Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

Differential alcohol dehydrogenase and malate dehydrogenase isozyme expression in long-term callus tissue cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH) and mitochondrial malate dehydrogenase (mMDH) isozymes were tested as markers to study the effect of a high kinetin concentration on isozyme phenotypes and on the development ofCereus peruvianus callus tissue culture. Three-year-old callus tissues were used as samples. Callus tissue samples grown on 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and on 4.0 and 8.0 mg/LN-(2-furanylmethyl)-1H-purine-6 amine (kinetin) were cut and transferred to fresh medium containing 4.0 mg/L 2,4-D and 4.0, 8.0, 16.0, and 32 mg/L kinetin combinations. The pattern of changes observed in the ADH and mMDH isozymes as well as the growth of callus tissues was independent of the concentrations tested. The various ADH and mMDH isozymes seem to be products of differential association of subunits of the twoAdh and twomMdh genes. Both genes are active throughout callus tissue development; however, gene expression changed with various callus culture conditions. This study addresses how long-term callus culture conditions affect constitutive and differential gene expression of theAdh andmMdh genes inC. peruvianus.     

Alcohol dehydrogenase (EC 1.1.1.1) isozymes as markers at 2,4-dichlorophenoxyacetic acid × kinetin combinations in callus cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH; EC 1.1.1.1) isozymes were investigated in tissue ofCereus peruvianus cultured in different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Five ADH isozymes were detected in starch gel and showed different patterns in seeds, seedlings, calli cultured at 32 and 22°C, and plants regenerated from calli cultured in three 2,4-D and kinetin combinations. Four phenotypes formed by different combinations of ADH-2, ADH-3, ADH-4, and ADH-5 were detected in calli cultured at 32°C and in plants regenerated from calli. ADH-1 isozyme was detected only in calli subcultured for 1 or 2 weeks at 22°C and was indicated as a marker of stress conditions that affect the growth ofC. peruvianus callus tissues in culture. ADH phenotypes with either a higher or a lower number of isozymes were detected in different proportions in the callus tissues cultured in media containing different 2,4-D and kinetin ratios. ADH isozyme patterns were found to be sensitive markers at the highest kinetin concentration or at high kinetin/2,4-D ratios. The results indicate a high correlation between the ADH isozyme patterns and the capacity for regeneration. Thus, ADH isozymes are indicated as good biochemical markers and as a powerful tool for monitoring studies ofC. peruvianus callus cultures.This research was supported by the CNPq.     

EXPERIMENTAL INDUCTION OF VASCULAR TISSUES IN CALLUS OF ANGIOSPERMS

Wetmore , Ralph H. (Harvard U., Cambridge, Mass.), and John P. Rier . Experimental induction of vascular tissues in callus of angiosperms . Amer. Jour. Bot. 50(5): 418–430. Illus. 1963.—Callus tissues in established maintenance culture lack morphological and physiological organization. Such callus consists of homogeneous parenchyma. Movement of auxin and sugar, therefore, must be along diffusion gradients. The only vascular tissues occurring in callus are induced. Experimental induction of vascular tissues has been successful in callus of 3 sp. of the Oleaceae: a tree, Fraxinus americana, and 2 shrubs, Syringa vulgaris and Ligustrum vulgare; another tree, Salix purpurea, var. lambertiana; a vine, Parthenocissus tricuspidata; and an herb, Helianthus tuberosus. In each of these species, an auxin (IAA or NAA in these studies) and a sugar (sucrose or glucose in these studies) prove necessary for the induction and complete differentiation of xylem and phloem in callus tissues. Varying concentrations of sugar alter the proportions of xylem to phloem: low concentrations, 1.5%–2.5%, favor xylem formation; high, 3%–4%, favor phloem. Middle concentrations, 2.5%–3.5%, favor the presence of xylem and phloem, usually with a cambium between. The almost universal association of xylem and phloem may have its explanation in this middle concentration of sugar. Grafting of apices into callus or direct application of appropriate concentrations of an auxin and a sugar in agar to the surface of callus causes nodules of vascular tissue to be formed, mostly in a circular pattern when seen in section transverse to the axis of orientation of the callus in the medium. The diameter of this circle varies directly with the auxin concentration at the place of application, 0.05 mg/liter giving a narrow, and 1 mg/liter, a wide pith. In individual nodules, xylem is characteristically oriented towards the center of the callus and the phloem towards the outside. Variable cross-sectional views of nodule distribution in calli under different treatments suggest experimental approaches to understanding stelar patterns. The induction and differentiation in callus of xylem and phloem tissues has no relation to conduction. Any use of vascular tissues can occur only after their induction.     

鲇鱼不同组织同工酶的组织特异性的初步研究

采用聚丙烯酰胺垂直凝胶电泳法(PAGE)对鲇鱼的9种组织的5种同工酶(LDH、MDH、ADH、SUDH和EST)进行了分析,结果表明:5种同工酶在各种组织均有不同的表达程度:在心、肝、卵巢和肌肉中的表达频率最高,在脑和眼中最低。LDH和MDH在各个组织中均为强表达,与以往研究结果比较,认为鲇鱼LDH同工酶不存在C位点。鲇鱼同工酶组织特异性较明显。     

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.     

准噶尔雅罗鱼组织同工酶表达特性

使用不连续PAGE法,分析了10尾准噶尔雅罗鱼(Leuciscus merzbacheri)眼睛、鳃、皮、背部肌肉、鳍和肝胰脏6种组织的10种同工酶(LDH,CCO,EST,CAT,POD,ME,MDH,G6PD,GDH,ADH)的差异表达,并对部分同工酶基因位点及表达酶谱表型进行了分析,以期为其种质资源保护和开发以及遗传育种等方面的研究提供基础资料。结果显示,10种同工酶中9种在6种组织中出现了明显的组织差异性,仅CCO在6种组织中的差异性较小。在对准噶尔雅罗鱼的10种同工酶的遗传多样性分析中,共记录到了21个基因位点,其中Est-1、Me-B、s-MDH、G6pd-A、G6pd-B和Adh-A为多态性基因位点。多态位点比例为:P=6/21=28.57%。     

乌鳢组织内三种同工酶的研究

本研究对乌鳢９种组织内的ＬＤＨ、ＭＤＨ和ＡＴＰａｓｅ同工酶作了分析，结果表明，乌鳢各组织内的ＬＤＨ、ＭＤＨ有明显的组织特异性，ＬＤＨ由两个基因编码。骨骼肌中的ｓ－ＭＤＨ也有两个基因编码。ＡＴＰａｓｅ同工酶存在于乌鳢的多种组织中，其基因编码数有待进一步探讨。     

Isozyme patterns in callus cultures and in plants regenerated from calli ofCereus peruvianus (Cactaceae)

Electrophoretic patterns for isocitrate dehydrogenase (IDH; EC 1.1.1.42), acid phosphatase (ACP; EC 3.1.3.2), peroxidase (PER; EC 1.11.1.7), and esterase (EST; EC 3.1.1.1) isozymes were determined inCereus peruvianus tissues and used as markers of genetic uniformity of calli and of the plants regenerated from callus cultures. One IDH, six ACP, six PER, and six EST isozymes were induced in cultured callus tissues in medium containing three 2,4-dichlorophenoxyacetic acid and kinetin combinations. Four ACP, two PER, and three EST isozymes were still present in all regenerated plantsin vitro and therefore can be used as markers of theC. peruvianus plants regenerated from callus tissues. The differential patterns of ACP and IDH isozymes and the similar zymograms for PER and EST isozymes presented by callus tissues were used in a comparison of callus tissues cultured for 2 years. The comparative analysis of zymograms within each enzyme system indicated a mean heterogeneity coefficient of 0.33 forC. peruvianus calli cultured for 2 years. Because of the isozyme variations, which developed in culture medium and were transferred to the regenerated plants, the IDH, ACP, PER, and EST enzyme systems can be considered to be good markers for investigating possible genetic variations in plant populations ofC. peruvianus obtainedin vitro from callus culture.This research was supported by the CNPq     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

Symplastic and apoplastic sugar contents in gall tissues and callus of the sumac (Rhus chinensis MILL.)

To elucidate the role of cell wall in interaction with gall-inducing organisms, symplastic and apoplastic sugar contents in different shapes of gall tissue of the sumac (Rhus chinensis Mill.) were compared with those of the callus. The gall tissues with vascular cylinders, intercellular spaces and callus were fractionated into symplastic [methanol (MeOH), hot water (HW), and starch] fractions and apoplastic [pectin, hemicellulose, trifluoroacetic acid (TFA)-soluble, and cellulose] fractions. Symplastic sugar content of gall tissues was higher than that of callus. In apoplastic (cell wall) fractions, the cellulose content of gall tissues was lower than that of callus, due to large amount of pectin with high ratio of uronic acid (UA) and hemicellulose with low ratio of UA. Analysis of neutral sugar component of the hemicellulosic, TFA-soluble fraction showed that arabinose (side chain) and galactose (backbone) of arabinogalactan were rich in gall tissues and callus. The gall tissues had higher glucose and lower xylose contents than the callus. These results suggest that the structure of cell wall polysaccharides of gall changed during its development with an increase in symplastic sugar contents. The feeding activities occuring in gall by the gall-inducers were discussed.     

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.     

Isozyme expression in F1 hybrids between carp and goldfish

Interspecific genetic differences in malate dehydrogenase (MDH), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and esterase (EST) isozymes in carp (Cyprinus carpio) and goldfish (Carassius auratus) were used to examine the allelic expressions in the hybrid between these species. A unique liver SOD and muscle LDH phenotype unambiguously identifies all presumed hybrid individuals. There was no evidence of F₂ or backcross phenotypes in hybrid individuals. Liver MDH and EST phenotypes in hybrids show a preferential expression of goldfish isozymes. Variation in the levels of carp liver MDH isozymes may result from the polymorphism of a regulatory mutation affecting isozyme expression, leading to gene silencing after duplication.This work was supported through NSERC (Canada) grants to James P. Bogart and John F. Leatherland.     

Effects of phenylhydrazine on the electrophoretic and isoelectric focusing behaviour of malate dehydrogenase isozymes in Ictalurus species (Ictaluridae,Teleostei)

Significant changes in MDH isozymes were noted in Ictalurus sp. after treatment with phenylhydrazine and these changes varied at different stages and in the various tissues. The alterations observed were related to the substance injected, exposure time and the fish's physiological state and ability to react. Thirteen days after exposure, most tissues were able to achieve full recovery of MDH isozyme activity, the sole exceptions being serum and white muscle, where recovery was only slight or non-existent. The results are correlated with MDH biological function and possible biotransformation of phenylhydrazine.     

The Role of Antioxidants for Initiation of Somatic Embryos with Conostephium pendulum (Ericaceae)

Using Conostephium pendulum as a 'model' species in the Ericaceae a study was instigated to investigate the role of oxidative stress on the embryogenic competency of the explant tissue and callus. Three antioxidants were examined as an explant pre-treatment and incorporated in the growth medium. Pre-treatment of explant tissues with tri-potassium citrate and citric acid significantly reduced callus necrosis, however this treatment significantly reduced somatic embryo production when compared to preparing tissue under sterile distilled water. A combination of tri-potassium citrate and citric acid incorporated in the medium significantly reduced phenolic browning, however this treatment had no significant effect on the number of somatic embryos formed suggesting that prevention of tissue browning may be feasible by reducing the contact with oxygen during excision of tissues.     

Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses,Hepatic Gene Expression,and Stress Protein Profiling

In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na⁺‐K⁺‐ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation–related genes expression and stress‐related proteins profiling in fish liver. Oxidative stress indices and Na⁺‐K⁺‐ATPase showed tissues‐specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF‐I axis and ion‐regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose‐dependent manner under TBT stress. In short, TBT‐induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long‐term experiments at lower concentrations will be necessary in the future.     

Salt Stress-induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania somnifera Dunal)

In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO₃, NaNO₃, and CaCl₂. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO₃ compared to other salts and the in vitro shoots appeared healthy at 50?mM concentration of NaCl and KNO_3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl₂ produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.     

Genetics and polymorphism of a duplicated malate dehydrogenase (MDH) locus in the grasshopperOxya japonica japonica (Orthoptera:Acrididae:Oxyinae)

A biochemical genetic study of the enzyme malate dehydrogenase (MDH) was conducted in the grasshopperOxya j. japonica. Analysis of MDH electrophoretic variation in this species of grasshopper shows that one of the two autosomal loci for MDH in grasshoppers, the Mdh-2 locus, controlling the anodal set of MDH isozymes, is duplicated. Results of breeding studies confirm this and the observed polymorphism at theMdh-2 locus in the two populations ofOxya j. japonica studied can be attributed to three forms of linked alleles at the duplicated locus in equilibrium in both populations. In this respect, all individuals of this species possess heterozygous allelic combinations at the duplicatedMdh-2 locus, which may account for the spread of the duplicated locus in the populations of this species of grasshopper.This research was supported by a grant (Vote F) from the University of Malaya, Kuala Lumpur.     

鳙鱼同工酶发育遗传学研究

采用淀粉或聚丙烯酰胺凝胶电泳法分析鳙鱼早期发育阶段(从未受精卵到卵黄吸尽期)及成体不同组织(脑、眼、心、肌、肾、肝)中六种同工酶(LDH,MDH,IDH,ADH,SDH,EST)的分化表达模式。鳙鱼同工酶基因的表达具有明显的组织特异性。早期发育阶段,ADH和SDH均无染色活性;LDH、MDH和IDH具有不同的发育变化谱式,而EST酶谱在整个早期发育阶段均无明显变化。与鲢、草鱼相比,鳙鱼早期发育过程中胚胎Ldh-A基因激活的时间被推迟。上述结果可为鳙鱼种群的生化遗传结构分析以及鳙鱼的人工育种提供基础资料。     

Physiological and Biochemical Characteristics and Response Patterns of Salinity Stress Responsive Genes (SSRGs) in Wild Quinoa (<i>Chenopodium quinoa</i> L.)

Cultivating salt-tolerant crops is a feasible way to effectively utilize saline-alkali land and solve the problem of underutilization of saline soils. Quinoa, a protein-comprehensive cereal in the plant kingdom, is an exceptional crop in terms of salt stress tolerance level. It seems an excellent model for the exploration of salt-tolerance mechanisms and cultivation of salt-tolerant germplasms. In this study, the seeds and seedlings of the quinoa cultivar Shelly were treated with different concentrations of NaCl solution. The physiological, biochemical characteristics and agronomic traits were investigated, and the response patterns of three salt stress-responsive genes (SSRGs) in quinoa were determined by real-time PCR. The optimum level of stress tolerance of quinoa cultivar Shelly was found in the range of 250–350 mM concentration of NaCl. Salt stress significantly induced expression of superoxide dismutase (SOD), peroxidase (POD), and particularly betaine aldehyde dehydrogenase (BADH). BADH was discovered to be more sensitive to salt stress and played an important role in the salt stress tolerance of quinoa seedlings, particularly at high NaCl concentrations, as it displayed upregulation until 24 h under 100 mM salt treatment. Moreover, it showed upregulation until 12 h under 250 mM salt stress. Taken together, these results suggest that BADH played an essential role in the salt-tolerance mechanism of quinoa. Based on the expression level and prompt response induced by NaCl, we suggest that the BADH can be considered as a molecular marker for screening salt-tolerant quinoa germplasm at the early stages of crop development. Salt treatment at different plant ontogeny or at different concentrations had a significant impact on quinoa growth. Therefore, an appropriate treatment approach needs to be chosen rationally in the process of screening salt-tolerant quinoa germplasm, which is useful to the utilization of saline soils. Our study provides a fundamental information to deepen knowledge of the salt tolerance mechanism of quinoa for the development of salt-tolerant germplasm in crop breeding programs.