Shin-Lon Ho

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an α-amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice α-amylase gene (αAmy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice α-amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.

Three Novel MYB Proteins with One DNA Binding Repeat Mediate Sugar and Hormone Regulation of α-Amylase Gene Expression

The expression of α-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. All α-amylase genes isolated from cereals contain a TATCCA element or its variants at positions ∼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 α-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 α-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 α-amylase gene expression in cereals.

The SnRK1A Protein Kinase Plays a Key Role in Sugar Signaling during Germination and Seedling Growth of Rice

Sugars repress α-amylase expression in germinating embryos and cell cultures of rice (Oryza sativa) through a sugar response complex (SRC) in α-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 αAmy3 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 αAmy3 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 αAmy3 SRC and playing a key role in regulating seed germination and seedling growth in rice.

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 Ca2+ 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

This work shows that not only sugar but also nitrogen and phosphate starvation signals converge with GA signaling to promote the co-nuclear import of MYBS1 and MYBGA and expression of a large set of GA-inducible but functionally distinct hydrolases, transporters, and regulators active in nutrient mobilization required for seedling growth in rice and barley. 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. Biotechnol. Bioeng. 2012; 109:1239–1247.

Molecular cloning and characterization of a novel starvation inducible MAP kinase gene in rice

Signaling pathways, specifically mitogen-activated protein kinase (MAPK) cascades, have been implicated in the regulation of stress and developmental signals in plants. Here, we report the characterization of a rice ( Oryza sativa L. cv. TN-67) MAPK gene, OsMAPK3, isolated by screening a sugar-starved treated rice cell cDNA library. The OsMAPK3gene was abundantly expressed in mature leaves and panicles of 3-month-old plants, but poorly in young leaves. Its developmental regulation was suggested by the fact that OsMAPK3mRNA levels changed during leaf differentiation and root development. Furthermore, the amount of this transcript increased markedly in sugar-starved or mannose-fed cells.

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.

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.