[4 + 2] Cycloaddition of Vinylphosphine Oxides to α-Oxy- o-xylylene as a route to Phosphorylated Naphthyl and Biaryl Scaffolds.

α-Oxy- o-xylylene, a highly reactive diene readily accessible from benzocyclobutenol, undergoes Diels-Alder reaction with vinylphosphine oxides, yielding the corresponding 2-phosphorylated 1-hydroxy-1,2,3,4-tetrahydronaphthalenes in excellent yields. Use of unsubstituted and trans-2-aryl-substituted vinylphosphine oxides leads to cycloadducts with complete regioselectivity and with cis/trans selectivity up to 19:1 in the most favorable case. In the case of P-stereogenic trans-2-aryl-substituted vinylphosphine oxides, a virtually complete chirality transfer from P to C can be achieved. Dehydration and aromatization of the obtained cycloadducts bearing the resolved P-stereogenic phosphinoyl groups can be carried out to afford the valuable P-stereogenic and axially chiral phosphorylated 1,2'-binaphthyl ring system. Cases of restricted rotation around Csp3-Csp2 single bond in some tetrahydronaphthalene cycloadducts have also been revealed.

Multiple circadian clock outputs regulate diel turnover of carbon and nitrogen reserves.

Plants accumulate reserves in the daytime to support growth at night. Circadian regulation of diel reserve turnover was investigated by profiling starch, sugars, glucose 6-phosphate, organic acids, and amino acids during a light-dark cycle and after transfer to continuous light in Arabidopsis wild types and in mutants lacking dawn (lhy cca1), morning (prr7 prr9), dusk (toc1, gi), or evening (elf3) clock components. The metabolite time series were integrated with published time series for circadian clock transcripts to identify circadian outputs that regulate central metabolism. (a) Starch accumulation was slower in elf3 and prr7 prr9. It is proposed that ELF3 positively regulates starch accumulation. (b) Reducing sugars were high early in the T-cycle in elf3, revealing that ELF3 negatively regulates sucrose recycling. (c) The pattern of starch mobilization was modified in all five mutants. A model is proposed in which dawn and dusk/evening components interact to pace degradation to anticipated dawn. (d) An endogenous oscillation of glucose 6-phosphate revealed that the clock buffers metabolism against the large influx of carbon from photosynthesis. (e) Low levels of organic and amino acids in lhy cca1 and high levels in prr7 prr9 provide evidence that the dawn components positively regulate the accumulation of amino acid reserves.

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day.

We investigated whether starch degradation occurs at the same time as starch synthesis in Arabidopsis (Arabidopsis thaliana) leaves in the light. Starch accumulated in a linear fashion for about 12 h after dawn, then accumulation slowed and content plateaued. Following decreases in light intensity, the rate of accumulation of starch declined in proportion to the decline in photosynthesis if the decrease occurred <10 h after dawn, but accumulation ceased or loss of starch occurred if the same decrease in light intensity was imposed more than 10 h after dawn. These changes in starch accumulation patterns after prolonged periods in the light occurred at both high and low starch contents and were not related to time-dependent changes in either the rate of photosynthesis or the partitioning of assimilate between starch and Suc, as assessed from metabolite measurements and 14CO2 pulse experiments. Instead, measurements of incorporation of 13C from 13CO2 into starch and of levels of the starch degradation product maltose showed that substantial starch degradation occurred simultaneously with synthesis at time points >14 h after dawn and in response to decreases in light intensity that occurred >10 h after dawn. Starch measurements in circadian clock mutants suggested that the clock influences the timing of onset of degradation. We conclude that the propensity for leaf starch to be degraded increases with time after dawn. The importance of this phenomenon for efficient use of carbon for growth in long days and for prevention of starvation during twilight is discussed.

Circadian, Carbon, and Light Control of Expansion Growth and Leaf Movement.

We used Phytotyping4D to investigate the contribution of clock and light signaling to the diurnal regulation of rosette expansion growth and leaf movement in Arabidopsis (Arabidopsis thaliana). Wild-type plants and clock mutants with a short (lhycca1) and long (prr7prr9) period were analyzed in a T24 cycle and in T-cycles that were closer to the mutants' period. Wild types also were analyzed in various photoperiods and after transfer to free-running light or darkness. Rosette expansion and leaf movement exhibited a circadian oscillation, with superimposed transients after dawn and dusk. Diurnal responses were modified in clock mutants. lhycca1 exhibited an inhibition of growth at the end of night and growth rose earlier after dawn, whereas prr7prr9 showed decreased growth for the first part of the light period. Some features were partly rescued by a matching T-cycle, like the inhibition in lhycca1 at the end of the night, indicating that it is due to premature exhaustion of starch. Other features were not rescued, revealing that the clock also regulates expansion growth more directly. Expansion growth was faster at night than in the daytime, whereas published work has shown that the synthesis of cellular components is faster in the day than at nighttime. This temporal uncoupling became larger in short photoperiods and may reflect the differing dependence of expansion and biosynthesis on energy, carbon, and water. While it has been proposed that leaf expansion and movement are causally linked, we did not observe a consistent temporal relationship between expansion and leaf movement.

Cellulose Synthesis and Cell Expansion Are Regulated by Different Mechanisms in Growing Arabidopsis Hypocotyls.

Plant growth is sustained by two complementary processes: biomass biosynthesis and cell expansion. The cell wall is crucial to both as it forms the majority of biomass, while its extensibility limits cell expansion. Cellulose is a major component of the cell wall and cellulose synthesis is pivotal to plant cell growth, and its regulation is poorly understood. Using periodic diurnal variation in Arabidopsis thaliana hypocotyl growth, we found that cellulose synthesis and cell expansion can be uncoupled and are regulated by different mechanisms. We grew Arabidopsis plants in very short photoperiods and used a combination of extended nights, continuous light, sucrose feeding experiments, and photosynthesis inhibition to tease apart the influences of light, metabolic, and circadian clock signaling on rates of cellulose biosynthesis and cell wall biomechanics. We demonstrate that cell expansion is regulated by protein-mediated changes in cell wall extensibility driven by the circadian clock. By contrast, the biosynthesis of cellulose is controlled through intracellular trafficking of cellulose synthase enzyme complexes regulated exclusively by metabolic signaling related to the carbon status of the plant and independently of the circadian clock or light signaling.

Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation.

The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes.

Nanostructured carbon materials decorated with organophosphorus moieties: synthesis and application.

A new synthetic approach for the production of carbon nanomaterials (CNM) decorated with organophosphorus moieties is presented. Three different triphenylphosphine oxide (TPPO) derivatives were used to decorate oxidized multiwalled carbon nanotubes (ox-MWCNTs) and graphene platelets (GPs). The TPPOs chosen bear functional groups able to react with the CNMs by Tour reaction (an amino group), nitrene cycloaddition (an azido group) or CuAAC reaction (one terminal C-C triple bond). All the adducts were characterized by FTIR, Raman spectroscopy, TEM, XPS, elemental analysis and ICP-AES. The cycloaddition of nitrene provided the higher loading on ox-MWCNTs and GPs as well, while the Tour approach gave best results with nanotubes (CNTs). Finally, we investigated the possibility to reduce the TPPO functionalized CNMs to the corresponding phosphine derivatives and applied one of the materials produced as heterogeneous organocatalyst in a Staudinger ligation reaction.

The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions.

Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound.

Photoperiod-dependent changes in the phase of core clock transcripts and global transcriptional outputs at dawn and dusk in Arabidopsis.

Plants use the circadian clock to sense photoperiod length. Seasonal responses like flowering are triggered at a critical photoperiod when a light-sensitive clock output coincides with light or darkness. However, many metabolic processes, like starch turnover, and growth respond progressively to photoperiod duration. We first tested the photoperiod response of 10 core clock genes and two output genes. qRT-PCR analyses of transcript abundance under 6, 8, 12 and 18 h photoperiods revealed 1-4 h earlier peak times under short photoperiods and detailed changes like rising PRR7 expression before dawn. Clock models recapitulated most of these changes. We explored the consequences for global gene expression by performing transcript profiling in 4, 6, 8, 12 and 18 h photoperiods. There were major changes in transcript abundance at dawn, which were as large as those between dawn and dusk in a given photoperiod. Contributing factors included altered timing of the clock relative to dawn, light signalling and changes in carbon availability at night as a result of clock-dependent regulation of starch degradation. Their interaction facilitates coordinated transcriptional regulation of key processes like starch turnover, anthocyanin, flavonoid and glucosinolate biosynthesis and protein synthesis and underpins the response of metabolism and growth to photoperiod.

Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure.

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell(-1)) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell(-1)) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible.

Selenitetriglycerides-Redox-active agents.

BACKGROUND: Human prostate cancer (hPCa) is the most commonly diagnosed cancer in elderly men and is the second leading cause of male cancer death. Data from epidemiological, eco-environmental, nutritional prevention and clinical trials suggest that selenium Se(IV) can prevent prostate cancer. Selol, a new organic semisynthetic derivative of Se(IV), is a mixture of selenitetriglycerides. This mixture is non-toxic and non-mutagenic, and after po treatment - 56-times less toxic (in mice) than sodium selenite. It exhibits strong anti-cancer activity in vitro in many cancer cell lines and can overcome the cell resistance to doxorubicin. Selol seems a promising compound for prostate cancer therapy. MATERIALS AND METHODS: The aim of the present study is the evaluation of Selol's influence on intracellular redox state (Eh) of prostatic tumors and the liver in androgen-dependent hPCa-bearing mice, and extracellular redox state in serum of these mice. RESULTS AND CONCLUSIONS: The anticancer activity of Selol involves perturbation of the redox regulation in the androgen dependent hPCa (LNCaP) cells, but not in healthy cells. After Selol treatment, intracellular Eh has increased in tumors from -223 mV to -175 mV, while in serum it has decreased (-82 mV vs -113 mV). It shows significant changes Eh in the extra- and intracellular environment. The difference decreases from 141 mV to 62 mV. The changes suggest that a tumor cell was probably directed toward apoptosis. This is exemplified in a significant decrease in cancer tumor mass by approx. 17% after the three weeks of Selol administration.

Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf starch synthesis in different photoperiods.

Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5'-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated.

Multiscale digital Arabidopsis predicts individual organ and whole-organism growth.

Understanding how dynamic molecular networks affect whole-organism physiology, analogous to mapping genotype to phenotype, remains a key challenge in biology. Quantitative models that represent processes at multiple scales and link understanding from several research domains can help to tackle this problem. Such integrated models are more common in crop science and ecophysiology than in the research communities that elucidate molecular networks. Several laboratories have modeled particular aspects of growth in Arabidopsis thaliana, but it was unclear whether these existing models could productively be combined. We test this approach by constructing a multiscale model of Arabidopsis rosette growth. Four existing models were integrated with minimal parameter modification (leaf water content and one flowering parameter used measured data). The resulting framework model links genetic regulation and biochemical dynamics to events at the organ and whole-plant levels, helping to understand the combined effects of endogenous and environmental regulators on Arabidopsis growth. The framework model was validated and tested with metabolic, physiological, and biomass data from two laboratories, for five photoperiods, three accessions, and a transgenic line, highlighting the plasticity of plant growth strategies. The model was extended to include stochastic development. Model simulations gave insight into the developmental control of leaf production and provided a quantitative explanation for the pleiotropic developmental phenotype caused by overexpression of miR156, which was an open question. Modular, multiscale models, assembling knowledge from systems biology to ecophysiology, will help to understand and to engineer plant behavior from the genome to the field.

Adjustment of carbon fluxes to light conditions regulates the daily turnover of starch in plants: a computational model.

In the light, photosynthesis provides carbon for metabolism and growth. In the dark, plant growth depends on carbon reserves that were accumulated during previous light periods. Many plants accumulate part of their newly-fixed carbon as starch in their leaves in the day and remobilise it to support metabolism and growth at night. The daily rhythms of starch accumulation and degradation are dynamically adjusted to the changing light conditions such that starch is almost but not totally exhausted at dawn. This requires the allocation of a larger proportion of the newly fixed carbon to starch under low carbon conditions, and the use of information about the carbon status at the end of the light period and the length of the night to pace the rate of starch degradation. This regulation occurs in a circadian clock-dependent manner, through unknown mechanisms. We use mathematical modelling to explore possible diurnal mechanisms regulating the starch level. Our model combines the main reactions of carbon fixation, starch and sucrose synthesis, starch degradation and consumption of carbon by sink tissues. To describe the dynamic adjustment of starch to daily conditions, we introduce diurnal regulators of carbon fluxes, which modulate the activities of the key steps of starch metabolism. The sensing of the diurnal conditions is mediated in our model by the timer α and the "dark sensor"ß, which integrate daily information about the light conditions and time of the day through the circadian clock. Our data identify the ß subunit of SnRK1 kinase as a good candidate for the role of the dark-accumulated component ß of our model. The developed novel approach for understanding starch kinetics through diurnal metabolic and circadian sensors allowed us to explain starch time-courses in plants and predict the kinetics of the proposed diurnal regulators under various genetic and environmental perturbations.

Arabidopsis coordinates the diurnal regulation of carbon allocation and growth across a wide range of photoperiods.

In short photoperiods, plants accumulate starch more rapidly in the light and degrade it more slowly at night, ensuring that their starch reserves last until dawn. To investigate the accompanying changes in the timing of growth, Arabidopsis was grown in a range of photoperiods and analyzed for rosette biomass, photosynthesis, respiration, ribosome abundance, polysome loading, starch, and over 40 metabolites at dawn and dusk. The data set was used to model growth rates in the daytime and night, and to identify metabolites that correlate with growth. Modeled growth rates and polysome loading were high in the daytime and at night in long photoperiods, but decreased at night in short photoperiods. Ribosome abundance was similar in all photoperiods. It is discussed how the amount of starch accumulated in the light period, the length of the night, and maintenance costs interact to constrain growth at night in short photoperiods, and alter the strategy for optimizing ribosome use. Significant correlations were found in the daytime and the night between growth rates and the levels of the sugar-signal trehalose 6-phosphate and the amino acid biosynthesis intermediate shikimate, identifying these metabolites as hubs in a network that coordinates growth with diurnal changes in the carbon supply.

Genome-wide association mapping of leaf metabolic profiles for dissecting complex traits in maize.

The diversity of metabolites found in plants is by far greater than in most other organisms. Metabolic profiling techniques, which measure many of these compounds simultaneously, enabled investigating the regulation of metabolic networks and proved to be useful for predicting important agronomic traits. However, little is known about the genetic basis of metabolites in crops such as maize. Here, a set of 289 diverse maize inbred lines was genotyped with 56,110 SNPs and assayed for 118 biochemical compounds in the leaves of young plants, as well as for agronomic traits of mature plants in field trials. Metabolite concentrations had on average a repeatability of 0.73 and showed a correlation pattern that largely reflected their functional grouping. Genome-wide association mapping with correction for population structure and cryptic relatedness identified for 26 distinct metabolites strong associations with SNPs, explaining up to 32.0% of the observed genetic variance. On nine chromosomes, we detected 15 distinct SNP-metabolite associations, each of which explained more then 15% of the genetic variance. For lignin precursors, including p-coumaric acid and caffeic acid, we found strong associations (P values to ) with a region on chromosome 9 harboring cinnamoyl-CoA reductase, a key enzyme in monolignol synthesis and a target for improving the quality of lignocellulosic biomass by genetic engineering approaches. Moreover, lignin precursors correlated significantly with lignin content, plant height, and dry matter yield, suggesting that metabolites represent promising connecting links for narrowing the genotype-phenotype gap of complex agronomic traits.