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71.
Background
The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. 相似文献72.
Wolfgang R Engelsberger Alexander Erban Joachim Kopka Waltraud X Schulze 《Plant methods》2006,2(1):14-11
Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that
address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population.
Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic
mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled
cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement.
Protein extracts from unlabeled and from standardized 15N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental
variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together
ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy
has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of
labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic 15N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen
focused metabolomics. 相似文献
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Molchanov AG 《Tree physiology》2000,20(17):1137-1148
Absorption and utilization of photosynthetically active radiation (PAR) were investigated in Scots pine (Pinus sylvestris L.) and birch (Betula pendula Roth.) stands that were 41 years old at the end of the experimental period. Canopy depth of the Scots pine stand was about half that of the birch stand (6.5 versus 11.0 m), but absorption of PAR was similar in the two stands. The Scots pine forest canopy, with a leaf area index of 8.9, absorbed 90% of the incoming PAR (APAR), whereas the birch forest canopy, with a leaf area index of 5.9, absorbed 92% of APAR. During maximum foliage development, the upper Scots pine canopy absorbed more PAR than the upper birch canopy (75 versus 66%). The upper, middle and lower layers of the Scots pine canopy contained 37, 48 and 15% of the total needle surface area, respectively. The corresponding distribution of foliage surface area in the three layers of the birch canopy was 50, 30 and 20%, respectively. Measurements of photosynthetic rate were combined with estimates of leaf area index and stand phytomass to determine rates of primary production on a sunny day, a cloudy day, and on an annual basis. The energy equivalents of short- and long-term carbon gain were used with determinations of APAR to calculate photosynthetic utilization efficiency. Throughout the growing season, photosynthetic utilization efficiency of APAR in the upper canopy layer of the Scots pine forest was almost twice that in the lower canopy layer. In the birch forest, photosynthetic utilization efficiency was greater in the lower canopy layer than in the upper canopy layer. In all cases, utilization efficiency was higher in the birch stand than in the Scots pine stand (52 versus 29 J kJ(-1)). Taking account of respiration of the non-photosynthetic parts of each stand (night respiration of needles or leaves; respiration of branches, trunk and roots), estimated utilization efficiency of APAR for net primary production was 11 J kJ(-1) for Scots pine and 19 J kJ(-1) for birch. Solar conversion ratios, expressed as whole-plant net primary productivity per unit of APAR for the growing season, were 0.81 g MJ(-1) for Scots pine and 0.93 g MJ(-1) for birch. 相似文献
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Aluminum (Al) distribution among several cellular fractions was investigated in root tips of seedlings of one Al-resistant and one Al-sensitive family of slash pine (Pinus elliottii Engelm.) and loblolly pine (Pinus taeda L.) grown in nutrient solution containing 100 microM AlCl3 (pH 4) for 167 h. Aluminum present in 5-mm-long root tips was fractionated into cell-wall-labile (desorbed in 0.5 mM citric acid), cell-wall-bound (retained after filtering disrupted cells through 20-microm mesh) and symplasmic (filtrate following cell disruption) fractions. When averaged across both species, 12% of Al absorbed by root tips appeared in the symplasmic fraction and 88% in the apoplasmic fraction (55% as cell-wall-labile, and 33% as cell-wall-bound). On a fresh mass basis, total Al in root tips was lower in loblolly pine than in slash pine, lower in the Al-resistant slash pine family than in the Al-sensitive slash pine family, and lower in the Al-resistant families than in the Al-sensitive families across species. Although the data support the hypothesis that Al-resistant plants limit Al uptake to root apices, they do not exclude other mechanisms of Al resistance. Differential Al resistance between the species and between slash pine families may also be associated with the size of the total non-labile and cell-wall-labile Al fractions, respectively. We were unable to identify the basis for differential Al resistance in loblolly pine. 相似文献