DataSheet_1_Gene expression variations and allele-specific expression of two rice and their hybrid in caryopses at single-nucleus resolution.docx

Seeds are an indispensable part of the flowering plant life cycle and a critical determinant of agricultural production. Distinct differences in the anatomy and morphology of seeds separate monocots and dicots. Although some progress has been made with respect to understanding seed development in Arabidopsis, the transcriptomic features of monocotyledon seeds at the cellular level are much less understood. Since most important cereal crops, such as rice, maize, and wheat, are monocots, it is essential to study transcriptional differentiation and heterogeneity during seed development at a finer scale. Here, we present single-nucleus RNA sequencing (snRNA-seq) results of over three thousand nuclei from caryopses of the rice cultivars Nipponbare and 9311 and their intersubspecies F1 hybrid. A transcriptomics atlas that covers most of the cell types present during the early developmental stage of rice caryopses was successfully constructed. Additionally, novel specific marker genes were identified for each nuclear cluster in the rice caryopsis. Moreover, with a focus on rice endosperm, the differentiation trajectory of endosperm subclusters was reconstructed to reveal the developmental process. Allele-specific expression (ASE) profiling in endosperm revealed 345 genes with ASE (ASEGs). Further pairwise comparisons of the differentially expressed genes (DEGs) in each endosperm cluster among the three rice samples demonstrated transcriptional divergence. Our research reveals differentiation in rice caryopsis from the single-nucleus perspective and provides valuable resources to facilitate clarification of the molecular mechanism underlying caryopsis development in rice and other monocots.</p

DataSheet_2_Gene expression variations and allele-specific expression of two rice and their hybrid in caryopses at single-nucleus resolution.xlsx

Seeds are an indispensable part of the flowering plant life cycle and a critical determinant of agricultural production. Distinct differences in the anatomy and morphology of seeds separate monocots and dicots. Although some progress has been made with respect to understanding seed development in Arabidopsis, the transcriptomic features of monocotyledon seeds at the cellular level are much less understood. Since most important cereal crops, such as rice, maize, and wheat, are monocots, it is essential to study transcriptional differentiation and heterogeneity during seed development at a finer scale. Here, we present single-nucleus RNA sequencing (snRNA-seq) results of over three thousand nuclei from caryopses of the rice cultivars Nipponbare and 9311 and their intersubspecies F1 hybrid. A transcriptomics atlas that covers most of the cell types present during the early developmental stage of rice caryopses was successfully constructed. Additionally, novel specific marker genes were identified for each nuclear cluster in the rice caryopsis. Moreover, with a focus on rice endosperm, the differentiation trajectory of endosperm subclusters was reconstructed to reveal the developmental process. Allele-specific expression (ASE) profiling in endosperm revealed 345 genes with ASE (ASEGs). Further pairwise comparisons of the differentially expressed genes (DEGs) in each endosperm cluster among the three rice samples demonstrated transcriptional divergence. Our research reveals differentiation in rice caryopsis from the single-nucleus perspective and provides valuable resources to facilitate clarification of the molecular mechanism underlying caryopsis development in rice and other monocots.</p

Data_Sheet_1_SWELLMAP 2, a phyB-Interacting Splicing Factor, Negatively Regulates Seedling Photomorphogenesis in Arabidopsis.docx

Light-triggered transcriptome reprogramming is critical for promoting photomorphogenesis in Arabidopsis seedlings. Nonetheless, recent studies have shed light on the importance of alternative pre-mRNA splicing (AS) in photomorphogenesis. The splicing factors splicing factor for phytochrome signaling (SFPS) and reduced red-light responses in cry1cry2 background1 (RRC1) are involved in the phytochrome B (phyB) signaling pathway and promote photomorphogenesis by controlling pre-mRNA splicing of light- and clock-related genes. However, splicing factors that serve as repressors in phyB signaling pathway remain unreported. Here, we report that the splicing factor SWELLMAP 2 (SMP2) suppresses photomorphogenesis in the light. SMP2 physically interacts with phyB and colocalizes with phyB in photobodies after light exposure. Genetic analyses show that SMP2 antagonizes phyB signaling to promote hypocotyl elongation in the light. The homologs of SMP2 in yeast and human belong to second-step splicing factors required for proper selection of the 3' splice site (3'SS) of an intron. Notably, SMP2 reduces the abundance of the functional REVEILLE 8 a (RVE8a) form, probably by determining the 3'SS, and thereby inhibits RVE8-mediated transcriptional activation of clock genes containing evening elements (EE). Finally, SMP2-mediated reduction of functional RVE8 isoform promotes phytochrome interacting factor 4 (PIF4) expression to fine-tune hypocotyl elongation in the light. Taken together, our data unveil a phyB-interacting splicing factor that negatively regulates photomorphogenesis, providing additional information for further mechanistic investigations regarding phyB-controlled AS of light- and clock-related genes.</p

Table_4_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.XLSX

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p

Image_3_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.JPEG

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p

Table_10_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.xlsx

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p

Table_5_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.XLSX

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p

Table_1_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.DOCX

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p

Table_8_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.XLSX

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p

Image_4_Analysis of the Transcriptional Dynamics of Regulatory Genes During Peanut Pod Development Caused by Darkness and Mechanical Stress.JPEG

Peanut is an oil crop with important economic value that is widely cultivated around the world. It blooms on the ground but bears fruit underground. When the peg penetrates the ground, it enters a dark environment, is subjected to mechanical stress from the soil, and develops into a normal pod. When a newly developed pod emerges from the soil, it turns green and stops growing. It has been reported that both darkness and mechanical stress are necessary for normal pod development. In this study, we investigated changes in gene expression during the reverse process of peg penetration: developmental arrest caused by pod (Pattee 3 pods) excavation. Bagging the aerial pods was used to simulate loss of mechanical pressure, while direct exposure of the aerial pods was used to simulate loss of both mechanical pressure and darkness. After the loss of mechanical stress and darkness, the DEGs were significantly enriched in photosynthesis, photosynthesis–antenna proteins, plant–pathogen interaction, DNA replication, and circadian rhythm pathways. The DNA replication pathway was enriched by down-regulated genes, and the other four pathways were enriched by upregulated genes. Upregulated genes were also significantly enriched in protein ubiquitination and calmodulin-related genes, highlighting the important role of ubiquitination and calcium signaling in pod development. Further analysis of DEGs showed that phytochrome A (Phy A), auxin response factor 9 (IAA9), and mechanosensitive ion channel protein played important roles in geocarpy. The expression of these two genes increased in subterranean pods but decreased in aerial pods. Based on a large number of chloroplast-related genes, calmodulin, kinases, and ubiquitin-related proteins identified in this study, we propose two possible signal transduction pathways involved in peanut geocarpy, namely, one begins in chloroplasts and signals down through phosphorylation, and the other begins during abiotic stress and signals down through calcium signaling, phosphorylation, and ubiquitination. Our study provides valuable information about putative regulatory genes for peanut pod development and contributes to a better understanding of the biological phenomenon of geocarpy.</p