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.     

SHB1 plays dual roles in photoperiodic and autonomous flowering

Flowering was initiated by the integration of environmental signals such as day-length with the internal development status in Arabidopsis, a facultative long-day plant. The photoperiodic flowering involves two key components, CONSTANS and FT, whereas the autonomous flowering is operated through a central quantitative floral repressor, FLC, and several other genes that act upstream of FLC. SOC1 acts downstream to integrate the flowering signals from the two pathways. Here, we report that SHB1 plays dual roles in both photoperiodic and autonomous flowering. shb1-D, a gain-of-function mutant, flowered early and shb1, a loss-of-function allele, flowered late under both long days and short days. The shb1-D mutation activated the expression of CO, FT, and SOC1 under both long and short days, and however, the co-2 mutation attenuated the shb1-D activated expression of FT and SOC1 only under long days but not short days. The shb1-D or shb1 mutations also reduced and increased, respectively, the expression of FLC under both long and short days. Transgenic remedy of FLC to wide-type level in shb1-D background also reverted shb1-D flowering and FT or SOC1 expression to wild type mostly under short days. Furthermore, the shb1-D suppression on FLC expression is likely operated through LD as ld-3 blocked this suppression and SHB1 appears to act upstream of LD. In summary, SHB1 represents signaling steps that regulate CO expression in leaves and LD or FLC expression in either leaves or shoot apical meristem, contributing to a threshold expression of SOC1 in shoot apical meristem for floral initiation.     

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.     

Seed-specific expression of seven Arabidopsis promoters

Seeds contain storage compounds, from various carbohydrates to proteins and lipids, which are synthesized during seed development. For the purposes of many plant researches or commercial applications, developing promoter systems expressing specifically in seeds or in particular constituents or tissues/compartments of seeds are indispensable. To screen genes dominantly or specifically expressed in seed tissues, we analyzed Arabidopsis ATH1 microarray data open to the public. Thirty-two candidate genes were selected and their expressions in seed tissues were confirmed by RT-PCR. Finally, seven genes were selected for promoter analysis. The promoters of seven genes were cloned into pBI101 vector and transformed into Arabidopsis to assay histochemical β-glucuronidase (GUS) activity. We found that Pro-at3g03230 promoter drove GUS expression in a chalazal endosperm, Pro-at4g27530:GUS expressed in both chalazal endosperm and embryo, Pro-at4g31830 accelerated GUS expression both in radicle and procambium, Pro-at5g10120 and Pro-at5g16460 drove GUS expression uniquely in embryo, Pro-at5g53100:GUS expressed only in endosperm, and Pro-at5g54000 promoted GUS expression in both embryo and inner integument. These promoters can be used for expressing any genes in specific seed tissues for practical application.     

Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis

We use Arabidopsis thaliana as a model to investigate coordination of cell proliferation and cell elongation in the three components that develop side by side in the seed. Two of these, the embryo and its nurturing annex, the endosperm, are placed under zygotic control and develop within the seed integument placed under maternal control. We show that integument cell proliferation and endosperm growth are largely independent from each other. By contrast, prevention of cell elongation in the integument by the mutation transparent testa glabra2 (ttg2) restricts endosperm and seed growth. Conversely, endosperm growth controlled by the HAIKU (IKU) genetic pathway modulates integument cell elongation. Combinations of TTG2 defective seed integument with reduction of endosperm size by iku mutations identify integument cell elongation and endosperm growth as the primary regulators of seed size. Our results strongly suggest that a cross talk between maternal and zygotic controls represents the primary regulator of the coordinated control of seed size in Arabidopsis.     

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.     

Maternal Gametophyte Effects on Seed Development in Maize

Flowering plants, like placental mammals, have an extensive maternal contribution toward progeny development. Plants are distinguished from animals by a genetically active haploid phase of growth and development between meiosis and fertilization, called the gametophyte. Flowering plants are further distinguished by the process of double fertilization that produces sister progeny, the endosperm and the embryo, of the seed. Because of this, there is substantial gene expression in the female gametophyte that contributes to the regulation of growth and development of the seed. A primary function of the endosperm is to provide growth support to its sister embryo. Several mutations in Zea mays subsp. mays have been identified that affect the contribution of the mother gametophyte to the seed. The majority affect both the endosperm and the embryo, although some embryo-specific effects have been observed. Many alter the pattern of expression of a marker for the basal endosperm transfer layer, a tissue that transports nutrients from the mother plant to the developing seed. Many of them cause abnormal development of the female gametophyte prior to fertilization, revealing potential cellular mechanisms of maternal control of seed development. These effects include reduced central cell size, abnormal architecture of the central cell, abnormal numbers and morphology of the antipodal cells, and abnormal egg cell morphology. These mutants provide insight into the logic of seed development, including necessary features of the gametes and supporting cells prior to fertilization, and set up future studies on the mechanisms regulating maternal contributions to the seed.     

Increased Maternal Genome Dosage Bypasses the Requirement of the FIS Polycomb Repressive Complex 2 in Arabidopsis Seed Development

Seed development in flowering plants is initiated after a double fertilization event with two sperm cells fertilizing two female gametes, the egg cell and the central cell, leading to the formation of embryo and endosperm, respectively. In most species the endosperm is a polyploid tissue inheriting two maternal genomes and one paternal genome. As a consequence of this particular genomic configuration the endosperm is a dosage sensitive tissue, and changes in the ratio of maternal to paternal contributions strongly impact on endosperm development. The FERTILIZATION INDEPENDENT SEED (FIS) Polycomb Repressive Complex 2 (PRC2) is essential for endosperm development; however, the underlying forces that led to the evolution of the FIS-PRC2 remained unknown. Here, we show that the functional requirement of the FIS-PRC2 can be bypassed by increasing the ratio of maternal to paternal genomes in the endosperm, suggesting that the main functional requirement of the FIS-PRC2 is to balance parental genome contributions and to reduce genetic conflict. We furthermore reveal that the AGAMOUS LIKE (AGL) gene AGL62 acts as a dosage-sensitive seed size regulator and that reduced expression of AGL62 might be responsible for reduced size of seeds with increased maternal genome dosage.     

The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin–dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of MINISEED3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways.     

Miniature Seed6, encoding an endoplasmic reticulum signal peptidase,is critical in seed development

Endoplasmic reticulum (ER) type I signal peptidases (ER SPases I) are vital proteases that cleave signal peptides from secreted proteins. However, the specific function of ER SPase I in plants has not been genetically characterized, and the substrate is largely unknown. Here, we report the identification of a maize (Zea mays) miniature seed6 (mn6) mutant. The loss-of-function mn6 mutant exhibited severely reduced endosperm size. Map-based cloning and molecular characterization indicated that Mn6 is an S26-family ER SPase I, with Gly102 (box E) in Mn6 critical for protein function during processing. Mass spectrometric and immunoprecipitation analyses revealed that Mn6 is predominantly involved in processing carbohydrate synthesis-related proteins, including the cell wall invertase miniature seed1 (Mn1), which is specifically expressed in the basal endosperm transfer layer. RNA and protein expression levels of Mn1 were both significantly downregulated in the mn6 mutant. Due to the significant reduction in cell wall invertase activity in the transfer cell layer, mutation of Mn6 caused dramatic defects in endosperm development. These results suggest that proper maturation of Mn1 by Mn6 may be a crucial step for proper seed filling and maize development.

Miniature seed6 (Mn6), involved in maize (Zea mays) seed development, is necessary for processing the cell wall invertase Mn1, which is specifically expressed in the basal endosperm transfer layer.