A Versatile High Throughput Screening Platform for Plant Metabolic Engineering Highlights the Major Role of ABI3 in Lipid Metabolism Regulation

Traditional functional genetic studies in crops are time consuming, complicated andcannot be readily scaled up. The reason is that mutant or transformed crops need tobe generated to study the effect of gene modifications on specific traits of interest.However, many crop species have a complex genome and a long generation time. Asa result, it usually takes several months to over a year to obtain desired mutants ortransgenic plants, which represents a significant bottleneck in the development of newcrop varieties. To overcome this major issue, we are currently establishing a versatileplant genetic screening platform, amenable to high throughput screening in almost anycrop species, with a unique workflow. This platform combines protoplast transformationand fluorescence activated cell sorting. Here we show that tobacco protoplasts canaccumulate high levels of lipid if transiently transformed with genes involved in lipidbiosynthesis and can be sorted based on lipid content. Hence, protoplasts can be usedas a predictive tool for plant lipid engineering. Using this newly established strategy, wedemonstrate the major role ofABI3in plant lipid accumulation. We anticipate that thisworkflow can be applied to numerous highly valuable metabolic traits other than storagelipid accumulation. This new strategy represents a significant step toward screeningcomplex genetic libraries, in a single experiment and in a matter of days, as opposed toyears by conventional means.This work was partly funded through the CSIRO Synthetic Biology Future Science Platform and the CSIRO Research Office CERC Postdoctoral Fellowship schem

The Road to Servomechanisms: The Influence of Cybernetics on Hayek from the Sensory Order to the Social Order

This paper explores the ways in which c ybernetics influenced the works of F. A. Hayek from the late 1940s onwar d. It shows that the concept of negative feedback, borrowed from cybernetics, was central to Hayek's attempt of giving an explanation of the principle to the emergence of human purposive behavior. Next, the paper discusses Ha yek's later uses of cybernetic ideas in his works on the spontaneous formation of social orders. Finally, Hayek's view on the appropriate scope of the use of cybernetics is considered

PrCYP707A1, an ABA catabolic gene, is a key component of Phelipanche ramosa seed germination in response to the strigolactone analogue GR24

After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8\u27-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2A, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

An integrated bioinformatics analysis reveals divergent evolutionary pattern of oil biosynthesis in high- and low-oil plants

Seed oils provide a renewable source of food, biofuel and industrial raw materials that is important for humans. Although many genes and pathways for acyl-lipid metabolism have been identified, little is known about whether there is a specific mechanism for high-oil content in high-oil plants. Based on the distinct differences in seed oil content between four high-oil dicots (20~50%) and three low-oil grasses (<3%), comparative genome, transcriptome and differential expression analyses were used to investigate this mechanism. Among 4,051 dicot-specific soybean genes identified from 252,443 genes in the seven species, 54 genes were shown to directly participate in acyl-lipid metabolism, and 93 genes were found to be associated with acyl-lipid metabolism. Among the 93 dicot-specific genes, 42 and 27 genes, including CBM20-like SBDs and GPT2, participate in carbohydrate degradation and transport, respectively. 40 genes highly up-regulated during seed oil rapid accumulation period are mainly involved in initial fatty acid synthesis, triacylglyceride assembly and oil-body formation, for example, ACCase, PP, DGAT1, PDAT1, OLEs and STEROs, which were also found to be differentially expressed between high- and low-oil soybean accessions. Phylogenetic analysis revealed distinct differences of oleosin in patterns of gene duplication and loss between high-oil dicots and low-oil grasses. In addition, seed-specific GmGRF5, ABI5 and GmTZF4 were predicted to be candidate regulators in seed oil accumulation. This study facilitates future research on lipid biosynthesis and potential genetic improvement of seed oil content

Measuring Macroprudential Risk Through Financial Fragility: A Minskyan Approach

This paper presents a method to capture the growth of financial fragility within a country and across countries. This is done by focusing on housing finance in the United States, the United Kingdom, and France. Following the theoretical framework developed by Hyman P. Minsky, the paper focuses on the risk of amplification of shock via a debt deflation instead of the risk of a shock per se. Thus, instead of focusing on credit risk, for example, financial fragility is defined in relation to the means used to service debts, given credit risk and all other sources of shocks. The greater the expected reliance on capital gains and debt refinancing to meet debt commitments, the greater the financial fragility, and so the higher the risk of debt deflation induced by a shock if no government intervention occurs. In the context of housing finance, this implies that the growth of subprime lending was not by itself a source of financial fragility; instead, it was the change in the underwriting methods in all sectors of the mortgage markets that created a financial situation favorable to the emergence of a debt deflation. Stated alternatively, when nonprime and prime mortgage lending moved to asset-based lending instead of income-based lending, the financial fragility of the economy grew rapidly