Comparative Transcriptome Analysis Reveals Candidate Genes Related to Structural and Storage Carbohydrate Biosynthesis in Kelp Saccharina japonica (Laminariales, Phaeophyceae).

Saccharina japonica is a brown macroalga that has been commercially cultivated in China for almost a century. As a natural raw material, it is widely used in the food and pharmaceutical industries, and it may potentially be useful for biofuel production. However, little is known about the genes involved in carbohydrate biosynthesis, and their regulation is less understood. In this study, the analysis of growth traits and alginate and mannitol contents suggested that sporophyte development could be divided into four stages. Accordingly, we performed transcriptome analysis of the S. japonica sporophyte. In total, 589 million clean reads were generated, and 4,514 novel genes were identified. Gene expression analysis revealed that 2,542 genes were differentially expressed. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that these genes were significantly enriched in "Carbon metabolism," "Photosynthesis," and "Photosynthesis-antenna proteins" pathways, which are important for metabolism of various carbohydrates during sporophyte development. Systematic analysis identified the genes encoding enzymes for the biosynthesis of cell wall carbohydrates (including alginate, fucoidan, and cellulose) and cytoplasm storage carbohydrates (mannitol, laminarin, and trehalose). Among them, some key genes associated with carbohydrate content were further identified based on detailed expression profiling, representing good candidates for further functional studies. This study provides a global view of the carbohydrate metabolism process and an important resource for functional genomics studies in S. japonica. The results obtained lay the basis for elucidating the molecular mechanism of carbohydrate biosynthesis and for genetic breeding of carbohydrates-related traits in kelp.

Development of FRET-based dual-excitation ratiometric fluorescent pH probes and their photocaged derivatives.

Dual-excitation ratiometric fluorescent probes allow the measurement of fluorescence intensities at two excitation wavelengths, which should provide a built-in correction for environmental effects. However, most of the small-molecule dual-excitation ratiometric probes that have been reported thus far have shown rather limited separation between the excitation wavelengths (20-70 nm) and/or a very small molar absorption coefficient at one of the excitation wavelengths. These shortcomings can lead to cross-excitation and thus to errors in the measurement of fluorescence intensities and ratios. Herein, we report a FRET-based molecular strategy for the construction of small-molecule dual-excitation ratiometric probes in which the donor and acceptor excitation bands exhibit large separations between the excitation wavelengths and comparable excitation intensities, which is highly desirable for determining the fluorescence intensities and signal ratios with high accuracy. Based on this strategy, we created a coumarin-rhodamine FRET platform that was then employed to develop the first class of FRET-based dual-excitation ratiometric pH probes that have two well-resolved excitation bands (excitation separations>160 nm) and comparable excitation intensities. In addition, these pH probes may be considered as in a kind of "secured ratioing mode". As a further application of these pH probes, the dual-excitation ratiometric pH probes were transformed into the first examples of photocaged dual-excitation ratiometric pH probes to improve the spatiotemporal resolution. It is expected that the modular nature of our FRET-based molecular strategy should render it applicable to other small-molecule dual-dye energy-transfer systems based on diverse fluorescent dyes for the development of a wide range of dual-excitation ratiometric probes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.

Photocontrollable analyte-responsive fluorescent probes: a photocaged copper-responsive fluorescence turn-on probe.

Analyte-responsive fluorescent probes are valuable chemical tools for dissecting complex living systems. However, the major shortcoming of fluorescent probes is that once they enter the cells, control over them is basically lost. It is critical to regulate fluorescent probes in a spatial and temporal manner, as functions of biomolecules are spatiotemporal. On the other hand, light can be manipulated in time and in the application site, so the photocaging technique allows researchers to control the biomolecules of interest in a temporal and spatial fashion. Herein, we propose for the first time the combination of the merits of sensing and photocaging technologies, which may afford the caging version of analyte-responsive fluorescent probes, referred to as photocontrollable analyte-responsive fluorescent probes (PCAFPs). These "smart" fluorescent probes apparently have the intrinsic advantage of spatiotemporal control when compared to traditional fluorescent probes, as the "sensing activity" of PCAFPs is photocontrollable. This should enable biologists to interrogate complex biological systems in a spatial and temporal manner with an innovative chemical tool. In this work, for proof of concept, we report the rational design, synthesis, photocontrollable sensing in solution and in living cells, and mechanistic studies of a molecular prototype of PCAFP for copper as the first paradigm of this new class of smart fluorescent probes. We believe that PCAFPs represent a substantial breakthrough in the sensing and photocaging fields, and that the general concept of PCAFPs should be broadly applicable for a wide variety of biologically relevant species.

A sensitive and selective fluorescent thiol probe in water based on the conjugate 1,4-addition of thiols to alpha,beta-unsaturated ketones.

Compound 1 was designed and synthesized as a new fluorescent thiol probe. Probe 1 was constructed on the basis of the conjugate 1,4-addition of thiols to alpha,beta-unsaturated ketones. Notably, probe 1 has suitable water solubility, which allows the sensing assay to be performed in water. Probe 1 is highly sensitive for thiols with a 211-fold fluorescence dynamic range and a low detection limit of 9.25x10(-7) M. The major features of probe 1 also include a high selectivity for thiols over other relevant biological species, excitation and emission in the visible region, rapid functioning at pH 7.4, and a good linear relationship between the fluorescence signal and the thiol concentration. Accordingly, these desirable characteristics may render probe 1 as potentially useful for biological applications.

Improving seedless kelp (Saccharina japonica) during its domestication by hybridizing gametophytes and seedling-raising from sporophytes.

Dongfang no.7 (Saccharina japonica) was bred and maintained by hybridizing gametophytes, self-crossing the best individuals, selecting the best self-crossing line and seedling-raising from yearly reconstructed sporophytes. It increased the air dry yield by 43.2% in average over 2 widely farmed controls. Dongfang no.7 was seedling-raised from bulked sporophytes reconstructed from its representative gametophyte clones. Such strategy ensured it against variety contamination due to possible cross fertilization and occasional mixing and inbred depletion due to self-crossing number-limited sporophytes year after year. It derived from an intraspecific hybrid through 4 rounds of self-crossing and selection and retained a certain degree of genetic heterozygosity, thus being immune to inbred depletion due to purification of unknown detrimental alleles. Most importantly, it can be farmed in currently available system as the seedlings for large scale culture can be raised from reconstructed Dongfang no.7 sporophytes. Breeding and maintaining Dongfang no.7 provided a model that other varieties of kelp (S. japonica) and brown algae may follow during their domestication.

A novel ratiometric fluorescent Fe3+ sensor based on a phenanthroimidazole chromophore.

Phenanthroimidazole derivative 1 has been developed as a rare example of ratiometric fluorescent sensors for Fe(3+). Interestingly, upon treatment with Fe(3+), the sensor displayed a ratiometric fluorescent response with an enhancement of the ratios of emission intensities at 440 and 500 nm from 0.36 to 3.24. The detection range of the sensor for Fe(3+) is in the 1.0x10(-5)-1.5x10(-4) M concentration range and the detection limit is 5.26x10(-6) M. In addition, the sensor showed good selectivity to Fe(3+) with the selectivity coefficients (K(Fe3+) = S(Fe3+)/S(0) of Fe(3+) over other metal ions tested in the range of 5-68.

A ratiometric fluorescent probe for cysteine and homocysteine displaying a large emission shift.

4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzaldehyde 1 was rationally designed as a novel ratiometric fluorescent probe for cysteine and homocysteine. Upon addition of cysteine or homocysteine, notably, the probe displayed a very large (125 nm) hypsochromic shift in emission due to switching off intramolecular charge transfer. This large emission wavelength shift may allow probe 1 to be employed for quantitatively detecting Cys/Hcy.