Daniel S. Jones

Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: Evidence for distinct sex-dependent chromatin and epigenetic states before fertilization

The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, whereas the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells and pollen vegetative cells from Oryza sativa (rice), and identified transcripts for approximately 36 000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three-quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in the expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions, and their contributions to zygote and seed development.

Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception

The Catharanthus roseus Receptor‐Like Kinase 1‐like (CrRLK1L) family of 17 receptor‐like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin‐like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co‐factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception.

Gene Family Analysis of the Arabidopsis NF-YA Transcription Factors Reveals Opposing Abscisic Acid Responses During Seed Germination

In the plant kingdom, each of the NUCLEAR FACTOR-Y (NF-Y) transcription factor families, NF-YA, NF-YB, and NF-YC, has undergone a great expansion compared to the animal kingdom. For example, Arabidopsis thaliana has 10 members of each gene family compared to only one in humans. Progress towards understanding the significance of this expansion is limited due to a lack of studies looking at the complete gene family during plant development. In the current study, transgenic overexpression lines were created for all 10 Arabidopsis NF-YA genes and examined for general development and alterations in abscisic acid (ABA)-mediated seed germination. NF-YA overexpression typically led to severe growth retardation and developmental defects, which extended from embryogenesis through to adult plants. Although overexpression of all NF-YA family members consistently led to growth retardation, some transgenic lines were hypersensitive to ABA during germination while others were hyposensitive. The opposing germination phenotypes were associated with the phylogenetic relationships between the NF-YA members. In addition, ABA marker genes were misregulated and ABA induction of gene expression was reduced in the overexpressors. Collectively, this study demonstrates that although NF-Ys have retained high degrees of similarity, they have evolved unique and sometimes opposing roles during plant development.

NUCLEAR FACTOR Y, subunit A (NF-YA) proteins positively regulate flowering and act through FLOWERING LOCUS T

Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal “CORE” elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.

The male germline of angiosperms: repertoire of an inconspicuous but important cell lineage

The male germline of flowering plants constitutes a specialized lineage of diminutive cells initiated by an asymmetric division of the initial microspore cell that sequesters the generative cell from the pollen vegetative cell. The generative cell subsequently divides to form the two male gametes (non-motile sperm cells) that fuse with the two female gametophyte target cells (egg and central cells) to form the zygote and endosperm. Although these male gametes can be as little as 1/800th of the volume of their female counterpart, they encode a highly distinctive and rich transcriptome, translate proteins, and display a novel suite of gamete-distinctive control elements that create a unique chromatin environment in the male lineage. Sperm-expressed transcripts also include a high proportion of transposable element-related sequences that may be targets of non-coding RNA including miRNA and silencing elements from peripheral cells. The number of sperm-encoded transcripts is somewhat fewer than the number present in the egg cell, but are remarkably distinct compared to other cell types according to principal component and other analyses. The molecular role of the male germ lineage cells is just beginning to be understood and appears more complex than originally anticipated.

The Zygotic Transition Is Initiated in Unicellular Plant Zygotes with Asymmetric Activation of Parental Genomes

The zygotic transition, from a fertilized egg to an embryo, is central to animal and plant reproduction. Animal embryos depend upon maternally provided factors until zygotic genome activation (ZGA). In plants, the timing and parental genome contributions to ZGA are unresolved. Here, we use the flowering plant Oryza sativa (rice) to characterize transcriptomes of time-staged isogenic and hybrid zygotes following fertilization. Large-scale transcriptomic changes were observed in unicellular zygotes, including upregulation of S-phase genes, a characteristic of ZGA. The parental contributions to ZGA were highly asymmetric. Zygotic transcription was primarily from the maternal genome and included genes for basic cellular processes. Transcription of the paternal genome was highly restricted but unexpectedly included genes encoding putative pluripotency factors expressed at the onset of ZGA. Thus, distinct transcriptional activities are exhibited by the parental genomes during the initiation of embryogenesis, which presumptively derive from divergent pre-zygotic transcriptional states established in the gametes.

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution

MLOs can substitute for NORTIA function in pollen tube reception if they localize to a Golgi compartment prior to pollen tube arrival and retain the ability to homooligomerize. Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana

In flowering plants, cell-cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane-bound proteins associated with the endomembrane system and the cell surface. While many synergid-expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane-bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co-localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI-anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane-associated trafficking within a haploid cell actively involved in polar transport.

Arabidopsis thaliana MLO genes are expressed in discrete domains during reproductive development

Key messageMLOs in Plant Reproduction.AbstractThe MILDEW RESISTANCE LOCUS-O (MLO) protein family, comprised of 15 members, plays roles in diverse cell–cell communication processes such as powdery mildew susceptibility, root thigmomorphogenesis, and pollen tube reception. The NORTIA (NTA, AtMLO7) gene is expressed in the synergid cells of the female gametophyte where it functions in intercellular communication with the pollen tube. Discrepancies between previously published promoter::GUS and promoter::gene-GUS constructs expression patterns led us to explore the regulation of NTA expression. Here we found via NTApro::gNTA-GUS truncations that sequences within the NTA gene negatively regulate its expression in the stomata and carpel walls. This led to the hypothesis that other MLO family members may also have additional regulatory sequences within the gene. MLOpro::gMLO-GUS constructs were examined for each family member focusing specifically on flowers in order to determine whether other MLOs could play a role in reproductive cell–cell communication. Notably, several MLOs were expressed in the pollen, in the stigma, in the pollinated style, and in the synergids and central cell. These findings indicate that other MLOs in addition to NTA could play a role in reproduction. Previous studies on the MLO family showed that phylogenetically related MLOs had redundant functions in powdery mildew infection and root thigmomorphogenesis; however, MLO expression in reproductive tissues did not strictly follow phylogenetic relationships, indicating that MLOs from different evolutionary origins may have been recruited for function in sexual reproduction.

Cell type-dependent localization of MLO proteins

ABSTRACT Mildew resistance locus O (MLO) proteins are transmembrane proteins that mediate cell-cell communication in plants. We recently demonstrated the importance of subcellular localization to MLO function during pollen tube reception. NORTIA (NTA), the MLO protein involved in this process, localizes to the Golgi of the synergid cell before interaction with the pollen tube. MLO proteins that can substitute for NTAs function in this pathway all partially localize with the same Golgi marker in the synergid cell. In this study, we report that MLO subcellular localization is cell type-dependent, with different distributions of some MLOs observed when ectopically expressed in the epidermal cells of tobacco and Arabidopsis compared to synergids. This dependency may be due to co-factors that influence MLO function within a given cell type, providing an intriguing new target for understanding MLO distribution and subsequent function in their respective processes.