Cell-Permeable Ubiquitin and Histone Tools for Studying Post-translational Modifications.

Protein post-translational modifications (PTMs) regulate nearly all biological processes in eukaryotic cells, and synthetic PTM protein tools are widely used to detect the activity of the related enzymes and identify the interacting proteins in cell lysates. Recently, the study of these enzymes and the interacting proteome has been accomplished in live cells using cell-permeable PTM protein tools. In this concept, we will introduce cell penetrating techniques, the syntheses of cell-permeable PTM protein tools, and offer some future perspective.

An alanine to valine mutation of glutamyl-tRNA reductase enhances 5-aminolevulinic acid synthesis in rice.

KEY MESSAGE: An alanine to valine mutation of glutamyl-tRNA reductase's 510th amino acid improves 5-aminolevulinic acid synthesis in rice. 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrroles and plays an important role in plant growth regulation. ALA is synthesized from glutamate, catalyzed by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde aminotransferase (GSAT). In Arabidopsis, ALA synthesis is the rate-limiting step in tetrapyrrole production via GluTR post-translational regulations. In rice, mutations of GluTR and GSAT homologs are known to confer chlorophyll deficiency phenotypes; however, the enzymatic activity of rice GluRS, GluTR, and GSAT and the post-translational regulation of rice GluTR have not been investigated experimentally. We have demonstrated that a suppressor mutation in rice partially reverts the xantha trait. In the present study, we first determine that the suppressor mutation results from a G → A nucleotide substitution of OsGluTR (and an A → V change of its 510th amino acid). Protein homology modeling and molecular docking show that the OsGluTRA510V mutation increases its substrate binding. We then demonstrate that the OsGluTRA510V mutation increases ALA synthesis in Escherichia coli without affecting its interaction with OsFLU. We further explore homologous genes encoding GluTR across 193 plant species and find that the amino acid (A) is 100% conserved at the position, suggesting its critical role in GluTR. Thus, we demonstrate that the gain-of-function OsGluTRA510V mutation underlies suppression of the xantha trait, experimentally proves the enzymatic activity of rice GluRS, GluTR, and GSAT in ALA synthesis, and uncovers conservation of the alanine corresponding to the 510th amino acid of OsGluTR across plant species.

An ABA-serotonin module regulates root suberization and salinity tolerance.

Suberin in roots acts as a physical barrier preventing water/mineral losses. In Arabidopsis, root suberization is regulated by abscisic acid (ABA) and ethylene in response to nutrient stresses. ABA also mediates coordination between microbiota and root endodermis in mineral nutrient homeostasis. However, it is not known whether this regulatory system is common to plants in general, and whether there are other key molecule(s) involved. We show that serotonin acts downstream of ABA in regulating suberization in rice and Arabidopsis and negatively regulates suberization in rice roots in response to salinity. We show that ABA represses transcription of the key gene (OsT5H) in serotonin biosynthesis, thus promoting root suberization in rice. Conversely, overexpression of OsT5H or supplementation with exogenous serotonin represses suberization and reduces tolerance to salt stress. These results identify an ABA-serotonin regulatory module controlling root suberization in rice and Arabidopsis, which is likely to represent a general mechanism as ABA and serotonin are ubiquitous in plants. These findings are of significant importance to breeding novel crop varieties that are resilient to abiotic stresses and developing strategies for production of suberin-rich roots to sequestrate more CO2 , helping to mitigate the effects of climate change.

Mutations of the Genomes Uncoupled 4 Gene Cause ROS Accumulation and Repress Expression of Peroxidase Genes in Rice.

The Genomes Uncoupled 4 (GUN4) is one of the retrograde signaling genes in Arabidopsis and its orthologs have been identified in oxygenic phototrophic organisms from cyanobacterium to higher plants. GUN4 is involved in tetrapyrrole biosynthesis and its mutation often causes chlorophyll-deficient phenotypes with increased levels of reactive oxygen species (ROS), hence it has been speculated that GUN4 may also play a role in photoprotection. However, the biological mechanism leading to the increased ROS accumulation in gun4 mutants remains largely unknown. In our previous studies, we generated an epi-mutant allele of OsGUN4 (gun4 epi ), which downregulated its expression to ∼0.5% that of its wild-type (WT), and a complete knockout allele gun4-1 due to abolishment of its translation start site. In the present study, three types of F2 plant derived from a gun4-1/gun4 epi cross, i.e., gun4-1/gun4-1, gun4-1/gun4 epi and gun4 epi /gun4 epi were developed and used for further investigation by growing them under photoperiodic condition (16 h/8 h light/dark) with low light (LL, 100 µmol photons m-2 s-1) or high light (HL, 1000 µmol photons m-2 s-1). The expression of OsGUN4 was light responsive and had two peaks in the daytime. gun4-1/gun4-1-F2 seeds showed defective germination and died within 7 days. Significantly higher levels of ROS accumulated in all types of OsGUN4 mutants than in WT plants under both the LL and HL conditions. A comparative RNA-seq analysis of WT variety LTB and its gun4 epi mutant HYB led to the identification of eight peroxidase (PRX)-encoding genes that were significantly downregulated in HYB. The transcription of these eight PRX genes was restored in transgenic HYB protoplasts overexpressing OsGUN4, while their expression was repressed in LTB protoplasts transformed with an OsGUN4 silencing vector. We conclude that OsGUN4 is indispensable for rice, its expression is light- and oxidative-stress responsive, and it plays a role in ROS accumulation via its involvement in the transcriptional regulation of PRX genes.

OsKEAP1 Interacts with OsABI5 and Its Downregulation Increases the Transcription of OsABI5 and the ABA Response Genes in Germinating Rice Seeds.

Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) is the key antioxidant system in animals. In a previous study, we identified a probable KEAP1 ortholog in rice, OsKEAP1, and demonstrated that the downregulation of OsKEAP1 could alter the redox system and impair plant growth, as well as increase the susceptibility to abscisic acid (ABA) in seed germination. However, no NRF2 orthologs have been identified in plants and the mechanism underlying the phenotype changes of downregulated oskeap1 mutants is yet unknown. An in silico search showed that OsABI5 is the gene that encodes a protein with the highest amino acid identity score (38.78%) to NRF2 in rice. In this study, we demonstrated that, via yeast two-hybrids analysis and bimolecular fluorescence complementation assays, OsKEAP1 interacted with OsABI5 via its Kelch repeat domain in the nucleus. In germinating seeds, the expression of OsKEAP1 was significantly downregulated in oskeap1-1 (39.5% that of the wild-type (WT)) and oskeap1-2 (64.5% that of WT), while the expression of OsABI5 was significantly increased only in oskeap1-1 (247.4% that of WT) but not in oskeap1-2 (104.8% that of WT). ABA (0.5 µM) treatment significantly increased the expression of OsKEAP1 and OsABI5 in both the oskeap1 mutants and WT, and 4 days post treatment, the transcription level of OsABI5 became significantly greater in oskeap1-1 (+87.2%) and oskeap1-2 (+55.0%) than that in the WT. The ABA-responsive genes (OsRab16A and three late embryogenesis abundant genes), which are known to be activated by OsABI5, became more responsive to ABA in both oskeap1 mutants than in the WT. The transcript abundances of genes that regulate OsABI5, e.g., OsSnRK2 (encodes a kinase that activates OsABI5), OsABI1, and OsABI2 (both encode proteins binding to OsSnRK2 and are involved in ABA signaling) were not significantly different between the two oskeap1 mutants and the WT. These results demonstrated that OsKEAP1 played a role in the ABA response in rice seed germination via regulating OsABI5, which is the key player in the ABA response. In-depth analyses of the components and their action mode of the KEAP1-NRF2 and ABA signaling pathways suggested that OsKEAP1 likely formed a complex with OsABI5 and OsKEG, and OsABI5 was ubiquitinated by OsKEG and subsequently degraded under physiological conditions; meanwhile, under oxidative stress or with increased an ABA level, OsABI5 was released from the complex, phosphorylated, and transactivated the ABA response genes. Therefore, OsKEAP1-OsABI5 bore some resemblance to KEAP1-NRF2 in terms of its function and working mechanism.

Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.).

The Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) module is a key component in the detoxification and antioxidant system in animals, which plays crucial roles in cell homeostasis and cytoprotection, and consequently in carcinogenesis and disease development. However, this system seems to have diverged throughout evolution across different organisms, and the question of whether a similar system exists in plants has thus far remained unresolved. In this study, a KEAP1 ortholog was identified in rice (Oryza sativa L., OsKEAP1) and its properties were characterized via in silico and laboratory studies. To reveal OsKEAP1's function, two knockdown mutants, oskeap1-1 and oskeap1-2, were generated by targeted mutagenesis in the 5' untranslated region (UTR) using the CRISPR-Cas9 system. In silico analysis showed that OsKEAP1 has a Kelch-repeat domain which is identical to those of animals and a plant-specific development and cell death (DCD) domain in place of the broad-complex, tramtrack, bric-a-brac (BTB) domain found in animals. Orthologs of OsKEAP1 are present across plant species and all have the DCD domain and the Kelch-repeat domain. OsKEAP1 was proven to be localized to both the cytoplasm and nucleus, in contrast to the exclusive cytoplasm localization of animal KEAP1. Single nucleotide insertions in the 5' UTR significantly reduced the transcription level of OsKEAP1 in the oskeap1-1 and oskeap1-2 mutants. The oskeap1 mutations greatly impaired plant growth and development, resulting in significant declines in a majority of agronomic and yield-related traits, i.e., plant height, panicle length, grain number per plant, and seed-set rate. The downregulation of OsKEAP1 increased the levels of H2O2, malondialdehyde, and proline while significantly decreasing the expression of two catalase genes in seedlings grown under normal and salt-stressed conditions. The changes in the above phenotypes are either positively or negatively correlated with the degree of OsKEAP1 downregulation. Altogether, we identified a probable KEAP1 ortholog in rice, revealed its unique subcellular localization, and demonstrated its important functions in vegetative and reproductive growth via regulation of the antioxidant response in plants.

Gene editing: an instrument for practical application of gene biology to plant breeding.

Plant breeding is well recognized as one of the most important means to meet food security challenges caused by the ever-increasing world population. During the past three decades, plant breeding has been empowered by both new knowledge on trait development and regulation (e.g., functional genomics) and new technologies (e.g., biotechnologies and phenomics). Gene editing, particularly by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and its variants, has become a powerful technology in plant research and may become a game-changer in plant breeding. Traits are conferred by coding and non-coding genes. From this perspective, we propose different editing strategies for these two types of genes. The activity of an encoded enzyme and its quantity are regulated at transcriptional and post-transcriptional, as well as translational and post-translational, levels. Different strategies are proposed to intervene to generate gene functional variations and consequently phenotype changes. For non-coding genes, trait modification could be achieved by regulating transcription of their own or target genes via gene editing. Also included is a scheme of protoplast editing to make gene editing more applicable in plant breeding. In summary, this review provides breeders with a host of options to translate gene biology into practical breeding strategies, i.e., to use gene editing as a mechanism to commercialize gene biology in plant breeding.

Generation and Characterization of a Soybean Line with a Vernonia galamensis Diacylglycerol Acyltransferase-1 Gene and a myo-Inositol 1-Phosphate Synthase Knockout Mutation.

Soybean (Glycine max) meal is an important protein source. Soybean meal with lower phytate and oligosaccharides improves meal quality. A single recessive mutation in soybean myo-inositol 1-phosphate synthase (Gm-lpa-TW75-1) confers a seed phenotype with low phytate and increased inorganic phosphate. The mutant was crossed with high oil lines expressing a diacylglycerol acyltransferase1 (DGAT) gene from Vernonia galamensis (VgD). Gm-lpa-TW75-1 X VgD, designated GV, has 21%, and 22% oil and 41% and 43% protein from field and greenhouse seed production, respectively. No significant differences were found in mineral concentrations except for Fe which was 229 µg/g dry mass for GV followed by 174.3 for VgD and 162 for Gm-lpa-TW75-1. Phosphate (Pi) is higher in Gm-lpa-TW75-1 as expected at 5 mg/g, followed by GV at 1.6 mg/g whereas Jack, VgD, and Taiwan75 have about 0.3 mg/g. The Gm-lpa-TW75-1 line has the lowest phytate concentration at 1.4 mg/g followed by GV with 1.8 mg/g compared to Taiwan75, VgD, and Jack with 2.5 mg/g. This work describes a high oil and protein soybean line, GV, with increased Pi and lower phytate which will increase the nutritional value for human and animal feed.

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice.

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

Genome-wide identification, evolution and expression analysis of cyclic nucleotide-gated channels in tobacco (Nicotiana tabacum L.).

Tobacco (Nicotiana tabacum) serve as the top leading commercial, non-food, and model crop worldwide. Cyclic nucleotide-gated channels (CNGCs) are ligand-gated, calcium-permeable, divalent, cation-selective channels, involved in important biological functions. Here, we systematically characterized thirty-five CNGC genes in the genome of Nicotiana tabacum, and classified into four phylogenetic groups. Evolutionary analysis showed that NtabCNGC family of N. tabacum originated from the parental genome of N. sylvestris and N. tomentosiformis, and further expanded via tandem and segmental duplication events. Tissue-specific expression analysis showed that twenty-three NtabCNGC genes are involved in the development of various tobacco tissues. Subsequent RT-qPCR analyses indicated that these genes are sensitive towards external abiotic and biotic stresses. Notable performances were exhibited by group-I and IV CNGC genes against black shank, Cucumber mosaic virus, Potato virus Y, cold, drought, and cadmium stresses. Our analyses also suggested that NtabCNGCs can be regulated by phosphorylation and miRNAs, and multiple light, temperature, and pathogen-responsive cis-acting regulatory elements present in promotors. These results will be useful for elaborating the biological roles of NtabCNGCs in tobacco growth and development.

High-resolution melting-based TILLING of γ ray-induced mutations in rice.

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis.

Rice is one of the world's most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control1,2. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals3, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice2. Serotonin and salicylic acid derive from chorismate4. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin5. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.

Comprehensive genomic analysis of the CNGC gene family in Brassica oleracea: novel insights into synteny, structures, and transcript profiles.

BACKGROUND: The cyclic nucleotide-gated ion channel (CNGC) family affects the uptake of cations, growth, pathogen defence, and thermotolerance in plants. However, the systematic identification, origin and function of this gene family has not been performed in Brassica oleracea, an important vegetable crop and genomic model organism. RESULTS: In present study, we identified 26 CNGC genes in B. oleracea genome, which are non-randomly localized on eight chromosomes, and classified into four major (I-IV) and two sub-groups (i.e., IV-a and IV-b). The BoCNGC family is asymmetrically fractioned into the following three sub-genomes: least fractionated (14 genes), most fractionated-I (10), and most fractionated-II (2). The syntenic map of BoCNGC genes exhibited strong relationships with the model Arabidopsis thaliana and B. rapa CNGC genes and provided markers for defining the regions of conserved synteny among the three genomes. Both whole-genome triplication along with segmental and tandem duplications contributed to the expansion of this gene family. We predicted the characteristics of BoCNGCs regarding exon-intron organisations, motif compositions and post-translational modifications, which diversified their structures and functions. Using orthologous Arabidopsis CNGCs as a reference, we found that most CNGCs were associated with various protein-protein interaction networks involving CNGCs and other signalling and stress related proteins. We revealed that five microRNAs (i.e., bol-miR5021, bol-miR838d, bol-miR414b, bol-miR4234, and bol-miR_new2) have target sites in nine BoCNGC genes. The BoCNGC genes were differentially expressed in seven B. oleracea tissues including leaf, stem, callus, silique, bud, root and flower. The transcript abundance levels quantified by qRT-PCR assays revealed that BoCNGC genes from phylogenetic Groups I and IV were particularly sensitive to cold stress and infections with bacterial pathogen Xanthomonas campestris pv. campestris, suggesting their importance in abiotic and biotic stress responses. CONCLUSION: Our comprehensive genome-wide analysis represents a rich data resource for studying new plant gene families. Our data may also be useful for breeding new B. oleracea cultivars with improved productivity, quality, and stress resistance.

The xantha Marker Trait Is Associated with Altered Tetrapyrrole Biosynthesis and Deregulated Transcription of PhANGs in Rice.

The xantha marker trait, which is controlled by a down-regulating epi-mutation of OsGUN4, has been applied to the production of hybrid rice. However, the molecular basis for the ability of xantha mutants to attain high photosynthetic capacity even with decreased chlorophyll contents has not been characterized. In the present study, we observed that the total chlorophyll content of the xantha mutant was only 27.2% of that of the wild-type (WT) plants. However, the xantha mutant still accumulated 59.9% of the WT δ-aminolevulinic acid content, 72.8% of the WT Mg-protoporphyrin IX content, and 63.0% of the WT protochlorophyllide a content. Additionally, the protoporphyrin IX and heme contents in the mutant increased to 155.0 and 160.0%, respectively, of the WT levels. A search for homologs resulted in the identification of 124 rice genes involved in tetrapyrrole biosynthesis and photosynthesis. With the exception of OsGUN4, OsHO-1, and OsHO-2, the expression levels of the genes involved in tetrapyrrole biosynthesis were significantly higher in the xantha mutant than in the WT plants, as were all 72 photosynthesis-associated nuclear genes. In contrast, there were no differences between the xantha mutant and WT plants regarding the expression of all 22 photosynthesis-associated chloroplast genes. Furthermore, the abundance of 1O2 and the expression levels of 1O2-related genes were lower in the xantha mutant than in the WT plants, indicating 1O2-mediated retrograde signaling was repressed in the mutant plants. These results suggested that the abundance of protoporphyrin IX used for chlorophyll synthesis decreased in the mutant, which ultimately decreased the amount of chlorophyll in the xantha mutant. Additionally, the up-regulated expression of photosynthesis-associated nuclear genes enabled the mutant to attain a high photosynthetic capacity. Our findings confirm that OsGUN4 plays an important role in tetrapyrrole biosynthesis and photosynthesis in rice. GUN4, chlorophyll synthesis pathways, and photosynthetic activities are highly conserved in plants and hence, novel traits (e.g., xantha marker trait) may be generated in other cereal crops by modifying the GUN4 gene.

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10-6-9.8×10-6 in the six M2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

Genome-wide profiling of genetic variation in Agrobacterium-transformed rice plants.

Agrobacterium-mediated transformation has been widely used in producing transgenic plants, and was recently used to generate "transgene-clean" targeted genomic modifications coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Although tremendous variation in morphological and agronomic traits, such as plant height, seed fertility, and grain size, was observed in transgenic plants, the underlying mechanisms are not yet well understood, and the types and frequency of genetic variation in transformed plants have not been fully disclosed. To reveal the genome-wide variation in transformed plants, we sequenced the genomes of five independent T0 rice plants using next-generation sequencing (NGS) techniques. Bioinformatics analyses followed by experimental validation revealed the following: (1) in addition to transfer-DNA (T-DNA) insertions, three transformed plants carried heritable plasmid backbone DNA of variable sizes (855-5216 bp) and in different configurations with the T-DNA insertions (linked or apart); (2) each transgenic plant contained an estimated 338-1774 independent genetic variations (single nucleotide variations (SNVs) or small insertion/deletions); and (3) 2-6 new Tos17 insertions were detected in each transformed plant, but no other transposable elements or bacterial genomic DNA.

Development and molecular characterization of a doubled haploid population derived from a hybrid between japonica rice and wide compatible indica rice.

Doubled haploid (DH) populations, particularly those from subspecies crosses possessing the wide compatible gene S5n , are important germplasm resources for rice genetic studies and breeding, but their feature and potential have not been fully assessed and explored. In the present study, we produced a DH population from the hybrid of japonica 668B and wide compatible indica T23. Genotyping of the S5 locus with allele-specific markers for ORF3, ORF4 and ORF5 revealed a potential recombination hot spot in the ORF3-ORF4 region. Haplotyping analysis revealed that 21/34 subspecies specific Indel markers segregated in distortion in the DH population, with a few lines having indica alleles either extremely low (1.7%) or high (98.3%), with little effect of the S5 allele. While DH lines with the S5n allele had higher frequency of indica alleles, no effect of the S5n allele was observed on all agronomic traits but flowering time. Taken together, the present study advanced understanding of the genetics of wide crosses in general, and DH production in particular between the two rice subspecies, and the new DH population generated will become a useful resource for rice genetic study and breeding in the future.

Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa rice.

The OsLpa1 gene (LOC_Os57400) was identified to be involved in phytic acid (PA) metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter::GUS transgenic plants (GUS: ß-glucuronidase); GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts (OsLpa1.1, OsLpa1.2, OsLpa1.3) are produced due to alternative splicing; quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa all tissues. OsLpa1.2 is predominant in germinating seeds (about 5 times that of OsLpa1.1), but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF (day after flowering) seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein (GFP) in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.

Genome-wide Association Mapping of Quantitative Trait Loci (QTLs) for Contents of Eight Elements in Brown Rice (Oryza sativa L.).

An association mapping of quantitative trait loci (QTLs) regulating the concentrations of eight elements in brown rice (Oryza sativa L.) was performed using USDA mini-core subset cultivated in two different environments. In addition, correlation between the grain elemental concentrations was also studied. A total of 60 marker loci associated with 8 grain elemental concentrations were identified, and these loci were clustered into 37 genomic regions. Twenty new QTLs were found to be associated with important elements such as Zn, Fe, and P, along with others. Fe concentration was associated with the greatest number of markers in two environments. In addition, several important elemental/metal transporter genes were identified in a few mapped regions. Positive correlation was observed within all grain elemental concentrations. In summary, the results provide insight into the genetic basis of rice grain element accumulation and may help in the identification of genes associated with the accumulation of Zn, Fe, and other essential elements in rice.