Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Detection of DNA polymorphism among pea cultivars using RAPD technique

An influence of some Random Amplified Polymorphic DNA (RAPD) reaction factors on resulting banding pattern and the ability of RAPD technique to detect DNA polymorphism among six economically important pea cultivars was tested. Relatively high level of DNA polymorphism among peas was observed, using polyacrylamide/urea gels and silver staining. Altogether 13 arbitrarily designed primers produced 313 amplification products. In addition 59 polymorphisms were found. These polymorphisms can serve as potential genetic markers. RAPD data were processed using cluster analysis and plotted as dendrogram. Each tested cultivar was clearly distinguished from the others. Moreover,Pisum sativum andP. sativum subsp.arvense cultivars were separated into 2 different clusters, according to their systematic relationships.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

Plasmodesmal widening accompanies the short-term increase in symplasmic phloem unloading in pea root tips under osmotic stress

Summary Root tips ofPisum sativum seedlings were exposed to 350 mM mannitol, which was shown to effect a transient but dramatic increase in phloem unloading, and investigated by electron microscopy. After chemical fixation and embedding, extremely thin sections of the root extension zone were examined. Outer, inner, and desmotubule diameters of 830 primary plasmodesmata in transverse walls of cortical cells were measured. Statistical analysis indicated that the majority of plasmodesmata had no neck constriction during osmoregulation. Compared to controls, a highly significant increase in mean plasmodesmata diameter was found, but the desmotubule diameter remained unchanged. Both loss of neck constriction and widening of the cytoplasmic sleeve indicate an increase in effective passage area of plasmodesmata. Spokes between plasma membrane and desmotubule were preserved. Continued exposure of the root tips to mannitol led to a return to control values for plasmodesmal diameters. In contrast to these responses, plasmolysis of cortical cells by 1,000 mM sucrose, diminishing phloem unloading, was accompanied by a reduction in those plasmodesmata classified as open. This is the first report showing a correlation between the ultrastructure of plasmodesmata and the rate of symplasmic transport. The role of the different plasmodesmal components in controlling the passage area of symplasmic transport is discussed.     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Fate of the nucleolar vacuole during resumption of cell cycle in pea cotyledonary buds

Summary Meristematic cells of pea cotyledonary buds blocked in G_0–1 state contain a small nucleolus with a large central clear area surrounded by a fibrillar rim. The nucleolar structure varies according to the cell cycle from the G_0–1-blocked state until the first mitoses occurring between 24 and 27h after removal of the main stem. In order to better identify and understand the role of the central area in the nucleolar function, its content was investigated by cytochemical and terminal deoxynucleotidyl transferase-immunogold methods. The central area showed the characteristics of a vacuole commonly constituted of the condensed chromatin, ribonucleoprotein granules, and lack of argyrophilic proteins. 3 h after decapitation, a thickening of the fibrillar rim occurred, accompanied by an increase of granules in the vacuole. After 6h, the unique vacuole broke up into two to four small vacuoles in which the granules are more abundant. After 12 h the nucleolus acquired compact structure with few minute vacuoles dispersed over the fibrillar component. During the whole cell cycle, the condensed chromatin is always observed in the vacuole. Our findings suggest that the appearance of the vacuoles is subsequent to the output of preribosomes from nucleolus. These vacuoles might play a role in condensation and decondensation of the chromatin.     

Investigations of the seed protein content of several pea genotypes grown in two different years

Summary Seventeen X-ray and neutron induced mutants of the commercial variety Dippes gelbe Victoria were analyzed with regard to their seed protein percentage. The interaction of genotypic and year effects in 1975 (normal weather conditions) and 1976 (extremely hot and dry) was also taken into consideration. To avoid undiscoverable environmental bias, the plants were grown in a nonstandard three-dimensional layout. Biometric analysis was done by using the theory of the general linear model with a formula-processing computer program. In the first year, significant genetically caused differences were found in the material. The bifurcated mutant 157A was especially of considerable interest because an improved protein content was combined with relative good yield. In the second year, no significant differences between the mutants were revealed, but all genotypes showed a similar good protein value of about 27%.     

Thylakoid and grana formation during the development of pea chloroplasts,illuminated by white,red, and blue low intensity light

Summary We analyzed the formation of thylakoids and grana during the development of pea chloroplasts, illuminated by white, red and blue low intensity light. The total length of granal and intergranal thylakoids, and the length of granal thylakoids per unit area of plastid section were measured. Initially the greatest increase in length of granal thylakoids and the highest incidence of grana with large thylakoid content occurred in red light. On the other hand, with illumination times of over 12 hours blue light appeared to be more efficient in stimulating grana formation and thylakoid growth.     

Biochemical and genetic comparison of two nitrate reductase-deficient pea mutants disturbed in the cofactor

Two nitrate reductase (NaR)-deficient mutants of pea (Pisum sativum L.), E₁ and A300, both disturbed in the molybdenum cofactor function and isolated, respectively, from cv Rondo and cv Juneau, were tested for allelism and were compared in biochemical and growth characteristics. The F₁ plants of the cross E₁ × A300 possessed NaR and xanthine dehydrogenase (XDH) activities comparable to those of the wild types, indicating that these mutants belong to different complementation groups, representing two different loci. Therefore, mutant E₁ represents, besides mutant A300 and the allelic mutants A317 and A334, a third locus governing NaR and is assigned the gene destignation nar 3. In comparison with the wild types, cytochrome c reductase activity was increased in both mutants. The mutants had different cytochrome c reductase distribution patterns, indicating that mutant A300 could be disturbed in the ability to dimerize NaR apoprotein monomers, and mutant E₁ in the catalytic function of the molybdenum cofactor. In growth characteristics studied, A300 did not differ from the wild types, whereas fully grown leaves of mutant E₁ became necrotic in soil and in liquid media containing nitrate.     

Proximity of an ARS consensus sequence to a replication origin of pea (Pisum sativum)

The replication origin (ori-r9) of the 9.0 kb rDNA repeats of pea (Pisum sativum, cv. Alaska) was cloned and found to reside in a 1.5 kb fragment of the non-transcribed spacer region located between the 25 S and 18 S genes. Labeled rDNA rich in replication forks, from cells positioned at the G₁/S phase boundary, was used to map ori-r9 by hybridization procedures. Ori-r9 is in a 210-base fragment that is 1.6 kb from the 5 end of the 18 S gene and about 1.5 kb from the 3 end of the 25 S gene. The same procedures, using labeled synthetic ARS consensus sequence as a probe, showed than an ARS consensus sequence is located 3 to ori-r9 in a 710-base fragment. An ARS consensus sequence is, therefore, adjacent to ori-r9 but not coincidental with it.     

Photosynthetic and yield responses of an old and a modern winter wheat cultivars to short-term ozone exposure

In order to study the responses of winter wheat cultivars released in different years to short-term high O₃ exposure, an old cultivar (‘Nongda 311’, released in 1960s) and a modern one (‘Yannong 19’, released in 1990s) were treated with an O₃ exposure (145 ± 12 mm³ m⁻³, 4 h d⁻¹ for 3 d) shortly after anthesis stage (> 50 % main stems blossomed). During the O₃ exposure, light-saturated photosynthetic rate (P _N) and stomatal conductance (g _s) of both cultivars decreased considerably. Elevated O₃ did not decrease dark-adapted maximum photochemical efficiency, but induced significant reduction in actual photochemical efficiency and thereby considerably increase in non-photochemical quenching. P _N, g _s of the modern cultivar ‘Yannong 19’ decreased more than the older one ‘Nongda 311’, indicating the former exhibited higher sensitivity to O₃ than the latter. After O₃ exposure, P _N, g _s and chlorophyll (Chl) content in flag leaf decreased more quickly than control, indicating induction of faster premature leaf senescence. As a result, the short-term O₃ exposure caused substantial yield loss, with larger reduction in ‘Yannong 19’ (−19.2 %) than in ‘Nongda 311’ (−8.4 %). Our results indicated that high O₃ exposure at grain filling stage would have greater negative impacts on the high yielding modern cultivar relative to the old one with lower yield.     

Fasciation in pea: Basic principles of morphogenesis

A study of fasciated pea Pisum sativum L. (Fabaceae) mutant Shtambovy in comparison with the wild type (Nemchinovsky cultivar) has shown that fasciation is a result of abnormal cohesion of axial or other structures which arise in a superfluous amount due to uncontrolled meristic processes. In some cases, the organs with the same number and position as in the wild type can be fascinated. Subsequent defasciation and some features of tissue differentiation suggest that the meristem of a fasciated shoot retains a certain degree of discreteness which reflects its complex structure. The number and position of leaves in a node is a function of the diameter of the leaf primordium inhibitory zone, size of the shoot apical meristem, and number of bundles in a shoot. In the absence of the apex proliferative activity combined with the reduction of phyllomes in the upper nodes, abnormal cohesion of the second order axes, racemes, can take place. As a result, inflorescences of special type develop.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.