Genome-wide survey of the RIP domain family in Oryza sativa and their expression profiles under various abiotic and biotic stresses

Ribosome-inactivating proteins (RIPs) are N-glycosidases that inhibit protein synthesis by depurinating rRNA. Despite their identification more than 25 years ago, little is known about their biological functions. Here, we report a genome-wide identification of the RIP family in rice based on the complete genome sequence analysis. Our data show that rice genome encodes at least 31 members of this family and they all belong to type 1 RIP genes. This family might have evolved in parallel to species evolution and genome-wide duplications represent the major mechanism for this family expansion. Subsequently, we analyzed their expression under biotic (bacteria and fungus infection), abiotic (cold, drought and salinity) and the phytohormone ABA treatment. These data showed that some members of this family were expressed in various tissues with differentiated expression abundances whereas several members showed no expression under normal growth conditions or various environmental stresses. On the other hand, the expression of many RIP members was regulated by various abiotic and biotic stresses. All these data suggested that specific members of the RIP family in rice might play important roles in biotic and abiotic stress-related biological processes and function as a regulator of various environmental cues and hormone signaling. They may be potentially useful in improving plant tolerance to various abiotic and biotic stresses by over-expressing or suppressing these genes.     

Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells

Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrient-induced insulin secretion from isolated beta-cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated beta-cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phosphoenolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion.     

PCR-identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics

The saline pond microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's (basal) medium under laboratory conditions was confirmed by amplifying the chromosomal DNA of the microalga by PCR with specific primers MA1 and MA2. Seaweed extracts obtained from Sargassum wightii and Ulva lactuca were amended separately at 1.0%, 1.5%, 2.0% and 2.5% levels to the basal medium in order to assess their potential on the growth and concentration of pigments, viz. Chl a, Chl b and beta-carotene of the alga. beta-Carotene was isolated and visible absorption spectrum was taken at 443 and 475 nm confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene isomers. Maximum yield, highest division rate (mu) and highest pigment concentrations were observed in the cells grown in 1.5% S. wightii and 2.0% U. lactuca amended medium and these cells were subjected to DAPI staining. The results of epifluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear area of the microalgae.     

Large-scale systematic study on stability of the Ds element and timing of transposition in rice

Activator/Dissociation (Ac/Ds) transposon mutagenesis is a widely used tool for gene identification; however, several reports on silencing of the Ac/Ds element in starter lines and in stable transposants question the applicability of such an approach in later generations. We have performed a systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare). High somatic and germinal transposition frequencies observed in earlier generations were maintained as late as T4 and T5 generations; thus the propagation of parental lines did not induce transposon silencing. Moreover, the stably transposed Ds element was active even at the F5 generation, since Ac could remobilize the Ds element as indicated by the footprint analysis of several revertants. Expression of the bar gene was monitored from F3 to F6 generations in >1,000 lines. Strikingly, substantial transgene silencing was not observed in any of the generations tested. We analyzed the timing of transposition during rice development and provide evidence that Ds is transposed late after tiller formation. The possibility, that the independent events could be the result of secondary transposition, was ruled out by analyzing potential footprints by reciprocal PCR. Our study validates the Ac/Ds system as a tool for large-scale mutagenesis in rice, since the Ds elements were active in the starter and insertion lines even in the later generations. We propose that harvesting rice seeds using their panicles is an alternative way to increase the number of independent transposants due to post-tillering transposition.     

The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.     

Identification and Characterization of RcMADS1, an AGL24 Ortholog from the Holoparasitic Plant Rafflesia cantleyi Solms-Laubach (Rafflesiaceae)

Rafflesia, a holoparasitic genus that produces the largest flower in the world is characterized by the absence of leaves, stem and other macroscopic organs. To better understand the molecular regulation of flower development in this genus we isolated and characterized a floral MADS-box gene, namely, RcMADS1 from Rafflesia cantleyi. Heterologous expression analysis in Arabidopsis was chosen because Rafflesia is not amenable to genetic manipulations. RcMADS1 shares sequence similarity with AGAMOUS-LIKE 24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) of Arabidopsis. Ectopic expression of RcMADS1 in Arabidopsis caused early flowering and conversion of sepals and petals into leaf-like structures, and carpels into inflorescences. In 35S::RcMADS1 plants SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), a downstream target gene of AGL24, was upregulated. 35S::RcMADS1 plants exhibit early flowering and conversion of the floral meristem into inflorescence meristem, as in 35S::AGL24 plants. Similar to AGL24, RcMADS1 could rescue the late flowering phenotypes of agl24-1 and FRIGIDA, but not the early flowering of svp-41. Based on these results, we propose that RcMADS1 is a functional ortholog of Arabidopsis AGL24.     

Exploitation of Dunaliella for β-carotene production

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.     

Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d

We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.