Differences in reward removal efficiency and its consequences for seed germination in the ant-dispersed sedge <Emphasis Type="Italic">Carex tristachya</Emphasis> (Cyperaceae)

Reward removal is an essential step for seed dispersal mutualism because residual rewards inhibit germination. Nevertheless, variation in the reward removal efficiency (RRE) among dispersers and its consequences for germination have rarely been reported. In this study, we compared the RREs of two sympatric seed-dispersing ants, Formica japonica and Pheidole noda, using seeds of the ant-dispersed sedge Carex tristachya. Then, we conducted seed sowing experiments in a non-heated glasshouse to evaluate the effect of RRE on the percentage and speed of germination. The majority (85%) of seeds handled by F. japonica had residual elaiosomes, while elaiosomes were completely removed from all seeds handled by P. noda, demonstrating that P. noda has much higher RRE than F. japonica. The seed sowing experiments revealed that RRE, defined by the presence or absence of residual elaiosomes, was not associated with the percentage germination within a year. However, high RRE seeds with no residual elaiosomes germinated significantly faster than low RRE seeds with residual elaiosomes. Similarly, artificial removal of elaiosomes from C. tristachya seeds accelerated germination speed without affecting germination percentage. These results suggest that RRE is one of the most important parameters determining the effectiveness of a seed dispersal agent.     

Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct

A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153A_{M. aq.}-CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l^–1 ω-hydroxy dodecanoic from 10 g l^–1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l^–1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.     

Post-translational regulation of the Arabidopsis circadian clock through selective proteolysis and phosphorylation of pseudo-response regulator proteins

The circadian clock controls the period, phasing, and amplitude of processes that oscillate with a near 24-h rhythm. One core group of clock components in Arabidopsis that controls the pace of the central oscillator is comprised of five PRR (pseudo-response regulator) proteins whose biochemical function in the clock remains unclear. Peak expression of TOC1 (timing of cab expression 1)/PRR1, PRR3, PRR5, PRR7, and PRR9 are each phased differently over the course of the day and loss of any PRR protein alters period. Here we show that, together with TOC1, PRR5 is the only other likely proteolytic substrate of the E3 ubiquitin ligase SCF(ZTL) within this PRR family. We further demonstrate a functional significance for the phosphorylated forms of PRR5, TOC1, and PRR3. Each PRR protein examined is nuclear-localized and is differentially phosphorylated over the circadian cycle. The more highly phosphorylated forms of PRR5 and TOC1 interact best with the F-box protein ZTL (ZEITLUPE), suggesting a mechanism to modulate their proteolysis. In vivo degradation of both PRR5 and ZTL is inhibited by blue light, likely the result of blue light photoperception by ZTL. TOC1 and PRR3 interact in vivo and phosphorylation of both is necessary for their optimal binding in vitro. Additionally, because PRR3 and ZTL both interact with TOC1 in vivo via the TOC1 N terminus, taken together these data suggest that the TOC1/PRR3 phosphorylation-dependent interaction may protect TOC1 from ZTL-mediated degradation, resulting in an enhanced amplitude of TOC1 cycling.