Yingguo Zhu

The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162

This work identifies the pentatricopeptide repeat protein RF9 and finds that it restores fertility in cytoplasmic male sterile (CMS) Hong-Lian rice by interacting with a Gly-rich protein as part of a complex that processes the CMS-associated transcript atp6-orfH79, thereby restoring fertility. The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene–mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS–Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration.

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.

A comparative study of genetic relationships among the AA-genome Oryza species using RAPD and SSR markers

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.

Overexpression of SBPase enhances photosynthesis against high temperature stress in transgenic rice plants

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, CO2 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 CO2 assimilation to high temperature stress. The results suggested that overexpression of SBPase might be an effective method for enhancing high temperature tolerance of plants.

Programmed cell death during microgenesis in a Honglian CMS line of rice is correlated with oxidative stress in mitochondria

The key role of mitochondria in the integration of apoptosis and oxidative stress in mammals has been documented. In plants, mitochondria are implicated in cytoplasmic male sterility (CMS), programmed cell death (PCD), and oxidative stress. However, to date there has been no evidence presented of the interplay among CMS, PCD and oxidative stress. In this study, we demonstrate that the pollen abortion of Honglian CMS line of rice (Oryza sativa L.) displays a PCD phenotype. A premature loss of microspores was accompanied by several biochemical markers of apoptosis. Analysis of mitochondria revealed that during the PCD process there was disruption of the inner mitochondrial membrane potential in microspores. This disruption was correlated with excess production of reactive oxygen species and down-regulation of the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase in mitochondria. The excess accumulation of ROS spanned three stages from pollen mother cell to early-uninucleate stage, and was followed by the occurrence of PCD at meiosis. Our data suggest that the microspores suffered from severe oxidative stress during pollen development. It is likely the chronic oxidative stress triggered the tissue-specific PCD and consequently resulted in the abortion of microspores.

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

Abstract 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.

Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice

One of the cytoplasmic male sterility (CMS) types used for hybrid rice (Oryza sativa L.) production in China is the Honglian (HL)-CMS. Previous studies suggested that pollen abortion of the sterile plants was resulted from a special programmed cell death (PCD) program started at meiosis in the microspores. To elucidate the molecular basis of the pollen abortion, we compared the biochemical and physiological properties such as content of reactive oxygen species (ROS), ATP, NADH, total glutathione and ascorbate acid, the activities of dehydroascrbate reductase, glutathione reductase, ascorbate peroxides and superoxide dismutase, and the integrity of mitochondrial genome DNA isolated from an HL-CMS line, Yuetai A and its maintainer line, Yuetai B. Our results indicated that the mitochondria of the HL-CMS line suffered from a serious oxidative stress during microspores development. Oxidative stress induced by abnormal increased ROS at meiosis stage resulted in the depletion of ATP and NADH, and the degradation of mitochondrial genomic DNA. This suggests that the presence of redox signal originated in mitochondria affects the rest of the cell. Therefore, it is possible that the abortion of premature microspores in HL-CMS line is induced by the chronic oxidative stress in mitochondria in the early stage of pollen development.

Overexpression of sedoheptulose-1,7-bisphosphatase enhances photosynthesis and growth under salt stress in transgenic rice plants

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC3.1.3.37) was increased in the transgenic rice cultivar zhonghua11 (Oryza sativa L. ssp. japonica) by overexpressing OsSbp cDNA from the rice cultivar 9311 (Oryza sativa ssp. indica). This genetic engineering enabled the transgenic plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance of transgenic rice plants to salt stress at the young seedlings stage. Moreover, CO2 assimilation in transgenic rice plants was significantly more tolerant to salt stress than in wild-type plants. The analysis of chlorophyll fluorescence and the activity of SBPase indicated that the enhancement of photosynthesis in salt stress was not related to the function of PSII but to the activity of SBPase. Western-blot analysis showed that salt stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, this association was much more prominent in wild-type plants than in transgenic plants. Results suggested that under salt stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase by providing more regeneration of the acceptor molecule ribulose-1,5-bisphosphate in the soluble stroma and by preventing the sequestration of Rubisco activase to the thylakoid membrane from the soluble stroma, and, thus, enhanced the tolerance of photosynthesis to salt stress. Results suggested that overexpression of SBPase was an effective method for enhanncing salt tolerance in rice.

Proteomic Analysis of Rice Endosperm Cells in Response to Expression of hGM-CSF

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.

ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice.

Cytoplasmic male sterility (CMS) has attracted great interest because of its application in crop breeding. Despite increasing knowledge of CMS, not much is understood about its molecular mechanisms. Previously, orfH79 was cloned and identified as the CMS gene in Honglian rice, but how the ORFH79 protein causes pollen abortion is still unknown. Through bacterial two-hybrid library screening, P61, a subunit of the mitochondrial electron transport chain (ETC) complex III, was selected as a candidate that interacts with ORFH79. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (coIP) assays verified their interaction inside mitochondria. Blue native polyacrylamide gel electrophoresis (BN-PAGE) and western blotting showed ORF79 and P61 colocalized in mitochondrial ETC complex III of CMS lines. Compared with the maintainer line, Yuetai B (YB), a significant decrease of enzyme activity was detected in mitochondrial complex III of the CMS line, Yuetai A (YA), which resulted in decreased ATP concentrations and an increase in the reactive oxygen species (ROS) content. We propose that the CMS protein, ORFH79, can bind to complex III and decrease its enzyme activity through interaction with P61. This defect results in energy production dysfunction and oxidative stress in mitochondria, which may work as retrograde signals that lead to abnormal pollen development.