Effects of exogenous ABA and H₂O₂ on seed germination of different transgenic lines.

<p>Sterilized seeds were germinated at 28°C on sterile filter papers in the petri dishes containing different concentrations of ABA (20 µM) or H₂O₂ (20 mM) or both. Germination (based on radicals >2 mm) was recorded at 72 h after imbibition. Fifty seeds per genotype were used. Data shown are means of three biological replications±SE. Asterisks (*) indicate significant difference (<i>P</i><0.05) between the seed germination of transgenic lines compared with the wildtype.</p

<i>OsRACK1</i> Is Involved in Abscisic Acid- and H₂O₂-Mediated Signaling to Regulate Seed Germination in Rice (<i>Oryza sativa</i>, L.)

<div><p>The receptor for activated C kinase 1 (RACK1) is one member of the most important WD repeat–containing family of proteins found in all eukaryotes and is involved in multiple signaling pathways. However, compared with the progress in the area of mammalian RACK1, our understanding of the functions and molecular mechanisms of RACK1 in the regulation of plant growth and development is still in its infancy. In the present study, we investigated the roles of rice RACK1A gene (<i>OsRACK1A</i>) in controlling seed germination and its molecular mechanisms by generating a series of transgenic rice lines, of which <i>OsRACK1A</i> was either over-expressed or under-expressed. Our results showed that <i>OsRACK1A</i> positively regulated seed germination and negatively regulated the responses of seed germination to both exogenous ABA and H₂O₂. Inhibition of ABA biosynthesis had no enhancing effect on germination, whereas inhibition of ABA catabolism significantly suppressed germination. ABA inhibition on seed germination was almost fully recovered by exogenous H₂O₂ treatment. Quantitative analyses showed that endogenous ABA levels were significantly higher and H₂O₂ levels significantly lower in <i>OsRACK1A</i>-down regulated transgenic lines as compared with those in wildtype or <i>OsRACK1A</i>-up regulated lines. Quantitative real-time PCR analyses showed that the transcript levels of <i>OsRbohs</i> and amylase genes, <i>RAmy1A</i> and <i>RAmy3D</i>, were significantly lower in <i>OsRACK1A</i>-down regulated transgenic lines. It is concluded that <i>OsRACK1A</i> positively regulates seed germination by controlling endogenous levels of ABA and H₂O₂ and their interaction.</p></div

Photoelectrochemical Sensor with a Z‑Scheme Fe₂O₃/CdS Heterostructure for Sensitive Detection of Mercury Ions

Mercury (Hg2+) is a highly toxic element and can seriously affect human health. This work proposed a photoelectrochemical (PEC) sensor with a Z-scheme Fe2O3/CdS heterostructure and two thymine-rich DNA strands (DNA-1 and Au@DNA-2) for sensitive detection of Hg2+. The light excitation of the Fe2O3/CdS composite accelerated the electron transfer among Fe2O3, CdS, and the electrode to produce a stable photocurrent response. Upon the recognition of Hg2+ to thymine bases (T) in two DNA strands to form a stable T-Hg2+-T biomimetic structure, the photocurrent response increased with the increasing concentration of Hg2+ due to the opening of electronic transmission channels from Au nanoparticles to Fe2O3/CdS nanocomposite. Under the optimal conditions screened by the Box–Behnken experiments, the proposed PEC sensor showed excellent analytical performance for Hg2+ detection with high sensitivity, a detection limit of 0.20 pM at a signal-to-noise ratio of 3, high selectivity, a detectable concentration range of 1 pM–100 nM, and acceptable stability. The good recovery and low relative standard deviation for the analysis of Hg2+ in lake and tap water samples demonstrated the potential application of the designed Z-scheme Fe2O3/CdS heterostructure in the PEC detection of heavy metal ions

Comparison of relative H₂O₂ contents among different transgenic rice lines and the effect of ABA treatment on it in imbibed seeds.

<p>For measurement of endogenous H₂O₂ levels of imbibed seeds, twenty seeds were homogenized in 500 µl phosphate buffer (20 mM K₂HPO₄, pH 6.5). After centrifugation, 50 µl of the supernatant was incubated with 0.2 U ml⁻¹ horseradish peroxidase and 100 µM Amplex Red reagent (10-acetyl-3,7-dihydrophenoxazine) at room temperature for 30 min in darkness. The fluorescence was quantified using EpochTM Microplate Reader (BioTek) (excitation at 560 nm and emission at 590 nm). Data shown are means of three biological replications±SE. Asterisks (*) indicate significant difference (<i>P</i><0.05) between the relative levels of transgenic lines compared with the wildtype (A) or 0 µM ABA (B).</p

Expressional profile of OsRACK1A genes (A) and proteins (B) in selected transgenic rice lines.

<p>A, <i>OsRACK1A</i> expression was monitored in seedlings of wildtype (non-transgenic lines, NTL), <i>OsRACK1A</i> over-expressed transgenic lines (OeTL), anti-sense transgenic lines (AsTL) and RNA-interfered transgenic lines (RiTLs), respectively. Relative expression levels were calculated and normalized with respect to <i>OsActin7</i> (LOC_Os11g06390). Results shown are means of three biological replications±SE. Asterisks (*) indicate significant difference (<i>P</i><0.05) between the levels of expression of transgenic lines compared with the wildtype (NTL). B, <i>OsRACK1A</i> expression was analyzed by incubating isolated proteins with polyclonal antibodies against OsRACK1A or OseEF1-α (as reference).</p

Effects of different treatments on seed germination of different transgenic lines.

<p>Sterilized seeds were germinated at 28°C on sterile filter papers in the petri dishes containing different concentrations of ABA (20 µM), fluridone (20 µM), diniconazole (100 µM) or H₂O₂ (20 mM). Germination (based on radicals >2 mm) was recorded at the indicated time points. Fifty seeds per genotype were used. Data shown are means of three biological replications±SE. Asterisks (*) indicate significant difference (<i>P</i><0.05) between the seed germination of transgenic lines compared with the wildtype.</p

Expressional of <i>OsRbohs</i> in imbibed seeds of the selected transgenic rice lines.

<p>Expressions of <i>OsRbohs</i> were monitored in seeds of NTL, OeTL, AsTL and RiTL, respectively after 48 h of imbibition. Relative expression levels were measured by qRT-PCR and normalized with respect to <i>OsActin7</i> (LOC_Os11g06390). Results shown are means of three biological replications±SE. Asterisks (*) indicate significant difference (<i>P</i><0.05) between the levels of expression of transgenic lines compared with the NTL.</p

A working model showing proposed molecular steps of OsRACK1A-mediated signal transduction pathway.

<p>This model presents a schematic of OsRACK1A and other signal molecules involved in the proposed signal transduction pathway during seed germination. The results presented in this work combined with the results reported by Nakashima et al. (2008) to support this signal transduction. Under normal germination conditions, cytosol OsRACK1A moved towards plasma membrane and interacted with OsRAC1 to form a complex. The formed complex then activated downstream OsRboh, and as a result, stimulated H₂O₂ production and seed germination. When responding to external stimuli, which may induce ABA accumulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097120#pone.0097120.s004" target="_blank">Figure S4</a>; Zhu et al, 2009), excess ABA inhibited <i>OsRACK1</i> expression and blocked the formation of OsRACK1A-OsRAC1 complex, and as a consequence, suppressed the expression of <i>OsRbohs</i> and the production of H₂O₂ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097120#pone-0097120-g005" target="_blank">Figure 5</a>). Excess H₂O₂ in return suppressed <i>OsRACK1</i> expression to avoid its hazardous effect (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097120#pone.0097120.s005" target="_blank">Figure S5</a>). In addition, H₂O₂ may also stimulate ABA catabolism and alleviated the inhibitory effect on seed germination.</p

Comparison of endogenous ABA contents in imbibed seeds of different transgenic lines.

<p>ABA analysis was carried out using the radioimmumoassay (RIA) method. Thirty imbibed rice seeds were homogenized in 1 ml of distilled water and then shaken at 4°C overnight. The homogenates were centrifuged at 12,000 g for 10 min at 4°C and the supernatant were used directly for ABA assay. Values shown are means of three biological replications±SE. Asterisks (*) indicate significant difference (<i>P</i><0.05) between the endogenous ABA contents of transgenic lines compared with the wildtype.</p

Photoelectrochemical Immunosensor Based on a 1D Fe₂O₃/3D Cd-ZnIn_2.2S_<i>y</i> Heterostructure as a Sensing Platform for Ultrasensitive Detection of Neuron-Specific Enolase

Lung cancer is a high-mortality cancer related to the concentration of neuron-specific enolase (NSE). In this work, a sandwich-type photoelectrochemical (PEC) immunosensor was constructed for ultrasensitive detection of NSE, which is based on iron trioxide/indium zinc cadmium sulfide (Fe2O3/Cd-ZnIn2.2Sy) as a sensing platform and Ag-modified polyaniline (Ag@PANI) as a signal amplification label. The 1D Fe2O3 porous nanorods with a large specific surface area were synthesized by calcination of Fe-MIL-88A and etching of NaOH. To improve the photocurrent response, the 3D architecture Cd-ZnIn2.2Sy was combined with the 1D Fe2O3 porous nanorods to form a 1D Fe2O3/3D Cd-ZnIn2.2Sy heterostructure. Specifically, the Fe2O3/Cd-ZnIn2.2Sy heterostructure with a good energy level matching (the two can form a stepped energy level matching, which accelerates the transfer rate of electrons) can improve the separation efficiency of electron–hole pairs (e–/h+) under visible light irradiation, which enhances the photocurrent response. Ag@PANI has a strong electron transport capability and can be used as a secondary antibody marker for the signal amplification of the immunosensor. The sensor exhibits a good linear detection range of 100 fg/mL to 100 ng/mL with a low detection limit of 33.5 fg/mL. Moreover, the constructed sandwich-type PEC immunosensor shows good performance and possesses excellent specificity, selectivity, and stability over a period of 4 weeks for NSE detection. With these excellent properties, the immunosensor can be extended to analyze and diagnose other disease biomarkers