Transcriptome analysis reveals biosynthesis and regulation of flavonoid in common bean seeds during grain filling.

The Andean domesticated common beans (Phaseolus vulgaris) are significant sources of phenolic compounds associated with health benefits. However, the regulation of biosynthesis of these compounds during bean seed development remains unclear. To elucidate the gene expression patterns involved in the regulation of the flavonoid pathway, we conducted a transcriptome analysis of two contrasting Chilean varieties, Negro Argel (black bean) and Coscorron (white bean), at three developmental stages associated with seed color change, as well as different flavonoid compound accumulations. Our study reveals that phenolic compound synthesis initiates during seed filling, although it exhibits desynchronization between both varieties. We identified 10,153 Differentially Expressed Genes (DEGs) across all comparisons. The KEGG pathway 'Flavonoid biosynthesis' showed enrichment of induced DEGs in Negro Argel (PV172), consistent with the accumulation of delphinidin, petunidin, and malvidin hexosides in their seeds, while catechin glucoside, procyanidin and kaempferol derivatives were predominantly detected in Coscorrón (PV24). Furthermore, while the flavonoid pathway was active in both varieties, our results suggest that enzymes involved in the final steps, such as ANS and UGT, were crucial, inducing anthocyanin formation in Negro Argel. Additionally, during active anthocyanin biosynthesis, the accumulation of reserve proteins or those related to seed protection and germination was induced. These findings provide valuable insights and serve as a guide for plant breeding aimed at enhancing the health and nutritional properties of common beans.

Haploid induction: an overview of parental factor manipulation during seed formation.

In plants, in vivo haploid induction has gained increasing attention for its significant potential applications in crop breeding and genetic research. This strategy reduces the chromosome number in progeny after fertilization, enabling the rapid production of homozygous plants through double haploidization, contrasting with traditional inbreeding over successive generations. Haploidy typically initiates at the onset of seed development, with several key genes identified as paternal or maternal factors that play critical roles during meiosis, fertilization, gamete communication, and chromosome integrity maintenance. The insights gained have led to the development of efficient haploid inducer lines. However, the molecular and genetic mechanisms underlying these factors vary considerably, making it challenging to create broadly applicable haploidy induction systems for plants. In this minireview, we summarize recent discoveries and advances in paternal and maternal haploid induction factors, examining their current understanding and functionalities to further develop efficient haploid inducer systems through the application of parental factor manipulation.

The Integrated Analysis of miRNome and Degradome Sequencing Reveals the Regulatory Mechanisms of Seed Development and Oil Biosynthesis in Pecan (Carya illinoinensis).

Pecan seed oil is a valuable source of essential fatty acids and various bioactive compounds; however, the functions of microRNAs and their targets in oil biosynthesis during seed development are still unknown. Here, we found that the oil content increased rapidly in the three early stages in three cultivars, and that oleic acid was the predominant fatty acid component in the mature pecan embryos. We identified, analyzed, and validated the expression levels of miRNAs related to seed development and oil biosynthesis, as well as their potential target genes, using small RNA sequencing data from three stages (120, 135, and 150 days after flowering). During the seed development process, 365 known and 321 novel miRNAs were discovered. In total, 91 known and 181 novel miRNAs were found to be differentially expressed, and 633 target genes were further investigated. The expression trend analysis revealed that the 91 known miRNAs were classified into eight groups, approximately two-thirds of which were up-regulated, whereas most novel miRNAs were down-regulated. The qRT-PCR and degradome sequencing data were used to identify five miRNA- target pairs. Overall, our study provides valuable insights into the molecular regulation of oil biosynthesis in pecan seeds.

Transcription factor ZmNAC19 promotes embryo development in Arabidopsis thaliana.

KEY MESSAGE: Overexpression of ZmNAC19, a NAC transcription factor gene from maize, improves embryo development in transgenic Arabidopsis. NAC proteins are plant-specific transcription factors that are involved in multiple aspects of plant growth, development and stress response. Although functions of many NAC transcription factors have been elucidated, little is known about their roles in seed development. In this study, we report the function of a maize NAC transcription factor ZmNAC19 in seed development. ZmNAC19 is highly expressed in embryos of developing maize seeds. ZmNAC19 localizes to nucleus and exhibits transactivation activity in yeast cells. Overexpression of ZmNAC19 in Arabidopsis significantly increases seed size and seed yield. During 3 to 7 days after flowering, embryos of ZmNAC19-overexpression Arabidopsis lines developed faster compared to Col-0, while no visible differences were detected for their endosperms. Furthermore, overexpression of ZmNAC19 in Arabidopsis leads to increased transcription levels of two embryo development-related genes YUC1 and RGE1, and several elements proven to be binding sites of NAC transcription factors were observed in promoters of these two genes. Taken together, these results suggest that ZmNAC19 acts as a positive regulator in plant embryo development.

Peanut allergen characterization and allergenicity throughout development.

Introduction: Peanut allergy (PA) in children is a major concern. There is a need for better biological material for both diagnosis and oral immunotherapy (OIT) treatments. The unique state of seeds at early reproductive stages may affect the allergenicity of storage proteins, and impact clinical diagnostic and OIT protocols. The objective of this study was to evaluate the major allergen content in sequential seed developmental stages and monitor allergenicity via specific IgE binding quantification and skin prick testing. Methods: Seeds were collected from peanut plants and sorted into five developmental stages: initial (S1), developing (S2), full-size without coloration (S3), full-size with coloration (S4), and fully mature (S5) seeds. Samples were characterized by RNA-Seq, ELISA, and immunohistochemistry. Lyophilized, ground preparations were used for evaluation of skin test responses in sixty challenge-proven PA children. Results: Gene expression, protein content, and specific IgE binding of allergenic proteins increased throughout seed maturation and development. An expression bias towards the less allergenic A-genome copy of the major allergen Ara h 2 was found in earlier stages, especially in stage S2. Immunohistochemical staining showed that Ara h 2 is more dispersed in the cell and less accumulated within organized bodies at stage S2 versus stage S4. Significant differences were found in mean wheal responses between the commercial peanut extract (equivalent to stage S5) and stages S1 and S2, but not with stage S4, upon skin prick testing in subjects with PA. Discussion: The observed decrease in peanut-specific IgE binding of immature peanut seeds may be a result not only of decreased amounts of allergenic proteins, but also of profound changes in seed composition and conformation. This may be significant for developing a safer and more effective peanut OIT protocol.

Contextualized Metabolic Modelling Revealed Factors Affecting Isoflavone Accumulation in Soybean Seeds.

Isoflavones, secondary metabolites with numerous health benefits, are predominantly found in legume seeds, especially soybean; however, their contents in domesticated soybean seeds are highly variable. Wild soybeans are known for higher seed isoflavone contents than cultivars. Here we used experimental and modelling approaches on wild soybean (W05) and cultivated soybean (C08) to delineate factors influencing isoflavone accumulation. We found imported nutrients were converted into storage compounds, with isoflavone accumulation in W05 seeds being faster than in C08 ones. The isoflavone accumulation during seed development was simulated using context-specific cotyledon metabolic models of four developmental stages on cultivar C08, and the metabolic burden imposed by increasing biomass was evaluated. Trade-off analyses between biomass and isoflavone suggest that high biomass requirement in cultivars could limit the reallocation of resources for secondary metabolite production. Isoflavone production in mature seeds was also influenced by biomass compositions. Seeds with higher carbohydrate contents favour isoflavone production, while those with highest protein and oil contents had lowest isoflavone contents. Although seeds could synthesize isoflavones on their own, the predicted fluxes from biosynthesis alone were lower than the empirical levels. Shadow price analyses indicated that isoflavone accumulation depended on both intrinsic biosynthesis and direct contribution from the plant.

Temporal Gene Expression Profiles From Pollination to Seed Maturity in Sorghum Provide Core Candidates for Engineering Seed Traits.

Sorghum (Sorghum bicolor (L.) Moench) is a highly nutritional multipurpose millet crop. However, the genetic and molecular regulatory mechanisms governing sorghum grain development and the associated agronomic traits remain unexplored. In this study, we performed a comprehensive transcriptomic analysis of pistils collected 1-2 days before pollination, and developing seeds collected -2, 10, 20 and 30 days after pollination of S. bicolor variety M35-1. Out of 31 337 genes expressed in these stages, 12 804 were differentially expressed in the consecutive stages of seed development. These exhibited 10 dominant expression patterns correlated with the distinct pathways and gene functions. Functional analysis, based on the pathway mapping, transcription factor enrichment and orthology, delineated the key patterns associated with pollination, fertilization, early seed development, grain filling and seed maturation. Furthermore, colocalization with previously reported quantitative trait loci (QTLs) for grain weight/size revealed 48 differentially expressed genes mapping to these QTL regions. Comprehensive literature mining integrated with QTL mapping and expression data shortlisted 25, 17 and 8 core candidates for engineering grain size, starch and protein content, respectively.

PPR21 is involved in the splicing of nad2 introns via interacting with PPR-SMR1 and SPR2 and is essential to maize seed development.

Pentatricopeptide repeat (PPR) proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression. Here, we report the function of PPR21 in mitochondrial intron splicing and its role in maize kernel development. PPR21 is a typical P-type PPR protein targeted to mitochondria. The ppr21 mutants are arrested in embryogenesis and endosperm development, leading to embryo lethality. Null mutations of PPR21 reduce the splicing efficiency of nad2 intron 1, 2, and 4 and impair the assembly and activity of mitochondrial complex I. Previous studies show that the P-type PPR protein EMP12 is required for the splicing of identical introns. However, our protein interaction analyses reveal that PPR21 does not interact with EMP12. Instead, both PPR21 and EMP12 interact with the small MutS-related (SMR) domain-containing PPR protein 1 (PPR-SMR1) and the short P-type PPR protein 2 (SPR2). PPR-SMR1 interacts with SPR2, and both proteins are required for the splicing of many introns in mitochondria, including nad2 intron 1, 2, and 4. These results suggest that a PPR21-(PPR-SMR1/SPR2)-EMP12 complex is involved in the splicing of nad2 introns in maize mitochondria.

Persistent inner tepals and wings protect developing seeds of Rheum nanum from insect herbivory in Central Asian cold deserts.

Although the postdispersal functions of diaspore (fruit and its appendages) have been reported, little is known about their protective/defensive functions. In this context, diaspores with appendages (persistent inner tepals and/or fruit wings) that experienced predispersal herbivory by insects in natural populations of Rheum nanum were investigated, and the seed abortion percentage, seed and embryo masses, and germination of seeds from diaspores with different categories of insect herbivory were measured and compared. Predispersal insect herbivory of R. nanum diaspores was prevalent in the four investigated populations, but the percentage of diaspores with appendages (persistent inner tepals and and/or fruit wings) damaged by insects was significantly higher than that of diaspores with the pericarp damaged by insects. Seeds from diaspores with gnawed appendages experienced significantly less damage than those with gnawed pericarps. Importantly, we conclude that fruit appendages of R. nanum help to mechanically protect developing seeds from predispersal insect herbivory.

Identification of key genes involved in lignin and flavonoid accumulation during Tilia tuan seed maturation.

KEY MESSAGE: Transcriptomics and phenotypic data analysis identified 24 transcription factors (TFs) that play key roles in regulating the competitive accumulation of lignin and flavonoids. Tilia tuan Szyszyl. (T. tuan) is a timber tree species with important ecological and commercial value. However, its highly lignified pericarp results in a low seed germination rate and a long dormancy period. In addition, it is unknown whether there is an interaction between the biosynthesis of flavonoids and lignin as products of the phenylpropanoid pathway during seed development. To explore the molecular regulatory mechanism of lignin and flavonoid biosynthesis, T. tuan seeds were harvested at five stages (30, 60, 90, 120, and 150 days after pollination) for lignin and flavonoid analyses. The results showed that lignin accumulated rapidly in the early and middle stages (S1, S3, and S4), and rapid accumulation of flavonoids during the early and late stages (S1 and S5). High-throughput RNA sequencing analysis of developing seeds identified 50,553 transcripts, including 223 phenylpropanoid biosynthetic pathway genes involved in lignin accumulation grouped into 3 clusters, and 106 flavonoid biosynthetic pathway genes (FBPGs) grouped into 2 clusters. Subsequent WGCNA and time-ordered gene co-expression network (TO-GCN) analysis revealed that 24 TFs (e.g., TtARF2 and TtWRKY15) were involved in flavonoids and lignin biosynthesis regulation. The transcriptome data were validated by qRT-PCR to analyze the expression profiles of key enzyme-coding genes. This study revealed that there existed a competitive relationship between flavonoid and lignin biosynthesis pathway during the development of T. tuan seeds, that provide a foundation for the further exploration of molecular mechanisms underlying lignin and flavonoid accumulation in T. tuan seeds.

Transcriptomic landscape of seedstick in Arabidopsis thaliana funiculus after fertilisation.

BACKGROUND: In Angiosperms, the continuation of plant species is intricately dependent on the funiculus multifaceted role in nutrient transport, mechanical support, and dehiscence of seeds. SEEDSTICK (STK) is a MADS-box transcription factor involved in seed size and abscission, and one of the few genes identified as affecting funiculus growth. Given the importance of the funiculus to a correct seed development, allied with previous phenotypic observations of stk mutants, we performed a transcriptomic analysis of stk funiculi from floral stage 17, using RNA-sequencing, to infer on the deregulated networks of genes. RESULTS: The generated dataset of differentially expressed genes was enriched with cell wall biogenesis, cell cycle, sugar metabolism and transport terms, all in accordance with stk phenotype observed in funiculi from floral stage 17. We selected eight differentially expressed genes for transcriptome validation using qPCR and/or promoter reporter lines. Those genes were involved with abscission, seed development or novel functions in stk funiculus, such as hormones/secondary metabolites transport. CONCLUSION: Overall, the analysis performed in this study allowed delving into the STK-network established in Arabidopsis funiculus, fulfilling a literature gap. Simultaneously, our findings reinforced the reliability of the transcriptome, making it a valuable resource for candidate genes selection for functional genetic studies in the funiculus. This will enhance our understanding on the regulatory network controlled by STK, on the role of the funiculus and how seed development may be affected by them.

Understanding grain development in the Poaceae family by comparing conserved and distinctive pathways through omics studies in wheat and maize.

The Poaceae family, commonly known as the grass family, encompasses a diverse group of crops that play an essential role in providing food, fodder, biofuels, environmental conservation, and cultural value for both human and environmental well-being. Crops in Poaceae family are deeply intertwined with human societies, economies, and ecosystems, making it one of the most significant plant families in the world. As the major reservoirs of essential nutrients, seed grain of these crops has garnered substantial attention from researchers. Understanding the molecular and genetic processes that controls seed formation, development and maturation can provide insights for improving crop yield, nutritional quality, and stress tolerance. The diversity in photosynthetic pathways between C3 and C4 plants introduces intriguing variations in their physiological and biochemical processes, potentially affecting seed development. In this review, we explore recent studies performed with omics technologies, such as genomics, transcriptomics, proteomics and metabolomics that shed light on the mechanisms underlying seed development in wheat and maize, as representatives of C3 and C4 plants respectively, providing insights into their unique adaptations and strategies for reproductive success.

Genome-Wide Analyses of MADS-Box Genes Reveal Their Involvement in Seed Development and Oil Accumulation of Tea-Oil Tree (Camellia oleifera).

The seeds of Camellia oleifera produce high amount of oil, which can be broadly used in the fields of food, industry, and medicine. However, the molecular regulation mechanisms of seed development and oil accumulation in C. oleifera are unclear. In this study, evolutionary and expression analyses of the MADS-box gene family were performed across the C. oleifera genome for the first time. A total of 86 MADS-box genes (ColMADS) were identified, including 60 M-type and 26 MIKC members. More gene duplication events occurred in M-type subfamily (6) than that in MIKC subfamily (2), and SEP-like genes were lost from the MIKCC clade. Furthermore, 8, 15, and 17 differentially expressed ColMADS genes (DEGs) were detected between three developmental stages of seed (S1/S2, S2/S3, and S1/S3), respectively. Among these DEGs, the STK-like ColMADS12 and TT16-like ColMADS17 were highly expressed during the seed formation (S1 and S2), agreeing with their predicted functions to positively regulate the seed organogenesis and oil accumulation. While ColMADS57 and ColMADS07 showed increasing expression level with the seed maturation (S2 and S3), conforming to their potential roles in promoting the seed ripening. In all, these results revealed a critical role of MADS-box genes in the C. oleifera seed development and oil accumulation, which will contribute to the future molecular breeding of C. oleifera.

Confocal Imaging of Seeds.

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

BASIC PENTACYSTEINE1 regulates ABI4 by modification of two histone marks H3K27me3 and H3ac during early seed development of Medicago truncatula.

Introduction: The production of highly vigorous seeds with high longevity is an important lever to increase crop production efficiency, but its acquisition during seed maturation is strongly influenced by the growth environment. Methods: An association rule learning approach discovered MtABI4, a known longevity regulator, as a gene with transcript levels associated with the environmentally-induced change in longevity. To understand the environmental sensitivity of MtABI4 transcription, Yeast One-Hybrid identified a class I BASIC PENTACYSTEINE (MtBPC1) transcription factor as a putative upstream regulator. Its role in the regulation of MtABI4 was further characterized. Results and discussion: Overexpression of MtBPC1 led to a modulation of MtABI4 transcripts and its downstream targets. We show that MtBPC1 represses MtABI4 transcription at the early stage of seed development through binding in the CT-rich motif in its promoter region. To achieve this, MtBPC1 interacts with SWINGER, a sub-unit of the PRC2 complex, and Sin3-associated peptide 18, a sub-unit of the Sin3-like deacetylation complex. Consistent with this, developmental and heat stress-induced changes in MtABI4 transcript levels correlated with H3K27me3 and H3ac enrichment in the MtABI4 promoter. Our finding reveals the importance of the combination of histone methylation and histone de-acetylation to silence MtABI4 at the early stage of seed development and during heat stress.

Genomic decoding of Theobroma grandiflorum (cupuassu) at chromosomal scale: evolutionary insights for horticultural innovation.

BACKGROUND: Theobroma grandiflorum (Malvaceae), known as cupuassu, is a tree indigenous to the Amazon basin, valued for its large fruits and seed pulp, contributing notably to the Amazonian bioeconomy. The seed pulp is utilized in desserts and beverages, and its seed butter is used in cosmetics. Here, we present the sequenced telomere-to-telomere genome of cupuassu, disclosing its genomic structure, evolutionary features, and phylogenetic relationships within the Malvaceae family. FINDINGS: The cupuassu genome spans 423 Mb, encodes 31,381 genes distributed in 10 chromosomes, and exhibits approximately 65% gene synteny with the Theobroma cacao genome, reflecting a conserved evolutionary history, albeit punctuated with unique genomic variations. The main changes are pronounced by bursts of long-terminal repeat retrotransposons at postspecies divergence, retrocopied and singleton genes, and gene families displaying distinctive patterns of expansion and contraction. Furthermore, positively selected genes are evident, particularly among retained and dispersed tandem and proximal duplicated genes associated with general fruit and seed traits and defense mechanisms, supporting the hypothesis of potential episodes of subfunctionalization and neofunctionalization following duplication, as well as impact from distinct domestication process. These genomic variations may underpin the differences observed in fruit and seed morphology, ripening, and disease resistance between cupuassu and the other Malvaceae species. CONCLUSIONS: The cupuassu genome offers a foundational resource for both breeding improvement and conservation biology, yielding insights into the evolution and diversity within the genus Theobroma.

Genome-Wide Analysis of MADS-Box Gene Family Reveals CjSTK as a Key Regulator of Seed Abortion in Camellia japonica.

The plant MADS-box transcription factor family is a major regulator of plant flower development and reproduction, and the AGAMOUS-LIKE11/SEEDSTICK (AGL11/STK) subfamily plays conserved functions in the seed development of flowering plants. Camellia japonica is a world-famous ornamental flower, and its seed kernels are rich in highly valuable fatty acids. Seed abortion has been found to be common in C. japonica, but little is known about how it is regulated during seed development. In this study, we performed a genome-wide analysis of the MADS-box gene the in C. japonica genome and identified 126 MADS-box genes. Through gene expression profiling in various tissue types, we revealed the C/D-class MADS-box genes were preferentially expressed in seed-related tissues. We identified the AGL11/STK-like gene, CjSTK, and showed that it contained a typical STK motif and exclusively expressed during seed development. We found a significant increase in the CjSTK expression level in aborted seeds compared with normally developing seeds. Furthermore, overexpression of CjSTK in Arabidopsis thaliana caused shorter pods and smaller seeds. Taken together, we concluded that the fine regulation of the CjSTK expression at different stages of seed development is critical for ovule formation and seed abortion in C. japonica. The present study provides evidence revealing the regulation of seed development in Camellia.

An epiQTL underlying asexual seed formation in Arabidopsis.

KEY MESSAGE: The DNA methylation status at an epigenetic quantitative trait locus in the Arabidopsis chromosome 2 is linked to the formation of apomictic-like endosperms. Seed development in most angiosperms is coupled to fertilization of the maternal gametes by two sperm cells. However, apomictic species can reproduce asexually via seeds. This trait is of great agricultural interest, as it would fix complex genotypes and allow for pollen-independent seed production. However, engineering full apomixis requires three independent processes: apomeiosis, parthenogenesis and autonomous endosperm development. While the first two have been successfully engineered in some crops, the formation of autonomous endosperms remains a challenge. Although it is known that this trait is under epigenetic control, such as of DNA methylation, the underlying mechanisms remain mostly undiscovered. Here, using epigenetic recombinant inbred lines, we identified an epigenetic quantitative trait locus in the Arabidopsis chromosome 2, which correlates with permissiveness for the formation of asexual seeds: hypomethylation at this genomic region allows the formation of larger autonomous endosperms. Importantly, the methylation at this locus only correlates with asexual seed size, and not to the size of sexual seeds or that of other organs. With this, we aim to show that screening for epialleles is a promising strategy to uncover loci underlying relevant traits and could pave the way to identifying genes necessary for the engineering of apomixis.

Spatiotemporally distinct responses to mechanical forces shape the developing seed of Arabidopsis.

Organ morphogenesis depends on mechanical interactions between cells and tissues. These interactions generate forces that can be sensed by cells and affect key cellular processes. However, how mechanical forces, together with biochemical signals, contribute to the shaping of complex organs is still largely unclear. We address this question using the seed of Arabidopsis as a model system. We show that seeds first experience a phase of rapid anisotropic growth that is dependent on the response of cortical microtubule (CMT) to forces, which guide cellulose deposition according to shape-driven stresses in the outermost layer of the seed coat. However, at later stages of development, we show that seed growth is isotropic and depends on the properties of an inner layer of the seed coat that stiffens its walls in response to tension but has isotropic material properties. Finally, we show that the transition from anisotropic to isotropic growth is due to the dampening of cortical microtubule responses to shape-driven stresses. Altogether, our work supports a model in which spatiotemporally distinct mechanical responses control the shape of developing seeds in Arabidopsis.

UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development.

Seeds play a crucial role in plant reproduction, making it essential to identify genes that affect seed development. In this study, we focused on UDP-glucosyltransferase 71C4 (UGT71C4) in cotton, a member of the glycosyltransferase family that shapes seed width and length, thereby influencing seed index and seed cotton yield. Overexpression of UGT71C4 results in seed enlargement owing to its glycosyltransferase activity on flavonoids, which redirects metabolic flux from lignin to flavonoid metabolism. This shift promotes cell proliferation in the ovule via accumulation of flavonoid glycosides, significantly enhancing seed cotton yield and increasing the seed index from 10.66 g to 11.91 g. By contrast, knockout of UGT71C4 leads to smaller seeds through activation of the lignin metabolism pathway and redirection of metabolic flux back to lignin synthesis. This redirection leads to increased ectopic lignin deposition in the ovule, inhibiting ovule growth and development, and alters yield components, increasing the lint percentage from 41.42% to 43.40% and reducing the seed index from 10.66 g to 8.60 g. Our research sheds new light on seed size development and reveals potential pathways for enhancing seed yield.