Effect of acetylcholine on growth and isoperoxidases of the lentil(Lens culinaris) root

The effects of acetylcholine (Ach) on growth, the total peroxidase activity and the isoperoxidase spectrum of the roots ofLens culinaris were studied and compared with actual and earlier results obtained with an auxin (IAA) treatment. The general growth and peroxidase activity patterns of Ach treated roots and IAA treated ones showed many important similarities.     

Changes in the biochemical composition, structure, and function of pea leaf chloroplasts in iron deficiency and root anoxia

A combined effect of iron deficiency and root anoxia on the biochemical composition, function, and structure of pea leaf chloroplasts was studied. It was found that the chlorosis of apical leaves in response to iron deficiency was determined by the reduction of light-harvesting complexes I and II. Under root anoxia, complexes of the reaction centers of photosystems I and II degraded first. Weak activity was preserved even in yellow and white leaves under the effect of both factors. The ultrastructure of leaf chloroplasts gradually degraded. Initially, intergranal thylakoid sites were reduced, and the longitudinal orientation of grana was disturbed. However, yellow and white leaves still retained small thylakoids and grana. It is concluded that the degrading effects of iron deficiency and root anoxia on the complex composition and leaf chloroplast structure and function are additive because of their autonomous mechanisms.     

Supramolecular assemblies of a new class of nonplanar cationic metalloporphyrins and anionic metallophthalocyanines

The heteroaggregation behavior between a new class of nonplanar cationic β-octabrominated meso-alkylpyridinium zinc(II)-porphyrins (β-Br₈(ZnP)) and anionic tetrasulfonated metallophthalocyanines (MTSPc, M = Ni(II) and Cu(II)) has been studied by UV-Vis electronic spectroscopy, in dimethylsulfoxide (DMSO) solution. The heteroaggregate stoichiometry and the association constants were determined by means of Job plots. Dimers and unexpected trimers, taking into account the existence of axially coordinated DMSO molecules to the central metal in both β-Br₈(ZnP) and MTSPc complexes, are formed in solution. The spectroscopic properties of the heteroaggregates are markedly different from those observed in the correspondent planar cationic derivatives, the heteroaggregates showing major changes predominantly in the β-Br₈(ZnP) Soret band region and minor effects in the MTSPc Q bands. The observed changes in the Soret band region (red/blue shifts, decrease in the absorption intensities) depend on the nature of the alkyl substituent attached to the meso-pyridinium group. The greater versatility of the nonplanar porphyrins accommodating the meso-substituents in out-of-plane and in-plane conformations is proposed to explain the observed stoichiometries and the differences on the heteroaggregates spectroscopic properties for each β-Br₈ZnP compound. The likely conformations assumed by the meso-substituents in these β-Br₈(ZnP) compounds and its spectroscopic characteristics are in accordance with the participation of the substituents as the main factor on the extent of the observed red-shifted spectra in nonplanar porphyrins. The obtained association constants (K_IP) for the dimers and trimers are lower than those previously found for the similar planar cationic porphyrin systems, due to the lack of extensive π-π interactions and to the less effective approximation between the ionic groups, resulting in loosened heteroaggregates, particularly for the trimeric systems. Furthermore, the experimental results suggest that the NiTSPc is more distorted in DMSO solution than the CuTSPc derivative, favoring the interaction with the nonplanar β-Br₈(ZnP) compounds.     

Wheat invertases : characterization of cell wall-bound and soluble forms

Wheat coleoptiles have two distinct invertases, a soluble and a cell wall-bound form as indicated by results from cytochemical and biochemical studies. These enzyme activities differ in their pH optima, chromatographic behavior on diethylaminoethyl cellulose, kinetic properties, thermal stability, and response to light treatment. The soluble invertase was purified to near homogeneity by diethylaminoethyl-cellulose, concanavalin-A Sepharose, and Sephacryl S-300 chromatography. The overall purification was 175-fold with a recovery of about 26%. The holoenzyme has an apparent molecular weight of 158,000 and subunit molecular weight of 53,000 as estimated by polyacrylamide gel electrophoresis under denaturing conditions. Illumination of wheat seedlings caused an increase in the cell wall, but not the soluble, invertase activity.     

Maximum axial root growth pressure in pea seedlings: effects of measurement techniques and cultivars

Values of the maximum axial growth pressure (σ_max) of seedling pea (Pisum sativum L.) roots reported in the literature, obtained using different apparatuses and cultivars, range from 0.3 MPa to 1.3 MPa. To investigate possible reasons for this large range, we studied the effect of apparatus and cultivar on measurements of σ_max in peas. We describe four types of apparatus which can be used to measure axial root growth force and hence σ_max, and used them to measure σ_max in seedling pea roots using cultivar Meteor. Two of these apparatuses were also used to compare σ_max for three pea cultivars (Helka, Meteor and Greenfeast). Both cultivar and apparatus significantly affected σ_max , but there were greater differences between apparatuses than between the three cultivars. Estimating root cross-sectional area from the diameter of cross-sections, rather than from in situ measurements (i.e. measurements made with the root still in place in the apparatus) may explain these differences. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of tungstate on pea root growth and protein tyrosine phosphorylation

Tungsten belong to heavy metal group, which physiological, biochemical, and molecular action mechanisms are essentially unstudied despite metal wide application in light, heavy, and military industries and the gradual accumulation in the environment. Protein phosphorylation/dephosphorylation (one of the most important posttranslational modifications) is a highly conserved mechanism of intracellular signaling and regulation of many processes of cell activity. Protein tyrosine phosphorylation/dephosphorylation is required for the cell cycle processing, plant growth and differentiation. In this work, the effects of sodium tungstate on pea (Pisum sativum L. cv. Truzhenik) root growth, protein tyrosine phosphorylation, and phosphatase activity in the roots were studied. It was shown that sodium tungstate suppressed growth, changed the mitotic index in the root meristem, and delayed cells at some mitosis phases. Under the influence of tungstate, hydrogen peroxide accumulated in the roots and phosphatase activity was inhibited. It was established by two-dimension electrophoresis and immunoblotting with the highly specific to phosphotyrosine antibody (PY20) that tungstate treatment increased both the number of such proteins and their specific phosphorylation. It is supposed that the inhibition of protein tyrosine phosphatases was one of the reasons for tungstateinduced pea root growth inhibition.     

Kurloff cell lysosomal arylsulphatases: Presence of both cationic and highly anionic isoforms of the sole B class

Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or LeeVaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4–23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, ‘classical’ cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their K_m (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO₃ were also characteristic of this class of Asase. Finally, chondroitin4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.     

Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.     

Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT

Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.     

The effect of mechanical impedance on root growth in pea (Pisum sativum). II. Cell expansion and wall rheology during recovery

The aim of the present work was to determine the factors limiting growth in mechanically impeded roots. Pea roots were grown in compressed and uncompressed sand cores, and then removed and transferred to hydroponics. Root elongation was slowed in impeded sand cores and did not recover to the unimpeded rates until 60 h after transfer to the hydroponics system. Root diameter was greater in impeded roots, and only after 36 h in hydroponics was new root tissue produced of the same diameter as the unimpeded controls. The turgor pressure of the growing cells was measured with a turgor probe and was the same in both treatments. The slower elongation rate of the previously impeded roots was, therefore, the result of axial tightening of the cell walls. Cell length profiles suggested that axial cell wall tightening persisted in the unrestricted hydroponics system. Production of new cells in unrestricted conditions was required before root elongation returned to the unimpeded state. Osmotic potential was decreased by approximately 0.2 MPa in previously impeded roots compared with the unimpeded ones. This corresponds to a decrease in water potential of 0.2 MPa. These data are discussed in relation to regulation of cell extension, solute unloading and the penetration of compacted soils by roots.     

The rates of shoot and root growth in intact plants of pea mutants in leaf morphology

Isogenic lines of pea (Pisum sativum L.) with the genetically determined changes in leaf morphology, afila (af) and tendril-less (tl), were used to study the relationship between shoot and root growth rates. The time-course of shoot and root growth was followed during the pre-floral period in the intact plants grown under similar conditions. The af mutation produced afila leaves without leaflets, whereas in the case of the tl mutations, tendrils were substituted with leaflets, and acacia-like leaves were developed. Due to the changes in leaf morphology caused by these mutations, pea genotypes differed in leaf area: starting from day 7, the leaf area was lower in the af plants and larger in the tl plants as compared to the wild-type plants. Such divergence was amplified in the course of plant development and reached its maximum immediately before the transition to flowering. Plants of isogenic lines did not notably differ in stem surface areas. In spite of significant difference in total leaf area, the wild type and tl plants did not differ in leaf dry weight. Starting from leaf 9, the af plants lagged behind two leaflet-bearing genotypes (wild type and tl) in leaf dry weight, whereas stem dry weight was similar in the wild type and tl forms and slightly lower in the af plants. Root dry weights were practically similar in the wild type and tl plants until flowering. The reduction of leaf area in the af plants drastically reduced root dry weight. In other words, the latter index was related to the total weight and total area of leaves and stems. The correlation analysis demonstrated an extremely low relationship between leaf and stem area and dry weight and those of roots early in plant development (when plants develop five to seven leaves). Later, immediately before flowering (nine to eleven leaves), root weight was positively related to leaf weight and area; however, stem area and root weight did not correlate. Thus, in three genotypes (wild type, af, and tl), at the end of their vegetative growth phase, leaf and root biomass accumulated in proportion, independently of leaf area expansion.     

Synthesis and characterization of new cationic hydride complexes of rhodium(III)

New cationic hydride complexes of rhodium(III) with PR₃ and R-DAB ligands have been prepared and characterised. The tertiary phosphines employed were PPh₃, PMePh₂, PEt₃ and the R-DAB ligands, (RN:CR′CR′:NR), c-Hex-DAB, Ph-DAB, NH2-DAB-(CH₃,CH₃). Hexacoordinate-dihydride complexes, characterized by ¹H and ³¹P NMR, with stoichiometry [RhH₂(R-DAB)(PR₃)₂]X were obtained. Compounds with other stoichiometries (R-DAB/PR₃=1 or 2) are also possible. Preliminary studies of the catalytic activity in hydrogenation of olefins have been carried out.     

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.)

Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml^?1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml^?1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.     

The epithelial tight junction: Structure,function and preliminary biochemical characterization

Summary The tight junction, or zonula occludens (ZO), forms a semi-permeable barrier in the paracellular pathway in most vertebrate epithelia. The ZO is the apical-most member of a series of intercellular junctions, collectively known as the junctional complex, found at the interface of the apical and lateral cell surface. This structure not only restricts movement of substances around the cells, but may also serve as a fence acting to maintain the cell surface compositional polarity characteristic of epithelial cells. The morphology and physiology of the ZO have been well documented and are briefly reviewed here. The biochemistry of this important intercellular junction remains largely unknown, although a tight junction-specific polypeptide called ZO-1 has recently been identified. Preliminary observations regarding the role of this peripheral phosphoprotein in the biology of the ZO are presented.     

Separation and comparative characterization of the cationic protease and anionic protease from the culture medium of Thermoactinomyces vulgaris

The anionic protease component which frequently contaminates preparations of routinely isolated cationic protease (thermitase) from Thermoactinomyces vulgaris was purified, virtually to homogeneity, by rechromatography on controlled pore glass (CPG-10). Starting materials were column eluates with anionic protease, contaminated with residual thermitase activity. The purified anionic enzyme shares several properties with thermitase, such as size, sensitivity against phenylmethanesulfonyl fluoride and Hg2+, UV-spectral, immunological and pH behavior. On the other hand, the isoelectric point (at pH 6.5), temperature dependence (more heat stable) and enzymatic activity (less active) of anionic protease differ significantly from thermitase. At pH 8 or 6 and 25 degrees or 4 degrees C anionic protease is hydrolysed completely by thermitase. Like other protein substrates, anionic protease simultaneously acts as a stabilizer for thermitase. In contrast to thermitase, the anionic enzyme partially changes spontaneously during long-term storage at 4 degrees C and pH 6 to a cationic protein species endowed with proteolytic activity.