Identification Of Heavy Metal Transporters

Increased release of heavy metals of geogenic and anthropogenic origin has led to generation of multiple polluted sites in the USA and across the world that are waiting for efficient clean-up technologies. Phytoremediation uses plants to mitigate these types of environmental problems, and provides a cost-efficient and environmentally friendly alternative to existing remediation solutions. Application of phytoremediation relies on the understanding of mechanisms of heavy metal detoxification in plants. As one of the most important heavy metal detoxification mechanisms, the phytochelatin dependent pathway plays an essential role in detoxification and sequestration of heavy metals in plants, fungi and some nematodes. While the mechanism of phytochelatin biosynthesis has been well established, proteins mediating transport of phytochelatins and/or heavy metal phytochelatin complexes have eluded definition. My PhD research led to the following findings: firstly, it was found that Arabidopsis Oligopeptide Transporter 3 (OPT3) mediates transport of cadmium and contributes to shoot accumulation of glutathione and phytochelatins. Secondly, in collaboration with three other research groups, it was shown that an ATPbinding cassette transporter of Schizosaccharomyces pombe, Abc2, is the long-sought phytochelatin transporter on the vacuolar membrane. Identification of such transporter(s) greatly improves the understanding of heavy metal detoxification mechanisms and provides promising bioengineering target(s) for phytoremediation applications. Thirdly, an efficient reverse genetic method was developed to study the function of genes of interest through RNA interference in plant protoplasts. The protocol has been developed for Arabidopsis thaliana; however, since protoplasts can be isolated from different tissues and different plant species, direct transfer of synthetic double stranded RNA into protoplasts can be employed as a gene-silencing tool to study tissue specific processes in a variety of species, and can be adapted to a high-throughput format

Ectopic Expression of OLEOSIN 1 and Inactivation of GBSS1 Have a Synergistic Effect on Oil Accumulation in Plant Leaves

During the transformation of wild-type (WT) Arabidopsis thaliana, a T-DNA containing OLEOSIN-GFP (OLE1-GFP) was inserted by happenstance within the GBSS1 gene, resulting in significant reduction in amylose and increase in leaf oil content in the transgenic line (OG). The synergistic effect on oil accumulation of combining gbss1 with the expression of OLE1-GFP was confirmed by transforming an independent gbss1 mutant (GABI_914G01) with OLE1-GFP. The resulting OLE1-GFP/gbss1 transgenic lines showed higher leaf oil content than the individual OLE1-GFP/WT or single gbss1 mutant lines. Further stacking of the lipogenic factors WRINKLED1, Diacylglycerol O-Acyltransferase (DGAT1), and Cys-OLEOSIN1 (an engineered sesame OLEOSIN1) in OG significantly elevated its oil content in mature leaves to 2.3% of dry weight, which is 15 times higher than that in WT Arabidopsis. Inducible expression of the same lipogenic factors was shown to be an effective strategy for triacylglycerol (TAG) accumulation without incurring growth, development, and yield penalties

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts

Establishing RNA Interference as a Reverse-Genetic Approach for Gene Functional Analysis in Protoplasts1[C][OA]

Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Because protoplasts are nongrowing cells, effective RNAi-triggered gene silencing depends not only on a depletion of gene transcripts but also on turnover rates of corresponding polypeptides. Herein, we tested if transient RNAi in protoplasts would result in the depletion of a targeted polypeptide and, because protoplasts have a limited life span, if functional assays of RNAi knockout genes would be feasible in protoplasts. We showed that protoplasts transfection with an in vitro-synthesized dsRNA against Arabidopsis (Arabidopsis thaliana) β-glutamylcysteine synthase (ECS1), a key enzyme in the synthesis of glutathione, resulted in a 95% depletion of ECS1 transcript, a 72% decrease of ECS1 polypeptide, and a 60% drop in glutathione content. These results were comparable with those obtained upon analysis of Arabidopsis seedlings bearing the cad2-1 mutant allele of ECS1. We also improved the procedure for RNAi inactivation of several genes simultaneously. Finally, because we isolated protoplasts from tissues of 14-d-old seedlings instead of 1-month-old mature plants, the described procedure is rapid (as it only takes 20 d from seed planting to functional studies), suitable for analyzing multiple genes in parallel, and independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost- and space-efficient initial screening and selection of genes for subsequent in planta studies

Absolute Measurement of the Refractive Index of Water by a Mode-Locked Laser at 518 nm

In this paper, we demonstrate a method using a frequency comb, which can precisely measure the refractive index of water. We have developed a simple system, in which a Michelson interferometer is placed into a quartz-glass container with a low expansion coefficient, and for which compensation of the thermal expansion of the water container is not required. By scanning a mirror on a moving stage, a pair of cross-correlation patterns can be generated. We can obtain the length information via these cross-correlation patterns, with or without water in the container. The refractive index of water can be measured by the resulting lengths. Long-term experimental results show that our method can measure the refractive index of water with a high degree of accuracy—measurement uncertainty at 10−5 level has been achieved, compared with the values calculated by the empirical formula

Study on Plasmon-enhanced Photoemission from Au Nanoparticles and Cesiated Surfaces

The plasmon-enhanced photoemission based on Au nanoparticles  and cesiated surfaces is investigated. Using the continuous laser and linearly polarized picosecond pulsed laser with 532nm wavelength, the electron emission performance is recorded under a low bias voltage. The experiment results indicate that the improvement of quantum efficiency is attributed to the plasmon-enhanced light absorption and localized electric field. With the increase of laser intensity, the electron emission mechanism is transmitted from photon absorption mechanism to strong-field tunneling emission

Electrically Tunable Polymer Whispering-Gallery-Mode Laser

Microlasers hold great promise for the development of photonics and optoelectronics. At present, tunable microcavity lasers, especially regarding in situ dynamic tuning, are still the focus of research. In this study, we combined a 0.7Pb(Mg1/3Nb2/3)O3-0.3PbTiO3 (PMN-PT) piezoelectric crystal with a Poly [9,9-dioctylfluorenyl-2,7-diyl] (PFO) microring cavity to realize a high-quality, electrically tunable, whispering-gallery-mode (WGM) laser. The dependence of the laser properties on the diameter of the microrings, including the laser spectrum and quality (Q) value, was investigated. It was found that with an increase in microring diameter, the laser emission redshifted, and the Q value increased. In addition, the device effectively achieved a blueshift under an applied electric field, and the wavelength tuning range was 0.71 nm. This work provides a method for in situ dynamic spectral modulation of microcavity lasers, and is expected to provide inspiration for the application of integrated photonics technology

Determination of Enantiomeric Excess by Optofluidic Microlaser near Exceptional Point

Abstract Enantiomeric excess (ee) is an essential indicator of chiral drug purification in the pharmaceutical industry. However, to date the ee determination of unknown concentration enantiomers generally involves two separate techniques for chirality and concentration measurement. Here, a whispering‐gallery mode (WGM) based optofluidic microlaser near exceptional point to achieve the ee determination under unknown concentration with a single technique is proposed. Exceptional point induces the unidirectional WGM lasing, providing the optofluidic microlaser with the novel capability to measure chirality by polarization, in addition to wavelength‐based concentration detection. The dual‐parameters detection of optofluidic microlaser empowers it to achieve ee determination of various unknown enantiomers without additional concentration measurements, a feat that is challenging to accomplish with other methods. Featuring the sensitivity enhancement and miniature structure of the WGM sensors, the obtained chiroptical response of the present approach is ≈30‐fold higher than that of the conventional optical rotation‐based polarimeter, and the reagent consumption is reduced by three orders of magnitude