Coupling of Cell Division and Differentiation in Arabidopsis thaliana Cultured Cells with Interaction of Ethylene and ABA Signaling Pathways.

Recent studies indicate direct links between molecular cell cycle and cell differentiation machineries. Ethylene and abscisic acid (ABA) are known to affect cell division and differentiation, but the mechanisms of such effects are poorly understood. As ethylene and ABA signaling routes may interact, we examined their involvement in cell division and differentiation in cell tissue cultures derived from several Arabidopsis thaliana plants: wild type (Col-0), and ethylene-insensitive mutants etr1-1, ctr1-1, and ein2-1. We designed an experimental setup to analyze the growth-related parameters and molecular mechanisms in proliferating cells upon short exposure to ABA. Here, we provide evidence for the ethylene-ABA signaling pathways' interaction in the regulation of cell division and differentiation as follows: (1) when the ethylene signal transduction pathway is functionally active (Col-0), the cells actively proliferate, and exogenous ABA performs its function as an inhibitor of DNA synthesis and division; (2) if the ethylene signal is not perceived (etr1-1), then, in addition to cell differentiation (tracheary elements formation), cell death can occur. The addition of exogenous ABA can rescue the cells via increasing proliferation; (3) if the ethylene signal is perceived, but not transduced (ein2-1), then cell differentiation takes place-the latter is enhanced by exogenous ABA while cell proliferation is reduced; (4) when the signal transduction pathway is constitutively active, the cells begin to exit the cell cycle and proceed to endo-reduplication (ctr1-1). In this case, the addition of exogenous ABA promotes reactivation of cell division.

Highly active extracellular α-class carbonic anhydrase of Cyanothece sp. ATCC 51142.

Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of α-class in cyanobacterial cells. The enzyme activity was confirmed both in vivo in intact cells and in vitro, using the recombinant protein. CA activity in intact cells of Cyanothece sp. ATCC 51142 reached ∼0.6 Wilbur-Anderson units (WAU) per 1 mg of total cell protein, and it was inhibited by a specific CAs inhibitor, ethoxyzolamide. The genes cce_4328 (ecaA) and cce_0871 (ecaB), encoding two potential extracellular CAs of Cyanothece have been cloned, and the corresponding proteins EcaA and EcaB, representing CAs of α- and ß-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (∼1.1 × 104 WAU per 1 mg of target protein) was detected for the recombinant EcaA only. The presence of EcaA in the outer cellular layers of Cyanothece was confirmed by immunological analysis with antibodies raised against the recombinant protein. The absence of redox regulation of EcaA activity indicates that this protein does not possess a disulfide bond essential for some α-class CAs. The content and activity of EcaA in a fraction of periplasmic proteins was higher in Cyanothece cells grown at ambient concentration of CO2 (0.04%) compared to those grown at an elevated CO2 concentration (1.7%). At the same time, the level of ecaA gene mRNA varied insignificantly in response to changes in CO2 supply. Our results indicate that EcaA is responsible for CA activity of intact Cyanothece cells and point to its possible physiological role under low-CO2 conditions.

Nitric Oxide Has a Concentration-Dependent Effect on the Cell Cycle Acting via EIN2 in Arabidopsis thaliana Cultured Cells.

Ethylene is known to influence the cell cycle (CC) via poorly characterized roles whilst nitric oxide (NO) has well-established roles in the animal CC but analogous role(s) have not been reported for plants. As NO and ethylene signaling events often interact we examined their role in CC in cultured cells derived from Arabidopsis thaliana wild-type (Col-0) plants and from ethylene-insensitive mutant ein2-1 plants. Both NO and ethylene were produced mainly during the first 5 days of the sub-cultivation period corresponding to the period of active cell division. However, in ein2-1 cells, ethylene generation was significantly reduced while NO levels were increased. With application of a range of concentrations of the NO donor, sodium nitroprusside (SNP) (between 20 and 500 µM) ethylene production was significantly diminished in Col-0 but unchanged in ein2-1 cells. Flow cytometry assays showed that in Col-0 cells treatments with 5 and 10 µM SNP concentrations led to an increase in S-phase cell number indicating the stimulation of G1/S transition. However, at ≥20 µM SNP CC progression was restrained at G1/S transition. In the mutant ein2-1 strain, the index of S-phase cells was not altered at 5-10 µM SNP but decreased dramatically at higher SNP concentrations. Concomitantly, 5 µM SNP induced transcription of genes encoding CDKA;1 and CYCD3;1 in Col-0 cells whereas transcription of CDKs and CYCs were not significantly altered in ein2-1 cells at any SNP concentrations examined. Hence, it is appears that EIN2 is required for full responses at each SNP concentration. In ein2-1 cells, greater amounts of NO, reactive oxygen species, and the tyrosine-nitrating peroxynitrite radical were detected, possibly indicating NO-dependent post-translational protein modifications which could stop CC. Thus, we suggest that in Arabidopsis cultured cells NO affects CC progression as a concentration-dependent modulator with a dependency on EIN2 for both ethylene production and a NO/ethylene regulatory function.

Complex formation of Sn(II) with glycine: an IR, DTA/TGA and DFT investigation.

The novel Sn(Gly)2⋅H2O complex compound has been synthesized and characterized by TGA, IR and Raman spectroscopy. Molecular spectroscopy and ab initio simulation have given the evidence of glycine molecule being coordinated to Sn(II) as bidentate chelating ligand by oxygen atom of carboxyl group and nitrogen atom of amino group. Water molecule is bonded with amino and carboxylic groups by hydrogen bonds in the out sphere. The M06, TPSS, TPSSm, TPSSh and revTPSS density functionals have been tested for calculation of structural and vibrational data. The vibrational assignment of experimental IR and Raman and simulated spectra has been carried out. The TPSS and TPSSm density functionals and Def2-TZVP basis set have provided the most accurate results.

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation.

Complex formation of Sn(II) with L-cysteine: an IR, DTA/TGA and DFT investigation.

The novel complex of Sn(II) with L-cysteine (L-H2Cys) has been synthesized and characterized by elemental analysis, TGA and IR spectroscopy. Vibrational assignment and DFT/PBE0/def2-TZVP ab initio simulation give evidence of cysteine molecule being coordinated to Sn(II) as three-dentate chelating N,O,S-donor ligand. The four Perdew density functionals TPSS, PBE0, PBE, TPSSh have been tested to provide consistency of simulated and experimental IR spectra, the best result is provided by unweighted Hartree-Fock density functionals (PBE, TPSS). On the contrary, the Hartree-Fock weighted functionals (PBE0, TPPSh) provide the most accurate geometry optimization. Unharmonic frequencies are obtained via ab initio vibrational self-consistent field (PT2-VSCF) calculations at DFT/TPSS/Def2-TZVP level, the vibrational assignment of IR spectra has been carried out.

Nitric oxide in plants: an assessment of the current state of knowledge.

BACKGROUND AND AIMS: After a series of seminal works during the last decade of the 20th century, nitric oxide (NO) is now firmly placed in the pantheon of plant signals. Nitric oxide acts in plant-microbe interactions, responses to abiotic stress, stomatal regulation and a range of developmental processes. By considering the recent advances in plant NO biology, this review will highlight certain key aspects that require further attention. SCOPE AND CONCLUSIONS: The following questions will be considered. While cytosolic nitrate reductase is an important source of NO, the contributions of other mechanisms, including a poorly defined arginine oxidizing activity, need to be characterized at the molecular level. Other oxidative pathways utilizing polyamine and hydroxylamine also need further attention. Nitric oxide action is dependent on its concentration and spatial generation patterns. However, no single technology currently available is able to provide accurate in planta measurements of spatio-temporal patterns of NO production. It is also the case that pharmaceutical NO donors are used in studies, sometimes with little consideration of the kinetics of NO production. We here include in planta assessments of NO production from diethylamine nitric oxide, S-nitrosoglutathione and sodium nitroprusside following infiltration of tobacco leaves, which could aid workers in their experiments. Further, based on current data it is difficult to define a bespoke plant NO signalling pathway, but rather NO appears to act as a modifier of other signalling pathways. Thus, early reports that NO signalling involves cGMP-as in animal systems-require revisiting. Finally, as plants are exposed to NO from a number of external sources, investigations into the control of NO scavenging by such as non-symbiotic haemoglobins and other sinks for NO should feature more highly. By crystallizing these questions the authors encourage their resolution through the concerted efforts of the plant NO community.

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination.

BACKGROUNDS AND AIMS: In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. METHODOLOGY: Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H(+)-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. PRINCIPAL RESULTS: Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H(+)-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. CONCLUSIONS: Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due to the short duration. The retained physiological activity of vacuoles allows them to function rapidly as dormancy is lost and when external conditions permit. Cell vacuolation precedes cell elongation in both hypocotyl and radicle, and provides impetus for rapid germination.

Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the Cyanobacterium synechocystis.

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

Ethylene regulates monomeric GTP-binding protein gene expression and activity in Arabidopsis.

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.

Ethylene rapidly up-regulates the activities of both monomeric GTP-binding proteins and protein kinase(s) in epicotyls of pea.

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants.