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.

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.

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.

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.

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

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

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.

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.

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.

High-resolution crossover mapping reveals similarities and differences of male and female recombination in maize.

Meiotic crossovers (COs) are not uniformly distributed across the genome. Factors affecting this phenomenon are not well understood. Although many species exhibit large differences in CO numbers between sexes, sex-specific aspects of CO landscape are particularly poorly elucidated. Here, we conduct high-resolution CO mapping in maize. Our results show that CO numbers as well as their overall distribution are similar in male and female meioses. There are, nevertheless, dissimilarities at local scale. Male and female COs differ in their locations relative to transcription start sites in gene promoters and chromatin marks, including nucleosome occupancy and tri-methylation of lysine 4 of histone H3 (H3K4me3). Our data suggest that sex-specific factors not only affect male-female CO number disparities but also cause fine differences in CO positions. Differences between male and female CO landscapes indicate that recombination has distinct implications for population structure and gene evolution in male and in female meioses.

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.

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.

Transgenic Analysis Reveals 5' Abbreviated OsRGLP2 Promoter(s) as Responsive to Abiotic Stresses.

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5' deletion analysis of the promoter sequences was performed and the deletion fragments fused with the ß-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.

Gene Evolutionary Trajectories and GC Patterns Driven by Recombination in Zea mays.

Recombination occurring during meiosis is critical for creating genetic variation and plays an essential role in plant evolution. In addition to creating novel gene combinations, recombination can affect genome structure through altering GC patterns. In maize (Zea mays) and other grasses, another intriguing GC pattern exists. Maize genes show a bimodal GC content distribution that has been attributed to nucleotide bias in the third, or wobble, position of the codon. Recombination may be an underlying driving force given that recombination sites are often associated with high GC content. Here we explore the relationship between recombination and genomic GC patterns by comparing GC gene content at each of the three codon positions (GC1, GC2, and GC3, collectively termed GCx) to instances of a variable GC-rich motif that underlies double strand break (DSB) hotspots and to meiocyte-specific gene expression. Surprisingly, GCx bimodality in maize cannot be fully explained by the codon wobble hypothesis. High GCx genes show a strong overlap with the DSB hotspot motif, possibly providing a mechanism for the high evolutionary rates seen in these genes. On the other hand, genes that are turned on in meiosis (early prophase I) are biased against both high GCx genes and genes with the DSB hotspot motif, possibly allowing important meiotic genes to avoid DSBs. Our data suggests a strong link between the GC-rich motif underlying DSB hotspots and high GCx genes.

Immunolocalization on Whole Anther Chromosome Spreads for Male Meiosis.

Immunolocalization of cells undergoing meiosis has proven to be one of the most important tools to decipher chromatin-associated protein dynamics and causal relationships. Here, we describe a protocol established for maize which is easily adaptable to other plants, for example, with minor modifications to Arabidopsis as stated here. In contrast to many other protocols, the following protocol is based on fixation by a 3:1 mixture of ethanol and acetic acid. Spreading of cells is followed by freeze-shattering, protein antigenicity retrieval by a hot citrate buffer bath, antibody incubations and washes, and DNA staining.

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

Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and micro RNAs (miRNAs) directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolated male meiocytes from maize (Zea mays) to investigate sRNA and DNA methylation landscapes during zygotene, an early stage of meiosis during which steps of meiotic recombination and synapsis of paired homologous chromosomes take place. We discovered two novel miRNAs from meiocytes, zma-MIR11969 and zma-MIR11970, and identified putative target genes. Furthermore, we detected abundant phasiRNAs of 21 and 24 nt length. PhasiRNAs are phased small RNAs which occur in 21 or 24 nt intervals, at a few hundred loci, specifically in male reproductive tissues in grasses. So far, the function of phasiRNAs remained elusive. Data from isolated meiocytes now revealed elevated DNA methylation at phasiRNA loci, especially in the CHH context, suggesting a role for phasiRNAs in cis DNA methylation. In addition, we consider a role of these phasiRNAs in chromatin remodeling/dynamics during meiosis. However, this is not well supported yet and will need more additional data. Here, we only lay out the idea due to other relevant literature and our additional observation of a peculiar GC content pattern at phasiRNA loci. Chromatin remodeling is also indicated by the discovery that histone genes were enriched for sRNA of 22 nt length. Taken together, we gained clues that lead us to hypothesize sRNA-driven DNA methylation and possibly chromatin remodeling during male meiosis in the monocot maize which is in line with and extends previous knowledge.

The role of mitochondria in plant development and stress tolerance.

Eukaryotic cells require orchestrated communication between nuclear and organellar genomes, perturbations in which are linked to stress response and disease in both animals and plants. In addition to mitochondria, which are found across eukaryotes, plant cells contain a second organelle, the plastid. Signaling both among the organelles (cytoplasmic) and between the cytoplasm and the nucleus (i.e. nuclear-cytoplasmic interactions (NCI)) is essential for proper cellular function. A deeper understanding of NCI and its impact on development, stress response, and long-term health is needed in both animal and plant systems. Here we focus on the role of plant mitochondria in development and stress response. We compare and contrast features of plant and animal mitochondrial genomes (mtDNA), particularly highlighting the large and highly dynamic nature of plant mtDNA. Plant-based tools are powerful, yet underutilized, resources for enhancing our fundamental understanding of NCI. These tools also have great potential for improving crop production. Across taxa, mitochondria are most abundant in cells that have high energy or nutrient demands as well as at key developmental time points. Although plant mitochondria act as integrators of signals involved in both development and stress response pathways, little is known about plant mtDNA diversity and its impact on these processes. In humans, there are strong correlations between particular mitotypes (and mtDNA mutations) and developmental differences (or disease). We propose that future work in plants should focus on defining mitotypes more carefully and investigating their functional implications as well as improving techniques to facilitate this research.

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.