Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control.

A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates.

While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples.

Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco.

Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis.

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana.

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes.

A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis.

We report on the early response of Arabidopsis thaliana to the obligate biotrophic pathogen Plasmodiophora brassicae at the hormone and proteome level. Using a CYCB1;1::GUS construct, the re-initiation of infection-related cell division is shown from 4 days after inoculation on. Sensitivity to cytokinins and auxins as well as the endogenous hormone levels are evaluated.

Banana (Musa spp.) as a model to study the meristem proteome: acclimation to osmotic stress.

Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety.

Integration of gel-based proteome data with pProRep.

Unlabelled: pProRep is a web application integrating electrophoretic and mass spectral data from proteome analyses into a relational database. The graphical web-interface allows users to upload, analyse and share experimental proteome data. It offers researchers the possibility to query all previously analysed datasets and can visualize selected features, such as the presence of a certain set of ions in a peptide mass spectrum, on the level of the two-dimensional gel.

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells.

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide.

Zeatin accumulation and misexpression of a class I knox gene are intimately linked in the epiphyllous response of the interspecific hybrid EMB-2 (Helianthus annuus x H. tuberosus).

Epiphylly, occurring in a somaclonal variant (EMB-2) of the interspecific hybrid Helianthus annuus x H. tuberosus, was used to investigate molecular and cyto-physiological mechanisms that underlie cellular fate change. EMB-2 plants are characterized by profuse proliferation of shoot- and embryo-like structures on some leaves.

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle.

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants.

Cytoenzymological analysis of adenylyl cyclase activity and 3':5'-cAMP immunolocalization in chloroplasts of Nicotiana tabacum.

In this study a combination of cytoenzymological and immunocytochemical techniques was used in order to demonstrate the presence of cyclic nucleotide metabolism in chloroplasts of higher plants. Catalytic cytochemistry was used to localize adenylyl cyclase activity by means of electron microscope investigation on Nicotiana tabacum cv. Petit Havana leaf fragments.

A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis.

The changes in root system architecture (RSA) triggered by phosphate (P) deprivation were studied in Arabidopsis (Arabidopsis thaliana) plants grown for 14 d on 1 mM or 3 microM P. Two different temporal phases were observed in the response of RSA to low P. First, lateral root (LR) development was promoted between days 7 and 11 after germination, but, after day 11, all root growth parameters were negatively affected, leading to a general reduction of primary root (PR) and LR lengths and of LR density.

Identification of four adenosine kinase isoforms in tobacco By-2 cells and their putative role in the cell cycle-regulated cytokinin metabolism.

Adenosine kinase (ADK), a key enzyme in the regulation of the intracellular level of adenosine is also speculated to be responsible for the conversion of cytokinin ribosides to their respective nucleotides. To elucidate the role of ADK in the cytokinin metabolism of tobacco BY-2 cells (Nicotiana tabacum cv. "Bright Yellow-2"; TBY-2), we have identified and characterized the full-length cDNAs encoding four ADK isoforms of N.

Jasmonic acid prevents the accumulation of cyclin B1;1 and CDK-B in synchronized tobacco BY-2 cells.

Jasmonic acid (JA) plays a crucial role in plant fertility and defense responses. It exerts an inhibitory effect on plant growth when applied exogenously. This effect seems to be somehow related to a negative regulation of cell cycle progression in the meristematic tissues.

Metabolic fate of jasmonates in tobacco bright yellow-2 cells.

Jasmonic acid and methyl jasmonate play an essential role in plant defense responses and pollen development. Their levels are temporarily and spatially controlled in plant tissue. However, whereas jasmonate biosynthesis is well studied, metabolic pathways downstream of jasmonic acid are less understood.

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture.

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells.

The Rg-1 encoded regeneration capacity of tomato is not related to an altered cytokinin homeostasis.

•  Cytokinin (CK) metabolism was analyzed in tomato (Lycopersicon esculentum) Rg-1 hybrids during in vitro shoot organogenesis from root explants. •  Data were obtained by combining physicochemical analysis with quantification and in situ detection methods. •  Although exogenous zeatin is added in all classical regeneration protocols, we show here that regenerating (Rg ) tomato explants did not require an exogenous CK source for regeneration.

Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv.

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate.

Fast liquid chromatography coupled to electrospray tandem mass spectrometry peptide sequencing for cross-species protein identification.

Using a parallel microcolumn switching liquid chromatography set-up coupled to a quadrupole time-of-flight mass spectrometer, a rapid liquid chromatography/mass spectrometric (LC/MS) protein identification method is presented. Without prior sample clean-up up to 300 protein digest samples a day can be processed. Using data-directed acquisition, up to 10 fragmentation analyses for each protein sample can be acquired in the same chromatographic run that can be used for database searching.

Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition.

Understanding the complete picture of floral transition is still impaired by the fact that physiological studies mainly concern plant species whose genetics is poorly known, and vice versa. Arabidopsis thaliana has been successfully used to unravel signalling pathways by genetic and molecular approaches, but analyses are still required to determine the physiological signals involved in the control of floral transition. In this work, the putative role of cytokinins was investigated using vegetative plants of Arabidopsis (Columbia) induced to flower synchronously by a single 22 h long day.

New cytokinin metabolites in IPT transgenic Arabidopsis thaliana plants.

Two novel cytokinin metabolites were identified in transgenic Arabidopsis thaliana (L.) Heynh. plants containing the bacterial IPT gene under the transcriptional control of a heat-regulated promoter.

Suppression of a vegetative MADS box gene of potato activates axillary meristem development.

Potato MADS box 1 (POTM1) is a member of the SQUAMOSA-like family of plant MADS box genes isolated from an early stage tuber cDNA library. The RNA of POTM1 is most abundant in vegetative meristems of potato (Solanum tuberosum), accumulating specifically in the tunica and corpus layers of the meristem, the procambium, the lamina of new leaves, and newly formed axillary meristems. Transgenic lines with reduced levels of POTM1 mRNA exhibited decreased apical dominance accompanied by a compact growth habit and a reduction in leaf size.

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin.

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type.

Cytokinin affinity purification and identification of a tobacco BY-2 adenosine kinase.

Adenosine kinase is one of the enzymes potentially responsible for the formation of cytokinin nucleotides in plants. Using a zeatin affinity column a 40 kDa protein was isolated from tobacco Bright Yellow 2 (TBY-2) and identified by mass spectrometry as adenosine kinase. The ligand interaction reported here can be disrupted by several other adenine- but not guanine-based purine derivatives.