OsPRR37 confers an expanded regulation of the diurnal rhythms of the transcriptome and photoperiodic flowering pathways in rice.

The circadian clock enables organisms to rapidly adapt to the ever-changing environmental conditions that are caused by daily light/dark cycles. Circadian clock genes universally affect key agricultural traits, particularly flowering time. Here, we show that OsPRR37, a circadian clock gene, delays rice flowering time in an expression level-dependent manner. Using high-throughput mRNA sequencing on an OsPRR37 overexpressing transgenic line (OsPRR37-OE5) and the recipient parent Guangluai4 that contains the loss-of-function Osprr37, we identify 14,992 genes that display diurnal rhythms, which account for 52.9% of the transcriptome. Overexpressing OsPRR37 weakens the transcriptomic rhythms and alters the phases of rhythmic genes. In total, 3,210 differentially expressed genes (DEGs) are identified, among which 1,863 rhythmic DEGs show a correlation between the change of absolute amplitudes and the mean expression levels. We further reveal that OsPRR37 functions as a transcriptional repressor to repress the expression levels and amplitudes of day-phased clock genes. More importantly, OsPRR37 confers expanded regulation on the evening-phased rhythmic DEGs by repressing the morning-phased rhythmic DEGs. Further study shows that OsPRR37 expands its regulation on flowering pathways by repressing Ehd1. Thus, our results demonstrate an expanded regulation mechanism of the circadian clock on the diurnal rhythms of the transcriptome.

Global epigenomic analysis indicates that epialleles contribute to Allele-specific expression via Allele-specific histone modifications in hybrid rice.

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.

A SNP in OsMCA1 responding for a plant architecture defect by deactivation of bioactive GA in rice.

Plant architecture directly affects biomass in higher plants, especially grain yields in agricultural crops. In this study, we characterized a recessive mutant, plant architecture determinant (pad), derived from the Oryza sativa ssp. indica cultivar MH86. The mutant exhibited severe dwarf phenotypes, including shorter and stunted leaves, fewer secondary branches during both the vegetative and reproductive growth stages. Cytological studies revealed that pad mutant growth defects are primarily due to the inhibition of cell expansion. The PAD gene was isolated using a map-based cloning strategy. It encodes a plasma membrane protein OsMCA1 and a SNP responsible for a single amino acid change was found in the mutant. PAD was universally expressed in rice tissues from the vegetative to reproductive growth stages, especially in seedlings, nodes and rachillae. Quantitative real-time PCR analysis revealed that the most of the genes responding to gibberellin (GA) metabolism were up-regulated in pad mutant internodes. The endogenous GA content measurement revealed that the levels of GA1 were significantly decreased in the third internode of pad mutants. Moreover, a GA response assay suggested that OsMCA1/PAD might be involved in the regulation of GA metabolism and signal transduction. Our results revealed the pad is a loss-of-function mutant of the OsMCA1/PAD, leading to upregulation of genes related to GA deactivation, which decreased bioactive GA levels.

The OsSec18 complex interacts with P0(P1-P2)2 to regulate vacuolar morphology in rice endosperm cell.

BACKGROUND: Sec18p/N-ethylmaleimide-sensitive factor (NSF) is a conserved eukaryotic ATPase, which primarily functions in vesicle membrane fusion from yeast to human. However, the function of the OsSec18 gene, a homologue of NSF in rice, remains unknown. RESULTS: In the present study, we investigated the function of OsSec18 in rice and found that OsSec18 complements the temperature-sensitive phenotype and interferes with vacuolar morphogenesis in yeast. Overexpression of OsSec18 in rice decreased the plant height and 1000-grain weight and altered the morphology of the protein bodies. Further examination revealed that OsSec18 presented as a 290-kDa complex in rice endosperm cells. Moreover, Os60sP0 was identified a component of this complex, demonstrating that the OsSec18 complex contains another complex of P0(P1-P2)2 in rice endosperm cells. Furthermore, we determined that the N-terminus of OsSec18 can interact with the N- and C-termini of Os60sP0, whereas the C-terminus of OsSec18 can only interact with the C-terminus of Os60sP0. CONCLUSION: Our results revealed that the OsSec18 regulates vacuolar morphology in both yeast and rice endosperm cell and the OsSec18 interacts with P0(P1-P2)2 complex in rice endosperm cell.

The phenotypic predisposition of the parent in F1 hybrid is correlated with transcriptome preference of the positive general combining ability parent.

BACKGROUND: Sprague and Tatum (1942) introduced the concepts of general combining ability (GCA) and specific combining ability (SCA) to evaluate the breeding parents and F1 hybrid performance, respectively. Since then, the GCA was widely used in cross breeding for elite parent selection. However, the molecular basis of GCA remains to unknown. RESULTS: We studied the transcriptomes of three varieties and three F1 hybrids using RNA-Sequencing. Transcriptome sequence analysis revealed that the transcriptome profiles of the F1s were similar to the positive GCA-effect parent. Moreover, the expression levels of most differentially expressed genes (DEGs) were equal to the parent with a positive GCA effect. Analysis of the gene expression patterns of gibberellic acid (GA) and flowering time pathways that determine plant height and flowering time in rice validated the preferential transcriptome expression of the parents with positive GCA effect. Furthermore, H3K36me3 modification bias in the Pseudo-Response Regulators (PRR) gene family was observed in the positive GCA effect parents and demonstrated that the phenotype and transcriptome bias in the positive GCA effect parents have been epigenetically regulated by either global modification or specific signaling pathways in rice. CONCLUSIONS: The results revealed that the transcriptome profiles and DEGs in the F1s were highly related to phenotype bias to the positive GCA-effect parent. The transcriptome bias toward high GCA parents in F1 hybrids attributed to H3K36me3 modification both on global modification level and specific signaling pathways. Our results indicated the transcriptome profile and epigenetic modification level bias to high GCA parents could be the molecular basis of GCA.

Large-scale production of functional human serum albumin from transgenic rice seeds.

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.

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death.

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.

Global RNA sequencing reveals that genotype-dependent allele-specific expression contributes to differential expression in rice F1 hybrids.

BACKGROUND: Extensive studies on heterosis in plants using transcriptome analysis have identified differentially expressed genes (DEGs) in F1 hybrids. However, it is not clear why yield in heterozygotes is superior to that of the homozygous parents or how DEGs are produced. Global allele-specific expression analysis in hybrid rice has the potential to answer these questions. RESULTS: We report a genome-wide allele-specific expression analysis using RNA-sequencing technology of 3,637-3,824 genes from three rice F1 hybrids. Of the expressed genes, 3.7% exhibited an unexpected type of monoallelic expression and 23.8% showed preferential allelic expression that was genotype-dependent in reciprocal crosses. Those genes exhibiting allele-specific expression comprised 42.4% of the genes differentially expressed between F1 hybrids and their parents. Allele-specific expression accounted for 79.8% of the genes displaying more than a 10-fold expression level difference between an F1 and its parents, and almost all (97.3%) of the genes expressed in F1, but non-expressed in one parent. Significant allelic complementary effects were detected in the F1 hybrids of rice. CONCLUSIONS: Analysis of the allelic expression profiles of genes at the critical stage for highest biomass production from the leaves of three different rice F1 hybrids identified genotype-dependent allele-specific expression genes. A cis-regulatory mechanism was identified that contributes to allele-specific expression, leading to differential gene expression and allelic complementary effects in F1 hybrids.

Expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds.

Basic fibroblast growth factor (FGF-2) is an important member of the FGF gene family. It is widely used in clinical applications for scald and wound healing in order to stimulate cell proliferation. Further it is applied for inhibiting stem cell differentiation in cultures. Due to a shortage of plasma and low expression levels of recombinant rbFGF in conventional gene expression systems, we explored the production of recombinant rbFGF in rice grains (Oryza sativa bFGF, OsrbFGF). An expression level of up to 185.66 mg/kg in brown rice was obtained. A simple purification protocol was established with final recovery of 4.49% and resulting in a yield of OsrbFGF reaching up to 8.33 mg/kg OsrbFGF. The functional assay of OsrbFGF indicated that the stimulating cell proliferation activity on NIH/3T3 was the same as with commercialized rbFGF. Wound healing in vivo of OsrbFGF is equivalent to commercialized rbFGF. Our results indicate that rice endosperm is capable of expressing small molecular mass proteins, such as bFGF. This again demonstrates that rice endosperm is a promising system to express various biopharmaceutical proteins.

Comparative transcriptomes profiling of photoperiod-sensitive male sterile rice Nongken 58S during the male sterility transition between short-day and long-day.

BACKGROUND: Photoperiod-sensitive genic male sterile (PGMS) rice, Nongken 58S, was discovered in 1973. It has been widely used for the production of hybrid rice, and great achievements have been made in improving rice yields. However, the mechanism of the male sterility transition in PGMS rice remains to be determined. RESULTS: To investigate the transcriptome during the male sterility transition in PGMS rice, the transcriptome of Nongken 58S under short-day (SD) and long-day (LD) at the glume primordium differentiation and pistil/stamen primordium forming stages was compared. Seventy-three and 128 differentially expressed genes (DEGs) were identified at the glume primordium differentiation and pistil/stamen primordium forming stages, respectively. Five and 22 genes were markedly up-regulated (≥ 5-fold), and two and five genes were considerably down-regulated (≥ 5-fold) under SD during the male sterility transition. Gene ontology annotation and pathway analysis revealed that four biological processes and the circadian rhythms and the flowering pathways coordinately regulated the male sterility transition. Further quantitative PCR analysis demonstrated that the circadian rhythms of OsPRR1, OsPRR37, OsGI, Hd1, OsLHY and OsDof in leaves were obviously different between Nongken 58S and Nongken 58 under LD conditions. Moreover, both OsPRR37 and Hd1 in the inflorescence displayed differences between Nongken 58S and Nongken 58 under both LD and SD conditions. CONCLUSION: The results presented here indicate that the transcriptome in Nongken 58S was significantly suppressed under LD conditions. Among these DEGs, the circadian rhythm and the flowering pathway were involved in the male sterility transition. Furthermore, these pathways were coordinately involved in the male sterility transition in PGMS rice.

Expression of α-1,6-fucosyltransferase (FUT8) in rice grain and immunogenicity evaluation of plant-specific glycans.

Rice seed is a cost-effective bioreactor for the large-scale production of pharmaceuticals. However, convincing evidence of the immunogenicity of plant-specific glycans is still limited although plant-specific glycans are considered potential allergic antigens. In the present study, we found that the α-1,3-fucose content of the glycoprotein produced from rice seed was much lower than that in leaf, and conversely, a higher ß-1,2-xylose content was detected in seed than that in leaf. We detected the α-1,6-fucose content in the glutelin and recombinant human α1-antitrypsin (OsrAAT). The further results in a line containing AAT and FUT8 genes indicated that the α-1,6-fucose content of modified glycosylated recombinant α1-antitrypsin (mgOsrAAT) was 38.4%, while glutelin was only 6.8%. Interestingly, the α-1,3-fucose content of mgOsrAAT was significantly reduced by 59.8% compared with that of OsrAAT. Furthermore, we assessed the immunogenicity of OsrAAT, mgOsrAAT and human α1-antitrypsin (hAAT) using an animal system. The PCA results indicated no significant differences in the IgG, IgM and IgE titers among OsrAAT, mgOsrAAT and hAAT. Further studies revealed that those antibodies were mainly from α-1,3-fucose, but not from ß-1,2-xylose, indicating that α-1,3-fucose was the major immunogenic resource. Our results demonstrated that α-1,3-fucose contents in seed proteins was much less than that of leaf, and could not be a plant-specific glycan because it also exists in human proteins.

Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay.

The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100 % for a single locus and 92.3 % for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection.

OsPRR37 and Ghd7 are the major genes for general combining ability of DTH, PH and SPP in rice.

Artificial selection of high yield crops and better livestock is paramount importance in breeding programs. Selection of elite parents with preferred traits from a phalanx of inbred lines is extremely laborious, time-consuming and highly random. General combining ability (GCA) was proposed and has been widely used for the evaluation of parents in hybrid breeding for more than half a century. However, the genetic and molecular basis of GCA has been largely overlooked. Here, we present two pleotropic QTLs are accounting for GCA of days to heading (DTH), plant height (PH) and spikelet per panicle (SPP) using an F2-based NCII design, the BC3F2 population as well as a set of nearly isogenic lines (NILs) with five testers. Both GCA1 and GCA2 were loss-of-function gene in low-GCA parent and gain-of-function gene in high-GCA parent, encoding the putative Pseudo-Response Regulators, OsPRR37 and Ghd7, respectively. Overexpression of GCA1 in low-GCA parent significantly increases GCA effects in three traits. Our results demonstrate that two GCA loci associate with OsPRR37 and Ghd7 and reveal that the genes responsible for important agronomic traits could simultaneously account for GCA effects.

Expression and characterization of recombinant human alpha-antitrypsin in transgenic rice seed.

Human alpha-antitrypsin (AAT) is the most abundant circulating protease inhibitor in the human plasma. It is produced in the liver and exerts a primary physiological role as inhibitor for the neutrophil elastase in the lung. Individuals with one or several gene mutations in AAT causing reduction of the protein are related to lung, liver and pancreatic emphysema diseases and are treated lifelong with infusions of human plasma-derived AAT. Due to shortage of plasma and low expression levels of recombinant AAT in conventional gene expression systems, we explored the possibility to produce recombinant AAT in rice grains (Oryza sativa AAT, OsrAAT). An expression level of up to 2.24g/kg brown rice and a final recovery of purified 0.366g/kg OsrAAT has been obtained. OsrAAT has the same secondary structure and protease inhibitory activity as plasma-derived AAT (pAAT), but was highly heterogeneous with regard to glycan modifications. Thus 32.8% of OsrAAT were glycosylated and 67.2% were free of glycans as determined by MALDI-MS. Of the N-glycan structures 64.8% were vacuole-specific paucimannosidic molecules. Immune electron microscopy located OsrAAT in the endoplasmic reticulum lumen as precursor-accumulating (PAC)-like vesicle structures. The pharmacokinetic study indicated that the half-life of OsrAAT was prolonged, while the clearance rate was faster than that of pAAT in vivo. The results demonstrate that rice endosperm is a promising system to express this biopharmaceutical protein.