Differential expression of CuZn-superoxide dismutases in Pinus sylvestris needles exposed to SO2 and NO2

Mature Pinus sylvestris trees were exposed to air-pollution strees in an open field fumigation experiment. The trees were exposed to low-levels of sulphur dioxide and nitrogen oxides throughout the whole growing season (June to October 1991). Three age-classes (current, one- and two-year-old) of needles were collected from exposed and control trees and the expression of chloroplastic and cytosolic CuZn-superoxide dismutase (SOD; EC 1.15.1.1) genes was studied. The levels of chloroplastic and cytosolic CuZn-SOD mRNA were significantly (up to 2.3-fold) higher in exposed trees for all age-classes of needles as compared to control trees. No significant differences were observed between different needle age-classes in CuZn-SOD mRNA induction. The chloroplastic and cytosolic CuZn-SOD mRNA were induced to the same extent, however, the level of chloroplastic CuZn-SOD mRNA was higher than the cytosolic one. Small differences were observed in CuZn-SOD mRNA levels for individual trees both within exposed and control areas. Protein activity measurements of the CuZn-SOD isozymes did not reveal any significant differences between control and exposed trees. The results are discussed in relation to protection against oxidant stress from air pollutants.     

Structural organization, regulation, and expression of the chloroplastic superoxide dismutase Sod1 gene in maize.

A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined.     

Chloroplast photooxidation inhibits the expression of a set of nuclear genes

Summary Mutations or herbicides which inhibit the accumulation of carotenoid pigments in higher plants also result in the arrest of chloroplast development at a very early stage. The cause is extensive photooxidative damage within the chloroplast in the absence of protective carotenoids. Because the extent of photooxidation is dependent upon light intensity, normal chloroplast development can occur when carotenoid-deficient seedlings are grown in very dim light. Normal accumulation of chloroplastic and cytosolic mRNAs encoding chloroplast proteins proceeds only under permissive dim light conditions. Illumination with higher intensity light causes rapid chlorophyll photooxidation and the loss of two cytosolic mRNAs coding for proteins destined for the chloroplast, but does not affect another light-regulated cytosolic mRNA encoding a cytosolic protein. This experimental system may have uncovered a mechanism which coordinates the expression of genes in different cellular compartments.Abbreviations LHCP light-harvesting chlorophyll a/b protein - SSu small subunit - RuBP fibulose 1,5-bisphoshate - PEP phosphoenolpyruvate     

Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.     

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.     

Glutamine Synthetases of Higher Plants : Evidence for a Specific Isoform Content Related to Their Possible Physiological Role and Their Compartmentation within the Leaf

The chromatographic properties of glutamine synthetase isoforms have been investigated in a wide range of higher plant leaves and shoots using ion exchange chromatography. Different patterns of glutamine synthetase isoform content were observed. Among higher plants, four patterns or groups could be recognized. The first group is characterized by having only cytosolic glutamine synthetase, whereas the second group is distinguished by having only chloroplastic glutamine synthetase. The third group is characterized by cytosolic glutamine synthetase being a minor component of the total leaf glutamine synthetase activity. The fourth group is distinct from the other groups in having high cytosolic and chloroplast glutamine synthetase activity. Immunological studies have been undertaken on a few species from each group to identify unambiguously both cytosolic and chloroplastic glutamine synthetases.     

Manganese nutrition effects on tomato growth, chlorophyll concentration, and superoxide dismutase activity

The effects of Mn nutrition of tomato (Lycopersicon esculentum Mill.) seedlings on Mn-, Fe- and CuZn-superoxide dismutase (SOD, EC 1.15.1.1) enzymatic activities, metal translocation, chlorophyll concentration, and plant growth were tested using a bioassay system consisting of chelator-buffered nutrient culture with Mn2+ activities set to pMn (-log activity of Mn2+) of 6.6, 7.6, 8.6, and 9.6. The two middle levels resulted in optimal plant growth, whereas the two extreme levels resulted in a gradual decrease in chlorophyll concentration and slower plant growth. At the end of the experiment, 26 days after transfer to the Mn treatments, significant differences in shoot Mn concentration were manifested, from 10.5 mg kg(-1) in plants grown in pMn 9.6 to 207.4 mg kg(-1) in plants grown in pMn 6.6. Other element concentrations in the leaf suggest that growth inhibition and chlorophyll synthesis were affected primarily by manganese deficiency and excess. Twenty days after transfer of plants to the Mn treatments Mn-, Fe- and CuZn-SOD activities were assayed in young expanded leaf tissues by electrophoresis running gel. Whereas chloroplastic CuZn-SOD activity did not differ among Mn treatments, the cytosolic CuZn-SOD and mitochondrial Mn-SOD activities increased in both Mn-excess and Mn-deficient plants.     

The effect of needle age on the acceptability of Sitka spruce needles to the aphid,Elatobium abietinum (Walker)

Summary A comparison of the feeding behaviour of E. abietinum on current year needles and previous year needles of P. sitchensis revealed that during the summer months the aphids preferentially settled on previous year needles, this response not being evident in winter. Aphids on current year needles in summer took a much longer period of time to commence sap uptake than in winter, intake ceasing following a very short feeding period. On previous year needles sap uptake in December commenced after a shorter feeding period than in June. Analysis of total and soluble nitrogen levels in Sitka spruce needles showed that current year needles had initially higher levels during shoot elongation in May and early June, but that previous year needles had higher levels for most of the remainder of the year. Quantitative analyses of amino acids revealed that in current year needles the levels were generally lower than in previous year needles. Less marked proportional differences were observed between previous year needles in May and in July/August when the needles were unsuitable. Addition of amino acids in solution into cut current year shoots resulted in increased longevity on shoots containing introduced iso-leucine, histidine and methionine and revealed a general imbalance of the amino acids.     

A small family of novel CuZn-superoxide dismutases with high isoelectric points in hybrid aspen

Several CuZn-superoxide dismutases (SODs; EC 1.15.1.1) were cloned from hybrid aspen (Populus tremula L. x tremuloides Michx.). Two of the cloned genes encode representatives of a novel type of CuZn-SOD and we named it HipI-SOD because of its high isoelectric point (> or =9). The SODs were cloned by screening a cDNA library with a probe based on a Scots pine (Pinus sylvestris L.) CuZn-SOD that is predominantly located extracellularly. The expression pattern of HipI-SOD was examined using a Northern blot technique and compared with the expression patterns of cytosolic and chloroplastic SODs. Distinct expression patterns were observed for the three types of CuZn-SOD, with HipI-SODs showing strong expression in apical tissues. Southern blots as well as protein analysis suggest that these novel HipI-SODs belong to a small gene family, one member of which might be monomeric.     

Involvement of the chloroplast signal recognition particle cpSRP43 in acclimation to conditions promoting photooxidative stress in Arabidopsis

In this study, we have investigated the role of the CAO gene (coding for the chloroplast recognition particle cpSRP43) in the protection against and acclimation to environmental conditions that promote photooxidative stress. Deficiency of cpSRP43 in the Arabidopsis mutant chaos has been shown previously to lead to partial loss of a number of proteins of the photosystem II (PSII) antennae. In addition, as reported here, mutant plants have lower growth rates and reduced lignin contents under laboratory conditions. However, chaos seedlings showed significantly higher tolerance to photooxidative stress under both tightly controlled laboratory conditions and highly variable conditions in the field. This greater tolerance of chaos plants was manifested in less photooxidative damage together with faster growth recovery in young seedlings. It was also associated with a lower production of H2O2, lower ascorbate levels and less induction of ascorbate peroxidases. Under field conditions, chaos exhibited better overall photosynthetic performance and had higher survival rates. Expression of the CAO gene may be regulated by a light-dependent chloroplastic redox signalling pathway, and was inhibited during acclimation to high light and chilling temperatures, simultaneously with induction of ascorbate peroxidases. It is concluded that the presence/absence of the CAO gene has an impact on photo-produced H2O2, lignification in the hypocotyls and on the plant's susceptibility to photooxidative stress. Therefore, regulation of the CAO gene may be part of the plant's system for acclimation to high light and chilling temperatures.