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.

Programmable RNA N6 -methyladenosine editing with CRISPR/dCas13a in plants.

N6 -methyladenonsine (m6 A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m6 A modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable m6 A editing tools by fusing the m6 A writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual m6 A sites on mRNAs. We demonstrated that our m6 A editors could efficiently and specifically deposit and remove m6 A modifications on specific RNA transcripts in both Nicotiana benthamiana and Arabidopsis thaliana. Moreover, we found that targeting SHORT-ROOT (SHR) transcripts with a methylation editor could significantly increase its m6 A levels with limited off-target effects and promote its degradation. This leads to a boost in plant growth with enlarged leaves and roots, increased plant height, plant biomass, and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6 A editing tools can be applied to study the functions of individual m6 A modifications in plants, and may also have potential applications for future crop improvement.

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.

The MRE11-ATM-SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae.

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11-ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11-ATM-SOG1 pathway serves as a prime example of plant-pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

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.

Gibberellin signaling regulates lignin biosynthesis to modulate rice seed shattering.

The elimination of seed shattering was a key step in rice (Oryza sativa) domestication. In this paper, we show that increasing the gibberellic acid (GA) content or response in the abscission region enhanced seed shattering in rice. We demonstrate that SLENDER RICE1 (SLR1), the key repressor of GA signaling, could physically interact with the rice seed shattering-related transcription factors quantitative trait locus of seed shattering on chromosome 1 (qSH1), O. sativa HOMEOBOX 15 (OSH15), and SUPERNUMERARY BRACT (SNB). Importantly, these physical interactions interfered with the direct binding of these three regulators to the lignin biosynthesis gene 4-COUMARATE: COENZYME A LIGASE 3 (4CL3), thereby derepressing its expression. Derepression of 4CL3 led to increased lignin deposition in the abscission region, causing reduced rice seed shattering. Importantly, we also show that modulating GA content could alter the degree of seed shattering to increase harvest efficiency. Our results reveal that the "Green Revolution" phytohormone GA is important for regulating rice seed shattering, and we provide an applicable breeding strategy for high-efficiency rice harvesting.

Identification of natural allelic variation in TTL1 controlling thermotolerance and grain size by a rice super pan-genome.

Continuously increasing global temperatures present great challenges to food security. Grain size, one of the critical components determining grain yield in rice (Oryza sativa L.), is a prime target for genetic breeding. Thus, there is an immediate need for genetic improvement in rice to maintain grain yield under heat stress. However, quantitative trait loci (QTLs) endowing heat stress tolerance and grain size in rice are extremely rare. Here, we identified a novel negative regulator with pleiotropic effects, Thermo-Tolerance and grain Length 1 (TTL1), from the super pan-genomic and transcriptomic data. Loss-of-function mutations in TTL1 enhanced heat tolerance, and caused an increase in grain size by coordinating cell expansion and proliferation. TTL1 was shown to function as a transcriptional regulator and localized to the nucleus and cell membrane. Furthermore, haplotype analysis showed that hapL and hapS of TTL1 were obviously correlated with variations of thermotolerance and grain size in a core collection of cultivars. Genome evolution analysis of available rice germplasms suggested that TTL1 was selected during domestication of the indica and japonica rice subspecies, but still had much breeding potential for increasing grain length and thermotolerance. These findings provide insights into TTL1 as a novel potential target for the development of high-yield and thermotolerant rice varieties.

Comparative Metabolomics Combined with Physiological Analysis Revealed Cadmium Tolerance Mechanism in Indica Rice (Oryza sativa L.).

Cadmium (Cd) pollution reduces rice production and quality, putting food security and human health at risk. We conducted comparative physiology and metabolomic analyses in two indica rice ('NH199' and 'NH224') to elucidate the Cd-tolerance mechanism. Cd hampered rice growth, induced oxidative stress, and changed the metabolomics profiling of the root. The biochemical and physiological analysis demonstrated that NH224 exhibited a more potent Cd-tolerance ability than NH199. Cd was primarily distributed in root, and NH224 had a lower Cd translocation factor than NH199 by about 24%. The metabolomic analysis revealed 180 and 177 differentially accumulated metabolites between Cd-stressed seedlings and the controls in NH224 and NH199, respectively. In NH224, amino acids biosynthesis, hormone metabolism, lipids-related metabolism, phenylalanine metabolism, and phenylpropanoid biosynthesis pathways were more active and highly associated with antioxidant defense system, biosynthesis of the cell wall and phytochelatins, and maintenance of plasma membrane stability. These findings provide insights into the metabolic profiles of rice following Cd stress and the screening and breeding of Cd-tolerant rice varieties.

Enhancement of Heat and Drought Stress Tolerance in Rice by Genetic Manipulation: A Systematic Review.

As a result of global warming, plants are subjected to ever-increasing abiotic stresses including heat and drought. Drought stress frequently co-occurs with heat stress as a result of water evaporation. These stressors have adverse effects on crop production, which in turn affects human food security. Rice is a major food resource grown widely in crop-producing regions throughout the world. However, increasingly common heat and drought stresses in growth regions can have negative impacts on seedling morphogenesis, reproductive organ establishment, overall yield, and quality. This review centers on responses to heat and drought stress in rice. Current knowledge of molecular regulation mechanisms is summarized. We focus on approaches to cope with heat and drought stress, both at the genetic level and from an agricultural practice perspective. This review establishes a basis for improving rice stress tolerance, grain quality, and yield for human benefit.

Systematic Analysis of BELL Family Genes in Zizania latifolia and Functional Identification of ZlqSH1a/b in Rice Seed Shattering.

The loss of seed shattering is an important event in crop domestication, and elucidating the genetic mechanisms underlying seed shattering can help reduce yield loss during crop production. This study is the first to systematically identify and analyse the BELL family of transcription factor-encoding genes in Chinese wild rice (Zizania latifolia). ZlqSH1a (Zla04G033720) and ZlqSH1b (Zla02G027130) were identified as key candidate genes involved in seed shattering in Z. latifolia. These genes were involved in regulating the development of the abscission layer (AL) and were located in the nucleus of the cell. Over-expression of ZlqSH1a and ZlqSH1b resulted in a complete AL between the grain and pedicel and significantly enhanced seed shattering after grain maturation in rice. Transcriptome sequencing revealed that 172 genes were differentially expressed between the wild type (WT) and the two transgenic (ZlqSH1a and ZlqSH1b over-expressing) plants. Three of the differentially expressed genes related to seed shattering were validated using qRT-PCR analysis. These results indicate that ZlqSH1a and ZlqSH1b are involved in AL development in rice grains, thereby regulating seed shattering. Our results could facilitate the genetic improvement of seed-shattering behaviour in Z. latifolia and other cereal crops.

Compared analysis with a high-quality genome of weedy rice reveals the evolutionary game of de-domestication.

The weedy rice (Oryza sativa f. spontanea) harbors large numbers of excellent traits and genetic diversities, which serves as a valuable germplasm resource and has been considered as a typical material for research about de-domestication. However, there are relatively few reference genomes on weedy rice that severely limit exploiting these genetic resources and revealing more details about de-domestication events. In this study, a high-quality genome (~376.4 Mb) of weedy rice A02 was assembled based on Nanopore ultra-long platform with a coverage depth of about 79.3× and 35,423 genes were predicted. Compared to Nipponbare genome, 5,574 structural variations (SVs) were found in A02. Based on super pan-genome graph, population SVs of 238 weedy rice and cultivated rice accessions were identified using public resequencing data. Furthermore, the de-domestication sites of weedy rice and domestication sites of wild rice were analyzed and compared based on SVs and single-nucleotide polymorphisms (SNPs). Interestingly, an average of 2,198 genes about de-domestication could only be found by F ST analysis based on SVs (SV-F ST) while not by F ST analysis based on SNPs (SNP-F ST) in divergent region. Additionally, there was a low overlap between domestication and de-domestication intervals, which demonstrated that two different mechanisms existed in these events. Our finding could facilitate pinpointing of the evolutionary events that had shaped the genomic architecture of wild, cultivated, and weedy rice, and provide a good foundation for cloning of the superior alleles for breeding.

Combining GWAS, Genome-Wide Domestication and a Transcriptomic Analysis Reveals the Loci and Natural Alleles of Salt Tolerance in Rice (Oryza sativa L.).

Soil salinity poses a serious threat to the sustainable production of rice (Oryza sativa L.) throughout the world. Thus, the detection of loci and alleles responsible for salt tolerance is fundamental to accelerating the improvement of rice and producing the resilient varieties that will ensure future harvests. In this study, we collected a set of 191 mini-core rice populations from around the world, evaluated their salt tolerance based on plant growth and development phenotypes at the seedling stage, and divided a standard evaluation score (SES) of visual salt injury into five different grades. We used ∼3.82 million single nucleotide polymorphisms (SNPs) to identify 155 significant SNPs and 275 genes associated with salt sensitivity based on a genome-wide association study (GWAS) of SES. In particular, two candidate genes, ZFP179 and OsDSR2, were associated with salt tolerance, and OsHKT1;1 was co-detected in the entire GWAS of all the panels and indica. Additionally, we investigated the transcriptional changes in cultivars 93-11 and PA64s under normal and salinity stress conditions and found 517 co-upregulated and 223 co-downregulated genes. These differentially expressed genes (DEGs) were highly enriched in "response to chemical" and "stress" based on the gene ontology enrichment analysis. Notably, 30 candidate genes that were associated with the salt tolerance analysis were obtained by integrating GWAS and transcriptomic DEG analyses, including 13 cloned genes that had no reports of tolerance to salt and 17 candidate genes whose functions were unknown. To further explore these genes and their alleles, we performed haplotype analysis, genome-wide domestication detection, and transcriptome analysis to breed improved varieties. This data and the genetic resources provided will be valuable for the development of salt tolerant rice varieties.

A super pan-genomic landscape of rice.

Pan-genomes from large natural populations can capture genetic diversity and reveal genomic complexity. Using de novo long-read assembly, we generated a graph-based super pan-genome of rice consisting of a 251-accession panel comprising both cultivated and wild species of Asian and African rice. Our pan-genome reveals extensive structural variations (SVs) and gene presence/absence variations. Additionally, our pan-genome enables the accurate identification of nucleotide-binding leucine-rich repeat genes and characterization of their inter- and intraspecific diversity. Moreover, we uncovered grain weight-associated SVs which specify traits by affecting the expression of their nearby genes. We characterized genetic variants associated with submergence tolerance, seed shattering and plant architecture and found independent selection for a common set of genes that drove adaptation and domestication in Asian and African rice. This super pan-genome facilitates pinpointing of lineage-specific haplotypes for trait-associated genes and provides insights into the evolutionary events that have shaped the genomic architecture of various rice species.

A transcriptional regulator that boosts grain yields and shortens the growth duration of rice.

Complex biological processes such as plant growth and development are often under the control of transcription factors that regulate the expression of large sets of genes and activate subordinate transcription factors in a cascade-like fashion. Here, by screening candidate photosynthesis-related transcription factors in rice, we identified a DREB (Dehydration Responsive Element Binding) family member, OsDREB1C, in which expression is induced by both light and low nitrogen status. We show that OsDREB1C drives functionally diverse transcriptional programs determining photosynthetic capacity, nitrogen utilization, and flowering time. Field trials with OsDREB1C-overexpressing rice revealed yield increases of 41.3 to 68.3% and, in addition, shortened growth duration, improved nitrogen use efficiency, and promoted efficient resource allocation, thus providing a strategy toward achieving much-needed increases in agricultural productivity.

Chromosome-level genome assembly of Zizania latifolia provides insights into its seed shattering and phytocassane biosynthesis.

Chinese wild rice (Zizania latifolia; family: Gramineae) is a valuable medicinal homologous grain in East and Southeast Asia. Here, using Nanopore sequencing and Hi-C scaffolding, we generated a 547.38 Mb chromosome-level genome assembly comprising 332 contigs and 164 scaffolds (contig N50 = 4.48 Mb; scaffold N50 = 32.79 Mb). The genome harbors 38,852 genes, with 52.89% of the genome comprising repetitive sequences. Phylogenetic analyses revealed close relation of Z. latifolia to Leersia perrieri and Oryza species, with a divergence time of 19.7-31.0 million years. Collinearity and transcriptome analyses revealed candidate genes related to seed shattering, providing basic information on abscission layer formation and degradation in Z. latifolia. Moreover, two genomic blocks in the Z. latifolia genome showed good synteny with the rice phytocassane biosynthetic gene cluster. The updated genome will support future studies on the genetic improvement of Chinese wild rice and comparative analyses between Z. latifolia and other plants.

OsRLR4 binds to the OsAUX1 promoter to negatively regulate primary root development in rice.

Root architecture is one of the most important agronomic traits that determines rice crop yield. The primary root (PR) absorbs mineral nutrients and provides mechanical support; however, the molecular mechanisms of PR elongation remain unclear in rice. Here, the two loss-of-function T-DNA insertion mutants of root length regulator 4 (OsRLR4), osrlr4-1 and osrlr4-2 with longer PR, and three OsRLR4 overexpression lines, OE-OsRLR4-1/-2/-3 with shorter PR compared to the wild type/Hwayoung (WT/HY), were identified. OsRLR4 is one of five members of the PRAF subfamily of the regulator chromosome condensation 1 (RCC1) family. Phylogenetic analysis of OsRLR4 from wild and cultivated rice indicated that it is under selective sweeps, suggesting its potential role in domestication. OsRLR4 controls PR development by regulating auxin accumulation in the PR tip and thus the root apical meristem activity. A series of biochemical and genetic analyses demonstrated that OsRLR4 functions directly upstream of the auxin transporter OsAUX1. Moreover, OsRLR4 interacts with the TRITHORAX-like protein OsTrx1 to promote H3K4me3 deposition at the OsAUX1 promoter, thus altering its transcription level. This work provides insight into the cooperation of auxin and epigenetic modifications in regulating root architecture and provides a genetic resource for plant architecture breeding.

Twenty years of plant genome sequencing: achievements and challenges.

Publication of the complete genome sequence of Arabidopsis thaliana, the first plant reference genome, in December 2000 heralded the beginning of the plant genome era. Over the past 20 years reference genomes have been generated for hundreds of plant species, spanning non-vascular to flowering plants. Releasing these plant genomes has dramatically advanced studies in all disciplines of plant biology. Importantly, multiple reference-level genomes have been generated for the major crops and their progenitors, enabling the creation of pan-genomes and exploration of domestication history and natural variations that can be adopted by modern crop breeding. We summarize the progress of plant genome sequencing and the challenges of sequencing more complex plant genomes and generating pan-genomes.