Flowering plant reproduction.

Flowering plants, also known as angiosperms, emerged approximately 150 to 200 million years ago. Since then, they have undergone rapid and extensive expansion, now encompassing around 90% of all land plant species. The remarkable diversification of this group has been a subject of in-depth investigations, and several evolutionary innovations have been proposed to account for their success. In this primer, we will specifically focus on one such innovation: the advent of seeds containing endosperm.

Mannans: Structural carbohydrates produced during seed maturation in Euterpe edulis Martius, an Atlantic Forest species vulnerable to extinction.

Palm seedlings are visually selected from mature fruits in a slow process that leads to nonuniform germination and high embryo mortality. In this study, we determined the levels of monosaccharides, their crystallinity, and their role in the formation of Euterpe edulis endosperm during seed maturation. Seeds harvested from 108 to 262 days after anthesis (DAA) were analyzed morphologically, physiologically, and chemically to measure soluble and insoluble lignins, ashes, structural carbohydrates, degree of crystallinity, and endo-ß-mannanase. The seeds achieved maximum germination and vigor at 164 DAA. During the early stages, only compounds with a low structural order were formed. The contents of soluble and insoluble lignins, ashes, glucans, and galactans decreased during maturation. Those of mannans, the main structural carbohydrate in the endosperm, increased along with the degree of crystallinity, as suggested by a mannan-I-type X-ray diffraction pattern. Similarly, endo-ß-mannanase activity peaked at 262 DAA. The superior physiological outcome of seeds and seedlings at 164 DAA implies a 98-day shorter harvesting time. The state of mannans during seed maturation could be used as a marker to improve seedling production by E. edulis.

Sucrose-associated SnRK1a1-mediated phosphorylation of Opaque2 modulates endosperm filling in maize.

During maize endosperm filling, sucrose not only serves as a source of carbon skeletons for storage-reserve synthesis but also acts as a stimulus to promote this process. However, the molecular mechanisms underlying sucrose and endosperm filling are poorly understood. In this study, we found that sucrose promotes the expression of endosperm-filling hub gene Opaque2 (O2), coordinating with storage-reserve accumulation. We showed that the protein kinase SnRK1a1 can attenuate O2-mediated transactivation, but sucrose can release this suppression. Biochemical assays revealed that SnRK1a1 phosphorylates O2 at serine 41 (S41), negatively affecting its protein stability and transactivation ability. We observed that mutation of SnRK1a1 results in larger seeds with increased kernel weight and storage reserves, while overexpression of SnRK1a1 causes the opposite effect. Overexpression of the native O2 (O2-OE), phospho-dead (O2-SA), and phospho-mimetic (O2-SD) variants all increased 100-kernel weight. Although O2-SA seeds exhibit smaller kernel size, they have higher accumulation of starch and proteins, resulting in larger vitreous endosperm and increased test weight. O2-SD seeds display larger kernel size but unchanged levels of storage reserves and test weight. O2-OE seeds show elevated kernel dimensions and nutrient storage, like a mixture of O2-SA and O2-SD seeds. Collectively, our study discovers a novel regulatory mechanism of maize endosperm filling. Identification of S41 as a SnRK1-mediated phosphorylation site in O2 offers a potential engineering target for enhancing storage-reserve accumulation and yield in maize.

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.

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.

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.

Cytological characteristics of blueberry fruit development.

Using the blueberry cultivar "Powderblue" after pollination, fruits at different developmental stages were collected for study. The transverse and longitudinal diameters, individual fruit weight, and fruit water content were measured during their development. Employing tissue sectioning and microscopy techniques, we systematically studied the morphological features and anatomical structures of the fruits and seeds at various developmental stages, aiming to elucidate the cytological patterns during blueberry fruit development. The results of our study revealed that the "Powderblue" blueberry fruit growth and development followed a double "S" curve. Mature "Powderblue" blueberries were blue-black in color, elliptical in shape, with five locules, an inferior ovary, and an average fruit weight of 1.73 ± 0.17 g, and a moisture content of 78.865 ± 0.9%. Blueberry fruit flesh cells were densely arranged with no apparent intercellular spaces, and mesocarp cells accounted for 52.06 ± 7.4% of fruit cells. In the early fruit development stages, the fruit flesh cells were rapidly dividing, significantly increasing in number but without greatly affecting the fruit's morphological characteristics. During the later stages of fruit development, the expansion of the fruit flesh cells became prominent, resulting in a noticeable increase in the fruit's dimensions. Except for the epidermal cells, cells in all fruit tissues showed varying degrees of rupture as fruit development progressed, with the extent of cell rupture increasing, becoming increasingly apparent as the fruit gradually softened. Additionally, numerous brachysclereids (stone cells) appeared in the fruit flesh cells. Stone cells are mostly present individually in the fruit flesh tissue, while in the placental tissue, they often group together. The "Powderblue" blueberry seeds were light brown, 4.13 ± 0.42 mm long, 2.2 ± 0.14 mm wide, with each fruit containing 50-60 seeds. The "Powderblue" seeds mainly consisted of the seed coat, endosperm, and embryo. The embryo was located at the chalazal end in the center of the endosperm and was spatially separated. The endosperm, occupying the vast majority of the seed volume, comprised both the chalazal and outer endosperm, and the endosperm developed and matured before the embryo. As the seed developed, the seed coat was gradually lignified and consisted of palisade-like stone cells externally and epidermal layer cells internally.

Autophagy maintains endosperm quality during seed storage to preserve germination ability in Arabidopsis.

To preserve germination ability, plant seeds must be protected from environmental stresses during the storage period. Here, we demonstrate that autophagy, an intracellular degradation system, maintains seed germination ability in Arabidopsis thaliana. The germination ability of long-term (>5 years) stored dry seeds of autophagy-defective (atg) mutant and wild-type (WT) plants was compared. Long-term stored (old) seeds of atg mutants showed lower germination ability than WT seeds, although short-term stored (new) seeds of atg mutants did not show such a phenotype. After removal of the seed coat and endosperm from old atg mutant seeds, the embryos developed into seedlings. Autophagic flux was maintained in endosperm cells during the storage period, and autophagy defect resulted in the accumulation of oxidized proteins and accelerated endosperm cell death. Consistent with these findings, the transcripts of genes, ENDO-ß-MANNANASE 7 and EXPANSIN 2, which are responsible for degradation/remodeling of the endosperm cell wall during germination, were reduced in old atg mutant seeds. We conclude that autophagy maintains endosperm quality during seed storage by suppressing aging-dependent oxidative damage and cell death, which allows the endosperm to perform optimal functions during germination, i.e., cell wall degradation/remodeling, even after long-term storage.

In vitro regeneration of triploid from mature endosperm culture of Passiflora edulis "Mantianxing".

The regeneration of shoots from endosperm tissue is a highly effective method to obtain triploid plants. In this study, we elucidated the establishment of an in vitro regeneration system from endosperm culture for the production of Passiflora edulis "Mantianxing." The highest callus induction rate (83.33%) was obtained on the media supplemented with 1.0 mg/L TDZ. Meanwhile, the MS medium containing 1.0 mg/L 6-BA and 0.4 mg/L IBA gave the optimum 75% shoot bud induction. Chromosome analysis revealed that the chromosomal count of P. edulis "Mantianxing" regenerated from endosperm tissues was 27 (2n = 3x = 27), which indicated that shoots regenerated from endosperm tissues were triploids. Triploid P. edulis had more drought resistance than diploid plants. Our study provided a method for breeding of passion fruit by means of a stable and reproducible regeneration system from endosperm culture, leading to the generation of triploid plants.

Characterization, interfacial rheology, and storage stability of Pickering emulsions stabilized by complex of whey protein isolate fiber and zein derived from micro-endosperm maize.

In present study, we characterized the formation, interfacial rheology, and storage stability of emulsions stabilized by microendosperm maize-derived zein (M-Zein)/whey protein isolate fiber (WPIF) nanoparticles. Microendosperm maize is a newly developed, oleic acid-rich oilseed resource. Recent research has shown that M-Zein possesses unique hydrophobic properties. Combining it with WPIF may enhance its performance as a stabilizer. Optimization of weight ratios for M-Zein/WPIF composites, guided by particle size analysis, fluorescence spectroscopy, three-phase contact angle (θ), and interfacial rheological analysis, revealed that a 4: 6 mass ratio at pH 7 yielded favorable wettability (θ = 91.2°). Interfacial rheology analysis showed that the combination of WPIF reduced M-Zein's interfacial tension to 7.2 mN/m and 36.7 mN/m at oil-water and air-water interfaces, respectively. The M-Zein/WPIF complex exhibited an elastic protein layer at the oil-water interface. Further investigations into nanoparticle concentration, oil phase volume, and pH revealed that emulsions containing 3 % nanoparticles (w/w), 50 % oil phase volume, and pH 7 showed the best storage stability. This research highlights the development of M-Zein/WPIF composited nanoparticles with superior storage stability and interfacial rheology. Additionally, it introduces a novel application for M-Zein, which elevates the value proposition of microendosperm maize.

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.

Imaging the ER and Endomembrane System in Cereal Endosperm.

The cereal endosperm is a complex structure comprising distinct cell types, characterized by specialized organelles for the accumulation of storage proteins. Protein trafficking in these cells is complicated by the presence of several different storage organelles including protein bodies (PBs) derived from the endoplasmic reticulum (ER) and dynamic protein storage vacuoles (PSVs). In addition, trafficking may follow a number of different routes depending on developmental stage, showing that the endomembrane system is capable of massive reorganization. Thus, developmental sequences involve progressive changes of the endomembrane system of endosperm tissue and are characterized by a high structural plasticity and endosomal activity.Given the technical dexterity required to access endosperm tissue and study subcellular structures and SSP trafficking in cereal seeds, static images are the state of the art providing a bulk of information concerning the cellular composition of seed tissue. In view of the highly dynamic endomembrane system in cereal endosperm cells, it is reasonable to expect that live cell imaging will help to characterize the spatial and temporal changes of the endomembrane system. The high resolution achieved with electron microscopy perfectly complements the live cell imaging.We therefore established an imaging platform for TEM as well as for live cell imaging. Here, we describe the preparation of different cereal seed tissues for live cell imaging concomitant with immunolocalization studies and ultrastructure.

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.

Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development.

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific ß-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant's phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., ß-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low ß-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

Molecular basis and evolutionary drivers of endosperm-based hybridization barriers.

The endosperm, a transient seed tissue, plays a pivotal role in supporting embryo growth and germination. This unique feature sets flowering plants apart from gymnosperms, marking an evolutionary innovation in the world of seed-bearing plants. Nevertheless, the importance of the endosperm extends beyond its role in providing nutrients to the developing embryo by acting as a versatile protector, preventing hybridization events between distinct species and between individuals with different ploidy. This phenomenon centers on growth and differentiation of the endosperm and the speed at which both processes unfold. Emerging studies underscore the important role played by type I MADS-box transcription factors, including the paternally expressed gene PHERES1. These factors, along with downstream signaling pathways involving auxin and abscisic acid, are instrumental in regulating endosperm development and, consequently, the establishment of hybridization barriers. Moreover, mutations in various epigenetic regulators mitigate these barriers, unveiling a complex interplay of pathways involved in their formation. In this review, we discuss the molecular underpinnings of endosperm-based hybridization barriers and their evolutionary drivers.

A paternal signal induces endosperm proliferation upon fertilization in Arabidopsis.

In multicellular organisms, sexual reproduction relies on the formation of highly differentiated cells, the gametes, which await fertilization in a quiescent state. Upon fertilization, the cell cycle resumes. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that reinstate cell division in a fertilization-dependent manner are poorly understood in both animals and plants. Using Arabidopsis, we show that a sperm-derived signal induces the proliferation of a female gamete, the central cell, precisely upon fertilization. The central cell is arrested in S phase by the activity of the RETINOBLASTOMA RELATED1 (RBR1) protein. Upon fertilization, delivery of the core cell cycle component CYCD7;1 causes RBR1 degradation and thus S phase progression, ensuring the formation of functional endosperm and, consequently, viable seeds.

Decoding the gene regulatory network of endosperm differentiation in maize.

The persistent cereal endosperm constitutes the majority of the grain volume. Dissecting the gene regulatory network underlying cereal endosperm development will facilitate yield and quality improvement of cereal crops. Here, we use single-cell transcriptomics to analyze the developing maize (Zea mays) endosperm during cell differentiation. After obtaining transcriptomic data from 17,022 single cells, we identify 12 cell clusters corresponding to five endosperm cell types and revealing complex transcriptional heterogeneity. We delineate the temporal gene-expression pattern from 6 to 7 days after pollination. We profile the genomic DNA-binding sites of 161 transcription factors differentially expressed between cell clusters and constructed a gene regulatory network by combining the single-cell transcriptomic data with the direct DNA-binding profiles, identifying 181 regulons containing genes encoding transcription factors along with their high-confidence targets, Furthermore, we map the regulons to endosperm cell clusters, identify cell-cluster-specific essential regulators, and experimentally validated three predicted key regulators. This study provides a framework for understanding cereal endosperm development and function at single-cell resolution.

Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

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.