Protein patterns and superoxide dismutase activity in non-mycorrhizal and arbuscular mycorrhizalPisum sativum L. plants

There are few reports in relation to the role of specific proteins in the mycorrhizal symbiosis. Among the changes in the protein expression as a consequence of the arbuscular mycorrhizal symbiosis, only one case related to changes in superoxide dismutase (SOD; EC 1.15.1.1) activity has been reported in the red clover-Glomus mosseae symbiosis.In this paper, the symbiotic system formed by a leguminous plant,Pisum sativum, and the fungusGlomus mosseae is studied in terms of protein patterns and SOD activity in both mycorrhizal and non-mycorrhizal roots. Our results show that among the differential polypeptides separated by SDS-PAGE, one with a molecular weight of 32.0 kDa, and a protein with an isoelectric point of pI 4.9 appeared strongly expressed in mycorrhizal roots. A partial purification of the related polypeptide could be achieved by DEAE-cellulose chromatography. A higher SOD activity was also detected in mycorrhizal pea roots, although both mycorrhizal and non-mycorrhizal roots showed the same isoenzymatic pattern for SODs: two Mn-SODs (I and II) and two Cu,Zn-SODs (I and II) were detected, Cu,Zn-SOD I being the most abundant isozyme in both types of roots. A similar pattern of SOD isozymes (Mn-SODs I and II, and Cu,Zn-SODs I and II) was also found in nodules of mycorrhizal and non-mycorrhizal pea roots. However, in nodules Mn-SOD II was the main isozyme. The bacterial nature of this isozyme is postulated in this report.Dr. Justo Arines died on the 15th November, 1993 in Dijon (France), while he was attending a molecular biology course on mycorrhizas.     

Soluble and membrane symbiosis-related polypeptides associated with the development of arbuscular mycorrhizas in tomato (Lycopersicon esculentum)

To analyse the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expression, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) patterns of crude extracts, soluble and membrane proteins of tomato roots, either mycorrhizal and the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or non-mycorrhizal, have been compared. In the three fractions analysed, AM colonization induced up-regulation with down-regulation of the synthesis of polypeptides already present in tomato roots and induction of some new polypeptides. Separation of root extracts into soluble and membrane fractions allowed us to identify two soluble, and five membrane-bound, newly induced polypeptides in AM roots. Comparison of the protein patterns of AM roots with those of the external mycelium of G. mosseae showed that one of the newly induced polypeptides might correspond to a fungal polypeptide. By using this experimental approach, we have been able to detect 44 polypeptides that are differentially displayed in tomato roots as a consequence of the establishment of the AM symbiosis.     

Subcellular distribution of superoxide dismutase in leaves of ureide-producing leguminous plants

The subcellular localization of superoxide dismutase (SOD; EC. 1.15.1.1) was studied in leaves of two ureide-producing leguminous plants ( Phaseolus vulgaris L. cv. Contender and Vigna unguiculata [L.] Walp). In leaves of Vigna and Phaseolus , three superoxide dismutases were found, an Mn-SOD and two Cu, Zn-containing SODs (I and II). Chloroplasts, mitochondria, and peroxisomes were purified by differential and density-gradient centrifugation using either Percoll or sucrose gradients. The yields obtained in intact chloroplasts and peroxisomes from Vigna were considerably higher than those achieved for Phaseolus . Purified chloroplasts only contained the Cu, Zn-SOD II isozyme, but in mitochondria both Mn-SOD and Cu, Zn-SOD I isozymes were present. In purified peroxisomes no SOD activity was detected. The absence of SOD activity in leaf peroxisomes from Vigna contrasts with results reported for the amide-metabolizing legume Pisum sativum L. where the occurrence of Mn-SOD was demonstrated in leaf peroxisomes (del Río et al. 1983. Planta 158: 216–224; Sandalio et al. 1987. Plant Sci. 51: 1–8). This suggests that in leaf peroxisomes from Vigna plants the generation of O₂^- radicals under normal conditions probably does not take place.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

我国北部的七种VA菌根真菌

从北京和新疆地区某些栽培及野生植物根际分离出7种VA真菌:丽孢无梗囊霉Acaulospora elegans Trappe & Gerd.,地表球囊霉Glomus versiforme (karsten) Berch,隐球囊霉G.occultum Walker,透光球囊霉G.diaphanum Morton & Walker,摩西球囊霉G.mosseae(Nicol.& Gerd.)Gerd.& Trappe,缩球囊霉G.constrictum Trappe,和苏格兰球囊霉G.caledonium(Nicol.& Gerd.)Trappe & Gerd.。其中,地表球囊霉为我国新记录种。本文除描述其形态特征外,还介绍了孢壁组织化学反应及生境条件。     

In vitro synthesis of superoxide dismutases of rat liver

The syntheses of copper, zinc-superoxide dismutase (Cu,Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in vitro were studied. Both Cu,Zn-SOD and Mn-SOD were preferentially synthesized by free polysomes. Mn-SOD was synthesized as a large precursor (26,000 daltons), which was processed to the mature size (22,500 daltons) by in vitro incubation with a rat liver mitochondrial fraction. On the other hand, Cu,Zn-SOD was synthesized as the mature size product. It was shown that Cu,Zn-SOD and Mn-SOD synthesized in vitro represented 0.018% and 0.016% of the total translation products of free polysomes, respectively.     

Effects of drought on non-mycorrhizal and mycorrhizal maize: changes in the pools of non-structural carbohydrates, in the activities of invertase and trehalase, and in the pools of amino acids and imino acids

To study the response of non-mycorrhizal and mycorrhizal maize plants to drought, the changes in the pools of non-structural carbohydrates and amino acids were analysed in leaves and roots of two maize cvs. Plants well colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) (60% of root length infected) and comparable non-mycorrhizal plants were subjected to moderate drought stress by reducing the water supply. This stress induced a conspicuous increase in the trehalose pool in the mycorrhizal roots, probably because it was accumulated by the fungal symbiont. Furthermore, glucose and fructose were accumulated in leaves and roots of non-mycorrhizal plants but not in the mycorrhizal ones. Starch disappeared completely from the leaves of both mycorrhizal and non-mycorrhizal plants in response to drought. Activities of soluble acid invertase and trehalase were also measured. Acid invertase activity increased during drought in the leaves of both non-mycorrhizal and mycorrhizal plants whilst in the roots it was unaffected in non-mycorrhizal plants and decreased in the mycorrhizal ones. Without drought stress, trehalase activity was considerably higher in the leaves and roots of mycorrhizal plants than in those of non-mycorrhizal plants. It increased conspicuously during drought, primarily in the leaves of non-mycorrhizal plants. A drought-induced accumulation of amino acids as well as imino acids was found in roots and leaves of both mycorrhizal and non-mycorrhizal plants; leaves of mycorrhizal plants accumulated more imino acids than those of non-mycorrhizal ones. Our results show that drought stress and the presence of a mycorrhizal fungus have a considerable effect on carbon partitioning, imino acid and amino acid accumulation in maize plants.     

Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.     

Xyloglucanases in the interaction between saprobe fungi and the arbuscular mycorrhizal fungus Glomus mosseae

We studied the production of xyloglucanase enzymes of pea and lettuce roots in the presence of saprobe and arbuscular mycorrhizal (AM) fungi. The AM fungus Glomus mosseae and the saprobe fungi Fusarium graminearum, Fusarium oxysporum-126, Trichoderma harzianum, Penicillium chrysogenum, Pleurotus ostreatus and Aspergillus niger were used. G. mosseae increased the shoot and root dry weight of pea but not of lettuce. Most of the saprobe fungi increased the level of mycorrhization of pea and lettuce, but only P. chrysogenum and T. harzianum inoculated together with G. mosseae increased the dry weight of pea and lettuce respectively. The AM and saprobe fungi increased the production of xyloglucanases by plant roots. The level of xyloglucanase activities and the number of xyloglucanolytic isozymes in plants inoculated with G. mosseae and most of the saprobe fungi tested were higher than when both microorganisms were inoculated separately. The possible relationship between xylogucanase activities and the ability of AM and saprobe fungi to improve the dry weight and AM root colonization of plants was discussed.     

Influence of an arbuscular mycorrhizal fungus on the interaction of a binucleate Rhizoctonia species with Myc+ and Myc- pea roots

A binucleate Rhizoctonia (BNR) species was isolated from a clay loam soil on the Epoisses experimental station of INRA, Dijon and identified as belonging to the anastomosis Group A (AG-A). The BNR was inoculated to a Myc- Pisum sativum mutant (P53, sym30 locus) and its wild-type parent (cv Frisson) in the presence or absence of the arbuscular mycorrhizal fungus Glomus mosseae. The BNR had no significant effect on plant weight. Myc+ and Myc- roots were equally susceptible towards BNR and showed no localized cellular defense responses. The presence of BNR decreased significantly the percentage of root length colonized by G. mosseae and, inversely, G. mosseae reduced the number of BNR monilioid chains formed in root epidermal cells of the two pea genotypes. The pisatin concentration was increased significantly by BNR in both Myc+ and Myc- roots and by G. mosseae in the wild-type pea plants. The highest accumulation of pisatin was observed in Myc+ roots when both fungi were present.     

Polyamines in humic acid and their effect on radical growth of lettuce seedlings

Linum usitatissimum, Sorghum bicolor and Triticum aestivum plants were further colonised by the arbuscular mycorrhizal fungus, Glomus mosseae, during a four week period of hydroponic culture after a pre-culture period of three weeks with the fungus in perlite substrate. The viability of mycorrhizal colonisation of T. aestivum was indicated by an initial experiment where G. mosseae from mycorrhizal plants colonised non-mycorrhizal plants when the plants were grown together in the same hydroponic container using modified Long Ashton nutrient solution. Intermittant aeration of the plant roots (2 h periods, four times per day) provided a compromise between adequate aeration and minimal disturbance of the fungus. In a second experiment, two nutrient media, modified Long Ashton and modified Knop plus Hoagland medium were compared for culturing G. mosseae on T. aestivum. A significantly higher root dry weight was found for the mycorrhizal versus the non-mycorrhizal wheat plants in modified Long Ashton nutrient medium, which contained 10 µM P and an organic buffer. Modified Knop plus Hoagland nutrient medium contained a high P concentration (0.9 mM) and did not produce viable cultures of mycorrhizal colonisation. In a third experiment, modified Long Ashton medium was used for hydroponic culture of mycorrhizal L. usitatissimum, S. bicolor and T. aestivum. The root colonisation percentages for T. aestivum (73%), S. bicolor (36%) and L. usitatissimum (65%) were within the range of colonisation rates obtained with solid substrate culture in perlite. Viability of the mycorrhizal structures in hydroponic culture was assessed by monitoring activity of fungal succinate dehydrogenase and found to be similar to cultures in perlite. No difference in the P concentration of mycorrhizal and non-mycorrhizal plants was observed, possibly owing to the lack of diffusion limits for P in hydroponic solution. This report describes a system for the viable culture of G. mosseae with different plant species where a high mycorrhizal colonisation rate was produced under conditions of a short culture period using intermittent aeration, a low concentration of P supply and an organic buffer.     

Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).     

氯化钠胁迫下嫁接黄瓜叶片SOD和CAT mRNA基因表达及其活性

研究了NaCl胁迫下嫁接和自根黄瓜叶片Cu/Zn-SOD、Mn-SOD和CAT mRNA的表达与其酶活性变化及其MDA含量和电解质渗漏率变化.结果表明：在NaCl胁迫条件下,嫁接黄瓜叶片Cu/Zn-SOD mRNA、Mn-SOD mRNA和CAT mRNA的相对表达量均高于自根黄瓜,SOD、Cu/Zn-SOD、Mn-SOD和CAT活性也均高于自根黄瓜,说明与自根黄瓜相比,嫁接黄瓜叶片较高的Cu/Zn-SOD mRNA、Mn-SOD mRNA和CAT mRNA相对表达量是其维持较高Cu/Zn-SOD、Mn-SOD和CAT活性的重要原因;随着NaCl胁迫时间的延长,嫁接和自根黄瓜叶片Cu/Zn-SOD- mRNA、Mn-SOD mRNA和CAT mRNA的相对表达量均呈上升趋势,但其酶活性变化并不完全一致,说明还有其他因素参与相关酶活性的调控;嫁接黄瓜叶片MDA含量和电解质渗漏率均低于自根黄瓜,说明嫁接黄瓜具有较高的活性氧清除系统,可以减少活性氧物质的危害,提高其耐盐性.     

低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响

研究了低温胁迫下嫁接和自根黄瓜叶片Mn-SOD、Cu/Zn-SOD和CAT mRNA基因表达和酶活性变化及其与抗冷性的关系.结果表明:低温胁迫下,嫁接与自根黄瓜叶片Cu/Zn-SOD、Mn-SOD mRNA基因相对表达量变化分别与其Cu/Zn-SOD、Mn-SOD活性变化相吻合,而CATmRNA相对表达量变化与其CAT活性变化并不一致;嫁接黄瓜叶片Cu/Zn-SOD和Mn-SOD mRNA相对表达量及SOD、Cu/Zn-SOD和Mn-SOD活性均高于自根黄瓜,MDA含量和电解质渗漏率均低于自根黄瓜,嫁接黄瓜较高的SOD基因表达量调控的较高SOD活性是其抗冷性强于自根黄瓜的主要因素;嫁接黄瓜的功能叶CAT mRNA相对表达量略高于自根黄瓜,而幼叶CAT mRNA相对表达量低于后者,但两者CAT活性差异不大,说明低温胁迫对嫁接黄瓜叶片CAT mRNA相对表达量及CAT活性的影响不大.     

Superoxide dismutase of plant cell vacuoles

Metal-dependent superoxide dismutases (SOD; EC 1.15.1.1) are present in many cell compartments (mitochondria, plastids, nuclei, peroxisomes, endoplasmic reticulum, cell wall and cytosol). We have established that SOD is also localized in the central vacuole. Cyanide-sensitive Cu, Zn-SOD was found in the fraction of isolated vacuoles of red beet roots (Beta vulgaris L.). The enzyme was represented by three isoforms. Comparison of isoenzyme composition and the level of SOD activity in vacuoles, nuclei, plastids and mitochondria isolated from root cells has shown that Cu, Zn-SOD is present in vacuoles and nuclei, two SOD forms (Cu, Zn- and Fe-SOD) are present in plastids, and two SOD forms (Cu, Zn- and Mn-SOD) are present in mitochondria. Cu, Zn-SOD of organelles, unlike vacuolar Cu, Zn-SOD, had only one isoform. The level of enzyme activity from the vacuolar fraction was twice higher than the level of SOD activity from the fractions of isolated organelles. Previously it has been suggested that Cu, Zn-SOD may be localized on the vacuolar membrane or in the near-membrane space from the side of cytoplasm. Our tests have revealed the Cu, Zn-SOD activity in water-soluble extracts of isolated vacuole fractions in the absence of detergent, which may confirm localization of the enzyme inside the organelles.     

Nod factors and tri-iodobenzoic acid stimulate mycorrhizal colonization and affect carbohydrate partitioning in mycorrhizal roots of Lablab purpureus

Roots of Lablab purpureus (L.) Sweet were treated with tri -iodobenzoic acid (TIBA), kinetin or with nodulation factors (Nod factors) purified from Rhizobium sp. NGR234 and grown in the presence of a mycorrhizal inoculum ( Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe. Colonization by the mycorrhizal fungus was increased from <30% to c . 65% of root length when roots were treated with these growth regulators. Moreover, treatment of mycorrhizal L. purpureus roots with Nod factors or TIBA strongly induced sporocarp formation of Glomus mosseae . In parallel, the pool size of the fungal disaccharide trehalose was significantly affected in roots treated with TIBA and Nod factors alone, and with TIBA combined with all effectors, and increased from 0·06 mg g⁻¹ d. wt in control roots to up to 1·7 mg g⁻¹ d. wt (TIBA+kinetin). Conversely, the sucrose pool decreased from 5% d. wt to less than a half in roots treated with Nod factors. Activities of trehalase were significantly enhanced in mycorrhizal roots by the treatment with Nod factors or TIBA. When Nod factors and TIBA were added in combination, these activities were strongly enhanced suggesting synergism between these growth regulators.     

AM真菌种间差异对枳壳生长及耐热性效应的研究※

用地表球囊霉、莫西球囊霉、珠状巨孢球囊霉及其混合菌剂接种无菌根枳壳幼苗进行盆栽试验,25℃培养4个月,观察对枳壳菌根形成和营养生长的影响,在40℃高温胁迫30d,调查分析菌根枳壳的耐热性。试验结果表明:接种AM真菌的根系形成了20%～80%的菌根侵染率;菌根枳壳的苗高、苗质量、节间长、茎基粗、须根数量和须根长度等营养生长显著增加;叶片中的SOD,POD活性和根系活力显著增强,可溶性蛋白、可溶性糖含量显著升高,叶片中的MDA含量降低,膜透性显著变小,枳壳苗的耐热性显著提高;但是,AM真菌在促进枳壳苗菌根化、营养生长和提高耐热性方面存在着种间差异,地表球囊霉、莫西球囊霉、珠状巨孢球囊霉、混合菌剂与枳壳根系形成丛枝菌根的侵染率依次为20.4%±1.2%、61.8%±3.4%、85.7%±2.7%、83.3%±2.2%,促进枳壳苗营养生长提高枳壳苗耐热能力的AM真菌依次为:地表球囊霉<莫西球囊霉<珠状巨孢球囊霉<混合菌剂,认为珠状巨孢球囊霉和莫西球囊霉是枳壳耐高温胁迫菌根化育苗的重要优良菌种。     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.