Hybridization alters maternal and paternal genome contributions to early plant embryogenesis.

After fertilization, zygotic genome activation results in a transcriptionally competent embryo. Hybrid transcriptome experiments in Arabidopsis have concluded that the maternal and paternal genomes make equal contributions to zygotes and embryos, yet embryo defective (emb) mutants in the Columbia (Col) ecotype display early maternal effects. Here, we show that hybridization of Col with Landsberg erecta (Ler) or Cape Verde Islands (Cvi) ecotypes decreases the maternal effects of emb mutants. Reanalysis of Col/Ler and Col/Cvi transcriptomes confirmed equal parental contributions in Col/Cvi early embryos. By contrast, thousands of genes in Col/Ler zygotes and one-cell embryos were biallelic in one cross and monoallelic in the reciprocal cross, with analysis of intron reads pointing to active transcription as responsible for this parent-of-origin bias. Our analysis shows that, contrary to previous conclusions, the maternal and paternal genomes in Col/Ler zygotes are activated in an asymmetric manner. The decrease in maternal effects in hybrid embryos compared with those in isogenic Col along with differences in genome activation between Col/Cvi and Col/Ler suggest that neither of these hybrids accurately reflects the general trends of parent-of-origin regulation in Arabidopsis embryogenesis.

lncRNAs involved in the Shade Avoidance Syndrome (SAS) in Arabidopsis thaliana.

BACKGROUND: Plant long non-coding RNAs (lncRNAs) have important regulatory roles in responses to various biotic and abiotic stresses, including light quality. However, no lncRNAs have been specifically linked to the Shade Avoidance Response (SAS). RESULTS: To better understand the involvement of lncRNAs in shade avoidance, we examined RNA-seq libraries for lncRNAs with the potential to function in the neighbor proximity phenomenon in Arabidopsis thaliana (A. thaliana). Using transcriptomes generated from seedlings exposed to high and low red/far-red (R/FR) light conditions, we identified 13 lncRNA genes differentially expressed in cotyledons and 138 in hypocotyls. To infer possible functions for these lncRNAs, we used a 'guilt-by-association' approach to identify genes co-expressed with lncRNAs in a weighted gene co-expression network. Of 34 co-expression modules, 10 showed biological functions related to differential growth. We identified three potential lncRNAs co-regulated with genes related to SAS. T-DNA insertions in two of these lncRNAs were correlated with morphological differences in seedling responses to increased FR light, supporting our strategy for computational identification of lncRNAs involved in SAS. CONCLUSIONS: Using a computational approach, we identified multiple lncRNAs in Arabidopsis involved in SAS. T-DNA insertions caused altered phenotypes under low R/FR light, suggesting functional roles in shade avoidance. Further experiments are needed to determine the specific mechanisms of these lncRNAs in SAS.

Evolutionary divergence in embryo and seed coat development of U's Triangle Brassica species illustrated by a spatiotemporal transcriptome atlas.

The economically valuable Brassica species include the six related members of U's Triangle. Despite the agronomic and economic importance of these Brassicas, the impacts of evolution and relatively recent domestication events on the genetic landscape of seed development have not been comprehensively examined in these species. Here we present a 3D transcriptome atlas for the six species of U's Triangle, producing a unique resource that captures gene expression data for the major subcompartments of the seed, from the unfertilized ovule to the mature embryo and seed coat. This comprehensive dataset for seed development in tetraploid and ancestral diploid Brassicas provides new insights into evolutionary divergence and expression bias at the gene and subgenome levels during the domestication of these valued crop species. Comparisons of gene expression associated with regulatory networks and metabolic pathways operating in the embryo and seed coat during seed development reveal differences in storage reserve accumulation and fatty acid metabolism among the six Brassica species. This study illustrates the genetic underpinnings of seed traits and the selective pressures placed on seed production, providing an immense resource for continued investigation of Brassica polyploid biology, genomics and evolution.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Low nitrogen availability inhibits the phosphorus starvation response in maize (Zea mays ssp. mays L.).

BACKGROUND: Nitrogen (N) and phosphorus (P) are macronutrients essential for crop growth and productivity. In cultivated fields, N and P levels are rarely sufficient, contributing to the gap between realized and potential production. Fertilizer application increases nutrient availability, but is not available to all farmers, nor are current rates of application sustainable or environmentally desirable. Transcriptomic studies of cereal crops have revealed dramatic responses to either low N or low P single stress treatments. In the field, however, levels of both N and P may be suboptimal. The interaction between N and P starvation responses remains to be fully characterized. RESULTS: We characterized growth and root and leaf transcriptomes of young maize plants under nutrient replete, low N, low P or combined low NP conditions. We identified 1555 genes to respond to our nutrient treatments, in one or both tissues. A large group of genes, including many classical P starvation response genes, were regulated antagonistically between low N and P conditions. An additional experiment over a range of N availability indicated that a mild reduction in N levels was sufficient to repress the low P induction of P starvation genes. Although expression of P transporter genes was repressed under low N or low NP, we confirmed earlier reports of P hyper accumulation under N limitation. CONCLUSIONS: Transcriptional responses to low N or P were distinct, with few genes responding in a similar way to the two single stress treatments. In combined NP stress, the low N response dominated, and the P starvation response was largely suppressed. A mild reduction in N availability was sufficient to repress the induction of P starvation associated genes. We conclude that activation of the transcriptional response to P starvation in maize is contingent on N availability.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.

Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis.

Zygotic genome activation in metazoans typically occurs several hours to a day after fertilization, and thus maternal RNAs and proteins drive early animal embryo development. In plants, despite several molecular studies of post-fertilization transcriptional activation, the timing of zygotic genome activation remains a matter of debate. For example, two recent reports that used different hybrid ecotype combinations for RNA sequence profiling of early Arabidopsis embryo transcriptomes came to divergent conclusions. One identified paternal contributions that varied by gene, but with overall maternal dominance, while the other found that the maternal and paternal genomes are transcriptionally equivalent. Here we assess paternal gene activation functionally in an isogenic background, by performing a large-scale genetic analysis of 49 EMBRYO DEFECTIVE genes and testing the ability of wild-type paternal alleles to complement phenotypes conditioned by mutant maternal alleles. Our results demonstrate that wild-type paternal alleles for nine of these genes are completely functional 2 days after pollination, with the remaining 40 genes showing partial activity beginning at 2, 3 or 5 days after pollination. Using our functional assay, we also demonstrate that different hybrid combinations exhibit significant variation in paternal allele activation, reconciling the apparently contradictory results of previous transcriptional studies. The variation in timing of gene function that we observe confirms that paternal genome activation does not occur in one early discrete step, provides large-scale functional evidence that maternal and paternal genomes make non-equivalent contributions to early plant embryogenesis, and uncovers an unexpectedly profound effect of hybrid genetic backgrounds on paternal gene activity.

Convergent repression of miR156 by sugar and the CDK8 module of Arabidopsis Mediator.

In Arabidopsis, leaves produced during the juvenile vegetative phase are simple, while adult leaves are morphologically complex. The juvenile to adult transition is regulated by miR156, a microRNA that promotes juvenility by impeding the function of SPL transcription factors, which specify adult leaf traits. Both leaf derived sugars, as well as the Mediator Cyclin Dependent Kinase 8 (CDK8) module genes CENTER CITY (CCT)/MED12 and GRAND CENTRAL (GCT)/MED13, act upstream of miR156 to promote the juvenile to adult transition. However, it is not known whether sugar, CCT and GCT repress miR156 independently, as part of the same pathway, or in a cooperative manner. Here we show that sugar treatment can repress MIR156 expression in the absence of CCT or GCT. Both cct and the photosynthetic mutant chlorina1 (ch1) (which decreases sugar synthesis) exhibit extended juvenile development and increased MIR156A and MIR156C expression. Compared to ch1 and cct single mutants, the ch1 cct double mutant has a stronger effect on juvenile leaf traits, higher MIR156C levels, and a dramatic increase in MIR156A. Our results show that sugar and the CDK8 module are capable of regulating MIR156 independently, but suggest they normally act together in a synergistic manner.

Arabidopsis thaliana miRNAs promote embryo pattern formation beginning in the zygote.

miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.

Mediator: A key regulator of plant development.

Mediator is a multiprotein complex that regulates transcription at the level of RNA pol II assembly, as well as through regulation of chromatin architecture, RNA processing and recruitment of epigenetic marks. Though its modular structure is conserved in eukaryotes, its subunit composition has diverged during evolution and varies in response to environmental and tissue-specific inputs, suggesting different functions for each subunit and/or Mediator conformation. In animals, Mediator has been implicated in the control of differentiation and morphogenesis through modulation of numerous signaling pathways. In plants, studies have revealed roles for Mediator in regulation of cell division, cell fate and organogenesis, as well as developmental timing and hormone responses. We begin this review with an overview of biochemical mechanisms of yeast and animal Mediator that are likely to be conserved in all eukaryotes, as well as a brief discussion of the role of Mediator in animal development. We then present a comprehensive review of studies of the role of Mediator in plant development. Finally, we point to important questions for future research on the role of Mediator as a master coordinator of development.

The Arabidopsis Mediator CDK8 module genes CCT (MED12) and GCT (MED13) are global regulators of developmental phase transitions.

Temporal coordination of developmental programs is necessary for normal ontogeny, but the mechanism by which this is accomplished is still poorly understood. We have previously shown that two components of the Mediator CDK8 module encoded by CENTER CITY (CCT; Arabidopsis MED12) and GRAND CENTRAL (GCT; Arabidopsis MED13) are required for timing of pattern formation during embryogenesis. A morphological, molecular and genomic analysis of the post-embryonic phenotype of gct and cct mutants demonstrated that these genes also promote at least three subsequent developmental transitions: germination, vegetative phase change, and flowering. Genetic and molecular analyses indicate that GCT and CCT operate in parallel to gibberellic acid, a phytohormone known to regulate these same three transitions. We demonstrate that the delay in vegetative phase change in gct and cct is largely due to overexpression of miR156, and that the delay in flowering is due in part to upregulation of FLC. Thus, GCT and CCT coordinate vegetative and floral transitions by repressing the repressors miR156 and FLC. Our results suggest that MED12 and MED13 act as global regulators of developmental timing by fine-tuning the expression of temporal regulatory genes.

Mutations in two non-canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects.

SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well-studied BRAHMA and SPLAYED ATPases, as well as two closely related non-canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication-coupled chromatin assembly are discussed.

The MED12-MED13 module of Mediator regulates the timing of embryo patterning in Arabidopsis.

The Arabidopsis embryo becomes patterned into central and peripheral domains during the first few days after fertilization. A screen for mutants that affect this process identified two genes, GRAND CENTRAL (GCT)and CENTER CITY (CCT). Mutations in GCT and CCT delay the specification of central and peripheral identity and the globular-to-heart transition, but have little or no effect on the initial growth rate of the embryo. Mutant embryos eventually recover and undergo relatively normal patterning, albeit at an inappropriate size. GCT and CCT were identified as the Arabidopsis orthologs of MED13 and MED12 - evolutionarily conserved proteins that act in association with the Mediator complex to negatively regulate transcription. The predicted function of these proteins combined with the effect of gct and cct on embryo development suggests that MED13 and MED12 regulate pattern formation during Arabidopsis embryogenesis by transiently repressing a transcriptional program that interferes with this process. Their mutant phenotype reveals the existence of a previously unknown temporal regulatory mechanism in plant embryogenesis.

Analysis of Global Gene Expression in Maize (Zea mays) Vegetative and Reproductive Tissues That Differ in Accumulation of Starch and Sucrose.

Carbon allocation between vegetative and reproductive tissues impacts cereal grain production. Despite great agricultural importance, sink-source relationships have not been fully characterized at the early reproductive stages in maize. Here, we quantify the accumulation of non-structural carbohydrates and patterns of gene expression in the top internode of the stem and the female inflorescence of maize at the onset of grain filling (reproductive stage R1). Top internode stem and female inflorescence tissues of the Puma maize inbred line were collected at reproductive stage R1 (without pollination) and non-structural carbohydrates were quantified by spectrophotometry. The female inflorescence accumulated starch at higher levels than the top internode of the stem. Global mRNA transcript levels were then evaluated in both tissues by RNA sequencing. Gene expression analysis identified 491 genes differentially expressed between the female inflorescence and the top stem internode. Gene ontology classification of differentially expressed genes showed enrichment for sucrose synthesis, the light-dependent reactions of photosynthesis, and transmembrane transporters. Our results suggest that sugar transporters play a key role in sugar partitioning in the maize stem and reveal previously uncharacterized differences between the female inflorescence and the top internode of the stem at early reproductive stages.

The pho1;2a'-m1.1 allele of Phosphate1 conditions misregulation of the phosphorus starvation response in maize (Zea mays ssp. mays L.).

Plant PHO1 proteins play a central role in the translocation and sensing of inorganic phosphate. The maize (Zea mays ssp. mays) genome encodes two co-orthologs of the Arabidopsis PHO1 gene, designated ZmPho1;2a and ZmPho1;2b. Here, we report the characterization of the transposon footprint allele Zmpho1;2a'-m1.1, which we refer to hereafter as pho1;2a. The pho1;2a allele is a stable derivative formed by excision of an Activator transposable element from the ZmPho1;2a gene. The pho1;2a allele contains an 8-bp insertion at the point of transposon excision that disrupts the reading frame and is predicted to generate a premature translational stop. We show that the pho1;2a allele is linked to a dosage-dependent reduction in Pho1;2a transcript accumulation and a mild reduction in seedling growth. Characterization of shoot and root transcriptomes under full nutrient, low nitrogen, low phosphorus, and combined low nitrogen and low phosphorus conditions identified 1100 differentially expressed genes between wild-type plants and plants carrying the pho1;2a mutation. Of these 1100 genes, 966 were upregulated in plants carrying pho1;2a, indicating the wild-type PHO1;2a to predominantly impact negative gene regulation. Gene set enrichment analysis of the pho1;2a-misregulated genes revealed associations with phytohormone signaling and the phosphate starvation response. In roots, differential expression was broadly consistent across all nutrient conditions. In leaves, differential expression was largely specific to low phosphorus and combined low nitrogen and low phosphorus conditions. Of 276 genes upregulated in the leaves of pho1;2a mutants in the low phosphorus condition, 153 were themselves induced in wild-type plants with respect to the full nutrient condition. Our observations suggest that Pho1;2a functions in the fine-tuning of the transcriptional response to phosphate starvation through maintenance and/or sensing of plant phosphate status.

Asymmetric gene expression in grain development of reciprocal crosses between tetraploid and hexaploid wheats.

Production of viable progeny from interploid crosses requires precise regulation of gene expression from maternal and paternal chromosomes, yet the transcripts contributed to hybrid seeds from polyploid parent species have rarely been explored. To investigate the genome-wide maternal and paternal contributions to polyploid grain development, we analyzed the transcriptomes of developing embryos, from zygote to maturity, alongside endosperm in two stages of development, using reciprocal crosses between tetraploid and hexaploid wheats. Reciprocal crosses between species with varied levels of ploidy displayed broad impacts on gene expression, including shifts in alternative splicing events in select crosses, as illustrated by active splicing events, enhanced protein synthesis and chromatin remodeling. Homoeologous gene expression was repressed on the univalent D genome in pentaploids, but this suppression was attenuated in crosses with a higher ploidy maternal parent. Imprinted genes were identified in endosperm and early embryo tissues, supporting predominant maternal effects on early embryogenesis. By systematically investigating the complex transcriptional networks in reciprocal-cross hybrids, this study presents a framework for understanding the genomic incompatibility and transcriptome shock that results from interspecific hybridization and uncovers the transcriptional impacts on hybrid seeds created from agriculturally-relevant polyploid species.

Developmental and genomic architecture of plant embryogenesis: from model plant to crops.

Embryonic development represents an important reproductive phase of sexually reproducing plant species. The fusion of egg and sperm produces the plant zygote, a totipotent cell that, through cell division and cell identity specification in early embryogenesis, establishes the major cell lineages and tissues of the adult plant. The subsequent morphogenesis phase produces the full-sized embryo, while the late embryogenesis maturation process prepares the seed for dormancy and subsequent germination, ensuring continuation of the plant life cycle. In this review on embryogenesis, we compare the model eudicot Arabidopsis thaliana with monocot crops, focusing on genome activation, paternal and maternal regulation of early zygote development, and key organizers of patterning, such as auxin and WOX transcription factors. While the early stages of embryo development are apparently conserved among plant species, embryo maturation programs have diversified between eudicots and monocots. This diversification in crop species reflects the likely effects of domestication on seed quality traits that are determined during embryo maturation, and also assures seed germination in different environmental conditions. This review describes the most important features of embryonic development in plants, and the scope and applications of genomics in plant embryo studies.

Identification of the maize Mediator CDK8 module and transposon-mediated mutagenesis of ZmMed12a.

Mediator is a conserved transcriptional co-activator that links transcription factors bound at enhancer elements to RNA Polymerase II. Mediator-RNA Polymerase II interactions can be sterically hindered by the Cyclin Dependent Kinase 8 (CDK8) module, a submodule of Mediator that acts to repress transcription in response to discrete cellular and environmental cues. The CDK8 module is conserved in all eukaryotes and consists of 4 proteins: CDK8, CYCLIN C (CYCC), MED12, and MED13. In this study, we have characterized the CDK8 module of Mediator in maize using genomic, molecular and functional resources. The maize genome contains single copy genes for Cdk8, CycC, and Med13, and two genes for Med12. Analysis of expression data for the CDK8 module demonstrated that all five genes are broadly expressed in maize tissues, and change their expression in response to phosphate and nitrogen limitation. We performed Dissociation (Ds) insertional mutagenesis, recovering two independent insertions in the ZmMed12a gene, one of which produces a truncated transcript. Our molecular identification of the maize CDK8 module, assays of CDK8 module expression under nutrient limitation, and characterization of transposon insertions in ZmMed12a establish the basis for molecular and functional studies of the role of these important transcriptional regulators in development and nutrient homeostasis in Zea mays.

EMS Mutagenesis of Arabidopsis Seeds.

The ethylating agent ethyl methanesulfonate (EMS) is widely used for inducing random point mutations. In Arabidopsis, treatment with EMS causes GC-to-AT transitions with great efficiency: it has been estimated that a population of 50,000 well-mutagenized plants harbors one or more transitions in almost every GC pair of the genome. These properties, combined with ease of use, make EMS a mutagen of choice for genetic screens. Here, we describe a protocol for mutagenizing Arabidopsis seed with EMS. In addition, we briefly consider the germ line sectors typically induced by this treatment, and approaches for estimating the rate of induced mutations.

Genetic Screens to Target Embryo and Endosperm Pathways in Arabidopsis and Maize.

The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet. Arabidopsis and maize have evolved different types of seeds, opening a range of experimental opportunities. Development of the Arabidopsis embryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: in Arabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments.