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Ovules of higher plants are the precursors of seeds. Ovules emerge from placental tissue inside the gynoecium of flowers. Three elements, funiculus, chalaza, and nucellus, can be distinguished along the proximal–distal axis of the outgrowing radially symmetrical ovule primordium. The asymmetric initiation of the outer integument marks the switch to adaxial–abaxial development, which leads to the formation of a bilaterally symmetrical ovule. The putative transcriptional regulator NOZZLE ( NZZ ) plays a role in mediating this transition by controlling the timing of expression of the putative transcriptional regulator INNER NO OUTER ( INO ) in an abaxial domain of the chalaza, from where the outer integument initiates. Integument formation depends on the homeobox gene WUSCHEL ( WUS ), which is expressed in the nucellus and is sufficient to induce integuments non-cell autonomously from a region adjacent to its expression domain. In this study, we describe the expression pattern of the homeobox-leucine zipper gene PHABULOSA ( PHB ) during ovule development, demonstrating that adaxial–abaxial polarity is established from the very beginning of ovule development. Furthermore, we examined the expression pattern of PHB , INO , and WUS in ovules of plants, which are affected in integument initiation and thus defective in the transition from proximal–distal to adaxial–abaxial development. We found that NZZ is required to restrict PHB expression to the distal chalaza, from where the inner integument initiates. PHB expression is not established in the distal chalaza of two mutants, aintegumenta ( ant ) and wus , which fail to form integuments. Furthermore, we suggest that one mechanism by which WUS controls integument formation is by establishing the chalaza and that outer and inner integument identity determination depends on additional region-specific factors. In addition, we present evidence that NZZ is essential for the normal nucellar expression pattern of WUS . Thus, both WUS and PHB affect processes downstream of NZZ action during the transition from proximal–distal to adaxial–abaxial ovule development.

Somatic polyploidization is recognized as a means to increase gene expression levels in highly active metabolic cells. The most common mechanisms are endoreplication, endomitosis and cell fusion. In animals and plants the nuclei of multinucleate cells are usually prevented from fusing. Here, we report that the nuclei from the syncytial cyst of the chalazal endosperm of Arabidopsis thaliana (L.) Heynh. are polyploid with some intermediate ploidy levels that cannot be attributed to endoreplication, suggesting nuclear fusion. Analysis of isolated nuclei, together with fluorescent in situ hybridization (FISH), revealed that nuclei from the chalazal endosperm are two or three times bigger than the nuclei from the peripheral endosperm and have a corresponding increase in ploidy. Together with the consistent observation of adjoined nuclei, we propose that nuclear fusion contributes, at least in part, to the process of polyploidization in the chalazal endosperm. Confocal analysis of intact seeds further suggested that free nuclei from the peripheral endosperm get incorporated into the chalazal cyst and likely participate in nuclear fusions.

The life cycle of flowering plants alternates between a diploid sporophytic and a haploid gametophytic generation. After fertilization of each the egg and central cells by one male gamete, the development of both fertilization products occurs coordinated with the maternally derived seed coat and carpel tissues forming the fruit. The reproduction program is likely to involve the concerted activity of many genes. To identify genes with specific functions during reproduction, we have analyzed the expression profile of more than 22,000 genes present on the Arabidopsis ATH1 microarray during three stages of flower and fruit development. We found 1,886 genes regulated during reproductive development and 1,043 genes that were specifically expressed during reproduction. When compared to cells from an Arabidopsis suspension culture, S-phase genes were underrepresented and G2 and M-phase genes were strongly enriched in the set of specific genes, indicating that important functions during reproduction are exerted in the G2 and M phases of the cell cycle. Many potential signaling components, such as receptor-like protein kinases, phosphatases, and transcription factors, were present in both groups of genes. Members of the YABBY, MADS box, and Myb transcription factor families were significantly overrepresented in the group of specific genes, revealing an important role of these families during reproduction. Furthermore, we found a significant enrichment of predicted secreted proteins smaller than 15 kD that could function directly as signaling molecules or as precursors for peptide hormones. Our study provides a basis for targeted reverse-genetic approaches aimed to identify key genes of reproductive development in plants.

Transposon activity is known to cause chromosome rearrangements in the host genome. Surprisingly, extremely little is known about Dissociation ( Ds )-induced chromosome rearrangements in Arabidopsis , where Ds is intensively used for insertional mutagenesis. Here, we describe three Arabidopsis mutants with reduced fertility and propose that excision of a hybrid Ds element induced a large genomic deletion flanking Ds . In the mutants anat and haumea , the deletion mechanism consists of a local Ds transposition from replicated into unreplicated DNA followed by Ds excision, where one end of the newly transposed element and one end of the Ds transposon at the donor site served as substrate for transposase. Excision of this hybrid element reminiscent of a macrotransposon leads to loss of the chromosomal piece located between the two ends, including one full Ds element and the flanking genomic sequence. This mechanism was found to be responsible for several other deletions and occurs at a genetically trackable frequency. Thus, it could be applied to efficiently generate deletions of various sizes in the vicinity of any existing Ds element present in the genome. In the mutant tons missing , a mechanism that involves endogenous repetitive sequences caused a large flanking deletion at a position unlinked to the starter locus. Our study of Ds transposition in Arabidopsis revealed previously undescribed mechanisms that lead to large genomic deletions flanking Ds elements, which may contribute to genome dynamics and evolution.

Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for anchoring proteins to the cell surface in eukaryotes. GPI anchors are synthesized in the endoplasmic reticulum via the sequential addition of monosaccharides, fatty acids, and phosphoethanolamines to phosphatidylinositol. Deficiencies in GPI biosynthesis lead to embryonic lethality in animals and to conditional lethality in eukaryotic microbes by blocking cell growth, cell division, or morphogenesis. We report the genetic and phenotypic analysis of insertional mutations disrupting SETH1 and SETH2, which encode Arabidopsis homologs of two conserved proteins involved in the first step of the GPI biosynthetic pathway. seth1 and seth2 mutations specifically block male transmission and pollen function. This results from reduced pollen germination and tube growth, which are associated with abnormal callose deposition. This finding suggests an essential role for GPI anchor biosynthesis in pollen tube wall deposition or metabolism. Using transcriptomic and proteomic approaches, we identified 47 genes that encode potential GPI-anchored proteins that are expressed in pollen and demonstrated that at least 11 of these proteins are associated with pollen membranes by GPI anchoring. Many of the identified candidate proteins are homologous with proteins involved in cell wall synthesis and remodeling or intercellular signaling and adhesion, and they likely play important roles in the establishment and maintenance of polarized pollen tube growth.

Seed development in angiosperms initiates after double fertilization, leading to the formation of a diploid embryo and a triploid endosperm. The active repression of precocious initiation of certain aspects of seed development in the absence of fertilization requires the Polycomb group proteins MEDEA (MEA), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and FERTILIZATION-INDEPENDENT SEED2. Here we show that the Arabidopsis WD-40 domain protein MSI1 is present together with MEA and FIE in a 600 kDa complex and interacts directly with FIE. Mutant plants heterozygous for msi1 show a seed abortion ratio of 50% with seeds aborting when the mutant allele is maternally inherited, irrespective of a paternal wild-type or mutant MSI1 allele. Further more, msi1 mutant gametophytes initiate endosperm development in the absence of fertilization at a high penetrance. After pollination, only the egg cell becomes fertilized, the central cell starts dividing prior to fertilization, resulting in the formation of seeds containing embryos surrounded by diploid endosperm. Our results establish that MSI1 has an essential function in the correct initiation and progression of seed development.

To identify genes that play an important gametophytic role during pollen development, an Arabidopsis transposon (DsE) mutagenised population was screened for marker segregation ratio distortion. We report the characterisation of a male gametophytic mutant termed halfman (ham) that results in an aborted pollen phenotype in mature anthers. The genetic transmission efficiency of DsE was 6.6% through the male and 98.4% through the female, which suggested that HAM may encode essential male-specific component(s) required for pollen development. Molecular analysis of the insertion site revealed a single copy of the DsE element inserted into the second exon of the RLK5 gene that was adjacent to a large (~150 kb) genomic deletion. The deleted region is predicted to encode 38 genes and to include one or more genes with important function(s) during pollen maturation and seed development.

The last decade provided the plant science community with the complete genome sequence of Arabidopsis thaliana and rice, tools to investigate the function of potentially every plant gene, methods to dissect virtually any aspect of the plant life cycle, and a wealth of information on gene expression and protein function. Focusing on Arabidopsis as a model system has led to an integration of the plant sciences that triggered the development of new technologies and concepts benefiting plant research in general. These enormous changes led to an unprecedented increase in our understanding of the genetic basis and molecular mechanisms of developmental, physiological and biochemical processes, some of which will be discussed in this article.

The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis, called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.

Correction for ‘High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes’ by Chengzhi Hu et al., Lab Chip, 2017, 17, 671–680.

Genomic imprinting refers to a form of epigenetic gene regulation whereby alleles are differentially expressed in a parent-of-origin-dependent manner. Imprinting evolved independently in flowering plants and in therian mammals in association with the elaboration of viviparity and a placental habit. Despite the striking differences in plant and animal reproduction, genomic imprinting shares multiple characteristics between them. In both groups, imprinted expression is controlled, at least in part, by DNA methylation and chromatin modifications in cis-regulatory regions, and many maternally and paternally expressed genes display complementary dosage-dependent effects during embryogenesis. This suggests that genomic imprinting evolved in response to similar selective pressures in flowering plants and mammals. Nevertheless, there are important differences between plant and animal imprinting. In particular, genomic imprinting has been shown to be more flexible and evolutionarily labile in plants. In mammals, imprinted genes are organized mainly in highly conserved clusters, whereas in plants they occur in isolation throughout the genome and are affected by local gene duplications. There is a large degree of intra- and inter-specific variation in imprinted gene expression in plants. These differences likely reflect the distinct life cycles and the different evolutionary dynamics that shape plant and animal genomes.

Over 70 years ago, increased spontaneous mutation rates were observed in Drosophila spp. hybrids, but the genetic basis of this phenomenon is not well understood. The model plant Arabidopsis (Arabidopsis thaliana) offers unique opportunities to study the types of mutations induced upon hybridization and the frequency of their occurrence. Understanding the mutational effects of hybridization is important, as many crop plants are grown as hybrids. Besides, hybridization is important for speciation and its effects on genome integrity could be critical, as chromosomal rearrangements can lead to reproductive isolation. We examined the rates of hybridization-induced point and frameshift mutations as well as homologous recombination events in intraspecific Arabidopsis hybrids using a set of transgenic mutation detector lines that carry mutated or truncated versions of a reporter gene. We found that hybridization alters the frequency of different kinds of mutations. In general, Columbia (Col) � Cape Verde Islands and Col � C24 hybrid progeny had decreased T?G and T?A transversion rates but an increased C?T transition rate. Significant changes in frameshift mutation rates were also observed in some hybrids. In Col � C24 hybrids, there is a trend for increased homologous recombination rates, except for the hybrids from one line, while in Col � Cape Verde Islands hybrids, this rate is decreased. The overall genetic distance of the parents had no influence on mutation rates in the progeny, as closely related accessions on occasion displayed higher mutation rates than accessions that are separated farther apart. However, reciprocal hybrids had significantly different mutation rates, suggesting parent-of-origin-dependent effects on the mutation frequency. � 2014 American Society of Plant Biologists. All rights reserved.

The FG (female gametophyte) of flowering plants (angiosperms) is a simple highly polar structure composed of only a few cell types. The FG develops from a single cell through mitotic divisions to generate, depending on the species, four to 16 nuclei in a syncytium. These nuclei are then partitioned into three or four distinct cell types. The mechanisms underlying the specification of the nuclei in the FG has been a focus of research over the last decade. Nevertheless, we are far from understanding the patterning mechanisms that govern cell specification. Although some results were previously interpreted in terms of static positional information, several lines of evidence now show that local interactions are important. In the present article, we revisit the available data on developmental mutants and cell fate markers in the light of theoretical frameworks for biological patterning. We argue that a further dissection of the mechanisms may be impeded by the combinatorial and dynamical nature of developmental cues. However, accounting for these properties of developing systems is necessary to disentangle the diversity of the phenotypic manifestations of the underlying molecular interactions.

SummarySperm delivery for double fertilization of flowering plants relies on interactions between the pollen tube (PT) and two synergids, leading to programmed cell death (PCD) of the PT and one synergid. The mechanisms underlying the communication among these cells during PT reception is unknown. We discovered that the synergids control this process by coordinating their distinct calcium signatures in response to the calcium dynamics and growth behavior of the PT. Induced and spontaneous aberrant calcium responses in the synergids abolish the two coordinated PCD events. Components of the FERONIA (FER) signaling pathway are required for initiating and modulating these calcium responses and for coupling the PCD events. Intriguingly, the calcium signatures are interchangeable between the two synergids, implying that their fates of death and survival are determined by reversible interactions with the PT. Thus, complex intercellular interactions involving a receptor kinase pathway and calcium-mediated signaling control sperm delivery in plants.

Cellular context can be crucial when studying developmental processes as well as responses to environmental variation. Several different tools have been developed in recent years to isolate specific tissues or cell types. Laser-assisted microdissection (LAM) allows for the isolation of such specific tissue or single cell-types purely based on morphology and cytology. This has the advantage that (1) cell types that are rare can be isolated from heterogeneous tissue, (2) no marker line with cell type-specific expression needs to be established, and (3) the method can be applied to non-model species and species that are difficult to genetically transform. The rapid development of next-generation sequencing (NGS) approaches has greatly advanced the possibilities to perform molecular analyses in diverse organisms. However, there is a mismatch between currently available cell isolation tools and their applicability to non-model organisms. Therefore, LAM will become increasingly popular in the study of diverse agriculturally or ecologically relevant plant species. Here, we describe a protocol that has been successfully used for LAM to isolate either whole floral organs or even single cell types in plants, e.g., Arabidopsis thaliana, Boechera spp., or tomato.

Cellular context can be crucial when studying developmental processes as well as responses to environmental variation. Several different tools have been developed in recent years to isolate specific tissues or cell types. Laser-assisted microdissection (LAM) allows for the isolation of such specific tissue or single cell-types purely based on morphology and cytology. This has the advantage that (1) cell types that are rare can be isolated from heterogeneous tissue, (2) no marker line with cell type-specific expression needs to be established, and (3) the method can be applied to non-model species and species that are difficult to genetically transform. The rapid development of next-generation sequencing (NGS) approaches has greatly advanced the possibilities to perform molecular analyses in diverse organisms. However, there is a mismatch between currently available cell isolation tools and their applicability to non-model organisms. Therefore, LAM will become increasingly popular in the study of diverse agriculturally or ecologically relevant plant species. Here, we describe a protocol that has been successfully used for LAM to isolate either whole floral organs or even single cell types in plants, e.g., Arabidopsis thaliana, Boechera spp., or tomato.

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

Apomixis, or asexual reproduction through seeds, is a natural trait that could have an immense positive impact on crop production. Apomictic breeding strategies could allow the fixation and indefinite propagation of any desired genotype, however complex. Apomicts display a wide variety of developmental mechanisms, which can be viewed as a short-circuiting of sexual development. Gametophytic and sporophytic apomixis are distinguished by the developmental origin of apomictically derived embryos. Genetic studies suggest that individual elements of gametophytic apomixis, such as apomeiosis and parthenogenesis, are either controlled by one or two dominant Mendelian factors. As recombination around apomeiosis loci is suppressed, it is currently not known how complex these loci are. Much less is known regarding the genetic control of sporophytic apomixis but initial studies suggest a complex genetic control. Genetic analyses of sexual reproduction in plant model systems have identified genes that, when mutated, display elements of apomixis. Such studies help in the identification of candidate genes and promoters that can be used for the de novo engineering of apomixis through biotechnology. Molecular genetic studies in apomictic and sexual systems will generate the knowledge necessary for the engineering of conditional apomixis technology. Approaches encouraging collaboration and widespread dissemination of the acquired knowledge will constitute the most innovative route to the development, deployment and acceptance of apomixis technology in agriculture.

Long before promiscuity was discovered to bear significant risk, many flowering plants had partially abandoned the pleasures of their sexual life to evolve one of the most intriguing reproductive alternatives found in nature. Apomixis is an asexual method of reproduction through seed that

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.

In higher plants, seed development requires maternal gene activity in the haploid (gametophytic) as well as diploid (sporophytic) tissues of the developing ovule. The Arabidopsis thaliana gene MEDEA (MEA) encodes a SET-domain protein of the Polycomb group that regulates cell proliferation by exerting a gametophytic maternal control during seed development. Seeds derived from female gametocytes (embryo sacs) carrying a mutant mea allele abort and exhibit cell proliferation defects in both the embryo and the endosperm. In this study we show that the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA alleles. Paternally inherited MEA alleles are transcriptionally silent in both the young embryo and endosperm. Mutations at the decrease in DNA methylation1 (ddm1) locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and exhibit some of the abnormalities found in aborting mea seeds. Our results indicate that the maintenance of the genomic imprint at the mea locus requires zygotic DDM1 activity. Because DDM1 encodes a putative chromatin remodeling factor, chromatin structure is likely to be interrelated with genomic imprinting in Arabidopsis.

The reproductive system determines the way in which gametes develop and interact to form a new organism. Therefore, it exerts the primary level of control of genotypic frequencies in plant populations, and plays a fundamental role in plant breeding. A basic understanding of plant reproductive development will completely transform current breeding strategies used for seed production. Apomixis is an asexual form of reproduction in which embryogenesis occurs in a cell lineage lacking both meiosis and fertilization, and that culminates in the formation of viable progeny genetically identical to the mother plant. The transfer of apomixis into sexual crops will allow the production of self-perpetuating improved hybrids, and the fixation of any desired heterozygous genotype. The initiation of apomictic development invariably takes place at early stages of ovule ontogeny, before the establishment of the megagametophytic phase. The developmental versatility associated with megagametophyte formation suggests that the genetic and molecular regulation of apomixis is intimately related to the regulation of sexuality. Differences between the initiation of sexual and apomictic development may be determined by regulatory genes that act during megasporogenesis, and that control events leading to the formation of unreduced female gametophytes. To test this hypothesis, we are isolating and characterizing genes that act during megasporogenesis inArabidopsis thaliana and investigating their potential role in the induction of apomixis. We are using a recently established transposon-based enhancer detection and gene trap insertional mutagenesis system that allows the identification of genes based on their expression patterns. An initial screen of transposants has yielded over 20 lines conferring restricted GUS expression during early ovule development. We have obtained the sequence of genomic fragments flanking the transposon insertion. Several have homology to genes playing important roles in plant and animal development. They include cell cycle regulators, enzymes involved in callose hydrolysis, leucine-rich repeat protein kinase receptors, and expressed sequence tags (ESTs) of unknown function. Independently, a genetic screen allows the identification of female sterile mutants defective in megasporogenesis. Results from these experiments will improve our basic understanding of reproductive development in plants, and will set the basis for a sustained effort in plant germ line biotechnology, a first step toward a flexible transfer of apomixis into a large variety of sexual crops.

The gametophytic maternal effect mutant medea ( mea ) shows aberrant growth regulation during embryogenesis in Arabidopsis thaliana . Embryos derived from mea eggs grow excessively and die during seed desiccation. Embryo lethality is independent of the paternal contribution and gene dosage. The mea phenotype is consistent with the parental conflict theory for the evolution of parent-of-origin–specific effects. MEA encodes a SET domain protein similar to Enhancer of zeste , a member of the Polycomb group. In animals, Polycomb group proteins ensure the stable inheritance of expression patterns through cell division and regulate the control of cell proliferation.

The plant life cycle alternates between a diploid and a haploid generation, the spore-producing sporophyte and the gamete-producing gametophyte. Unlike in animals, where meiotic products differentiate directly into gametes, the haploid spores of plants divide mitotically to form a multicellular haploid organism. The differentiation of gametes occurs later in the development of the gametophyte.

It has become increasingly apparent that the extracellular matrix (ECM), which in plants corresponds to the cell wall, can influence intracellular activities in ways that go far beyond their supposedly passive mechanical support. In plants, growing cells use mechanisms sensing cell wall integrity to coordinate cell wall performance with the internal growth machinery to avoid growth cessation or loss of integrity. How this coordination precisely works is unknown. Previously, we reported that in the tip-growing pollen tube the ANXUR receptor-like kinases (RLKs) of the CrRLK1L subfamily are essential to sustain growth without loss of cell wall integrity in Arabidopsis. Here, we show that over-expression of the ANXUR RLKs inhibits growth by over-activating exocytosis and the over-accumulation of secreted cell wall material. Moreover, the characterization of mutations in two partially redundant pollen-expressed NADPH oxidases coupled with genetic interaction studies demonstrate that the ANXUR RLKs function upstream of these NADPH oxidases. Using the H2O2-sensitive HyPer and the Ca2+-sensitive YC3.60 sensors in NADPH oxidase-deficient mutants, we reveal that NADPH oxidases generate tip-localized, pulsating H2O2 production that functions, possibly through Ca2+ channel activation, to maintain a steady tip-focused Ca2+ gradient during growth. Our findings support a model where ECM-sensing receptors regulate reactive oxygen species production, Ca2+ homeostasis, and exocytosis to coordinate ECM-performance with the internal growth machinery., PLoS Biology, 11 (11), ISSN:1544-9173, ISSN:1545-7885

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

BackgroundSexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids.ResultsWe have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors.ConclusionOur comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of floral morphology. These data will serve as an invaluable resource for research in orchid floral biology, enabling studies into the molecular mechanisms of pollinator attraction and speciation.

Plant populations show phenotypic diversity, which may be caused by genetic and epigenetic variation. It has recently been shown that new epigenetic variants are generated at a higher rate than genetic variants and several studies have shown that epigenetic variation can be influenced by the environment. Although the heritability of environmentally induced epigenetic traits has gained increasing interest in past years, it is still not clear whether and to what extent induced epigenetic changes have a role in ecology and evolution. Some reports on model and nonmodel species support the possibility of adaptive epigenetic alleles, indicating that epigenetic variants are subject to natural selection. However, most of these studies rely solely on phenotypic data and no information is available about the underlying mechanisms. Thus, the role of inherited epigenetic variation for plant adaptation is unclear and further investigations are required to gain insights into the significance of epigenetic variation for ecological and evolutionary processes. Here, we review mechanisms of epigenetic regulation, epigenetic responses to environmental challenges, their inheritance, and their implication for adaptation and plant evolution.

Meiosis is a key step in sexual plant reproduction. Cytological analysis of meiosis enables hypotheses of gene function during meiosis to be confirmed or discarded. Observation of meiosis in the female reproductive organs in many higher plants is technically challenging because the cells are enclosed deep within the nucellus and ovule tissues. In this chapter, we describe a simple staining procedure using Schiff's reagent optimized for use with confocal microscopy to observe meiosis within intact ovules of Zea mays (maize). Using this procedure we present images taken by confocal laser scanning microscopy of wild-type meiotic cells in whole-mount maize ovules.

Background Plant parasitism represents an extraordinary interaction among flowering plants: parasitic plants use a specialized organ, the haustorium, to invade the host vascular system to deprive host plants of water and nutrients. Various compounds present in exudates of host plants trigger haustorium development. The two most effective haustorium inducing factors (HIFs) known for the parasitic plant Triphysaria versicolor (T. versicolor) are peonidin, an antioxidant flavonoid, and 2,6-dimethoxybenzoquinone (DMBQ), an oxidative stress agent. To date, two genes involved in haustorium initiation in T. versicolor have been identified: TvQR1, a quinone oxidoreductase that generates the active HIF from DMBQ, and TvPirin, a transcription co-factor that regulates several other DMBQ- responsive and –non-responsive genes. While the expression of these genes in response to DMBQ is well characterized, their expression in response to peonidin is not. In addition, the pattern of polymorphisms in these genes is unknown, even though nucleotide changes in TvQR1 and TvPirin may have contributed to the ability of T. versicolor to develop haustoria. To gain insights into these aspects, we investigated their transcriptional responses to HIFs and non-HIF and their natural nucleotide diversity. Results Here we show that TvQR1 and TvPirin are transcriptionally upregulated by both DMBQ and peonidin in T. versicolor roots. Yet, while TvQR1 also responded to juglone, a non-HIF quinone with toxicity comparable to that of DMBQ, TvPirin did not. We further demonstrate that TvPirin encodes a protein shorter than the one previously reported. In the T. versicolor natural population of Northern California, TvQR1 exhibited remarkably higher molecular diversity and more recombination events than TvPirin, with the highest non-synonymous substitution rate in the substrate recognition and catalytic domain of the TvQR1 protein. Conclusion Our results suggest that TvQR1 and TvPirin have most likely evolved highly distinct roles for haustorium formation. Unlike TvPirin, TvQR1 might have been under diversifying selection to maintain a diverse collection of polymorphisms, which might be related to the recognition of an assortment of HIF and non-HIF quinones as substrates for successful haustorial establishment in a wide range of host plants.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

The plant life cycle alternates between a diploid sporophytic and a haploid gametophytic generation. The female gametophyte (FG) of flowering plants is typically formed through three syncytial mitoses, followed by cellularisation that forms seven cells belonging to four cell types. The specification of cell fates in the FG has been suggested to depend on positional information provided by an intrinsic auxin concentration gradient. The goal of this study was to develop mathematical models that explain the formation of this gradient in a syncytium. Two factors were proposed to contribute to the maintenance of the auxin gradient in Arabidopsis FGs: polar influx at early stages and localised auxin synthesis at later stages. However, no gradient could be generated using classical, one-dimensional theoretical models under these assumptions. Thus, we tested other hypotheses, including spatial confinement by the large central vacuole, background efflux and localised degradation, and investigated the robustness of cell specification under different parameters and assumptions. None of the models led to the generation of an auxin gradient that was steep enough to allow sufficiently robust patterning. This led us to re-examine the response to an auxin gradient in developing FGs using various auxin reporters, including a novel degron-based reporter system. In agreement with the predictions of our models, auxin responses were not detectable within the FG of Arabidopsis or maize, suggesting that the effects of manipulating auxin production and response on cell fate determination might be indirect.

Meiosis is a key step in sexual plant reproduction. Cytological analysis of meiosis enables hypotheses of gene function during meiosis to be confirmed or discarded. Observation of meiosis in the female reproductive organs in many higher plants is technically challenging because the cells are enclosed deep within the nucellus and ovule tissues. In this chapter, we describe a simple staining procedure using Schiff's reagent optimized for use with confocal microscopy to observe meiosis within intact ovules of Zea mays (maize). Using this procedure we present images taken by confocal laser scanning microscopy of wild-type meiotic cells in whole-mount maize ovules.

The maternally expressed Arabidopsis thaliana Polycomb group protein MEDEA (MEA) controls expression of the MADS-box gene PHERES1 (PHE1). Here, we show that PHE1 is mainly paternally expressed but maternally repressed and that this maternal repression of PHE1 breaks down in seeds lacking maternal MEA activity. Because Polycomb group proteins control parental imprinting in mammals as well, the independent recruitment of similar protein machineries for the imprinting of genes is a notable example of convergent evolution.

We have characterized the Drosophila homologue of the proto-oncogenic RAC protein kinase (DRAC-PK). The DRAC-PK gene gives rise to two transcripts with the same coding potential, generated by the use of two different polyadenylation signals. Each transcript encodes two polypeptides because of the presence of a weaker initiator ACG codon, upstream from the major AUG, such that the larger protein contains an N-terminal extension. Like the human isoforms, DRAC-PKs possess a novel signaling region, the pleckstrin homology domain. DRAC-PK proteins have a similar expression pattern, being regulated both maternally and zygotically, and are expressed throughout Drosophila development. Antisera specific for recombinant DRAC-PK and for its C terminus detected two polypeptides of 66 and 85 kDa in Drosophila extracts. The antirecombinant antisera also recognized a polypeptide of 120 kDa from Drosophila, which apparently shared an epitope related to DRAC-PK sequences. The role of p120 appears to be restricted compared with that of DRAC-PK, since it was not detected in larvae or adult flies. There was no spatial restriction of DRAC-PK expression during embryogenesis, suggesting that localized activation might be a regulatory mechanism for its function. DRAC-PK possesses an intrinsic kinase activity that is approximately 8-fold higher in adult flies than in 0-3-h embryos undergoing rapid mitotic cycles.

Expression profiling of single cells can yield insights into cell specification, cellular differentiation processes, and cell type-specific responses to environmental stimuli. Recent work has established excellent tools to perform genome-wide expression studies of individual cell types, even if the cells of interest occur at low frequency within an organ. We review the advances and impact of gene expression studies of rare cell types, as exemplified by recently gained insights into the development and function of the angiosperm female gametophyte. The detailed transcriptional characterization of different stages during female gametophyte development has significantly helped to improve our understanding of cellular specification or cell–cell communication processes. Next-generation sequencing approaches — used increasingly for expression profiling — will now allow for comparative approaches that focus on agriculturally, ecologically or evolutionarily relevant aspects of plant reproduction.

Successful pollination depends on cell-cell communication and rapid cellular responses. In Arabidopsis, the pollen grain lands on a dry stigma, where it hydrates, germinates and grows a pollen tube that delivers the sperm cells to the female gametophyte to effect double fertilization. Various studies have emphasized that a mature, dehydrated pollen grain contains all the transcripts and proteins required for germination and initial pollen tube growth. Therefore, it is important to explore the role of post-translational modifications (here phosphorylation), through which many processes induced by pollination are probably controlled. We report here a phosphoproteomic study conducted on mature Arabidopsis pollen grains with the aim of identifying potential targets of phosphorylation. Using three enrichment chromatographies, a broad coverage of pollen phosphoproteins with 962 phosphorylated peptides corresponding to 598 phosphoproteins was obtained. Additionally, 609 confirmed phosphorylation sites were successfully mapped. Two hundred and seven of 240 phosphoproteins that were absent from the PhosPhAt database containing the empirical Arabidopsis phosphoproteome showed highly enriched expression in pollen. Gene ontology (GO) enrichment analysis of these 240 phosphoproteins shows an over-representation of GO categories crucial for pollen tube growth, suggesting that phosphorylation regulates later processes of pollen development. Moreover, motif analyses of pollen phosphopeptides showed an over-representation of motifs specific for Ca²⁺/calmodulin-dependent protein kinases, mitogen-activated protein kinases, and binding motifs for 14-3-3 proteins. Lastly, one tyrosine phosphorylation site was identified, validating the TDY dual phosphorylation motif of mitogen-activated protein kinases (MPK8/MPK15). This study provides a solid basis to further explore the role of phosphorylation during pollen development.

Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20–90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM-based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.

Ultraconserved elements (UCEs), stretches of DNA that are identical between distantly related species, are enigmatic genomic features whose function is not well understood. First identified and characterized in mammals, UCEs have been proposed to play important roles in gene regulation, RNA processing, and maintaining genome integrity. However, because all of these functions can tolerate some sequence variation, their ultraconserved and ultraselected nature is not explained. We investigated whether there are highly conserved DNA elements without genic function in distantly related plant genomes. We compared the genomes of Arabidopsis thaliana and Vitis vinifera; species that diverged ∼115 million years ago (Mya). We identified 36 highly conserved elements with at least 85% similarity that are longer than 55 bp. Interestingly, these elements exhibit properties similar to mammalian UCEs, such that we named them UCE-like elements (ULEs). ULEs are located in intergenic or intronic regions and are depleted from segmental duplications. Like UCEs, ULEs are under strong purifying selection, suggesting a functional role for these elements. As their mammalian counterparts, ULEs show a sharp drop of A+T content at their borders and are enriched close to genes encoding transcription factors and genes involved in development, the latter showing preferential expression in undifferentiated tissues. By comparing the genomes of Brachypodium distachyon and Oryza sativa, species that diverged ∼50 Mya, we identified a different set of ULEs with similar properties in monocots. The identification of ULEs in plant genomes offers new opportunities to study their possible roles in genome function, integrity, and regulation.

Plant Anti-Insect Armaments Because individual plants are unable to relocate, they are subject to extreme selection by the insects feeding upon them. One means by which plants suppress herbivory is to produce toxic compounds to deter feeding (see the Perspective by Hare ). Agrawal et al. (p. 113 ) compared pesticide–treated or untreated evening primroses. Over 5 years of pesticide treatment, the production of defensive chemicals in the fruit reduced and flowering times shifted, and the primrose's competitive ability against dandelions improved. Züst et al. (p. 116 ) examined large-scale geographic patterns in a polymorphic chemical defense locus in the model plant Arabidopsis thaliana and found that it is matched by changes in the relative abundance of two specialist aphids. Thus, herbivory has strong and immediate effects on the local genotypic composition of plants and traits associated with herbivore resistance.

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin-dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of miniseed3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways.

Reproduction is a crucial step in the life cycle of plants. The male and female germline lineages develop in the reproductive organs of the flower, which in higher plants are the anthers and ovules, respectively. Development of the germline lineage initiates from a dedicated sporophytic cell that undergoes meiosis to form spores that subsequently give rise to the gametophytes through mitotic cell divisions. The mature male and female gametophytes harbour the male (sperm cells) and female gametes (egg and central cell), respectively. Those unite during double fertilization to initiate embryo and endosperm development in sexually reproducing higher plants. While cytological changes involved in development of the germline lineages have been well characterized in a number of species, investigation of the transcriptional basis underlying their development and the specification of the gametes proved challenging. This is largely due to the inaccessibility of the cells constituting the germline lineages, which are enclosed by sporophytic tissues. Only recently, these technical limitations could be overcome by combining new methods to isolate the relevant cells with powerful transcriptional profiling methods, such as microarrays or high-throughput sequencing of RNA. This review focuses on these technical advances and the new insights gained from them concerning the transcriptional basis and molecular mechanisms underlying germline development.

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from

Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple-sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development.