Background Rice ( Oryza sativa ) straw is a common waste product that represents a considerable amount of bound energy. This energy can be used for biogas production, but the rate and level of methane produced from rice straw is still low. To investigate the potential for an increased biogas production from rice straw, we have here utilized WRINKLED1 (WRI1), a plant AP2/ERF transcription factor, to increase triacylglycerol (TAG) biosynthesis in rice plants. Two forms of Arabidopsis thaliana WRI1 were evaluated by transient expression and stable transformation of rice plants, and transgenic plants were analyzed both for TAG levels and biogas production from straw. Results Both full-length AtWRI1, and a truncated form lacking the initial 141 amino acids (including the N-terminal AP2 domain), increased fatty acid and TAG levels in vegetative and reproductive tissues of Indica rice. The stimulatory effect of the truncated AtWRI1 was significantly lower than that of the full-length protein, suggesting a role for the deleted AP2 domain in WRI1 activity. Full-length AtWRI1 increased TAG levels also in Japonica rice, indicating a conserved effect of WRI1 in rice lipid biosynthesis. The bio-methane production from rice straw was 20% higher in transformants than in the wild type. Moreover, a higher producing rate and final yield of methane was obtained for rice straw compared with rice husks, suggesting positive links between methane production and a high amount of fatty acids. Conclusions Our results suggest that heterologous WRI1 expression in transgenic plants can be used to improve the metabolic potential for bioenergy purposes, in particular methane production.

Rice cultivation is the primary source of anthropogenic methane (CH4), which dramatically impacts global climate change. A growing body of evidence shows that optimizing photosynthate distribution is important for increasing rice yields and mitigating CH4 emissions. Therefore, the molecular rice breeding with a barley HvSUSIBA2 gene that confers elevated photosynthate flux to grains, is predicted to enhance rice yield and mitigate CH4 emissions in paddies. Here, in a series of field experiments with differences in growing season and rice variety, we show that SUSIBA2 rice reduced CH4 emissions from paddies. SUSIBA2 rice grown in the early rice season and late rice season showed similar mitigation effects, with reduction rates of 50.98% for early rice and 50.97% for late rice. The reduction rate of SUSIBA2 rice during the winter rice season was significantly lower (22.26%) than those of other rice seasons. The reduction rates also varied between rice varieties, and SUSIBA2 japonica rice showed a more significant CH4 mitigation effect than SUSIBA2 indica rice. Further yield-scaled CH4 emission analyses indicated that the SUSIBA2 effect did not mitigate CH4 emissions at the expense of yield. Compared with the wild type, SUSIBA2 rice significantly reduced soil organic carbon properties and the abundance of CH4-related microbes, and altered methanogenic and methanotrophic communities, indicating that SUSIBA2 rice released less carbon to the soil, which reduced CH4 production. Furthermore, a comparison of microbial communities between SUSIBA2 japonica and indica rice revealed different responses of methanogenic and methanotrophic communities, which may partly explain their differences in growth performance and CH4 mitigation effect. Thus, our results show that SUSIBA2 rice substantially reduces CH4 emissions and that SUSIBA2 can potentially mitigate the CH4 emissions of japonica and indica rice under distinct cultivation conditions.

There is an urgent demand for low-arsenic rice in the global market, particularly for consumption by small children. Soils in Uppsala, Sweden, contain low concentrations of arsenic (As). We hypothesize that if certain japonica paddy rice varieties can adapt to the cold climate and long day length in Uppsala and produce normal grains, such a variety could be used for organic production of low-arsenic rice for safe rice consumption. A japonica paddy rice variety, "Heijing 5," can be cultivated in Uppsala, Sweden, after several years' adaptation, provided that the rice plants are kept under a simple plastic cover when the temperature is below 10°C. Uppsala-adapted "Heijing 5" has a low concentration of 0.1 mg per kg and high protein content of 12.6% per dry weight in brown rice grain, meaning that it thus complies with all dietary requirements determined by the EU and other countries for small children. The high protein content is particularly good for small children in terms of nutrition. Theoretically, Uppsala-adapted "Heijing 5" can produce a yield of around 5100 kg per ha, and it has a potential for organic production. In addition, we speculate that cultivation of paddy rice can remove nitrogen and phosphorus from Swedish river water and reduce nutrient loads to the Baltic Sea and associated algae blooms.

Grain size is an important factor in rice yield. Several genes related to grain size have been reported, but most of them are determined by quantitative trail loci (QTL) traits. Gene D26 is a novel site mutation of OsGRAS19 and involved in the brassinosteroid (BR) signalling pathway. However, whether D26 is involved in the process of rice reproductive development remains unclear. Here, gene cloning and functional analysis revealed that D26 has an obvious regulatory effect on grain size. Overexpression or CRISP/Cas9 mutant of D26 also showed that grain size was positively influenced. Cellular analyses show that D26 modulates grain size by promoting cell division and regulating the cell number in the upper epidermis of the glume. The overexpression results further suggest that the level of D26 expression positively impacts grain length and leaf angles and that the expression of several known grain size genes is involved in the regulation. Based on our results, D26, as a transcription factor, effectively improves rice grain shape.

Genetically engineered (GE) rice endogenous epsps (5-enolpyruvoylshikimate-3-phosphate synthase) gene overexpressing EPSPS can increase glyphosate herbicide-resistance of cultivated rice. This type of epsps transgene can enhance the fecundity of rice crop-weed hybrid offspring in the absence of glyphosate, stimulating great concerns over undesired environmental impacts of transgene flow to populations of wild relatives. Here, we report the substantial alteration of phenology and fitness traits in F-1-F-3 crop-wild hybrid descendants derived from crosses between an epsps GE rice line and two endangered wild rice (Oryza rufipogon) populations, based on the common-garden field experiments. Under the glyphosate-free condition, transgenic hybrid lineages showed significantly earlier tillering and flowering, as well as increased fecundity and overwintering survival/regeneration abilities. In addition, a negative correlation was observed between the contents of endogenous EPSPS of wild, weedy, and cultivated rice parents and fitness differences caused by the incorporation of the epsps transgene. Namely, a lower level of endogenous EPSPS in the transgene-recipient populations displayed a more pronounced enhancement in fitness. The altered phenology and enhanced fitness of crop-wild hybrid offspring by the epsps transgene may cause unwanted environmental consequences when this type of glyphosate-resistance transgene introgressed into wild rice populations through gene flow.

Ecological impact caused by transgene flow from genetically engineered(GE) crops to their wild relatives is largely determined by the fitness effect brought by a transgene. To estimate such impact is critical for the ecological risk assessment prior to the commercialization of GE crops. We produced F1 and F2hybrid descendants from crosses of two insect-resistant GE rice lines(Bt, Bt/Cp TI)and their non-GE rice parent with a wild rice(Oryza rufipogon) population to estimate the transgenic fitness. Insect damages and life-cycle fitness of GE and non-GE crop–wild hybrid descendants as well as their wild parent were examined in a common-garden experiment. No significant differences in insect damages were observed between the wild rice parent and GE hybrid descendants under highinsect pressure. The wild parent showed significantly greater relative survival-regeneration ratios than its GE and non-GE hybrid descendants under both high- and low-insect pressure. However, more seeds were produced in GE hybrid descendants than their non-GE counterparts under high-insect pressure. Given that the introduction of Bt and Bt/Cp TI transgenes did not provide greater insect resistance to crop–wild hybrid descendants than their wild parent, we predict that transgene flow from GE insect-resistant rice to wild rice populations may not cause considerable ecological risks.

Ecological impact of transgene flow into populations of wild/weedy relatives is associated with fitness effects in hybrid progeny. Most studies assessing fitness effects focus essentially on early-generation hybrid progeny. However, whether the transgenes remain effective and durable in advanced generations of hybrid progeny remains unclear. We conducted a common garden experiment with F5–F7 hybrid progeny derived from crosses between insect-resistant transgenic (Bt/CpTI) rice and weedy rice, to examine their insect resistance and fitness effects of transgenes on progeny. Hybrid progeny were grown under different insect pressures and cultivation modes where insect damage and fitness-related traits were measured in the same growth season. Plants with transgenes showed significantly lower insect damage (10% vs. 32%) and higher fecundity (551 vs. 392 seeds/plant) than those without transgenes in F5–F7 populations, suggesting the efficacy of transgenes for insect resistance. Fitness benefits of the transgenes were similar among the F5–F7 populations, indicating the stability of transgenic effects. A positive correlation between insect index and fecundity change was detected, stressing the important role of ambient insect pressures in assessing fitness effects caused by insect-resistance transgenes. Our results have important implications for assessing ecological impacts caused by transgene flow to wild/weedy relatives. For cost-effectiveness, the experimental estimation of fitness effects is probably sufficient based on data from hybrids in early generations. Given that fitness effects of insect-resistance transgenes are associated with ambient insect pressure, ecological risk assessment on transgene flow should consider this variable in experimental design, reasonably reflecting actual situations in wild/weedy populations.

Expression of a barley transcription factor SUSIBA2 in rice generates a plant with high-starch content and low-methane emissions by conferring a shift in the carbon flux that favours the allocation of photosynthates to aboveground biomass rather than to the roots.

Background Rice seed shattering is an important domestication syndrome encoded by a gene named as SH4 . The coding region of SH4 has been well studied regarding its function and roles in evolution. However, its promoter has not been identified, which limited our understanding of the detailed regulatory mechanisms of this gene. It is therefore critical to characterize the promoter and study its expression pattern. Results We analyzed the 5′ upstream sequences of this gene and identified a ~2.6 kb fragment with typical promoter features, which was designated as pSH4. The promoter contained a number of cis -acting elements related to abscisic acid (ABA) and a CpG island that were characteristics of multiple tissue-specific expression. We isolated and ligated pSH4 to the β-glucuronidase (GUS) reporter gene, and transformed it into a japonica rice cultivar to determine the multiple expression pattern of SH4 . Histochemical location and fluorescence analyses of GUS activity of transgenic plants indicated multiple tissue-specific expression of pSH4 in the seed-pedicel junction region of mature panicles (with highest level), stems, coleoptiles of germinated seeds, and scutella of mature seeds. Conclusions The multiple tissue-specific expression pSH4 is categorized as a spatiotemporal promoter that drives the expression of the SH4 gene in different rice tissues, in addition to the seed-pedicel junction region. Our findings suggest that SH4 may have additional functions in the growth and development of rice, apart from its major role in seed shattering.

Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression.

Transgene flow from genetically engineered (GE) crops to non-GE varieties and wild relatives via pollen-mediated gene flow (PMGF) may create food and environmental biosafety concerns. Assessing the level of PMGF from GE crops is required before commercialization. Whether the level of PMGF estimated at relatively small scales can sufficiently represent the actual scenario at large production scales remains unresolved. Here, we estimated average PMGF frequencies from three insect-resistant GE rice lines to their non-GE counterparts at four scales ranging from 9 to 576?m2, having the number of GE to non-GE plants constantly at the ratio of 8:1. Based on nearly 1.3 million examined seedlings from non-GE rice plots, very low frequencies (<0.1%) of transgene flow were detected. The highest frequencies were found in plots at the smallest scales. Scale had a significantly negative effect on the frequency of PMGF in rice, with decreased gene flow at increased scale. An extended PMGF model could well represent the experimental data. Field experiments at relatively small scales combined with mathematical modelling could provide useful prediction on the level of rice transgene flow at large production scales. This is probably applicable for other crop species with wind- and self-pollination.

Gene flow from transgenic crops allows novel traits to spread to sexually compatible weeds. Traits such as resistance to insects may enhance the fitness of weeds, but few studies have tested for these effects under natural field conditions. We created F(2) and F(3) crop-weed hybrid lineages of genetically engineered rice (Oryza sativa) using lines with two transgene constructs, cowpea trypsin inhibitor (CpTI) and a Bt transgene linked to CpTI (Bt/CpTI). Experiments conducted in Fuzhou, China, demonstrated that CpTI alone did not significantly affect fecundity, although it reduced herbivory. In contrast, under certain conditions, Bt/CpTI conferred up to 79% less insect damage and 47% greater fecundity relative to nontransgenic controls, and a 44% increase in fecundity relative to the weedy parent. A small fitness cost was detected in F(3) progeny with Bt/CpTI when grown under low insect pressure and direct competition with transgene-negative controls. We conclude that Bt/CpTI transgenes may introgress into co-occurring weedy rice populations and contribute to greater seed production when target insects are abundant. However, the net fitness benefits that are associated with Bt/CpTI could be ephemeral if insect pressure is lacking, for example, because of widespread planting of Bt cultivars that suppress target insect populations.

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice ( Oryza rufipogon ), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F 1 hybrids with common wild rice and F 2 progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F 1 hybrids and F 2 progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.

Genetically modified (GM) rice with enhanced agronomic traits and pharmaceutical uses are ready for widespread adoption. Little is known about isolation requirements for achieving stringent transgene confinement in rice. To investigate the extent of pollen-mediated crop-to-crop transgene flow, we conducted a field experiment with four plot-size treatments of adjacent GM and nonGM rice (Oryza sativa) in China. Three insect-resistant GM rice (Bt/CpTI) and nonGM isogenic lines were used in the study. The hygromycin-resistance transgene (hpt) marker was used to screen seeds from the nonGM rice rows at different distance intervals from GM rice plots. Based on the examination of > 2.1 million germinated seeds, we found a dramatic reduction in transgene frequencies with increasing distance from the GM crop, ranging from c. 0.28% at 0.2 m to < 0.01% at 6.2 m. In addition, different plot size did not significantly affect the frequencies of gene flow. In conclusion, pollen-mediated crop-to-crop transgene flow in rice can be maintained at negligible levels with short spatial isolation. The model can also be applied to other crops with self- and wind-pollination.

Crop-to-crop transgene flow will affect seed purity of non-GM rice varieties, leading to unwanted consequences. To assess the maximum probability of transgene outflow in rice (Oryza sativa), gene flow experiments were conducted with three cultivation patterns with different mixed-planting proportions of adjacent GM and non-GM rice at two sites in Fujian and Hainan Provinces of China. Three GM rice lines containing two insect-resistance genes (Bt/CpTI) and their non-GM counterparts were used in the experiments to allow natural hybridization to occur. A hygromycin resistance gene was used as a selective marker for identifying hybrids. Based on the examination of > 645 700 geminated seeds, the result showed low frequencies (0.05-0.79%) of transgene flow from GM to non-GM rice at close spacing, although with significant variation among mixed-planting proportions. It is concluded that rice transgene flow will occur at a very low frequency (< 1.0%), even if the GM rice is planted at close spacing with non-GM rice, and high densities of GM rice cultivated in the neighborhood of non-GM rice will increase the probability of outcrossing with the non-GM rice.