Overexpression of OsHsp 18.0 in rice enhanced tolerance to heavy metal stress.

KEY MESSAGE: OsHsp18.0 plays a key role in cross-protection of rice seedlings from damages to photochemical systems and cellular membranes, caused by Cd and Cu stresses.

Sugar starvation-regulated MYBS2 and 14-3-3 protein interactions enhance plant growth, stress tolerance, and grain weight in rice.

Autotrophic plants have evolved distinctive mechanisms for maintaining a range of homeostatic states for sugars. The on/off switch of reversible gene expression by sugar starvation/provision represents one of the major mechanisms by which sugar levels are maintained, but the details remain unclear. α-Amylase (αAmy) is the key enzyme for hydrolyzing starch into sugars for plant growth, and it is induced by sugar starvation and repressed by sugar provision. αAmy can also be induced by various other stresses, but the physiological significance is unclear. Here, we reveal that the on/off switch of αAmy expression is regulated by 2 MYB transcription factors competing for the same promoter element. MYBS1 promotes αAmy expression under sugar starvation, whereas MYBS2 represses it. Sugar starvation promotes nuclear import of MYBS1 and nuclear export of MYBS2, whereas sugar provision has the opposite effects. Phosphorylation of MYBS2 at distinct serine residues plays important roles in regulating its sugar-dependent nucleocytoplasmic shuttling and maintenance in cytoplasm by 14-3-3 proteins. Moreover, dehydration, heat, and osmotic stress repress MYBS2 expression, thereby inducing αAmy3 Importantly, activation of αAmy3 and suppression of MYBS2 enhances plant growth, stress tolerance, and total grain weight per plant in rice. Our findings reveal insights into a unique regulatory mechanism for an on/off switch of reversible gene expression in maintaining sugar homeostatic states, which tightly regulates plant growth and development, and also highlight MYBS2 and αAmy3 as potential targets for crop improvement.

A CCR4-associated factor 1, OsCAF1B, confers tolerance of low-temperature stress to rice seedlings.

KEY MESSAGE: Rice is an important crop in the world. However, little is known about rice mRNA deadenylation, which is an important regulation step of gene expression at the post-transcriptional level. The CCR4-NOT1 complex contains two key components, CCR4 and CAF1, which are the main cytoplasmic deadenylases in eukaryotic cells. Expression of OsCAF1B was tightly coupled with low-temperature exposure. In the present study, we investigated the function of OsCAF1B in rice by characterizing the molecular and physiological responses to cold stress in OsCAF1B overexpression lines and dominant-negative mutant lines. Our results demonstrate that OsCAF1B plays an important role in growth and development of rice seedlings at low temperatures. Rice is a tropical and subtropical crop that is sensitive to low temperature, and activates a complex gene regulatory network in response to cold stress. Poly(A) tail shortening, also termed deadenylation, is the rate-limiting step of mRNA degradation in eukaryotic cells. CCR4-associated factor 1 (CAF1) proteins are important enzymes for catalysis of mRNA deadenylation in eukaryotes. In the present study, the role of a rice cold-induced CAF1, OsCAF1B, in adaptation of rice plants to low-temperature stress was investigated. Expression of OsCAF1B was closely linked with low-temperature exposure. The increased survival percentage and reduced electrolyte leakage exhibited by OsCAF1B overexpression transgenic lines subjected to cold stress indicate that OsCAF1B plays a positive role in rice growth under low ambient temperature. The enhancement of cold tolerance by OsCAF1B in transgenic rice seedlings involved OsCAF1B deadenylase gene expression, and was associated with elevated expression of late-response cold-related transcription factor genes. In addition, the expression level of OsCAF1B was higher in a cold-tolerant japonica rice cultivar than in a cold-sensitive indica rice cultivar. The results reveal a hitherto undiscovered function of OsCAF1B deadenylase gene expression, which is required for adaptation to cold stress in rice.

DEAD-Box RNA Helicase 42 Plays a Critical Role in Pre-mRNA Splicing under Cold Stress.

Low temperature is an important environmental stress that adversely affects rice (Oryza sativa) growth and productivity. Splicing of pre-mRNA is a crucial posttranscriptional regulatory step in gene expression in plants and is sensitive to temperature. DEAD-box RNA helicases belong to an RNA helicase family involved in the rearrangement of ribonucleoprotein complexes and the modification of RNA structure and are therefore involved in all aspects of RNA metabolism. In this study, we demonstrate that the rate of pre-mRNA splicing is reduced in rice at low temperatures and that the DEAD-box RNA Helicase42 (OsRH42) is necessary to support effective splicing of pre-mRNA during mRNA maturation at low temperatures. OsRH42 expression is tightly coupled to temperature fluctuation, and OsRH42 is localized in the splicing speckles and interacts directly with U2 small nuclear RNA. Retarded pre-mRNA splicing and plant growth defects were exhibited by OsRH42-knockdown transgenic lines at low temperatures, thus indicating that OsRH42 performs an essential role in ensuring accurate pre-mRNA splicing and normal plant growth under low ambient temperature. Unexpectedly, our results show that OsRH42 overexpression significantly disrupts the pre-mRNA splicing pathway, causing retarded plant growth and reducing plant cold tolerance. Combined, these results indicate that accurate control of OsRH42 homeostasis is essential for rice plants to respond to changes in ambient temperature. In addition, our study presents the molecular mechanism of DEAD-box RNA helicase function in pre-mRNA splicing, which is required for adaptation to cold stress in rice.

A CCR4 Association Factor 1, OsCAF1B, Participates in the αAmy3 mRNA Poly(A) Tail Shortening and Plays a Role in Germination and Seedling Growth.

Poly(A) tail (PAT) shortening, also termed deadenylation, is the rate-limiting step of mRNA degradation in eukaryotic cells. The carbon catabolite repressor 4-associated factor 1s (CAF1s) were shown to be one of the major enzymes for catalyzing mRNA deadenylation in yeast and mammalian cells. However, the functions of CAF1 proteins in plants are poorly understood. Herein, a sugar-upregulated CAF1 gene, OsCAF1B, is investigated in rice. Using gain-of-function and dominant-negative mutation analysis, we show that overexpression of OsCAF1B resulted in an accelerated α-amylase gene (αAmy3) mRNA degradation phenomenon, while ectopic expression of a form of OsCAF1B that had lost its deadenylase activity resulted in a delayed αAmy3 mRNA degradation phenomenon in transgenic rice cells. The change in αAmy3 mRNA degradation in transgenic rice is associated with the altered lengths of the αAmy3 mRNA PAT, indicating that OsCAF1B acts as a negative regulator of αAmy3 mRNA stability in rice. Additionally, we found that overexpression of OsCAF1B retards seed germination and seedling growth. These findings indicate that OsCAF1B participates in sugar-induced αAmy3 mRNA degradation and deadenylation and acts a negative factor for germination and seedling development.

Novel interaction between CCR4 and CAF1 in rice CCR4-NOT deadenylase complex.

KEY MESSAGE: Rice is an important crop in the world. However, little is known about rice mRNA deadenylation, which is an important regulation step of gene expression at the post-transcriptional level. The CCR4-NOT1 complex contains two key components, CCR4 and CAF1, which are the main cytoplasmic deadenylases in eukaryotic cells. In yeast and humans, CCR4 can interact with CAF1 via its N-terminal LRR domain. However, no CCR4 protein containing N-terminal LRR motifs have been found in plants. In this manuscript, we demonstrate a novel pattern of interaction between OsCCR4 and OsCAF1 in the rice CCR4-NOT complex, and that OsCAF1 acts as a bridge between OsCCR4 and OsNOT1 in this complex. Our results revealed that the Mynd-like domain at the N-terminus of rice CCR4 proteins and the PXLXP motif at the rice CAF1 N-terminus play critical roles in OsCCR4-OsCAF1 interaction. Deadenylation, also called poly(A) tail shortening, is the first rate-limiting step in general cytoplasmic mRNA degradation in eukaryotic cells. Carbon catabolite repressor (CCR)4 and CCR4-associated factor (CAF)1 in the CCR4-NOT complex function in mRNA poly(A) tail shortening. CCR4s contain N-terminal leucine-rich repeat (LRR) motifs that interact with CAF1s in yeast, fruit fly and mammals. In silico analysis has not identified any plant CCR4 proteins that contain LRR motifs. Here, two rice CCR4 homologous genes, OsCCR4a and OsCCR4b, were identified. The isolated recombinant exonuclease-endonuclease-phosphatase domain of OsCCR4a and OsCCR4b exhibited 3'-5' exonuclease activity in vitro, and point mutation of a catalytic residue in this domain disrupted the deadenylase activity. Both OsCCR4a and OsCCR4b fluorescent fusion proteins were localized in the rice cytoplasm and nucleus, and both associated with processing bodies via their N-terminus. Binding analyses showed that OsCCR4a and OsCCR4b directly interacted with three rice CAF1 family members: OsCAF1A, OsCAF1G and OsCAF1H. The zf-MYND-like domain at the N terminus of rice CCR4 and the PXLXP motif of rice CAF1 play critical roles in OsCCR4-OsCAF1 interaction. OsCAF1 proteins, but not OsCCR4 proteins, can interact with the MIG4G domain of rice OsNOT1. Our studies thus reveal a hitherto undiscovered novel interaction pattern that connects OsCCR4 and OsCAF1 in the rice CCR4-NOT complex.

Two MYB-related transcription factors play opposite roles in sugar signaling in Arabidopsis.

KEY MESSAGE: Sugar regulation of gene expression has profound effects at all stages of the plant life cycle. Although regulation at the transcriptional level is one of the most prominent mechanisms by which gene expression is regulated, only a few transcription factors have been identified and demonstrated to be involved in the regulation of sugar-regulated gene expression. OsMYBS1, an R1/2-type MYB transcription factor, has been demonstrated to be involved in sugar- and hormone-regulated α-amylase gene expression in rice. Arabidopsis contains two OsMYBS1 homologs. In the present study, we investigate MYBS1 and MYBS2 in sugar signaling in Arabidopsis. Our results indicate that MYBS1 and MYBS2 play opposite roles in regulating glucose and ABA signaling in Arabidopsis during seed germination and early seedling development. MYB proteins have been classified into four subfamilies: R2R3-MYB, R1/2-MYB, 3R-MYB, and 4R-MYB. An R1/2-type MYB transcription factor, OsMYBS1, has been demonstrated to be involved in sugar- and hormone-regulated α-amylase genes expression in rice. In this study, two genes homologous to OsMYBS1, MYBS1 and MYBS2, were investigated in Arabidopsis. Subcellular localization analysis showed that MYBS1 and MYBS2 were localized in the nucleus. Rice embryo transient expression assays indicated that both MYBS1 and MYBS2 could recognize the sugar response element, TA-box, in the promoter and induced promoter activity. mybs1 mutant exhibited hypersensitivity to glucose, whereas mybs2 seedlings were hyposensitive to it. MYBS1 and MYBS2 are involved in the control of glucose-responsive gene expression, as the mybs1 mutant displayed increased expression of a hexokinase gene (HXK1), chlorophyll a/b-binding protein gene (CAB1), ADP-glucose pyrophosphorylase gene (APL3), and chalcone synthase gene (CHS), whereas the mybs2 mutant exhibited decreased expression of these genes. mybs1 also showed an enhanced response to abscisic acid (ABA) in the seed germination and seedling growth stages, while mybs2 showed reduced responses. The ABA biosynthesis inhibitor fluridone rescued the mybs1 glucose-hypersensitive phenotype. Moreover, the mRNA levels of three ABA biosynthesis genes, ABA1, NCED9, and AAO3, and three ABA signaling genes, ABI3, ABI4, and ABI5, were increased upon glucose treatment of mybs1 seedlings, but were decreased in mybs2 seedlings. These results indicate that MYBS1 and MYBS2 play opposite roles in regulating glucose and ABA signaling in Arabidopsis during seed germination and early seedling development.

SnRK1A-interacting negative regulators modulate the nutrient starvation signaling sensor SnRK1 in source-sink communication in cereal seedlings under abiotic stress.

In plants, source-sink communication plays a pivotal role in crop productivity, yet the underlying regulatory mechanisms are largely unknown. The SnRK1A protein kinase and transcription factor MYBS1 regulate the sugar starvation signaling pathway during seedling growth in cereals. Here, we identified plant-specific SnRK1A-interacting negative regulators (SKINs). SKINs antagonize the function of SnRK1A, and the highly conserved GKSKSF domain is essential for SKINs to function as repressors. Overexpression of SKINs inhibits the expression of MYBS1 and hydrolases essential for mobilization of nutrient reserves in the endosperm, leading to inhibition of seedling growth. The expression of SKINs is highly inducible by drought and moderately by various stresses, which is likely related to the abscisic acid (ABA)-mediated repression of SnRK1A under stress. Overexpression of SKINs enhances ABA sensitivity for inhibition of seedling growth. ABA promotes the interaction between SnRK1A and SKINs and shifts the localization of SKINs from the nucleus to the cytoplasm, where it binds SnRK1A and prevents SnRK1A and MYBS1 from entering the nucleus. Our findings demonstrate that SnRK1A plays a key role regulating source-sink communication during seedling growth. Under abiotic stress, SKINs antagonize the function of SnRK1A, which is likely a key factor restricting seedling vigor.

A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis.

DEAD-box RNA helicases belong to an RNA helicase family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation and RNA decay. This study investigated a DEAD-box RNA helicase, AtRH7/PRH75, in Arabidopsis. Expression of AtRH7/PRH75 was ubiquitous; however, the levels of mRNA accumulation were increased in cell division regions and were induced by cold stress. The phenotypes of two allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled auxin-related developmental defects that were exhibited in several ribosomal protein mutants, and were more severe under cold stress. Northern blot and circular reverse transcription-PCR (RT-PCR) analyses indicated that unprocessed 18S pre-rRNAs accumulated in the atrh7 mutants. The atrh7 mutants were hyposensitive to the antibiotic streptomycin, which targets ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. In addition, the atrh7-2 and atrh7-3 mutants displayed cold hypersensitivity and decreased expression of CBF1, CBF2 and CBF3, which might be responsible for the cold intolerance. The present study indicated that AtRH7 participates in rRNA biogenesis and is also involved in plant development and cold tolerance in Arabidopsis.

Two highly similar DEAD box proteins, OsRH2 and OsRH34, homologous to eukaryotic initiation factor 4AIII, play roles of the exon junction complex in regulating growth and development in rice.

BACKGROUND: The exon junction complex (EJC), which contains four core components, eukaryotic initiation factor 4AIII (eIF4AIII), MAGO/NASHI (MAGO), Y14/Tsunagi/RNA-binding protein 8A, and Barentsz/Metastatic lymph node 51, is formed in both nucleus and cytoplasm, and plays important roles in gene expression. Genes encoding core EJC components have been found in plants, including rice. Currently, the functional characterizations of MAGO and Y14 homologs have been demonstrated in rice. However, it is still unknown whether eIF4AIII is essential for the functional EJC in rice. RESULTS: This study investigated two DEAD box RNA helicases, OsRH2 and OsRH34, which are homologous to eIF4AIII, in rice. Amino acid sequence analysis indicated that OsRH2 and OsRH34 had 99 % identity and 100 % similarity, and their gene expression patterns were similar in various rice tissues, but the level of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings. From bimolecular fluorescence complementation results, OsRH2 and OsRH34 interacted physically with OsMAGO1 and OsY14b, respectively, which indicated that both of OsRH2 and OsRH34 were core components of the EJC in rice. To study the biological roles of OsRH2 and OsRH34 in rice, transgenic rice plants were generated by RNA interference. The phenotypes of three independent OsRH2 and OsRH34 double-knockdown transgenic lines included dwarfism, a short internode distance, reproductive delay, defective embryonic development, and a low seed setting rate. These phenotypes resembled those of mutants with gibberellin-related developmental defects. In addition, the OsRH2 and OsRH34 double-knockdown transgenic lines exhibited the accumulation of unspliced rice UNDEVELOPED TAPETUM 1 mRNA. CONCLUSIONS: Rice contains two eIF4AIII paralogous genes, OsRH2 and OsRH34. The abundance of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings, suggesting that the OsRH2 is major eIF4AIII in rice. Both OsRH2 and OsRH34 are core components of the EJC, and participate in regulating of plant height, pollen, and seed development in rice.

A single-repeat MYB transcription repressor, MYBH, participates in regulation of leaf senescence in Arabidopsis.

Leaf senescence, the final stage of leaf development, is regulated tightly by endogenous and environmental signals. MYBS3, a MYB transcription factor with a single DNA-binding domain, mediates sugar signaling in rice. Here we report that an Arabidopsis MYBS3 homolog, MYBH, plays a critical role in developmentally regulated and dark-induced leaf senescence by repressing transcription. Expression of MYBH was enhanced in older and dark-treated leaves. Gain- and loss-of-function analysis indicated that MYBH was involved in the onset of leaf senescence. Plants constitutively overexpressing MYBH underwent premature leaf senescence and showed enhanced expression of leaf senescence marker genes. In contrast, the MYBH mutant line, mybh-1, exhibited a delayed-senescence phenotype. The EAR repression domain was required for MYBH-regulated leaf senescence. Overexpression and knockout of MYBH repressed and enhanced auxin-responsive gene expression, respectively. MYBH repressed the auxin-amido synthase genes DFL1/GH3.6 and DFL2/GH3.10, which regulate auxin homoeostasis, by binding directly to the TA box in each of their regulatory regions. An auxin-responsive phenotype was enhanced in MYBH overexpression lines and reduced in mybh knockout lines. Overexpression of MYBH enhanced gene expression of SAUR36, an auxin-promoted leaf senescence key regulator, and accelerated ABA- and ethylene-induced leaf senescence in transgenic Arabidopsis plants. Our results suggest that the role of MYBH in controlling auxin homeostasis accounts for its capacity to participate in regulation of age- and darkness-induced leaf senescence in Arabidopsis.

A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty.

Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water-use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.

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.

Divergence of the expression and subcellular localization of CCR4-associated factor 1 (CAF1) deadenylase proteins in Oryza sativa.

Deadenylation, also called poly(A) tail shortening, is the first, rate-limiting step in the general cytoplasmic mRNA degradation in eukaryotic cells. The CCR4-NOT complex, containing the two key components carbon catabolite repressor 4 (CCR4) and CCR4-associated factor 1 (CAF1), is a major player in deadenylation. CAF1 belongs to the RNase D group in the DEDD superfamily, and is a protein conserved through evolution from yeast to humans and plants. Every higher plant, including Arabidopsis and rice, contains a CAF1 multigene family. In this study, we identified and cloned four OsCAF1 genes (OsCAF1A, OsCAF1B, OsCAF1G, and OsCAF1H) from rice. Four recombinant OsCAF1 proteins, rOsCAF1A, rOsCAF1B, rOsCAF1G, and rOsCAF1H, all exhibited 3'-5' exonuclease activity in vitro. Point mutations in the catalytic residues of each analyzed recombinant OsCAF1 proteins were shown to disrupt deadenylase activity. OsCAF1A and OsCAF1G mRNA were found to be abundant in the leaves of mature plants. Two types of OsCAF1B mRNA transcript were detected in an inverse expression pattern in various tissues. OsCAF1B was transient, induced by drought, cold, abscisic acid, and wounding treatments. OsCAF1H mRNA was not detected either under normal conditions or during most stress treatments, but only accumulated during heat stress. Four OsCAF1-reporter fusion proteins were localized in both the cytoplasm and nucleus. In addition, when green fluorescent protein fused with OsCAF1B, OsCAF1G, and OsCAF1H, respectively, fluorescent spots were observed in the nucleolus. OsCAF1B fluorescent fusion proteins were located in discrete cytoplasmic foci and fibers. We present evidences that OsCAF1B colocalizes with AtXRN4, a processing body marker, and AtKSS12, a microtubules maker, indicating that OsCAF1B is a component of the plant P-body and associate with microtubules. Our findings provide biochemical evidence that OsCAF1 proteins may be involved in the deadenylation in rice. The unique expression patterns of each OsCAF1 were observed in various tissues when undergoing abiotic stress treatments, implying that each CAF1 gene in rice plays a specific role in the development and stress response of a plant.

Expression of a gene encoding a rice RING zinc-finger protein, OsRZFP34, enhances stomata opening.

By oligo microarray expression profiling, we identified a rice RING zinc-finger protein (RZFP), OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. As compared with the wild type, rice and Arabidopsis with OsRZFP34 overexpression showed increased relative stomata opening even with ABA treatment. Furthermore, loss-of-function mutation of OsRZFP34 and AtRZFP34 (At5g22920), an OsRZFP34 homolog in Arabidopsis, decreased relative stomata aperture under nonstress control conditions. Expressing OsRZFP34 in atrzfp34 reverted the mutant phenotype to normal, which indicates a conserved molecular function between OsRZFP34 and AtRZFP34. Analysis of water loss and leaf temperature under stress conditions revealed a higher evaporation rate and cooling effect in OsRZFP34-overexpressing Arabidopsis and rice than the wild type, atrzfp34 and osrzfp34. Thus, stomata opening, enhanced leaf cooling, and ABA insensitivity was conserved with OsRZFP34 expression. Transcription profiling of transgenic rice overexpressing OsRZFP34 revealed many genes involved in OsRZFP34-mediated stomatal movement. Several genes upregulated or downregulated in OsRZFP34-overexpressing plants were previously implicated in Ca(2+) sensing, K(+) regulator, and ABA response. We suggest that OsRZFP34 may modulate these genes to control stomata opening.

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.

Improving pharmaceutical protein production in Oryza sativa.

Application of plant expression systems in the production of recombinant proteins has several advantages, such as low maintenance cost, absence of human pathogens, and possession of complex post-translational glycosylation capabilities. Plants have been successfully used to produce recombinant cytokines, vaccines, antibodies, and other proteins, and rice (Oryza sativa) is a potential plant used as recombinant protein expression system. After successful transformation, transgenic rice cells can be either regenerated into whole plants or grown as cell cultures that can be upscaled into bioreactors. This review summarizes recent advances in the production of different recombinant protein produced in rice and describes their production methods as well as methods to improve protein yield and quality. Glycosylation and its impact in plant development and protein production are discussed, and several methods of improving yield and quality that have not been incorporated in rice expression systems are also proposed. Finally, different bioreactor options are explored and their advantages are analyzed.

A plant protein farnesylation system in prokaryotic cells reveals Arabidopsis AtJ3 produced and farnesylated in E. coli maintains its function of protecting proteins from heat inactivation.

BACKGROUND: Protein farnesylation involves the addition of a 15-carbon polyunsaturated farnesyl group to proteins whose C-terminus ends with a CaaX motif. This post-translational protein modification is catalyzed by a heterodimeric protein, i.e., farnesyltransferase (PFT), which is composed of an α and a ß subunit. Protein farnesylation in plants is of great interest because of its important roles in the regulation of plant development, responses to environmental stresses, and defense against pathogens. The methods traditionally used to verify whether a protein is farnesylated often require a specific antibody and involve isotope labeling, a tedious and time-consuming process that poses hazardous risks. RESULTS: Since protein farnesylation does not occur in prokaryotic cells, we co-expressed a known PFT substrate (i.e., AtJ3) and both the α and ß subunits of Arabidopsis PFT in E. coli in this study. Farnesylation of AtJ3 was detected using electrophoretic mobility using SDS-PAGE and confirmed using mass spectrometry. AtJ3 is a member of the heat shock protein 40 family and interacts with Arabidopsis HSP70 to protect plant proteins from heat-stress-induced denaturation. A luciferase-based protein denaturation assay demonstrated that farnesylated AtJ3 isolated from E. coli maintained this ability. Interestingly, farnesylated AtJ3 interacted with E. coli HSP70 as well and enhanced the thermotolerance of E. coli. Meanwhile, AtFP3, another known PFT substrate, was farnesylated when co-expressed with AtPFTα and AtPFTß in E. coli. Moreover, using the same strategy to co-express rice PFT α and ß subunit and a potential PFT target, it was confirmed that OsDjA4, a homolog of AtJ3, was farnesylated. CONCLUSION: We developed a protein farnesylation system for E. coli and demonstrated its applicability and practicality in producing functional farnesylated proteins from both mono- and dicotyledonous plants.