Epigenetic Control of Plant Response to Heavy Metals.

Plants are sessile organisms that must adapt to environmental conditions, such as soil characteristics, by adjusting their development during their entire life cycle. In case of low-distance seed dispersal, the new generations are challenged with the same abiotic stress encountered by the parents. Epigenetic modification is an effective option that allows plants to face an environmental constraint and to share the same adaptative strategy with their progeny through transgenerational inheritance. This is the topic of the presented review that reports the scientific progress, up to date, gained in unravelling the epigenetic response of plants to soil contamination by heavy metals and metalloids, collectively known as potentially toxic elements. The effect of the microbial community inhabiting the rhizosphere is also considered, as the evidence of a transgenerational transfer of the epigenetic status that contributes to the activation in plants of response mechanisms to soil pollution.

Dissecting postharvest chilling injuries in pome and stone fruit through integrated omics.

Lowering the storage temperature is an effective method to extend the postharvest and shelf life of fruits. Nevertheless, this technique often leads to physiological disorders, commonly known as chilling injuries. Apples and pears are susceptible to chilling injuries, among which superficial scald is the most economically relevant. Superficial scald is due to necrotic lesions of the first layers of hypodermis manifested through skin browning. In peaches and nectarines, chilling injuries are characterized by internal symptoms, such as mealiness. Fruits with these aesthetic or compositional/structural defects are not suitable for fresh consumption. Genetic variation is a key factor in determining fruit susceptibility to chilling injuries; however, physiological, or technical aspects such as harvest maturity and storage conditions also play a role. Multi-omics approaches have been used to provide an integrated explanation of chilling injury development. Metabolomics in pome fruits specifically targets the identification of ethylene, phenols, lipids, and oxidation products. Genomics and transcriptomics have revealed interesting connections with metabolomic datasets, pinpointing specific genes linked to cold stress, wax synthesis, farnesene metabolism, and the metabolic pathways of ascorbate and glutathione. When applied to Prunus species, these cutting-edge approaches have uncovered that the development of mealiness symptoms is linked to ethylene signaling, cell wall synthesis, lipid metabolism, cold stress genes, and increased DNA methylation levels. Emphasizing the findings from multi-omics studies, this review reports how the integration of omics datasets can provide new insights into understanding of chilling injury development. This new information is essential for successfully creating more resilient fruit varieties and developing novel postharvest strategies.

The Italian Research on the Molecular Characterization of Maize Kernel Development.

The study of the genetic control of maize seed development and seed-related pathways has been one of the most important themes approached by the Italian scientific community. Maize has always attracted the interest of the Italian community of agricultural genetics since its beginning, as some of its founders based their research projects on and developed their "schools" by adopting maize as a reference species. Some of them spent periods in the United States, where maize was already becoming a model system, to receive their training. In this manuscript we illustrate the research work carried out in Italy by different groups that studied maize kernels and underline their contributions in elucidating fundamental aspects of caryopsis development through the characterization of maize mutants. Since the 1980s, most of the research projects aimed at the comprehension of the genetic control of seed development and the regulation of storage products' biosyntheses and accumulation, and have been based on forward genetics approaches. We also document that for some decades, Italian groups, mainly based in Northern Italy, have contributed to improve the knowledge of maize genomics, and were both fundamental for further international studies focused on the correct differentiation and patterning of maize kernel compartments and strongly contributed to recent advances in maize research.

Evidence of chromatin and transcriptional dynamics for cold development in peach flower bud.

In temperate zones, fruit trees regulate their annual growth cycle to seasonal environmental changes. During the cold season, growth is limited by both environmental and genetic factors. After the exposure to low temperature and fulfillment of chilling requirements, mild temperatures promote the growth and flowering. However, an insufficient chilling exposure may lead to nonuniform blooming, with a negative impact on fruit set. To gain insights into flower development in the fruit tree buds, peach is an interesting model, the flower and vegetative bud being distinct organs. To understand how flower bud development is regulated, we integrated cytological observations and epigenetic and chromatin genome-wide data with transcriptional changes to identify the main regulatory factors involved in flower development during chilling accumulation. We demonstrated that growth cessation does not occur in peach flower buds during chilling accumulation, but that there are changes in transcript abundance of key genes of hormone metabolism and flower bud development, distribution of histone modifications (H3K4me3 and H3K27me3) and DNA methylation. Altogether, our findings indicate that during the cold season the flower bud is in a nondormant state and that the chilling experience allows flower differentiation to be completed.

Exploitation of epigenetic variation of crop wild relatives for crop improvement and agrobiodiversity preservation.

Crop wild relatives (CWRs) are recognized as the best potential source of traits for crop improvement. However, successful crop improvement using CWR relies on identifying variation in genes controlling desired traits in plant germplasms and subsequently incorporating them into cultivars. Epigenetic diversity may provide an additional layer of variation within CWR and can contribute novel epialleles for key traits for crop improvement. There is emerging evidence that epigenetic variants of functional and/or agronomic importance exist in CWR gene pools. This provides a rationale for the conservation of epigenotypes of interest, thus contributing to agrobiodiversity preservation through conservation and (epi)genetic monitoring. Concepts and techniques of classical and modern breeding should consider integrating recent progress in epigenetics, initially by identifying their association with phenotypic variations and then by assessing their heritability and stability in subsequent generations. New tools available for epigenomic analysis offer the opportunity to capture epigenetic variation and integrate it into advanced (epi)breeding programmes. Advances in -omics have provided new insights into the sources and inheritance of epigenetic variation and enabled the efficient introduction of epi-traits from CWR into crops using epigenetic molecular markers, such as epiQTLs.

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in peach reproductive tissues.

BACKGROUND: Perennial fruit trees display a growth behaviour characterized by annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning but also the progression of the reproductive cycle depending upon the impact of climate conditions. Additionally, current developments in high-throughput technologies have impacted Rosaceae tree research while investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among epigenetic mechanisms, chromatin remodelling mediated by histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression. Chromatin immunoprecipitation is an effective technique to investigate chromatin dynamics in plants. This technique is generally applied for studies on chromatin states and enrichment of post-transcriptional modifications (PTMs) in histone proteins. RESULTS: Peach is considered a model organism among climacteric fruits in the Rosaceae family for studies on bud formation, dormancy, and organ differentiation. In our work, we have primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach (Prunus persica). Subsequently, we focused our investigations on the role of two chromatin marks, namely the trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) in modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as our model system. CONCLUSIONS: We present general strategies to optimize ChIP protocols for buds and mesocarp tissues of peach and analyze the correlation between gene expression and chromatin mark enrichment/depletion. The procedures proposed may be useful to evaluate any involvement of histone modifications in the regulation of gene expression during bud dormancy progression and core ripening in fruits.

Sucrose Metabolism and Transport in Grapevines, with Emphasis on Berries and Leaves, and Insights Gained from a Cross-Species Comparison.

In grapevines, as in other plants, sucrose and its constituents glucose and fructose are fundamentally important and carry out a multitude of roles. The aims of this review are three-fold. First, to provide a summary of the metabolism and transport of sucrose in grapevines, together with new insights and interpretations. Second, to stress the importance of considering the compartmentation of metabolism. Third, to outline the key role of acid invertase in osmoregulation associated with sucrose metabolism and transport in plants.

Meta-analysis of RNA-Seq studies reveals genes with dominant functions during flower bud endo- to eco-dormancy transition in Prunus species.

In deciduous fruit trees, entrance into dormancy occurs in later summer/fall, concomitantly with the shortening of day length and decrease in temperature. Dormancy can be divided into endodormancy, ecodormancy and paradormancy. In Prunus species flower buds, entrance into the dormant stage occurs when the apical meristem is partially differentiated; during dormancy, flower verticils continue their growth and differentiation. Each species and/or cultivar requires exposure to low winter temperature followed by warm temperatures, quantified as chilling and heat requirements, to remove the physiological blocks that inhibit budburst. A comprehensive meta-analysis of transcriptomic studies on flower buds of sweet cherry, apricot and peach was conducted, by investigating the gene expression profiles during bud endo- to ecodormancy transition in genotypes differing in chilling requirements. Conserved and distinctive expression patterns were observed, allowing the identification of gene specifically associated with endodormancy or ecodormancy. In addition to the MADS-box transcription factor family, hormone-related genes, chromatin modifiers, macro- and micro-gametogenesis related genes and environmental integrators, were identified as novel biomarker candidates for flower bud development during winter in stone fruits. In parallel, flower bud differentiation processes were associated to dormancy progression and termination and to environmental factors triggering dormancy phase-specific gene expression.

Novel and emerging biotechnological crop protection approaches.

Traditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for non-model plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation.

Regulation of Fruit Growth in a Peach Slow Ripening Phenotype.

Consumers' choices are mainly based on fruit external characteristics such as the final size, weight, and shape. The majority of edible fruit are by tree fruit species, among which peach is the genomic and genetic reference for Prunus. In this research, we used a peach with a slow ripening (SR) phenotype, identified in the Fantasia (FAN) nectarine, associated with misregulation of genes involved in mesocarp identity and showing a reduction of final fruit size. By investigating the ploidy level, we observed a progressive increase in endoreduplication in mesocarp, which occurred in the late phases of FAN fruit development, but not in SR fruit. During fruit growth, we also detected that genes involved in endoreduplication were differentially modulated in FAN compared to SR. The differential transcriptional outputs were consistent with different chromatin states at loci of endoreduplication genes. The impaired expression of genes controlling cell cycle and endocycle as well as those claimed to play a role in fruit tissue identity result in the small final size of SR fruit.

Strigolactones and Auxin Cooperate to Regulate Maize Root Development and Response to Nitrate.

In maize, nitrate regulates root development thanks to the coordinated action of many players. In this study, the involvement of strigolactones (SLs) and auxin as putative components of the nitrate regulation of lateral root (LR) was investigated. To this aim, the endogenous SL content of maize root in response to nitrate was assessed by liquid chromatography with tandem mass Spectrometry (LC-MS/MS) and measurements of LR density in the presence of analogues or inhibitors of auxin and SLs were performed. Furthermore, an untargeted RNA-sequencing (RNA-seq)-based approach was used to better characterize the participation of auxin and SLs to the transcriptional signature of maize root response to nitrate. Our results suggested that N deprivation induces zealactone and carlactonoic acid biosynthesis in root, to a higher extent if compared to P-deprived roots. Moreover, data on LR density led to hypothesize that the induction of LR development early occurring upon nitrate supply involves the inhibition of SL biosynthesis, but that the downstream target of SL shutdown, besides auxin, also includes additional unknown players. Furthermore, RNA-seq results provided a set of putative markers for the auxin- or SL-dependent action of nitrate, meanwhile also allowing to identify novel components of the molecular regulation of maize root response to nitrate. Globally, the existence of at least four different pathways was hypothesized: one dependent on auxin, a second one mediated by SLs, a third deriving from the SL-auxin interplay, and a last one attributable to nitrate itself through further downstream signals. Further work will be necessary to better assess the reliability of the model proposed.

Epigenetics: possible applications in climate-smart crop breeding.

To better adapt transiently or lastingly to stimuli from the surrounding environment, the chromatin states in plant cells vary to allow the cells to fine-tune their transcriptional profiles. Modifications of chromatin states involve a wide range of post-transcriptional histone modifications, histone variants, DNA methylation, and activity of non-coding RNAs, which can epigenetically determine specific transcriptional outputs. Recent advances in the area of '-omics' of major crops have facilitated identification of epigenetic marks and their effect on plant response to environmental stresses. As most epigenetic mechanisms are known from studies in model plants, we summarize in this review recent epigenetic studies that may be important for improvement of crop adaptation and resilience to environmental changes, ultimately leading to the generation of stable climate-smart crops. This has paved the way for exploitation of epigenetic variation in crop breeding.

Nitrate and Ammonium Affect the Overall Maize Response to Nitrogen Availability by Triggering Specific and Common Transcriptional Signatures in Roots.

Nitrogen (N) is an essential macronutrient for crops. Plants have developed several responses to N fluctuations, thus optimizing the root architecture in response to N availability. Nitrate and ammonium are the main inorganic N forms taken up by plants, and act as both nutrients and signals, affecting gene expression and plant development. In this study, RNA-sequencing was applied to gain comprehensive information on the pathways underlying the response of maize root, pre-treated in an N-deprived solution, to the provision of nitrate or ammonium. The analysis of the transcriptome shows that nitrate and ammonium regulate overlapping and distinct pathways, thus leading to different responses. Ammonium activates the response to stress, while nitrate acts as a negative regulator of transmembrane transport. Both the N-source repress genes related to the cytoskeleton and reactive oxygen species detoxification. Moreover, the presence of ammonium induces the accumulation of anthocyanins, while also reducing biomass and chlorophyll and flavonoids accumulation. Furthermore, the later physiological effects of these nutrients were evaluated through the assessment of shoot and root growth, leaf pigment content and the amino acid concentrations in root and shoot, confirming the existence of common and distinct features in response to the two nitrogen forms.

Epigenetic signatures of stress adaptation and flowering regulation in response to extended drought and recovery in Zea mays.

During their lifespan, plants respond to a multitude of stressful factors. Dynamic changes in chromatin and concomitant transcriptional variations control stress response and adaptation, with epigenetic memory mechanisms integrating environmental conditions and appropriate developmental programs over the time. Here we analyzed transcriptome and genome-wide histone modifications of maize plants subjected to a mild and prolonged drought stress just before the flowering transition. Stress was followed by a complete recovery period to evaluate drought memory mechanisms. Three categories of stress-memory genes were identified: i) "transcriptional memory" genes, with stable transcriptional changes persisting after the recovery; ii) "epigenetic memory candidate" genes in which stress-induced chromatin changes persist longer than the stimulus, in absence of transcriptional changes; iii) "delayed memory" genes, not immediately affected by the stress, but perceiving and storing stress signal for a delayed response. This last memory mechanism is described for the first time in drought response. In addition, applied drought stress altered floral patterning, possibly by affecting expression and chromatin of flowering regulatory genes. Altogether, we provided a genome-wide map of the coordination between genes and chromatin marks utilized by plants to adapt to a stressful environment, describing how this serves as a backbone for setting stress memory.

Chromatin dynamics during interphase and cell division: similarities and differences between model and crop plants.

Genetic information in the cell nucleus controls organismal development and responses to the environment, and finally ensures its own transmission to the next generations. To achieve so many different tasks, the genetic information is associated with structural and regulatory proteins, which orchestrate nuclear functions in time and space. Furthermore, plant life strategies require chromatin plasticity to allow a rapid adaptation to abiotic and biotic stresses. Here, we summarize current knowledge on the organization of plant chromatin and dynamics of chromosomes during interphase and mitotic and meiotic cell divisions for model and crop plants differing as to genome size, ploidy, and amount of genomic resources available. The existing data indicate that chromatin changes accompany most (if not all) cellular processes and that there are both shared and unique themes in the chromatin structure and global chromosome dynamics among species. Ongoing efforts to understand the molecular mechanisms involved in chromatin organization and remodeling have, together with the latest genome editing tools, potential to unlock crop genomes for innovative breeding strategies and improvements of various traits.

Morphological Changes and Expressions of AOX1A, CYP81D8, and Putative PFP Genes in a Large Set of Commercial Maize Hybrids Under Extreme Waterlogging.

Waterlogging is a severe abiotic stressor causing significant growth impairment and yield losses in many crops. Maize is highly sensitive to the excess of water, and against the background of climate change there is an urgent need for deeper insights into the mechanisms of crop adaptation to waterlogging. In the present study, changes in maize morphology at the 4-5 leaf stage and the expression of three candidate genes for flooding tolerance in plants subjected to six continuous days of waterlogging were recorded in 19 commercial hybrids and in the inbred line B73, with the aim of investigating the current variability in cultivated hybrids and identifying useful morphological and molecular markers for screening tolerant genotypes. Here it was demonstrated that root parameters (length, area, biomass) were more impaired by waterlogging than shoot parameters (shoot height and biomass). Culm height generally increased in stressed plants (by up to +24% vs. controls), while shoot biomass was significantly reduced in only two hybrids. Root biomass was reduced in all the hybrids, by an average of 30%, and significantly in 7 hybrids, while root length and area were even more severely reduced, by 30-55% vs. controls, depending on the hybrid. The earlier appearance of aerial roots seemed to be associated with greater root injuries. In leaves, the transcript of the PFP enzyme (phosphofructokinase), which is involved in glycolytic reactions, was markedly up-regulated (up to double the values) in half the waterlogged hybrids, but down-regulated in the others. The transcript of CYP81D8 (ROS-related proteins) in waterlogged plants exhibited relevant increases or strong decreases in level, depending on the hybrid. The transcript of the AOX1A gene, coding for a mitochondrial respiratory electron transport chain-related protein, was markedly down-regulated in all the treated hybrids. Expression analysis of these genes under extreme waterlogging only partially correlate with the shoot and root growth impairments observed, and AOX1A seems to be the most informative of them.

Control of Maize Vegetative and Reproductive Development, Fertility, and rRNAs Silencing by HISTONE DEACETYLASE 108.

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from acetylated histone tails that consequently interact more closely with DNA, leading to chromatin state refractory to transcription. Zea mays HDA108 belongs to the Rpd3/HDA1 HDAC family and is ubiquitously expressed during development. The newly isolated hda108/hda108 insertional mutant exhibited many developmental defects: significant reduction in plant height, alterations of shoot and leaf development, and alterations of inflorescence patterning and fertility. Western blot analyses and immunolocalization experiments revealed an evident increase in histone acetylation, accompanied by a marked reduction in H3K9 dimethylation, in mutant nuclei. The DNA methylation status, in the CHG sequence context, and the transcript level of ribosomal sequences were also affected in hda108 mutants, while enrichment in H3 and H4 acetylation characterizes both repetitive and nonrepetitive transcriptional up-regulated loci. RNA-Seq of both young leaf and anthers indicated that transcription factor expression is highly affected and that the pollen developmental program is disrupted in hda108 mutants. Crosses between hda108/hda108 and epiregulator mutants did not produce any double mutant progeny indicating possible genetic interactions of HDA108 with distinct epigenetic pathways. Our findings indicate that HDA108 is directly involved in regulation of maize development, fertility, and epigenetic regulation of genome activity.

De Novo Identification of sRNA Loci and Non-coding RNAs by High-Throughput Sequencing.

Non-coding RNA transcripts, such as long non-coding RNAs, miRNAs, siRNAs, and transposon-originating transcripts, are involved in the regulation of RNA stability, protein translation, and/or the modulation of chromatin states. RNA-Seq can be used to catalog this diversity of novel transcripts and a joint analysis of these transcriptomic data can provide useful insights into epigenetic regulation of dynamic responses such as the stress response, which may not be deciphered from individual analysis of single transcript categories. Here, we present a protocol that allows the identification and analysis of small RNAs and long non-coding RNAs, together with the comparison of these species between different sample types.