Rice SNF2 family helicase ENL1 is essential for syncytial endosperm development

The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human Plk1‐Interacting Checkpoint Helicase (PICH), which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1‐Venus (enhanced yellow fluorescent protein (YFP)) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1‐Venus is also detected on a thread‐like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue‐dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice.     

Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in Arabidopsis

Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size.     

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.     

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 rice LEC1-like transcription factor OsNF-YB9 interacts with SPK,an endosperm-specific sucrose synthase protein kinase,and functions in seed development

LEAFY COTYLEDON1 (LEC1), a NUCLEAR FACTOR-Y (NF-Y) family member, plays a critical role in embryogenesis and seed development in Arabidopsis. Previous studies have shown that rice OsNF-YB9 and OsNF-YB7 are homologous to Arabidopsis LEC1. However, the functions of LEC1-like genes in rice remain unclear. Here we report that OsNF-YB9 and OsNF-YB7 display sub-functionalization in rice. We demonstrate that OsNF-YB7 is expressed mainly in the embryo, whereas OsNF-YB9 is preferentially expressed in the developing endosperm. Heterologous expression of either OsNF-YB9 or OsNF-YB7 in Arabidopsis lec1-1 was able to complement the lec1-1 defects. We failed to generate osnf-yb7 homozygous mutants due to lethality caused by OsNF-YB7 defects. Loss of OsNF-YB9 function caused abnormal seed development: seeds were longer, narrower and thinner and exhibited a higher chalkiness ratio. Furthermore, the expression of genes related to starch synthesis was deregulated in osnf-yb9. OsNF-YB9 could interact with SPK, a sucrose synthase protein kinase that is predominantly expressed in rice endosperm. Knockout of SPK resulted in chalky seeds similar to those observed in the osnf-yb9 mutants. Ectopic expression of OsNF-YB9 in both rice and Arabidopsis resulted in unhealthy plants with small seeds. Taken together, these results suggest a critical role for OsNF-YB9 in rice seed development.     

An interpretation of genomic balance in seed formation in interspecific hybrids of Solanum

Summary To investigate the mechanisms of seed failure in intraspecific and interspecific crosses of Solanum two diploid, S. commersonii and Group Phureja, and one tetraploid species, S. acaule, species were crossed and the seeds were analyzed for embryo and endosperm development. Many seeds of certain crosses observed seven days after pollinations were found to contain abnormal embryos and degenerating endosperms. In some cases seeds contained an embryo with no endosperm, or an endosperm with no embryo. Other interspecific crosses which were predicted to fail actually produced seeds with normally developed embryos and endosperms. To further characterize the intra- and interspecific embryos and endosperms the nuclear DNA was measured. There are several ways to explain the ploidy levels of embryos and endosperms among the crosses, the occurrence of unreduced gametes in some cases and genomic instability in other cases. The latter resulted in chromosome loss at meiotic and mitotic divisions. Genomic balance in interspecific seeds is critical to both embryo and endosperm development.Scientific Journal Series Article No. 14636 of the Minnesota Experiment Station     

The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development

We describe in this report a novel class of mutants that should facilitate the identification of genes required for progression through the mitotic cell cycle during seed development in angiosperms. Three non-allelic titan ( ttn ) mutants with related but distinct phenotypes are characterized. The common feature among these mutants is that endosperm nuclei become greatly enlarged and highly polyploid. The mutant embryo is composed of a few giant cells in ttn1 , several small cells in ttn2 , and produces a normal plant in ttn3 . Condensed chromosomes arrested at prophase of mitosis are found in the free nuclear endosperm of ttn1 and ttn2 seeds. Large mitotic figures with excessive numbers of chromosomes are visible in ttn3 endosperm. The ttn1 mutation appears to disrupt cytoskeletal organization because endosperm nuclei fail to migrate to the chalazal end of the seed. How double fertilization leads to the establishment of distinct patterns of mitosis and cytokinesis in the embryo and endosperm is a central question in plant reproductive biology. Molecular isolation of TITAN genes should help to answer this question, as well as related issues concerning cell cycle regulation, chromosome movement and endosperm identity in angiosperms.     

Fructokinase is required for carbon partitioning to cellulose in aspen wood

DNA damage checkpoints delay mitotic cell‐cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant‐specific cyclin‐dependent kinase (CDK) that is required for the G₂/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G₂ arrest gives cells time to repair damaged DNA. G₂ arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down‐regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.     

Apomixis in <Emphasis Type="Italic">Eulaliopsis binata</Emphasis>: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

The progamic phase, embryo and endosperm development in an intraspecific Tulipa gesneriana L. cross and in the incongruent interspecific cross T. gesneriana × T. agenensis DC.

 The development of the embryo and endosperm has been investigated in an intraspecific Tulipa gesneriana cross and in the incongruent cross T. gesneriana ×T. agenensis at intervals of 10 days, from 12 to 82 days after pollination (DAP). In both tulip crosses, the zygote gives rise to an apparently undifferentiated cell mass, the proembryonal cell mass, on which a suspensor then develops. Subsequently, a globular embryo is formed on top of the suspensor. This embryo finally elongates, giving rise to a spindle-shaped embryo. The cellular endosperm fills the whole embryo sac in mature seeds, except for a region immediately around the embryo. In both crosses, aberrant developments were found. In the intraspecific T. gesneriana cross, the pollen tubes did not open in a number of ovules. In other ovules, the pollen tubes seemed to have opened, but an embryo or endosperm was not found or only endosperm was observed. In the cross T. gesneriana ×T. agenensis, fewer pollen tubes entered the ovules than in the intraspecific T. gesneriana cross. The ovules with embryo and endosperm formation of the incongruent interspecific cross showed, in general, retarded development in comparison with the intraspecific T. gesneriana cross. The first globular embryos and spindle-shaped embryos were found at the later fixation dates and the relatively lower number of spindle-shaped embryos at 82 DAP had a shorter average length. The number of ovules with deformations in embryo and/or endosperm development was also higher in the cross T. gesneriana × T. agenensis in comparison with the intraspecific T. gesneriana cross. Between 87% and 100% of the ovules with embryo and endosperm development showed normal development in the intraspecific T. gesneriana cross, while in the incongruent interspecific cross, from 22 DAP, between 17% and 56% of the ovules showed normal development. Of those ovules with aberrations in embryo and/or endosperm formation, about 80% had a deformed endosperm, of which more than 50% also contained a deformed embryo. Embryos of the incongruent cross might be saved by the application of embryo rescue techniques. Received: 10 December 1996 / Revision accepted: 23 April 1997     

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8 days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8‐DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1‐altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over‐representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage‐specific and SHB1‐induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.