Rice has two distinct classes of protein kinase genes related to SNF1 of Saccharomyces cerevisiae, which are differently regulated in early seed development

We have isolated five cDNA clones (osk1–5) for protein kinases from rice which are related to SNF1 protein kinase of Saccharomyces cerevisiae. Based on the sequence homology, these cDNAs can be classified into two groups, group 1 (osk1) and group 2 (osk2–5). The products of these genes were demonstrated to be functional SNF1-related protein kinases by in vitro and in vivo experiments. Recombinant proteins expressed from both groups of genes were fully active as protein kinases and could phosphorylate SAMS peptide, a substrate specific for the SNF1/AMPK family, as well as themselves (autophosphorylation). Moreover, expression of osk3 cDNA in yeast snf1 mutants restored SNF1 function. Northern blot analyses showed differential expression of these two gene groups; group 1 is expressed uniformly in growing tissues (young roots, young shoots, flowers, and immature seeds), whereas group 2 is strongly expressed in immature seeds. SNF1-related protein kinases have been reported from different plant species, such as rye, barley, Arabidopsis, tobacco, and potato, while the type of gene strongly expressed in immature seeds is known only in cereals such as rye, barley, and, from our findings, in rice. Expression levels of the group 2 genes were further analyzed in seeds during seed maturation. Expression is transiently increased in the early stages of seed maturation and then decreases. The expression peak precedes those of the sbe1 and waxy genes, which are involved in starch synthesis in rice. Taken together, these findings suggest that group 2 OSK genes play important roles in the early stages of endosperm development in rice seeds.     

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4?×?10⁴ to 2.1?×?10⁶?CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100 %), followed by Penicillium chrysogenum (62 %), Aspergillus niger (54 %), and Aspergillus flavus (44 %). Citrinin was present in 100 % samples (0.23–20.65 mg/kg), aflatoxin in 92 % samples (0.61–77.33 μg/kg) and Ochratoxin-A in 100 % samples (0.23–2.48 μg/kg); 100 % citrinin and 76.09 % aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.     

Diversity of the bacterial community in the rice rhizosphere managed under conventional and no-tillage practices

Bacterial diversity in the rice rhizosphere at different rice growth stages, managed under conventional and no-tillage practices, was explored using a culture-based approach. Actinobacteria are among the bacterial phyla abundant in the rice rhizosphere. Their diversity was further examined by constructing metagenomic libraries based on the 16S rRNA gene, using actinobacterial- and streptomycete-specific polymerase chain reaction (PCR) primers. The study included 132 culturable strains and 125 clones from the 16S rRNA gene libraries. In conventional tillage, there were 38% Proteobacteria, 22% Actinobacteria, 33% Firmicutes, 5% Bacteroidetes, and 2% Acidobacteria, whereas with no-tillage management there were 63% Proteobacteria, 24% Actinobacteria, 6% Firmicutes, and 8% Bacteroidetes as estimated using the culture-dependent method during the four stages of rice cultivation. Principal coordinates analysis was used to cluster the bacterial communities along axes of maximal variance. The different growth stages of rice appeared to influence the rhizosphere bacterial profile for both cultivation practices. Novel clones with low similarities (89–97%) to Actinobacteria and Streptomyces were retrieved from both rice fields by screening the 16S rRNA gene libraries using actinobacterial- and streptomycete-specific primers. By comparing the actinobacterial community retrieved by culture-dependent and molecular methods, it was clear that a more comprehensive assessment of microbial diversity in the rice rhizosphere can be obtained using a combination of both techniques than by using either method alone. We also succeeded in culturing a number of bacteria that were previously described as unculturable. These were in a phylogenetically deep lineage when compared with related cultivable genera.     

Regulation of the protein and gene expressions of ethylene biosynthesis enzymes under different temperature during peach fruit ripening

Ethylene has profound effect on fruit development and ripening, and the role of ethylene biosynthesis enzymes involving 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and S-Adenosyl-l-methionine synthetase (SAMS) in peach fruit (cv. Xiahui-8) was characterized under 25 and 4 °C, respectively. All these enzymes in ethylene synthesis pathway were identified using 2-DE and real-time PCR. Both protein and gene expressions of ACO and SAMS were much higher at 25 °C than at 4 °C. Among five members of ACS family, PpaACS4 may belong to system II ethylene biosynthesis, while PpaACS3 involved in system I during development stage, and low temperature can induce PpaACS1 expression. The ethylene release and low expressions of proteins and genes of most enzymes indicated that low temperature can effectively postpone ripening stage by reducing ethylene evolution. High gene expression of PpaSAMS did not cause excessive expression of SAMS protein under low temperature, and over-expression of PpaACS1 at low temperature still did not induce increase of ethylene production. The mechanism underlying the phenomenon about how temperature affects ethylene release was also discussed.     

Accumulation of arsenic in soil and rice under wetland condition in Bangladesh

Shallow tube well (STW) water, often contaminated with arsenic (As), is used extensively in Bangladesh for irrigating rice fields in the dry season, leading to potential As accumulation in soils. In the current study the consequences of arsenic from irrigation water and direct surface (0–15 cm) soil application were studied under field conditions with wetland rice culture over 2 years. Twenty PVC cylinders (30-cm length and 30-cm diameter) were installed in field plots to evaluate the mobility and vertical distribution of soil As, As mass balance, and the resulting influences on rice yield and plant-As concentration in Boro (dry season) and transplanted (T.) Aman (wet season) rice over the 2-year growth cycle. Treatments included irrigation-water As concentrations of 0, 1 and 2 mg L^?1 (Boro season only) and soil-As concentrations of 10 and 20 mg kg^?1. Following the 2-year cropping sequence the major portion (39.3–47.6%) of the applied arsenic was retained within the rooting zone at 0–15 cm depth, with 14.7–19.5% of the total applied As at the 5–10 cm and 10–15 cm soil depths compared to 1.3–3.6% at the 35–40 cm soil depth. These results indicate the relatively low mobility of applied As and the likely continued detrimental accumulation of As within the rooting zone. Arsenic addition in either irrigation water or as soil-applied As resulted in yield reductions from 21 to 74 % in Boro rice and 8 to 80 % in T. Aman rice, the latter indicating the strong residual effect of As on subsequent crops. The As concentrations in rice grain (0.22 to 0.81 µg g^?1), straw (2.64 to 12.52 µg g^?1) and husk (1.20 to 2.48 µg g^?1) increased with increasing addition of As. These results indicate the detrimental impacts of continued long-term irrigation with As-contaminated water on agricultural sustainability, food security and food quality in Bangladesh. A critical need exists for the development of crop and water management strategies to minimize potential As hazard in wetland rice production.     

Apple aminopropyl transferase, MdACL5 interacts with putative elongation factor 1-alpha and S-adenosylmethionine synthase [corrected

Several lines of evidence suggest different allocations of the physiological roles of aminopropyl transferase genes, SPMS and ACL5 in plants. To get deeper insights into the physiological role of apple ACL5 (MdACL5), we performed yeast two-hybrid (Y2H) assay to identify proteins which interact with MdACL5. After intense screening processes, including the swapping of the bait and prey vectors and in vitro coimmunoprecipitation, we identified three MdACL5-interacting proteins: putative translation elongation factor 1A (eEF-1A), putative S-adenosyl-l-methionine synthetase (SAMS) and an unknown protein. Results from Y2H and RNA gel blot analysis suggested the involvement of MdACL5 and eEF-1A or SAMS complexes in the plant growth and development of the organized tissues and/or organs.     

Egg parasitism of the yellow rice borer,Scirpophaga incertulas [Lep.: Pyralidae] in Bangladesh deepwater rice

Studies on deepwater rice in Bangladesh from 1977 to 1980 showed that 61–89% of the egg masses of the yellow rice borer,Scirpophaga incertulas (Walker), were attacked by hymenopterous parasites. Egg parasitism increased from 43–48% in broods 2–4 (May–August) to 64–88% in broods 5–6 (September–November). Parasitism was reduced in broods 4 and 5 in 1979 (because of lower host densities) and in 1980 (possibly due to extreme late flooding). Telenomus rowani (Gahan), active throughout the year, parasitized 64% of the eggs in the masses it attacked, 32 larvae/mass surviving.Tetrastichus schoenobii Ferrière first appearing at medium host densities, increased rapidly to attack half of the brood 5 and 6 masses. More than 98% of the eggs in the attacked masses were parasitized, leaving only 1.6 surviving larvae/mass. In 1979 its appearance was delayed by 10 weeks and fewer masses were parasitized due to lower host densities.Trichogramma japonicum Ashmead andTelenomus sp. (a new record) were less important. Multiple parasitism of masses rose from 7% in brood 2 to 28% in brood 5. Egg parasites clearly reduce the numbers ofS. incertulas, a major pest, and thus probably improve the yields of deepwater rice.     

Antifungal effect and mechanism of chitosan against the rice sheath blight pathogen, Rhizoctonia solani

The antifungal properties and mechanism of three types of chitosan against the rice sheath blight pathogen, Rhizoctonia solani, were evaluated. Each chitosan had strong antifungal activity against R. solani and protected rice seedlings from sheath blight, in particular, two types of acid-soluble chitosan caused a 60–91?% inhibition in mycelial growth, 31–84?% inhibition of disease incidence, and 66–91?% inhibition in lesion length. The mechanism of chitosan in protection of rice from R. solani pathogen was attributed to direct destruction of the mycelium, evidenced by scanning and transmission electron microscopic observations and pathogenicity testing; indirect induced resistance was evidenced by the changes in the activities of the defense-related phenylalanine ammonia lyase, peroxidase and polyphenol oxidase in rice seedling. To our knowledge, this is the first report on the antifungal activity of chitosan against rice R. solani.     

Genotype and environment effects on rice (Oryza sativa L.) grain arsenic concentration in Bangladesh

Genetic analysis of 38 rice varieties released by the Bangladesh Rice Research Institute (BRRI) identified 34 as indica, 2 as admixed between indica and aus, and 4 as belonging to the aromatic/Group V subpopulation. Indica varieties developed for the two major rice-growing seasons, the wet monsoon (aman) and the dry winter (boro), were not genetically differentiated. The Additive Main Effect and Multiplicative Interaction (AMMI) model was used to assess the effect of genotype (G), environment (E) and genotype-environment interaction (GEI) on grain arsenic (As) concentration when these rice varieties were grown at ten BRRI research stations located across diverse agro-ecological zones in Bangladesh. G, E and GEI, significantly influenced grain As concentration in both seasons. Overall, E accounted for 69%–80%, G 9%–10% and GEI 10%–21% of the observed variability in grain As. One site, Satkhira had the highest mean grain As concentration and the largest interaction principle component analysis (IPCA) scores in both seasons, indicating maximum interaction with genotypes. Site effects were more pronounced in the boro than in the aman season. The soil level of poorly crystalline Fe-oxide (AOFe), the ratio of AOFe to associated As, soil phosphate extractable As and soil pH were important sub-components of E controlling rice grain As concentration. Irrespective of environment, the mean grain As concentration was significantly higher in the boro (0.290 mg As kg^?1) than in the aman (0.154 mg As kg^?1) season (p?<?0.0001), though the reasons for this are unclear. Based on mean grain As concentration and stability across environments, the variety BR3 is currently the best choice for the boro season, while BR 23 and BRRI dhan 38 are the best choices for the aman season. Popular varieties BR 11 (aman) and BRRI dhan 28 and 29 (boro) had grain As concentrations close to the mean value and were fairly stable across environments, while high-yielding, short-duration aman season varieties (BRRI dhan 32, 33 and 39) developed for intensified cropping had relatively high grain As concentrations. Results suggest that genetic approaches to reducing As in rice grain will require the introduction of novel genetic variation and must be accompanied by appropriate management strategies to reduce As availability and uptake by rice.     

Identification of quantitative trait loci for resistance to rice black-streaked dwarf virus disease and small brown planthopper in rice

Small brown planthopper (SBPH) and its transmitted rice black-streaked dwarf virus disease (RBSDVD) cause serious damage to rice (Oryza sativa L.) production. Though breeding of resistant cultivars is believed to be one of the most important strategies for RBSDVD management, few high-resistance lines have been found to date. In the present study, we identified an indica variety, 9194, that is highly resistant to RBSDVD and analyzed the quantitative trait loci (QTLs) underlying this resistance . In total, four QTLs for RBSDVD resistance, viz. qRBSDV3, qRBSDV6, qRBSDV9, and qRBSDV11, were identified. Among them, qRBSDV6, qRBSDV9, and qRBSDV11 with LOD (logarithm [base 10] of odds) scores of 4.42–4.48, 2.11–7.26, and 5.01–7.16 were repeatedly detected in 2 years, accounting for 10.3–16.7%, 8.3–35.5%, and 20.0–31.1% of the total phenotypic variation, respectively. Further, introgression of single- or multiple-resistance QTLs into a susceptible rice variety by marker-assisted selection (MAS) indicated that stacking the QTLs could progressively enhance RBSDVD resistance, suggesting that these QTLs act additively. The same population was also used for QTL mapping of SBPH resistance. Four QTLs, viz. qSBPH1, qSBPH5, qSBPH8, and qSBPH9, with LOD scores of 2.72, 2.78, 2.15, and 2.85 were detected, explaining 13.7%, 11.0%, 12.0%, and 21.0% of the phenotypic variation, respectively. The identification of RBSDVD and SBPH resistance QTLs, and the development of single and multiple genes with pyramided lines, in this study provides innovative resources for molecular breeding of resistant rice cultivars.     

Temporal distribution of larval Chironomidae (Diptera) in experimental rice fields in Penang,Malaysia

The distribution and abundance of chironomid larvae were investigated in experimental rice fields at the Bukit Merah Agricultural Experimental Research Station, Penang, Northern Malaysia. The mean density of chironomid larvae during the period of the study (including two cultivation seasons) was 121 larvae m^?2. Chironomus kiiensis (48.6%) was the most abundant species, while four other species, Polypedilum trigonus (4.8%), Tanytarsus formosanus (22.6%), Tanypus punctipennis (13.9%), and Clinotanypus sp. (10.1%) occurred less frequently, although they were present during all wet phases of rice cultivation. Generally, water depth, rice plant height, conductivity, and nitrate–nitrogen content of the water only weakly influenced the abundance of the larvae. Other environmental parameters such as pH, dissolved oxygen, daytime water temperature, total suspended solids, phosphate, and sediment total organic matter did not significantly affect larval populations. The community structure of chironomids in this rice agroecosystem followed the dynamic changes of the field including agronomic practices, patterns of water availability, and phases of rice plant growth.     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

Disruption of the methionine cycle and reduced cellular gluthathione levels underlie potex–potyvirus synergism in Nicotiana benthamiana

Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex–potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S‐adenosyl‐l ‐methionine synthetase (SAMS) and S‐adenosyl‐l ‐homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post‐infiltration (dpi) and upregulation of (–)‐strand gRNA accumulation at 9 dpi were observed in the SAHH‐silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX‐infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX‐infected plants caused an even stronger reduction in GSH levels than did SAMS + SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase‐silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex–potyvirus mixed infection.     

Over-expression of PsGPD,a mushroom glyceraldehyde-3-phosphate dehydrogenase gene,enhances salt tolerance in rice plants

Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192–196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1–T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars.     

Polluting macrophytes Colombian lake Fúquene used as substrate by edible fungus Pleurotus ostreatus

Invasive aquatic plants from Lake Fúquene (Cundinamarca, Colombia), water hyacinth (Eichhornia crassipes C. Mart.) and Brazilian elodea (Egeria densa Planch.) have been removed mechanically from the lake and can be used for edible mushrooms production. The growth of the oyster mushroom (Pleurotus ostreatus) on these aquatic macrophytes was investigated in order to evaluate the possible use of fruiting bodies and spent biomass in food production for human and animal nutrition, respectively. Treatments included: water hyacinth, Brazilian elodea, sawdust, rice hulls and their combinations, inoculated with P. ostreatus at 3 %. Water hyacinth mixed with sawdust stimulated significantly fruiting bodies production (P = 3.3 × 10^?7) with 71 % biological efficacy, followed by water hyacinth with rice husk (55 %) and elodea with rice husk (48 %), all of these have protein contents between 26 and 47 %. Loss of lignin (0.9–21.6 %), cellulose (3.7–58.3 %) and hemicellulose (1.9–53.8 %) and increment in vitro digestibility (16.7–139.3 %) and reducing sugars (73.4–838.4 %) were observed in most treatments. Treatments spent biomass presented Relative Forage Values (RFV) from 46.1 to 232.4 %. The results demonstrated the fungus degrading ability and its potential use in aquatic macrophytes conversion biomass into digestible ruminant feed as added value to the fruiting bodies production for human nutrition.     

Nitrogen release from incorporated 15N-labelled Chinese milk vetch (Astragalus sinicus L.) residue and its dynamics in a double rice cropping system

Background and aims

Chinese milk vetch (Astragalus sinicus L. CMV), a leguminous cover crop, has been shown to provide N benefits to rice crops, but little is known about the pathway of incorporated CMV and its N dynamics. In this study, effects of CMV under different application treatments (incorporated alone, applied in conjunction with urea fertilizer and applied with ryegrass (Lolium multiflorum Lam.)) on N dynamics, rice yields and N uptake were investigated and compared with those of chemical fertilizer (CF) and no fertilizer (NF) in a double rice cropping system.

Methods

Nitrogen release from incorporated CMV residue was quantified by using a bag method. Nitrogen dynamics of CMV were evaluated by using ¹⁵N-labelled fresh CMV tops and compared with those of CF (¹⁵N-labelled urea).

Results

CMV residue decomposition pattern and its N release pattern followed a single exponential decay model, with 87.8–89.5 % of the applied CMV decomposed and 95.1–96.1 % of the original N released in the double rice season (177 days after fertilizer application). CMV treatments had higher rice N uptake efficiency than CF (39.2–51.3 % vs. 29.9 %) at the sum of early and late rice seasons. Rice yield, N accumulation and mineral fertilizer efficiency in CMV treated treatments were higher than those in CF. After two consecutive rice seasons the amounts of residual N remained in the soil were higher in the CMV treated fields than in CF (29.4–33.2 % vs. 14.1 %).

Conclusions

CMV can be considered an efficient N source alternative to chemical fertilizer in double rice cropping systems.