RGA1 Negatively Regulates Thermo-tolerance by Affecting Carbohydrate Metabolism and the Energy Supply in Rice.

BACKGROUND: Signal transduction mediated by heterotrimeric G proteins, which comprise the α, ß, and γ subunits, is one of the most important signaling pathways in rice plants. RGA1, which encodes the Gα subunit of the G protein, plays an important role in the response to various types of abiotic stress, including salt, drought, and cold stress. However, the role of RGA1 in the response to heat stress remains unclear. RESULTS: The heat-resistant mutant ett1 (enhanced thermo-tolerance 1) with a new allele of the RGA1 gene was derived from an ethane methyl sulfonate-induced Zhonghua11 mutant. After 45 °C heat stress treatment for 36 h and recovery for 7 d, the survival rate of the ett1 mutants was significantly higher than that of wild-type (WT) plants. The malondialdehyde content was lower, and the maximum fluorescence quantum yield of photosystem II, peroxidase activity, and hsp expression were higher in ett1 mutants than in WT plants after 12 h of exposure to 45 °C. The RNA-sequencing results revealed that the expression of genes involved in the metabolism of carbohydrate, nicotinamide adenine dinucleotide, and energy was up-regulated in ett1 under heat stress. The carbohydrate content and the relative expression of genes involved in sucrose metabolism indicated that carbohydrate metabolism was accelerated in ett1 under heat stress. Energy parameters, including the adenosine triphosphate (ATP) content and the energy charge, were significantly higher in the ett1 mutants than in WT plants under heat stress. Importantly, exogenous glucose can alleviate the damages on rice seedling plants caused by heat stress. CONCLUSION: RGA1 negatively regulates the thermo-tolerance in rice seedling plants through affecting carbohydrate and energy metabolism.

RGA1 alleviates low-light-repressed pollen tube elongation by improving the metabolism and allocation of sugars and energy.

Low-light stress compromises photosynthetic and energy efficiency and leads to spikelet sterility; however, the effect of low-light stress on pollen tube elongation in the pistil remains poorly understood. The gene RGA1, which encodes a Gα-subunit of the heterotrimeric G-protein, enhanced low-light tolerance at anthesis by preventing the cessation of pollen tube elongation in the pistil of rice plants. In this process, marked increases in the activities of acid invertase (INV), sucrose synthase (SUS) and mitochondrial respiratory electron transport chain complexes, as well as the relative expression levels of SUTs (sucrose transporter), SWEETs (sugars will eventually be exported transporters), SUSs, INVs, CINs (cell-wall INV 1), SnRK1A (sucrose-nonfermenting 1-related kinase 1) and SnRK1B, were observed in OE-1 plants. Accordingly, notable increases in contents of ATP and ATPase were presented in OE-1 plants under low-light conditions, while they were decreased in d1 plants. Importantly, INV and ATPase activators (sucrose and Na2 SO3 , respectively) increased spikelet fertility by improving the energy status in the pistil under low-light conditions, and the ATPase inhibitor Na2 VO4 induced spikelet sterility and decreased ATPase activity. These results suggest that RGA1 could alleviate the low-light stress-induced impairment of pollen tube elongation to increase spikelet fertility by promoting sucrose unloading in the pistil and improving the metabolism and allocation of energy.

Abscisic Acid Improves Rice Thermo-Tolerance by Affecting Trehalose Metabolism.

Heat stress that occurs during the flowering stage severely decreases the rice (Oryza sativa L.) seed-setting rate. This damage can be reversed by abscisic acid (ABA), through effects on reactive oxygen species, carbohydrate metabolism, and heat shock proteins, but the exact role of trehalose and ATP in this process remains unclear. Two rice genotypes, namely, Zhefu802 (heat-resistant plant, a recurrent parent) and its near-isogenic line (faded green leaf, Fgl, heat-sensitive plant), were subjected to 38 °C heat stress after being sprayed with ABA or its biosynthetic inhibitor, fluridone (Flu), at the flowering stage. The results showed that exogenous ABA significantly increased the seed-setting rate of rice under heat stress, by 14.31 and 22.40% in Zhefu802 and Fgl, respectively, when compared with the H2O treatment. Similarly, exogenous ABA increased trehalose content, key enzyme activities of trehalose metabolism, ATP content, and F1Fo-ATPase activity. Importantly, the opposite results were observed in plants treated with Flu. Therefore, ABA may improve rice thermo-tolerance by affecting trehalose metabolism and ATP consumption.

Oligomeric Proanthocyanidins Confer Cold Tolerance in Rice through Maintaining Energy Homeostasis.

Oligomeric proanthocyanidins (OPCs) are abundant polyphenols found in foods and botanicals that benefit human health, but our understanding of the functions of OPCs in rice plants is limited, particularly under cold stress. Two rice genotypes, named Zhongzao39 (ZZ39) and its recombinant inbred line RIL82, were subjected to cold stress. More damage was caused to RIL82 by cold stress than to ZZ39 plants. Transcriptome analysis suggested that OPCs were involved in regulating cold tolerance in the two genotypes. A greater increase in OPCs content was detected in ZZ39 than in RIL82 plants under cold stress compared to their respective controls. Exogenous OPCs alleviated cold damage of rice plants by increasing antioxidant capacity. ATPase activity was higher and poly (ADP-ribose) polymerase (PARP) activity was lower under cold stress in ZZ39 than in RIL82 plants. Importantly, improvements in cold tolerance were observed in plants treated with the OPCs and 3-aminobenzamide (PARP inhibitor, 3ab) combination compared to the seedling plants treated with H2O, OPCs, or 3ab alone. Therefore, OPCs increased ATPase activity and inhibited PARP activity to provide sufficient energy for rice seedling plants to develop antioxidant capacity against cold stress.

Acid invertase confers heat tolerance in rice plants by maintaining energy homoeostasis of spikelets.

Heat stress impairs both pollen germination and pollen tube elongation, resulting in pollination failure caused by energy imbalance. Invertase plays a critical role in the maintenance of energy homoeostasis; however, few studies investigated this during heat stress. Two rice cultivars with different heat tolerance, namely, TLY83 (heat tolerant) and LLY722 (heat susceptible), were subjected to heat stress. At anthesis, heat stress significantly decreased spikelet fertility, accompanied by notable reductions in pollen germination on stigma and pollen tube elongation in ovule, especially in LLY722. Acid invertase (INV), rather than sucrose synthase, contributed to sucrose metabolism, which explains the different tolerances of both cultivars. Under heat stress, larger enhancements in NAD(H), ATP, and antioxidant capacity were found in TLY83 compared with LLY722, whereas a sharp reduction in poly(ADP-ribose) polymerase (PARP) activity was found in the former compared with the latter. Importantly, exogenous INV, 3-aminobenzamide (a PARP inhibitor), sucrose, glucose, and fructose significantly increased spikelet fertility under heat stress, where INV activity was enhanced and PARP activity was inhibited. Therefore, INV can balance the energy production and consumption to provide sufficient energy for pollen germination and pollen tube growth under heat stress.

An EGFP-marked recombinant lactobacillus oral tetravalent vaccine constitutively expressing α, ε, ß1, and ß2 toxoids for Clostridium perfringens elicits effective anti-toxins protective immunity.

Clostridium perfringens is a common opportunistic pathogen endangering livestock and poultry breeds. Here, using enhanced green fluorescent protein as screening marker, a recombinant lactobacillus tetravalent vaccine constitutively expressing α, ε, ß1, and ß2 toxoids of C. perfringens was developed, and its immunogenicity in mice was investigated via oral administration. This probiotic vaccine could effectively induce antigen-specific secretory IgA (sIgA)-based mucosal and IgG-based humoral immune responses, and significantly high levels (p< 0.05) of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and IFN-γ were produced in immunized mice. Moreover, lymphoproliferation and percentage of CD4+ and CD8+ T cells significantly increased in mice of the probiotic vaccine group. Challenge experiments were performed in mice with C. perfringens toxinotypes A, C, and D crude toxins to evaluate protection efficiency of the probiotic vaccine, using a commercial inactivated C. perfringens vaccine made by C. perfringens toxinotypes A, C, and D as vaccine control. We observed 80% protection rate in the probiotic vaccine group, which was higher than commercial vaccine group, whereas all mice in control groups died and obvious histopathological changes were observed in liver, spleen, kidney, and intestines of mice. Significantly, we compared the immunogenicity and protection efficiency of lactobacillus constitutive expression system and lactobacillus inducible expression system, and results showed that lactobacillus constitutive expression system has obvious advantages. Our study clearly demonstrated that the probiotics vaccine could effectively induce mucosal, humoral, and cellular immunity, and provide effective protection against C. perfringens toxins, suggesting a promising strategy for the development of oral vaccine against C. perfringens.

Dendritic Cell Targeting of Bovine Viral Diarrhea Virus E2 Protein Expressed by Lactobacillus casei Effectively Induces Antigen-Specific Immune Responses via Oral Vaccination.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer's patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

N-linked glycosylation sites in G protein of infectious hematopoietic necrosis virus (IHNV) affect its virulence and immunogenicity in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis in salmonid fish, resulting in substantial economic losses to the aquaculture industry worldwide. The G protein, which harbors the major antigenic determinants of IHNV, is an envelope glycoprotein that plays an important role in both pathogenicity and immunogenicity of IHNV. Previous studies have demonstrated that changes to viral glycosylation sites may affect replication and immunogenicity, but little is known about the specific contributions of G protein glycosylation to IHNV replication and pathogenicity. In this study, we predicted four N-linked glycosylation sites at position 56, 379, 401, and 438 Asp (N) in G protein, and using a reverse genetics system developed in our laboratory, constructed nine recombinant viruses with single, triple, or quadruple glycosylation site disruptions using alanine substitutions in the following combinations: rIHNV-N56A, rIHNV-N379A, rIHNV-N401A, rIHNV-N438A, rIHNV-N56A-N379A-N401A, rIHNV-N56A-N379A-N438A, rIHNV-N56A-N401A-N438A, rIHNV-N379A-N401A-N438A, and rIHNV-N56A-N379A-N401A-N438A. Our results confirmed that all four asparagines are sites of N-linked glycosylation, and Western blot confirmed that mutation of each predicted N-glycosylation sited impaired glycosylation. Among the nine recombinant IHNVs, replication levels decreased significantly in vitro and in vivo in the triple and quadruple mutants that combined mutation of asparagines 401 and 438, indicating the importance of glycosylation at these sites for efficient replication. Moreover, juvenile rainbow trout mortality after challenge by each of the nine mutants showed that, while eight mutants suffered almost 100% cumulative mortality over 30 days, the mutant with a single alanine substitution at position 438 resulted in cumulative mortality of less than 50% over 30 days. This mutant also elicited specific anti-IHNV IgM production earlier than other mutants, suggesting that glycosylation of asparagine 438 may be important for viral immune escape. In conclusion, our study reveals the effect of G protein glycosylation on the pathogenicity and immunogenicity of IHNV and provides a foundation for developing a live-attenuated vaccine.

Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice.

A premature senescence and death 128 (psd128) mutant was isolated from an ethyl methane sulfonate-induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six-leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence-related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map-based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA-type ATPase with 809 amino-acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype.

CD133+ subpopulation of the HT1080 human fibrosarcoma cell line exhibits cancer stem-like characteristics.

The cancer stem cell (CSC) theory holds that a minority population within tumors possesses stem cell properties of self-renewal and multilineage differentiation capacity and provides the initiating cells from which tumors are derived and sustained. However, verifying the existence of these CSCs has been a significant challenge. The CD133 antigen is a pentaspan membrane glycoprotein proposed to be a CSC marker for cancer-initiating subpopulations in the brain, colon and various other tissues. Here, CD133+ cells were obtained and characterized from the HT1080 cell line to determine the utility of this marker for isolating CSCs from human fibrosarcoma cells. In this study, CD133+ cells were separated from HT1080 cells using magnetic beads and characterized for their proliferation rate and resistance to chemotherapeutic drugs, cisplatin and doxorubicin, by MTS assay. Relative expression of tumor-associated genes Sox2, Oct3/4, Nanog, c-Myc, Bmi-1 and ABCG2 was measured by real-time polymerase chain reaction (PCR). Clonal sphere formation and the ability of CD133+ cells to initiate tumors in BALB/c nude mice was also evaluated. We found that CD133+ cells showed a high proliferation rate, increased resistance to chemotherapy drugs and overexpression of tumor-associated genes compared with these features in CD133- cells. Additionally, CD133+ cells were able to form spherical clusters in serum-free medium with high clonogenic efficiency, indicating a significantly greater tumor-initiating potential when compared with CD133- cells. These findings indicate that CD133+ cells identified within the HT1080 human fibrosarcoma cell line possess many CSC properties and may facilitate the development of improved therapies for fibrosarcoma.

The CD133+ subpopulation of the SW982 human synovial sarcoma cell line exhibits cancer stem-like characteristics.

Several soft tissues sarcomas have been reported to contain cancer stem-like cells (CSCs) or tumor-initiating cells, based on their ability to initiate and sustain tumor growth. However, these cells have not yet been identified in the human synovial sarcoma cell line SW982. CD133, a surface glycoprotein specific to stem and progenitor cells, has been described as a CSC marker in different tumor types. In the present study, we identified a CSC subpopulation in SW982 cells using the CD133 cell surface marker. CD133-positive (CD133(+)) cells were identified in SW982 cells (8.59%); these cells showed an increased ability to form spherical colonies and could self-renew in serum-starved culture conditions, compared to CD133-negative (CD133(-)) cells. Real-time PCR analysis of stemness genes revealed that the CD133+ subpopulation expresses higher levels of Bmi1, c-Myc, Nanog, Oct3/4 and Sox2. CD133(+) cells showed increased resistance to cisplatin (CDDP) and doxorubicin (DXR), possibly due to upregulation of the ABCG2 drug transporter gene. In vivo studies revealed that the CD133(+) subpopulation is highly tumorigenic. These findings indicate that CD133(+) SW982 cells have characteristics similar to CSCs. This discovery may lead to the development of novel therapies that specifically target CD133(+) synovial sarcoma CSCs.