The roles of a novel CDKB/KRP/FB3 cell cycle core complex in rice gametes and initiation of embryogenesis.

The cell cycle controls division and proliferation of all eukaryotic cells and is tightly regulated at multiple checkpoints by complexes of core cell cycle proteins. Due to the difficulty in accessing female gametes and zygotes of flowering plants, little is known about the molecular mechanisms underlying embryogenesis initiation despite the crucial importance of this process for seed crops. In this study, we reveal three levels of factors involved in rice zygotic cell cycle control and characterize their functions and regulation. Protein-protein interaction studies, including within zygote cells, and in vitro biochemical analyses delineate a model of the zygotic cell cycle core complex for rice. In this model, CDKB1, a major regulator of plant mitosis, is a cyclin (CYCD5)-dependent kinase; its activity is coordinately inhibited by two cell cycle inhibitors, KRP4 and KRP5; and both KRPs are regulated via F-box protein 3 (FB3)-mediated proteolysis. Supporting their critical roles in controlling the rice zygotic cell cycle, mutations in KRP4, KRP5 and FB3 result in the compromised function of sperm cells and abnormal organization of female germ units, embryo and endosperm, thus significantly reducing seed-set rate. This work helps reveal regulatory mechanisms controlling the zygotic cell cycle toward seed formation in angiosperms.

Somatic embryo initiation by rice BABY BOOM1 involves activation of zygote-expressed auxin biosynthesis genes.

Plant embryogenesis results from the fusion of male and female gametes but can also be induced in somatic cells. The molecular pathways for embryo initiation are poorly understood, especially in monocots. In rice, the male gamete expressed BABY BOOM1 (OsBBM1) transcription factor functions as an embryogenic trigger in the zygote and can also promote somatic embryogenesis when ectopically expressed in somatic tissues. We used gene editing, transcriptome profiling, and chromatin immunoprecipitation to determine the molecular players involved in embryo initiation downstream of OsBBM1. We identify OsYUCCA (OsYUC) auxin biosynthesis genes as direct targets of OsBBM1. Unexpectedly, these OsYUC targets in zygotes are expressed only from the maternal genome, whereas the paternal genome exclusively provides functional OsBBM1 to initiate embryogenesis. Induction of somatic embryogenesis by exogenous auxin requires OsBBM genes and downstream OsYUC targets. Ectopic OsBBM1 initiates somatic embryogenesis without exogenous auxins but requires functional OsYUC genes. Thus, an OsBBM-OsYUC module is a key player for both somatic and zygotic embryogenesis in rice. Zygotic embryo initiation involves a partnership of male and female genomes, through which paternal OsBBM1 activates maternal OsYUC genes. In somatic embryogenesis, exogenous auxin triggers OsBBM1 expression, which then activates endogenous auxin biosynthesis OsYUC genes.

High-frequency synthetic apomixis in hybrid rice.

Introducing asexual reproduction through seeds - apomixis - into crop species could revolutionize agriculture by allowing F1 hybrids with enhanced yield and stability to be clonally propagated. Engineering synthetic apomixis has proven feasible in inbred rice through the inactivation of three genes (MiMe), which results in the conversion of meiosis into mitosis in a line ectopically expressing the BABYBOOM1 (BBM1) parthenogenetic trigger in egg cells. However, only 10-30% of the seeds are clonal. Here, we show that synthetic apomixis can be achieved in an F1 hybrid of rice by inducing MiMe mutations and egg cell expression of BBM1 in a single step. We generate hybrid plants that produce more than 95% of clonal seeds across multiple generations. Clonal apomictic plants maintain the phenotype of the F1 hybrid along successive generations. Our results demonstrate that there is no barrier to almost fully penetrant synthetic apomixis in an important crop species, rendering it compatible with use in agriculture.

Acquisition of a complex root microbiome reshapes the transcriptomes of rice plants.

Soil microorganisms can colonize plant roots and assemble in communities engaged in symbiotic relationships with their host. Though the compositional dynamics of root-associated microbiomes have been extensively studied, the host transcriptional response to these communities is poorly understood. Here, we developed an experimental system by which rice plants grown under axenic conditions can acquire a defined endosphere microbiome. Using this setup, we performed a cross-sectional characterization of plant transcriptomes in the presence or absence of a complex microbial community. To account for compositional variation, plants were inoculated with soil-derived microbiomes harvested from three distinct agricultural sites. Soil microbiomes triggered a major shift in the transcriptional profiles of rice plants that included the downregulation of one-third to one-fourth of the families of leucine-rich repeat receptor-like kinases and nucleotide-binding leucine-rich repeat receptors expressed in roots. Though the expression of several genes was consistent across all soil sources, a large fraction of this response was differentially impacted by soil type. These results demonstrate the role of root microbiomes in sculpting the transcriptomes of host plants and highlight the potential involvement of the two main receptor families of the plant immune system in the recruitment and maintenance of an endosphere microbiome.

Resetting of the 24-nt siRNA landscape in rice zygotes.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells-the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

Prolonged drought imparts lasting compositional changes to the rice root microbiome.

Microbial symbioses can mitigate drought stress in crops but harnessing these beneficial interactions will require an in-depth understanding of root microbiome responses to drought cycles. Here, by detailed temporal characterization of root-associated microbiomes of rice plants during drought stress and recovery, we find that endosphere communities remained compositionally altered after rewatering, with prolonged droughts leading to decreased resilience. Several endospheric Actinobacteria were significantly enriched during drought and for weeks after rewatering. Notably, the most abundant endosphere taxon during this period was a Streptomyces, and a corresponding isolate promoted root growth. Additionally, drought stress disrupted the temporal dynamics of late-colonizing microorganisms, permanently altering the normal successional trends of root microbiota. These findings reveal that severe drought results in enduring impacts on rice root microbiomes, including enrichment of taxonomic groups that could shape the recovery response of the host, and have implications relevant to drought protection strategies using root microbiota.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Bioactive diterpenoids impact the composition of the root-associated microbiome in maize (Zea mays).

Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize (Zea mays) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 (anther ear 2), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.

Plant zygote development: recent insights and applications to clonal seeds.

In flowering plants, haploid gametes - an egg cell and a sperm cell fuse to form the first diploid cell - the zygote. The zygote is the progenitor stem cell that gives rise to all the embryonic and post embryonic tissues and organs. Unlike animals, both maternal and paternal gene products participate in the initial development of zygotes in plants. Here, we discuss recent advances in understanding of the zygotic transition and embryo initiation in angiosperms, including the role of parental contributions to gene expression in the zygote. We further discuss utilization of this knowledge in agricultural biotechnology through synthetic apomixis. Parthenogenesis obtained by manipulation of embryogenic factors, combined with mutations that bypass meiosis, enables clonal propagation of hybrid crops through seeds.

Coordinated Activation of ARF1 GTPases by ARF-GEF GNOM Dimers Is Essential for Vesicle Trafficking in Arabidopsis.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

Comparative Analysis of Root Microbiomes of Rice Cultivars with High and Low Methane Emissions Reveals Differences in Abundance of Methanogenic Archaea and Putative Upstream Fermenters.

Rice cultivation worldwide accounts for ∼7 to 17% of global methane emissions. Methane cycling in rice paddies is a microbial process not only involving methane producers (methanogens) and methane metabolizers (methanotrophs) but also other microbial taxa that affect upstream processes related to methane metabolism. Rice cultivars vary in their rates of methane emissions, but the influence of rice genotypes on methane cycling microbiota has been poorly characterized. Here, we profiled the rhizosphere, rhizoplane, and endosphere microbiomes of a high-methane-emitting cultivar (Sabine) and a low-methane-emitting cultivar (CLXL745) throughout the growing season to identify variations in the archaeal and bacterial communities relating to methane emissions. The rhizosphere of the high-emitting cultivar was enriched in methanogens compared to that in the low emitter, whereas the relative abundances of methanotrophs between the cultivars were not significantly different. Further analysis of cultivar-sensitive taxa identified families enriched in the high emitter that are associated with methanogenesis-related processes. The high emitter had greater relative abundances of sulfate-reducing and iron-reducing taxa which peak earlier in the season than methanogens and are necessary to lower soil oxidation reduction potential before methanogenesis can occur. The high emitter also had a greater abundance of fermentative taxa which produce methanogenesis precursors (acetate, CO2, and H2). Furthermore, the high emitter was enriched in taxa related to acetogenesis which compete with methanogens for CO2 and H2 These taxa were enriched in a spatio-specific manner and reveal a complex network of microbial interactions on which plant genotype-dependent factors can act to affect methanogenesis and methane emissions.IMPORTANCE Rice cultivation is a major source of anthropogenic emissions of methane, a greenhouse gas with a potentially severe impact on climate change. Emission variation between rice cultivars suggests the feasibility of breeding low-emission rice, but there is a limited understanding of how genotypes affect the microbiota involved in methane cycling. Here, we show that the root microbiome of the high-emitting cultivar is enriched both in methanogens and in taxa associated with fermentation, iron, and sulfate reduction and acetogenesis, processes that support methanogenesis. Understanding how cultivars affect microbes with methanogenesis-related functions is vital for understanding the genetic basis for methane emission in rice and can aid in the development of breeding programs that reduce the environmental impact of rice cultivation.

Genome-wide redistribution of 24-nt siRNAs in rice gametes.

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

Soil domestication by rice cultivation results in plant-soil feedback through shifts in soil microbiota.

BACKGROUND: Soils are a key component of agricultural productivity, and soil microbiota determine the availability of many essential plant nutrients. Agricultural domestication of soils, that is, the conversion of previously uncultivated soils to a cultivated state, is frequently accompanied by intensive monoculture, especially in the developing world. However, there is limited understanding of how continuous cultivation alters the structure of prokaryotic soil microbiota after soil domestication, including to what extent crop plants impact soil microbiota composition, and how changes in microbiota composition arising from cultivation affect crop performance. RESULTS: We show here that continuous monoculture (> 8 growing seasons) of the major food crop rice under flooded conditions is associated with a pronounced shift in soil bacterial and archaeal microbiota structure towards a more consistent composition, thereby domesticating microbiota of previously uncultivated sites. Aside from the potential effects of agricultural cultivation practices, we provide evidence that rice plants themselves are important drivers of the domestication process, acting through selective enrichment of specific taxa, including methanogenic archaea, in their rhizosphere that differ from those of native plants growing in the same environment. Furthermore, we find that microbiota from soils domesticated by rice cultivation contribute to plant-soil feedback, by imparting a negative effect on rice seedling vigor. CONCLUSIONS: Soil domestication through continuous monoculture cultivation of rice results in compositional changes in the soil microbiota, which are in part driven by the rice plants. The consequences include a negative impact on plant performance and increases in greenhouse gas emitting microbes.

[Screening and identification of CKI1 upstream transcription regulators in Arabidopsis].

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.

Step-by-step protocols for rice gamete isolation.

KEY MESSAGE: A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented. Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.

A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.

The molecular pathways that trigger the initiation of embryogenesis after fertilization in flowering plants, and prevent its occurrence without fertilization, are not well understood1. Here we show in rice (Oryza sativa) that BABY BOOM1 (BBM1), a member of the AP2 family2 of transcription factors that is expressed in sperm cells, has a key role in this process. Ectopic expression of BBM1 in the egg cell is sufficient for parthenogenesis, which indicates that a single wild-type gene can bypass the fertilization checkpoint in the female gamete. Zygotic expression of BBM1 is initially specific to the male allele but is subsequently biparental, and this is consistent with its observed auto-activation. Triple knockout of the genes BBM1, BBM2 and BBM3 causes embryo arrest and abortion, which are fully rescued by male-transmitted BBM1. These findings suggest that the requirement for fertilization in embryogenesis is mediated by male-genome transmission of pluripotency factors. When genome editing to substitute mitosis for meiosis (MiMe)3,4 is combined with the expression of BBM1 in the egg cell, clonal progeny can be obtained that retain genome-wide parental heterozygosity. The synthetic asexual-propagation trait is heritable through multiple generations of clones. Hybrid crops provide increased yields that cannot be maintained by their progeny owing to genetic segregation. This work establishes the feasibility of asexual reproduction in crops, and could enable the maintenance of hybrids clonally through seed propagation5,6.

Recent advances in understanding female gametophyte development.

The haploid female gametophyte (embryo sac) is an essential reproductive unit of flowering plants, usually comprising four specialized cell types, including the female gametes (egg cell and central cell). The differentiation of these cells relies on spatial signals which pattern the gametophyte along a proximal-distal axis, but the molecular and genetic mechanisms by which cell identities are determined in the embryo sac have long been a mystery. Recent identification of key genes for cell fate specification and their relationship to hormonal signaling pathways that act on positional cues has provided new insights into these processes. A model for differentiation can be devised with egg cell fate as a default state of the female gametophyte and with other cell types specified by the action of spatially regulated factors. Cell-to-cell communication within the gametophyte is also important for maintaining cell identity as well as facilitating fertilization of the female gametes by the male gametes (sperm cells).

Reproductive Long Intergenic Noncoding RNAs Exhibit Male Gamete Specificity and Polycomb Repressive Complex 2-Mediated Repression.

Long noncoding RNAs (lncRNAs) have been characterized extensively in animals and are involved in several processes, including homeobox gene expression and X-chromosome inactivation. In comparison, there has been much less detailed characterization of plant lncRNAs, and the number of distinct lncRNAs encoded in plant genomes and their regulation by developmental and epigenetic mechanisms remain largely unknown. Here, we analyzed transcriptome data from Asian rice (Oryza sativa) and identified 6,309 long intergenic noncoding RNAs (lincRNAs), focusing on their expression in reproductive tissues and organs. Most O. sativa lincRNAs were expressed in a highly tissue-specific manner, with an unexpectedly high fraction specifically expressed in male gametes. Mutation of a component of the Polycomb Repressive Complex2 (PRC2) resulted in derepression of another large class of lincRNAs, whose expression is correlated with H3K27 trimethylation in developing panicles. Overlap with the sperm cell-specific lincRNAs suggests that epigenetic repression of lincRNAs in the panicles was partially relieved in the male germline. Expression of a subset of lincRNAs also showed modulation by drought in reproductive tissues. Comparison with other cereal genomes showed that the lincRNAs generally have low levels of conservation at both the sequence and structural levels. Use of a novelty detection support vector machine model enabled the detection of nucleotide sequence and structural homology in ∼10% and ∼4% of the lincRNAs in genomes of purple false brome (Brachypodium distachyon) and maize (Zea mays), respectively. This is the first study to report on a large number of lncRNAs that are targets of repression by PRC2 rather than mediating regulation via PRC2. That the vast majority of the lincRNAs reported here do not overlap with those of other rice studies indicates that these are a significant addition to the known lincRNAs in rice.

The gymnosperm ortholog of the angiosperm central cell-specification gene CKI1 provides an essential clue to endosperm origin.

A defining feature of angiosperms is double fertilization involving the female gametophyte central cell and formation of a nutrient-storing tissue called endosperm. The route for the evolutionary origin of endosperm from a gymnosperm ancestor, particularly the molecular steps involved, has remained elusive. Recently, the histidine kinase gene Cytokinin-Independent 1 (CKI1), an activator of cytokinin signaling, was described as a key to specification of the endosperm precursor central cell in Arabidopsis. Here, we have investigated the function and expression of a putative ortholog of CKI1 in the gymnosperm Ginkgo biloba. We demonstrate that Ginkgo CKI1 can partially rescue an Arabidopsis cki1 mutant and promote weak activation of the cytokinin signaling pathway in the Arabidopsis embryo sac, but does not confer central cell specification. Ginkgo CKI1 is expressed in both male and female gametophytes of Ginkgo. In the latter, it is expressed in the ventral canal cell, which is sister to the egg cell in the archegonium. As in Arabidopsis, Ginkgo CKI1 is not expressed in the egg cell. The similarities in expression patterns of CKI1 in Ginkgo and Arabidopsis female gametophytes suggest that extant gymnosperms possess an essential component of the molecular machinery required for angiosperm endosperm development, and provide new insights into endosperm origin from a gymnospermous ancestor.