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.

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.

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.

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.

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.

Oat species and interspecific amphiploids show predominance of diploid nuclei in the syncytial endosperm.

Apart from apomictic types, the Polygonum-type eight-nuclear embryo sac is considered to be dominant in grasses. A triploid endosperm is formed as a result of double fertilisation. This study showed, for the first time, the dominance of diploid nuclei in the syncytial stage of the central cell of embryo sac in oat species and amphiploids. The dominance of diploid nuclei, which were the basis for the formation of polyploid nuclei, was weaker in amphiploids due to aneuploid events. The genomic in situ hybridisation method applied in the study did not distinguish the maternal and paternal haploid nuclei of embryo sac. However, this method demonstrated the lack of a set of genomes of one haploid nucleus. Embryological analyses of the initial stages of oat endosperm development revealed a fertilised egg cell, and two polar nuclei differing in size. It can be assumed that the formation of diploid oat endosperm occurred after the fusion of one polar nucleus and the nucleus of a male gamete, while the second polar nucleus gave rise to 1n nuclei. The levels of ploidy of syncytial nuclei were not influenced by both aneuploid events and correlated with pollen developmental anomalies. The differences in the analysed cytogenetic events distinguished amphiploids and their parental species in the ordination space.

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.

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.

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.

Paternally imprinted LATE-FLOWERING2 transcription factor contributes to paternal-excess interploidy hybridization barriers in wheat.

Interploidy hybridization between hexaploid and tetraploid genotypes occurred repeatedly during genomic introgression events throughout wheat evolution, and is commonly employed in wheat breeding programs. Hexaploid wheat usually serves as maternal parent because the reciprocal cross generates progeny with severe defects and poor seed germination, but the underlying mechanism is poorly understood. Here, we performed detailed analysis of phenotypic variation in endosperm between two interploidy reciprocal crosses arising from tetraploid (Triticum durum, AABB) and hexaploid wheat (Triticum aestivum, AABBDD). In the paternal- versus the maternal-excess cross, the timing of endosperm cellularization was delayed and starch granule accumulation in the endosperm was repressed, causing reduced germination percentage. The expression profiles of genes involved in nutrient metabolism differed strongly between these endosperm types. Furthermore, expression patterns of parental alleles were dramatically disturbed in interploidy versus intraploidy crosses, leading to increased number of imprinted genes. The endosperm-specific TaLFL2 showed a paternally imprinted expression pattern in interploidy crosses partially due to allele-specific DNA methylation. Paternal TaLFL2 binds to and represses a nutrient accumulation regulator TaNAC019, leading to reduced storage protein and starch accumulation during endosperm development in paternal-excess cross, as confirmed by interploidy crosses between tetraploid wild-type and clustered regularly interspaced palindromic repeats (CRISPR) - CRISPR-associated protein 9 generated hexaploid mutants. These findings reveal a contribution of genomic imprinting to paternal-excess interploidy hybridization barriers during wheat evolution history and explains why experienced breeders preferentially exploit maternal-excess interploidy crosses in wheat breeding programs.

An elastic proteinaceous envelope encapsulates the early Arabidopsis embryo.

Plant external surfaces are often covered by barriers that control the exchange of molecules, protect from pathogens and offer mechanical integrity. A key question is when and how such surface barriers are generated. Post-embryonic surfaces have well-studied barriers, including the cuticle, and it has been previously shown that the late Arabidopsis thaliana embryo is protected by an endosperm-derived sheath deposited onto a primordial cuticle. Here, we show that both cuticle and sheath are preceded by another structure during the earliest stages of embryogenesis. This structure, which we named the embryonic envelope, is tightly wrapped around the embryonic surface but can be physically detached by cell wall digestion. We show that this structure is composed primarily of extensin and arabinogalactan O-glycoproteins and lipids, which appear to form a dense and elastic crosslinked embryonic envelope. The envelope forms in cuticle-deficient mutants and in a mutant that lacks endosperm. This embryo-derived envelope is therefore distinct from previously described cuticle and sheath structures. We propose that it acts as an expandable diffusion barrier, as well as a means to mechanically confine the embryo to maintain its tensegrity during early embryogenesis.

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.

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.

Plant development: How to kill the endosperm.

Using molecular markers and genetic analysis of mutant phenotypes, a new study reveals that endosperm elimination in plant seeds is under control of the programmed cell death pathway.

Endosperm cell death promoted by NAC transcription factors facilitates embryo invasion in Arabidopsis.

In flowering plants, two fertilization products develop within the limited space of the seed: the embryo and the surrounding nutritive endosperm. The final size of the endosperm is modulated by the degree of embryo growth. In Arabidopsis thaliana, the endosperm expands rapidly after fertilization, but later gets invaded by the embryo that occupies most of the seed volume at maturity, surrounded by a single remaining aleurone-like endosperm layer.1,2,3,4 Embryo invasion is facilitated by the endosperm-expressed bHLH-type transcription factor ZHOUPI, which promotes weakening of endosperm cell walls.5,6 Endosperm elimination in zou mutants is delayed, and embryo growth is severely affected; the endosperm finally collapses around the dwarf embryo, causing the shriveled appearance of mature zou seeds.5,6,7 However, whether ZHOUPI facilitates mechanical endosperm destruction by the invading embryo or whether an active programmed cell death (PCD) process causes endosperm elimination has been subject to debate.2,8 Here we show that developmental PCD controlled by multiple NAC transcription factors in the embryo-adjacent endosperm promotes gradual endosperm elimination. Misexpressing the NAC transcription factor KIRA1 in the entire endosperm caused total endosperm elimination, generating aleurone-less mature seeds. Conversely, dominant and recessive higher-order NAC mutants led to delayed endosperm elimination and impaired cell corpse clearance. Promoting PCD in the zhoupi mutant partially rescued its embryo growth defects, while the endosperm in a zhoupi nac higher-order mutant persisted until seed desiccation. These data suggest that a combination of cell wall weakening and PCD jointly facilitates embryo invasion by an active auto-elimination of endosperm cells.

The transcription factors ZmNAC128 and ZmNAC130 coordinate with Opaque2 to promote endosperm filling in maize.

Endosperm filling in maize (Zea mays), which involves nutrient uptake and biosynthesis of storage reserves, largely determines grain yield and quality. However, much remains unclear about the synchronization of these processes. Here, we comprehensively investigated the functions of duplicate NAM, ATAF1/2, and CUC2 (NAC)-type transcription factors, namely, ZmNAC128 and ZmNAC130, in endosperm filling. The gene-edited double mutant zmnac128 zmnac130 exhibits a poorly filled kernel phenotype such that the kernels have an inner cavity. RNA sequencing and protein abundance analysis revealed that the expression of many genes involved in the biosynthesis of zein and starch is reduced in the filling endosperm of zmnac128 zmnac130. Further, DNA affinity purification and sequencing combined with chromatin-immunoprecipitation quantitative PCR and promoter transactivation assays demonstrated that ZmNAC128 and ZmNAC130 are direct regulators of 3 (16-, 27-, and 50-kD) γ-zein genes and 6 important starch metabolism genes (Brittle2 [Bt2], pullulanase-type starch debranching enzyme [Zpu1], granule-bound starch synthase 1 [GBSS1], starch synthase 1 [SS1], starch synthase IIa [SSIIa], and sucrose synthase 1 [Sus1]). ZmNAC128 and ZmNAC130 recognize an additional cis-element in the Opaque2 (O2) promoter to regulate its expression. The triple mutant zmnac128 zmnac130 o2 exhibits extremely poor endosperm filling, which results in more than 70% of kernel weight loss. ZmNAC128 and ZmNAC130 regulate the expression of the transporter genes sugars that will eventually be exported transporter 4c (ZmSWEET4c), sucrose and glucose carrier 1 (ZmSUGCAR1), and yellow stripe-like2 (ZmYSL2) and in turn facilitate nutrient uptake, while O2 plays a supporting role. In conclusion, ZmNAC128 and ZmNAC130 cooperate with O2 to facilitate endosperm filling, which involves nutrient uptake in the basal endosperm transfer layer (BETL) and the synthesis of zeins and starch in the starchy endosperm (SE).

zma-miR159 targets ZmMYB74 and ZmMYB138 transcription factors to regulate grain size and weight in maize.

Endosperm cell number is critical in determining grain size in maize (Zea mays). Here, zma-miR159 overexpression led to enlarged grains in independent transgenic lines, suggesting that zma-miR159 contributes positively to maize grain size. Targeting of ZmMYB74 and ZmMYB138 transcription factor genes by zma-miR159 was validated using 5' RACE and dual-luciferase assay. Lines in which ZmMYB74 was knocked out using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) presented a similar enlarged grain phenotype as those with zma-miR159 overexpression. Downstream genes regulating cell division were identified through DNA affinity purification sequencing using ZmMYB74 and ZmMYB138. Our results suggest that zma-miR159-ZmMYB modules act as an endosperm development hub, participating in the division and proliferation of endosperm cells.