit maturation in the grapevine. Furthermore, the demonstration that whole transcriptome sequencing can be used to identify candidate splicing events and approximate primary microRNA transcript coordinates represents a significant step towards the large-scale elucidation of mechanisms regulating the expression of microRNAs at the transcriptional and post-transcriptional levels.

High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera.

BACKGROUND: MicroRNAs are short (approximately 21 base) single stranded RNAs that, in plants, are generally coded by specific genes and cleaved specifically from hairpin precursors. MicroRNAs are critical for the regulation of multiple developmental, stress related and other physiological processes in plants. The recent annotation of the genome of the grapevine (Vitis vinifera L.) allowed the identification of many putative conserved microRNA precursors, grouped into multiple gene families. RESULTS: Here we use oligonucleotide arrays to provide the first indication that many of these microRNAs show differential expression patterns between tissues and during the maturation of fruit in the grapevine. Furthermore we demonstrate that whole transcriptome sequencing and deep-sequencing of small RNA fractions can be used both to identify which microRNA precursors are expressed in different tissues and to estimate genomic coordinates and patterns of splicing and alternative splicing for many primary miRNA transcripts. CONCLUSION: Our results show that many microRNAs are differentially expressed in different tissues and during fruit maturation in the grapevine. Furthermore, the demonstration that whole transcriptome sequencing can be used to identify candidate splicing events and approximate primary microRNA transcript coordinates represents a significant step towards the large-scale elucidation of mechanisms regulating the expression of microRNAs at the transcriptional and post-transcriptional levels.

Cloning and characterization of small non-coding RNAs from grape.

Small non-coding RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are effectors of regulatory pathways underlying plant development, metabolism, and responses to biotic and abiotic stresses. To address the nature and functions of these regulators in grapevine (Vitis vinifera L.), we have produced a small RNA library from mixed-stage grape berries. Thirteen conserved miRNAs belonging to nine miRNA families, a non-conserved miRNA, and four putative non-conserved miRNAs were isolated, and their expression and targets are described. Experimentally validated targets of non-conserved miRNAs and putative miRNAs included three genes encoding NB-LRR proteins and a gene encoding a heavy metal ion transport/detoxification protein. Of the endogenous and pathogen-derived siRNAs that were also isolated, four endogenous siRNAs mapped to genes encoding RD22-like proteins and two to a gene encoding a cytokinin synthase. The siRNA id65 targeted the cytokinin synthase gene transcript with antisense complementarity, and was specifically expressed in mature berries, in which, by contrast, expression of the cytokinin synthase gene was strongly repressed. 5' RACE revealed that the transcript of this gene was processed in 21 nucleotide increments from the id65 cleavage site, and that further cleavage was mediated by secondary siRNAs in cis. These results indicate that grapevine miRNA- and siRNA-mediated regulatory circuits have evolved to comprise processes associated with defence and fruit ripening, and broaden the range of small RNA-mediated regulation, which was previously associated with auxin, ABA, gibberellins and jasmonate, to encompass cytokinin metabolism.

Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana.

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

Characterization of five microRNA families in maize.

In recent years, microRNAs (miRNAs) have polarized the interest of the scientific community as a new category of gene expression regulators, present in both plants and animals. Plant miRNAs are involved in processes such as plant development, organ identity, and stress response. Nonetheless, knowledge of their functions is still incomplete, and it is conceivable that further new processes in which they are involved will be discovered. For these reasons, structural and functional characterization of MIR genes, that are also in crop species such as Zea mays L., becomes instrumental in addressing genetic and molecular mechanisms controlling phenotype determination and phenotypic adaptation to growing conditions. The present study contributes to the characterization of five miRNA families in maize, from the determination of their expression pattern in different maize tissues and genotypes, to the identification of putative targets by bioinformatic means and subsequent experimental validation of three targets by modified 5' RACE experiments. Furthermore, 30 different MIR genes belonging to these five miRNA families were analysed by their attribution to maize chromosomes using oat-maize addition lines and by investigating their phylogenetic relationship with genes from other cereals. In particular, sequence homology was determined by the reciprocal best BLAST hit approach, to define groups of homologous genes between maize, rice, and sorghum.