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Chronic pain is the principal clinical manifestation of knee osteoarthritis (KOA) and an essential indicator of the diagnosis and treatment effect. Changes in brain functional activity are related with chronic pain in KOA. Bushen Zhuangjin Decoction (BZD) has been proved to reduce inflammation of arthritis, improve cartilage degeneration and analgesia, but whether it plays a role through the change of brain function activity is not clear. Here, three experiments were performed: (1) network pharmacology evaluation to discover the potential targets of BZD to relieve pain in KOA; (2) verification of the therapeutic effects of BZD treatment on KOA pain with histomorphology, behavioral assessments, suspension chip analysis, and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) assays; and (3) functional magnetic resonance imaging to explore the effects of BZD treatment on brain function associated to KOA. The analgesic effect of BZD on KOA was found to be related to the neurotransmitters of pain signals through network pharmacology and the therapeutic effect of BZD on KOA pain was verified in vivo, and related to neuropeptides and neurotransmitters. Functional magnetic resonance imaging showed that BZD treatment could reverse the regional homogeneity/amplitude of low-frequency fluctuation analysis in pain-related brain regions of KOA, suggesting that the analgesic mechanism of BZD is related to neural regulation. This study confirmed the key position of pain-related neuromodulation mechanisms in the analgesic therapy of BZD and provide a theoretical basis for the treatment of KOA pain with BZD as a traditional Chinese medical.

Summary Rice is a facultative short day (SD) plant. In addition to serving as a model plant for molecular genetic studies of monocots, rice is a staple crop for about half of the world’s population. Heading date is a critical agronomic trait, and many genes controlling heading date have been cloned over the last 2 decades. The mechanism of flowering in rice from recognition of day length by leaves to floral activation in the shoot apical meristem has been extensively studied. In this review, we summarise current progress on transcriptional and post‐transcriptional regulation of heading date in rice, with emphasis on post‐translational modifications of key regulators, including Heading date 1 (Hd1), Early heading date 1 (Ehd1), Grain number, plant height, and heading date7 (Ghd7). The contribution of heading date genes to heterosis and the expansion of rice cultivation areas from low‐latitude to high‐latitude regions are also discussed. To overcome the limitations of diverse genetic backgrounds used in heading date studies and to gain a clearer understanding of flowering in rice, we propose a systematic collection of genetic resources in a common genetic background. Strategies in breeding adapted cultivars by rational design are also discussed.

Chlorophyll (Chl) biosynthesis is essential for photosynthesis and plant growth. Glutamyl-tRNA reductase (GluTR) catalyzes glutamyl-tRNA into glutamate-1-semialdehyde (GSA) and initiates the chlorophyll biosynthesis. Even though the main role of GluTR has been established, the effects caused by natural variations in its corresponding gene remain largely unknown. Here, we characterized a spontaneous mutant in paddy field with Chl biosynthesis deficiency, designated as cbd1. With intact thylakoid lamellar structure, the cbd1 plant showed light green leaves and reduced Chl and carotenoids (Cars) content significantly compared to the wild type. By map-based gene cloning, the mutation was restricted within a 57-kb region on chromosome 10, in which an mPingA miniature inverted-repeat transposable element (MITE) inserted in the promoter region of OsHemA gene. Both leaf color and the pigment contents in cbd1 were recovered in a complementation test, confirming OsHemA was responsible for the mutant phenotype. OsHemA was uniquely predicted to encode GluTR and its expression level was dramatically repressed in cbd1. Transient transformation in protoplasts demonstrated that GluTR localized in chloroplasts and a signal peptide exists in its N-terminus. A majority of Chl biosynthesis genes, except for POR and CHLG, were down-regulated synchronously by the repression of OsHemA, suggesting that an attenuation occurred in the Chl biosynthesis pathway. Interestingly, we found major agronomic traits involved in rice yield were statistically unaffected, except for the number of full grains per panicle was increased in cbd1. Collectively, OsHemA plays an essential role in Chl biosynthesis in rice and its weak allele can adjust leaf color and Chls content without compromise to rice yield.

The mortality rate associated with hepatocellular carcinoma (HCC) is the third highest among all digestive system tumors. However, the causes of HCC development and the underlying mechanisms have remained to be fully elucidated. In the present bioinformatics study, genetic markers were identified and their association with HCC was determined. The mRNA expression datasets GSE87630, GSE74656 and GSE76427 were downloaded from the Gene Expression Omnibus (GEO) database. A total of 96 differentially expressed genes (DEGs) were screened from the 3 GEO datasets, including 25 upregulated and 71 downregulated genes. DEGs were uploaded to the database for Annotation, Visualization and Integrated Discovery to screen for enriched Gene Ontology terms in various categories and the Search Tool for the Retrieval of Interacting Genes/Proteins was used to identify the interactions and functions of the DEGs. A total of 3 genetic markers were identified in a stepwise pathway and functional analysis in a previous study. The association of the genetic markers with prognosis was analysed using the UALCAN online analysis tool. Regression analysis was also performed to identify the relationship between HCC grade and disease recurrence and the expression of genetic markers using The Cancer Genome Atlas HCC dataset. In addition, the expression of the 3 genetic markers in HCC tissues was determined using reverse transcription-quantitative PCR, the Oncomine database and the Human Protein Atlas database. The expression levels of the 3 genetic markers cyclin B2 (CCNB2), nucleolar and spindle-associated protein 1 (NUSAP1) and thymidine kinase 1 (TK1) were significantly correlated with each other and high mRNA expression of CCNB2 was significantly associated with poor overall survival of patients with HCC. Receiver operating characteristic curve analysis indicated that NUSAP1 and TK1 were capable of distinguishing between recurrent and non-recurrent HCC. Furthermore, CCNB2, NUSAP1 and TK1 were highly correlated with the HCC grade. It was also indicated that the mRNA expression of CCNB2, NUSAPA and TK1 was increased in primary HCC tissues when compared with that in adjacent tissues. The present study identified that the CCNB2, NUSAP1 and TK1 genes may serve as prognostic markers for HCC, and may be of value from the perspectives of basic research and clinical treatment of HCC.

BACKGROUND At the present, cancer patients are experiencing symptoms that can seriously influence their quality of life. There are many mobile health applications in Internet medical services to manage those symptoms. However, further research is needed to see whether mobile health can provide benefits to cancer patients. OBJECTIVE The meta-analysis aimed to value the benefits of mHealth for depression, anxiety, pain, fatigue, and insomnia outcomes of cancer patients. METHODS We searched PubMed, CINHAL, Web of Science, Cochrane library Trials, EMBASE database to select random clinic trials that investigated the benefits of mHealth for cancer patients, from the date of inception to 17 April 2021. RESULTS Eight random clinic trials were eligible for this meta-analysis. Collectively, mHealth group has 1641 participants, the control group has 1463 participants. The meta-analysis revealed that the mHealth intervention improves significantly cancer patients’ fatigue(SMD −0.19, 95% CI −0.32 to −0.05），P= .007), insomnia(SMD −0.26, 95% CI −0.44 to −0.08, P= .005), depression(SMD −0.22, 95% CI −0.32 to −0.13，P CONCLUSIONS mHealth intervention may significantly improve depression, anxiety, fatigue, and insomnia outcomes of cancer patients, but have no significant effort on pain. CLINICALTRIAL This meta-analysis was registered with The International Prospective Register of Systematic Reviews (PROSPERO) , registration number is: CRD42021272537.

Ubiquitination and deubiquitination are reversible processes that play crucial roles in regulating organ size in plants. However, information linking deubiquitination and seed size in rice (Oryza sativa) is limited. Here, we characterized a dominant large-grain mutant, large grain1-D (lg1-D), with a 30.8% increase in seed width and a 34.5% increase in 1,000-grain weight relative to the wild type. The lg1-D mutant had more cells oriented in the lateral direction of the spikelet hull compared with the wild type. Map-based cloning showed that LG1 encodes a constitutively expressed ubiquitin-specific protease15 (OsUBP15) that possesses deubiquitination activity in vitro. Loss-of-function and down-regulated expression of OsUBP15 produced narrower and smaller grains than the control. A set of in vivo experiments indicated that the mutant Osubp15 had enhanced protein stability relative to wild-type OsUBP15. Further experiments verified that OsDA1 directly interacted with OsUBP15. Genetic data indicated that OsUBP15 and GRAIN WIDTH 2 (GW2) were not independent in regulating grain width and size. In summary, we identified OsUBP15 as a positive regulator of grain width and size in rice and provide a promising strategy for improvement of grain yield by pyramiding OsUBP15 and gw2.

FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.

Brassinosteroids play a negative role in rice defense against infestation by the brown planthopper through contrasting effects on the salicylic acid and jasmonic acid pathways., Improved knowledge of the interactions between plants and insects will facilitate better insect control in crops. Brassinosteroids (BRs) play a vital role in plant growth, developmental processes, and responses to pathogen infection, but the role of BRs in interactions between plants and insects remain largely unknown. In this study, we characterized a negative role of BRs in rice defense against brown planthopper (BPH, Nilaparvata lugens) and examined its underlying mechanisms. We found that BPH infestation suppressed the BR pathway while successively activating the salicylic acid (SA) and jasmonic acid (JA) pathways. In addition, BR-overproducing mutants and plants treated with 24-epibrassinolide (BL) showed increased susceptibility to BPH, whereas BR-deficient mutants were more resistant than the wild-type. BRs down-regulated the expression of genes related to the SA pathway and reduced SA content while genes related to the JA pathway were up-regulated and JA content increased after BPH infestation. Furthermore, BR-mediated suppression of the SA pathway was impaired both in JA-deficient and JA-insensitive mutants. Our results demonstrate that BRs promote the susceptibility of rice plants to BPH by modulating the SA and JA pathways.

A large volume sample stacking (LVSS) method in micellar electrokinetic chromatography (MEKC) with diode array detector was developed for the simultaneous separation and analysis of five compounds: protocatechuic acid, protocatechuic aldehyde, caffeic acid, syringetin and vanillin in Cibotium barometz. The electrophoretic separation was performed in a 10 mM sodium dodecyl sulfate (SDS) and 50 mM sodium borax-sodium dihydrogen phosphate system (pH = 8.5) with 10% methanol at a separation voltage of 30 kV after optimizing the typical parameters. The detection limits were from 32 pg to 65 pg, which were around 12–27 times lower than MEKC, and 500 times less than reported methods. Finally, the established method was validated to be applicable for the determination of protocatechuic acid and caffeic acid in Cibotium barometz. This proposed method is expected to facilitate the quality control of Cibotium barometz.

Brassinosteroids (BRs) regulate several aspects of plant growth and development. Although extensive studies have shown that BR signaling is conservative in higher plants, the molecular mechanism of regulating plant architecture in rice still remains to be explored. Here, we characterized a rice mutant named top bending panicle1 (tbp1). Compared to wild type, tbp1 mutant plants displayed semi-dwarf stature, erect leaves, small and round grains, as well as more tillers. Remarkably, the panicles of tbp1 plants were shorter and denser, and the tops of the panicles were curved by rolling of the base of flag leaves, which was later verified as due to reduced bulliform cell numbers. Map-based cloning, together with transgenic complementation and RNA-interference tests, revealed that TBP1 is a member of the somatic embryogenesis receptor kinases (SERKs) family involved in BR signaling. Furthermore, bimolecular fluorescence complementation and co-immunoprecipitation analysis demonstrated that a substitution at 61st amino acid (His61Leu) in the tbp1 mutant may result in a reduction of the interaction between TBP1 and OsBRI1 (BR receptor in rice). Taken together, our results demonstrate that TBP1 plays a significant role in regulating plant architecture via the brassinosteroid signaling pathway.

Cereal opaque-kernel mutants are ideal genetic materials for studying the mechanism of starch biosynthesis and amyloplast development. Here we isolated and identified two allelic floury endosperm 8 (flo8) mutants of rice, named flo8-1 and flo8-2. In the flo8 mutant, the starch content was decreased and the normal physicochemical features of starch were altered. Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution and an 8-bp insertion occurred in UDP-glucose pyrophosphorylase 1 (Ugp1) gene in flo8-1 and flo8-2, respectively. Complementation of the flo8-1 mutant restored normal seed appearance by expressing full length coding sequence of Ugp1. RT-qPCR analysis revealed that Ugp1 was ubiquitously expressed. Mutation caused the decreased UGPase activity and affected the expression of most of genes associated with starch biosynthesis. Meanwhile, western blot and enzyme activity analyses showed the comparability of protein levels and enzyme activity of most tested starch biosynthesis related genes. Our results demonstrate that Ugp1 plays an important role for starch biosynthesis in rice endosperm.

Differentiation from proplastids into chloroplasts is a light- and energy-dependent process. How this process is regulated is still poorly understood at the molecular level. We herein report a new putative plastidial adenine nucleotide transporter, BRITTLE1-3 (referred to as OsBT1-3), encoded by the rice (Oryza sativa) White Stripe Leaf 2 (WSL2) gene. Loss of OsBT1-3 function results in defective chloroplast biogenesis, severely reduced photosynthetic efficiency, and finally a white stripe leaf phenotype in the first four leaves. The expression levels of genes related to chlorophyll biosynthesis and photosynthesis are drastically reduced, accompanied with over accumulation of reactive oxygen species (ROS) in the wsl2 mutant. OsBT1-3 is targeted to the chloroplasts and it expresses in almost all tissues in plants, especially in young leaves. OsBT1-3 consists of 419 amino acids and exhibits features of all mitochondrial carrier proteins, including a typical transmembrane-spanning domain and a highly conserved sequence motif designated as the ‘mitochondrial energy transfer signatures’. Phylogenetic analysis shows that OsBT1-3 is a putative plastidial adenine nucleotide transporter and is most closely related to ZmBT1-2. Together, these observations suggest that the new putative adenine nucleotide transporter, OsBT1-3, plays an essential role in regulating chloroplast biogenesis and maintenance of ROS homeostasis during rice seedling de-etiolation.

WSL4 encodes a KCS6 protein which is required for cuticular wax accumulation in rice. Very long chain fatty acids (VLCFAs) are essential precursors for cuticular wax biosynthesis. VLCFA biosynthesis occurs in the endoplasmic reticulum and requires the fatty acid elongase (FAE) complex. The β-ketoacyl-coenzyme A synthase (KCS) catalyzes the first step of FAE-mediated VLCFA elongation. Here we characterized the Wax Crystal-Sparse Leaf 4 (WSL4) gene involved in leaf cuticular wax accumulation in rice. The wsl4 mutant displayed a pleiotropic phenotype including dwarfism, less tiller numbers and reduced surface wax load. Map-based cloning and nucleotide sequencing results revealed that wsl4 carried a single nucleotide substitution in the second exon of a putative KCS6 gene, encoding one subunit of the FAE complex for VLCFAs. Genetic complementation confirmed that the mutation in WSL4 was responsible for the phenotype of wsl4. WSL4 was constitutively expressed in various rice tissues and localized in the endoplasmic reticulum. Both WSL4-RNAi transgenic lines and WSL4 knocked-out mutants exhibited wax-deficient phenotypes similar to the wsl4 mutant. These data indicate that WSL4 is required for cuticular wax accumulation in rice.

Oryza longistaminata originates from African wild rice and contains valuable traits conferring tolerance to biotic and abiotic stress. However, interspecific crosses between O. longistaminata and Oryza sativa cultivars are hindered by reproductive barriers. To dissect the mechanism of interspecific hybrid sterility, we developed a near-isogenic line (NIL) using indica variety RD23 as the recipient parent and O. longistaminata as the donor parent. Both pollen and embryo sac semi-sterility were observed in F1 hybrids between RD23 and NIL. Cytological analysis demonstrated that pollen abortion in F1 hybrids occurred at the early bi-nucleate stage due to a failure of the first mitosis in microspores. Partial embryo sacs in the F1 hybrids were defective during the functional megaspore formation stage. Most notably, nearly half of the male or female gametes were aborted in heterozygotes S40iS40l, regardless of their genotypes. Thus, S40 was indicated as a one-locus sporophytic sterility gene controlling both male and female fertility in hybrids between RD23 and O. longistaminata. A population of 16 802 plants derived from the hybrid RD23/NIL-S40 was developed to fine-map S40. Finally, the S40 locus was delimited to an 80-kb region on the short arm of chromosome 1 in terms with reference sequences of cv. 93-11. Eight open reading frames (ORFs) were localized in this region. On the basis of gene expression and genomic sequence analysis, ORF5 and ORF8 were identified as candidate genes for the S40 locus. These results are helpful in cloning the S40 gene and marker-assisted transferring of the corresponding neutral allele in rice breeding programs.

Highlight Loss-of-function of the rice eEF1A-like protein SPL33 causes a lesion-mimic phenotype by triggering programmed cell death, and increases disease resistance, likely through activating defense pathways., Lesion-mimic mutants are useful to dissect programmed cell death and defense-related pathways in plants. Here we identified a new rice lesion-mimic mutant, spotted leaf 33 (spl33) and cloned the causal gene by a map-based cloning strategy. SPL33 encodes a eukaryotic translation elongation factor 1 alpha (eEF1A)-like protein consisting of a non-functional zinc finger domain and three functional EF-Tu domains. spl33 exhibited programmed cell death-mediated cell death and early leaf senescence, as evidenced by analyses of four histochemical markers, namely H2O2 accumulation, cell death, callose accumulation and TUNEL-positive nuclei, and by four indicators, namely loss of chlorophyll, breakdown of chloroplasts, down-regulation of photosynthesis-related genes, and up-regulation of senescence-associated genes. Defense responses were induced in the spl33 mutant, as shown by enhanced resistance to both the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae and by up-regulation of defense response genes. Transcriptome analysis of the spl33 mutant and its wild type provided further evidence for the biological effects of loss of SPL33 function in cell death, leaf senescence and defense responses in rice. Detailed analyses showed that reactive oxygen species accumulation may be the cause of cell death in the spl33 mutant, whereas uncontrolled activation of multiple innate immunity-related receptor genes and signaling molecules may be responsible for the enhanced disease resistance observed in spl33. Thus, we have demonstrated involvement of an eEF1A-like protein in programmed cell death and provided a link to defense responses in rice.

Rice black-streaked dwarf virus disease (RBSDVD) transmitted by small brown planthopper (SBPH, Laodelphax striatellus) is one of the most destructive viral diseases in rice (Oryza sativa L.). It is urgent to explore resistance resources and genes to breed resistant cultivars for controlling this disease. In the present study, linkage analysis was performed using an RIL (recombinant inbred line) population including 181 lines and two quantitative trait loci (QTLs) underlying resistance to RBSDVD, qRBSDVD5 and qRBSDVD6 were detected on chromosomes 5 and 6. Furthermore, 1070 sequenced diverse rice accessions collected from 72 countries or regions were evaluated for resistance to RBSDVD. Moreover, a significantly associated locus was also detected in the same interval of qRBSDVD6 by genome-wide association study (GWAS). Candidate genes were predicted in the significantly association interval with RBSDVD resistance. Among the five haplotypes of LOC_Os06g03120, encoding an aspartyl protease precursor, haplotype 1 showed higher resistance than the other four. The study provides a powerful resource for elucidating the mechanism underlying RBSDVD resistance and breeding resistance rice cultivars by molecular-assisted selection (MAS).

The root system in monocotyledonous plants is largely composed of postembryonic shoot-borne roots named crown roots, which are important for nutrients and water uptake. The molecular mechanism underlying regulation of crown root development is not fully explored. In this study, we characterized a rice (Oryza sativa) mutant defective in crown root formation, designated as crown rootless6 (crl6). Histological analysis showed that CRL6 influences crown root formation by regulating primordial initiation and development. Map-based cloning and subsequent complementation tests verified that the CRL6 gene encodes a member of the large chromodomain, helicase/ATPase, and DNA-binding domain (CHD) family protein. Realtime RT-PCR analysis showed that CRL6 was most highly expressed in the stem base region where crown roots initiated. In addition, auxin-action inhibited phenotype was observed during crl6 development. The expressions of OsIAA genes were down-regulated in crl6. Our results provide evidence that CRL6 plays an important role in crown root development in rice via auxin-related signaling pathway.

Leaf-color mutants are ideal genetic materials for understanding the mechanism of chloroplast development and chlorophyll (Chl) biosynthesis. Here we isolated and identified a new leaf-color mutant of rice, named white-stripe leaf3 (wsl3), from a 60Co-irradiated mutant pool. The wsl3 mutant displayed a visible white-stripe leaf in both young seedlings and flag leaves of mature plant. Chl content in homozygous wsl3 mutant was approximately 47% of that in the wild type. Besides, chloroplast development in the mutant was severely arrested. By a map-based cloning strategy, the wsl3 gene was finely confined to a 50.8 kb region on chromosome 1. Moreover, a 9-bp deletion was identified in the genomic region of LOC_Os01g01920, which encodes an HD (histidine and aspartic acid) domaincontaining protein. Genetic complementation confirmed that LOC_Os01g01920 could recover the lesion of wsl3 mutation. Real-time PCR analyses showed that the expression levels of WSL3 were the highest in young and flag leaves among various tissues, and most of the genes associated with Chl biosynthesis were significantly down-regulated in the wsl3 mutant. Meanwhile, in contrast to many nuclear gene-encoded phage-type RNA polymerase(s) (NEP) transcribed genes were up-regulated, most of plastid-encoded bacterialtype RNA polymerase (PEP) transcribed genes were downregulated. These results demonstrated that the WSL3 gene, as an HD domain-containing protein, is involved in chl biosynthesis and chloroplast development in rice.

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.

Additional file 6: Table S3. Primers used for vector construction.

Loss of function of a mitochondrial complex I subunit (OsNDUFA9) causes abnormal embryo development and affects starch synthesis by altering the expression of starch synthesis-related genes and proteins. Proton-pumping NADH: ubiquinone oxidoreductase (also called complex I) is thought to be the largest and most complicated enzyme of the mitochondrial respiratory chain. Mutations of complex I subunits have been revealed to link with a number of growth inhibitions in plants. However, the function of complex I subunits in rice remains unclear. Here, we isolated a rice floury endosperm mutant (named flo13) that was embryonic lethal and failed to germinate. Semi-thin sectioning analysis showed that compound starch grain development in the mutant was greatly impaired, leading to significantly compromised starch biosynthesis and decreased 1000-grain weight relative to the wild type. Map-based cloning revealed that FLO13 encodes an accessory subunit of complex I protein (designated as OsNDUFA9). A single nucleotide substitution (G18A) occurred in the first exon of OsNDUFA9, introducing a premature stop codon in the flo13 mutant gene. OsNDUFA9 was ubiquitously expressed in various tissues and the OsNDUFA9 protein was localized to the mitochondria. Quantitative RT-PCR and protein blotting indicated loss of function of OsNDUFA9 altered gene expression and protein accumulation associated with respiratory electron chain complex in the mitochondria. Moreover, transmission electron microscopic analysis showed that the mutant lacked obvious mitochondrial cristae structure in the mitochondria of endosperm cell. Our results demonstrate that the OsNDUFA9 subunit of complex I is essential for embryo development and starch synthesis in rice endosperm.

Highlight The OsMSH4/OsMSH5 heterodimer interacts with type A and C RPA heterotrimer complexes during second-end capture to regulate crossing over during meiosis I in rice., Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4–green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.

Heterosis is a phenomenon whereby hybrids of inbred lines produce favourable phenotypes that exceed those of their parents. Traits of interest are higher yield and stronger stress tolerance. The two-line super-hybrid rice ‘Liangyoupei9’ (LYP9) shows superiority to both its elite inbred line ‘93-11’ and ‘Pei'ai64s’ (‘PA64s’) parents and conventional hybrids. However, the genetic basis of its hybrid vigour, especially yield determination, remains elusive. In the present study, a set of 156 chromosome segment substitution lines (CSSLs) carrying overlapping segments from ‘PA64s’ in a genetic background of ‘93-11’ were constructed and planted in six environments. Three major agronomic traits, viz. panicle length (PL), heading date (HD) and plant height (PH), and five yield-related traits, viz. grain weight per panicle (GWP), number of grains per panicle (GPP), 1000-grain weight (TGW), seed set (SS) and number of panicles of per plant (PPP), were evaluated over 3 years. Quantitative trait loci (QTL) analysis was conducted using a likelihood ratio test based on stepwise regression. Forty-six putative QTL distributed on 11 chromosomes were detected in more than one year. Remarkably, GWP of four CSSLs carrying positive yield QTL outperformed the recurrent parent ‘93-11’ by more than 15%, in at least two environments. These results indicate that CSSLs are effective in identifying yield-associated traits, and lines harbouring such QTL will be rich in resources for future molecular breeding programmes.

A two-line super-hybrid rice (Oryza sativa L.) variety [Liangyoupei9 (LYP9)] demonstrated superiority over its both parents, viz. elite inbred lines 93-11 and Pei-ai64S (PA64S), as well as other conventional hybrids, and had long been exploited in China. However, the genetic basis of its leaf-related traits, supposed to be an important component for yield potential, remains elusive. Here, initially a set of chromosome segment substitution lines (CSSLs) was constructed, in which the genome of Pei-ai64S has been introgressed into the background of 93-11. This set was developed by marker aided selection, based on 123 polymorphic SSR markers. The introgressed chromosomal segments presented in the 156 CSSLs covered 96.46% of Pei-ai64S genome. Afterwards, the CSSLs were deployed to assess the genetic basis of leaf size (length and width) and chlorophyll content of top three leaves across five different environments. The CSSLs showed transgressive segregation for all of the traits, and significant correlations were detected among most of the traits. A total of 27 quantitative trait loci (QTL) were identified on ten chromosomes, and three QTL cluster affecting related traits were found on chromosome 3, 6, and 8, respectively. Remarkably, two key QTLs, qALW3-1 and qALW3-2, both controlling the antepenultimate leaf width, were identified in all five environments, and their effect were further validated by CSSLs harboring the two QTL alleles. Our results indicate that developing CSSLs is a powerful tool for genetic dissection of quantitative traits. Meanwhile, the QTLs controlling leaf-related traits uncovered here provide useful information for marker-assisted selection in improving the performance of leaf morphology and photosynthetic ability.

By using a thermogravimetric analyser under argon atmosphere, the pyrolysis process and the kinetic model of phoenix tree residues (the little stem, middle stem, and leaf) at a 30 °C min−1 heating rate and the phoenix tree mix at three different heating rates (10 °C min−1, 30 °C min−1, and 50 °C min−1) were examined. The catalyst and the co-pyrolysis samples were at a 30 °C min−1 heating rate. The catalysts were Na2CO3, ZnCl2 and CaO in a mass fraction of 5 %. The experimental results revealed that the phoenix tree residues pyrolysis process consisted of three stages: dehydration stage, main pyrolysis stage, and the slow decomposition of residues. As the heating rate increased, the pyrolysis characteristic temperature of the phoenix tree grew, there was a backward-shift of the pyrolysis rate curve, and the mass loss rate gradually increased. The phoenix tree residues’ activation energy changed throughout the whole pyrolysis process, and the pyrolysis temperature ranges of the three main components (cellulose, hemicellulose, and lignin) existed in overlapping phenomenon. As compared to the little stem, middle stem, and leaf, the phoenix tree mix was more likely to be pyrolysed under the same heating rate. Different catalysts had a different impact on the pyrolysis: ZnCl2 moved the start point of the reaction to the lower temperatures, but did not speed up the reaction; Na2CO3 speeded up the reaction without changing the start point of the reaction; CaO speeded up the reaction, moved the start point of the reaction to higher temperatures.

Switchgrass (Panicum virgatum L.) has potential to be a major cellulosic bioenergy crop. Selection for late flowering plants will extend the vegetative growth, likely resulting in larger biomass yields. However, the genetic structure for reproductive maturity in switchgrass is undefined. Accordingly, the major objective of this study was to identify genomic regions associated with reproductive development. Two lowland populations, one consisting of 176 progeny from NL94 (♀) × SL93 (♂) and a first-generation self-fertilized population of 265 progeny from NL94, were field established in a randomized complete block design with three replications at two Oklahoma locations in 2011. Phenotypic data of reproductive maturity in the populations were collected in 2012 and 2013. Significant genetic variation for reproductive maturity was observed within the two populations. Broad-sense heritabilities were 0.46 to 0.77 and 0.28 to 0.74 for the hybrid and selfed populations, respectively. A linkage map with 178 simple sequence repeat (SSR) markers of the hybrid population constructed in this study and a pre-existing linkage map of 439 SSR markers in the selfed population were used for quantitative trait loci (QTL) characterization. QTL analyses revealed that reproductive maturity was controlled by multiple genomic regions. The QTL regions between nfsg-125 and PVE-781/782 on linkage group (LG) 2b, between PVGA-1727/1728 and PVGA-1201/1202 on LG 3b, and between PVCAG-2503/2504 and PVAAG-3253/3254 on LG 7a were associated with reproductive maturity in both populations. The markers linked to the significant QTL could be used to accelerate the development of switchgrass germplasm with later flowering to increase biomass yield.

Proper chloroplast development and chlorophyll biosynthesis are essential for the photoautotrophic plants. The insertion of magnesium (Mg2+) into protoporphyrin IX (Proto), catalyzed by magnesium chelatase (Mg-chelatase), is the first committed step of chlorophyll biosynthesis. In dicot plants, a proposed model revealed that Mg-chelatase I subunit (CHLI) and Mg-chelatase D subunit (CHLD) can interact directly; however, their relation remains elusive in rice, a monocot model plant. In this study, we characterized a chlorophyll-deficiency mutant, etiolated leaf and lethal (ell), which displayed a yellow leaf in young seedlings and became lethal after three-leaf stage. Chlorophyll content in homozygous ell mutant was approximately 1 % of that in the wild type. Besides, chloroplast development in the mutant was entirely arrested and no thylakoid structure was observed. By map-based cloning, the ell locus was delimited to a 3.9-Mb interval in chromosome 3. A single-base mutation (G529C) in OsCHLI was identified, leading to an amino acid substitution (G177R) in a highly conserved region. Compared with the wild type, more Proto but less magnesium protoporphyrin IX (Mg-Proto) was measured in the ell mutant. Using protoplast transfection and callus transformation, we found that exogenous OsCHLI could consistently recover the lesion of chloroplast in the ell mutant. By subcellar localization analysis, OsCHLI was detected in the chloroplast. Despite the secondary structure of OsCHLI that was predicted to be altered in the mutant, the point mutation did not affect subcellular localization. Real-time PCR demonstrated that the ell mutation induced significantly transcriptional downregulation of the photosynthesis-associated nuclear and plastid genes. Additionally, yeast-two-hybrid experiments indicated that the single amino acid substitution blocked the intrinsic interaction between OsCHLI and OsCHLD. Moreover, OsCHLI showed physical interactions with some thioredoxins (TRXs), suggesting a similar regulatory mechanism of Mg-chelatase activity in both monocot and dicot plants.

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. Two segregating populations with N22 (indica) as a common parent, viz. a set of 122 backcross-inbred lines (BILs) derived from the backcross Nanjing35 (japonica)/N22//Nanjing35 and another population comprising 189 recombinant inbred lines (RILs) from the cross of USSR5 (japonica) and N22, were studied to detect quantitative trait loci (QTL) controlling seed storability. Germination percentage (GP) was used to evaluate seed storability after aging treated under three different conditions, viz. natural, artificial and combined aging treatments. A total of seven QTLs were identified on chromosomes 1, 2, 5, 6 and 9. Among them, a major QTL, qSSn-9, was common in the two populations. In contrast, four QTLs (qSSnj-2-1, qSSn-2-2, qSSn-5 and qSSn-6) were detected in BILs and the QTL qSSn-1 was identified in RILs, which was a new QTL for seed storability. The N22-derived alleles increased the seed storability at all the loci except qSSnj-2-1. We also investigated the effect of QTLs using five selected lines with high storability from BILs and verified qSSn-5 with a near-isogenic line (NIL). These results provide an opportunity for pyramiding or map-based cloning major QTLs for seed storability in rice.

Small brown planthopper (SBPH) and its transmitted rice black-streaked dwarf virus disease (RBSDVD) cause serious damage to rice (Oryza sativa L.) production. Though breeding of resistant cultivars is believed to be one of the most important strategies for RBSDVD management, few high-resistance lines have been found to date. In the present study, we identified an indica variety, 9194, that is highly resistant to RBSDVD and analyzed the quantitative trait loci (QTLs) underlying this resistance . In total, four QTLs for RBSDVD resistance, viz. qRBSDV3, qRBSDV6, qRBSDV9, and qRBSDV11, were identified. Among them, qRBSDV6, qRBSDV9, and qRBSDV11 with LOD (logarithm [base 10] of odds) scores of 4.42–4.48, 2.11–7.26, and 5.01–7.16 were repeatedly detected in 2 years, accounting for 10.3–16.7%, 8.3–35.5%, and 20.0–31.1% of the total phenotypic variation, respectively. Further, introgression of single- or multiple-resistance QTLs into a susceptible rice variety by marker-assisted selection (MAS) indicated that stacking the QTLs could progressively enhance RBSDVD resistance, suggesting that these QTLs act additively. The same population was also used for QTL mapping of SBPH resistance. Four QTLs, viz. qSBPH1, qSBPH5, qSBPH8, and qSBPH9, with LOD scores of 2.72, 2.78, 2.15, and 2.85 were detected, explaining 13.7%, 11.0%, 12.0%, and 21.0% of the phenotypic variation, respectively. The identification of RBSDVD and SBPH resistance QTLs, and the development of single and multiple genes with pyramided lines, in this study provides innovative resources for molecular breeding of resistant rice cultivars.

Histone recognition is important for understanding the mechanisms of histone modification, which play a pivotal role in transcriptional regulation during plant development. Here, we identified three cysteine-tryptophan (CW)-domain containing zinc finger (ZF) proteins involved in histone recognition, namely OsCW-ZF3, OsCW-ZF5 and OsCW-ZF7. Protein sequence analysis showed that they have two unknown motifs in addition to the CW domain. All three OsCW-ZFs were expressed in aerial tissues, with relatively high levels in developing panicles. Subcellular localization revealed that the OsCW-ZFs target the cell nucleus and CW domains are not necessary for their nuclear localization. In contrast to OsCW-ZF3 and OsCW-ZF5 where the CW domains bind histone H3 lysine 4 with different methylated forms (H3K4me), the CW domain from OsCW-ZF7 recognizes only trimethylated histone H3 lysine 4 (H3K4me3). Analysis of mutant suggested that three conserved tryptophan residues in the CW domain are essential for binding to H3K4me. Further study found that OsCW-ZF7 interacts with TAFII20, a transcription initiation factor TFIID 20kDa subunit. Knockout of OsCW-ZF7 caused defective development of awns. This study provides new insights into our understanding of the CW domain and lays a foundation for further investigation of its roles in rice.