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Abstract Background Microarray technology has become a widely accepted and standardized tool in biology. The first microarray data analysis programs were developed to support pair-wise comparison. However, as microarray experiments have become more routine, large scale experiments have become more common, which investigate multiple time points or sets of mutants or transgenics. To extract biological information from such high-throughput expression data, it is necessary to develop efficient analytical platforms, which combine manually curated gene ontologies with efficient visualization and navigation tools. Currently, most tools focus on a few limited biological aspects, rather than offering a holistic, integrated analysis. Results Here we introduce PageMan, a multiplatform, user-friendly, and stand-alone software tool that annotates, investigates, and condenses high-throughput microarray data in the context of functional ontologies. It includes a GUI tool to transform different ontologies into a suitable format, enabling the user to compare and choose between different ontologies. It is equipped with several statistical modules for data analysis, including over-representation analysis and Wilcoxon statistical testing. Results are exported in a graphical format for direct use, or for further editing in graphics programs. PageMan provides a fast overview of single treatments, allows genome-level responses to be compared across several microarray experiments covering, for example, stress responses at multiple time points. This aids in searching for trait-specific changes in pathways using mutants or transgenics, analyzing development time-courses, and comparison between species. In a case study, we analyze the results of publicly available microarrays of multiple cold stress experiments using PageMan, and compare the results to a previously published meta-analysis. PageMan offers a complete user's guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/. Conclusion PageMan allows multiple microarray experiments to be efficiently condensed into a single page graphical display. The flexible interface allows data to be quickly and easily visualized, facilitating comparisons within experiments and to published experiments, thus enabling researchers to gain a rapid overview of the biological responses in the experiments.

Reports on the highlights of poster sessions held on September 23, 1989 at the 25th annual meeting of the European Association for the Study of Diabetes, which took place in Lisbon, Portugal. Effect of gliclazide on the fibrinolytic system of blood in 23 Type I diabetic patients; Investigation of secondary failure rate with oral agent use; Combination therapy with sulfonylurea agents and insulin.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H + -ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H + -ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Anthocyanins and carotenoids determine the diversity of potato tuber flesh pigmentation; here, the underlying chemical and genetic bases were elucidated by multiomics analyses. A total of 31 anthocyanins and 30 carotenoids were quantified in five differently pigmented tubers. Cyanidin and pelargonidin derivatives determined the redness, while malvidin, petunidin, and delphinidin derivatives contributed to purpleness. Violaxanthin derivatives determined the light-yellow color, while zeaxanthin and antheraxanthin derivatives further enhanced the deep-yellow deposition. Integrated transcriptome and proteome analyses identified that F3 '5' H highly enhanced anthocyanin biosynthesis in purple flesh and was responsible for metabolic divergence between red and purple samples. BCH2 significantly enhanced carotenoid biosynthesis in yellow samples and along with ZEP , NCED1 , and CCD1 genes determined metabolic divergence between light and deep-yellow samples. The weighted correlation network analysis constructed a regulatory network revealing the central role of AN1 in regulating anthocyanin biosynthesis, and 10 new transcription factors related to anthocyanin and carotenoid metabolism regulation were identified. Our findings provide targeted genes controlling tuber pigmentation, which will be meaningful for the genetic manipulation of tuber quality improvement.

The transition to grain agriculture restructured human societies, creating a new whole, an economic superorganism. Homo sapiens became expansionary, structurally interdependent in material life, and a duality between them and Earth was created that had not previously existed. Yet H. sapiens are not the only species to make the transition to agriculture. Cross-species comparisons create an opening for a movement toward a focus on the universal and powerful agricultural system as a unique expression of the evolution of species cooperation. This shifts the focus around human social evolution away from culture and toward the formation and power of the economic system that took hold with the cultivation of annual grains. The basic structure and dynamic to economic life that began with grain agriculture has endured for 10 000 years and the duality between humans and Earth established therein is now reaching an apogee with the spectre of climate change and the mass extinction of other species on Earth. In this light, the questions emerge: Is the agricultural revolution an evolutionary transition adequately captured in existing frameworks of human social evolution? Is the human capacity for culture sufficient to override the power and dynamic of the economic superorganism? This article is part of the theme issue 'Human socio-cultural evolution in light of evolutionary transitions'.

Spatial transcriptomics, which combine gene expression data with spatial information, has quickly expanded in recent years. With application of this method in liver research, our knowledge about liver development, regeneration, and diseases have been greatly improved. While this field is moving forward, a variety of problems still need to be addressed, including sensitivity, limited capacity to obtain exact single-cell information, data processing methods, as well as others. Methods like single-cell RNA sequencing (scRNA-seq) are usually used together with spatial transcriptome sequencing (ST-seq) to clarify cell-specific gene expression. In this review, we explore how advances of scRNA-seq and ST-seq, especially ST-seq, will pave the way to new opportunities to investigate fundamental questions in liver research. Finally, we will discuss the strengths, limitations, and future perspectives of ST-seq in liver research., (© 2023. The Author(s).)

Both the phosphorylation and dephosphorylation of plant proteins is involved in multiple biological processes, especially in regard to signal transduction. The identification of phosphopeptides from MS (mass spectrometry)-based methods and their subsequent quantification play an important role in plant phosphoproteomics analysis. Phosphopeptide(s) identification and label-free quantification can determine dynamic changes of phosphorylation events in plants. Both MaxQuant and Proteome Discoverer are professional software tools used to identify and quantify large-scale MS-based phosphoproteomic data. This chapter gives a detailed workflow of MaxQuant and Proteome Discoverer software to analyze large amounts of phosphoproteomic-related MS data for the identification and quantification of label-free plant phosphopeptides., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Plants absorb water and nutrients from soil through roots and transmit these resources through the xylem to the shoot. Roots therefore participate in information and material transduction as well as signal communication with the shoot. The importance of reversible protein phosphorylation in the regulation of plant growth and development has been amply demonstrated through decades of research. Here, we present a simple mass spectrometry-based shotgun phosphoproteomics protocol for Arabidopsis root tissue. Through this method, we can profile the Arabidopsis root phosphoproteome and construct signal networks of key proteins to better understand their roles in plant growth and development., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The transmembrane receptor kinase family is the largest protein kinase family in Arabidopsis. Many members of this family play critical roles in plant signaling pathways. However, many of these kinases have yet uncharacterized functions and very little is known about the direct substrates of these kinases. We have developed the "ShortPhos" method, an efficient and simple mass spectrometry (MS)-based phosphoproteomics protocol to perform comparative phosphopeptide profiling of knockout mutants of receptor-like kinases. Through this method, we are able to better understand the functional roles of plant kinases in the context of their signaling networks., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Since the Yucca Mountain project in the U.S. was defunded in 2010, the notion of disposing of spent nuclear fuel (SNF) in deep boreholes has been reinvigorated, most recently by private companies proposing to utilize lateral drilling technology to excavate boreholes for SNF disposal in sedimentary rock. It is claimed that this approach will alleviate site characterization efforts and expand the availability of potential disposal sites. However, long-term safety will hinge upon the prevalence of geochemically reducing, highly saline, and slow-flowing fluids around the waste emplacement zone, and to quantify these parameters in fluids sampled from depths >1 km will present a challenge. Regional data indicate only a narrow geographical extent of such conditions in the conterminous United States. Furthermore, models of radionuclide transport from disposal boreholes must take into account processes that may accelerate degradation of the canisters, plug, and SNF itself, such as radiolysis and attack by constituents of hydrothermal brines, coupled with hydrogeologic features that promote advective groundwater flow. This review summarizes some geologic considerations, most notably those related to geochemistry, that challenge the long-term safety case for deep borehole disposal of SNF.

Ultrasociality refers to the social organization of a few species, including humans and some social insects, having a complex division of labor, city-states, and an almost exclusive dependence on agriculture for subsistence. We argue that the driving forces in the evolution of these ultrasocial societies were economic. With the agricultural transition, species could directly produce their own food and this was such a competitive advantage that those species now dominate the planet. Once underway, this transition was propelled by the selection of within-species groups that could best capture the advantages of (1) actively managing the inputs to food production, (2) a more complex division of labor, and (3) increasing returns to larger scale and larger group size. Together these factors reoriented productive life and radically altered the structure of these societies. Once agriculture began, populations expanded as these economic drivers opened up new opportunities for the exploitation of resources and the active management of inputs to food production. With intensified group-level competition, larger populations and intensive resource exploitation became competitive advantages, and the "social conquest of Earth" was underway. Ultrasocial species came to dominate the earth's ecosystems. Ultrasociality also brought a loss of autonomy for individuals within the group. We argue that exploring the common causes and consequences of ultrasociality in humans and the social insects that adopted agriculture can provide fruitful insights into the evolution of complex human society.

We appreciate the depth and breadth of comments we received. They reflect the interdisciplinary challenge of our inquiry and reassured us of its broad interest. We believe that our target article and the criticisms, elaborations, and extensions of the commentators can be an important contribution to establishing human ultrasociality as a new field of social science inquiry. A few of the commentators questioned our definition of ultrasociality, and we begin our response with an elaboration of that definition and a defense of our argument that human ultrasociality began with agriculture. We then respond to the second major area of controversy, namely, our use of group selection to explain the economic drivers behind the agricultural transition. We then focus on the issue of human intentionality raised by the phenomenon of collective intelligence. The intriguing question is to what extent can an entire culture change its own destiny? We then address the issue of the division of labor raised by a number of commentators. The complex division of labor was both a driver and a defining characteristic of ultrasociality, even though it was present in simpler forms in earlier societies. The remaining issues addressed include energy and complexity, expansion and sustainability, and the accelerating evolution of human ultrasociality. These were raised by only a few commentators, but their importance warrants further elaboration.

Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.

With the development of high-throughput metabolic technologies, a plethora of primary and secondary compounds have been detected in the plant cell. However, there are still major gaps in our understanding of the plant metabolome. This is especially true with regards to the compartmental localization of these identified metabolites. Non-aqueous fractionation (NAF) is a powerful technique for the determination of subcellular metabolite distributions in eukaryotic cells, and it has become the method of choice to analyze the distribution of a large number of metabolites concurrently. However, the NAF technique produces a continuous gradient of metabolite distributions, not discrete assignments. Resolution of these distributions requires computational analyses based on marker molecules to resolve compartmental localizations. In this article we focus on expanding the computational analysis of data derived from NAF. Along with an experimental workflow, we describe the critical steps in NAF experiments and how computational approaches can aid in assessing the quality and robustness of the derived data. For this, we have developed and provide a new version (v1.2) of the BestFit command line tool for calculation and evaluation of subcellular metabolite distributions. Furthermore, using both simulated and experimental data we show the influence on estimated subcellular distributions by modulating important parameters, such as the number of fractions taken or which marker molecule is selected. Finally, we discuss caveats and benefits of NAF analysis in the context of the compartmentalized metabolome.

Here we describe an integrative protocol for metabolite extraction and the measurement of three cellular constituents, chlorophyll a, total protein, and glycogen from the same small volume of cyanobacterial cultures that can be used as alternative sample amount parameters for data adjustment in comparative metabolome studies. We conducted recovery experiments to assess the robustness and reproducibility of the measurements obtained for the cellular constituents. Also, we have chosen three profile-intrinsic parameters derived from gas chromatography-mass spectrometry (GC/MS) data in order to test their utility for spectral data adjustment. To demonstrate the relevance of these six parameters, we analyzed three cyanobacteria with greatly different morphologies, comprising a unicellular, a filamentous, and a filamentous biofilm-forming strain. Comparative analysis of GC/MS data from cultures grown under standardized conditions indicated that adjustment of the corresponding metabolite profiles by any of the measured cellular constituents or chosen intrinsic parameters led to similar results with respect to sample cohesion and strain separation. Twenty-one metabolites significantly enriched for the carbohydrate and amine superclasses are mainly responsible for strain separation, with a majority of the remaining metabolites contributing to sample group cohesion. Therefore, we conclude that any of the parameters tested in this study can be used for spectral data adjustment of cyanobacterial strains grown under controlled conditions. However, their use for the differentiation between different stresses or physiological states within a strain remains to be shown. Interestingly, both the adjustment approaches and statistical tests applied effected the detection of metabolic differences and their patterns among the analyzed strains.

Ecological economics remains unfinished in its effort to provide a framework for transforming the economy so that it is compatible with biophysical limits. Great strides have been made in valuing natural capital and ecosystem services and recognizing the need to limit the scale of economic activity, but the question of how to effectively transform the economy to limit the scale of economic activity remains unclear. To gain clarity about the institutional changes necessary to limit the scale of economic activity, it is essential that ecological economics understands the limitations of its neoclassical roots and expands its theoretical framework to include how markets are embedded in social and institutional structures. This has long been the domain of institutional economics and heterodox political economy., (© 2011 New York Academy of Sciences.)

The last century has seen the ascendance of a core economic model, which we will refer to as Walrasian economics. This model is driven by the psychological assumptions that humans act only in a self-referential and narrowly rational way and that production can be described as a self-contained circular flow between firms and households. These assumptions have critical implications for the way economics is used to inform conservation biology. Yet the Walrasian model is inconsistent with a large body of empirical evidence about actual human behavior, and it violates a number of basic physical laws. Research in behavioral science and neuroscience shows that humans are uniquely social animals and not self-centered rational economic beings. Economic production is subject to physical laws including the laws of thermodynamics and mass balance. In addition, some contemporary economic theory, spurred by exciting new research in human behavior and a wealth of data about the negative global impact of the human economy on natural systems, is moving toward a world view that places consumption and production squarely in its behavioral and biophysical context. We argue that abandoning the straightjacket of the Walrasian core is essential to further progress in understanding the complex, coupled interactions between the human economy and the natural world. We call for a new framework for economic theory and policy that is consistent with observed human behavior, recognizes the complex and frequently irreversible interaction between human and natural systems, and directly confronts the cumulative negative effects of the human economy on the Earth's life support systems. Biophysical economics and ecological economics are two emerging economic frameworks in this movement., (© 2010 Society for Conservation Biology.)

Metabolomics is the comprehensive analysis of the small molecules that compose an organism's metabolism. The main limiting step in microbial metabolomics is the requirement for fast and efficient separation of microbes from the culture medium under conditions in which metabolism is rapidly halted. In this article we compare three different sampling strategies, quenching, filtering, and centrifugation, for arresting the metabolic activities of two morphologically diverse cyanobacteria, the unicellular Synechocystis sp. PCC 6803 and the filamentous Nostoc sp. PCC 7120 for GC-MS analysis. We demonstrate that each sampling technique produces internally consistent and reproducible data, however, cold methanol-water quenching caused leakage and substantial loss of metabolites from various compound classes, while fast filtering and centrifugation produced quite similar metabolite pool sizes, even for metabolites with predicted high turnover. This indicates that cyanobacterial metabolic pools, as measured by GC-MS, do not show high turnover under standard growing conditions. As well, using stable (13)C labeling we show the biological origin of some of the consistently observed unknown analytes. With the development of these techniques, we establish the basis for broad scale comparative metabolite profiling of cyanobacteria.

VirB5 is a minor component of the extracellular T pilus determined by the Agrobacterium tumefaciens type IV secretion system. To identify proteins that interact with VirB5 during the pilus assembly process, we purified VirB5 as a recombinant fusion protein and, by using a gel overlay assay, we detected a 26-kDa interacting protein in Agrobacterium cell lysates. The VirB5-binding protein was purified from A. tumefaciens and identified as the cytokinin biosynthetic enzyme Tzs. The VirB5-Tzs interaction was confirmed using pulldown assays with purified proteins and the yeast two-hybrid system. An analysis of the subcellular localization in A. tumefaciens showed that Tzs was present in the soluble as well as the membrane fraction. Tzs was extracted from the membranes with the mild detergent dodecyl-beta-D-maltoside in complexes of different molecular masses, and this association was strongly reduced in the absence of VirB5. Using immunoelectron microscopy, we also detected Tzs on the Agrobacterium cell surface. A functional type IV secretion system was required for efficient translocation to the surface, but Tzs was not secreted into the cell supernatant. The fact that Tzs localizes on the cell surface suggests that it may contribute to the interaction of Agrobacterium with plants.

Background: Microarray technology has become a widely accepted and standardized tool in biology. The first microarray data analysis programs were developed to support pair-wise comparison. However, as microarray experiments have become more routine, large scale experiments have become more common, which investigate multiple time points or sets of mutants or transgenics. To extract biological information from such high-throughput expression data, it is necessary to develop efficient analytical platforms, which combine manually curated gene ontologies with efficient visualization and navigation tools. Currently, most tools focus on a few limited biological aspects, rather than offering a holistic, integrated analysis., Results: Here we introduce PageMan, a multiplatform, user-friendly, and stand-alone software tool that annotates, investigates, and condenses high-throughput microarray data in the context of functional ontologies. It includes a GUI tool to transform different ontologies into a suitable format, enabling the user to compare and choose between different ontologies. It is equipped with several statistical modules for data analysis, including over-representation analysis and Wilcoxon statistical testing. Results are exported in a graphical format for direct use, or for further editing in graphics programs.PageMan provides a fast overview of single treatments, allows genome-level responses to be compared across several microarray experiments covering, for example, stress responses at multiple time points. This aids in searching for trait-specific changes in pathways using mutants or transgenics, analyzing development time-courses, and comparison between species. In a case study, we analyze the results of publicly available microarrays of multiple cold stress experiments using PageMan, and compare the results to a previously published meta-analysis.PageMan offers a complete user's guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/., Conclusion: PageMan allows multiple microarray experiments to be efficiently condensed into a single page graphical display. The flexible interface allows data to be quickly and easily visualized, facilitating comparisons within experiments and to published experiments, thus enabling researchers to gain a rapid overview of the biological responses in the experiments.

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili.

Arabidopsis halleri ssp. halleri (accession Langelsheim) is a naturally selected zinc (Zn)- and cadmium-tolerant Zn hyperaccumulator. This plant differs strikingly from its close relative A. thaliana by accumulating Zn specifically in above-ground tissues. A. thaliana GeneChips were used in order to identify, on a transcriptome-wide scale, genes with a potential involvement in cellular metal uptake or detoxification in the shoots of A. halleri. Compared to A. thaliana, transcript abundance of several genes was found and confirmed to be substantially higher in A. halleri after 4 days of exposure to low as well as high Zn concentrations in the hydroponic culture medium. The identified candidate genes encode proteins closely related to the following A. thaliana proteins: AtZIP6, a putative cellular Zn uptake system and member of the zinc-regulated transporter (ZRT)-iron regulated transporter (IRT)-like protein (ZIP)-family of metal transporters, the putative P-type metal ATPase AtHMA3, the cation diffusion facilitator ZAT/AtCDF1, and the nicotianamine synthase AtNAS3. Heterologous expression in mutant strains of the yeast Saccharomyces cerevisiae suggested that AhHMA3, AhCDF1-3, and AhNAS3 can function in cellular Zn detoxification. Our data indicate that, at the transcript level, the Zn tolerance strategy of A. halleri involves high constitutive expression of metal homeostasis genes in the shoots to accommodate higher basal levels of Zn accumulation, and possibly to prepare for sudden increases in Zn influx into shoot cells. Furthermore, profiling of metal homeostasis gene transcripts in shoot and root tissues by real-time RT-PCR indicated that A. halleri and A. thaliana respond differently to changes in plant Zn status.

The plant pathogen Agrobacterium tumefaciens produces cytokinins upon induction of the virulence genes by secondary metabolites from wounded plants, and these hormones are believed to stimulate the infection process. To study the biosynthetic pathway, the tzs gene, encoding the Tzs (trans-zeatin-synthesizing) protein from A. tumefaciens, was cloned and the protein was overproduced and purified. Analysis of its reactivity with radioactively labeled substrate demonstrated conversion of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate, a product of the deoxyxylulose phosphate pathway, with AMP to zeatin riboside 5'-phosphate. This suggests that A. tumefaciens uses an alternative pathway of cytokinin biosynthesis, which had previously been hypothesized to operate in plants.

The VirB/D4 type IV secretion system of Agrobacterium tumefaciens translocates virulence factors (VirE2, VirF, and the VirD2-T-DNA complex) to plant cells. The membrane-bound translocation machinery consists of 12 proteins (VirB1-11 and VirD4) required for substrate translocation. Protein-protein interactions in the membranes were analyzed after extraction with the mild detergent dodecyl-beta-d-maltoside followed by separation under native conditions. Incubation of the membranes with increasing concentrations of the detergent differentially extracted virulence proteins. Separation of the solubilized proteins by blue native electrophoresis revealed cofractionation between two classes of protein complexes containing VirB7. The first class, consisting of major T-pilus component VirB2 and associated proteins VirB5 and VirB7, comigrated in the low molecular mass portion of the gel of about 100 kDa. The second class contains putative translocation complex core components VirB8, VirB9, and VirB10 in the high molecular mass portion of the gel larger than 232 kDa, as well as VirB7. Solubilized proteins were characterized further by gel filtration chromatography. This procedure separated T-pilus-associated proteins VirB2, VirB5, and VirB7 in the low molecular mass range from the other components of the translocation machinery and the substrates VirE2 and VirD2. Fractionation of VirB7-containing complexes (VirB7-VirB7 homodimers and VirB7-VirB9 heterodimers) suggested that they may link the T-pilus components to the core of the translocation machinery. Based on previously described VirB protein interactions and biochemical analysis of C58 wild type as well as of virB5 and virB6 deletion mutants, a model of T-pilus assembly in A. tumefaciens is suggested.

Phytochromes are photoreceptors that control many plant light responses. Phytochromes have two carboxyl-terminal structural domains called the PAS repeat domain and the histidine kinase-related domain. These domains are each related to bacterial histidine kinase domains, and biochemical studies suggest that phytochromes are light-regulated kinases. The PAS repeat domain is important for proper phytochrome function and can interact with putative signaling partners. We have characterized several new phytochrome B mutants in Arabidopsis that express phyB protein, three of which affect the histidine kinase-related domain. Point mutations in the histidine kinase-related domain cause phenotypes similar to those of null mutants, indicating that this domain is important for phyB signaling. However, a truncation that removes most of the histidine kinase-related domain results in a phyB molecule with partial activity, suggesting that this domain is dispensable. These results suggest that phytochromes evolved in modular fashion. We discuss possible functions of the histidine kinase-related domain in phytochrome signaling.

To determine the contribution of p53 loss to tumor progression, we have induced abnormal proliferation in the brain choroid plexus epithelium of transgenic mice using a SV40 T antigen fragment that perturbs pRB family function but does not affect p53 function. Tumors induced by this mutant develop slowly compared with those induced by wild-type T antigen. Suppressed tumor growth is directly attributable to p53 function, since rapid tumor development occurs when the T antigen fragment is expressed in p53-null mice. In p53-heterozygous mice, stochastic loss of the wild-type p53 allele results in the focal emergence of aggressive tumor nodules characteristic of tumor progression. In each case, aggressive tumor development in the absence of p53 function corresponds to a decrease in the level of apoptosis. These results provide in vivo evidence that p53-dependent apoptosis, occurring in response to oncogenic events, is a critical regulator of tumorigenesis.