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.

Plants provide a promising expression platform for producing recombinant proteins with several advantages in terms of high expression level, lower production cost, scalability, and safety and environment-friendly. Molecular pharming has been recognized as an emerging industry with strategic importance that could play an important role in economic development and healthcare in China. Here, this review represents the significant advances using transgenic rice endosperm as bioreactor to produce various therapeutic recombinant proteins in transgenic rice endosperm and large-scale production of OsrHSA, and discusses the challenges to develop molecular pharming as an emerging industry with strategic importance in China.

Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2 × 104 to 0.2 pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8 ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

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.

Oryza longistaminata, a perennial wild rice species with an AA genome, is characterized by the presence of rhizomatous stems. The rhizomatous trait in rice was previously shown to be quantitatively controlled by many genes, but the molecular mechanism related to rhizome development is still unknown. In the present study, expressed sequence tags (ESTs) generated from rhizome tips of O. longistaminata were collected and analyzed. A total of 10,283 complimentary deoxyribunucleic acid clones were randomly sequenced, which generated 10,136 raw sequences, and finally, 4,419 unisequences with diverse functional categories were generated. These unisequences were mapped onto the Oryza sativa genome, which revealed that 4,285 (96.97%) and 4,151 (93.94%) of the unisequences were alignable to the japonica and indica genomic sequences, respectively, with >80% sequence identity. Additionally, 41 unisequences showed four typical types of alternative splicing patterns. More than 600 simple sequence repeats were identified in these unisequences. A subset of unisequences were physically colocalized onto rhizome-related quantitative trait locus intervals in rice and sorghum, and one gene, OLRR1, was further confirmed to be enriched in the rhizome tip and young leaf by real-time polymerase chain reaction and in situ hybridization. Unisequences reported in this study provide valuable data for molecular dissection of the rhizomatous growth habit in O. longistaminata.

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 accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.

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 μg/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.

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 2.24 g/kg brown rice and a final recovery of purified 0.366 g/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.