Differential expression and cellular localization of ERKs during organogenic nodule formation from internodes of Humulus lupulus var. Nugget

The expression and subcellular localization of extracellular signal-regulated kinase 1 or 2 (ERK1/2) homologues (HLERK1/2) during the process of organogenic nodule formation in Humulus lupulus var. Nugget was studied using antibodies specific for ERK1 and ERK2, and for phosphorylated mitogen-activated protein kinases (MAPKs). The increase in HLERK levels, detected by Western blotting 12 hours after wounding suggests their involvement in response to the wounding treatment applied for morphogenesis induction. In dividing cambial cells, occurring in between 4 and 7 days after morphogenesis induction, as well as in dividing prenodular cells (15 days after induction) HLERK1 and/or 2 were localized in the nucleus. However, as soon as nodular cells start proliferating to form shoot meristems, HLERK1 and 2 were detected in the cytoplasm and not in the nucleus. The data reported account for a differential expression and activation of HLERK1 and HLERK2 throughout the process of nodule formation and plantlet regeneration. HLERK1 appears to be expressed in the stages of nodule formation and plantlet regeneration, playing a possible role in controlling cell proliferation and differentiation. HLERK2 may be induced as a response to reactive oxygen species (ROS) generated by wounding of internodes as its expression is reduced in liquid medium with less oxygen availability compared to solid medium. However, addition of a ROS inhibitor to the liquid medium does not result in a further decrease in the HLERK2 level.     

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.     

Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli

Organogenic calli induced from internodal segments were subcultured three times. Regenerated plants obtained from each subculture were analysed by molecular methods. No major genetic rearrangements were detected in the callus-derived plants since none of the amplified fragment-length polymorphism (AFLP) loci were found to be polymorphic. However, epigenetic changes due to a demethylation process were detected by methylation-sensitive amplified polymorphism (MSAP) technique. The results allowed inference of the possible relationship among the plants derived from different calli subcultures and the in vitro control. The plants recovered from the first and second callus subcultures clustered with the in vitro control pools in the phenogram while the regenerants from the third callus subculture showed the highest genetic distance with the controls. This is the first study reporting data about the genetic stability of callus-derived Humulus lupulus L. plants.     

Purification of a lipoxygenase from ungerminated barley. Characterization and product formation

Lipoxygenase was purified from ungerminated barley (variety 'Triumph'), yielding an active enzyme with a pI of 5.2 and a molecular mass of approximately 90 kDa. In addition to the 90 kDa band SDS-PAGE showed the presence of two further proteins of 63 kDa. Western blot analysis showed cross-reactivity of each of these proteins with polyclonal antisera against lipoxygenases from pea as well as from soybean, suggesting a close immunological relationship. The 63 kDa proteins appear to be inactive degradation products of the active 90-kDa enzyme. This barley lipoxygenase converts linoleic acid mainly into (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid, and arachidonic acid into (5S)-(6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eic osatetraenoic acid.     

Expression of lupin nodulin genes during root nodule development

Seven lupin cDNA clones were used to study the expression of corresponding genes during nodule development by Northern blots analysis. They include six nodulin cDNAs: pLLb (lupin leghemoglobin), pLN 13, pLN 21–27, pLN 281, pLN 50, pLNGS (nodule form of glutamine synthetase GS_n and root form of GS: pGS. The appearance of nodulin mRNAs during lupin nodule development showed that the nodulin sequences analysed represent a group of plant genes involved in the nitrogen fixation process rather than formation of nodule. This is based on the observation that they are activated at the time when the nodule has already been formed, prior to the onset of nitrogenase activity. The products of Lb, nodulin 21–67, the nodulin coded by pLN13 and the nodulin 281 genes appeared between 11 and 13 days after infection, whereas the nodulion coded by pLN50 and the nodule form of GS appeared 18 days after inoculation. Twenty-one days post-infection a dramatic increase in the transciption rates of all nodulin genes is observed. This phenomenon may be related to the onset of nitrogenase activity. The possible mechanism of two-step activation of nodulin genes is discussed.     

The influence of micelle formation on lipoxygenase kinetics.

The effects of a series of n-alcohols and n-carboxylic acids on lipoxygenase activity was studied. It was shown that to a large extent the effects of these compounds could be ascribed to physiochemical interaction with the substrate solution rather than a direct action on the enzyme itself. The effect of better substrate analogues such as stearate and oleate could also be ascribed to this effect. A type-2 lipoxygenase was found to have a very unusual velocity-substrate relationship which could be normalized by addition of calcium chloride in amounts stoichiometric with the substrate. An excess of calcium inhibited the enzyme. By comparison of results with linoleoyl sulphate/linoleoyl alcohol mixed micelles, an explanation for this unusual velocity-substrate activity is presented.     

Expression of host genes during root nodule development in soybeans

Summary Nine unique nodulin cDNA clones from soybean have been characterized with regard to the size of the RNA and the corresponding protein products. Based on the sequence homology between clones C51 and E27 and the multiple RNA species corresponding to clones D41 and E41, it is suggested that some of the nodulin genes represent members of small gene families. The amino acid sequence deduced from the nucleotide sequence of clones C51 and E27 revealed the presence of a signal peptide and no stop transfer signal, typical of membrane proteins, suggesting that the proteins encoded by these clones are localized in organelles and as such probably involved in ureide biosynthesis (Boland et al. 1982; Schubert and Boland 1984). Based on the timing of appearance of RNA corresponding to the nodulin clones and the pattern of their accumulation, at least three sets of nodulin genes are being represented here. Al1 the nodulin RNAs examined were made in Fix^- nodules formed by strain Ag168 (which does not make Cl component of nitrogenase) at a level comparable to that in Fix⁺ nodules and at a very reduced level in Fix^- nodules formed by strain HS124 (which show very few infected cells). It is concluded that all the nodulin genes examined here are induced independent of nitrogenase activity.     

Is lipoxygenase involved in the formation of ethylene from ACC?

Freezing or desiccation of winter rape leaves ( Brassica napus L. var. oleifera (cv. Górczanski) stimulated both lipoxygenase (EC 1.13.11.12) activity and ethylene formation during the post-stress period. The effect depended on the degree of membrane injury. In tissues showing injury less than 50% (as checked with the electrical conductivity method) both activities increased according to the degree of stress-induced damage. In leaves injured to a higher degree both activities decreased. Light and low temperature (5°C) inhibited the development of both lipoxygenase activity and ethylene formation in leaf disks stored for 20 h. Ethylene formation was also observed in a model system where soybean lipoxygenase was added to a mixture containing 1-aminocyclopropane-1-carboxylic acid and linoleic or linolenic acid as substrate for lipoperoxide formation. Changes in pH and temperature conditions of the incubation mixture caused similar differences in the lipoxygenase activity and ethylene formation. We propose that the stimulation of lipoxygenase-catalysed oxidation of polyunsaturated fatty acids (increasing free radical formation) leads to an increased ethylene production from ACC.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

Cessation of cytoplasmic streaming of Chara internodes during action potential

Cytoplasmic streaming of the Chara internode stops temporarilyat the peak of the action potential. Use of the technique ofvacuolar perfusion established that the sudden cessation ofcytoplasmic streaming is caused mainly by a temporary disappearanceof its motive force. Recovery of the rate of cytoplasmic streamingoccurs in parallel with that of the motive force. The ‘viscosity’of the cytoplasm remains almost unchanged during the whole periodof excitation except at the peak of the action potential. (Received February 1, 1968; )     

Feedback regulation of nodule formation in alfalfa

When high dosages of wild-type Rhizobium meliloti RCR2011 were inoculated at two different times, 24 h apart, onto either the primary roots of alfalfa (Medicago sativa L.) seedlings or onto lateral roots on opposite sides of a split-root system, the number of nodules generated by the second inoculum was much smaller than the number generated by the first inoculum. These results provide evidence that alfalfa has an active, systemic mechanism for feedback control of nodulation. Non-nodulating mutants and delayed, weakly nodulating mutants did not elicit a discernable suppression of nodulation by subsequently inoculated wild-type cells. An appreciable number of Rhizobium infections thus seem required to elicit the suppressive response. Mutants in nodulation regions II_b and II_a nodulated extensively in the initially susceptible region of the root, but nodule initiation by these mutants was 100–1000 times less efficient, respectively, than the parent. Nodules formed by these mutants emerged 1 d later than normal. The II_b mutants elicited a relatively strong suppression of nodulation in younger parts of the root, but region-II_a mutants elicited only a weak response. These results indicate that elicitation of the regulatory response need not be proportional to nodule formation and imply that genes in region II_a play an important role in elicitation. At high dosages, the region-II mutants induced the development of thick, short roots in a considerably higher percentage of plants than the wild-type bacteria. Nodules generated by wild-type isolates and region-II mutants did not emerge in strict acropetal sequence, probably because some infections developed more slowly than others. Prior exposure of the root to non-nodulating mutants resulted in nodulation by the parent in regions of the root otherwise too mature to be susceptible, indicating that exposure to these mutants may affect the sequence of root development.Abbreviations RT root tip - EH smallest emergent root hair - Tsr thick, short roots This is contribution No. 79-88 of the Ohio Agricultural Research and Development Center     

Expression of multiple delta-protocadherins during feather bud formation

The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.     

Some properties of the lipoxygenase from rabbit reticulocytes.

A lipoxygenase was enriched from the stoma-free supernatant of rabbit reticulocytes. The enzyme causes drastic deterioration of mitochondrial membranes. The release of matrix enzymes is paralleled by formation of products of lipid peroxidation. The enzyme reacts with isolated phospholipds and free cis-unsaturated fatty acids. Some properties were determined: molecular weight, isoelectric point, temperature and pH-dependence and Km value for linoleic acid. The enzyme is inhibited by reaction products and a variety of inhibitors, especially antioxidants and chelating agents.