The pan-tandem repeat map highlights multiallelic variants underlying gene expression and agronomic traits in rice.

Tandem repeats (TRs) are genomic regions that tandemly change in repeat number, which are often multiallelic. Their characteristics and contributions to gene expression and quantitative traits in rice are largely unknown. Here, we survey rice TR variations based on 231 genome assemblies and the rice pan-genome graph. We identify 227,391 multiallelic TR loci, including 54,416 TR variations that are absent from the Nipponbare reference genome. Only 1/3 TR variations show strong linkage with nearby bi-allelic variants (SNPs, Indels and PAVs). Using 193 panicle and 202 leaf transcriptomic data, we reveal 485 and 511 TRs act as QTLs independently of other bi-allelic variations to nearby gene expression, respectively. Using plant height and grain width as examples, we identify and validate TRs contributions to rice agronomic trait variations. These findings would enhance our understanding of the functions of multiallelic variants and facilitate rice molecular breeding.

A pan-TE map highlights transposable elements underlying domestication and agronomic traits in Asian rice.

Transposable elements (TEs) are ubiquitous genomic components and hard to study due to being highly repetitive. Here we assembled 232 chromosome-level genomes based on long-read sequencing data. Coupling the 232 genomes with 15 existing assemblies, we developed a pan-TE map comprising both cultivated and wild Asian rice. We detected 177 084 high-quality TE variations and inferred their derived state using outgroups. We found TEs were one source of phenotypic variation during rice domestication and differentiation. We identified 1246 genes whose expression variation was associated with TEs but not single-nucleotide polymorphisms (SNPs), such as OsRbohB, and validated OsRbohB's relative expression activity using a dual-Luciferase (LUC) reporter assays system. Our pan-TE map allowed us to detect multiple novel loci associated with agronomic traits. Collectively, our findings highlight the contributions of TEs to domestication, differentiation and agronomic traits in rice, and there is massive potential for gene cloning and molecular breeding by the high-quality Asian pan-TE map we generated.

Population-level exploration of alternative splicing and its unique role in controlling agronomic traits of rice.

Alternative splicing (AS) plays crucial roles in regulating various biological processes in plants. However, the genetic mechanisms underlying AS and its role in controlling important agronomic traits in rice (Oryza sativa) remain poorly understood. In this study, we explored AS in rice leaves and panicles using the rice minicore collection. Our analysis revealed a high level of transcript isoform diversity, with approximately one fifth of potential isoforms acting as major transcripts in both tissues. Regarding the genetic mechanism of AS, we found that the splicing of 833 genes in the leaf and 1,230 genes in the panicle was affected by cis-genetic variation. Twenty-one percent of these AS events could only be explained by large structural variations. Approximately 77.5% of genes with significant splicing quantitative trait loci (sGenes) exhibited tissue-specific regulation, and AS can cause 26.9% (leaf) and 23.6% (panicle) of sGenes to have altered, lost or gained functional domains. Additionally, through splicing-phenotype association analysis, we identified phosphate-starvation induced RING-type E3 ligase (OsPIE1; LOC_Os01g72480), whose splicing ratio was significantly associated with plant height. In summary, this study provides an understanding of AS in rice and its contribution to the regulation of important agronomic traits.

Uncovering key salt-tolerant regulators through a combined eQTL and GWAS analysis using the super pan-genome in rice.

For sessile plants, gene expression plays a pivotal role in responding to salinity stress by activating or suppressing specific genes. However, our knowledge of genetic variations governing gene expression in response to salt stress remains limited in natural germplasm. Through transcriptome analysis of the Global Mini-Core Rice Collection consisting of a panel of 202 accessions, we identified 22 345 and 27 610 expression quantitative trait loci associated with the expression of 7787 and 9361 eGenes under normal and salt-stress conditions, respectively, leveraging the super pan-genome map. Notably, combined with genome-wide association studies, we swiftly pinpointed the potential candidate gene STG5-a major salt-tolerant locus known as qSTS5. Intriguingly, STG5 is required for maintaining Na+/K+ homeostasis by directly regulating the transcription of multiple members of the OsHKT gene family. Our study sheds light on how genetic variants influence the dynamic changes in gene expression responding to salinity stress and provides a valuable resource for the mining of salt-tolerant genes in the future.

Rosa roxburghii Tratt Pomace Crude Extract Inactivates Cronobacter sakazakii Isolated from Powdered Infant Formula.

Cronobacter sakazakii is an important foodborne pathogen in powder infant formula (PIF). The objective of this study was to evaluate the inactivation effect of Rosa roxburghii Tratt pomace crude extract (RRPCE) on C. sakazakii isolated from PIF and to reveal the mechanism of action. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to evaluate the inhibitory activity of RRPCE against C. sakazakii. The inhibitory mechanism was revealed from the perspective of effects of RRPCE on intracellular adenosine 5'-triphosphate (ATP), reactive oxygen species (ROS), membrane potential, protein and nucleic acid leakage, and cell morphology of C. sakazakii. The inactivation effects of RRPCE on C. sakazakii in biofilms on stainless steel, tinplate, glass, silica gel, polyethylene terephthalate, and polystyrene to evaluate its potential as a natural disinfectant. The results showed that the MIC and MBC of RRPCE against C. sakazakii were 7.5 and 15 mg/mL, respectively. After treatments with RRPCE, intracellular ATP content decreased significantly while intracellular ROS level increased significantly (p < 0.05). The cell membrane depolarization, large leakage of proteins and nucleic acids, and severely damaged cell morphology also occurred in C. sakazakii treated with RRPCE. In addition, a 20-minute treatment with 2 MIC (15 mg/mL) of RRPCE could inactivate all C. sakazakii (from 6.10 to 6.40 CFU/mL) in biofilms on all six contact surfaces. Our findings suggest that RRPCE is ideal for the inactivation of C. sakazakii and has the potential to be used as a natural disinfectant for the inactivation of PIF packaging materials and containers.

mRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

A centromere map based on super pan-genome highlights the structure and function of rice centromeres.

Rice (Oryza sativa) is a significant crop worldwide with a genome shaped by various evolutionary factors. Rice centromeres are crucial for chromosome segregation, and contain some unreported genes. Due to the diverse and complex centromere region, a comprehensive understanding of rice centromere structure and function at the population level is needed. We constructed a high-quality centromere map based on the rice super pan-genome consisting of a 251-accession panel comprising both cultivated and wild species of Asian and African rice. We showed that rice centromeres have diverse satellite repeat CentO, which vary across chromosomes and subpopulations, reflecting their distinct evolutionary patterns. We also revealed that long terminal repeats (LTRs), especially young Gypsy-type LTRs, are abundant in the peripheral CentO-enriched regions and drive rice centromere expansion and evolution. Furthermore, high-quality genome assembly and complete telomere-to-telomere (T2T) reference genome enable us to obtain more centromeric genome information despite mapping and cloning of centromere genes being challenging. We investigated the association between structural variations and gene expression in the rice centromere. A centromere gene, OsMAB, which positively regulates rice tiller number, was further confirmed by expression quantitative trait loci, haplotype analysis and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 methods. By revealing the new insights into the evolutionary patterns and biological roles of rice centromeres, our finding will facilitate future research on centromere biology and crop improvement.

A rice variation map derived from 10 548 rice accessions reveals the importance of rare variants.

Detailed knowledge of the genetic variations in diverse crop populations forms the basis for genetic crop improvement and gene functional studies. In the present study, we analyzed a large rice population with a total of 10 548 accessions to construct a rice super-population variation map (RSPVM), consisting of 54 378 986 single nucleotide polymorphisms, 11 119 947 insertion/deletion mutations and 184 736 presence/absence variations. Assessment of variation detection efficiency for different population sizes revealed a sharp increase of all types of variation as the population size increased and a gradual saturation of that after the population size reached 10 000. Variant frequency analysis indicated that ∼90% of the obtained variants were rare, and would therefore likely be difficult to detect in a relatively small population. Among the rare variants, only 2.7% were predicted to be deleterious. Population structure, genetic diversity and gene functional polymorphism of this large population were evaluated based on different subsets of RSPVM, demonstrating the great potential of RSPVM for use in downstream applications. Our study provides both a rich genetic basis for understanding natural rice variations and a powerful tool for exploiting great potential of rare variants in future rice research, including population genetics and functional genomics.

B4GALNT1 promotes carcinogenesis by regulating epithelial-mesenchymal transition in hepatocellular carcinoma based on pan-cancer analysis.

BACKGROUND: ß-1,4-N-Acetyl galactosaminyltransferase1 (B4GALNT1) has been reported to play important roles in tumor progression and metastasis. Herein, we investigate the oncogenic roles of B4GALNT1 for hepatocellular carcinoma (HCC) based on immune microenvironment. METHODS: The Cancer Genome Atlas database and Genotype-Tissue Expression, cBioportal, ImmuCellAI, TIMER2 and other databases were searched to analyze the expression and clinical applications of B4GALNT1 in liver cancer patients. Kaplan-Meier survival analysis, Cox regression analysis, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis were utilized. Moreover, western blot assay, immune histochemistry staining, Cell Counting Kit-8 (CCK-8) assay, invasion and migration assay were performed to evaluate the function of B4GALNT1 in HCC. RESULTS: B4GALNT1 is overexpressed in 14 tumors, and the mRNA expression levels of B4GALNT1 were remarkably elevated in most tumor types, including HCC. In addition, B4GALNT1 expression was an independent prognostic factor, and a low expression of B4GALNT1 showed a better overall survival and disease-specific recurrence-free survival in patients with HCC. Gene set variation analysis indicated that B4GALNT1 presented a positive correlation with the epithelial-mesenchymal transition (EMT) pathway in HCC. B4GALNT1 expression was closely associated with immune activation genes in the HCC microenvironment. Moreover, B4GALNT1 expression was higher in HCC tissue than that in surrounding tissues. B4GALNT1 promoted the expression of apoptosis-related or EMT-related proteins and then decreased the expression of Bcl-2 and Bcl-xl in HCC cells, suggesting that B4GALNT1 knockdown significantly inhibited the proliferation and invasion of HCC cells. CONCLUSIONS: B4GALNT1 may promote HCC development through regulating the EMT pathway, which suggests that B4GALNT1 may serve as a promising predictive biomarker and a potential therapeutic target for HCC.

Genome-Wide Identification of Eucalyptus Heat Shock Transcription Factor Family and Their Transcriptional Analysis under Salt and Temperature Stresses.

Heat shock transcription factors (HSFs) activate heat shock protein gene expression by binding their promoters in response to heat stress and are considered to be pivotal transcription factors in plants. Eucalyptus is a superior source of fuel and commercial wood. During its growth, high temperature or other abiotic stresses could impact its defense capability and growth. Hsf genes have been cloned and sequenced in many plants, but rarely in Eucalyptus. In this study, we used bioinformatics methods to analyze and identify Eucalyptus Hsf genes, their chromosomal localization and structure. The phylogenetic relationship and conserved domains of their encoded proteins were further analyzed. A total of 36 Hsf genes were identified and authenticated from Eucalyptus, which were scattered across 11 chromosomes. They could be classified into three classes (A, B and C). Additionally, a large number of stress-related cis-regulatory elements were identified in the upstream promoter sequence of HSF, and cis-acting element analysis indicated that the expression of EgHsf may be regulated by plant growth and development, metabolism, hormones and stress responses. The expression profiles of five representative Hsf genes, EgHsf4, EgHsf9, EgHsf13, EgHsf24 and EgHsf32, under salt and temperature stresses were examined by qRT-PCR. The results show that the expression pattern of class B genes (EgHsf4, EgHsf24 and EgHsf32) was more tolerant to abiotic stresses than that of class A genes (EgHsf9 and EgHsf13). However, the expressions of all tested Hsf genes in six tissues were at different levels. Finally, we investigated the network of interplay between genes, and the results suggest that there may be synergistic effects between different Hsf genes in response to abiotic stresses. We conclude that the Hsf gene family played an important role in the growth and developmental processes of Eucalyptus and could be vital for maintaining cell homeostasis against external stresses. This study provides basic information on the members of the Hsf gene family in Eucalyptus and lays the foundation for the functional identification of related genes and the further investigation of their biological functions in plant stress regulation.

Comparative Expression Profiling of Snf2 Family Genes During Reproductive Development and Stress Responses in Rice.

Sucrose non-fermenting 2 (Snf2) protein family, as chromatin remodeling factors, is an enormous and the most diverse protein family, which contributes to biological processes of replication, transcription, and DNA repair using the energy of adenosine triphosphate (ATP) hydrolysis. The members of Snf2 family proteins have been well characterized in Arabidopsis, rice, and tomato. Although this family received significant attention, few genes were identified uniquely for their roles in mediating reproductive development and stress tolerance in rice. In the present study, we comprehensively analyzed the expression profiling of Snf2 genes during reproductive development and biotic/abiotic stresses. Our results showed that five proteins (OsCHR712/715/720/726/739) were mainly localized in the nucleus, while OsCHR715/739 were also slightly expressed in the cell membrane. There were abundant cis-acting elements in the putative promoter of Snf2 genes, including dehydration, MeJA, MYB binding site for drought, ABA-responsive, and stress-responsive element. Most of the genes were induced immediately after Magnaporthe oryzae infection at 12 h post-infection (hpi). About 55% of the total genes were upregulated under salt and drought stresses during the entire time, and 22-35% of the total genes were upregulated at 3 h. It was noteworthy that the seven genes (OsCHR705, OsCHR706, OsCHR710, OsCHR714, OsCHR721, OsCHR726, and OsCHR737) were upregulated, and one gene (OsCHR712) was downregulated under salt and drought stresses, respectively. The deficiency of OsCHR726 mutations displayed a hypersensitive phenotype under salt stress. These results will be significantly useful features for the validation of the rice Snf2 genes and facilitate understanding of the genetic engineering of crops with improved biotic and abiotic stresses.

OsDDM1b Controls Grain Size by Influencing Cell Cycling and Regulating Homeostasis and Signaling of Brassinosteroid in Rice.

Snf2 family proteins are the crucial subunits of chromatin-remodeling complexes (CRCs), which contributes to the biological processes of transcription, replication, and DNA repair using ATP as energy. Some CRC subunits have been confirmed to be the critical regulators in various aspects of plant growth and development and in epigenetic mechanisms such as histone modification, DNA methylation, and histone variants. However, the functions of Snf2 family genes in rice were poorly investigated. In this study, the relative expression profile of 40 members of Snf2 family in rice was studied at certain developmental stages of seed. Our results revealed that OsCHR741/OsDDM1b (Decrease in DNA methylation 1) was accumulated highly in the early developmental stage of seeds. We further analyzed the OsDDM1b T-DNA insertion loss-of-function of mutant, which exhibited dwarfism, smaller organ size, and shorter and wider grain size than the wild type (Hwayoung, HY), yet no difference in 1,000-grain weight. Consistent with the grain size, the outer parenchyma cell layers of lemma in osddm1b developed more cells with decreased size. OsDDM1b encoded a nucleus, membrane-localized protein and was distributed predominately in young spikelets and seeds, asserting its role in grain size. Meanwhile, the osddm1b was less sensitive to brassinosteroids (BRs) while the endogenous BR levels increased. We detected changes in the expression levels of the BR signaling pathway and feedback-inhibited genes with and without exogenous BR application, and the alterations of expression were also observed in grain size-related genes in the osddm1b. Altogether, our results suggest that OsDDM1b plays a crucial role in grain size via influencing cell proliferation and regulating BR signaling and homeostasis.

Characterization of auxin transporter AUX, PIN and PILS gene families in pineapple and evaluation of expression profiles during reproductive development and under abiotic stresses.

Polar auxin transport in plant is mediated by influx and efflux transporters, which are encoded by AUX/LAX, PIN and PILS genes, respectively. The auxin transporter gene families have been characterized in several species from monocots and eudicots. However, a genome-wide overview of auxin transporter gene families in pineapple is not yet available. In this study, we identified a total of threeAcAUX genes, 12 AcPIN genes, and seven AcPILS genes in the pineapple genome, which were variably located on 15 chromosomes. The exon-intron structure of these genes and properties of deduced proteins were relatively conserved within the same family. Most protein motifs were widespread in the AUX, PIN or PILS proteins, whereas a few motifs were absent in only one or two proteins. Analysis of the expression profiles of these genes elucidated that several genes exhibited either preferential or tissue-specific expression patterns in vegetative and/or reproductive tissues. AcAUX2 was specifically expressed in the early developmental ovules, while AcPIN1b and AcPILS2 were strongly expressed in stamens and ovules. AcPIN9b, AcPILS1, AcPILS6a, 6b and 6c were abundantly expressed in stamens. Furthermore, qRT-PCR results showed that several genes in these families were responsive to various abiotic stresses. Comparative analysis indicated that the genes with close evolutionary relationships among pineapple, rice and Arabidopsis exhibited similar expression patterns. Overexpression of the AcAUX1 in Arabidopsis rescued the phenotype in aux1-T, and resulted in increased lateral roots in WT. These results will provide new insights into auxin transporter genes of pineapple and facilitate our understanding of their roles in pineapple growth and development.

CELL CYCLE SEITCH 52 regulates tillering by interacting with LATERAL SUPPRESSOR in non-heading Chinese cabbage.

With the discovery of essential genes regulating tillering, such as MONOCULM 1 (MOC1) in rice and LATERAL SUPPRESSOR (LAS in Arabidopsis, LS in tomato), research on tillering mechanisms has made great progress; however, the study of tillering in non-heading Chinese cabbage (NHCC) is rare. Here, we report that BcLAS, as a member of the GRAS family, plays an important role in the tillering of NHCC during its vegetative growth. BcLAS was almost not expressed in other examed parts except leaf axils throughout life. When the expression of BcLAS was silenced utilizing virus-induced gene silencing (VIGS) technology, we found that the tiller number of 'Maertou' decreased sharply. In 'Suzhouqing', overexpression of BcLAS significantly promoted tillering. BcCCS52, the orthologue to CELL CYCLE SEITCH 52 (CCS52), interacts with BcLAS. Downregulation of the expression of BcCCS52 promoted tillering of 'Suzhouqing'; therefore, we conclude that BcCCS52 plays a negative role in tillering regulation. Our findings reveal the tillering regulation mechanism of NHCCs at the vegetative stage and report an orthologue of CCS52 regulating tillering in NHCC.

BcHTT4 Inhibits Branching of Non-Heading Chinese Cabbage at the Vegetative Stage.

Branching is speculated to contribute to the plant architecture and crop yield. As a quantitative trait, branching is regulated by multiple genes in non-heading Chinese cabbage (NHCC). Several related candidate genes have been discovered in previous studies on the branching of NHCC, but their specific functions and regulatory mechanisms still need to be verified and explored. In this study, we found that the expression of BcHTT4, the ortholog to HEAT-INDUCED TAS1 TARGET4 (HTT4) in Arabidopsis, was significantly different between 'Suzhouqing' (common type) and 'Maertou' (multiple shoot branching type) in NHCC, which was consistent with the previous transcriptome sequencing results. The silencing of BcHTT4 expression in non-heading Chinese cabbage promotes axillary bud growth at the vegetative stage. When BcHTT4 is overexpressed in Arabidopsis, branching will decrease. In further study, we found that BcHTT4 interacts with immunophilin BcFKBP13 in vivo and in vitro through yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays. Moreover, quantitative real-time PCR analysis showed that when the expression of BcHTT4 was silenced in 'Suzhouqing', the expression of BcFKBP13 also decreased significantly. Our findings reveal that BcHTT4 is involved in the branching mechanism and interacts with immunophilin BcFKBP13 in NHCC.

Genome-wide characterization and expression profiling of Eucalyptus grandis HD-Zip gene family in response to salt and temperature stress.

BACKGROUND: The HD-Zip transcription factors are unique to plants and play an essential role in plant growth, development and stress responses. The HD-Zip transcription factor family consists of a highly conserved homeodomain (HD) and a leucine zipper domain (LZ) domain. Although the HD-Zip gene family has been extensively studied in many plant species, a systematic study of the Eucalyptus HD-Zip family has not been reported until today. Here, we systematically identified 40 HD-Zip genes in Eucalyptus (Eucalyptus grandis). Besides, we comprehensively analyzed the HD-Zips of Eucalyptus by studying the homology, conserved protein regions, gene structure, 3D structure of the protein, location of the genes on the chromosomes and the expression level of the genes in different tissues. RESULTS: The HD-Zip family in Eucalyptus has four subfamilies, which is consistent with other plants such as Arabidopsis and rice. Moreover, genes that are in the same group tend to have similar exon-intron structures, motifs, and protein structures. Under salt stress and temperature stress, the Eucalyptus HD-Zip transcription factors show a differential expression pattern. CONCLUSIONS: Our findings reveal the response of HD-Zip transcription factors under salt and temperature stresses, laying a foundation for future analysis of Eucalyptus HD-Zip transcription factors.

The bZIP Transcription Factor GmbZIP15 Negatively Regulates Salt- and Drought-Stress Responses in Soybean.

Soybean (Glycine max), as an important oilseed crop, is constantly threatened by abiotic stress, including that caused by salinity and drought. bZIP transcription factors (TFs) are one of the largest TF families and have been shown to be associated with various environmental-stress tolerances among species; however, their function in abiotic-stress response in soybean remains poorly understood. Here, we characterized the roles of soybean transcription factor GmbZIP15 in response to abiotic stresses. The transcript level of GmbZIP15 was suppressed under salt- and drought-stress conditions. Overexpression of GmbZIP15 in soybean resulted in hypersensitivity to abiotic stress compared with wild-type (WT) plants, which was associated with lower transcript levels of stress-responsive genes involved in both abscisic acid (ABA)-dependent and ABA-independent pathways, defective stomatal aperture regulation, and reduced antioxidant enzyme activities. Furthermore, plants expressing a functional repressor form of GmbZIP15 exhibited drought-stress resistance similar to WT. RNA-seq and qRT-PCR analyses revealed that GmbZIP15 positively regulates GmSAHH1 expression and negatively regulates GmWRKY12 and GmABF1 expression in response to abiotic stress. Overall, these data indicate that GmbZIP15 functions as a negative regulator in response to salt and drought stresses.