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. Due to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Since siRNAs serve as both regulators and outputs of the epigenome, we performed here the successful characterization of small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibited extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation was accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ~70% of the zygote siRNA loci did 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 which are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci were 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.

Phylogenetic and Expression Analysis of CENH3 and APOLLO Genes in Sexual and Apomictic Boechera Species

Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO (Aspartate Glutamate Aspartate Aspartate histidine exonuclease) is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Also, we characterize the structure of CENH3, study expression levels of APOLLO and CENH3 in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes.

The Evolution and Expression Profiles of EC1 Gene Family during Development in Cotton

Fertilization is essential to sexual reproduction of flowering plants. EC1 (EGG CELL 1) proteins have a conserved cysteine spacer characteristic and play a crucial role in double fertilization process in many plant species. However, to date, the role of EC1 gene family in cotton is fully unknown. Hence, detailed bioinformatics analysis was explored to elucidate the biological mechanisms of EC1 gene family in cotton. In this study, we identified 66 genes in 10 plant species in which a total of 39 EC1 genes were detected from cotton genome. Phylogenetic analysis clustered the identified EC1 genes into three families (I-III) and all of them contain Prolamin-like domains. A good collinearity was observed in the synteny analysis of the orthologs from cotton genomes. Whole-genome duplication was determined to be one of the major impetuses for the expansion of the EC1 gene family during the process of evolution. qRT-PCR analysis showed that EC1 genes were highly expressed in reproductive tissues under multiple stresses, signifying their potential role in enhancing stress tolerance or responses. Additionally, gene interaction networks showed that EC1 genes may be involved in cell stress and response transcriptional regulator in the synergid cells and activate the expression of genes required for pollen tube guidance. Our results provide novel functional insights into the evolution and functional elucidation of EC1 gene family in cotton.

Overcoming Pre-Fertilization Barriers in Intertribal Crosses between Anemone coronaria L. and Ranunculus asiaticus L.

Hybridization in flowering plants depends, in the first place, on the delivery of pollen to a receptive stigma and the subsequent growth of pollen tubes through the style to the ovary, where the sperm nucleus of the pollen grain can ultimately fertilize the egg cell. However, reproductive failure is often observed in distant crosses and is caused by pre- and/or post-zygotic barriers. In this study, the reproductive pre-fertilization barriers of intertribal crosses between Anemone coronaria L. and Ranunculus asiaticus L., both belonging to the Ranunculaceae, were investigated. Despite the incongruity of intertribal crosses between A. coronaria and R. asiaticus having been of low intensity at the stigmatic level, interstylar obstructions of the pollen tube growth occurred, which confirmed the presence of pre-fertilization barriers. We show that these barriers could be partially bypassed by combining pollination with a stigma treatment. More specifically, a significantly higher ratio of the pollen tube length to the total style length and a better seed set were observed when the stigma was treated with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg.mL−1) together with the cytokinin kinetin (KIN, 0.5 mg.mL−1) 24 h after pollination, irrespective of the cross direction. More specifically, the stigma treatments with any form of auxin (combined or not combined with cytokinin) resulted in a full seed set, assuming an apomictic fruit set, because no pollination was needed to obtain these seeds.

An F-Actin Mega-Cable Is Associated With the Migration of the Sperm Nucleus During the Fertilization of the Polarity-Inverted Central Cell of Agave inaequidens

Asparagaceae’s large embryo sacs display a central cell nucleus polarized toward the chalaza, which means the sperm nucleus that fuses with it during double fertilization migrates an atypical long distance before karyogamy. Because of the size and inverted polarity of the central cell in Asparagaceae, we hypothesize that the second fertilization process is supported by an F-actin machinery different from the short-range F-actin structures observed in Arabidopsis and other plant models. Here, we analyzed the F-actin dynamics of Agave inaequidens, a classical Asparagaceae, before, during, and after the central cell fertilization. Several parallel F-actin cables, spanning from the central cell nucleus to the micropylar pole, and enclosing the vacuole, were observed. As fertilization progressed, a thick F-actin mega-cable traversing the vacuole appeared, connecting the central cell nucleus with the micropylar pole near the egg cell. This mega-cable wrapped the sperm nucleus in transit to fuse with the central cell nucleus. Once karyogamy finished, and the endosperm started to develop, the mega-cable disassembled, but new F-actin structures formed. These observations suggest that Asparagaceae, and probably other plant species with similar embryo sacs, evolved an F-actin machinery specifically adapted to support the migration of the fertilizing sperm nucleus within a large-sized and polarity-inverted central cell.

Method for preserving the viability of a chicken embryo with a shell defect in experiment

The egg has always been and remains an ideal object for conducting various scientific research. An egg is an isolated egg cell outside the mother’s body. Therefore, it is an ideal object for studying embryogenesis and performing various manipulations during embryogenesis and before the birth of a viable organism. The existing methods allow conducting experimental manipulations with the embryo in situ, inside the egg shells without damaging them. However, the achievement of ideal parameters for closing the defect of the fertilized egg shell in the experiment is the key to the successful completion of the experiment. Periods of embryogenesis, especially at the last stage, when osteogenesis occurs, require the presence of a sufficient amount of calcium ions in the metabolism of the developing chicken, which are vital for the formation of a full-fledged chicken.The aim: to develop an optimal method for closing the defect and fixing the egg shell after manipulation or during the experiment.Materials and methods. The experiment was carried out on fertile eggs of the breed of chickens – meat breed of broilers Ross-308 (ROSS-308), JSC Poultry Farm “NovoBaryshevskaya” (Koltsovo, Novosibirsk Region, Russian Federation). In the experiment, 120 fertilized eggs were used. Eggs weighing 60–70 g were incubated at a temperature of 37.5–38.0 °C and 50–55 % humidity. Comparative anatomical and physiological parameters were evaluated on the 7th, 14th, 20th day of incubation and on the 1st day of the postnatal period. In the experimental group, the shell defect was covered with a fragment of the egg shell of the donor. Incubation was carried out in an incubator – a household incubator “Nesushka” (Novosibirsk, Russian Federation).Results. The proposed method of fixing and closing the defect of the fertilized egg shell excludes the use of foreign materials that have an adverse effect on the development of the embryo. There were no anatomical and physiological deviations in the chicks of the study group when comparing the indicators with the parameters in the comparison group and the Hamburger – Hamilton classification.

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

Background Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis thaliana mutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome. Results To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating mutations at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. FastRed and FastGreen binary vectors can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) from A. thaliana or the constitutive promoter from Petroselinum crispum (PcUBi4-2). Available enzymes are “classical” Cas9 codon-optimized for A. thaliana and a recently reported, intron-containing version of Cas9 codon-optimized for Zea mays, zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containing zCas9i driven under egg-cell specific pEC1.2 promoter. Finally, we introduced convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system. Conclusion A rapid, simple and flexible CISPR/Cas9 cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher order A. thaliana mutants.

Taking the Wheel – de novo DNA Methylation as a Driving Force of Plant Embryonic Development

During plant embryogenesis, regardless of whether it begins with a fertilized egg cell (zygotic embryogenesis) or an induced somatic cell (somatic embryogenesis), significant epigenetic reprogramming occurs with the purpose of parental or vegetative transcript silencing and establishment of a next-generation epigenetic patterning. To ensure genome stability of a developing embryo, large-scale transposon silencing occurs by an RNA-directed DNA methylation (RdDM) pathway, which introduces methylation patterns de novo and as such potentially serves as a global mechanism of transcription control during developmental transitions. RdDM is controlled by a two-armed mechanism based around the activity of two RNA polymerases. While PolIV produces siRNAs accompanied by protein complexes comprising the methylation machinery, PolV produces lncRNA which guides the methylation machinery toward specific genomic locations. Recently, RdDM has been proposed as a dominant methylation mechanism during gamete formation and early embryo development in Arabidopsis thaliana, overshadowing all other methylation mechanisms. Here, we bring an overview of current knowledge about different roles of DNA methylation with emphasis on RdDM during plant zygotic and somatic embryogenesis. Based on published chromatin immunoprecipitation data on PolV binding sites within the A. thaliana genome, we uncover groups of auxin metabolism, reproductive development and embryogenesis-related genes, and discuss possible roles of RdDM at the onset of early embryonic development via targeted methylation at sites involved in different embryogenesis-related developmental mechanisms.