hsp23 and hsp26 exhibit distinct spatial and temporal patterns of constitutive expression in Drosophila adults

To determine differences in the patterns of expression of Drosophila small heat shock proteins (shsp) during normal development in the absence of stress, proteins obtained from head, thorax and gonads of young (0–12 h, 3 days), middle-aged (3–6 days) and 15- to 20-day-old adult flies were separated on SDS-PAGE gels and blotted with monoclonal antibodies against hsp23 and hsp26. hsp23 was found in the heads and gonads of young males and females. In contrast, the maximum expression of hsp26 was seen in gonads of young flies, and it was only lightly detected in the brain. The expression of both proteins decreased as flies aged. This age-related decrease was particularly striking for hsp23 in females. The immunoblot results obtained were complemented by immunostaining of thin parasagittal sections of whole fly bodies Hsp23 was found to be expressed in the brain, thoracic ganglion, fat body and gonads of young (0-12 h) males and females. On the other hand, hsp26 was essentially detected in ovaries and testes of these young flies. The analysis of the tissue expression of both proteins demonstrate that each shsp has a distinct cellular localization. In the central nervous system, hsp23 and hsp26 were present in the neurocytes of the brain and the thoracic ganglion. In addition, hsp23 (but not hsp26) was also detected in the central neuropile of these two organs. In testis, hsp26 was localized in the cytoplasm of spermatocytes and, probably, in the spermatid bundles. In contrast, hsp23 was detected at the periphery of cells (membranes). In ovorioles of newborn females the expression of hsp26 was stronger, and the maximum expression of hsp23 was only reached in older (2 days and more) flies. These results demonstrate that each shsp possesses a specific spatial and temporal pattern of expression in adults of Drosophila. The distinct tissue-specific and age-dependent expression of hsp23 and hsp26 suggests that these two proteins may have different functions in crucial organs of Drosophila. © 1993Wiley-Liss, Inc.     

Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.     

Gas exchange and nutrition patterns during the life cycle of an artificial wheat crop

The growth, mineral and shoot and root CO₂ exchange of wheat plants ( Triticum aestivum L. cv. Courtot) cultivated in growth chambers have been studied during the complete life cycle. The life cycle could be divided into 4 periods according to the patterns of CO₂ exchange: exponential increase, linear increase, stabilization and decline of photosynthesis. These patterns are analysed in relation to light interception, tillering, competition between plants and ageing of the leaves, all of which constitute successive limiting factors. Root metabolism seemed to be subordinated to the demand of the shoot for minerals. Ion uptake from the nutrient solution was particularly pronounced in young plants, which were higher in minerals and nitrogen than older ones. The ratios of K and P uptake varied with plant age, and the charge balance in ion exchange was equlibrated by H uptake. Rhythmic patterns appeared in all exchanges and varied with plant age. The highest amplitude of rhythm was found in root respiration. After taking the losses due to respiration and photorespiration into account, the maximum rate of photosynthesis approached the theoretical value calculated from the light energy absorbed.     

Changes in gene expression patterns during the sexual life cycle of Chlamydomonas reinhardtii

Significant differences in membrane fluidities, expressed as fluorescence anisotropies, are demonstrated between embryogenic (E) and non-embryogenic (NE) cell lines when cells in suspension culture are removed from auxin. Cells of an E and NE cell line of Asclepias tuberosa were grown for 21 days either with or without 2, 4-dichlorophenoxy acetic acid (2,4-D), cultures were sampled at various intervals and protoplast membrane (hydrophobic interiors) was labeled with 1, 6 diphenyl-1, 3, 5-hexatriene (DPH). No differences between cultures with and without 2,4-D were detected in the NE line. In contrast the E line rapidly developed differences in membrane fluidity over time. Such clear differences in the responses of E and NE lines in membrane fluidity indicated that this parameter could be a good predictor and marker for embryogenesis. Eight suspension cell lines of Asclepias and 2 of Daucus carota were tested. After 2 days on medium without auxin, every E cell line exhibited a positive change in anisotropy and became embryogenic, whereas NE cell lines exhibited much lower positive changes or even negative changes in anisotropy and never underwent embryogenesis. Such changes have been consistent in all cell lines tested and represent a marker for embryogenicity in suspension cell lines before morphological change becomes apparent after removal from auxin. Basic molecular membrane changes in embryogenesis are likely to be common among different culture systems and understanding them could be a major step in removing barriers to regenerating plants from cultured material.     

Genome-wide analysis of light-dependent transcript accumulation patterns during early stages of Arabidopsis seedling deetiolation

Light is among the most important exogenous factors that regulate plant development. To sense light quality, intensity, direction, and duration, plants have evolved multiple photoreceptors that enable the detection of photons from the ultraviolet B (UV-B) to the far-red spectrum. To study the effect of different light qualities on early gene expression, dark-grown Arabidopsis (Arabidopsis thaliana) seedlings were either irradiated with continuous far-red, red, or blue light or received pulses of red, UV-A, or UV-A/B light. The expression profiles of seedlings harvested at 45 min and 4 h were determined on a full genome level and compared with the profiles of dark controls. Data were used to identify light-regulated genes and to group these genes according to their light responses. While most of the genes were regulated by more than one light quality, a considerable number of UV-B-specific gene expression responses were obtained. An extraordinarily high similarity in gene expression patterns was obtained for samples that perceived continuous irradiation with either far-red or blue light for 4 h. Mutant analyses hint that this coincidence is caused by a convergence of the signaling cascades that regulate gene expression downstream of cryptochrome blue light photoreceptors and phytochrome A. Whereas many early light-regulated genes exhibited uniform responses to all applied light treatments, highly divergent expression patterns developed at 4 h. These data clearly indicate that light signaling during early deetiolation undergoes a switch from a rapid, but unspecific, response mode to regulatory systems that measure the spectral composition and duration of incident light.     

Climate-driven spatial and temporal patterns in peatland pool biogeochemistry

Peatland pools are freshwater bodies that are highly dynamic aquatic ecosystems because of their small size and their development in organic-rich sediments. However, our ability to understand and predict their contribution to both local and global biogeochemical cycles under rapidly occurring environmental change is limited because the spatiotemporal drivers of their biogeochemical patterns and processes are poorly understood. We used (1) pool biogeochemical data from 20 peatlands in eastern Canada, the United Kingdom, and southern Patagonia and (2) multi-year data from an undisturbed peatland of eastern Canada, to determine how climate and terrain features drive the production, delivering and processing of carbon (C), nitrogen (N), and phosphorus (P) in peatland pools. Across sites, climate (24%) and terrain (13%) explained distinct portions of the variation in pool biogeochemistry, with climate driving spatial differences in pool dissolved organic C (DOC) concentration and aromaticity. Within the multi-year dataset, DOC, carbon dioxide (CO₂), total N concentrations, and DOC aromaticity were highest in the shallowest pools and at the end of the growing seasons, and increased gradually from 2016 to 2021 in relation to a combination of increases in summer precipitation, mean air temperature for the previous fall, and number of extreme summer heat days. Given the contrasting effects of terrain and climate, broad-scale terrain characteristics may offer a baseline for the prediction of small-scale pool biogeochemistry, while broad-scale climate gradients and relatively small year-to-year variations in local climate induce a noticeable response in pool biogeochemistry. These findings emphasize the reactivity of peatland pools to both local and global environmental change and highlight their potential to act as widely distributed climate sentinels within historically relatively stable peatland ecosystems.     

Test for temporal or spatial restrictions in gene product function during the cell division cycle.

The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes. If so, the function of the gene product in question must be limited in time or in space. CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions. In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei. In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature. The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon. Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function. We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants. A particularly interesting example was provided by the CDC4 gene. Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy. Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not.     

Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection

The phytopathogenic bacterium Rhodococcus fascians provokes shoot meristem formation and malformations on aerial plant parts, mainly at the axils. The interaction is accompanied by bacterial colonization of the plant surface and tissues. Upon infection, the two bacterial loci required for full virulence, fas and att, were expressed only at the sites of symptom development, although their expression profiles differed both spatially and temporally. The att locus was expressed principally in bacteria located on the plant surface at early stages of infection. Expression of the fas locus occurred throughout infection, mainly in bacteria that were penetrating, or had penetrated, the plant tissues and coincided with sites of meristem initiation and proliferation. The implications for the regulation of virulence genes of R. fascians during plant infection are discussed.     

A characterization model with spatial and temporal resolution for life cycle impact assessment of photochemical precursors in the United States

Background, aim, and scope  Traditional life cycle impact assessment methodologies have used aggregated characterization factors, neglecting spatial and temporal variations in regional impacts like photochemical oxidant formation. This increases the uncertainty of the LCA results and diminishes the ease of decision-making. This study compares four common impact assessment methods, CML2001, Eco-indicator 99, TRACI, and EDIP2003, on their underlying models, spatial and temporal resolution, and the level at which photochemical oxidant impacts are calculated. A new characterization model is proposed that incorporates spatial and temporal differentiation. Materials and methods  A photochemical air quality modeling system (CAMx-MM5-SMOKE) is used to simulate the process of formation, transformation, transport, and removal of photochemical pollutants. Monthly characterization factors for individual US states are calculated at three levels along the cause–effect chain, namely, fate level, human and ecosystem exposure level, and human effect level. Results and discussion  The results indicate that a spatial variability of one order of magnitude and a temporal variability of two orders of magnitude exist in both the fate level and human exposure and effect level characterization factors for NO_x. The summer time characterization factors for NO_x are higher than the winter time factors. However, for anthropogenic VOC, the summer time factors are lower than the winter time in almost half of the states. This is due to the higher emission rates of biogenic VOCs in the summer. The ecosystem exposure factors for NO_x and VOC do not follow a regular pattern and show a spatial variation of about three orders of magnitude. They do not show strong correlation with the human exposure factors. Sensitivity analysis has shown that the effect of meteorology and emission inputs is limited to a factor of three, which is several times smaller than the variation seen in the factors. Conclusions  Uncertainties are introduced in the characterization of photochemical precursors due to a failure to consider the spatial and temporal variations. Seasonal variations in photochemical activity influence the characterization factors more than the location of emissions. The human and ecosystem exposures occur through different mechanisms, and impacts calculated at the fate level based only on ozone concentration are not a good indicator for ecosystem impacts. Recommendations and perspectives  Spatial and temporal differentiation account for fate and transport of the pollutant, and the exposure of and effect on the sensitive human population or ecosystem. Adequate resolution for seasonal and regional processes, like photochemical oxidant formation, is important to reduce the uncertainty in impact assessment and improve decision-making power. An emphasis on incorporating some form of spatial and temporal information within standard LCI databases and using adequately resolved characterization factors will greatly increase the fidelity of a standard LCA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity

Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.     

Arabidopsis cryptochrome 2 completes its posttranslational life cycle in the nucleus

CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light-dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light-dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light-dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light-dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light-dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.     

Changes in composition patterns of basic proteins in Chlorella cells during their life cycle

1. (1) Changes in the composition pattern of acid soluble basicproteins in Chlorella cells during their life cycle were studiedusing various methods of fractionation ; i. e., extraction,chromatography on CM-Sephadex column and electrophoresis onpolyacrylamide gel, etc.
2. (2) The content of basic proteinswas of the order of 1-2.5%of the dry weight of cells and showeda maximum at the D_n stageand a minimum at the formative stages(L₂, L₃). The change wasdue to basic proteins (I-HC1) whichwere insoluble in 0.14 MNaCl and were non-elutable with 2 MNaCl from CM-Sephadex. The0.14 M NaCl soluble basic proteincontent remained almost constantthroughout the life cycle.
3. (3) The I-HC1 fraction in question showed 20 bands and shouldersin electrophoresis on polyacrylamide gels. Some components showedremarkable changes in their relative quantities during the lifecycle, while others exhibited relatively little change.
4. (4)Preliminary fractionation experiments of subcellular componentsby a non-aqueous method showed that the basic proteins showingmarked changes in their relative amounts were present in a nucleus-richfraction, and those exhibiting little change were found in achloroplast-rich fraction, except one main component which showedcharacteristic behavior.

(Received June 22, 1968; )     

Microfluorimetric analysis of spatial and temporal patterns of immobilized cell growth

Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high-resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse-labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass-transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr-alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.     

Nutrition of winter wheat during the life cycle. I. Yield and accumulation of dry matter and minerals

Mineral uptake by winter wheat (Trilicum aestivum L. cv. Martonvasari 8) was studied throughout the life cycle. Accumulation of macronutrients (i.e. total nitrogen, phosphorus, potassium, sodium, magnesium and calcium) and the water content of roots and shoots of plants grown in complete nutrient solution were higher than those of plants grown in two types of soils. The supply of macronutrients was in some cases limiting for soil-grown plants as revealed by a comparison of available and accumulated amounts of these nutrients. Their supply was abundant, however, for solution-grown plants. This led to a doubling of grain yield for the latter plants with a three fold increase in accumulation of dry matter and a five-fold increase in fresh weight. The efficiency ratios of solution-grown plants to soil-grown plants were approximately 1 for N and Na, 0.5 for Mg and Ca, and 0.3 for P and K.     

Progression and specificity of protein oxidation in the life cycle of Arabidopsis thaliana

Protein carbonylation is an irreversible oxidative process leading to a loss of function of the modified proteins, and in a variety of model systems, including worms, flies, and mammals, carbonyl levels gradually increase with age. In contrast, we report here that in Arabidopsis thaliana an initial increase in protein oxidation during the first 20 days of the life cycle of the plant is followed by a drastic reduction in protein carbonyls prior to bolting and flower development. Protein carbonylation prior to the transition to flowering targets specific proteins such as Hsp70, ATP synthases, the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and proteins involved in light harvesting/energy transfer and the C2 oxidative photosynthetic carbon cycle. The precipitous fall in protein carbonyl levels is due to the specific reduction in the levels of oxidized proteins rather than an overall loss of chlorophyll and Rubisco associated with the senescence syndrome. The results are discussed in light of contemporary theories of aging in animals.