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ABSTRACT: We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.
PLoS ONE 01/2012; 7(8):e43072. · 4.09 Impact Factor
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ABSTRACT: Developmental mechanisms that enable perception of and response to the environment may enhance fitness. Ecophysiological traits typically vary depending on local conditions and contribute to resource acquisition and allocation, yet correlations may limit adaptive trait expression. Notably, photosynthesis and stomatal conductance vary diurnally, and the circadian clock, which is an internal estimate of time that anticipates diurnal light/dark cycles, may synchronize physiological behaviors with environmental conditions. Using recombinant inbred lines of Brassica rapa, we examined the quantitative-genetic architecture of ecophysiological and phenological traits and tested their association with the circadian clock. We also investigated how trait expression differed across treatments that simulated seasonal settings encountered by crops and naturalized populations. Many ecophysiological traits were correlated, and some correlations were consistent with expected biophysical constraints; for example, stomata jointly regulate photosynthesis and transpiration by affecting carbon dioxide and water vapor diffusion across leaf surfaces, and these traits were correlated. Interestingly, some genotypes had unusual combinations of ecophysiological traits, such as high photosynthesis in combination with low stomatal conductance or leaf nitrogen, and selection on these genotypes could provide a mechanism for crop improvement. At the genotypic and QTL level, circadian period was correlated with leaf nitrogen, instantaneous measures of photosynthesis, and stomatal conductance as well as with a long-term proxy (carbon isotope discrimination) for gas exchange, suggesting that gas exchange is partly regulated by the clock and thus synchronized with daily light cycles. The association between circadian rhythms and ecophysiological traits is relevant to crop improvement and adaptive evolution.
Genetics 07/2011; 189(1):375-90. · 4.01 Impact Factor
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ABSTRACT: Bioluminescence resonance energy transfer (BRET) has become a widely used technique to monitor protein-protein interactions. It involves resonance energy transfer between a bioluminescent donor and a fluorescent acceptor. Because the donor emits photons intrinsically, fluorescence excitation is unnecessary. Therefore, BRET avoids some of the problems inherent in fluorescence resonance energy transfer (FRET) approaches, such as photobleaching, autofluorescence, and undesirable stimulation of photobiological processes. In the past, BRET signals have generally been too dim to be effectively imaged. Newly available cameras that are more sensitive coupled to image splitter now enable BRET imaging in plant and mammalian cells and tissues. In addition, new substrates and enhanced luciferases enable brighter signals that allow even subcellular BRET imaging. Here, we report methods for BRET imaging of (1) localization of COP1 dimerization in plant cells and tissues and (2) subcellular distributions of interactions of the CCAAT/Enhancer Binding Protein α (C/EBPα) in single mammalian cells. We also discuss methods for the correction of BRET images for tissues that absorb light of different spectra. This progress should catalyze further applications of BRET for imaging and high-throughput assays.
Methods in molecular biology (Clifton, N.J.) 01/2011; 680:3-28.
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ABSTRACT: Circadian clocks provide temporal coordination by synchronizing internal biological processes with daily environmental cycles. To date, study of the plant circadian clock has emphasized Arabidopsis (Arabidopsis thaliana) as a model, but it is important to determine the extent to which this model applies in other species. Accordingly, we have investigated circadian clock function in Brassica rapa. In Arabidopsis, analysis of gene expression in transgenic plants in which luciferase activity is expressed from clock-regulated promoters has proven a useful tool, although technical challenges associated with the regeneration of transgenic plants has hindered the implementation of this powerful tool in B. rapa. The circadian clock is cell autonomous, and rhythmicity has been shown to persist in tissue culture from a number of species. We have established a transgenic B. rapa tissue culture system to allow the facile measurement and manipulation of clock function. We demonstrate circadian rhythms in the expression of several promoter:LUC reporters in explant-induced tissue culture of B. rapa. These rhythms are temperature compensated and are reset by light and temperature pulses. We observe a strong positive correlation in period length between the tissue culture rhythm in gene expression and the seedling rhythm in cotyledon movement, indicating that the circadian clock in B. rapa tissue culture provides a good model for the clock in planta.
Plant physiology 06/2010; 153(2):841-50. · 6.53 Impact Factor
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ABSTRACT: Dodd et al. (Reports, 14 December 2007, p. 1789) reported that the Arabidopsis circadian clock incorporates the signaling molecule cyclic adenosine diphosphate ribose (cADPR). In contrast, we found that there is no rhythm of cADPR levels nor are there any significant effects on the rhythm by cADPR overexpression, thus raising questions about the conclusions of Dodd et al.
Science 10/2009; 326(5950):230; author reply 230. · 31.20 Impact Factor
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ABSTRACT: The circadian coordination of organismal biology with the local temporal environment has consequences for fitness that may become manifest early in development. We directly explored the development of the Arabidopsis (Arabidopsis thaliana) clock in germinating seedlings by monitoring expression of clock genes. Clock function is detected within 2 d of imbibition (hydration of the dried seed). Imbibition is sufficient to synchronize individuals in a population in the absence of entraining cycles of light-dark or temperature, although light-dark and temperature cycles accelerate the appearance of rhythmicity and improve synchrony among individuals. Oscillations seen during the first 2 d following imbibition are dependent on the clock genes LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, ZEITLUPE, GIGANTEA, PSEUDO-RESPONSE REGULATOR7 (PRR7), and PRR9, although later circadian oscillations develop in mutants defective in each of these genes. In contrast to circadian rhythmicity, which developed under all conditions, amplitude was the only circadian parameter that demonstrated a clear response to the light environment; clock amplitude is low in the dark and high in the light. A circadian clock entrainable by temperature cycles in germinating etiolated seedlings may synchronize the buried seedling with the local daily cycles before emergence from the soil and exposure to light.
Plant physiology 08/2008; 147(3):1110-25. · 6.53 Impact Factor
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ABSTRACT: We have reported that Arabidopsis might have genetically distinct circadian oscillators in multiple cell-types.1 Rhythms of CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are 2.5 h longer in phytochromeB mutants in constant red light and in cryptocrome1 cry2 double mutant (hy4-1 fha-1) in constant blue light than the wild-type.2 However, we found that cytosolic free Ca(2+) ([Ca(2+)](cyt)) oscillations were undetectable in these mutants in the same light conditions.1 Furthermore, mutants of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) have short period rhythms of leaf movement but have arrhythmic [Ca(2+)](cyt) oscillations. More important, the timing of cab1-1 (toc1-1) mutant has short period rhythms of CAB2 promoter activity ( approximately 21 h) but, surprisingly, has a wild-type period for circadian [Ca(2+)](cyt) oscillations ( approximately 24 h). In contrast, toc1-2, a TOC1 loss-of-function mutant, has a short period of both CAB2 and [Ca(2+)](cyt) rhythms ( approximately 21 h). Here we discuss the difference between the phenotypes of toc1-1 and toc1-2 and how rhythms of CAB2 promoter activity and circadian [Ca(2+)](cyt) oscillations might be regulated differently.
Plant signaling & behavior 05/2008; 3(5):342-4.
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ABSTRACT: Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca(2+)](cyt)). To dissect the circadian Ca(2+)-signaling network, we monitored circadian [Ca(2+)](cyt) oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca(2+)](cyt). Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca(2+)](cyt), and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca(2+)](cyt) rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca(2+)](cyt). Oscillations of [Ca(2+)](cyt) and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca(2+)](cyt) and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca(2+)](cyt) and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.
The Plant Cell 12/2007; 19(11):3474-90. · 8.99 Impact Factor
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ABSTRACT: FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.
Proceedings of the National Academy of Sciences 07/2007; 104(24):10264-9. · 9.68 Impact Factor
