Comparison of meiotic transcriptomes of three maize inbreds with different origins reveals differences in cell cycle and recombination.

BACKGROUND: Cellular events during meiosis can differ between inbred lines in maize. Substantial differences in the average numbers of chiasmata and double-strand breaks (DSBs) per meiotic cell have been documented among diverse inbred lines of maize: CML228, a tropical maize inbred line, B73 and Mo17, temperate maize lines. To determine if gene expression might explain these observed differences, an RNA-Seq experiment was performed on CML228 male meiocytes which was compared to B73 and Mo17 male meiocytes, where plants were grown in the same controlled environment. RESULTS: We found that a few DSB-repair/meiotic genes which promote class I crossovers (COs) and the Zyp1 gene which limits newly formed class I COs were up-regulated, whereas Mus81 homolog 2 which promotes class II COs was down-regulated in CML228. Although we did not find enriched gene ontology (GO) categories directly related to meiosis, we found that GO categories in membrane, localization, proteolysis, energy processes were up-regulated in CML228, while chromatin remodeling, epigenetic regulation, and cell cycle related processes including meiosis related cell cycle processes were down-regulated in CML228. The degree of similarity in expression patterns between the three maize lines reflect their genetic relatedness: B73 and Mo17 had similar meiotic expressions and CML228 had a more distinct expression profile. CONCLUSIONS: We found that meiotic related genes were mostly conserved among the three maize inbreds except for a few DSB-repair/meiotic genes. The findings that the molecular players in limiting class I CO formation (once CO assurance is achieved) were up-regulated and those involved in promoting class II CO formation were down-regulated in CML228 agree with the lower chiasmata number observed in CML228 previously. In addition, epigenetics such as chromatin remodeling and histone modification might play a role. Transport and energy-related processes was up-regulated and Cyclin13 was down-regulated in CML228. The direction of gene expression of these processes agree with that previously found in meiotic tissues compared with vegetative tissues. In summary, we used different natural maize inbred lines from different climatic conditions and have shown their differences in expression landscape in male meiocytes.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Correction to: MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the 'Background'. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the 'Background' [1]. Thus, the following amendment to the Background section should be noted.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.

Correction to: The transcriptome landscape of early maize meiosis.

CORRECTION: Following publication of the original article [1], the authors reported that the number of genes overlaying the bar graph in Fig. 3A were incorrectly counted and inserted (i.e. including a title tile, and in inverse order). The corrected numbers are below and match with the listed genes supplied in Additional File: Table S2.

Not just gene expression: 3D implications of chromatin modifications during sexual plant reproduction.

DNA methylation and histone modifications are epigenetic changes on a DNA molecule that alter the three-dimensional (3D) structure locally as well as globally, impacting chromatin looping and packaging on a larger scale. Epigenetic marks thus inform higher-order chromosome organization and placement in the nucleus. Conventional epigenetic marks are joined by chromatin modifiers like cohesins, condensins and membrane-anchoring complexes to support particularly 3D chromosome organization. The most popular consequences of epigenetic modifications are gene expression changes, but chromatin modifications have implications beyond this, particularly in actively dividing cells and during sexual reproduction. In this opinion paper, we will focus on epigenetic mechanisms and chromatin modifications during meiosis as part of plant sexual reproduction where 3D management of chromosomes and re-organization of chromatin are defining features and prime tasks in reproductive cells, not limited to modulating gene expression. Meiotic chromosome organization, pairing and synapsis of homologous chromosomes as well as distribution of meiotic double-strand breaks and resulting crossovers are presumably highly influenced by epigenetic mechanisms. Special mobile small RNAs have been described in anthers, where these so-called phasiRNAs seem to direct DNA methylation in meiotic cells. Intriguingly, many of the mentioned developmental processes make use of epigenetic changes and small RNAs in a manner other than gene expression changes. Widening our approaches and opening our mind to thinking three-dimensionally regarding epigenetics in plant development holds high promise for new discoveries and could give us a boost for further knowledge.

The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony.

The transcriptome landscape of early maize meiosis.

BACKGROUND: A major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq. RESULTS: We detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis. CONCLUSIONS: Taken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.

The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.

In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.

RAD5A, RECQ4A, and MUS81 have specific functions in homologous recombination and define different pathways of DNA repair in Arabidopsis thaliana.

Complex DNA structures, such as double Holliday junctions and stalled replication forks, arise during DNA replication and DNA repair. Factors processing these intermediates include the endonuclease MUS81, helicases of the RecQ family, and the yeast SNF2 ATPase RAD5 and its Arabidopsis thaliana homolog RAD5A. By testing sensitivity of mutant plants to DNA-damaging agents, we defined the roles of these factors in Arabidopsis. rad5A recq4A and rad5A mus81 double mutants are more sensitive to cross-linking and methylating agents, showing that RAD5A is required for damage-induced DNA repair, independent of MUS81 and RECQ4A. The lethality of the recq4A mus81 double mutant indicates that MUS81 and RECQ4A also define parallel DNA repair pathways. The recq4A/mus81 lethality is suppressed by blocking homologous recombination (HR) through disruption of RAD51C, showing that RECQ4A and MUS81 are required for processing recombination-induced aberrant intermediates during replication. Thus, plants possess at least three different pathways to process DNA repair intermediates. We also examined HR-mediated double-strand break (DSB) repair using recombination substrates with inducible site-specific DSBs: MUS81 and RECQ4A are required for efficient synthesis-dependent strand annealing (SDSA) but only to a small extent for single-strand annealing (SSA). Interestingly, RAD5A plays a significant role in SDSA but not in SSA.

BRCC36A is epistatic to BRCA1 in DNA crosslink repair and homologous recombination in Arabidopsis thaliana.

BRCA1 is a well-known tumor suppressor protein in mammals, involved in multiple cellular processes such as DNA repair, chromosome segregation and chromatin remodeling. Interestingly, homologs of BRCA1 and several of its complex partners are also found in plants. As the respective mutants are viable, in contrast to mammalian mutants, detailed analyses of their biological role is possible. Here we demonstrate that the model plant Arabidopsis thaliana harbors two homologs of the mammalian BRCA1 interaction partner BRCC36, AtBRCC36A and AtBRCC36B. Mutants of both genes as well as the double mutants are fully fertile and show no defects in development. We were able to show that mutation of one of the homologs, AtBRCC36A, leads to a severe defect in intra- and interchromosomal homologous recombination (HR). A HR defect is also apparent in Atbrca1 mutants. As the Atbrcc36a/Atbrca1 double mutant behaves like the single mutants of AtBRCA1 and AtBRCC36A both proteins seem to be involved in a common pathway in the regulation of HR. AtBRCC36 is also epistatic to AtBRCA1 in DNA crosslink repair. Upon genotoxic stress, AtBRCC36A is transferred into the nucleus.

Characterization of a set of novel meiotically-active promoters in Arabidopsis.

BACKGROUND: Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis. RESULTS: In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants. CONCLUSIONS: We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.

BRCA2 is a mediator of RAD51- and DMC1-facilitated homologous recombination in Arabidopsis thaliana.

• Mutations in the breast cancer susceptibility gene 2 (BRCA2) are correlated with hereditary breast cancer in humans. Studies have revealed that mammalian BRCA2 plays crucial roles in DNA repair. Therefore, we wished to define the role of the BRCA2 homologs in Arabidopsis in detail. • As Arabidopsis contains two functional BRCA2 homologs, an Atbrca2 double mutant was generated and analyzed with respect to hypersensitivity to genotoxic agents and recombination frequencies. Cytological studies addressing male and female meiosis were also conducted, and immunolocalization was performed in male meiotic prophase I. • The Atbrca2 double mutant showed hypersensitivity to the cross-linking agent mitomycin C and displayed a dramatic reduction in somatic homologous recombination frequency, especially after double-strand break induction. The loss of AtBRCA2 also led to severe defects in male meiosis and development of the female gametophyte and impeded proper localization of the synaptonemal complex protein AtZYP1 and the recombinases AtRAD51 and AtDMC1. • The results demonstrate that AtBRCA2 is important for both somatic and meiotic homologous recombination. We further show that AtBRCA2 is required for proper meiotic synapsis and mediates the recruitment of AtRAD51 and AtDMC1. Our results suggest that BRCA2 controls single-strand invasion steps during homologous recombination in plants.

Profiling m6A RNA Modifications in Low Amounts of Plant Cells Using Maize Meiocytes.

RNA modifications can influence gene expression via multiple aspects such as RNA stability and alternative splicing. The most prominent RNA modification is m6A (N6-methyladenosine). Its profiling from low starting amounts of <100 cells is challenging. We describe here a complete workflow from cell isolation to data analysis that is based on using an RNA CUT&RUN-supported m6A-RIP (RNA immunoprecipitation) procedure and a subsequent adaptor-tagging library synthesis. Male meiocytes isolated from maize anthers were used as a test system to establish the protocol.

UV-B Irradiation Results in Inhibition of Hypocotyl Elongation, Cell Cycle Arrest, and Decreased Endoreduplication Mediated by miR5642.

UV-B as a component of natural solar radiation can induce damage and morphological development in plants. The UV-B response from germination and early development in seedlings is still largely unknown, with most studies focused on older, light-exposed seedlings. We used fluence response curves measuring hypocotyl length after UV-B exposure coupled with RNA-seq and sRNA-seq evaluation of the early seedling response in the model organism Arabidopsis thaliana. We identified miR5642 as a potential novel key regulator of UV-B responses. miR5642 is a noncanonical miRNA predicted to target previously known and unknown components involved in hypocotyl growth inhibition. These include (i) SMAX1, a signal transmitter for seedling germination and growth; (ii) ZAT1, an uncharacterized transcription factor; and (iii) membrane pores and transporters (VHA-E1, VHA-E3, EPSIN-LIKE and PIP1.4) implicated in cell elongation. In addition, HY5 and HYH, two homologous and redundant transcription factors involved in seedling photomorphogenesis, may interact with these newly identified components. Interestingly, UV-B-induced DNA photodimer formation seems to be the direct trigger leading to inhibition of hypocotyl growth through a combination of cellular decisions including cell cycle arrest, reduced endoreduplication and reduced cell elongation, and this inhibition appears to be modulated by miR5642 target genes.

Oxygen, secreted proteins and small RNAs: mobile elements that govern anther development.

Correct anther development is essential for male fertility and subsequently agricultural yield. Defects in anther development range from the early stage of stamen formation until the late stage of tapetum degeneration. In particular, the specification of the four distinct somatic layers and the inner sporogenous cells need perfect orchestration relying on precise cell-cell communication. Up to now, several signals, which coordinate the anther´s developmental program, have been identified. Among the known signals are phytohormones, environmental conditions sensed via glutaredoxins, several receptor-like kinases triggered by ligands like MAC1, and small RNAs such as miRNAs and the monocot-prevalent reproductive phasiRNAs. Rather than giving a full review on anther development, here we discuss anther development with an emphasis on mobile elements like ROS/oxygen, secreted proteins and small RNAs (only briefly touching on phytohormones), how they might act and interact, and what the future of this research area might reveal.

IER2-induced senescence drives melanoma invasion through osteopontin.

Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.

Isolating Male Meiocytes from Maize and Wheat for "-Omics" Analyses.

Genome-wide gene expression studies have become a routine approach due to the advances in sequencing technologies, their ease of use, and increasing affordability. Simultaneous investigation of small RNA expression adds further valuable information but is not adopted as widely yet. Both RNA-seq and small RNA-seq benefit from the use of specific cell types. Here, we describe a protocol for the isolation of male meiotic cells from maize or wheat plants, along with the application of downstream RNA sequencing, extendable to other -omics approaches.

Corrigendum: Novel Meiotic miRNAs and Indications for a Role of PhasiRNAs in Meiosis.

[This corrects the article DOI: 10.3389/fpls.2016.00762.].

A Critical Assessment of 60 Years of Maize Intragenic Recombination.

Until the mid-1950s, it was believed that genetic crossovers did not occur within genes. Crossovers occurred between genes, the "beads on a string" model. Then in 1956, Seymour Benzer published his classic paper describing crossing over within a gene, intragenic recombination. This result from a bacteriophage gene prompted Oliver Nelson to study intragenic recombination in the maize Waxy locus. His studies along with subsequent work by others working with maize and other organisms described the outcomes of intragenic recombination and provided some of the earliest evidence that genes, not intergenic regions, were recombination hotspots. High-throughput genotyping approaches have since replaced single gene intragenic studies for characterizing the outcomes of recombination. These large-scale studies confirm that genes, or more generally genic regions, are the most active recombinogenic regions, and suggested a pattern of crossovers similar to the budding yeast Saccharomyces cerevisiae. In S. cerevisiae recombination is initiated by double-strand breaks (DSBs) near transcription start sites (TSSs) of genes producing a polarity gradient where crossovers preferentially resolve at the 5' end of genes. Intragenic studies in maize yielded less evidence for either polarity or for DSBs near TSSs initiating recombination and in certain respects resembled Schizosaccharomyces pombe or mouse. These different perspectives highlight the need to draw upon the strengths of different approaches and caution against relying on a single model system or approach for understanding recombination.