Rice Cultivar Nipponbare Research Articles

Transcriptome-wide m6A methylation and metabolomic analysis reveal regulatory networks in rice roots under manganese stress

Rice (Oryza sativa) has a higher tolerance to manganese (Mn) stress than other cereals. However, the regulatory mechanisms governing Mn tolerance in rice remain poorly understood. In this work, seedlings of the rice cultivar Nipponbare were treated with 1.0 mM MnCl2 for 10 days before root samples were collected for transcriptome-wide N6-methyladenosine (m6A) methylation and metabolome profiling. In the presence of extra Mn, we identified 2050 significantly modified m6A peaks and 2549 differentially expressed genes (DEGs). These DEGs were linked to key signaling pathways such as MAPK signaling, calcium signaling, and peroxides. Among these, 282 DEGs showed differential m6A methylation peaks, including 29 transcription factors, indicating they might be key upstream regulators of the Mn toxicity response. Furthermore, metabolomic research indicated considerable metabolic alterations in rice roots under Mn stress, notably in purine metabolism, amino acid biosynthesis, and glycerophospholipid metabolic pathways. Almost half of the metabolites were lipids or lipid-like compounds, indicating a potential function in signal transduction and membrane biogenesis. The findings lead to a better understanding of regulatory networks in rice roots that aid in Mn stress tolerance.

Difference in responsiveness of expressions of starch synthesis-related genes to CRCT among rice cultivars

ABSTRACT CO2-Responsive CCT Protein (CRCT) is a transcription factor regulating the expression of starch synthesis-related genes in the vegetative organs of rice. Among six rice cultivars (Nipponbare, Koshihikari, Habataki, Takanari, Momiroman, Leafstar), the expression of CRCT was highly correlated with the starch content of leaf sheath in Nipponbare, but not in Koshihikari, Takanari and Momiroman during plant development. Meanwhile, the expressions of starch synthesis-related genes such as ADP-glucose pyrophosphorylase large subunit 1 was correlated with CRCT expression in Nipponbare, Momiroman, and Leafstar, but the correlations were ambiguous in Koshihikari, Habataki, and Takanari. In addition, the slopes of its regression lines in Nipponbare were larger than those cultivars. These results suggest that the influence of CRCT on starch synthesis varies among these cultivars. Additionally, the similar cultivar differences were observed when these gene expressions were analyzed after sucrose treatment in leaf blade. Some mutations in the promoter regions of the starch synthesis-related genes were identified among these cultivars. There were cultivars in which the presence of mutations correlated with decreased expression of starch synthesis genes in response to CRCT. Thus, these mutations can partially explain the responsiveness of the starch synthesis-related genes to CRCT, and difference in starch content of vegetative organs among rice cultivars.

An epi-allele of SMS causes Sanming dominant genic male sterility in rice.

Male sterility is an important trait in rice for hybrid rice (Oryza sativa) breeding. However, the factors involved in dominant male sterility are largely unknown. Here, we identified a gene from Sanming dominant genic male sterile rice, named Sanming dominant male sterility (SMS), and reported that an epi-allele of this locus contributes to male sterility. Segregation analysis attributed dominant male sterility to a single locus, SMS, which we characterized using a male-sterile near isogenic line (NIL) of rice cultivar 93-11. The SMS locus was heterozygous in the male-sterile 93-11 NIL, containing an epi-allele identical to that in 93-11, and an epi-allele identical to that in rice cultivar Nipponbare, which we refer to as SMS9 and SMSN, respectively. SMS9 is silent and hyper-methylated, whereas SMSN is expressed and hypo-methylated in the 93-11 NIL. Overexpressing SMSN led to male sterility. Mutations in SMS rescued the male sterility of the 93-11 NIL. Interestingly, we observed the duplication of SMSN in Nipponbare, but did not observe the duplication of SMS9 in 93-11. Together, these findings suggest that the reduced methylation and enhanced expression of the SMSN epi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.

MiRiQ Database: A Platform forIn Silico Rice Mutant Screening.

Genetic studies using mutant resources have significantly contributed to elucidating plant gene function. Massive mutant libraries sequenced by next-generation sequencing technology facilitate mutant identification and functional analysis of genes of interest. Here, we report the creation and release of an open-access database (https://miriq.agr.kyushu-u.ac.jp/index.php), called Mutation-induced Rice in Kyushu University (MiRiQ), designed for in silico mutant screening based on a whole-genome-sequenced mutant library. This database allows any user to easily find mutants of interest without laborious efforts such as large-scale screening by PCR. The initial version of the MiRiQ database (version 1.0) harbors a total of 1.6 million single-nucleotide variants (SNVs) and InDels of 721 M1 plants that were mutagenized by N-methyl-N-nitrosourea treatment of the rice cultivar Nipponbare (Oryza sativa ssp. japonica). The SNVs were distributed among 87% of all 35,630 annotated protein-coding genes of the Nipponbare genome and were predicted to induce missense and nonsense mutations. The MiRiQ database provides built-in tools, such as a search tool by keywords and JBrowse for mutation searches. Users can request mutant seeds in the M2 or M3 generations from a request form linked to this database. We believe that the availability of a wide range of gene mutations in this database will benefit the plant science community and breeders worldwide by accelerating functional genomic research and crop improvement.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Growth and DNA Methylation Alteration in Rice (Oryza sativa L.) in Response to Ozone Stress.

With the development of urban industrialization, the increasing ozone concentration (O3) at ground level stresses on the survival of plants. Plants have to adapt to ozone stress. DNA methylation is crucial for a rapid response to abiotic stress in plants. Little information is known regarding the epigenetic response of DNA methylation of plants to O3 stress. This study is designed to explore the epigenetic mechanism and identify a possible core modification of DNA methylation or genes in the plant, in response to O3 stress. We investigated the agronomic traits and genome-wide DNA methylation variations of the Japonica rice cultivar Nipponbare in response to O3 stress at three high concentrations (80, 160, and 200 nmol·mol-1), simulated using open-top chambers (OTC). The flag leaf length, panicle length, and hundred-grain weight of rice showed beneficial effects at 80 nmol·mol-1 O3 and an inhibitory effect at both 160 and 200 nmol·mol-1 O3. The methylation-sensitive amplified polymorphism results showed that the O3-induced genome-wide methylation alterations account for 14.72-15.18% at three different concentrations. Our results demonstrated that methylation and demethylation alteration sites were activated throughout the O3 stress, mainly at CNG sites. By recovering and sequencing bands with methylation alteration, ten stress-related differentially amplified sequences, widely present on different chromosomes, were obtained. Our findings show that DNA methylation may be an active and rapid epigenetic response to ozone stress. These results can provide us with a theoretical basis and a reference to look for more hereditary information about the molecular mechanism of plant resistance to O3 pollution.

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

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.

Creating high-resistant starch rice by simultaneous editing of SS3a and SS3b.

Creating high-resistant starch rice by simultaneous editing of SS3a and SS3b.

Metabolomic analysis reveals the molecular responses to copper toxicity in rice (Oryza sativa)

Copper (Cu) is one of the essential microelements and widely participates in various pathways in plants, but excess Cu in plant cells could induce oxidative stress and harm plant growth. Rice (Oryza sativa) is a main crop food worldwide. The molecular mechanisms of rice in response to copper toxicity are still not well understood. In this study, two-week-old seedlings of the rice cultivar Nipponbare were treated with 100 μM Cu2+ (CuSO4) in the external solution for 10 days. Physiological analysis showed that excess Cu significantly inhibited the growth and biomass of rice seedlings. After Cu treatment, the contents of Mn and Zn were significantly reduced in the roots and shoots, while the Fe content was significantly increased in the roots. Meanwhile, the activities of antioxidant enzymes including SOD and POD were dramatically enhanced after Cu treatment. Based on metabolomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, 695 metabolites were identified in rice roots. Among these metabolites, 123 metabolites were up-regulated and 297 were down-regulated, respectively. The differential metabolites (DMs) include carboxylic acids and derivatives, benzene and substituted derivatives, carbonyl compounds, cinnamic acids and derivatives, fatty acyls and organ nitrogen compounds. KEGG analysis showed that these DMs were mainly enriched in TCA cycle, purine metabolism and starch and sucrose metabolism pathways. Many intermediates in the TCA cycle and purine metabolism were down-regulated, indicating a perturbed carbohydrate and nucleic acid metabolism. Taken together, the present study provides new insights into the mechanism of rice roots to Cu toxicity.

Lack of Tocopherol Inhibits Rice Growth by Triggering an Ectopic Stress Response and the Accumulation of DELLA Protein

Although tocopherols are essential for rice development, the molecular details by which their absence affects development remain to be determined. To study how tocopherols function during rice development, we performed a transcriptome deep sequencing (RNA-seq) analysis of the rice cultivar Nipponbare (Nip) and the tocopherol-deficient mutant small grain and dwarf 1-2 (sgd1-2). We identified 563 differentially expressed genes that were enriched in Gene Ontology categories associated with metabolism, stress, cellular responses, and transcriptional regulation. We determined that the total fatty acid composition of Nip and sgd1-2 was comparable, although cell membrane penetrability in sgd1-2 was significantly higher than in Nip under optimal growth conditions, indicating that tocopherol deficiency induces cell membrane damage. The expression levels of dehydration-responsive element binding 1 (DREB1) genes and free proline content in sgd1-2 were also higher than those in Nip. We also showed that the DELLA protein SLENDER RICE1 (SLR1) accumulated in sgd1-2, resulting in significant changes in the global transcriptome. Our study confirms that the lack of tocopherol accumulation in rice induced ectopic stress responses and limited growth by enhancing SLR1 abundance through increasing SLR1 transcript levels. These results provide new insights into tocopherol during rice development.

Transcriptome-wide analysis of m6A methylation reveals genetic responses to cadmium stress at germination stage in rice

Excess cadmium (Cd) seriously suppresses plant growth and development. Rice (Oryza sativa L.) is more susceptible to Cd toxicity than other crops. N6-methyladenosine (m6A) modifications play important regulatory roles in plant adaption to various stresses. However, the post-transcriptional RNA modification in response to Cd stress is still largely unclear in rice. In this study, seeds of the rice cultivar Nipponbare were treated with 0.5 μM CdCl2 for 10 days at germination stage, and then the roots were sampled for the transcriptome-m6A methylation profiling. Here, 1920 differential peaks (DPs) of m6A modifications and 1710 differentially expressed genes (DEGs) were identified in the roots after Cd treatment. Among them, root cell expansion suppressor OsRePRPs were remarkably up-regulated, while auxin/cytokinin-related genes were differentially modified by m6A under Cd stress including OsYUCCA1, OsCKX4, OsRR6, and OsEIL1. Besides, peroxidases (PRXs), metallothioneins (MTs), glutathione S-transferases (GSTs) and sulfate transporter SULTRs were modified by m6A and significantly up-regulated, which might be related to the Cd detoxification via the cell wall modifications and Cd chelate formation. Moreover, several m6A modified transcription factors and MAPK signaling genes showed obviously increased expression, consequently participating in the Cd-response regulatory network as the upstream components. The present study proposes a working model for the m6A modification and transcriptional regulation in rice during germination, and also provides new insights into how plants alleviate Cd toxicity.

N6-methyladenosine methylation analysis reveals transcriptome-wide expression response to salt stress in rice roots

The N6-methyladenosine (m6A) modifications play crucial regulatory roles in plant yield development, nutritional growth and stress adaption. Rice ( Oryza sativa ) is relatively sensitive to salt stress among cereal crops, while its regulatory mechanism conferring salt tolerance is still unclear. Here, we performed transcriptome-wide m6A analysis in roots of rice cultivar Nipponbare under salt conditions (0 and 75 mM NaCl). After salt treatments for 4 d, a total of 18,115 m6A peak callings of 12,516 genes and 22,052 peaks of 14,235 genes were identified under salt and control conditions, respectively. These m6A peaks were mostly enriched in CDS and 3′UTR regions. Totally, 346 m6A differentially peaks (DPs) and 1522 differentially expressed genes (DEGs) were identified in response to salt stress, which were mainly enriched in "MAPK signaling module" and "metal ion binding". Among them, the m6A related genes including 2 writers ( MTB and TRM4A ), 1 reader ( CPSF30 ) and 1 eraser ( ALKBH9A ). The m6A modified salt-responding genes including HAK4 , CIPK06 , RBOHH , Myb10 , ERF067 and other transcription factors might participate in transcriptional regulation or multiple Ca 2+ , ABA and ROS signal pathways. The m6A level and gene expression of these genes were further confirmed by m6A-IP-qPCR and qRT-PCR, respectively, which showed a high consistency with the m6A-seq and transcriptome data. This study provides epigenetic responses of molecular regulatory mechanisms of salt tolerance in rice. ● Identified 346 salt-induced m6A DPs and 1522 DEGs in rice roots. ● Most m6A DPs were enriched in stop codon and CDS related to gene length. ● Proposed the roles of m6A modified salt-responding genes in transcriptional regulation and multiple signal pathways. ● Performed m6A-IP-qPCR and qRT-PCR experiments to validate the accuracy of data.

Chromosome-Level Genome Assembly of a Fragrant Japonica Rice Cultivar 'Changxianggeng 1813' Provides Insights into Genomic Variations between Fragrant and Non-Fragrant Japonica Rice.

East Asia has an abundant resource of fragrant japonica rice that is gaining increasing interest among both consumers and producers. However, genomic resources and in particular complete genome sequences currently available for the breeding of fragrant japonica rice are still scarce. Here, integrating Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C methods, we presented a high-quality chromosome-level genome assembly (~378.78 Mb) for a new fragrant japonica cultivar ‘Changxianggeng 1813’, with 31,671 predicated protein-coding genes. Based on the annotated genome sequence, we demonstrated that it was the badh2-E2 type of deletion (a 7-bp deletion in the second exon) that caused fragrance in ‘Changxianggeng 1813’. Comparative genomic analyses revealed that multiple gene families involved in the abiotic stress response were expanded in the ‘Changxianggeng 1813’ genome, which further supported the previous finding that no generalized loss of abiotic stress tolerance associated with the fragrance phenotype. Although the ‘Changxianggeng 1813’ genome showed high genomic synteny with the genome of the non-fragrant japonica rice cultivar Nipponbare, a total of 289,970 single nucleotide polymorphisms (SNPs), 96,093 small insertion-deletion polymorphisms (InDels), and 8690 large structure variants (SVs, >1000 bp) were identified between them. Together, these genomic resources will be valuable for elucidating the mechanisms underlying economically important traits and have wide-ranging implications for genomics-assisted breeding in fragrant japonica rice.

Proteomic Analysis Reveals Salicylic Acid as a Pivotal Signal Molecule in Rice Response to Blast Disease Infection.

Rice blast disease caused by a fungus, Magnaporthe grisea, is one of the most destructive diseases in rice production worldwide, and salicylic acid (SA) can efficiently decrease the damage of M. grisea. Here, we combined the 2-Dimensional-Liquid Chromatography and the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (2D-LC-MALDI-TOF-TOF MS) techniques to compare and identify differentially expressed labelled proteins by the isobaric tags for relative and absolute quantitation (iTRAQ) between the blast-resistant cultivar Minghui and the susceptible rice cultivar Nipponbare in response to blast fungus infection. The group samples were treated with salicylic acid and compared to control samples. A total of 139 DEPs from the two cultivars showed either more than a two-fold change or alternating regulation patterns. Protein functionality analysis also exhibited that these proteins are involved in a wide range of molecular functions including: energy-related activity (30%), signal transduction (11%), redox homeostasis (15%), amino acid and nitrogen metabolism (4%), carbohydrate metabolism (5%), protein folding and assembly (10%), protein hydrolysis (9%), protein synthesis (12%), and other unknown functions (4%). Specifically, we demonstrated that exogenous treatment with salicylic acid promoted recovery in both rice cultivars from Magnaporthe grisea infection by enhancing: the regulation of signal transduction, increasing energy conversion and production through the regulation of the glycolytic pathway, and other various biochemical processes. These findings may facilitate future studies of the molecular mechanisms of rice blast resistance.

Genome Editing of Rice eIF4G Loci Confers Partial Resistance to Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is a serious constraint in Chinese rice production. Breeding disease-resistant varieties through multigene aggregation is considered an effective way to control diseases, but few disease-resistant resources have been characterized thus far. To develop novel resources for resistance to RBSDV through CRISPR/Cas9-mediated genome editing, a guide RNA sequence targeting exon 1 of eIF4G was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via Agrobacterium-mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 generation. Sequence analysis of the eIF4G target region from T1 transgenic plants identified 3 bp deletion mutants, and analysis of the predicted amino acid sequence identified one amino acid deletion in mutants that possess near full-length eIF4G. Furthermore, our data suggest that eIF4G may plays an important role in rice normal development, as there were no eIF4G knock-out homozygous mutants in T1 generation plants. When homozygous mutant lines were inoculated with RBSDV, they exhibited enhanced tolerance to virus infection, without visibly affecting plant growth and development. However, the eif4g mutant plants showed the same sensitivity to rice stripe virus (RSV) infection as wild-type plants. Notably, the wild-type and mutant N-termini of eIF4G interacted directly with RBSDV P8 in yeast and in planta. Additionally, compared to wild-type plants, the eIF4G transcript level was reduced twofold in the mutant plants. These results indicate that site-specific mutation of rice eIF4G successfully conferred partial resistance specific to RBSDV associated with less transcription of eIF4G in mutants. Therefore, this study demonstrates that the novel eIF4G alleles generated by CRISPR/Cas9 represent valuable disease-resistant resources that can be used to develop RBSDV-resistant varieties.

The Interaction between Rice Genotype and Magnaporthe oryzae Regulates the Assembly of Rice Root-Associated Microbiota

BackgroundUtilizating the plant microbiome to enhance pathogen resistance in crop production is an emerging alternative to the use of chemical pesticides. However, the diversity and structure of the microbiota, and the assembly mechanisms of root-associated microbial communities of plants are still poorly understood.ResultsWe invstigated the microbiota of the root endosphere and rhizosphere soils of the rice cultivar Nipponbare (NPB) and its Piz-t-transgenic line (NPB-Piz-t) when infected with the filamentous fungus Magnaporthe oryzae (M. oryzae) isolate KJ201, using 16S rRNA and internal transcribed spacer 1 (ITS1) amplicon sequencing. The rhizosphere soils showed higher bacterial and fungal richness and diversity than the endosphere except for fungal richness in the rhizosphere soils of the mock treatment. Bacteria richness and diversity increased in the endospheric communities of NPB and Piz-t under inoculation with KJ201 (referred to as ‘NPB-KJ201’ and ‘Piz-t-KJ201’, respectively) compared with the corresponding mock treatments, with the NPB-KJ201 showing the highest diversity in the four bacterial endocompartments. In contrast, fungal richness and diversity decreased in the endospheric communities of NPB-KJ201 and Piz-t-KJ201, relative to the corresponding mock treatments, with NPB-KJ201 and Piz-t-KJ201 having the lowest richness and diversity, respectively, across the four fungal endocompartments. Principal component analysis (PCA) indicated that the microbiota of Piz-t-KJ201 of root endophytes were mostly remarkablely distinct from that of NPB-KJ201. Co-occurrence network analysis revealed that the phyla Proteobacteria and Ascomycota were the key contributors to the bacterial and fungal communities, respectively. Furthermore, a comparative metabolic analysis showed that the contents of tryptophan metabolism and indole alkaloid biosynthesis were significantly lower in the Piz-t-KJ201 plants.ConclusionsIn this study, we compared the diversity, composition, and assembly of microbial communities associated with the rhizosphere soils and endosphere of Piz-t-KJ201 and NPB-KJ201. On the basis of the different compositions, diversities, and assemblies of the microbial communities among different compartments, we propose that the host genotype and inoculation pattern of M. oryzae played dominant roles in determining the microbial community assemblage. Further metabolomics analysis revealed that some metabolites may influence changes in bacterial communities. This study improves our understanding of the complex interactions between rice and M. oryzae, which could be useful in developing new strategies to improve rice resistance through the manipulation of soil microorganisms.

Development of a multiplex PCR system to discriminate the alleles of the brown planthopper resistance locus BPH1 in rice (Oryza sativa L.)

The brown planthopper, Nilaparvata lugens Stal (Hemiptera: Delphacidae), is a serious insect pest of rice (Oryza sativa L.). It is known that several wild rice and some Indica Group rice cultivars carry brown planthopper resistance genes. The brown planthopper resistance locus BPH1 has been used for the development of brown planthopper resistance rice in Southeast Asia. Bph1 and Bph2 are considered to be different resistance alleles in the same BPH1 locus, and each resistance allele has a different specificity against different brown planthopper biotypes. The first and second intronic DNA sequences in the BPH1 locus, which are derived from the Indica Group rice cultivar ADR52, wild rice Oryza officinalis and the Japonica Group rice cultivar Nipponbare, were analyzed to find insertion and deletion (InDel) polymorphisms. Based on this InDel polymorphisms information, we developed a multiplex polymerase chain reaction (PCR) method that enables the simultaneous identification of three alleles from the BPH1 locus: Bph1, Bph2 and a Japonica-type allele. Our method will be useful for future breeding research because it facilitates the classification of allele types in the BPH1 locus.

Effects of light-emitting diodes on tissue culture plantlets and seedlings of rice (Oryza sativa L.)

Light-emitting diodes (LEDs) are a new light source with low energy consumption and high photoelectric conversion efficiency, and they can satisfy the energy-saving needs of plant culture systems. However, the effects of LED light sources on rice tissue culture and rice seedling cultivation are poorly understood. This study aimed to evaluate the effects of LEDs on the growth of tissue culture plantlets and seedlings of the rice (Oryza sativa L.) cultivar Nipponbare. The best light source for rice tissue culture was different from that for rice seedling cultivation. Blue (B) LED light was the most appropriate light for rice tissue culture. Under a B LED light, the time required for callus proliferation, differentiation and regeneration was the shortest, and the frequency of plantlet initiation, differentiation and regeneration was the highest. A blue:red (B:R)=1:1 LED light facilitated the growth of rice seedlings and produced the highest chlorophyll and carotenoid contents and photosynthetic rates in the rice seedlings. Abundant photosynthetic products were more effectively generated in the rice seedlings under the B:R=1:1 LED and R LED lights than under the B LED light. B LED light is the most appropriate light for rice tissue culture plantlets and can be used as an alternative light source for rice tissue culture, and B:R=1:1 LED light facilitated the cultivation of robust rice seedlings and can be used as the primary light source for rice factory seedling cultivation.

Inactivation of retrotransposon Tos17 Chr.7 in rice cultivar Nipponbare through CRISPR/Cas9-mediated gene editing.

Retrotransposons are mobile genetic elements capable of transposition via reverse transcription of RNA intermediates. Rice cultivar Nipponbare contains two nearly identical genomic copies of Tos17, an endogenous copia-like LTR retrotransposon, on chromosomes 7 (Tos17 Chr.7) and 10 (Tos17 Chr.10), respectively. Previous studies demonstrated that only Tos17 Chr.7 is active in transposition during tissue culture. Tos17 Chr.7 has been extensively used for insertional mutagenesis as a tool for functional analysis of rice genes. However, Tos17 Chr.7 transposition might generate somaclonal mutagenesis with undesirable traits during rice transformation, which would affect the evaluation or application of transgenes. In this study, we generated a Tos17 Chr.7 knockout mutant D873 by using CRISPR/Cas9 gene editing system. The gene-edited allele of Tos17 Chr.7 in D873, designated as Tos17 D873, has an 873-bp DNA deletion in the pol gene of Tos17 Chr.7, which caused the deletion of the GAG-pre-integrase domain and the integrase core domain. Although the transcription of Tos17 D873 was activated in D873 calli, no transposition of Tos17 D873 was detected in the regenerated D873 plants. The results demonstrate that the GAG-pre-integrase domain and the integrase core domain are essential for Tos17 Chr.7 transposition and the deletion of the two domains could be not complemented by other LTR retrotransposons in rice genome. As the Tos17 Chr.7-derived somaclonal mutagenesis is blocked in the D873 plants, the generation of the Tos17 D873 allele will be helpful in production of transgenic rice plants for gene function study and genetic engineering. Similar approach can be used to inactivate other retrotransposons in crop breeding.

Production of novel beneficial alleles of a rice yield-related QTL by CRISPR/Cas9.

Production of novel beneficial alleles of a rice yield-related QTL by CRISPR/Cas9.