Rice Cells Research Articles

The codon optimised gene produces an active human basic fibroblastic growth factor in rice cell suspension culture

The coding sequence of human basic fibroblast growth factor (hbFGF) was optimised for expression in rice. An expression cassette was constructed by fusing the PCR-amplified RAmy3D promoter, along with its 5’UTR, 3’UTR, and terminator sequences, to the codon-optimised hbFGF sequence. This cassette was inserted into the pCAMBIA1304 shuttle vector, which also contained the RAmy3D signal peptide. Agrobacterium tumefaciens strain LBA 4404 was used to transform rice callus. Among the transformed lines, the callus expressing the highest level of bFGF (38.1 mg/kg fresh weight) was identified via ELISA and selected for establishing a cell suspension culture. Expression and secretion of the recombinant bFGF into the culture medium were observed three days after incubating the transgenic rice cells in sucrose-free medium. The presence of recombinant bFGF was confirmed through Western blot and SDS-PAGE analyses. Furthermore, the rice-derived bFGF effectively stimulated the proliferation of NIH/3T3 cells, demonstrating a comparable biological activity to that of commercial bFGF.

Basic design of artificial membrane-less organelles using condensation-prone proteins in plant cells

Membrane-less organelles, formed by the condensation of biomolecules, play a pivotal role in eukaryotes. Artificial membrane-less organelles and condensates are effective tools for the creation of new cellular functions. However, it is poorly understood how to control the properties that affect condensate function, particularly in plants. Here, we report the construction of model artificial condensates using the condensation-prone proteins OsJAZ2 and AtFCA in a transient assay using rice (Oryza sativa) cells, and how condensate properties, such as subcellular localization, protein mobility, and size can be altered. We showed that proteins of interest can be recruited to condensates using nanobodies or chemically induced dimerization. Furthermore, by combining two types of condensation-prone proteins, we demonstrated that artificial hybrid condensates with heterogeneous material properties could be constructed. Finally, we showed that modified artificial condensates can be constructed in transgenic Arabidopsis thaliana plants. These results provide a framework for the basic design of synthetic membrane-less organelles in plants.

PAM-relaxed and temperature-tolerant CRISPR-Mb3Cas12a single transcript unit systems for efficient singular and multiplexed genome editing in rice, maize, and tomato.

Class 2 Type V-A CRISPR-Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T-rich genomic regions, complementing the widely used CRISPR-Cas9 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs-Mb3Cas12a, PrCas12a, and HkCas12a-in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine-tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature-tolerant variants: Mb3Cas12a-R (D172R) and Mb3Cas12a-RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a-RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a-RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T0 lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications.

OsHRZ1 negatively regulates rice resistant to Magnaporthe oryzae infection by targeting OsVOZ2.

Rice blast disease caused by Magnaporthe oryzae significantly reduces yield production. Blast resistance is closely associated with iron (Fe) status, but the mechanistic basis linking iron status to immune function in rice remains largely unknown. Here, iron-binding haemerythrin RING ubiquitin ligases OsHRZ1 was confirmed to play key roles in iron-mediated rice blast resistance. The expression of OsHRZ1 was suppressed by M. oryzae inoculation and high iron treatment. Both mutants of OsHRZ1 enhanced rice resistance to M. oryzae. OsPR1a was up-regulated in OsHRZ1 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and Co-IP assay results indicated that OsHRZ1 interacts with Vascular Plant One Zinc Finger 2 (OsVOZ2) in the nucleus. Additionally, the vitro ubiquitination assay indicated that OsHRZ1 can ubiquitinate OsVOZ2 and mediate the degradation of OsVOZ2. The mutants of OsVOZ2 showed reduced resistance to M. oryzae and down-regulated the expression of OsPR1a. Yeast one-hybrid, EMSA, and dual-luciferase reporter assay results indicated that OsVOZ2 directly binds to the promoter of OsPR1a, activating its expression. In summary, OsHRZ1 plays an important role in rice disease resistance by mediated degradation of OsVOZ2 thus shaping PR gene expression dynamics in rice cells. This highlights an important link between iron signaling and rice pathogen defenses.

The invertase gene PWIN1 confers chilling tolerance of rice at the booting stage via mediating pollen development.

Pollen fertility is a primary regulator of grain yield and is highly susceptible to cold and other environmental stress. We revealed the roles of rice cell wall invertase gene PWIN1 in pollen development and chilling tolerance. We uncovered its preferential expression in microspores and bicellular pollen and identified its knock-down and knock-out mutants. pwin1 mutants produced a higher proportion of abnormal pollen than wild-type plants. The contents of sucrose, glucose, and fructose were increased, while ATP content and primary metabolism activity were reduced in the mutant pollen. Furthermore, the loss of function of PWIN1 coincided with an increase in SnRK1 activity and a decrease in TOR activity. Under chilling conditions, pwin1 mutants displayed significantly reduced pollen viability and seed-setting rate, while overexpressing PWIN1 notably increased pollen viability and seed-setting rate as compared with the wild-type, indicating that PWIN1 is essential for rice pollen development and grain yield under cold stress. This study provides insights into the molecular mechanisms underlying rice pollen fertility during chilling stress, and a new module to improve chilling tolerance of rice at the booting stage by molecular design.

Carbohydrate-active enzymes involved in rice cell wall metabolism.

Plant cell walls are complex, multifunctional structures, built up of polysaccharides and proteins. The configuration and abundance of cell wall constituents determine cellular elongation and plant growth. The emphasis of this review is on rice, a staple crop with economic importance, serving as model for grasses/cereals. Recent advancements have contributed to a better understanding of the grass/cereal cell wall. This review brings together the current knowledge about the organisation and metabolism of the rice cell wall, and addresses gaps and missing information connected to the cell wall of rice and the enzymes involved. Several cell wall fractions, including cellulose, mixed-linkage glucans and glucuronoarabinoxylans, are well-understood in rice and other grasses/grains. Conversely, there are still open questions and missing links when it comes down to xyloglucans, glucomannans, pectin, lignin and arabinogalactan proteins. There is still a large and untapped potential to identify carbohydrate-active enzymes (CAZymes), to characterise their activity and to elucidate their involvement in the metabolism of the mentioned cell wall fractions. With this review, we demonstrate the current state and demarcate the research areas with potential for further investigations.

Bacillus amyloliquefaciens LM-1 Affects Multiple Cell Biological Processes in Magnaporthe oryzae to Suppress Rice Blast.

Magnaporthe oryzae, one of the most destructive rice pathogens, causes significant losses during the rice harvest every year. Bacillus amyloliquefaciens has been explored in many crops as a potential biocontrol agent. However, the mechanisms of B. amyloliquefaciens controled rice blast are not fully understood. Here, a biocontrol strain LM-1, isolated from a contaminated medium, was identified as B. amyloliquefaciens using morphological observation, physiological and biochemical tests, and 16S rDNA sequencing. LM-1 inhibited the growth and pathogenicity of M. oryzae and Bipolaris oryzae (Breda de Haan) Shoem. The mycelia of M. oryzae co-cultured with LM-1 were enlarged and broken by fluorescence microscopy using calcofluor white. LM-1 inhibited the mycelia of M. oryzae from producing conidia. Genes itu, srf, and fenB were detected in LM-1. Furthermore, the supernatant of LM-1 interfered with the appressorium formation of M. oryzae, blocked conidial cell death, and reduced autophagy degradation but did not affect the normal germination of rice seeds and seeding growth. Additionally, we observed hypersensitivity reactions, reactive oxygen species, and iron accumulation reduction in rice cells inoculated with supernatant. Our study reveals that LM-1 has a control effect on rice blast and affects cell wall integrity, sporulation, appressorium formation, cell death, and autophagy.

Comparing the Mechanical Properties of Rice Cells and Protoplasts under PEG6000 Drought Stress Using Double Resonator Piezoelectric Cytometry.

Plant cells' ability to withstand abiotic stress is strongly linked to modifications in their mechanical characteristics. Nevertheless, the lack of a workable method for consistently tracking plant cells' mechanical properties severely restricts our comprehension of the mechanical alterations in plant cells under stress. In this study, we used the Double Resonator Piezoelectric Cytometry (DRPC) method to dynamically and non-invasively track changes in the surface stress (ΔS) generated and viscoelasticity (storage modulus G' and loss modulus G″) of protoplasts and suspension cells of rice under a drought stress of 5-25% PEG6000. The findings demonstrate that rice suspension cells and protoplasts react mechanically differently to 5-15% PEG6000 stress, implying distinct resistance mechanisms. However, neither of them can withstand 25% PEG6000 stress; they respond mechanically similarly to 25% PEG6000 stress. The results of DRPC are further corroborated by the morphological alterations of rice cells and protoplasts observed under an optical microscope. To sum up, the DRPC technique functions as a precise cellular mechanical sensor and offers novel research tools for the evaluation of plant cell adversity and differentiating between the mechanical reactions of cells and protoplasts under abiotic stress.

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.

High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

A pharmacological approach to investigating effector translocation in rice-Magnaporthe oryzae interactions

ABSTRACT Fungal pathogens deliver effector proteins into living plant cells to suppress plant immunity and control plant processes that are needed for infection. During plant infection, the devastating rice blast fungus, Magnaporthe oryzae, forms the specialized biotrophic interfacial complex (BIC), which is essential for effector translocation. Cytoplasmic effectors are first focally secreted into BICs, and subsequently packaged into dynamic membranous effector compartments (MECs), then translocated via clathrin-mediated endocytosis (CME) into the host cytoplasm. This study demonstrates that clathrin-heavy chain inhibitors endosidin-9 (ES9) and endosidin-9–17 (ES9–17) blocked the internalization of the fluorescently labeled effectors Bas1 and Pwl2 in rice cells, leading to swollen BICs lacking MECs. In contrast, ES9–17 treatment had no impact on the localization pattern of the apoplastic effector Bas4. This study provides further evidence that cytoplasmic effector translocation occurs by CME in BICs, suggesting a potential role for M. oryzae effectors in co-opting plant endocytosis.

Alginate Oligosaccharides Alleviate Salt Stress in Rice Seedlings by Regulating Cell Wall Metabolism to Maintain Cell Wall Structure and Improve Lodging Resistance.

Salt stress is one of the major abiotic stresses that damage the structure and composition of cell walls. Alginate oligosaccharides (AOS) have been advocated to significantly improve plant stress tolerance. The metabolic mechanism by which AOS induces salt tolerance in rice cell walls remains unclear. Here, we report the impact of AOS foliar application on the cell wall composition of rice seedlings using the salt-tolerant rice variety FL478 and the salt-sensitive variety IR29. Data revealed that salt stress decreased biomass, stem basal width, stem breaking strength, and lodging resistance; however, it increased cell wall thickness. In leaves, exogenous AOS up-regulated the expression level of OSCESA8, increased abscisic acid (ABA) and brassinosteroids (BR) content, and increased β-galacturonic activity, polygalacturonase activity, xylanase activity, laccase activity, biomass, and cellulose content. Moreover, AOS down-regulated the expression levels of OSMYB46 and OSIRX10 and decreased cell wall hemicellulose, pectin, and lignin content to maintain cell wall stability under salt stress. In stems, AOS increased phenylalamine ammonia-lyase and tyrosine ammonia-lyase activities, while decreasing cellulase, laccase, and β-glucanase activities. Furthermore, AOS improved the biomass and stem basal width and also enhanced the cellulose, pectin, and lignin content of the stem, As a result, increased resistance to stem breakage strength and alleviated salt stress-induced damage, thus enhancing the lodging resistance. Under salt stress, AOS regulates phytohormones and modifies cellulose, hemicellulose, lignin, and pectin metabolism to maintain cell wall structure and improve stem resistance to lodging. This study aims to alleviate salt stress damage to rice cell walls, enhance resistance to lodging, and improve salt tolerance in rice by exogenous application of AOS.

Phytoalexin sakuranetin attenuates endocytosis and enhances resistance to rice blast

Phytoalexin sakuranetin functions in resistance against rice blast. However, the mechanisms underlying the effects of sakuranetin remains elusive. Here, we report that rice lines expressing resistance (R) genes were found to contain high levels of sakuranetin, which correlates with attenuated endocytic trafficking of plasma membrane (PM) proteins. Exogenous and endogenous sakuranetin attenuates the endocytosis of various PM proteins and the fungal effector PWL2. Moreover, accumulation of the avirulence protein AvrCO39, resulting from uptake into rice cells by Magnaporthe oryzae, was reduced following treatment with sakuranetin. Pharmacological manipulation of clathrin-mediated endocytic (CME) suggests that this pathway is targeted by sakuranetin. Indeed, attenuation of CME by sakuranetin is sufficient to convey resistance against rice blast. Our data reveals a mechanism of rice against M. oryzae by increasing sakuranetin levels and repressing the CME of pathogen effectors, which is distinct from the action of many R genes that mainly function by modulating transcription.

Biochemical mechanisms underlying iron plaque-mediated phosphorus accumulation and uptake in rice roots

The iron oxyhydroxides of iron plaque on the surface of rice root are crucial for the uptake of nutrition elements, especially phosphorus (P), but the effects of iron oxyhydroxides of iron plaque on the accumulation and uptake of P remain largely unknown. In this study, we investigated the regulatory mechanism of iron plaque on P uptake in rice via hydroponics of whole plant and simulation of iron oxyhydroxides-coated suspension cells in rice. The hydroponic experiment results showed that the presence of iron plaque increased the P content in rice shoots. The simulation experiment results further confirmed that after iron plaque coating, the P contents in the whole cell and on the cell wall were significantly increased from 5.16 mg/g and 2.73 mg/g to 8.85 mg/g and 5.27 mg/g, respectively. In addition, our data also showed that iron plaque coating led to an increase in cell surface potentials from −380 ± 40 mV to −200 ± 30 mV, thus promoting the adsorption of more P. Taken together, this study demonstrated that the iron plaque coating increased the surface potential of the cells, thus enhancing cellular P enrichment, eventually promoting P efficient adsorption in rice. Deciphering these regulatory mechanisms provide an insight into P biogeochemical cycling at the soil-plant interface and offer theoretical basis and practical references for the improvement of P bioavailability in rice production.

Biochemical Characterization of Rice Xylan Biosynthetic Enzymes in Determining Xylan Chain Elongation and Substitutions.

Grass xylan consists of a linear chain of β-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or glucuronic acid (GlcA)/methylglucuronic acid (MeGlcA) residues, and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remains elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto (Araf)-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution patterns of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3’ splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 μg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter–signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins.Key points• The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2• The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins• The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity

Lysine acetylation of histone acetyltransferase adaptor protein ADA2 is a mechanism of metabolic control of chromatin modification in plants.

Histone acetylation is a predominant active chromatin mark deposited by histone acetyltransferases (HATs) that transfer the acetyl group from acetyl coenzyme A (acetyl-CoA) to lysine ε-amino groups in histones. GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) is one of the best-characterized HATs and functions in association with several adaptor proteins such as ADA2 within multiprotein HAT complexes. ADA2-GCN5 interaction increases GCN5 binding to acetyl-CoA and stimulates its HAT activity. It remains unclear whether the HAT activity of GCN5 (which acetylates not only histones but also cellular proteins) is regulated by acetyl-CoA levels, which vary greatly in cells under different metabolic and nutrition conditions. Here we show that the ADA2 protein itself is acetylated by GCN5 in rice cells. Lysine acetylation exposes ADA2 to a specific E3 ubiquitin ligase and reduces its protein stability. In rice plants, ADA2 protein accumulation reversely parallels its lysine acetylation and acetyl-CoA levels, both of which are dynamically regulated under varying growth conditions. Stress-induced ADA2 accumulation could stimulate GCN5 HAT activity to compensate for the reduced acetyl-CoA levels for histone acetylation. These results indicate that ADA2 lysine acetylation that senses cellular acetyl-CoA variations is a mechanism to regulate HAT activity and histone acetylation homeostasis in plants under changing environments.

Hijacking of the methylglyoxal detoxification pathway: a new tactic of Xoo pathogenesis in rice.

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight (BB), has developed a unique strategy to infect rice by hijacking the host's methylglyoxal (MG) detoxification pathway. This results in an over-accumulation of MG, which facilitates tissue colonization and evasion of host's immune responses. While MG role in abiotic stresses is well-documented, its involvement in biotic stresses has not been extensively explored. Recently, Fu et al. (2024) provided the first evidence of MG role in promoting Xoo pathogenesis in rice. This new virulence strategy contributes to the pathogen's remarkable adaptability and survival. In this mechanism of hijacking of MG detoxification pathway, Xoo induces OsWRKY62.1 to inhibit OsGLY II expression, leading to MG overaccumulation in infected rice cells. This excess MG hinders plant cell organelle function, creating a favorable environment for Xoo by compromising the rice defense system. In this article, we have presented our perspectives on how the BB pathogen adapts its virulence mechanisms to infect and cause disease in rice.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+-NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

OsbZIP38/87-mediated activation of OsHXK7 improves the viability of rice cells under hypoxic conditions

Maintenance of energy metabolism is critical for rice (Oryza sativa) tolerance under submerged cultivation. Here, OsHXK7 was the most actively induced hexokinase gene in the embryos of hypoxically germinating rice seeds. Suspension-cultured cells established from seeds of T-DNA null mutants for the OsHXK7 locus did not regrow after 3-d-hypoxic stress and showed increased susceptibility to low-oxygen stress—in terms of viability—and decreased alcoholic fermentation activities compared to those of the wild-type. The promoter element containing the TGACG-motif, a well-known target site for the basic leucine zipper (bZIP) transcription factors, was responsible for sugar regulation of the OsHXK7 promoter activity. Systematic screening of the OsbZIP genes showing the similar expression patterns to that of OsHXK7 in the transcriptomic datasets produced two bZIP genes, OsbZIP38 and 87, belonging to the S1 bZIP subfamily as the candidate for the activator for this gene expression. Gain- and loss-of-function experiments through transient expression assays have demonstrated that these two bZIP proteins are indeed involved in the induction of OsHXK7 expression under starvation or low-energy conditions. Our finding suggests that C/S1 bZIP network-mediated hypoxic deregulation of sugar-responsive genes may work in concert for the molecular adaptation of rice cells to submergence.

Production and characterization of basic fibroblast growth factor protein in rice suspension cultures

The basic fibroblast growth factor (bFGF) plays a major role in cell growth, survival, and other mitogenic activities. General applications of this protein involve tissue repair, morphogenesis, embryonic development, cellular growth, etc. Due to its importance and high demand, research has been done on producing recombinant bFGF in different expression platforms. We have produced bFGF in transgenic rice (Oryza sativa) cells growing in liquid suspension culture. The gene construct consists of the rice alpha-amylase (RAmy) 3D sugar-inducible promotor and rice glutelin secretion signal peptide for controlled production and release of the bFGF protein by manipulating the sugar concentration for batch or semi-continuous bioreactor runs. A thermal shift assay was performed on the purified rice cell produced bFGF (rbFGF) and the results were compared with E. coli derived bFGF. The purified rbFGF protein from rice cells was tested for propagating human-induced pluripotent stem cells (hiPSCs) up to eight passages. Furthermore, the biological activity measured in both crude lysate and purified rbFGF showed a similar pattern of the extracellular signal-regulated kinase (ERK) activity compared to E. coli derived bFGF (control), indicating that rbFGF is active before purification and the protein activity is not inhibited by other components of the lysate. From the current results, the rice-produced rbFGF protein is biologically active and could serve as a viable product for various applications.