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Multiferroic CoFe2O4 (CFO)/Bi0.97Ce0.03FeO3 (BCFO) coaxial nanotubes were prepared by a sol-gel template method. Transmission electron microscopy revealed that the coaxial nanotubes featured with inner CFO and outer BCFO nanotubes. Selected area electron diffraction confirmed the coexistence of spinel CFO and perovskite BCFO phases in the coaxial nanotubes. Vibrating sample magnetometer measurements showed that the saturated magnetization of the coaxial nanotubes was 3.3 emu/g, smaller than that of CFO nanotubes. The P–E hysteresis loop of the coaxial nanotubes was of poor shape due to possible high conductivity in the inner CFO nanotubes. Dielectric measurements exhibited that the dielectric constant of the coaxial nanotubes decreased while the dielectric loss increased due probably to the small dielectric constant and high conductivity in the inner CFO nanotube. Ferroelectric and magnetic properties were simultaneously demonstrated in the CFO/BCFO coaxial nanotubes.

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.

A QTL, qHY-8, has been resolved into a single Mendelian locus controlling heading date and yield-related traits in rice. The trait-performance of the three genotypes for the QTL in the BC5F2 or BC4F2 populations (with cultivar Teqing or YuetaiB as recurrent parents) indicated that the Lemont (donor parent) allele at qHY-8 locus showed a positive and pleiotropic additive effect on heading date (HD), plant height (PH), panicle length (PL), and spikelets per panicle (SPP).The QTL’s additive effects varied for different traits according to its heritabilities, it showed a largest effect on HD with a heritability of 98%, secondly on PL with a heritability of 70–87%, then on PH with a heritability of 56–61%, and lastly on SPP with a heritability of 45–47%. In addition, the QTL showed a weaker dominant effect, significant only on HD and PL. With an advanced BC5F2 population, qHY-8 was firstly mapped to a 299 kb genomic region by recessive-class analysis. Then, with a set of segment substitution lines, substitution mapping located qHY-8 within a chromosomal region between markers ID3468 and ID4891, corresponding to 31.4 kb on the genome sequence of Nipponbare. Sequence analysis of an ORF Os08g07740 which encodes a histone-like CCAAT-box binding transcription factor suggested that the ORF would be tentatively referred to as the first strong candidate gene. All these results demonstrated that qHY-8 is a pleiotropic QTL for heading date and multiple yield-related traits and would be of great value to the improvement of rice productivity.

A pollen-expressed gene from rice, OsPCBP, that was previously cloned and identified encodes a novel calmodulin-binding protein. Here, we investigated its functioning in pollen development. Overexpression of OsPCBP in transgenic rice plants did not lead to phenotypic changes but did delay anther formation by about 1 week. Cytological observations at different stages revealed that microspores from the OsPCBP double-stranded RNA interference plants developed normally until the binucleate stage, but about half were later aborted. Meanwhile, the anther microstructure of those plants was unaffected. Further examination of OsPCBP expression in transgenic lines via RT-PCR showed that a significant reduction in transcripts was correlated with this abortion phenotype. Our experimental results provide convincing evidence that OsPCBP protein plays an important role during the late stage of pollen development.

Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.

Although the characterization of genes associated with cytoplasmic male sterility (CMS) and fertility restoration (Rf) has been well documented, the evolutionary relationship between nuclear Rf and CMS factors in mitochondria in Oryza species is still less understood. Here, 41 accessions from 7 Oryza species with AA genome were employed for analyzing the evolutionary relationships between the CMS factors and Rf candidates on chromosome 10. The phylogenetic tree based on restriction fragment length polymorphism patterns of CMS-associated mitochondrial genes showed that these 41 Oryza accessions fell into 3 distinct groups. Another phylogenetic tree based on PCR profiles of the nuclear Rf candidates on chromosome 10 was also established, and three groups were distinctively grouped. The accessions in each subgroup/group of the two phylogenetic trees are well parallel to each other. Furthermore, the 41 investigated accessions were test-crossed with Honglian (gametophytic type) and Wild-abortive (sporophytic type) CMS, and 5 groups were classified according to their restoring ability. The accessions in the same subgroup of the two phylogenetic trees shared similar fertility restoring pattern. Therefore, we conclude that the CMS-associated mitotypes are compatible to the Rf candidate-related nucleotypes, CMS and Rf have a parallel evolutionary relation in the Oryza species.

Cytoplasmic male sterility (CMS) has often been associated with mitochondrial dysfunction. In this report, the heterogeneity of mitochondria was analyzed in both Honglian (HL) CMS (YtA) rice seedlings and those of its corresponding maintainers (YtB) by flow cytometry and staining with rhodamine-123 (Rh-123). Both lines revealed two distinct fluorescence populations: high fluorescence populations (HFP) and light fluorescence populations (LFP), and a somewhat lower LFP/HFP ratio was detected in conjunction with the higher reactive oxygen species (ROS) content in YtA. In addition, use of the specific effector hydrogen peroxide (H2O2) demonstrated a correlation between the LFP/HFP ratio and ROS levels in both lines. Higher ROS content caused a more swift decrease of F(0)F(1)-ATPase activity and ATP contents in YtA than those in YtB, which accompanied with an obvious decline of the LFP/HFP ratio in YtA. Furthermore, a mitochondrial genomic DNA smear was detected by pulsed field gel electrophoresis. Taken together, these results implied that HL-CMS line rice seedlings and those of its corresponding maintainer have different proportion of Rh-123 staining mitochondria populations, which may be accounted for by ROS contents on the basis of ATPase activity and ATP contents.

Honglian (HL) cytoplasmic male sterility (CMS) is one of the rice CMS types and has been widely used in hybrid rice production in China. The CMS line (Yuetai A, YTA) has a Yuetai B (maintainer line, YTB) nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of Yuetai B. The fertility of hybrid (HL-6) was restored by restorer gene in nuclear genome of restorer line (9311). We used isotope-code affinity tag (ICAT) technology to perform the protein profiling of uninucleate stage rice anther and identify the CMS-HL related proteins. Two separate ICAT analyses were performed in this study: (1) anthers from YTA versus anthers from YTB, and (2) anthers from YTA versus anthers from HL-6. Based on the two analyses, a total of 97 unique proteins were identified and quantified in uninucleate stage rice anther under the error rate of less than 10%, of which eight proteins showed abundance changes of at least twofold between YTA and YTB. Triosephosphate isomerase, fructokinase II, DNA-binding protein GBP16 and ribosomal protein L3B were over-expressed in YTB, while oligopeptide transporter, floral organ regulator 1, kinase and S-adenosyl-L: -methionine synthetase were over-expressed in YTA. Reduction of the proteins associated with energy production and lesser ATP equivalents detected in CMS anther indicated that the low level of energy production played an important role in inducing CMS-HL.

Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames (ORF), orfH79 is a mitochondria chimeric gene being responsible for the CMS trait in Honglian (HL) rice. Weakly expressed ORFH79 strongly inhibits the growth of yeast cells. In addition, the content of reactive oxygen species (ROS) in the transformants that expressed ORFH79 was increased by 31%, and ATP was decreased by 41% compared with the control. These results showed ORFH79 peptide is toxic to yeast cells.

Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is used clinically to treat leucopenia typically caused by cancer chemotherapy or radiotherapy. This study used multiple strategies to obtain very high expression levels of OsrhGM-CSF (14 microg/seed) in rice endosperm. Electron micrographs of immunogold-labeled transgenic endosperm showed that rhGM-CSF was not only localized in protein bodies but was also distributed in the apoplast. A biological activity assay indicated that OsrhGM-CSF stimulated the growth of TF-1 cells in vitro. In addition, the transgene was used to effectively treat leucopenia by oral administration of the unprocessed transgenic grains. In cyclophosphamide-induced leucopenic mice, transgenic seeds produced a 27% (t=0.021) gain in leukocytes after 14 days feeding. Even in non-leucopenic mice, leukocyte gain was 37% (t=0.002) more than that of mice fed non-transgenic seeds. This study provides a novel approach to the use of oral unprocessed transgenic OsrhGM-CSF seeds to treat leucopenia.

Being the crucial step for rice transgenic manipulation, callus culture from mature seeds is severely restricted by browning of induced calli, especially in the case of indica (Oryza Sativa L.) rice. Once this browning occurs, the callus will die and no embryonic calli can be obtained for regeneration. Here we report an induction procedure that overcomes callus browning was found. To clarify the inheritance pattern of callus browning, two reciprocal crosses F2 and two backcrosses BC1 were made between indica cultivar inbred lines 93-11 and YueTaiB (YTB) which produced normal and browning respectively in the same induction medium. The ratio of browning to normal in the reciprocal F2 and backcross (BC1) populations tested was approximately 1:3 and 1:1, respectively, these results indicate that callus browning is controlled by one single chromosomal locus which is tentatively named Ic1 (Induced callus 1). The genetic mapping of this locus was carried out using microsatellite markers (SSR) in a 216 extremely browning F2 seed callus. The analysis of genetic linkage indicated that one single locus that mapped to chromosome 1 was correlated to callus browning, and the closest marker in this study was mapped within 1.9 cM from the target locus.

The hybrid rice (Oryza sativa L.) breeding that was initiated in China in the 1970s led to a great improvement in rice productivity. In general, it increases the grain yield by over 20% to the inbred rice varieties, and now hybrid rice has been widely introduced into Africa, Southern Asia and America. These hybrid varieties are generated through either three-line hybrid and two-line hybrid systems; the former is derived from cytoplasmic male sterility (CMS) and the latter derived from genic male sterility (GMS). There are three major types of CMS (HL, BT and WA) and two types of GMS (photoperiod-sensitive (PGMS) and temperature-sensitive (TGMS)). The BT- and HL-type CMS genes are characterized as orf79 and orfH79, which are chimeric toxic genes derived from mitochondrial rearrangement. Rf3 for CMS-WA is located on chromosome 1, while Rf1, Rf4, Rf5 and Rf6 correspond to CMS-BT, CMS-WA and CMS-HL, located on chromosome 10. The Rf1 gene for BT-CMS has been cloned recently, and encodes a mitochondria-targeted PPR protein. PGMS is thought to be controlled by two recessive loci on chromosomes 7 and 12, whereas nine recessive alleles have been identified for TGMS and mapped on different chromosomes. Attention is still urgently needed to resolve the molecular complexity of male sterility to assist rice breeding.

Starch biosynthesis, a very critical event during pollen development, is not well understood in terms of gene regulation and intracellularcontrols. Using the rice HL-type cytoplasmic male sterile (HL-CMS) line with starch deficiency in pollen and two related male-fertile lines (Oryza sativa L.), we have studied two specific developmental stages: 'early', that is, before or during pollen mitosis I, without starch accumulation, and 'late', the actively starch-filling postpollen mitosis I phase. Real-time reverse transcription-PCR analysis showed temporal changes in the expression patterns of six metabolic genes involved in the sucrose-starch pathway, i.e. sucrose synthase (EC 2.4.1.13), Rsus3; acid invertase (EC 3.2.1.26), OSINV2; phosphoglucomutase (EC 5.4.2.2), OsPGM; uridine diphosphate-glucose pyrophosphorylase (EC 2.7.7.9), OsUGP, a subunit of adenosine diphosphate-glucose pyrophosphorylase (AGPase) (EC 2.2.7.2), OsAGPL3; soluble starch synthase (EC 2.4.1.21), OsSSI and three transporter genes [sucrose transporter, OsSUT3; hexose transporter, OSMST7 and plasma membrane H + -adenosine triphosphatase (ATPase) (EC 3.6.1.35), OSA2]. Regarding metabolic enzyme activities, lower activity of sucrose synthase was detected at 'late' compared with 'early' stage in the three rice lines; invertase and other downstream enzymes had higher activities at 'late' stage in male-fertile but not male-sterile lines. Meanwhile, significantly reduced sugars and ATP levels were detected at 'early' and 'late' stages in male-sterile compared with male-fertile tissues. Furthermore, transgenic data showed that AGPase gene can stimulate starch accumulation in pollen of male-sterile host lines, showing the importance of AGPase in the metabolic pathway. Taken together, these data suggest that the combined effects of the reduced energy supply and sugar levels in starch biosynthesis may lead to the observed temporal changes in gene expression, and eventually to pollen sterility.

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of a rice plants 9,311 (Oryza sativa L.) cDNA in rice plants zhonghua11 (Oryza sativa L.). The genetic engineering enabled the plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO(2) assimilation of transgenic plants was significantly more tolerant to high temperature than that of wild-type plants. The analyses of chlorophyll fluorescence and the content and activation of SBPase indicated that the enhancement of photosynthesis to high temperature was not related to the function of photosystem II but to the content and activation of SBPase. Western blotting analyses showed that high temperature stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than that in transgenic plants. The results in this study suggested that under high temperature stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fraction and thus enhanced the tolerance of CO(2) assimilation to high temperature stress. The results suggested that overexpression of SBPase might be an effective method for enhancing high temperature tolerance of plants.

Rice ( Oryza sativa L.) cultivars of strong seedling and early vigor is one of the most important factors associated with the increase of yield potential. With 285 recombinant inbred lines derived from a rice cross between a tropical japonica var. Lemont and an indica var. Teqing with a higher level of vigor than Lemont, seedling/early vigor related traits (plant height, dry weight and seed weight) during 14–44 days post sowing were assessed at an interval of 10 days. Time-related QTL mapping identified 18 QTL or chromosomal regions for the three traits by using a linkage map consisting of 222 marker loci. Of the regions, four produced significant effects on the traits at three measuring stages while the others showed effects only at one or two stages. Comparison of the effect magnitudes indicated that the region qSEV-5-2 on chromosome 5 showed a major effect on seedling/early vigor during stages 1–3 but by stage 4, the major role was substituted by the region qSEV-3-4 on chromosome 3. Five regions simultaneously showed effects on two or three vigor related traits. For each of the five regions, comparison of its LOD peak locations and LOD curve shapes detected for different traits to some extent suggested that within each of the three regions ( qSEV-3-1 , qSEV-3-4 and qSEV-5-2 ), there could be a single locus showing a pleiotropical effect on both plant height and dry weight while within each of the three regions ( qSEV-2-2 , qSEV-4-2 and qSEV-5-2 ), there could be at least two linked loci, one showing effect on seed weight and the other on plant height. The chromosomal regions or QTL identified in the current study were compared with those previously reported for the other traits.

Honglian (HL) is one of the three major types of cytoplasmic male sterility (CMS) of rice (Oryza sativa L) that has been commercially used in hybrid production. In previous studies, the CMS in HL is shown to be associated with a chimeric gene orfH79 that is cotranscribed with an extra atp6 in mitochondria. This study demonstrated that the intact F0F1-ATPase in HL CMS line was specifically reduced in both of its protein quantity and enzyme activity, whereas its F1 sector was not affected. It implies that the F0 sector presents a labile linkage with F1. In the presence of fertility restorer gene, F0F1-ATPase can be recovered. Furthermore, orfH79 transcripts were preferentially polyadenylated, and consequently degraded rapidly in florets of the restored hybrid plants, indicating that the atp6-orfH79 is involved in the sterile phenotype. With inhibition of cytochrome pathway of electron transfer chain, the biomass of the sterile plants grown in dark was significantly lower than that of the fertile lines. However, the respiration measurements showed an increase in the electron transferring capacity in the sterile plants, suggesting that the reduction of biomass in sterile line was caused by the disruption of F0F1-ATPase.

Strategies to increase rice productivity to meet the global demand have been the main concern of breeders around the world. Although a growing number of functional genes related to crop yield have been characterized, our understanding of its associated regulatory pathways is limited. Using rice as a model, we find that blocking miR396 greatly increases grain yield by modulating development of auxiliary branches and spikelets through direct induction of the growth regulating factor 6 (OsGRF6) gene. The upregulation of OsGRF6 results in the coordinated activation of several immediate downstream biological clades, including auxin (IAA) biosynthesis, auxin response factors, and branch and spikelet development-related transcription factors. This study describes a conserved microRNA (miRNA)-dependent regulatory module that integrates inflorescence development, auxin biosynthesis and signalling pathways, and could potentially be used in engineering high-yield crop plants. The regulatory pathway associated with crop yield remains poorly understood. Now researchers have found that blocking miR396 dramatically increases rice grain yield by inducing the GRF6 gene and subsequently activating the auxin pathway and development-related genes.

Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3–5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.

The drought-induced 19 protein family consists of several atypical Cys2/His2-type zinc finger proteins in plants and plays an important role in abiotic stress. In this study, we found that overexpressing OsDi19-4 in rice altered the expression of a series of abscisic acid (ABA)-responsive genes, resulting in strong ABA-hypersensitive phenotypes including ABA-induced seed germination inhibition, early seedling growth inhibition and stomatal closure. On the contrary, OsDi19-4 knockdown lines were less sensitive to ABA. Additionally, OsCDPK14 was identified to interact with OsDi19-4 and be responsible for the phosphorylation of OsDi19-4, and the phosphorylation of OsDi19-4 was further enhanced after the treatment of ABA. Apart from these, OsDi19-4 was shown to directly bind to the promoters of OsASPG1 and OsNAC18 genes, two ABA-responsive genes, and regulate their expression. Transient expression assays confirmed the direct regulation role of OsDi19-4, and the regulation was further enhanced by the increased phosphorylation of OsDi19-4 after the treatment of ABA. Taken together, these data demonstrate that OsDi19-4 acts downstream of OsCDPK14 to positively regulate ABA response by modulating the expression of ABA-responsive genes in rice.

In response to the challenge of food security in the new century, heterosis exploitation is being intensified as an effective way to increase grain yield of rice. Thus, breeding of new cytoplasmic male sterility (CMS) is necessary so as to prevent homog- enization of hybrid rice in commercial breeding programs. As the rapid development of molecular biology, molecular marker based selection can not only increase the efficiency and accuracy for breeding, but also provide us the possibility to create new CMS by genotypic identification. In this study, we devel- opedanovel CMSricebyscreeningricemalesterility- inducing cytoplasm and maintainer candidates using mitotype-specific and nuclear molecular markers, respectively. Field trials reveal that the newly bred CMS line has good general combining ability and special combining abilityfor generation of hybrid rice, which means that the molecular marker system provides us an efficient and practicable approach for development of new CMS in rice breeding programs.

Exploitation of heterosis in crops has contributed greatly to improvement in global food and energy production. In spite of the pervasive importance of heterosis, a complete understanding of its mechanisms has remained elusive. In this study, a small test-crossed rice population was constructed to investigate the formation mechanism of heterosis for thirteen traits. The results of the relative midparent heterosis and modes of inheritance of all investigated traits demonstrated that additive effects were the foundation of heterosis for complex traits in a hierarchical structure, and multiplicative interactions among the component traits were the framework of heterosis in complex traits. Furthermore, new balances between unit traits and related component traits provided hybrids with the opportunity to achieve an optimal degree of heterosis for complex traits. This study dissected heterosis of both reproductive and vegetative traits from the perspective of hierarchical structure for the first time. Additive multiplicative interactions of component traits were proven to be the origin of heterosis in complex traits. Meanwhile, more attention should be paid to component traits, rather than complex traits, in the process of revealing the mechanism of heterosis.

Background For heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants. Results We used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study. Conclusions Our results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1454-z) contains supplementary material, which is available to authorized users.

Allele analysis of the pedigree of Guichao 2. The colored materials were selected for this study; the green varieties harbored the AP9 allele and the yellow varieties contained the AP2 allele. (DOC 157 kb)

Primers used in this study. (DOC 30 kb)

Basic information on the wild rice varieties. The number ahead of each material indicated the serial number in the field in 2012, and the alleles were established based on the different amino acid sequences among the wild rice varieties. (DOC 38 KB)

Amino acid variations of the different alleles in O. rufipogon. The number in the first line indicates the variable amino acid sites of the GN1A protein and the GN1A alleles are in brackets; “-” indicates the deletion of the corresponding amino acid, and “&” indicates the stop codon of the protein. (DOC 68 kb)

Germplasm variety information and their alleles of Gn1a. “NO” in the first line indicates the serial number in the field; SPP, TGW, PY, SSR and EP indicate the number of spikelets per panicle, thousand-grain weight, seed setting rate and effective panicles per plant, respectively; “/” indicates that the data were not available. (DOC 266 KB)

Plant height and tiller number are two important characters related to yield in rice (Oriza sativa L.). Zhenshan97 x Minghui63 recombinant inbred lines were employed to dissect the genetic basis of development of plant height and tiller number using conditional and unconditional composite interval mapping approaches. The traits were normally distributed with transgressive segregation in both directions. Increasingly negative correlations were observed between tiller number and plant height at five consecutive growth stages. A total of 23 and 24 QTL were identified for tiller number and plant height, respectively. More QTL were detected by conditional mapping than by conventional mapping. Different QTL/genes apparently controlled the traits at different developmental stages. Three genomic regions were identified as putative co-located QTL, which showed opposite additive effects on tiller number and plant height. Furthermore, in the period reaching maximum tiller number, the expression of QTL for tiller number was active, whereas that of QTL for plant height was inactive. These facts provided a possible genetic explanation for the negative correlations between the traits. The research demonstrates conditional mapping to be superior to conventional mapping for this type of research. Implications of the results for hybrid rice improvement are discussed.

Crop growth rate is a dynamic character that determines the final yield in cereal crops. The objective of the current study is to reveal its genetic basis and to genetically understand its relationships with grain yield and yield related traits like heading date, grain-filling duration, 1000-grain weight and plant height in rice ( Oryza sativa L.). With 258 recombinant inbred lines (RILs) derived from a cross between cv. Lemont and cv. Teqing, time-related QTL mapping was conducted. Eight QTL for crop growth rate were identified at four consecutive growth periods. These QTL individually explained 5–29% of the trait variation. The Teqing alleles increased the trait values at all the four QTL detected before heading, while Lemont contributed alleles to increase the trait values at three of the other four QTL mapped after heading, which was in agreement with the observation that Teqing was the high-value parent before heading, while Lemont became the high-value parent after heading. No common QTL was observed at different growth periods. These results implied that crop growth rate of rice might be controlled by different genes (QTL) during different time intervals. A total of 22 QTL were identified for the other five traits, and the amount of variation explained by individual QTL ranged from 3% to 26%. The interval RM25–CSU754 on chromosome 8 was identified to produce a major effect on multiple traits such as crop growth rate IV, grain yield, heading date, grain-filling duration, plant heights II and IV, which provided a genetic explanation for the close correlations among the traits. The implications of the results for further improvement in rice yield potential were discussed.

Phosphoribulokinase (PRKase, EC 2.7.1.19) plays an important role in regulating the flow of sugar through the Calvin cycle. To investigate its regulatory character and expression pattern, the gene encoding PRKase in rice was cloned by RACE. A full-length cDNA with an open reading frame of 1212 bp encoding 403 amino acids residues was obtained from an indica rice variety, 9311. The OsPrk is a single locus gene in the rice genome. It is localized in the region of 28.32 approximately 28.33 Mb on the Chromosome 2, flanked by the genetic markers RM450 and MRG0168. RT-PCR analysis revealed that OsPrk gene was expressed in all the tissues we tested and OsPrk transcript level could be dramatically boosted by light illumination. Its expression was down-regulated by externally applied NaCl, ABA, MeJA and glucose over 24 hr, whereas it was up-regulated by GA after 24 hr treatments. These results also indicated that OsPrk gene expression is modulated by these factors at multiple levels.

Sedoheptulose-1, 7-bisphosphatase (SBPase, EC 3.1.3.37) regulates the flow of carbon through the Calvin cycle. To investigate its regulatory character and expression pattern, the gene encoding SBPase in rice has been cloned by RACE. A full-length cDNA with an open reading frame of 1179 bp that encodes 392 amino acid residues was obtained from an indica rice variety, 93-11. The OsSbp is a single locus gene in the rice genome. It is localized in the region of 8.42 - 8.59 Mb on the short arm of chromosome 4, flanked by markers R288 and S11182. RT-PCR analysis revealed that OsSbp gene is expressed in all the tissues we tested, and OsSbp transcript level can be dramatically boosted by light illumination. Its expression is down-regulated by externally applied NaCl, ABA, MeJA and glucose over 24 h, whereas up-regulated by GA after 24 h treatments. These results indicated that OsSbp gene expression is modulated by these factors at multiple levels.

In order to estimate genetic relationships of the AA-genome Oryza species, RAPD and SSR analyses were performed with 45 accessions, including 13 cultivated varieties (eight Oryza sativa and five Oryza glaberrima) and 32 wild accessions (nine Oryza rufipogon, seven Oryza nivara, three Oryza glumaepatula, four Oryza longistaminata, six Oryza barthii, and three Oryza meridionalis). A total of 181 clear and repeatable bands were amplified from 27 selected RAPD primers, and 101 alleles were detected from 29 SSR primer pairs. The dendrogram constructed using UPGMA from a genetic-similarity matrix based on the RAPD data supported the clustering of distinct five groups with a few exceptions: O. rufipogon/ O. nivara/ O. meridionalis, O. barthii/ O. glaberrima, O. glumaepatula, O. sativa and O. longistaminata. The dendrogram based on the SSR analysis showed a more-complicated genetic variation pattern, but the O. longistaminata and O. barthii/ O. glaberrima accessions were consistently separated from all other accessions, indicating significant differentiation of the African AA-genome Oryza species. For accessions in the O. rufipogon/ O. nivara/ O. sativa complex, it is apparent that geographical isolation has played an important role in differentiation of the Asian AA-genome Oryza taxa. It is also demonstrated from this study that both RAPD and SSR analyses are powerful methods for detecting polymorphisms among the different AA-genome Oryza accessions. However, the RAPD analysis provides a more-informative result in terms of the overall genetic relationships at the species level compared to the SSR analysis. The SSR analysis effectively reveals diminutive variation among accessions or individuals within the same species, given approximately the same number of primers or primer-pairs used in the studies.

Summary • Post-pollination competition is reported here in cultivated rice (Oryza sativa) and a perennial wild rice (O. rufipogon) to investigate the occurrence of crop-to-wild gene flow. • Wild and cultivated rice (variety Minghui-63) were grown in a common garden in Hunan province, China, and crop-specific genetic markers were used to detect hybridization following hand-pollinations. Using 11 sequential pollination treatments, the effects of the relative timing of pollination on the success of foreign pollen was investigated. • Foreign pollen from the crop resulted in lower pollen germination, fewer pollen tubes per style, and a significant reduction of seed set, demonstrating a disadvantage of foreign pollen even in the absence of pollen competition. When 1 : 1 pollen mixtures were applied, only 2% of the resulting seeds were hybrids, revealing a much stronger disadvantage of foreign pollen when competing with conspecific pollen. Testing the effects of the relative timing of pollination on the success of foreign pollen suggested that conspecific pollen is often more successful than foreign pollen. Nonetheless, hybridization is possible following the deposition of pollen mixtures, especially when foreign pollen arrives earlier than conspecific pollen. • Pollen competition between wild and cultivated rice could slow the rate of crop-to-wild gene flow, but even if pollen competition was ubiquitous it would not prevent gene flow from the crop.

Agrobacterium tumefaciens-mediated genetic transformation is a powerful tool for plant research, but it can be labor-intensive and time-consuming. Here, we report a protoplast-based approach to study nucleo-cytoplasmic interactions, such as cytoplasmic male sterility/fertility restoration (CMS/Rf) and organellar RNA editing. To test the system, we transfected the fertility restorer gene Rf5, which is involved in the rice HL-CMS/Rf system, into rice protoplasts prepared from the HL-CMS line. As the Rf5 protein accumulated in the transformed protoplasts, the CMS-associated transcripts were endonucleolytically cleaved. There were much lower levels of the CMS-associated protein ORFH79 in the transfected protoplasts than in the mock-transfected protoplasts. Next, we used a dsRNA-mediated gene silencing approach to down-regulate the pentatricopeptide protein gene MPR25, which participates in RNA editing of the organellar transcript nad5. The editing efficiency of mitochondrial transcripts of nad5 at nucleotide 1580 was much lower in the transfected protoplasts than in the mock-transfected protoplasts. Together, these results show that protoplast is a simple and efficient system to study interactions between the nucleus and organelles.

Quantitative real-time polymerase chain reaction (qPCR) is one of the most accurate and widely used methods for gene expression analysis. However, the choice of reference genes for normalization is critical for accurate quantification of gene expression. As development of genomics, mining large-scale datasets such as microarray and RNA-sequencing data becomes a new approach for exploitation of new reference genes. In this study, we analyzed an RNA-sequencing dataset of rice anther and 167 microarray datasets involving different tissues and developing stages of rice anthers and pollens. We selected 12 candidate genes and other 5 reference genes, including ACT1, eEF-1α, GAPDH, Exp2, and CCDC72 used in previous studies, and evaluated their expression in eight tissues and different developmental stages of anthers in rice variety 9311 and Yuetai. UPF3, eIF4A-3, GAPDH, and PPP6 were identified as the most suitable reference genes for qPCR analysis of anther development in rice. The new candidate reference genes showed more stable expression than the traditionally used reference genes. These results provide a set of reliable reference genes for studies in rice anther developmental process.

Lepidopteran insect pests are the main class of pests causing significant damage to crop plant yields. Insecticidal scorpion peptides exhibit toxicity specific for insects. Here, we report that a peptide LMX, optimized from the insect-specific scorpion neurotoxin LqhIT2, showed high levels of activity against rice leaf folder in vitro and in planta. Oral ingestion of LMX protein led to a significant decrease in feeding on rice leaves, repression of larval growth and development, delay in molting, and increase in larval lethality. Compared with LqhIT2 protein, the stability and insecticidal efficacy of LMX was better. Meanwhile, biochemical analysis showed that LMX protein ingestion dramatically decreased ecdysone content in rice leaf folder larvae, and down-regulated enzymatic activities of the detoxification system (α-naphthyl acetate esterase and glutathione S-transferase), the digestive system (tryptase and chymotrypsin), and the antioxidant system (catalase). These changes were tightly correlated with the dosage of LMX protein. Transgene analysis showed that the rate of leaf damage, and the number of damaged tillers and leaves in the transgenic line were greatly reduced relative to wild type plants and empty vector plants. Based on these observations, we propose that the insect-specific scorpion neurotoxin peptide LMX is an attractive and effective alternative molecule for the protection of rice from rice leaf folder.

A novel approach for the differential display of mitochondria RNA has been described. The total mitochondria RNAs are ligated to a single-strand linker with T4 RNA ligase and then reversely transcripted into the first strands of cDNA. The first strands of cDNA that are ligated to another single-strand linker also with T4 RNA ligase can be directly used as the templates of AFLP. After preamplification, selective amplification and then separating the selectively amplified products on 5% PAGE gel, the display patterns of mtRNA can be generated and the RNAs that represent the differentially expressed mitochondria genes may be identified. This new procedure has been used to analyze the total RNAs that were prepared from the mitochondria of CMS line, maintainer line and F1 hybrid in wild abortive type rice; and one differential band has been detected and then verified.

The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F2 population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus.

Rice anther and leaf mRNAs from two cytoplasmic male sterile lines (Maxie A and Congguong 41A) were compared with those from their maintainers and F(1) hybrids by using mRNA differential display to study gene expression pattern in sterile anther during pollen abortion. Anther cDNA differential bands between sterile and fertile plants were more than those of leaf. It was indicated that the expression of fertility gene(s) in the anther was more activable and sufficient than in the leaf. The gene transcript pattern in one of different types of anther was not only associated with its pollen sterility degree but also with its stage of pollen abortion. The anthers with full or partial sterility or with early abortion produced more cDNA differential bands than those fertile or late abortion anthers. In twelve recovered differential cDNA fragments, two were probably associated with male sterility, i. e. one is (AB(4)A(5)), which was specifically expressed in the sterile anther, and the other (AB(3)B(2)), which contained some sequence homologous to a mitochondrial gene (coxII) and whose expression was partially suppressed in the sterile anther.

To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby.

The glycine-rich proteins (GRP) containing RNA recognition motifs (RRM) are involved in the regulation of transcriptional and/or post-transcriptional events. Previous studies have established that GRP162 plays an important role in the restoration of fertility in Honglian cytoplasmic male sterile (HL-CMS) rice. In this study, the ion binding properties of rGRP162 were tested by isothermal titration calorimetry (ITC) and electrophoretic mobility shift assay (EMSA) was performed to test the interaction. Circular dichroism (CD) was carried out to detect the alteration of secondary structure in the presence and absence of Cu(2+). Furthermore, two RRM containing proteins, AtRBP45A and AtRBP47A, were expressed to validate the interaction. Results showed Cu(2+) and Fe(3+) bound GRP162, whereas Ca(2+), Mn(2+), Mg(2+) and K(+) did not. EMSA confirmed that interaction with Cu(2+) interrupted the biological activity of GRP162 by disrupting the secondary structure of the protein based on the results of CD. Moreover, the RNA binding activities of rAtRBP45A and rAtRBP47A were also impaired in the presence of Cu(2+). Data suggest that Cu(2+) in excess may disrupt RNA-binding proteins containing RRM that are essential for post-transcriptional regulation and may impair the development of plants or animals.

Mitochondria are essential organelles in cells not only because they supply over 90% of the cell's energy but also because their dysfunction is associated with disease. Owing to the importance of mitochondria, there are many questions about mitochondria that must be answered. Cytoplasmic male sterility (CMS) is a mysterious natural phenomenon, and the mechanism of the origin of CMS is unknown. Despite successful utilization of CMS and restoration of fertility (Rf) in practice, the underlying mechanisms of these processes remain elusive. This review summarizes the genes involved in CMS and Rf, with a special focus on recent studies reporting the mechanisms of the CMS and Rf pathways, and concludes with potential working models.

Rice false smut, a fungal disease caused by Ustilaginoidea virens is becoming a severe detriment to rice production worldwide. However, little is known about the molecular response of rice to attacks by the smut pathogen. In this article, we define the initial infection process as having three stages: initial colonization on the pistil (stage 1, S1), amplification on the anther (stage 2, S2) and sporulation in the anther chambers (stage 3, S3). Based on the transcriptome of rice hosts in response to U. virens in two separate years, we identified 126, 204, and 580 specific regulated genes in their respective stages S1, S2, and S3, respectively, by excluding common expression patterns in other openly biotic/abiotic databases using bioinformatics. As the disease progresses, several stage-specific biological processes (BP) terms were distinctively enriched: “Phosphorylation” in stage S1, “PCD” in S2, and “Cell wall biogenesis” in S3, implying a concise signal cascade indicative of the tactics that smut pathogens use to control host rice cells during infection. 113 regulated genes were coexpressed among the three stages. They shared highly conserved promoter cis-element in the promoters in response to the regulation of WRKY and Myb for up-regulation, and ABA and Ca2+ for down regulation, indicating their potentially critical roles in signal transduction during rice-U. virens interaction. We further analyzed seven highly regulated unique genes; four were specific to pollen development, implying that pollen-related genes play critical roles in the establishment of rice susceptibility to U. virens. To my knowledge, this is the first report about probing of molecular response of rice to smut pathogen infection, which will greatly expand our understanding of the molecular events surrounding infection by rice false smut.

Katanin, a microtubule-severing enzyme, consists of two subunits: the catalytic subunit P60, and the regulatory subunit P80. In several species, P80 functions in meiotic spindle organization, the flagella biogenesis, the neuronal development, and the male gamete production. However, the P80 function in higher plants remains elusive. In this study, we found that there are three katanin P80 orthologs (OsKTN80a, OsKTN80b, and OsKTN80c) in Oryza sativa L. Overexpression of OsKTN80a caused the retarded root growth of rice seedlings. Further investigation indicates that the retained root growth was caused by the repressed cell elongation in the elongation zone and the stalled cytokinesis in the division zone in the root tip. The in vivo examination suggests that OsKTN80a acts as a microtubule stabilizer. We prove that OsKTN80a, possibly associated with OsKTN60, is involved in root growth via regulating the cell elongation and division.

The restoration fertility complex (RFC) was previously identified in Honglian (HL)-cytoplasmic male sterility (CMS) rice (Oryza sativa), and glycine-rich protein 162 (GRP162) is responsible for binding to the CMS-associated transcript atp6-orfH79. Here, we engineered a recombinant GRP162 containing the mitochondrial transit peptide, termed Mt-GRP162, as an artificial restorer of fertility (Rf) gene. Mt-GRP162 was confirmed to bind to CMS-associated RNA and to localize to the mitochondria. The transgenic plants showed restored fertility with partially functional pollen. We found that the expression of ORFH79 decreased in transgenic plants, while the expression of atp6-orfH79 was not changed. These findings indicate that Mt-GRP162 restores fertility by suppressing the expression of the cytotoxic protein ORFH79 at the post-transcriptional level rather than via the cleavage of atp6-orfH79 in the presence of RFC. These findings contribute to our understanding of the mechanisms of restoration through diverse pathways.