Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Dynamics of Endogenous Auxin and Its Role in Somatic Embryogenesis Induction and Progression in Cork Oak.

Somatic embryogenesis (SE) is a feasible in vitro regeneration system with biotechnological applications in breeding programs, although, in many forest species, SE is highly inefficient, mainly due to their recalcitrance. On the other hand, SE represents a valuable model system for studies on cell reprogramming, totipotency acquisition, and embryogenic development. The molecular mechanisms that govern the transition of plant somatic cells to embryogenic cells are largely unknown. There is increasing evidence that auxins mediate this transition and play a key role in somatic embryo development, although data on woody species are very limited. In this study, we analyzed the dynamics and possible role of endogenous auxin during SE in cork oak (Quercus suber L.). The auxin content was low in somatic cells before cell reprogramming, while it increased after induction of embryogenesis, as revealed by immunofluorescence assays. Cellular accumulation of endogenous auxin was also detected at the later stages of somatic embryo development. These changes in auxin levels correlated with the expression patterns of the auxin biosynthesis (QsTAR2) and signaling (QsARF5) genes, which were upregulated after SE induction. Treatments with the inhibitor of auxin biosynthesis, kynurenine, reduced the proliferation of proembryogenic masses and impaired further embryo development. QsTAR2 and QsARF5 were downregulated after kynurenine treatment. Our findings indicate a key role of endogenous auxin biosynthesis and signaling in SE induction and multiplication, as well as somatic embryo development of cork oak.

Small molecule inhibitors of mammalian GSK-3ß promote in vitro plant cell reprogramming and somatic embryogenesis in crop and forest species.

Plant in vitro regeneration systems, such as somatic embryogenesis, are essential in breeding; they permit propagation of elite genotypes, production of doubled-haploids, and regeneration of whole plants from gene editing or transformation events. However, in many crop and forest species, somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3ß (GSK-3ß), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, a representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis genes FUS3, LEC2, and AGL15. Plant GSK-3 kinase BIN2 is a master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1, and BZR1 were up-regulated and the BAS1 catabolic gene was repressed, indicating activation of the BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening up the way for new strategies using GSK-3ß inhibitors that could be extended to other species.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Advances in Plant Regeneration: Shake, Rattle and Roll.

Some plant cells are able to rebuild new organs after tissue damage or in response to definite stress treatments and/or exogenous hormone applications. Whole plants can develop through de novo organogenesis or somatic embryogenesis. Recent findings have enlarged our understanding of the molecular and cellular mechanisms required for tissue reprogramming during plant regeneration. Genetic analyses also suggest the key role of epigenetic regulation during de novo plant organogenesis. A deeper understanding of plant regeneration might help us to enhance tissue culture optimization, with multiple applications in plant micropropagation and green biotechnology. In this review, we will provide additional insights into the physiological and molecular framework of plant regeneration, including both direct and indirect de novo organ formation and somatic embryogenesis, and we will discuss the key role of intrinsic and extrinsic constraints for cell reprogramming during plant regeneration.

Modulation of autophagy and protease activities by small bioactive compounds to reduce cell death and improve stress-induced microspore embryogenesis initiation in rapeseed and barley.

Microspore embryogenesis is a powerful biotechnological tool that is very useful in crop breeding for the rapid production of haploid and double-haploid embryos and plants. In this in vitro system, the haploid microspore is reprogrammed by the application of specific stress treatments. A high level of cell death after the stress is a major factor that greatly reduces embryogenesis yield at its initial stages. Autophagy is a degradation pathway that is present in all eukaryotes and plays key roles in a range of processes, including stress responses. Many proteases participate in autophagy and cell death; among them, cathepsins are the most abundant enzymes with a role in plant senescence and programmed cell death (PCD). Moreover, although plant genomes do not contain homologues of caspases, caspase 3-like activity (main executioner protease of animal cell death) has been detected in many plant PCD processes. Recent studies by our group in barley microspore cultures reported that the stress treatment required for inducing microspore embryogenesis (cold treatment), also produced reactive oxygen species (ROS) and cell death, concomitantly with the induction of autophagy, as well as cathepsin-like and caspase 3-like proteolytic activities. In the present study, we report new data on microspore embryogenesis of rapeseed that indicate, as in barley, activation of cell death and autophagy processes after the inductive stress. The results revealed that treatments modulating autophagy and proteases produced the same effect in the two plant systems, regardless of the stress applied, cold in barley or heat in rapeseed. Pharmacological treatments with small bioactive compounds that inhibit ROS, autophagy and specific cell death-proteases led to reduced cell death and an increased embryogenesis initiation rate in both, barley and rapeseed. Taken together, these findings open up new intervention pathways by modulating autophagy and proteases, which are very promising in terms of increasing the efficiency of in vitro microspore embryogenesis systems for biotechnological applications in crop breeding.

Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with ß-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

The epigenetic memory of temperature during embryogenesis modifies the expression of bud burst-related genes in Norway spruce epitypes.

MAIN CONCLUSION: Epigenetic memory affects the timing of bud burst phenology and the expression of bud burst-related genes in genetically identical Norway spruce epitypes in a manner usually associated with ecotypes. In Norway spruce, a temperature-dependent epigenetic memory established during embryogenesis affects the timing of bud burst and bud set in a reproducible and predictable manner. We hypothesize that the clinal variation in these phenological traits, which is associated with adaptation to growth under frost-free conditions, has an epigenetic component. In Norway spruce, dehydrins (DHNs) have been associated with extreme frost tolerance. DHN transcript levels decrease gradually prior to flushing, a time when trees are highly sensitive to frost. Furthermore, EARLY BUD BREAK 1 genes (EBB1) and the FT-TFL1-LIKE 2-gene (PaFTL2) were previously suggested to be implied in control of bud phenology. Here we report an analysis of transcript levels of 12 DHNs, 3 EBB1 genes and FTL2 in epitypes of the same genotype generated at different epitype-inducing temperatures, before and during spring bud burst. Earlier flushing of epitypes originating from embryos developed at 18 °C as compared to 28 °C, was associated with differential expression of these genes between epitypes and between buds and last year's needles. The majority of these genes showed significantly different expressions between epitypes in at least one time point. The general trend in DHN expression pattern in buds showed the expected reduction in transcript levels when approaching flushing, whereas, surprisingly, transcript levels peaked later in needles, mainly at the moment of bud burst. Collectively, our results demonstrate that the epigenetic memory of temperature during embryogenesis affects bud burst phenology and expression of the bud burst-related DHN, EBB1 and FTL2 genes in genetically identical Norway spruce epitypes.

Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in Norway spruce.

MAIN CONCLUSION: A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and histones, as well as sRNAs, are pivotal for the establishment of the epigenetic memory. As a forest tree species with long generation times, Norway spruce is remarkably well adapted to local environmental conditions despite having recently, from an evolutionary perspective, recolonized large areas following the last glaciation. In this species, there is an enigmatic epigenetic memory of the temperature conditions during embryogenesis that allows rapid adaptation to changing environment. We used a transcriptomic approach to investigate the molecular mechanisms underlying the formation of the epigenetic memory during somatic embryogenesis in Norway spruce. Nine mRNA libraries were prepared from three epitypes of the same genotype resulting from exposure to epitype-inducing temperatures of 18, 23 and 28 °C. RNA-Seq analysis revealed more than 10,000 differentially expressed genes (DEGs). The epitype-inducing conditions during SE were accompanied by marked transcriptomic changes for multiple gene models related to the epigenetic machinery. Out of 735 putative orthologs of epigenetic regulators, 329 were affected by the epitype-inducing temperatures and differentially expressed. The majority of DEGs among the epigenetic regulators was related to DNA and histone methylation, along with sRNA pathways and a range of putative thermosensing and signaling genes. These genes could be the main epigenetic regulators involved in formation of the epigenetic memory. We suggest considerable expansion of gene families of epigenetic regulators in Norway spruce compared to orthologous gene families in Populus and Arabidopsis. Obtained results provide a solid basis for further genome annotation and studies focusing on the importance of these candidate genes for the epigenetic memory formation.

EARLY BUD-BREAK1 (EBB1) defines a conserved mechanism for control of bud-break in woody perennials.

Bud-break is an environmentally and economically important trait in trees, shrubs and vines from temperate latitudes. Poor synchronization of bud-break timing with local climates can lead to frost injuries, susceptibility to pests and pathogens and poor crop yields in fruit trees and vines. The rapid climate changes outpace the adaptive capacities of plants to respond through natural selection. This is particularly true for trees which have long generation cycle and thus the adaptive changes are significantly delayed. Therefore, to devise appropriate breeding and conservation strategies, it is imperative to understand the molecular underpinnings that govern dormancy mechanisms. We have recently identified and characterized the poplar EARLY BUD-BREAK 1 (EBB1) gene. EBB1 is a positive regulator of bud-break and encodes a transcription factor from the AP2/ERF family. Here, using comparative and functional genomics approaches we show that EBB1 function in regulation of bud-break is likely conserved across wide range of woody perennial species with importance to forestry and agriculture.

The GRAS gene family in pine: transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots.

BACKGROUND: Adventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown. RESULTS: Here, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata's SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or non-existent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation. CONCLUSIONS: The GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots.