Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells.

BACKGROUND: Transgenic plant suspension cells show economic potential for the production of valuable bioproducts. The sugar starvation-inducible rice αAmy3 promoter, together with its signal peptide, is widely applied to produce recombinant proteins in rice suspension cells. The OsMYBS2 transcription factor was shown recently to reduce activation of the αAmy3 promoter by competing for the binding site of the TA box of the αAmy3 promoter with the potent OsMYBS1 activator. In this study, rice suspension cells were genetically engineered to silence OsMYBS2 to enhance the production of recombinant proteins. RESULTS: The mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene was controlled by the αAmy3 promoter and expressed in OsMYBS2-silenced transgenic rice suspension cells. Transcript levels of the endogenous αAmy3 and the transgene mGM-CSF were increased in the OsMYBS2-silenced suspension cells. The highest yield of recombinant mGM-CSF protein attained in the OsMYBS2-silenced transgenic suspension cells was 69.8 µg/mL, which is 2.5-fold that of non-silenced control cells. The yield of recombinant mGM-CSF was further increased to 118.8 µg/mL in cultured cells derived from homozygous F5 seeds, which was 5.1 times higher than that of the control suspension cell line. CONCLUSIONS: Our results demonstrate that knockdown of the transcription factor gene OsMYBS2 increased the activity of the αAmy3 promoter and improved the yield of recombinant proteins secreted in rice cell suspension cultures.

Effects of OsCDPK1 on the Structure and Physicochemical Properties of Starch in Developing Rice Seeds.

Overexpression of a constitutively active truncated form of OsCDPK1 (OEtr) in rice produced smaller seeds, but a double-stranded RNA gene-silenced form of OsCDPK1 (Ri) yielded larger seeds, suggesting that OsCDPK1 plays a functional role in rice seed development. In the study presented here, we propose a model in which OsCDPK1 plays key roles in negatively controlling the grain size, amylose content, and endosperm appearance, and also affects the physicochemical properties of the starch. The dehulled transgenic OEtr grains were smaller than the dehulled wild-type grains, and the OEtr endosperm was opaque and had a low amylose content and numerous small loosely packed polyhedral starch granules. However, the OEtr grain sizes and endosperm appearances were not affected by temperature, which ranged from low (22 °C) to high (31 °C) during the grain-filling phase. In contrast, the transgenic Ri grains were larger, had higher amylose content, and had more transparent endosperms filled with tightly packed polyhedral starch granules. This demonstrates that OsCDPK1 plays a novel functional role in starch biosynthesis during seed development and affects the transparent appearance of the endosperm. These results improve our understanding of the molecular mechanisms through which the grain-filling process occurs in rice.

Enhancing the abiotic stress tolerance of plants: from chemical treatment to biotechnological approaches.

Abiotic stresses affect crop plants and cause decreases in plant quality and productivity. Plants can overcome environmental stresses by activating molecular networks, including signal transduction, stress perception, metabolite production and expressions of specific stress-related genes. Recent research suggests that chemical priming is a promising field in crop stress management because plants can be primed by chemical agents to increase their tolerance to various environmental stresses. We present a concept to meet this objective and protect plants through priming of existing defense mechanisms avoiding manipulation of the genome. In addition, recent developments in plant molecular biology include the discovery of genes related to stress tolerance, including functional genes for protecting cells and regulatory genes for regulating stress responses. Therefore, enhancing abiotic stress tolerance using a transgenic approach to transfer these genes into plant genomes has attracted more investigations. Both chemical priming agents and genetic engineering can enhance regulatory and functional genes in plants and increase stress tolerance of plants. This review summarizes the latest findings of chemical priming agents and major achievements in molecular approaches that can potentially enhance the abiotic stress tolerance of plants.

Overexpression of a constitutively active truncated form of OsCDPK1 confers disease resistance by affecting OsPR10a expression in rice.

The rice pathogenesis-related protein OsPR10a was scarcely expressed in OsCDPK1-silenced (Ri-1) rice, which was highly sensitive to pathogen infection. After inoculating the leaves with bacterial blight (Xanthomonas oryzae pv. oryzae; Xoo), we found that the expression of OsPR10a was up- and down-regulated in OEtr-1 (overexpression of the constitutively active truncated form of OsCDPK1) and Ri-1 rice plants, respectively. OsPR10a and OsCDPK1 showed corresponding expression patterns and were up-regulated in response to the jasmonic acid, salicylic acid and Xoo treatments, and OsPR1 and OsPR4 were significantly up-regulated in OEtr-1. These results suggest that OsCDPK1 may be an upstream regulator involved in rice innate immunity and conferred broad-spectrum of disease resistance. Following the Xoo inoculation, the OEtr-1 and Ri-1 seedlings showed enhanced and reduced disease resistance, respectively. The dihybrid rice Ri-1/OsPR10a-Ox not only bypassed the effect of OsCDPK1 silencing on the susceptibility to Xoo but also showed enhanced disease resistance and, consistent with Ri-1 phenotypes, increased plant height and grain size. Our results reveal that OsCDPK1 plays novel key roles in the cross-talk and mediation of the balance between stress response and development and provides a clue for improving grain yield and disease resistance simultaneously in rice.

Multiple Patterns of Regulation and Overexpression of a Ribonuclease-Like Pathogenesis-Related Protein Gene, OsPR10a, Conferring Disease Resistance in Rice and Arabidopsis.

An abundant 17 kDa RNase, encoded by OsPR10a (also known as PBZ1), was purified from Pi-starved rice suspension-cultured cells. Biochemical analysis showed that the range of optimal temperature for its RNase activity was 40-70°C and the optimum pH was 5.0. Disulfide bond formation and divalent metal ion Mg2+ were required for the RNase activity. The expression of OsPR10a::GUS in transgenic rice was induced upon phosphate (Pi) starvation, wounding, infection by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), leaf senescence, anther, style, the style-ovary junction, germinating embryo and shoot. We also provide first evidence in whole-plant system, demonstrated that OsPR10a-overexpressing in rice and Arabidopsis conferred significant level of enhanced resistance to infection by the pathogen Xoo and Xanthomona campestris pv. campestris (Xcc), respectively. Transgenic rice and Arabidopsis overexpressing OsPR10a significantly increased the length of primary root under phosphate deficiency (-Pi) condition. These results showed that OsPR10a might play multiple roles in phosphate recycling in phosphate-starved cells and senescing leaves, and could improve resistance to pathogen infection and/or against chewing insect pests. It is possible that Pi acquisition or homeostasis is associated with plant disease resistance. Our findings suggest that gene regulation of OsPR10a could act as a good model system to unravel the mechanisms behind the correlation between Pi starvation and plant-pathogen interactions, and also provides a potential application in crops disease resistance.

Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella.

Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings.

Germination followed by seedling growth constitutes two essential steps in the initiation of a new life cycle in plants, and in cereals, completion of these steps is regulated by sugar starvation and the hormone gibberellin. A calcium-dependent protein kinase 1 gene (OsCDPK1) was identified by differential screening of a cDNA library derived from sucrose-starved rice suspension cells. The expression of OsCDPK1 was found to be specifically activated by sucrose starvation among several stress conditions tested as well as activated transiently during post-germination seedling growth. In gain- and loss-of-function studies performed with transgenic rice overexpressing a constitutively active or RNA interference gene knockdown construct, respectively, OsCDPK1 was found to negatively regulate the expression of enzymes essential for GA biosynthesis. In contrast, OsCDPK1 activated the expression of a 14-3-3 protein, GF14c. Overexpression of either constitutively active OsCDPK1 or GF14c enhanced drought tolerance in transgenic rice seedlings. Hence, our studies demonstrated that OsCDPK1 transduces the post-germination Ca(2+) signal derived from sugar starvation and GA, refines the endogenous GA concentration and prevents drought stress injury, all essential functions to seedling development at the beginning of the life cycle in rice.

Convergent starvation signals and hormone crosstalk in regulating nutrient mobilization upon germination in cereals.

Germination is a unique developmental transition from metabolically quiescent seed to actively growing seedling that requires an ensemble of hydrolases for coordinated nutrient mobilization to support heterotrophic growth until autotrophic photosynthesis is established. This study reveals two crucial transcription factors, MYBS1 and MYBGA, present in rice (Oryza sativa) and barley (Hordeum vulgare), that function to integrate diverse nutrient starvation and gibberellin (GA) signaling pathways during germination of cereal grains. Sugar represses but sugar starvation induces MYBS1 synthesis and its nuclear translocation. GA antagonizes sugar repression by enhancing conuclear transport of the GA-inducible MYBGA with MYBS1 and the formation of a stable bipartite MYB-DNA complex to activate the α-amylase gene. We further discovered that not only sugar but also nitrogen and phosphate starvation signals converge and interconnect with GA to promote the conuclear import of MYBS1 and MYBGA, resulting in the expression of a large set of GA-inducible but functionally distinct hydrolases, transporters, and regulators associated with mobilization of the full complement of nutrients to support active seedling growth in cereals.

Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture.

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice.

Sugars repress alpha-amylase expression in germinating embryos and cell cultures of rice (Oryza sativa) through a sugar response complex (SRC) in alpha-amylase gene promoters and its interacting transcription factor MYBS1. The Snf1 protein kinase is required for the derepression of glucose-repressible genes in yeast. In this study, we explored the role of the yeast Snf1 ortholog in rice, SnRK1, in sugar signaling and plant growth. Rice embryo transient expression assays indicated that SnRK1A and SnRK1B act upstream and relieve glucose repression of MYBS1 and alphaAmy3 SRC promoters. Both SnRK1s contain N-terminal kinase domains serving as activators and C-terminal regulatory domains as dominant negative regulators of SRC. The accumulation and activity of SnRK1A was regulated by sugars posttranscriptionally, and SnRK1A relieved glucose repression specifically through the TA box in SRC. A transgenic RNA interference approach indicated that SnRK1A is also necessary for the activation of MYBS1 and alphaAmy3 expression under glucose starvation. Two mutants of SnRK1s, snrk1a and snrk1b, were obtained, and the functions of both SnRK1s were further studied. Our studies demonstrated that SnRK1A is an important intermediate in the sugar signaling cascade, functioning upstream from the interaction between MYBS1 and alphaAmy3 SRC and playing a key role in regulating seed germination and seedling growth in rice.

A rice gene activation/knockout mutant resource for high throughput functional genomics.

Using transfer DNA (T-DNA) with functions of gene trap and gene knockout and activation tagging, a mutant population containing 55,000 lines was generated. Approximately 81% of this population carries 1-2 T-DNA copies per line, and the retrotransposon Tos17 was mostly inactive in this population during tissue culture. A total of 11,992 flanking sequence tags (FSTs) have been obtained and assigned to the rice genome. T-DNA was preferentially ( approximately 80%) integrated into genic regions. A total of 19,000 FSTs pooled from this and another T-DNA tagged population were analyzed and compared with 18,000 FSTs from a Tos17 tagged population. There was difference in preference for integrations into genic, coding, and flanking regions, as well as repetitive sequences and centromeric regions, between T-DNA and Tos17; however, T-DNA integration was more evenly distributed in the rice genome than Tos17. Our T-DNA contains an enhancer octamer next to the left border, expression of genes within genetics distances of 12.5 kb was enhanced. For example, the normal height of a severe dwarf mutant, with its gibberellin 2-oxidase (GA2ox) gene being activated by T-DNA, was restored upon GA treatment, indicating GA2ox was one of the key enzymes regulating the endogenous level of GA. Our T-DNA also contains a promoterless GUS gene next to the right border. GUS activity screening facilitated identification of genes responsive to various stresses and those regulated temporally and spatially in large scale with high frequency. Our mutant population offers a highly valuable resource for high throughput rice functional analyses using both forward and reverse genetic approaches.

Three novel MYB proteins with one DNA binding repeat mediate sugar and hormone regulation of alpha-amylase gene expression.

The expression of alpha-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. All alpha-amylase genes isolated from cereals contain a TATCCA element or its variants at positions approximately 90 to 150 bp upstream of the transcription start sites. The TATCCA element was shown previously to be an important component of the GA response complex and the sugar response complex of alpha-amylase gene promoters. In the present study, three cDNA clones encoding novel MYB proteins with single DNA binding domains were isolated from a rice suspension cell cDNA library and designated OsMYBS1, OsMYBS2, and OsMYBS3. Gel mobility shift experiments with OsMYBSs showed that they bind specifically to the TATCCA element in vitro. Yeast one-hybrid experiments demonstrated that OsMYBS1 and OsMYBS2 bind to the TATCCA element and transactivate a promoter containing the TATCCA element in vivo. Transient expression assays with barley half-seeds showed that OsMYBS1 and OsMYBS2 transactivate a promoter containing the TATCCA element when sugar is provided, whereas OsMYBS3 represses transcription of the same promoter under sugar starvation. Transient expression assays also showed that these three OsMYBSs cooperate with a GA-regulated transcription factor, HvMYBGa, in the transactivation of a low-pI barley alpha-amylase gene promoter in the absence of GA. Two-hybrid experiments with barley half-seeds showed that OsMYBS1 is able to form a homodimer. The present study demonstrates that differential DNA binding affinity, promoter transactivation ability, dimerization, and interactions with other protein factors determine the biological function of OsMYBSs. This study also suggests that common transcription factors are involved in the sugar and hormonal regulation of alpha-amylase gene expression in cereals.