In the social amoebae Dictyostelium discoideum, periodic synthesis and release of extracellular cyclic AMP (cAMP) guides cell aggregation and commitment to form fruiting bodies. It is unclear whether these oscillations represent an intrinsic property of individual cells or if they only exist as a population-level phenomenon. Here we show by live-cell imaging of intact cell populations that pulses originate from a discrete location despite constant exchange of cells to and from the region. In a perfusion chamber, both isolated single cells and cell populations switch from quiescence to rhythmic activity depending on the level of extracellular cAMP. A quantitative analysis shows that stochastic pulsing of individual cells below the threshold concentration of extracellular cAMP plays a critical role in the onset of collective behavior.

Glycyrrhizin, a major bioactive compound derived from the underground parts of Glycyrrhiza (licorice) plants, is a triterpene saponin that possesses a wide range of pharmacological properties and is used worldwide as a natural sweetener. Because of its economic value, the biosynthesis of glycyrrhizin has received considerable attention. Glycyrrhizin is most likely derived from the triterpene ␤-amyrin, an initial product of the cyclization of 2,3-oxidosqualene. The subsequent steps in glycyrrhizin biosynthesis are believed to involve a series of oxidative reactions at the C-11 and C-30 positions, followed by glycosyl transfers to the C-3 hydroxyl group; however, no genes encoding relevant oxidases or glycosyltransferases have been identified. Here we report the successful identification of CYP88D6, a cytochrome P450 monooxygenase (P450) gene, as a glycyrrhizin-biosynthetic gene, by transcript profilingbased selection from a collection of licorice expressed sequence tags (ESTs). CYP88D6 was characterized by in vitro enzymatic activity assays and shown to catalyze the sequential two-step oxidation of ␤-amyrin at C-11 to produce 11-oxo-␤-amyrin, a possible biosynthetic intermediate between ␤-amyrin and glycyrrhizin. CYP88D6 coexpressed with ␤-amyrin synthase in yeast also catalyzed in vivo oxidation of ␤-amyrin to 11-oxo-␤-amyrin. CYP88D6 expression was detected in the roots and stolons by RT-PCR; however, no amplification was observed in the leaves or stems, which is consistent with the accumulation pattern of glycyrrhizin in planta. These results suggest a role for CYP88D6 as a ␤-amyrin 11-oxidase in the glycyrrhizin pathway.expressed sequence tag ͉ medicinal plant ͉ secondary metabolite ͉ monooxygenase ͉ isoprenoid

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton b-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding b-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-b-amyrin (a possible biosynthetic intermediate between b-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-b-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of b-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

Potatoes (Solanum tuberosum) contain a-solanine and a-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.

In both randomly moving Dictyostelium and mammalian cells, phosphatidylinositol (3,4,5)-trisphosphate and F-actin are known to propagate as waves at the membrane and act to push out the protruding edge. To date, however, the relationship between the wave geometry and the patterns of amoeboid shape change remains elusive. Here, by using phase map analysis, we show that morphology dynamics of randomly moving Dictyostelium discoideum cells can be characterized by the number, topology, and position of spatial phase singularities, i.e., points that represent organizing centers of rotating waves. A single isolated singularity near the cellular edge induced a rotational protrusion, whereas a pair of singularities supported a symmetric extension. These singularities appeared by strong phase resetting due to de novo nucleation at the back of preexisting waves. Analysis of a theoretical model indicated excitability of the system that is governed by positive feedback from phosphatidylinositol (3,4,5)-trisphosphate to PI3-kinase activation, and we showed experimentally that this requires F-actin. Furthermore, by incorporating membrane deformation into the model, we demonstrated that geometries of competing waves explain most of the observed semiperiodic changes in amoeboid morphology.reaction-diffusion | oscillations | excitable media | self-organization | PTEN

Nutrient-deprived Dictyostelium amoebae aggregate to form a multicellular structure by chemotaxis, moving towards propagating waves of cyclic AMP that are relayed from cell to cell. Organizing centres are not formed by founder cells, but are dynamic entities consisting of cores of outwardly rotating spiral waves that self-organize in a homogeneous cell population. Spiral waves are ubiquitously observed in chemical reactions as well as in biological systems. Although feedback control of spiral waves in spatially extended chemical reactions has been demonstrated in recent years, the mechanism by which control is achieved in living systems is unknown. Here we show that mutants of the cyclic AMP/protein kinase A pathway show periodic signalling, but fail to organize coherent long-range wave territories, owing to the appearance of numerous spiral cores. A theoretical model suggests that autoregulation of cell excitability mediated by protein kinase A acts to optimize the number of signalling centres.

Triterpenoid saponins are a diverse group of natural products in plants and are considered defensive compounds against pathogenic microbes and herbivores. Because of their various beneficial properties for humans, saponins are used in wide-ranging applications in addition to medicinally. Saponin biosynthesis involves three key enzymes: oxidosqualene cyclases, which construct the basic triterpenoid skeletons; cytochrome P450 monooxygenases, which mediate oxidations; and uridine diphosphate-dependent glycosyltransferases, which catalyze glycosylations. The discovery of genes committed to saponin biosynthesis is important for the stable supply and biotechnological application of these compounds. Here, we review the identified genes involved in triterpenoid biosynthesis, summarize the recent advances in the biotechnological production of useful plant terpenoids, and discuss the bioengineering of plant triterpenoids.

Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.