Kyeong-Hwan Lee

Characterization of tryptamine 5-hydroxylase and serotonin synthesis in rice plants

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.

The CONSTANS-like 4 transcription factor, AtCOL4, positively regulates abiotic stress tolerance through an abscisic acid-dependent manner in Arabidopsis

The precise roles of the B-box zinc finger family of transcription factors in plant stress are poorly understood. Functional analysis was performed on AtCOL4, an Arabidopsis thaliana L. CONSTANS-like 4 protein that is a putative novel transcription factor, and which contains a predicted transcriptional activation domain. Analyses of an AtCOL4 promoter-β-glucuronidase (GUS) construct revealed substantial GUS activity in whole seedlings. The expression of AtCOL4 was strongly induced by abscisic acid (ABA), salt, and osmotic stress. Mutation in atcol4 resulted in increased sensitivity to ABA and salt stress during seed germination and the cotyledon greening process. In contrast, AtCOL4-overexpressing plants were less sensitive to ABA and salt stress compared to the wild type. Interestingly, in the presence of ABA or salt stress, the transcript levels of other ABA biosynthesis and stress-related genes were enhanced induction in AtCOL4-overexpressing and WT plants, rather than in the atcol4 mutant. Thus, AtCOL4 is involved in ABA and salt stress response through the ABA-dependent signaling pathway. Taken together, these findings provide compelling evidence that AtCOL4 is an important regulator for plant tolerance to abiotic stress.

Fabrication of PDMS through-holes using the MIMIC method and the surface treatment by atmospheric-pressure CH4/He RF plasma

This note presents a simple fabrication process for patterning micro through-holes in a PDMS layer by a combination of the micromolding in capillaries (MIMIC) method and the surface treatment by atmospheric-pressure CH4/He RF plasma. The fabrication process is confirmed by forming micro through-holes with various shapes including circle, C-shape, open microfluidic channel and hemisphere. All micro through-holes of various shapes in a wide range of diameters and heights are well fabricated by the proposed method. Also, a 3D micromixer containing a PDMS micro through-hole layer formed by the proposed method is built and its performance is tested as another practical demonstration of the proposed fabrication method. Therefore, we believe that the proposed fabrication process will build a PDMS micro through-hole layer in a simple and easy way and will contribute to developing highly efficient multi-layered microfluidic systems, which may require PDMS micro through-hole layers.

Rapid detection of Norovirus from Fresh Lettuce Using Immunomagnetic Separation and a Quantum Dots Assay

Current molecular methods that include PCR have been used to detect norovirus in many food samples. However, the protocols require removing PCR inhibitors and incorporate time-consuming concentration steps to separate virus from analyte for rapid and sensitive detection of norovirus. We developed an immunomagnetic separation (IMS) and a quantum dots (QDs) assay to detect norovirus eluted from fresh lettuce with Tris buffer containing 1% beef extract (pH 9.5). IMS facilitated viral precipitation with a 10-min incubation, whereas virus concentration using polyethylene glycol (PEG) requires more than 3 h and an additional high-speed centrifugation step to precipitate virus before reverse transcription PCR (RT-PCR) analysis. The fluorescence intensity of QDs was detected qualitatively on norovirus dilutions of 10(-1) to 10(-3) in a stool suspension (100 RT-PCR units/ml). The results suggest that a fluorescence assay based on IMS and QDs is valid for detecting norovirus qualitatively according to fluorescent signal intensity within the same virus detection limit produced by IMS-RT-PCR and PEG-RT-PCR.

A Melon Fruit Grading Machine Using a Miniature VIS/NIR Spectrometer: 1. Calibration Models for the Prediction of Soluble Solids Content and Firmness

th , 2012; Revised: June 27 th , 2012; Accepted: June 29 th , 2012 Purpose: This study was conducted to investigate the potential of interactance mode of NIR spectroscopy technology for the estimation of soluble solids content (SSC) and firmness of muskmelons. Methods: Melon samples were taken from local greenhouses in three different harvesting seasons (experiments 1, 2, and 3). The fruit attributes were measured at the 6 points on an equator of each sample where the spectral data were collected. The prediction models were developed using the original spectral data and the spectral data sets preproces sed by 20 methods. The performance of the models was compared. Results: In the prediction of SSC, the highest coefficient of determination (Rcv 2 ) values of the cross-validation was 0.755 (standard error of prediction, SEP=0.89°Brix) with the preprocessing of normalization with range in experiment 1. The highest coefficient of determination in the robustness tests, Rrt 2 =0.650 (SEP=1.03 °Brix), was found when the best model of experiment 3 was evaluated with the data set of experiment 2. The best Rcv 2 for the prediction of firmness was 0.715 (SEP=3.63 N) when no preprocessing was applied in experiment 1. The highest Rrt 2 was 0.404 (SEP=5.30 N) when the best model of experiment 3 was applied to the data set of experiment 1. Conclusions: From the test results, it can be concluded that the interactance mode of VIS/NIR spectroscopy te chnology has a great potential to measure SSC and firmness of thick-skinned muskmelons.

AtSKIP functions as a mediator between cytokinin and light signaling pathway in Arabidopsis thaliana

Key messageAtSKIP participated in cytokinin-regulated leaf initiation. Putative phosphorylated AtSKIP (AtSKIPDD) displayed the opposite function in the leaf development from AtSKIP transgenic seedlings.AbstractAtSKIP, as a multiple protein, is involved in many physiological processes, such as flowering, cell cycle regulator, photomorphogenesis and stress tolerance. However, the mechanism of AtSKIP in these processes is unclear. Here, we identify one gene, AtSKIP, which is associated with cytokinin-regulated leaf growth process in Arabidopsis. The expression of AtSKIP was regulated by cytokinin. Leaf development in AtSKIP overproduced seedlings was independent of light, but promoted by cytokinin, and phosphorylation of AtSKIP (AtSKIPDD) partially interfered with AtSKIP function as a positive regulator in cytokinin signaling, indicative of true leaf formation, and the defects of AtSKIPDD in the true leaf formation could be recovered to some extent by the addition of cytokinin. Moreover, different cytokinin-responsive gene AuthenticResponseRegulator7 (ARR7) promoter-GUS activity further proved that expression of AtSKIP or AtSKIPDD altered endogenous cytokinin signaling in plants. Together, these data indicate that AtSKIP participates in cytokinin-regulated promotion of leaf growth in photomorphogenesis, and that phosphorylation interferes with AtSKIP normal function.

A Melon Fruit Grading Machine Using a Miniature VIS/NIR Spectrometer: 2. Design Factors for Optimal Interactance Measurement Setup

th , 2012; Revised: June 27 th , 2012; Accepted: June 29 th , 2012 Purpose: In near infrared spectroscopy, interactance configuration of a light source and a spectrometer probe can provide more information regarding fruit internal attributes, compared to reflectance and transmittance configuration. However, there is no through study on the parameters of interactance measurement setup. The objective of this study was to investigate the effect of the parameters on the estimation of soluble solids content (SSC) and firmness of muskmelons. Methods: Melon samples were taken from greenhouses at three different harvesting seasons. The prediction models were developed at three distances of 2, 5, and 8 cm between the light source and the spectrometer probe, three measurement points of 2, 3, and 6 evenly distributed on each sample, and different number of fruit samples for calibration models. The performance of the models was compared. Results: In the test at the three distances, the best results were foun d at a 5 cm distance. The coefficient of determination (Rcv 2 ) values of the cross-validation were 0.717 (standard error of prediction, SEP=1.16 °Brix) and 0.504 (SEP=4.31 N) for the estimation of SSC and firmness, respectively. The minimum measurement point required to fully represent the spectral characteristics of each fruit sample was 3. The highest Rcv 2 values were 0.736 (SEP=0.87 °Brix) and 0.644 (SEP=4.16 N) for the estimation of SSC and firmness, respectively. The performance of the models began to be saturated when 60 fruit samples were used for developing calibration models. The highest Rcv 2 of 0.713 (SEP=0.88 °Brix) and 0.750 (SEP=3.30 N) for the estimation of SSC and firmness, respectively, were achieved. Conclusions: The performance of the prediction models was quite different according to the condition of interactance measurement setup. In designing a fruit grading machine with interactance configuration, the parameters for interactance measurement setup should be chosen carefully.

PCA22 acts as a suppressor of atrzf1 to mediate proline accumulation in response to abiotic stress in Arabidopsis

Highlight PCA22 negatively regulates the drought response through the modulation of osmolytic components and regulates pollen tube length in Arabidopsis.

Comparison of Performance of Measuring Method of VIS/NIR Spectroscopic Spectrum to Predict Soluble Solids Content of 'Shingo' Pear

Three modes of VIS/NIR spectroscopic measurement (interactance and two modes of transmission) were compared for their ability to estimate soluble solids content (SSC) of ‘Shingo’ pear non-destructively. The two transmission modes are named as full- and semi-transmission, where full-transmission stands for passing of light through abdomen of pear and semi-transmission is for transit of light mainly through flesh of pear. For comparison of the modes, prediction models developed from the collected spectroscopic data by the three modes were developed and tested for comparison of their performance. Partial least square regression (PSLR) was used to develop the models and various pre-processing methods were applied to develop models of high accuracy. The experiment was repeated three times with pears produced in different regions. The experiments resulted that selection of pre-processing is very important to attain accurate models, and multiplicative scatter correction (MSC) was selected as a pre-processor of high accuracy for the three modes of spectroscopic measurement in every experiment. Except for MSC, different group of pre-processing methods were selected for the three modes of measurement in every experiment without any tendency to the tested modes of measurement and pears of different produced region. Root-mean-square error of prediction (RMSEP) of prediction models of the three modes of measurement using prepreocessor of MSC were compared for their ability to estimate SSC. The models resulted in ranges of 0.37~0.57°Brix, 0.65~0.72°Brix, 0.39~0.51°Brix for interactance, full- and semi-transmission, respectively. As shown, modes of semi-transmission and interactance resulted about the same level of prediction accuracy and were noted as modes of high performance to predict SSC.

Optimization of the ninhydrin reaction and development of a multiwell plate-based high-throughput proline detection assay

We developed a high-throughput technique for highly sensitive measurement of trace amounts of proline, an indicator of drought stress in plants, using an optimized proline-ninhydrin reaction. In order to do this, proline detection time was minimized by omitting phosphoric acid from the ninhydrin reagent. Chromophore extraction using toluene was also omitted, thus lowering the risks to environment and human health, and allowing the use of readily available polystyrene plates. Proline detection sensitivity was assessed based on the concentration of sulfosalicylic acid in the solution, which indicated that 1% sulfosalicylic acid yielded the best sensitivity and linearity. These findings were applied to a multiwell plate-based multiplex analysis using a dry oven for the simultaneous analysis of a large number of drought-stressed plant samples with trace amounts of proline. The results showed that proline could be effectively detected in plants grown in soil with water content under 5%, demonstrating its potential for diagnosing drought early. The proposed multiwell plate-based multiplex assay is expected to be useful in manifold agricultural applications.