Spectroscopic characterization of quinone-site mutants of the bacterial photosynthetic reaction center

Site-specific mutations in the quinone binding sites of the photosynthetic reaction center (RC) protein complexes of Rhodobacter (R.) capsulatus caused pronounced effects on sequential electron transfer. Conserved residues that break the twofold symmetry in this region of the RC – M246Ala and M247Ala in the QA binding pocket, and L212Glu and L213Asp in the QB binding pocket – were targeted. We constructed a QB-site mutant, L212Glu-L213Asp Ala-Ala, and a QA-site mutant, M246Ala–M247Ala Glu-Asp, to partially balance the differences in charge distribution normally found between the two quinone binding sites. In addition, two photocompetent revertants were isolated from the photosynthetically-incompetent M246Glu-M247Asp mutant: M246Ala–M247Asp and M246Gly–M247Asp. Sequential electron transfer was investigated by continuous light excitation and time-resolved electron paramagnetic resonance (EPR), and time-resolved optical techniques. Several lines of EPR evidence suggested that the forward electron transfer rate to QA, kQ, was slowed in those strains containing altered QA sites. The slower rates of secondary electron transfer were confirmed by time-resolved optical results with the M246Glu-M247Asp mutations in the QA site resulting in a dramatically lowered secondary electron transfer efficiency [kQ < (2 ns)-1] in comparison with either the native R. capsulatus RC or the QB site mutant [kQ (200 ps)-1]. Secondary electron transfer in the two revertants was intermediate between that of the native RC and the QA mutant. The P+ QA- PQA charge recombination rates were also changed in the strains that carried altered QA sites. We show that local mutations in the QA site, presumably through local electrostatic changes, significantly alter binding and electron transfer properties of QA.     

Are rice chromosomes components of a holocentric chromosome ancestor?

Comparative genomics reveals that cereal genomes are composed of similar genomic building blocks (linkage blocks). By stacking these blocks in a unique order, it is possible to construct a single ancestral chromosome which can be cleaved to give the basic structure of the 56 different chromosomes found in wheat, rice, maize, sorghum, millet and sugarcane. The borders of linkage blocks are defined by cereal centromeric and telomeric sites. However, a number of studies have shown that telomeric heterochromatin has neocentromeric activity, implying that linkage blocks are in fact defined by centromeric-like sites with conserved sequences. The structure of the ancestral cereal genome thus resembles a holocentric chromosome, which is the chromosome structure shared by the closest relatives of the Gramineae, the Cypericeae and Juncaceae.     

Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis

Summary: Mouse embryos homozygous for the allele eed^{l7Rn5‐3354SB} of the Polycomb Group gene embryonic ectoderm development (eed) display a gastrulation defect in which epiblast cells move through the streak and form extraembryonic mesoderm derivatives at the expense of development of the embryo proper. Here we demonstrate that homozygous mutant ES cells have the capacity to differentiate embryonic cell types both in vitro as embryoid bodies and in vivo as chimeric embryos. In chimeric embryos, eed mutant cells can respond to wild‐type signals and participate in normal gastrulation movements. These results indicate a non–cell‐autonomous function for eed. Evidence of mutant cell exclusion from the forebrain and segregation within somites, however, suggests that eed has cell‐autonomous roles in aspects of organogenesis. A requirement for eed in the epiblast during embryonic development is supported by the fact that high‐contribution chimeras could not be rescued by a wild‐type extraembryonic environment. genesis 31:142–146, 2001. © 2001 Wiley‐Liss, Inc.     

The effect of concentrated smoke products on the restoration of highly disturbed mineral sands in southeast Victoria

Summary Recent studies have recognized the potential of broad‐scale surface application of smoke compounds for enhancing germination from the soil seed‐bank in fire‐prone vegetation communities. Results suggest that smoke technology may play, in the future, a significant role in the restoration and management of areas supporting indigenous vegetation. An important step in the development of smoke‐based restoration tools is the conduct of in situ field trials in a range of geographical locations and environmental conditions. However, most of the published work on the effectiveness of smoke products in promoting seedbank germination has been conducted at sites in southwestern Australia. The present study examines the effect of commercially available smoke‐water products on the regeneration of a highly disturbed former mine‐site at the Royal Botanic Gardens Cranbourne, in southeastern Victoria. Various combinations of concentrated smoke products and topsoil harvested from a nearby heathy woodland community were applied to exposed, uniform mineral sands to test their effect on seedling density and species richness of regrowth. The trials showed that after 12 months a number of common, herbaceous species including Austrodanthonia setacea, Opercularia varia and Platysace heterophylla were recorded in significantly higher numbers in areas treated with a commercial smoke‐water. However, there was no overall improvement in the density of seedlings or the richness of species as a result of the application of the smoke products. Similarly, total seedling density and species richness were not affected by the addition of topsoil, either alone or in combination with smoke products.     

GROWTH RATE VARIATION IN THE N:P REQUIREMENT RATIO OF PHYTOPLANKTON1

Theoretical considerations predict that the cell N:P ratio at transition from nitrogen limitation to phosphorus limitation of phytoplankton growth (critical ratio, R_c) varies, as a function of population growth rate. This prediction is confirmed by experimental, data from the literature along with new experimental data for the marine, prymnesiophyte Pavlova lutheri (Droop) Green. R_c passes through a maximum at intermediate growth rates for the three phytoplankton species for which data, are available, but there is significant interspecific variability in its value. There is no theoretical or experimental evidence to support the idea that the ratio of subsistence N and P cell quotas is equal to R_c over the range of growth rates, or that the subsistence quota ratio equals the ratio of the N and P cell quotas minus a storage fraction. Examination of N:P composition ratios can be used to determine which nutrient is limiting, but cannot be used to determine relative growth rates or competitive advantage between species limited by the same nutrient. Growth rates are determined by environmental conditions and by the cell quota of the limiting nutrient, without reference to the cell quota of the non-limiting nutrient.     

Light-, nitrogen-, and phosphorus-limited growth of Phaeodactylum tricornutum Bohlin strain TFX-1: Chemical composition,carbon partitioning,and the diel periodicity of physiological processes

Phaeodactylum tricomutum Bohlin (strain TFX-1) was grown under light-, nitrogen-, and phosphorus-limited conditions in continuous or semicontinuous cultures under a 12L-12D light regime. The C, N, and P contents were determined at each steady state, as was the partitioning of cellular organic carbon into protein, lipids, polysaccharides, and metabolic intermediates. All determinations were made at the beginning and again at the end of the light period. The rates of nutrient assimilation and of synthesis of biochemical constituents during the light and dark periods were calculated from the above data, and the periodicities of these processes characterized. The elemental composition of the cells was different under each limitation. In particular, phosphorus limitation severely restricted the ability of the cell to store nitrogen in non-protein forms. Biochemical composition and the diel periodicity of cellular processes also differed between limitations. Nutrient uptake was most strongly periodic under light limitation. Protein synthesis showed increased periodicity under nitrogen limitation, relative to the other limitations, while the periodicity of lipid synthesis was reduced under phosphate limitation. Polysaccharide was synthesized at high rates during the light period and consumed in the dark under all limitations.     

Whole cell segmentation in solid tissue sections.

BACKGROUND: Understanding the cellular and molecular basis of tissue development and function requires analysis of individual cells while in their tissue context. METHODS: We developed software to find the optimum border around each cell (segmentation) from two-dimensional microscopic images of intact tissue. Samples were labeled with a fluorescent cell surface marker so that cell borders were brighter than elsewhere. The optimum border around each cell was defined as the border with an average intensity per unit length greater that any other possible border around that cell, and was calculated using the gray-weighted distance transform. Algorithm initiation requiring the user to mark two points per cell, one approximately in the center and the other on the border, ensured virtually 100% correct segmentation. Thereafter segmentation was automatic. RESULTS: The method was highly robust, because intermittent labeling of the cell borders, diffuse borders, and spurious signals away from the border do not significantly affect the optimum path. Computer-generated cells with increasing levels of added noise showed that the approach was accurate provided the cell could be detected visually. CONCLUSIONS: We have developed a highly robust algorithm for segmenting images of surface-labeled cells, enabling accurate and quantitative analysis of individual cells in tissue.