Su-May Yu

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.

A Novel Class of Gibberellin 2-Oxidases Control Semidwarfism, Tillering, and Root Development in Rice

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2β-hydroxylation: a larger class of C19 GA2oxs and a smaller class of C20 GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C20 GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C20 GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C20 GA2oxs were found to cause less severe GA-defective phenotypes than C19 GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C20 GA2oxs.

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.

Sugar Response Sequence in the Promoter of a Rice α-Amylase Gene Serves as a Transcriptional Enhancer

Expression of α-amylase genes in both rice suspension cells and germinating embryos is repressed by sugars and the mechanism involves transcriptional regulation. The promoter of a rice α-amylase gene αAmy3 was analyzed by both loss- and gain-of-function studies and the major sugar response sequence (SRS) was located between 186 and 82 base pairs upstream of the transcription start site. The SRS conferred sugar responsiveness to a minimal promoter in an orientation-independent manner. It also converted a sugar-insensitive rice actin gene promoter into a sugar-sensitive promoter in a dose-dependent manner. Linker-scan mutation studies identified three essential motifs: the GC box, the G box, and the TATCCA element, within the SRS. Sequences containing either the GC box plus G box or the TATCCA element each mediated sugar response, however, they acted synergistically to give a high level glucose starvation-induced expression. Nuclear proteins from rice suspension cells binding to the TATCCA element in a sequence-specific and sugar-dependent manner were identified. The TATCCA element is also an important component of the gibberellin response complex of the α-amylase genes in germinating cereal grains, suggesting that the regulation of α-amylase gene expression by sugar and hormone signals may share common regulatory machinery.

A Novel MYBS3-dependent Pathway Confers Cold Tolerance in Rice

Rice (Oryza sativa) seedlings are particularly sensitive to chilling in early spring in temperate and subtropical zones and in high-elevation areas. Improvement of chilling tolerance in rice may significantly increase rice production. MYBS3 is a single DNA-binding repeat MYB transcription factor previously shown to mediate sugar signaling in rice. In this study, we observed that MYBS3 also plays a critical role in cold adaptation in rice. Gain- and loss-of-function analyses indicated that MYBS3 was sufficient and necessary for enhancing cold tolerance in rice. Transgenic rice constitutively overexpressing MYBS3 tolerated 4°C for at least 1 week and exhibited no yield penalty in normal field conditions. Transcription profiling of transgenic rice overexpressing or underexpressing MYBS3 led to the identification of many genes in the MYBS3-mediated cold signaling pathway. Several genes activated by MYBS3 as well as inducible by cold have previously been implicated in various abiotic stress responses and/or tolerance in rice and other plant species. Surprisingly, MYBS3 repressed the well-known DREB1/CBF-dependent cold signaling pathway in rice, and the repression appears to act at the transcriptional level. DREB1 responded quickly and transiently while MYBS3 responded slowly to cold stress, which suggests that distinct pathways act sequentially and complementarily for adapting short- and long-term cold stress in rice. Our studies thus reveal a hitherto undiscovered novel pathway that controls cold adaptation in rice.

Mutant resources in rice for functional genomics of the grasses.

Rice ( Oryza sativa ) is the reference genome for the grasses, including cereals. The complete genome sequence lays the foundation for comparative genomics to the other grasses based on genome structure and individual gene function ([Devos, 2005][1]; [International Rice Genome Sequencing Project,

Coordinated Responses to Oxygen and Sugar Deficiency Allow Rice Seedlings to Tolerate Flooding

The protein kinase CIPK15 integrates the response to hypoxia with sugar signaling to allow submerged rice seedlings to grow. Surviving Submergence Although plants need water to survive, too much of a good thing can be devastating: Submergence in water limits the diffusion of oxygen and thereby aerobic metabolism and energy production. Therefore, flooding represents a potential disaster that can wipe out crops. Lee et al. investigated the mechanisms that allow rice seedlings to survive flooding and discovered that CIPK15 [calcineurin B–like (CBL)–interacting protein kinase]—a protein kinase previously implicated in mediating various stress responses—integrates the response to hypoxia with that to sugar depletion to regulate anaerobic carbohydrate metabolism under flooded conditions. Thus, rice seedlings survive—and continue to grow—even when submerged in water. Flooding is a widespread natural disaster that leads to oxygen (O2) and energy deficiency in terrestrial plants, thereby reducing their productivity. Rice is unusually tolerant to flooding, but the underlying mechanism for this tolerance has remained elusive. Here, we show that protein kinase CIPK15 [calcineurin B–like (CBL)–interacting protein kinase] plays a key role in O2-deficiency tolerance in rice. CIPK15 regulates the plant global energy and stress sensor SnRK1A (Snf1-related protein kinase 1) and links O2-deficiency signals to the SnRK1-dependent sugar-sensing cascade to regulate sugar and energy production and to enable rice growth under floodwater. Our studies contribute to understanding how rice grows under the conditions of O2 deficiency necessary for growing rice in irrigated lowlands.

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.

SUGARS ACT AS SIGNAL MOLECULES AND OSMOTICA TO REGULATE THE EXPRESSION OF ALPHA -AMYLASE GENES AND METABOLIC ACTIVITIES IN GERMINATING CEREAL GRAINS

The molecular mechanisms that initiate and control the metabolic activities of seed germination are largely unknown. Sugars may play important roles in regulating such metabolic activities in addition to providing an essential carbon source for the growth of young seedlings and maintaining turgor pressure for the expansion of tissues during germination. To test this hypothesis, we investigated the physiological role of sugars in the regulation of α-amylase gene expression and carbohydrate metabolism in embryo and endosperm of germinating rice seeds. RNA gel blot analysis revealed that in the embryo and aleurone cells, expression of four α-amylase genes was differentially regulated by sugars via mechanisms beyond the well-known hormonal control mechanism. In the aleurone cells, expression of these α-amylase genes was regulated by gibberellins produced in the embryo and by osmotically active sugars. In the embryo, expression of two α-amylase genes and production of gibberellins were transient, and were probably induced by depletion of sugars in the embryo upon imbibition, and suppressed by sugars influx from the endosperm as germination proceeded. The differential expression of the four α-amylase genes in the embryo and aleurone cells was probably due to their markedly different sensitivities to changes in tissue sugar levels. Our study supports a model in which sugars regulate the expression of α-amylase genes in a tissue-specific manner: via a feedback control mechanism in the embryo and via an osmotic control mechanism in the aleurone cells. An interactive loop among sugars, gibberellins, and α-amylase genes in the germinating cereal grain is proposed.

Signal Peptide-Dependent Targeting of a Rice α-Amylase and Cargo Proteins to Plastids and Extracellular Compartments of Plant Cells

α-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether α-amylases are localized in plastids where starch is stored. To study the subcellular localization of α-amylases in plant cells, a rice (Oryza sativa) α-amylase, αAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of αAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated αAmy3 to be cotranslationally imported and processed in canine microsomes. αAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to αAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized αAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the αAmy3 SP was further studied by gain-of-function analyses. β-Glucuronidase (GUS) and green fluorescence protein fused with or without the αAmy3 SP was expressed in transgenic tobacco or rice. The αAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice α-amylase, αAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.