A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition.

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time-consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co-introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic-resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high-throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant-specific DNA herbicides.

A low-methane rice with high-yield potential realized via optimized carbon partitioning.

Global rice cultivation significantly contributes to anthropogenic methane emissions. The methane emissions are caused by methane-producing microorganisms (methanogenic archaea) that are favoured by the anoxic conditions of paddy soils and small carbon molecules released from rice roots. However, different rice cultivars are associated with differences in methane emission rates suggesting that there is a considerable natural variation in this trait. Starting from the hypothesis that sugar allocation within a plant is an important factor influencing both yields and methane emissions, the aim of this study was to produce high-yielding rice lines associated with low methane emissions. In this study, the offspring (here termed progeny lines) of crosses between a newly characterized low-methane rice variety, Heijing 5, and three high-yielding elite varieties, Xiushui, Huayu and Jiahua, were selected for combined low-methane and high-yield properties. Analyses of total organic carbon and carbohydrates showed that the progeny lines stored more carbon in above-ground tissues than the maternal elite varieties. Also, metabolomic analysis of rhizospheric soil surrounding the progeny lines showed reduced levels of glucose and other carbohydrates. The carbon allocation, from roots to shoots, was further supported by a transcriptome analysis using massively parallel sequencing of mRNAs that demonstrated elevated expression of the sugar transporters SUT-C and SWEET in the progeny lines as compared to the parental varieties. Furthermore, measurement of methane emissions from plants, grown in greenhouse as well as outdoor rice paddies, showed a reduction in methane emissions by approximately 70 % in the progeny lines compared to the maternal elite varieties. Taken together, we report here on three independent low-methane-emission rice lines with high yield potential. We also provide a first molecular characterisation of the progeny lines that can serve as a foundation for further studies of candidate genes involved in sugar allocation and reduced methane emissions from rice cultivation.

Characterisation of a low methane emission rice cultivar suitable for cultivation in high latitude light and temperature conditions.

Rice cultivation on paddy soil is commonly associated with emissions of methane, a greenhouse gas, but rice varieties may differ in their actual level of emissions. This study analysed methane emissions associated with 22 distinct rice genotypes, using gas chromatography, and identified the cultivar Heijing 5 from northern China as a potential low-methane rice variety. To confirm this and to examine whether Heijing 5 can perform similarly at higher latitudes, Heijing 5 was cultivated in field trials in China (lat. 32° N) and Sweden (lat. 59° N) where (i) methane emissions were measured, (ii) methanogen abundance in the rhizosphere was determined using quantitative PCR, and (iii) the concentrations of nutrients in water and of heavy metals in rice grain and paddy soil were analysed. The results demonstrated that the low-methane rice cultivar Heijing 5 can successfully complete an entire growth period at high-latitude locations such as central Sweden. Massively parallel sequencing of mRNAs identified candidate genes involved in day length and cold acclimatisation. Cultivation of Heijing 5 in central Sweden was also associated with relatively low heavy metal accumulation in rice grains and lowered nutrient losses to neighbouring water bodies.

High fructan barley lines produced by selective breeding may alter ß-glucan and amylopectin molecular structure.

Six cross-bred barley lines developed by a breeding strategy with the target to enhance the fructan synthesis activity and reduce the fructan hydrolysis activity were analyzed together with their parental lines, and a reference line (Gustav) to determine whether the breeding strategy also affected the content and molecular structure of amylopectin and ß-glucan. The highest fructan and ß-glucan content achieved in the novel barley lines was 8.6 % and 12 %, respectively (12.3-fold and 3.2-fold higher than in Gustav). The lines with low fructan synthesis activity had higher starch content, smaller building blocks in amylopectin, and smaller structural units of ß-glucans than the lines with high-fructan synthesis activity. Correlation analysis confirmed that low starch content was associated with high amylose, fructan, and ß-glucan content, and larger building blocks in amylopectin.

Improved bioenergy value of residual rice straw by increased lipid levels from upregulation of fatty acid biosynthesis.

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.

Achieving of high-diet-fiber barley via managing fructan hydrolysis.

High fructan content in the grain of cereals is an important trait in agriculture such as environmental resilience and dietary fiber food production. To understand the mechanism in determining final grain fructan content and achieve high fructan cereal, a cross breeding strategy based on fructan synthesis and hydrolysis activities was set up and have achieved barley lines with 11.8% storage fructan in the harvested grain. Our study discovered that high activity of fructan hydrolysis at later grain developmental stage leads to the low fructan content in mature seeds, simultaneously increasing fructan synthesis at early stage and decreasing fructan hydrolysis at later stage through crossing breeding is an efficient way to elevate grain diet-fiber content. A good correlation between fructan and beta glucans was also discovered with obvious interest. Field trials showed that the achieved high fructan barley produced over seven folds higher fructan content than control barley and pull carbon-flux to fructan through decreasing fructan hydrolysis without disruption starch synthesis will probably not bring yield deficiency.

[6b genes: the important effective factors relative to tumor formation in plants].

In recent years, the functional mechanisms of the oncogenens from Agrobacterium in plants were received more and more attentions. 6b genes, derived from the T-DNA fragment, are vital carcinogenesis factors of plants and belong to rolB genes family. In plants, 6b genes can affect phytohormone levels and carbohydrate contents, and can also cause accumulation of secondary metabolites, as well as change the relative genes expression. The specific mechanisms behind these impacts remain to be researched in-depth. In this paper, the function, structure, activity, and acting mode of the 6b genes were summarized, which provide a theoretical foundation for further study and application of these functional genes.

Adaptation of Rice to the Nordic Climate Yields Potential for Rice Cultivation at Most Northerly Site and the Organic Production of Low-Arsenic and High-Protein Rice.

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.

T-6b allocates more assimilation product for oil synthesis and less for polysaccharide synthesis during the seed development of Arabidopsis thaliana.

BACKGROUND: As an Agrobacterium tumefaciens T-DNA oncogene, T-6b induces the development of tumors and the enation syndrome in vegetative tissues of transgenic plants. Most of these effects are related to increases in soluble sugar contents. To verify the potential roles of T-6b in the distribution of carbon in developing seeds, not in vegetative tissues, we fused an endosperm-specific promoter to the T-6b gene for expression in transgenic Arabidopsis thaliana plants. RESULTS: The expression of T-6b in reproductive organs did not induce the development of the enation syndrome, and moreover, promoted endosperm expansion, which increased the total seed biomass by more than 10%. Additionally, T-6b also increased oil content in mature seeds by more than 10% accompanied with the decrease of starch and mucilage content at the same time. CONCLUSIONS: T-6b enhances seed biomass and helps oil biosynthesis but not polysaccharides in reproductive organs without disturbing vegetative growth and development. Our findings suggest T-6b may be very useful for increasing oil production in biodiesel plants.

A Dual-Promoter Gene Orchestrates the Sucrose-Coordinated Synthesis of Starch and Fructan in Barley.

Sequential carbohydrate synthesis is important for plant survival because it guarantees energy supplies for growth and development during plant ontogeny and reproduction. Starch and fructan are two important carbohydrates in many flowering plants and in human diets. Understanding this coordinated starch and fructan synthesis and unraveling how plants allocate photosynthates and prioritize different carbohydrate synthesis for survival could lead to improvements to cereals in agriculture for the purposes of greater food security and production quality. Here, we report a system from a single gene in barley employing two alternative promoters, one intronic/exonic, to generate two sequence-overlapping but functionally opposing transcription factors, in sensing sucrose, potentially via sucrose/glucose/fructose/trehalose 6-phosphate signaling. The system employs an autoregulatory mechanism in perceiving a sucrose-controlled trans activity on one promoter and orchestrating the coordinated starch and fructan synthesis by competitive transcription factor binding on the other promoter. As a case in point for the physiological roles of the system, we have demonstrated that this multitasking system can be exploited in breeding barley with tailored amounts of fructan to produce healthy food ingredients. The identification of an intron/exon-spanning promoter in a hosting gene, resulting in proteins with distinct functions, adds to the complexity of plant genomes.