Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants

40S ribosome biogenesis co-factors are essential for gametophyte and embryo development

Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants

Functional analysis of CDKA;1, the Arabidopsis thaliana homologue of the p34cdc2 protein kinase

CYCLIN-DEPENDENT KINASEs (CDKs) are the central gatekeepers of cell cycle progression and conserved in all eukaryotes. In this study, the Arabidopsis thaliana master cell cycle regulator CDKA;1 was functionally analyzed. CDKA;1 is a single gene in Arabidopsis and homologous to the human Cdk1 and the yeast cdc2/CDC28. Screening of two T-DNA insertion mutant collections resulted in the isolation of two independent cdka;1 null mutant alleles, which displayed the same phenotype. CDKA;1 was found to be required for both the sporophytic and the male gametophytic generations of the flowering plant Arabidopsis. While during sporophyte development, heterozygous mutant plants were unaffected, homozygous cdka;1 mutants were not viable and died as young embryos. During male gametophyte (pollen) development, the lack of CDKA;1 function caused a cell cycle arrest in the G2 phase prior to the last mitotic division. This cell cycle defect led to cdka;1 mutant pollen with only one instead of the usual two sperm cells. Nevertheless, the mutant cdka;1 pollen was viable and could fertilize the female gametophyte (embryo sac). Because cdka;1 pollen grains had only one instead of two sperm cells, they only performed single fertilization and thus, disrupted the double fertilization event characteristic of flowering plants. Interestingly, the cdka;1 mutant single fertilization exclusively targeted the egg cell, leaving the progenitor of the endosperm, the central cell, unfertilized. However, upon cdka;1 fertilization of the egg cell, not only the embryo started to develop, but the unfertilized central cell nucleus also began to divide. This onset of endosperm development without fertilization revealed a hitherto unrecognized endosperm proliferation signal emitted from the fertilization of the egg cell. The autonomous endosperm in cdka;1-fertilized seeds only underwent up to five nuclear division cycles before it stopped proliferating, followed by an early abortion of the whole seed. Thus, the cdka;1 mutant belongs to a rare class of paternal effect mutants that cause seed abortion irrespective of the genetic constitution of the female partner. In order to enhance endosperm proliferation in cdka;1-fertilized seeds, cdka;1 pollen was crossed to various fis-class mutants. These mutants are defective in the maternally inherited FIS-complex, a Polycomb-group repressive complex controlling genomic imprinting in the endosperm. In fis-class mutants, autonomous endosperm develops in the absence of fertilization. When fertilized, the fis-class mutant endosperm over-proliferates and due to a maternal effect these seeds abort later during development. The endosperm development in cdka;1-fertilized fis-mutant seeds was substantially enhanced and led to a partial rescue of the cdka;1-mediated seed abortion. Unexpectedly, the maternally conferred seed abortion caused by fis-class mutants was also partially reversed, producing viable seeds among the fis-class x cdka;1 offspring. This rescue was characterized by a down-regulated expression of the MADS-box transcription factor PHERES1, a downstream target of FIS-complex repression which is highly over-expressed in fertilized fis-class mutants. The down-regulation of PHERES1 in fis-class x cdka;1 endosperm suggests that the lack of paternal expression in combination with the defective gene repression of fis-class mutants results in a more balanced gene dosage of PHERES1 and potentially other genes of which the dosage is pivotal for regular seed development. These results indicate that the FIS-complex is not essential for endosperm development, but is important to harmonize maternal and paternal gene expression by the control of imprinting in the female genome. Furthermore, these data demonstrate that the paternal genome is not required for functional endosperm development if maternally derived genomic imprinting is bypassed due to mutations in the FIS-complex. The finding that a solely maternally derived endosperm can sustain seed development supports a hypothesis raised by Eduard Strasburger, who proposed in 1900 that the endosperm of flowering plants is of female gametophytic origin and that central cell fertilization might have evolved as a trigger to start endosperm proliferation

Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase e, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability

애기장대 DEMETER 유전자의 전사 조절과 DNA demethylase 상동유전자군의 상호 작용에 대한 연구

학위논문(박사)--서울대학교 대학원 :자연과학대학 생명과학부,2020. 2. 최연희.The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana. DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation is confined to the companion cell of the egg, the central cell. Here, I show that in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time; immediately following separation of the companion cell lineage from the germline. I define transcriptional regulatory elements of DME using reporter genes, showing that a small region within the DME gene controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, I found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combine predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. Besides I provide evidence of the minimal promoters specific binding in yeast by a BPC and an HD-ZIP transcription factor. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation. There are several differences in dme mutant depending on mutation site and their ecotype. To identify what make the differences, I crossed mutants having different allele or ecotype. As a result of the crosses, I found a genomic region that seemed to be beneficial in overcoming seed abortion. In Arabidopsis there are three homologous genes of DME; REPRESSOR OF SILENCING1 (ROS1), DEMETER-LIKE (DML) 2 and 3. These family genes are expressed in all sporophytic tissues. To address the contribution of DNA demethylation to plant life cycle and interaction between demethylases, using crosses of mutants of DNA demethylase family, I found evidence of interaction of DNA demethylase family.DEMETER (DME) DNA glycosylase는 base-excision repair pathway를 통해 DNA demethylation을 개시하고 애기장대에서의 생식에 필수적이다. DME-매개 DNA demethylation은 짧고 AT-rich하며 nucleosome이 적은 euchromatic TE 목표로 하며, 인접한 유전자의 발현에 영향을 미치고 배유유전자의 각인을 유도한다. 암배우체에서 DME 발현과 그에 따른 게놈 전체 DNA demethylation은 난자의 동반 세포인 중심 세포에 국한된다. 이 연구에서 나는 수배우체의 DME 발현은 정자의 동반 세포인 vegetative cell에 제한되며 발현하는 시기도 정밀하게 제한됨을 밝혔다. 리포터 유전자를 사용하여 DME의 전사 조절 요소를 찾았고, DME 유전자의 조절 인자가 암수배우체의 동반 세포에서의 발현을 제어하는 것을 보였다. 이 조절 인자를 가진 프로모터에 의한 DME의 발현은 dme-2 돌연변이의 종자유산표현형을 극복하고 DME와 관련된 DNA methylation을 회복하기에 충분하다. 이 최소 프로모터에서 나는 DME의 발현에 필요한 enhancer 서열을 발견하고, 공개된 데이터베이스를 통해 이 모티프에 결합할 수 있는 후보 단백질들의 목록을 제시한다. 또한 yeast 2 hybrid 실험을 통해 프로모터의 전사 조절 인자에 BPC와 HD-ZIP 전사 인자가 결합함을 보였다. 이 연구결과는 DNA demethylation이 어떻게 조절되어 유전자각인을 유도하고 유해한 요소인 TE을 효과적으로 제한하는지를 알려준다. 돌연변이 부위와 그 에코 타입에 따라 dme 돌연변이 체에는 몇 가지 차이점이 있다. 차이점을 확인하기 위해, 다른 에코 타입을 가진 돌연변이체를 교배 시켰다. 교배의 결과, 나는 종자 유산표현형을 극복하는 데 도움이 될 것으로 보이는 게놈 영역을 발견했다. 애기 장대에는 DME의 homologue가 3 개의 존재한다. REPRESSOR OF SILENCING1 (ROS1), DEMETER-LIKE (DML) 2 놔 3이 그것이다. DNA demethylation이 식물 생애주기에 미치는 영향과 demethylase 사이의 상호 작용을 알기 위해, DNA demethylase들의 돌연변이 체의 교배를 사용했고 그를 통해 DNA homologue 사이의 상호 작용의 증거를 발견 하였다.1. CHAPTER I. CONTROL OF DEMETER DNA DEMETHYLASE GENE TRANSCRIPTION IN MALE AND FEMALE GAMETE COMPANION CELLS IN ARABIDOPSIS THALIANA １ 1.1 INTRODUCTION ２ 1.2 MATERIALS AND METHODS ７ 1.2.1. Plant materials and growth conditions ７ 1.2.2. Recombinant Plasmid Construction ７ 1.2.3. Histochemical GUS Staining, GFP fluorescence and Microscopy ８ 1.2.4. Gene expression analysis ９ 1.2.5. Identification of DME Regulatory Regions – TUO vector series １０ 1.2.6. Identification of DME Regulatory Regions – Element deletion １１ 1.2.7. Identification of DME Regulatory Regions – Element substitution １１ 1.2.8. Yeast one-hybrid assay １１ 1.2.9. 5 Rapid amplification of cDNA ends (5 RACE) analysis １２ 1.2.10. Bisulfite sequencing library construction. １２ 1.3. RESULTS １8 1.3.1. DME is Expressed Specifically in the Companion Cell of the Male Gametophyte after Separation of the Sperm Cell Lineage. １8 1.3.2. The DME Promoter Lies within the DME Transcriptional Unit and Contains Both Positive and Negative Regulatory Elements. ２4 1.3.3. Expressing DME Polypeptide in the Central Cell with a Minimal Reproductive Promoter Rescues Seed Abortion and Aberrant DNA methylation associated with the dme-2 mutation. ３4 1.3.4. A 357 bp Region of the DME Transcriptional Unit is both Necessary and Sufficient to Generate the Appropriate DME Expression Profile during Female Gametophyte Development. ４8 1.3.5. DME Expression in Sporophytic Tissues Is Regulated by Distinct DNA Sequences. ５7 1.3.6. Sequence Substitution Inside the SPE Region Abolishes Sporophytic Expression, and Binds the BPC3 Transcription Factor ６4 1.3.7. Overlapping 15 and 47 Base Pair Regions Are Necessary for DME Expression in the Central and Vegetative Cells, Respectively. ６9 1.3.8. The 15 bp CCE Sequence, Shared by the VCE, Is Required for DME Expression and Is Predicted to Bind Several Key Transcription Factors. 72 1.4. DISCUSSION ７9 2. CHAPTER II. INTERACTION BETWEEN DNA DEMETHYLASE FAMILY MEMBERS IN ARABIDOPSIS THALIANA ８8 2.1 INTRODUCTION ８9 2.2 MATERIALS AND METHODS 92 2.2.1. Plant materials and growth conditions 92 2.2.2. Gene expression analysis 92 2.3 RESULTS 94 2.3.1 Strong allele dme-2 homozygous mutants are able to be generate by cross with weak allele dme-1 mutants. 94 2.3.2. Backcrossing of Ler dme-2 homozygous mutant with Col-gl dme-2 heterozygous mutant is not able to eliminate Ler genome. 103 2.3.3. Mutant allele of DNA demethylase family gene can rescue dme-mediated seed abortion partially, and abolish the rescue 111 2.3.4. It is altered that interaction of ros1-3, dml2-1 and dml3-1 in +46 cDME; dme-2 Col background １25 2.4 DISCUSSION 133 3. REFERENCES 140 4. ABSTRACT IN KOREAN 국문초록 148Docto

애기장대에서 DEMETER와 상호작용하는 인자의 선별

학위논문(석사)--서울대학교 대학원 :자연과학대학 생명과학부,2019. 8. 최연희.DNA methylation and demethylation plays significant roles in regulating gene expression on variable stress reactions and TEs silencing. A DEMETER (DME), an active DNA demethylase in plants, is expressed in the central cell of the female gametophyte in Arabidopsis required for seed development and has glycosylase activity that can actively remove 5-mC that is replaced by cytosine via base excision pathway. DME induces maternal allele expression of the imprinted MEDEA (MEA) polycomb gene and the DNA glycosylase activity of DME leads to the DNA demethylation on its targets. In plants, there is no massive methylation reprogramming in embryo like mammal but instead, the companion cells whose DNA contents are not inherited to the next generation go through global demethylation and the hypomethylated state is maintained in late endosperm stage in which DME is no longer expressed. Despite all these distinct significances, little is known about the interactors that associate with DME. Here, to identify the interacting partners of DME, I used Bimolecular Fluorescence Complementation (BiFC). From 83 genes that have been confirmed by the Yeast Two-Hybrid system that interact with DME, I primarily chose 18 candidates to test interaction. While examining these 18 candidates by BiFC, AT5G37930, AT5G60980, AT1G70620, AT5G23090 and the C terminal part of AT1G20960 showed fluorescent signals when it was co-transfected with DME in Arabidopsis Col-0 protoplasts. These interactors contain either E3-ubiquitin ligase activity, RNA binding motif, homologous feature of transcription factor which is related to histone acetylation, or are related to RNA splicing. To further understand its relation with DME in plants one candidate, At1g20960 that is related to RNA splicing, was chosen and its mutant was crossed with dme mutant for phenotyping analyses. dme mutant allele transmission, segregation and seed abortion ratio shown in dme single mutant were not changed in double mutants. Therefore, by doing further experiments, using different candidates, this study would give some specific and clear perspectives and contribute to widen the knowledge by identifying a novel protein involved in the DME demethylation pathway in plants.DNA의 메틸화와 디메틸화는 유전자의 발현을 조절하여 생물체가 직면하는 변화하는 주변 환경과 다양한 스트레스 상황에 대한 적절한 반응을 가능하게 하며 전이 인자의 발현을 억제하는 등의 역할을 하므로 매우 중요하다. DEMETER(DME)는 식물에서 작용하는 DNA 글리코실화 효소로 애기장대 암배우체의 중심세포에서 발현하며 종자의 발달에 필수적이다. DME는 염기절제회복방식을 통해 5-mC를 직접 제거하고 시토신으로 치환하는 방식의 디메틸화를 매개한다. 식물의 배에서는 포유동물과 같은 대규모의 메틸레이션 리프로그래밍이 일어나지 않는 대신 다음 세대로 유전 정보가 전달되지 않는 생식세포나 배의 주변 세포에서 전반적인 디메틸레이션이 일어나고 이러한 하이포메틸레이션 상태가 DME가 더 이상 발현하지 않는 후기 배주까지 유지된다. 이러한 중요성에도 불구하고 DME의 디메틸레이션 과정에서 DME와 상호작용하는 인자들에 대한 연구는 많이 알려진 바가 없다. 본 연구에서는 DME와 상호작용하는 인자들을 밝히기 위하여 이분자형광상보기법(Bimolecular Fluorescence Complementation)을 이용하였다. 효모이중잡종화(Yeast Two-Hybrid)를 이용한 선행연구를 통해 밝혀진 DME와 상호작용하는 83개의 유전자 중 18개의 후보 유전자를 우선적으로 선정하였고 그 중 AT5G37930, AT5G60980, AT1G70620, AT5G23090, 그리고 AT1G20960의 C 말단부가 애기장대 야생종(Col-0)의 원형질체에 DME와 함께 트랜스펙션되었을 때 형광 신호를 보이는 것을 확인할 수 있었다. 이 유전자들은 E3 유비퀴틴 라이게이즈 활성을 가지거나 RNA 결합 모티브를 가지며 히스톤 아세틸레이션과 관계된 전사인자의 상동 유전자를 포함한다. 추가적으로 이분자형광상보기법을 통해 밝혀진 유전자들과 DME의 관계에 대한 이해를 위해 RNA 스플라이싱에 관여하는 Brr2a를 암호화하는 유전자인 At1g20960을 이용하여 돌연변이 연구를 진행하였다. At1g20960의 T-DNA 삽입 돌연변이체인 emb1507-1 식물체를 ¬dme-2 돌연변이체와 교배하여 얻은 이중이형접합 돌연변이를 이용하여 종자의 발달단계 및 종자유산 여부와 돌연변이 대립유전자의 분리비, 트랜스미션의 변화를 중심으로 표현형을 분석한 결과 dme-2 단일 이형접합돌연변이체와 비교했을 때 해당 표현형들에서의 주목할만한 변화를 관찰할 수는 없었다. 때문에 식물에서 DME의 디메틸레이션 과정에 관여하는 새로운 인자를 밝혀냄으로써 이에 대한 구체적인 이해를 더하기 위해서는 추가적인 실험을 통해 분자생물학적 관점에서의 표현형 분석이 필요하며 At1g20960 외에 나머지 후보 유전자들에 대한 연구 역시 필요하다.ABSTRACT ⅰ TABLES OF CONTENTS ⅲ LIST OF FIGURES ⅴ LIST OF TABLES ⅵ BACKGROUNDS 1 1. DNA Methylation in Arabidopsis 1 2. DNA demethylation and DEMETER 4 3. Purpose of this Study 7 CHAPTER ONE: Interaction Partner Screening for DEMETER using Bimolecular Fluorescence Complementation(BiFC) Assay 8 1. Introduction 9 2. Materials and methods 12 2.1. Plant material and growth conditions 12 2.2. Previous yeast two-hybrid data 12 2.3. List-up for BiFC assay 15 2.4. Cloning for BiFC assay 15 2.5. BiFC assay 21 3. Results and discussion 23 3.1. Cloning for BiFC assay 23 3.2. 5 genes were identified as interactors of DME through BiFC 25 CHAPTER TWO: Phenotypic Study for the DME interactors 31 1. Introduction 32 2. Materials and methods 35 2.1. Plant material and growth conditions 35 2.2. Seed-set analysis and whole-mount clearing 35 3. Results and discussion 38 3.1. EMB/emb1507-1 seed phenotype 38 3.2. EMB/emb1507-1;DME/dme-2 double heterozygote mutant seed phenotype 38 3.3. Transmission of emb and dme alleles in EMB/emb1507-1;DME/dme-2 mutant plants 41 CONCLUSION 43 REFERENCE 46 ABSTRACT IN KOREAN 52Maste

Genetic and cytological analyses of four partial-sterile mutants in soybean (Glycine max L. Merr.)

Four partial-sterile soybean mutants recovered from a transposon tagging study were the subject of this research. Soybean partial-sterile mutants 1, 2, 3, and 4 (PS-1, PS-2, PS-3, and PS-4) are characterized by reduced number of seed per pods. The objectives were to study the inheritance, linkage, allelism, and reproductive biology of the PS\u27s mutants. For inheritance and linkage tests the PS\u27s mutants were crossed to Harosoy-w[subscript]4, and to chlorophyll-deficient mutants CD-1 and CD-5, also recovered from the tagging study. For allelism tests reciprocal crosses were made between PS\u27s mutants. The gene in PS-1 is a single recessive gene, which was inherited in a 3:1 ratio. The genes in PS-2, PS-3, and PS-4 were inherited in a 1:1 ratio. Reciprocal crosses made between normal plants from PS-2, PS-3, PS-4 with \u27BSR 101\u27 indicated that the PS\u27s were homozygous for normal chromosome structure. Linkage results from F[subscript]2 and F[subscript]3 generations indicated that the gene for partial sterility in the PS\u27s was not linked to the w[subscript]4 locus or to the CD-1 or CD-5 mutants. Allelism test showed that the gene in PS-1 was nonallelic to the gene in PS-2, PS-3, and PS-4. The allelism test also indicated that the gene for partial sterility in PS-2, PS-3, and PS-4 was not transmitted to the next generation when PS-2, PS-3 and PS-4 mutants were used as female parent. Results of pollen grains stained with iodine potassium iodide, differential staining, and fluorescein diacetate, from partial-sterile plants of PS\u27s mutants, indicated no difference in stainability, morphology, and fluorescence compared to pollen grains from normal plants. These results suggested that the pollen grains from PS\u27s mutants were fully viable. Megagametogenesis indicated that the ovule abortion in PS-1 mutant was due to abnormalities associated with polar nuclei/secondary endosperm nucleus. The failure of double fertilization, the absence of endosperm development and lack of nutrients in the PS-1 ovule mutant lead to early embryo abortion. PS-2, PS-3, and PS-4 had normal megagametogenesis which was identical to ovule development in normal plants. At anthesis, the embryo sac had the egg apparatus, with two synergids and egg cell, and the secondary endosperm nucleus. Thus the lack of fertilization in these mutants was probably due a specific gene already imprinted in the gametes and it was not due to failure of pollen-tube growth

GAMETOPHYTE AND SPOROPHYTE CROSSTALK DURING FERTILIZATION IN ARABIDOPSIS

Arabidopsis thaliana seeds comprise three tissues, which are genetically and functionally distinct. However, in order to ensure a correct seed development, a communication and coordination of them is strictly necessary. The seed coat is a five-layer tissue surrounding the female gametophyte. In the stk abs double mutant only four layers form this tissue: the endothelium, the innermost one, does not differentiate. Since the endothelium is a sporophytic layer directly in contact with the gametophyte before the fertilization and with the endosperm after the fertilization, we hypothesized a possible role of this layer in the communication between those tissues. In this work, the role of the endothelium has been investigated, as well as the role of the STK and ABS, the two MADS box transcription factors required for endothelium differentiation. An additional interesting phenotype of the stk abs double mutant is the excessive amount of starch accumulating in the central cell of the female gametophyte. This might causes the partial sterility described for the double mutant. Combining stk abs with the gpt1 mutant, whose mature ovules do not accumulate starch, we found a partial recovery of the phenotype; this result support the idea that the excess of starch in the double mutant could physically prevent the correct fertilization process

DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.

Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING 1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion