Mutants in the imprinted PICKLE RELATED 2 gene suppress seed abortion of fertilization independent seed class mutants and paternal excess interploidy crosses in Arabidopsis

Endosperm cellularization is essential for embryo development and viable seed formation. Loss of function of the FERTILIZATION INDEPENDENT SEED (FIS) class Polycomb genes, which mediate trimethylation of histone H3 lysine27 (H3K27me3), as well as imbalanced contributions of parental genomes interrupt this process. The causes of the failure of cellularization are poorly understood. In this study we identified PICKLE RELATED 2 (PKR2) mutations which suppress seed abortion in fis1/mea by restoring endosperm cellularization. PKR2, a paternally expressed imprinted gene (PEG), encodes a CHD3 chromatin remodeler. PKR2 is specifically expressed in syncytial endosperm and its maternal copy is repressed by FIS1. Seed abortion in a paternal genome excess interploidy cross was also partly suppressed by pkr2. Simultaneous mutations in PKR2 and another PEG, ADMETOS (ADM), additively rescue the seed abortion in fis1 and in the interploidy cross, suggesting that PKR2 and ADM modulate endosperm cellularization independently and reproductive isolation between plants of different ploidy is established by imprinted genes. Genes upregulated in fis1 and downregulated in the presence of pkr2 are enriched in glycosyl‐hydrolyzing activity, while genes downregulated in fis1 and upregulated in the presence of pkr2 are enriched with microtubule motor activity, consistent with the cellularization patterns in fis1 and the suppressor line. The antagonistic functions of FIS1 and PKR2 in modulating endosperm development are similar to those of PICKLE (PKL) and CURLY LEAF (CLF), which antagonistically regulate root meristem activity. Our results provide further insights into the function of imprinted genes in endosperm development and reproductive isolation.     

Comparative genetic analysis of the Aegilops longissima and Ae. sharonensis genomes with common wheat

Aegilops longissima Schw. et Musch. (2n= 2x=14, S^lS^l) and Aegilops sharonensis Eig. (2n=2x=14, S^lS^l) are diploid species belonging to the section Sitopsis in the tribe Triticeae and potential donors of useful genes for wheat breeding. A comparative genetic map was constructed of the Ae. longissima genome, using RFLP probes with known location in wheat. A high degree of conserved colinearity was observed between the wild diploid and basic wheat genome, represented by the D genome of cultivated wheat. Chromosomes 1S^l, 2S^l, 3S^l, 5S^l and 6S^l are colinear with wheat chromosomes 1D, 2D, 3D, 5D and 6D, respectively. The analysis confirmed that chromosomes 4S^l and 7S^l are translocated relative to wheat. The short arms and major part of the long arms are homoeologous to most of wheat chromosomes 4D and 7D respectively, but the region corresponding to the distal segment of 7D was translocated from 7S^lL to the distal region of 4S^lL. The map and RFLP markers were then used to analyse the genomes and added chromosomes in a set of ’Chinese Spring’ (CS)/Ae. longissima chromosome additions. The study confirmed the availability of disomic CS/Ae. longissima addition lines for chromosomes 1S^l, 2S^l, 3S^l, 4S^l and 5S^l. An as yet unpublished set of Ae. sharonensis chromosome addition lines were also available for analysis. Due to the gametocidal nature of Ae. sharonensis chromosomes 2S^l and 4S^l, additions 1S^l, 3S^l, 5S^l, 6S^l and 7S^l were produced in a (4D)4S^l background, and 2S^l and 4S^l in a euploid wheat background. The analysis also confirmed that the 4/7 translocation found in Ae. longissima was not present in Ae. sharonensis although the two wild relatives of wheat are considered to be closely related. The phenotypes of the Ae. sharonensis addition lines are described in an Appendix. Received: 28 September 2000 / Accepted: 19 January 2001     

Diploidization and genome size change in allopolyploids is associated with differential dynamics of low‐ and high‐copy sequences

Recent advances have highlighted the ubiquity of whole‐genome duplication (polyploidy) in angiosperms, although subsequent genome size change and diploidization (returning to a diploid‐like condition) are poorly understood. An excellent system to assess these processes is provided by Nicotiana section Repandae, which arose via allopolyploidy (approximately 5 million years ago) involving relatives of Nicotiana sylvestris and Nicotiana obtusifolia. Subsequent speciation in Repandae has resulted in allotetraploids with divergent genome sizes, including Nicotiana repanda and Nicotiana nudicaulis studied here, which have an estimated 23.6% genome expansion and 19.2% genome contraction from the early polyploid, respectively. Graph‐based clustering of next‐generation sequence data enabled assessment of the global genome composition of these allotetraploids and their diploid progenitors. Unexpectedly, in both allotetraploids, over 85% of sequence clusters (repetitive DNA families) had a lower abundance than predicted from their diploid relatives; a trend seen particularly in low‐copy repeats. The loss of high‐copy sequences predominantly accounts for the genome downsizing in N. nudicaulis. In contrast, N. repanda shows expansion of clusters already inherited in high copy number (mostly chromovirus‐like Ty3/Gypsy retroelements and some low‐complexity sequences), leading to much of the genome upsizing predicted. We suggest that the differential dynamics of low‐ and high‐copy sequences reveal two genomic processes that occur subsequent to allopolyploidy. The loss of low‐copy sequences, common to both allopolyploids, may reflect genome diploidization, a process that also involves loss of duplicate copies of genes and upstream regulators. In contrast, genome size divergence between allopolyploids is manifested through differential accumulation and/or deletion of high‐copy‐number sequences.     

Molecular Mechanisms of HMW Glutenin Subunits from 1Sl Genome of Aegilops longissima Positively Affecting Wheat Breadmaking Quality

A wheat cultivar “Chinese Spring” chromosome substitution line CS-1S^l(1B), in which the 1B chromosome was substituted by 1S^l from Aegilops longissima, was developed and found to possess superior dough and breadmaking quality. The molecular mechanism of its super quality conformation is studied in the aspects of high molecular glutenin genes, protein accumulation patterns, glutenin polymeric proteins, protein bodies, starch granules, and protein disulfide isomerase (PDI) and PDI-like protein expressions. Results showed that the introduced HMW-GS 1S^l×2.3* and 1S^ly16* in the substitution line possesses long repetitive domain, making both be larger than any known x- and y-type subunits from B genome. The introduced subunit genes were also found to have a higher level of mRNA expressions during grain development, resulting in more HMW-GS accumulation in the mature grains. A higher abundance of PDI and PDI-like proteins was observed which possess a known function of assisting disulfide bond formation. Larger HMW-GS deposited in protein bodies were also found in the substitution line. The CS substitution line is expected to be highly valuable in wheat quality improvement since the novel HMW-GS are located on chromosome 1S^l, making it possible to combine with the known superior D×5+Dy10 subunits encoded by Glu-D1 for developing high quality bread wheat.     

Chromosome structure of Triticum longissimum relative to wheat

Homoeologous pairing at meiotic metaphase I was analyzed in T. longissimum x T. aestivum hybrids in order to reconfirm the homoeologous relationships of T. longissimum chromosomes to wheat. Hybrids between T. longissimum and Chinese Spring carrying the Ph1 gene or theph1b mutation, which showed low and high pairing levels, respectively, were used. Chromosome arms associated at metaphase I were identified by C-banding. The homoeology of chromosomes 1S ^l , 2S ^l , 3S ^l , 5S ^l and 6S ^l to wheat group 1,2, 3, 5, and 6 chromosomes, respectively, was confirmed. Chromsome arms 4S ^l S and 7S ^l S showed normal homoeologous relationships to wheat. The 4S ^l L arm carries a translocated segment from 7S ^l L relative to wheat. The 7S ^l L arm seldom paired, likely because this arm lost a relatively long segment and received a very short segment in the interchange with 4S ^l L. Available data suggest that translocation 4S ^l L/7S ^l L arose in the evolution of T. longissimum, which implies that this species was not the donor of the B genome of wheat.     

G9α‐dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis‐related genes in foetal mice

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol‐induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis‐related genes, and histone H3K9me3 hypomethylation was observed in alcohol‐treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol‐induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol‐induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis‐related genes (MEF2C, Cx43, ANP and β‐MHC) was observed in alcohol‐exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down‐regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and β‐MHC overexpression. These findings suggest that G9α histone methyltransferase‐mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol‐induced abnormal expression cardiomyogenesis‐related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.     

Gluten quality of bread wheat is associated with activity of RabD GTPases

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER‐to‐Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down‐regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT‐PCR to be down‐regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE‐HPLC found that a smaller proportion of HMW‐GS and large proportion of LMW‐GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS‐PAGE was seen in the transgenic grain.     

ADP‐glucose pyrophosphorylase genes,associated with kernel weight,underwent selection during wheat domestication and breeding

ADP‐glucose pyrophosphorylase, comprising two small subunits and two large subunits, is considered a key enzyme in the endosperm starch synthesis pathway in wheat (Triticum aestivum L.). Two genes, TaAGP‐S1‐7A and TaAGP‐L‐1B, were investigated in this study. Haplotypes of these genes were associated with thousand kernel weight (TKW) in different populations. Mean TKWs of favoured haplotypes were significantly higher than those of nonfavoured ones. Two molecular markers developed to distinguish these haplotypes could be used in molecular breeding. Frequencies of favoured haplotypes were dramatically increased in cultivars released in China after the 1940s. These favoured haplotypes were also positively selected in six major wheat production regions globally. Selection of AGP‐S1 and AGP‐L‐1B in wheat mainly occurred during and after hexaploidization. Strong additive effects of the favoured haplotypes of with other genes for starch synthesis were also detected in different populations.     

GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice

Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss‐of‐function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild‐type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss‐of‐function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo–endosperm size control, the genetic network of the related genes should be elucidated.     

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine‐tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late‐flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1‐like1 lsd1‐like2 (ldl1 ldl2). We found that the early‐flowering mutants sdg25, atx1 and clf interact antagonistically with the late‐flowering mutant sdg26, whereas the late‐flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS‐LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late‐flowering phenotype.     

Imprinting in Plants and Its Underlying Mechanisms

Genomic imprinting (or imprinting) refers to an epigenetic phenomenon by which the allelic expression of a gene depends on the parent of origin. It has evolved independently in placental mammals and flowering plants. In plants, imprinting is mainly found in endosperm. Recent genome-wide surveys in Arabidopsis, rice, and maize identified hundreds of imprinted genes in endosperm. Since these genes are of diverse functions, endosperm development is regulated at different regulatory levels. The imprinted expression of only a few genes is conserved between Arabidopsis and monocots, suggesting that imprinting evolved quickly during speciation. In Arabidopsis, DEMETER (DME) mediates hypomethylation in the maternal genome at numerous loci (mainly transposons and repeats) in the central cell and results in many differentially methylated regions between parental genomes in the endosperm, and subsequent imprinted expression of some genes. In addition, histone modification mediated by Polycomb group (PcG) proteins is also involved in regulating imprinting. DME-induced hypomethylated alleles in the central cell are considered to produce small interfering RNAs (siRNAs) which are imported to the egg to reinforce DNA methylation. In parallel, the activity of DME in the vegetative cell of the male gametophyte demethylates many regions which overlap with the demethylated regions in the central cell. siRNAs from the demethylated regions are hypothesized to be also transferred into sperm to reinforce DNA methylation. Imprinting is partly the result of genome-wide epigenetic reprogramming in the central cell and vegetative cell and evolved under different selective pressures.