Chalkiness and premature controlled by energy homeostasis in OsNAC02 Ko-mutant during vegetative endosperm development.

BACKGROUND: Chalkiness is a common phenotype induced by various reasons, such as abiotic stress or the imbalance of starch synthesis and metabolism during the development period. However, the reason mainly for one gene losing its function such as NAC (TFs has a large family in rice) which may cause premature is rarely known to us. RESULTS: The Ko-Osnac02 mutant demonstrated an obviously early maturation stage compared to the wild type (WT) with 15 days earlier. The result showed that the mature endosperm of Ko-Osnac02 mutant exhibited chalkiness, characterized by white-core and white-belly in mature endosperm. As grain filling rate is a crucial factor in determining the yield and quality of rice (Oryza sativa, ssp. japonica), it's significant that mutant has a lower amylose content (AC) and higher soluble sugar content in the mature endosperm. Interestingly among the top DEGs in the RNA sequencing of N2 (3DAP) and WT seeds revealed that the OsBAM2 (LOC_Os10g32810) expressed significantly high in N2 mutant, which involved in Maltose up-regulated by the starch degradation. As Prediction of Protein interaction showed in the chalky endosperm formation in N2 seeds (3 DAP), seven genes were expressed at a lower-level which should be verified by a heatmap diagrams based on DEGs of N2 versus WT. The Tubulin genes controlling cell cycle are downregulated together with the MCM family genes MCM4 ( ↓), MCM7 ( ↑), which may cause white-core in the early endosperm development. In conclusion, the developing period drastically decreased in the Ko-Osnac02 mutants, which might cause the chalkiness in seeds during the early endosperm development. CONCLUSIONS: The gene OsNAC02 which controls a great genetic co-network for cell cycle regulation in early development, and KO-Osnac02 mutant shows prematurity and white-core in endosperm.

Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation.

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm.

Transcriptomic analysis of developing sorghum grains to detect genes related to cell wall biosynthesis and remodelling.

OBJECTIVE: Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important grain produced in the world. Interest for cultivating sorghum is increasing all over the world in the context of climate change, due to its low input and water requirements. Like other cultivated cereals, sorghum has significant nutritional value thanks to its protein, carbohydrate and dietary fiber content, these latter mainly consisting of cell wall polysaccharides. This work describes for the first time a transcriptomic analysis dedicated to identify the genes involved in the biosynthesis and remodelling of cell walls both in the endosperm and outer layers of sorghum grain during its development. Further analysis of these transcriptomic data will improve our understanding of cell wall assembly, which is a key component of grain quality. DATA DESCRIPTION: This research delineates the steps of our analysis, starting with the cultivation conditions and the grain harvest at different stages of development, followed by the laser microdissection applied to separate the endosperm from the outer layers. It also describes the procedures implemented to generate RNA libraries and to obtain a normalized and filtered table of transcript counts, and finally determine the number of putative cell wall-related genes already listed in literature.

Lipidomics studies reveal dynamic changes in polar lipids of developing endosperm of oat and wheat varieties with differing oil contents.

Polar lipids have biosynthetic pathways which intersect and overlap with triacylglycerol biosynthesis; however, polar lipids have not been well characterized in the developing endosperms of oat with high oil accumulation. The polar lipids in endosperms of oat and wheat varieties having different oil contents were analyzed and compared at different developmental stages. Our study shows that the relative contents of polar lipid by mass were decreased more slowly in wheat than in oat. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, which showed similar abundance and gradual decreases during endosperm development in oat and wheat, while lysophospholipids were noticeably higher in oat. Monogalactosyldiacylglycerol showed a gradual increase in wheat and a decrease in oat during endosperm development. The relative contents of some polar lipid species and their unsaturation index were significantly different in their endosperms. These characteristics of polar lipids might indicate an adaption of oat to accommodate oil accumulation.

Auxin receptor OsTIR1 mediates auxin signaling during seed filling in rice.

Cereal endosperm represents the most important source of the world's food. Nevertheless, the molecular mechanisms behind sugar import into rice (Oryza sativa) endosperm and their relationship with auxin signaling are poorly understood. Here, we report that auxin transport inhibitor response 1 (TIR1) plays an essential role in rice grain yield and quality via modulating sugar transport into endosperm. The fluctuations of OsTIR1 transcripts parallel to the early stage of grain expansion among those of the 5 TIR1/AFB (auxin-signaling F-box) auxin co-receptor proteins. OsTIR1 is abundantly expressed in ovular vascular trace, nucellar projection, nucellar epidermis, aleurone layer cells, and endosperm, providing a potential path for sugar into the endosperm. Compared to wild-type (WT) plants, starch accumulation is repressed by mutation of OsTIR1 and improved by overexpression of the gene, ultimately leading to reduced grain yield and quality in tir1 mutants but improvement in overexpression lines. Of the rice AUXIN RESPONSE FACTOR (ARF) genes, only the OsARF25 transcript is repressed in tir1 mutants and enhanced by overexpression of OsTIR1; its highest transcript is recorded at 10 d after fertilization, consistent with OsTIR1 expression. Also, OsARF25 can bind the promoter of the sugar transporter OsSWEET11 (SWEET, sugars will eventually be exported transporter) in vivo and in vitro. arf25 and arf25/sweet11 mutants exhibit reduced starch content and seed size (relative to the WTs), similar to tir1 mutants. Our data reveal that OsTIR1 mediates sugar import into endosperm via the auxin signaling component OsARF25 interacting with sugar transporter OsSWEET11. The results of this study are of great significance to further clarify the regulatory mechanism of auxin signaling on grain development in rice.

Cell-type proteomic and metabolomic resolution of early and late grain filling stages of wheat endosperm.

The nutritional value of wheat grains, particularly their protein and metabolite composition, is a result of the grain-filling process, especially in the endosperm. Here, we employ laser microdissection (LMD) combined with shotgun proteomics and metabolomics to generate a cell type-specific proteome and metabolome inventory of developing wheat endosperm at the early (15 DAA) and late (26 DAA) grain-filling stages. We identified 1803 proteins and 41 metabolites from four different cell types (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE) and endosperm transfer cells (ETCs). Differentially expressed proteins were detected, 67 in the AL, 31 in the SA, 27 in the SE and 50 in the ETCs between these two-time points. Cell-type accumulation of specific SUT and GLUT transporters, sucrose converting and starch biosynthesis enzymes correlate well with the respective sugar metabolites, suggesting sugar upload and starch accumulation via nucellar projection and ETC at 15 DAA in contrast to the later stage at 26 DAA. Changes in various protein levels between AL, SA and ETC support this metabolic switch from 15 to 26 DAA. The distinct spatial and temporal abundances of proteins and metabolites revealed a contrasting activity of nitrogen assimilation pathways, e.g. for GOGAT, GDH and glutamic acid, in the different cell types from 15 to 26 DAA, which can be correlated with specific protein accumulation in the endosperm. The integration of cell-type specific proteome and metabolome data revealed a complex metabolic interplay of the different cell types and a functional switch during grain development and grain-filling processes.

Dynamic transcriptome landscape of maize pericarp development.

As a maternal tissue, the pericarp supports and protects for other components of seed, such as embryo and endosperm. Despite the importance of maize pericarp in seed, the genome-wide transcriptome pattern throughout maize pericarp development has not been well characterized. Here, we developed RNA-seq transcriptome atlas of B73 maize pericarp development based on 21 samples from 5 days before fertilization (DBP5) to 32 days after fertilization (DAP32). A total of 25 346 genes were detected in programming pericarp development, including 1887 transcription factors (TFs). Together with pericarp morphological changes, the global clustering of gene expression revealed four developmental stages: undeveloped, thickening, expansion and strengthening. Coexpression analysis provided further insights on key regulators in functional transition of four developmental stages. Combined with non-seed, embryo, endosperm, and nucellus transcriptome data, we identified 598 pericarp-specific genes, including 75 TFs, which could elucidate key mechanisms and regulatory networks of pericarp development. Cell wall related genes were identified that reflected their crucial role in the maize pericarp structure building. In addition, key maternal proteases or TFs related with programmed cell death (PCD) were proposed, suggesting PCD in the maize pericarp was mediated by vacuolar processing enzymes (VPE), and jasmonic acid (JA) and ethylene-related pathways. The dynamic transcriptome atlas provides a valuable resource for unraveling the genetic control of maize pericarp development.

DEFECTIVE KERNEL 56 functions in mitochondrial RNA editing and maize seed development.

Proper seed development is essential for achieving grain production, successful seed germination, and seedling establishment in maize (Zea mays). In the past few decades, pentatricopeptide repeat (PPR) proteins have been proven to play an essential role in regulating the development of maize kernels through posttranscriptional RNA modification of mitochondrial genes. However, the underlying mechanisms remain largely unknown. Here, we characterized a mutant of DEFECTIVE KERNEL 56 (DEK56) with defective kernels that exhibited arrested development of both the embryo and endosperm. Accordingly, we isolated DEK56 through a map-based cloning strategy and found that it encoded an E subgroup PPR protein located in the mitochondria. Dysfunction of DEK56 resulted in altered cytidine (C)-to-uridine (U) editing efficiency at 48 editing sites across 21 mitochondrial transcripts. Notably, the editing efficiency of the maturase-related (matR)-1124 site was substantially reduced or abolished in the dek56 mutant. Furthermore, we found that the splicing efficiency of NADH dehydrogenase subunit 4 (nad4) Introns 1 and 3 was substantially reduced in dek56 kernels, which might be a consequence of the defective MatR function. Through a protein-protein interaction test, we hypothesized that DEK56 carries out its function by recruiting the PPR-DYW protein PPR motif, coiled-coil, and DYW domain-containing protein 1 (PCW1). This interaction is facilitated by Multiple Organellar RNA Editing Factors (ZmMORFs) and Glutamine-Rich Protein 23 (ZmGRP23). Based on these findings, we developed a working model of PPR-mediated mitochondrial processing that plays an essential role in the development of maize kernels. The present study will further broaden our understanding of PPR-mediated seed development and provide a theoretical basis for maize improvement.

Triacylglycerol lipase, OsSG34, plays an important role in grain shape and appearance quality in rice.

Optimal grain-appearance quality is largely determined by grain size. To date, dozens of grain size-related genes have been identified. However, the regulatory mechanism of slender grain formation is not fully clear. We identified the OsSG34 gene by map-based cloning. A 9-bp deletion on 5'-untranslated region of OsSG34, which resulted in the expression difference between the wild-type and sg34 mutant, led to the slender grains and good transparency in sg34 mutant. OsSG34 as an α/ß fold triacylglycerol lipase affected the triglyceride content directly, and the components of cell wall indirectly, especially the lignin between the inner and outer lemmas in rice grains, which could affect the change in grain size by altering cell proliferation and expansion, while the change in starch content and starch granule arrangement in endosperm could affect the grain-appearance quality. Moreover, the OsERF71 was identified to directly bind to cis-element on the mutant site, thereby regulating the OsSG34 expression. Knockout of three OsSG34 homologous genes resulted in slender grains as well. The study demonstrated OsSG34, involved in lipid metabolism, affected grain size and quality. Our findings suggest that the OsSG34 gene could be used in rice breeding for high yield and good grain-appearance quality via marker-assisted selection and gene-editing approaches.

The conservation of allelic DNA methylation and its relationship with imprinting in maize.

Genomic imprinting refers to allele-specific expression of genes depending on parental origin, and it is regulated by epigenetic modifications. Intraspecific allelic variation for imprinting has been detected; however, the intraspecific genome-wide allelic epigenetic variation in maize and its correlation with imprinting variants remain unclear. Here, three reciprocal hybrids were generated by crossing Zea mays inbred lines CAU5, B73, and Mo17 in order to examine the intraspecific conservation of the imprinted genes in the kernel. The majority of imprinted genes exhibited intraspecific conservation, and these genes also exhibited interspecific conservation (rice, sorghum, and Arabidopsis) and were enriched in some specific pathways. By comparing intraspecific allelic DNA methylation in the endosperm, we found that nearly 15% of DNA methylation existed as allelic variants. The intraspecific whole-genome correlation between DNA methylation and imprinted genes indicated that DNA methylation variants play an important role in imprinting variants. Disruption of two conserved imprinted genes using CRISPR/Cas9 editing resulted in a smaller kernel phenotype. Our results shed light on the intraspecific correlation of DNA methylation variants and variation for imprinting in maize, and show that imprinted genes play an important role in kernel development.

Type I MADS-box transcription factor TaMADS-GS regulates grain size by stabilizing cytokinin signalling during endosperm cellularization in wheat.

Grain size is one of the important traits in wheat breeding programs aimed at improving yield, and cytokinins, mainly involved in cell division, have a positive impact on grain size. Here, we identified a novel wheat gene TaMADS-GS encoding type I MADS-box transcription factor, which regulates the cytokinins signalling pathway during early stages of grain development to modulate grain size and weight in wheat. TaMADS-GS is exclusively expressed in grains at early stage of seed development and its knockout leads to delayed endosperm cellularization, smaller grain size and lower grain weight. TaMADS-GS protein interacts with the Polycomb Repressive Complex 2 (PRC2) and leads to repression of genes encoding cytokinin oxidase/dehydrogenases (CKXs) stimulating cytokinins inactivation by mediating accumulation of the histone H3 trimethylation at lysine 27 (H3K27me3). Through the screening of a large wheat germplasm collection, an elite allele of the TaMADS-GS exhibits higher ability to repress expression of genes inactivating cytokinins and a positive correlation with grain size and weight, thus representing a novel marker for breeding programs in wheat. Overall, these findings support the relevance of TaMADS-GS as a key regulator of wheat grain size and weight.

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana.

BACKGROUND: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. RESULTS: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. CONCLUSIONS: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-ß-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

Mutation in Polycomb repressive complex 2 gene OsFIE2 promotes asexual embryo formation in rice.

Prevention of autonomous division of the egg apparatus and central cell in a female gametophyte before fertilization ensures successful reproduction in flowering plants. Here we show that rice ovules of Polycomb repressive complex 2 (PRC2) Osfie1 and Osfie2 double mutants exhibit asexual embryo and autonomous endosperm formation at a high frequency, while ovules of single Osfie2 mutants display asexual pre-embryo-like structures at a lower frequency without fertilization. Earlier onset, higher penetrance and better development of asexual embryos in the double mutants compared with those in Osfie2 suggest that the autonomous endosperm facilitated asexual embryo development. Transcriptomic analysis showed that male genome-expressed OsBBM1 and OsWOX8/9 were activated in the asexual embryos. Similarly, the maternal alleles of the paternally expressed imprinted genes were activated in the autonomous endosperm, suggesting that the egg apparatus and central cell convergently adopt PRC2 to maintain the non-dividing state before fertilization, possibly through silencing of the maternal alleles of male genome-expressed genes.

Transcriptome-wide identification and characterization of genes exhibit allele-specific imprinting in maize embryo and endosperm.

BACKGROUND: Genomic imprinting refers to a subset of genes that are expressed from only one parental allele during seed development in plants. Studies on genomic imprinting have revealed that intraspecific variations in genomic imprinting expression exist in naturally genetic varieties. However, there have been few studies on the functional analysis of allele-specific imprinted genes. RESULTS: Here, we generated three reciprocal crosses among the B73, Mo17 and CAU5 inbred lines. Based on the transcriptome-wide analysis of allele-specific expression using RNA sequencing technology, 305 allele-specific imprinting genes (ASIGs) were identified in embryos, and 655 ASIGs were identified in endosperms from three maize F1 hybrids. Of these ASIGs, most did not show consistent maternal or paternal bias between the same tissue from different hybrids or different tissues from one hybrid cross. By gene ontology (GO) analysis, five and eight categories of GO exhibited significantly higher functional enrichments for ASIGs identified in embryo and endosperm, respectively. These functional categories indicated that ASIGs are involved in intercellular nutrient transport, signaling pathways, and transcriptional regulation of kernel development. Finally, the mutation and overexpression of one ASIG (Zm305) affected the length and width of the kernel. CONCLUSION: In this study, our data will be helpful in gaining further knowledge of genes exhibiting allele-specific imprinting patterns in seeds. The gain- and loss-of-function phenotypes of ASIGs associated with agronomically important seed traits provide compelling evidence for ASIGs as crucial targets to optimize seed traits in crop plants.

Dynamics of imprinted genes and their epigenetic mechanisms in castor bean seed with persistent endosperm.

Genomic imprinting refers to parent-of-origin-dependent gene expression and primarily occurs in the endosperm of flowering plants, but its functions and epigenetic mechanisms remain to be elucidated in eudicots. Castor bean, a eudicot with large and persistent endosperm, provides an excellent system for studying the imprinting. Here, we identified 131 imprinted genes in developing endosperms and endosperm at seed germination phase of castor bean, involving into the endosperm development, accumulation of storage compounds and specially seed germination. Our results showed that the transcriptional repression of maternal allele of DNA METHYLTRANSFERASE 1 (MET1) may be required for maternal genome demethylation in the endosperm. DNA methylation analysis showed that only a small fraction of imprinted genes was associated with allele-specific DNA methylation, and most of them were closely associated with constitutively unmethylated regions (UMRs), suggesting a limited role for DNA methylation in controlling genomic imprinting. Instead, histone modifications can be asymmetrically deposited in maternal and paternal genomes in a DNA methylation-independent manner to control expression of most imprinted genes. These results expanded our understanding of the occurrence and biological functions of imprinted genes and showed the evolutionary flexibility of the imprinting machinery and mechanisms in plants.