Seeing the unseen in characterizing RNA editome during rice endosperm development.

Rice (Oryza sativa L.) endosperm is essential to provide nutrients for seed germination and determine grain yield. RNA editing, a post-transcriptional modification essential for plant development, unfortunately, is not fully characterized during rice endosperm development. Here, we perform systematic analyses to characterize RNA editome during rice endosperm development. We find that most editing sites are C-to-U CDS-recoding in mitochondria, leading to increased hydrophobic amino acids and changed structures of mitochondrial proteins. Comparative analysis of RNA editome reveals that CDS-recoding sites present higher editing frequencies with lower variabilities and their resultant recoded amino acids tend to exhibit stronger evolutionary conservation across many land plants. Furthermore, we classify mitochondrial genes into three groups, presenting distinct patterns in terms of CDS-recoding events. Besides, we conduct genome-wide screening to detect pentatricopeptide repeat (PPR) proteins and construct PPR-RNA binding profiles, yielding candidate PPR editing factors related to rice endosperm development. Taken together, our findings provide valuable insights for deciphering fundamental mechanisms of rice endosperm development underlying RNA editing machinery.

Upstream regulator of genomic imprinting in rice endosperm is a small RNA-associated chromatin remodeler.

Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small RNAs (sRNAs) mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in the sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its likely paternal imprinted nature. These important features are modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines, we observe defects in endosperm development and a loss of around 70% of all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms are also dependent on OsCLSY3. We observed that many imprinted genes and seed development-associated genes are under the control of OsCLSY3. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

Understanding the amylose biosynthesis and regulation mechanisms in Tartary buckwheat by the endosperm transcriptome.

Starch serves as a crucial energy source for both plants and humans, predominantly synthesized and stored in endosperms, tubers, rhizomes, and cotyledons. Given the significant role of amylose in determining the quality of starchy crops, optimizing its content has become a key objective in current crop breeding efforts. Tartary buckwheat, a dicotyledonous plant, notably accumulates high levels of amylose in its endosperm, surpassing common cereals like rice and maize. However, the mechanisms underlying amylose accumulation, distribution, and regulation in Tartary buckwheat remain unclear. Here, amylose content was determined across various tissues and organs of Tartary buckwheat, identifying with the endosperm as the primary site for its biosynthesis and accumulation. RNA sequencing analysis of endosperms from different developmental stages identified 35 genes potentially involved in starch biosynthesis, with 13 genes showing high endosperm-specific expression, suggesting crucial roles in starch biosynthesis. Additionally, the transcription factor FtNF-YB2, which was specifically highly expressed in the endosperm, was discovered to enhance amylose synthesis. Moreover, promoters with potential endosperm-specific activity were identified, advancing our understanding of amylose regulation. Additionally, this study also demonstrates that brassinosteroids (BR) positively influence amylose biosynthesis in Tartary buckwheat endosperm. These findings provide essential insights into the mechanisms of understanding amylose biosynthesis, accumulation and regulation in Tartary buckwheat, offering significant implications for future breeding strategies.

Genome-Wide Identification of microRNAs Associated with Starch Biosynthesis and Endosperm Development in Foxtail Millet.

Foxtail millet is one of the oldest crops, and its endosperm contains up to 70% of starch. Grain filling is an important starch accumulation process associated with foxtail millet yield and quality. However, the molecular mechanisms of grain filling in foxtail millet are relatively unclear. Here, we investigate the genes and regulated miRNAs associated with starch synthesis and metabolism in foxtail millet using high-throughput small RNA, mRNA and degradome sequencing. The regulation of starch synthesis and quality is carried out mainly at the 15 DAA to 35 DAA stage during grain filling. The DEGs between waxy and non-waxy foxtail millet were significant, especially for GBSS. Additionally, ptc-miR169i_R+2_1ss21GA, fve-miR396e_L-1R+1, mtr-miR162 and PC-5p-221_23413 regulate the expression of genes associated with the starch synthesis pathway in foxtail millet. This study provides new insights into the molecular mechanisms of starch synthesis and quality formation in foxtail millet.

Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley.

KEY MESSAGE: The hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm. The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

Preferentially expressed endosperm genes reveal unique activities in wheat endosperm during grain filling.

BACKGROUND: Bread wheat (Triticum aestivum L.) endosperm contains starch and proteins, which determine the final yield, quality, and nutritional value of wheat grain. The preferentially expressed endosperm genes can precisely provide targets in the endosperm for improving wheat grain quality and nutrition using modern bioengineering technologies. However, the genes specifically expressed in developing endosperms remain largely unknown. RESULTS: In this study, 315 preferentially expressed endosperm genes (PEEGs) in the spring wheat landrace, Chinese Spring, were screened using data obtained from an open bioinformatics database, which reveals a unique grain reserve deposition process and special signal transduction in a developing wheat endosperm. Furthermore, transcription and accumulation of storage proteins in the wheat cultivar, XC26 were evaluated. The results revealed that 315 PEEG plays a critical role in storage protein fragment deposition and is a potential candidate for modifying grain quality and nutrition. CONCLUSION: These results provide new insights into endosperm development and candidate genes and promoters for improving wheat grain quality through genetic engineering and plant breeding techniques.

Transport and spatio-temporal conversion of sugar facilitate the formation of spatial gradients of starch in wheat caryopses.

Wheat grain starch content displays large variations within different pearling fractions, which affecting the processing quality of corresponding flour, while the underlying mechanism on starch gradient formation is unclear. Here, we show that wheat caryopses acquire sugar through the transfer of cells (TCs), inner endosperm (IE), outer endosperm (OE), and finally aleurone (AL) via micro positron emission tomography-computed tomography (PET-CT). To obtain integrated information on spatial transcript distributions, developing caryopses are laser microdissected into AL, OE, IE, and TC. Most genes encoding carbohydrate transporters are upregulated or specifically expressed, and sugar metabolites are more highly enriched in the TC group than in the AL group, in line with the PET-CT results. Genes encoding enzymes in sucrose metabolism, such as sucrose synthase, beta-fructofuranosidase, glucose-1-phosphate adenylyltransferase show significantly lower expression in AL than in OE and IE, indicating that substrate supply is crucial for the formation of starch gradients. Furthermore, the low expressions of gene encoding starch synthase contribute to low starch content in AL. Our results imply that transcriptional regulation represents an important means of impacting starch distribution in wheat grains and suggests breeding targets for enhancing specially pearled wheat with higher quality.

Simultaneous knockout of cytosolic and plastidial disproportionating enzymes disrupts grain setting and filling in rice.

Rice (Oryza sativa) plants contain plastidial and cytosolic disproportionating enzymes (DPE1 and DPE2). Our previous studies showed that DPE2 acts on maltose, the major product of starch degradation in pollens, releasing one glucose to fuel pollen tube growth and fertilization, whereas DPE1 participates in endosperm starch synthesis by transferring maltooligosyl groups from amylose to amylopectin, and removing excess short maltooligosaccharides. However, little is known about their integrated function. Here, we report that the coordinated actions of DPE1 and DPE2 contribute to grain setting and filling in rice. The dpe1dpe2 mutants could not be isolated from the progeny of heterozygous parental plants but were obtained via anther culture. Unlike that reported in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), the dpe1dpe2 rice plants grew normally but only yielded a small number of empty, unfilled seeds. In the dpe1dpe2 seeds, nutrient accumulation was substantially reduced, and dorsal vascular bundles were also severely malnourished. Zymogram analyses showed that changes in the activities of the major starch-synthesizing enzymes matched well with various endosperm phenotypes of mutant seeds. Mechanistically, DPE1 deficiency allowed normal starch mobilization in leaves and pollens but affected starch synthesis in endosperm, while DPE2 deficiency blocked starch degradation, resulting in substantially decreased levels of the sugars available for pollen tube growth and grain filling. Overall, our results demonstrate the great potential of DPE1-DPE2 as an important regulatory module to realize higher crop yields and present a promising target for regulating nutrient accumulation in cereal crop endosperm.

Environment found to explain the largest variance in physical and compositional traits in malting barley grain.

BACKGROUND: Starch is the most abundant constituent (dry weight) in the barley endosperm, followed by protein. Variability of compositional and potentially related physical traits due to genotype and environment can have important implications for the malting and brewing industry. This was the first study to assess the effects of genotype, environment, and their interaction (G × E) on endosperm texture, protein content, and starch traits corresponding to granule size, gelatinization, content, and composition, using a multi-environment variety trial in California, USA. RESULTS: Overall, environment explained the largest variance for all traits (ranging from 23.2% to 76.5%), except the endosperm texture traits wherein the G × E term explained the largest variance (45.0-86.5%). Our unique method to quantify the proportion of fine and coarse milled barley particles using laser diffraction showed a binomial distribution of endosperm texture. The number of small starch granules varied significantly (P-value < 0.05) across genotypes and environments. We observed negative correlations between total protein content and each of enthalpy (-0.70), total starch content (-0.54), and difference between offset and onset gelatinization temperature (-0.52). Furthermore, amylose to amylopectin ratio was positively correlated to volume of small starch granules (0.36). CONCLUSION: Our findings indicate that environment played a larger role in influencing the majority of starch-related physical and compositional traits. In contrast, variance in endosperm texture was largely explained by G × E. Maltsters would benefit from accounting for environmental contributions in addition to solely genotype when making sourcing decisions, especially with regards to total protein, total starch, enthalpy, and difference between offset and onset gelatinization temperature. © 2024 Society of Chemical Industry.

Characterization of a near isogenic barley line with high grain ß-amylase activity reveals a separation in the tight co-regulation of B-hordeins (Hor2) with endosperm-specific ß-amylase (Bmy1).

GSHO 2096 is a near isogenic barley line with extremely high grain ß-amylase activity, a desirable trait in the malting and brewing industry. High levels of grain ß-amylase activity are caused by a surge in endosperm-specific ß-amylase (Bmy1) gene expression during the early stages of grain development with high expression levels persisting throughout development. Origins of the high ß-amylase activity trait are perplexing considering GSHO 2096 is not supposed to have grain ß-amylase activity. GSHO 2096 is reported to be derived from a Bowman x Risø 1508 cross followed by recurrent backcrossing to Bowman (BC5). Risø 1508 carries a mutated form of the barley prolamin binding factor, which is responsible for Bmy1 expression during grain development. Thus, the pedigree of GSHO 2096 was explored to determine the potential origins of the high grain ß-amylase trait. Genotyping using the barley 50k iSelect SNP array revealed Bowman and GSHO 2096 were very similar (95.4 %) and provided evidence that both Risø 56 and 1508 are in the pedigree. Risø mutants 56 and 1508 both have perturbed hordein gene expression leading to a discernable pattern using SDS-PAGE. GSHO 2096 and Risø 56 have the same hordein pattern whereas Bowman and Risø 1508 have unique patterns. RNAseq revealed that Hor2 (B-hordein) gene expression was completely downregulated making it unique as the only known line with Bmy1 expression without Hor2 co-expression. Regardless of pedigree, GSHO 2096 remains an extremely valuable high ß-amylase activity line with potential utilization in breeding for malt quality.

Transcriptome and metabolome analyses reveal regulatory networks associated with nutrition synthesis in sorghum seeds.

Cereal seeds are vital for food, feed, and agricultural sustainability because they store and provide essential nutrients to human and animal food and feed systems. Unraveling molecular processes in seed development is crucial for enhancing cereal grain yield and quality. We analyze spatiotemporal transcriptome and metabolome profiles during sorghum seed development in the inbred line 'BTx623'. Morphological and molecular analyses identify the key stages of seed maturation, specifying starch biosynthesis onset at 5 days post-anthesis (dpa) and protein at 10 dpa. Transcriptome profiling from 1 to 25 dpa reveal dynamic gene expression pathways, shifting from cellular growth and embryo development (1-5 dpa) to cell division, fatty acid biosynthesis (5-25 dpa), and seed storage compounds synthesis in the endosperm (5-25 dpa). Network analysis identifies 361 and 207 hub genes linked to starch and protein synthesis in the endosperm, respectively, which will help breeders enhance sorghum grain quality. The availability of this data in the sorghum reference genome line establishes a baseline for future studies as new pangenomes emerge, which will consider copy number and presence-absence variation in functional food traits.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Multilayered epigenetic control of persistent and stage-specific imprinted genes in rice endosperm.

In angiosperms, epigenetic profiles for genomic imprinting are established before fertilization. However, the causal relationships between epigenetic modifications and imprinted expression are not fully understood. In this study, we classified 'persistent' and 'stage-specific' imprinted genes on the basis of time-course transcriptome analysis in rice (Oryza sativa) endosperm and compared them to epigenetic modifications at a single time point. While the levels of epigenetic modifications are relatively low in stage-specific imprinted genes, they are considerably higher in persistent imprinted genes. Overall trends revealed that the maternal alleles of maternally expressed imprinted genes are activated by DNA demethylation, while the maternal alleles of paternally expressed imprinted genes with gene body methylation (gbM) are silenced by DNA demethylation and H3K27me3 deposition, and these regions are associated with an enriched motif related to Tc/Mar-Stowaway. Our findings provide insight into the stability of genomic imprinting and the potential variations associated with endosperm development, different cell types and parental genotypes.

The biosynthesis of storage reserves and auxin is coordinated by a hierarchical regulatory network in maize endosperm.

Grain filling in maize (Zea mays) is intricately linked to cell development, involving the regulation of genes responsible for the biosynthesis of storage reserves (starch, proteins, and lipids) and phytohormones. However, the regulatory network coordinating these biological functions remains unclear. In this study, we identified 1744 high-confidence target genes co-regulated by the transcription factors (TFs) ZmNAC128 and ZmNAC130 (ZmNAC128/130) through chromatin immunoprecipitation sequencing coupled with RNA-seq analysis in the zmnac128/130 loss-of-function mutants. We further constructed a hierarchical regulatory network using DNA affinity purification sequencing analysis of downstream TFs regulated by ZmNAC128/130. In addition to target genes involved in the biosynthesis of starch and zeins, we discovered novel target genes of ZmNAC128/130 involved in the biosynthesis of lipids and indole-3-acetic acid (IAA). Consistently, the number of oil bodies, as well as the contents of triacylglycerol, and IAA were significantly reduced in zmnac128/130. The hierarchical regulatory network centered by ZmNAC128/130 revealed a significant overlap between the direct target genes of ZmNAC128/130 and their downstream TFs, particularly in regulating the biosynthesis of storage reserves and IAA. Our results indicated that the biosynthesis of storage reserves and IAA is coordinated by a multi-TFs hierarchical regulatory network in maize endosperm.

Epigenetic and transcriptional consequences in the endosperm of chemically induced transposon mobilization in Arabidopsis.

Genomic imprinting, an epigenetic phenomenon leading to parent-of-origin-specific gene expression, has independently evolved in the endosperm of flowering plants and the placenta of mammals-tissues crucial for nurturing embryos. While transposable elements (TEs) frequently colocalize with imprinted genes and are implicated in imprinting establishment, direct investigations of the impact of de novo TE transposition on genomic imprinting remain scarce. In this study, we explored the effects of chemically induced transposition of the Copia element ONSEN on genomic imprinting in Arabidopsis thaliana. Through the combination of chemical TE mobilization and doubled haploid induction, we generated a line with 40 new ONSEN copies. Our findings reveal a preferential targeting of maternally expressed genes (MEGs) for transposition, aligning with the colocalization of H2A.Z and H3K27me3 in MEGs-both previously identified as promoters of ONSEN insertions. Additionally, we demonstrate that chemically-induced DNA hypomethylation induces global transcriptional deregulation in the endosperm, leading to the breakdown of MEG imprinting. This study provides insights into the consequences of chemically induced TE remobilization in the endosperm, revealing that chemically-induced epigenome changes can have long-term consequences on imprinted gene expression.

Identification and analysis of nine new flo2 allelic mutants in rice.

FLO2 is involved in grain development and storage substance synthesis in rice, and therefore can regulate grain size and quality. In this study, we identified 4 new flo2 allelic mutants with nonsense and frameshift mutation in the exon of 6, 10, 11 and 21 and 5 new flo2 allelic mutants with alternative splicing and frameshift mutation at the splicing site of intron 13, 14, 16 and 17. Compared with wild-type rice, the outer endosperm of flo2 mutants was transparent, and the inner endosperm was floury. Different mutation sites and types of FLO2 significantly decreased kernel width, thickness and weight to some extent. The contents of storage protein, starch, amylose and amylopectin showed significant decrease at different levels among 9 flo2 mutants. The expressions of most storage protein synthesis genes and starch synthesis-related genes were significantly down-regulated, and exhibited different ranges of variation among different flo2 mutants. This study could add helpful information for the roles of flo2 alleles in rice quality regulation and provide abundant germplasm resources for rice quality breeding.

Deciphering the Transcriptional Regulatory Network Governing Starch and Storage Protein Biosynthesis in Wheat for Breeding Improvement.

Starch and seed storage protein (SSP) composition profoundly impact wheat grain yield and quality. To unveil regulatory mechanisms governing their biosynthesis, transcriptome, and epigenome profiling is conducted across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlight accessible promoter regions' crucial role as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. Integration of time-serial RNA-seq and ATAC-seq enables the construction of a hierarchical transcriptional regulatory network governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibit overlap with genetic regions associated with grain size and quality traits, and their functional significance is validated through expression-phenotype association analysis among wheat accessions and loss-of-function mutants. Functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrates its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. Excellent TaABI3-A1Hap1 with enhanced grain weight is selected during the breeding process in China, linked to altered expression levels. This study unveils key regulators, advancing understanding of SSP and starch biosynthesis regulation and contributing to breeding enhancement.

Insights into dynamic coenocytic endosperm development: Unraveling molecular, cellular, and growth complexity.

The endosperm, a product of double fertilization, is one of the keys to the evolution and success of angiosperms in conquering the land. While there are differences in endosperm development among flowering plants, the most common form is coenocytic growth, where the endosperm initially undergoes nuclear division without cytokinesis and eventually becomes cellularized. This complex process requires interplay among networks of transcription factors such as MADS-box, auxin response factors (ARFs), and phytohormones. The role of cytoskeletal elements in shaping the coenocytic endosperm and influencing seed growth also becomes evident. This review offers a recent understanding of the molecular and cellular dynamics in coenocytic endosperm development and their contributions to the final seed size.

Parental dialectic: Epigenetic conversations in endosperm.

Endosperm is a major evolutionary innovation of flowering plants, and its proper development critically impacts seed growth and viability. Epigenetic regulators have a key function in parental control of endosperm development. Notably, epigenetic regulation of parental genome dosage is a major determinant of seed development success, and disruption of this balance can produce inviable seed, as observed in some interploidy and interspecific crosses. These postzygotic reproduction barriers are also a potent driver of speciation. The molecular machinery and regulatory architecture governing endosperm development is proposed to have evolved under parental conflict. In this review, we emphasize parental conflict as a dialectic conflict and discuss recent findings about the epigenetic molecular machinery that mediates parental conflict in the endosperm.