Activation of phenylpropanoid pathway in legume plants exposed to heavy metals. Part II. Profiling of isoflavonoids and their glycoconjugates induced in roots of lupine (Lupinus luteus) seedlings treated with cadmium and lead

We examined changes in profiles of isoflavonoids in roots of lupine (Lupinus luteus L. cv. Juno) seedlings in response to treatment with two heavy metals: cadmium (at 10 mg/l) and lead (at 150 mg/l). Overall, 21 flavonoid conjugates were identified in root extracts, some of them with up to six positional isomers. The total amount of all isoflavonoids increased by about 15 % in cadmium-treated plants and by 46 % in lead-treated ones. Heavy metals markedly increased the content of two compounds: 2'-hydroxygenistein glucoside and 2'-hydroxygenistein 7-O-glucoside malonylated. Possible functions of the identified isoflavonoids in yellow lupine exposed to heavy metal stress are discussed.     

Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis

The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.     

MicroRNA mediated regulation of gene expression in response to heavy metals in plants

Journal of Plant Biochemistry and Biotechnology - Plants being sessile organisms are exposed to innumerable abiotic and biotic stresses on a daily basis. Heavy metal toxicity in plants results in...     

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.     

Regulation of phenylalanine ammonia-lyase activity in cell-suspension cultures of Petroselinum hortense. Apparent rates of enzyme synthesis and degradation.

The time courses for induced changes in the phenylalanine ammonia-lyase activity at five different stages during the growth cycle of cell-suspension cultures from parsley (Petroselinum hortense Hoffm.) were investigated. Large increases in the enzyme activity, induced either by irradiation or by dilution of a cell culture into fresh medium, were followed by an expotential decline to the initial low level. The maximum inducible level of specific enzyme activity varied within a range of about six-fold, depending on the mode of induction and the growth stage of the cell culture.     

Effects of nitrogen on the activity of antioxidant enzymes and gene expression in leaves of Populus plants subjected to cadmium stress

The research aimed to verify the important physiological effect of nitrogen (N) on plants exposed to cadmium (Cd). The poplar plants were grown in a Hoagland nutrient solution and treated with extra N, Cd, and N + Cd. After treatment, plant growth and chlorophyll content were recorded. The oxidative stress, the activity of antioxidant enzymes, and the expression of related genes were also examined. The results indicated the plants treated with sole Cd presented obvious toxicity symptoms, i.e. growth inhibition, reactive oxygen species accumulation, and chlorophyll content decrement. However, when N was added to the plants under Cd stress, plant growth was enhanced, chlorophyll synthesis was promoted, and the oxidative stress was alleviated. Further, the expression of antioxidant enzymes genes was upregulated by N. The results indicated that N partially reversed the toxic effect of Cd on poplar plants, which can provide new methodology to enhance the phytoremediation technology for heavy metal pollution soil.     

Effects of silicon nutrition on cadmium uptake, growth and photosynthesis of rice plants exposed to low-level cadmium

The effect of silicon (Si) nutrition on low-level cadmium (Cd) toxicity symptoms was investigated in hydroponically-grown rice seedlings (Oryza sativa L.). Silicon (0.0, 0.2, or 0.6 mM) was added when seedlings were 6 or 20 days old representing early (Si^E) or late (Si^L) Si treatment, respectively. Cadmium (0.0 or 2.5 μM) was added when seedlings were 6 days old. Measurements included generation of CO₂ and light response curves; chlorophyll fluorescence analysis; growth; and tissue-element content analysis. Our results showed that low-level Cd treatment generally inhibited growth and photosynthesis. However, the addition of 0.2 or 0.6 mM Si^E or Si^L significantly reduced root- and leaf-Cd content. Consequently, the addition of 0.6 mM Si^L significantly alleviated low-level Cd-induced inhibition of growth. Furthermore, 0.2 mM Si treatment significantly reduced g _s compared to 0.0 or 0.6 mM Si without inhibiting A, especially in +Cd plants, suggesting an increase in instantaneous water-use-efficiency (IWUE). Additionally, in +Cd plants, the addition of 0.6 mM Si^E significantly reduced F _o but increased F _v/F _m, while treatment with 0.2 mM Si^L significantly increased q_P, suggesting an increase in light-use-efficiency. We thus, propose that 0.6 mM Si^L treatment is required for the alleviation of low-level Cd-mediated growth inhibition. Furthermore, we suggest that 0.2 mM Si concentration might be close to the optimum requirement for maximum Si-induced increase in IWUE in rice plants, especially when under low-level Cd-stress. Our results also suggest that Si alleviates low-level Cd toxicity by improving light-use-efficiency.     

Alterations of the gene expression, lipid peroxidation, proline and thiol content along the barley root exposed to cadmium

Barley seedlings grown on filter paper moistened with 1mM Cd showed 50% root growth inhibition within 24h of exposure. The amount of cadmium after 24h Cd treatment was highest in the first 2mm-long apical root segment, while it was slightly higher in the fourth segment, 6-8mm behind the root tip, after 48h. In recovery experiments, when Cd-treated plants were transferred onto filter paper moistened with distilled water, a large amount of Cd was localised in the apoplast and considerable cell death was detected even though root growth was renewed. This indicates that cell death is likely an active physiological process that contributes to the removal of Cd from the root during root growth recovery. Elevated lipid peroxidation and thiol contents were detected in all individual segments of Cd-treated barley root. On the other hand, proline accumulation was disturbed during Cd stress, showing a significant decrease in all of the studied segments except the first. Cd-induced alteration in the expression of genes involved in metal signalling and detoxification and in drought and oxidative stress responses indicates that Cd-induced water and oxidative stress is responsible for the root growth inhibition, probably through an accelerated differentiation of root tissues.     

Effects of cadmium and lead stress on somatic embryogenesis of coniferous species. Part I: Evaluation of the genotype-dependent response

Somatic embryogenesis is an important biotechnological tool that has significant potential to be used in studies related to environmental stress. In this study, embryogenic cell masses of Abies alba and Picea abies were grown on media enriched with 50–500 µM cadmium (Cd²⁺) or lead (Pb²⁺). The effects of cadmium and lead were evaluated during the subsequent stages of somatic embryogenesis: proliferation, maturation, and germination. The following characteristics were evaluated: proliferation potential, cell viability, average number of somatic embryos obtained per 1 g of fresh weight, and morphology of the developed somatic embryos. The tested heavy metals significantly reduced the proliferation rate of A. alba and P. abies embryogenic cell masses. The highest tested cadmium concentration markedly slowed or stopped the growth of embryogenic cell masses in both species. Unexpectedly, the proliferation ratio remained fairly high for the P. abies cell lines treated with lead at all concentrations tested. During the maturation stage, the total number of somatic embryos declined under cadmium exposure. The formation of early precotyledonary and cotyledonary somatic embryos in both species was similarly reduced, although cadmium caused a higher death rate and was more toxic than lead. To the best of our knowledge, this is the first report to study the effects of heavy metals on A. alba embryogenic cell masses during the proliferation stage as well as on the maturation and germination processes of both species. This in vitro system can be used for testing the response of large sets of genotypes, and the best performing lines can be used in the future for in vivo performance tests of heavy metal-polluted soils.     

Comparison of CEL I gene expression and mismatch-cleavage activity in some Apiaceae plants

The most familiar enzyme in mutation screening through heteroduplex analysis, CEL I, has been isolated from celery of the Apiaceae family. In this study, in a search for new sources with the same or better enzymatic activity, we studied the mismatch-cleavage activity of plant juice extracts from several Apiaceae plants (celery, carrot, coriander, parsley, dill, and fennel). This study was then followed by investigation of the level of CEL I gene expression in these plants. Mismatch-cleavage activity of fennel and dill juice extracts was lower than that of celery juice extract, and levels of CEL I mRNA expression in these plants were substantially higher than in celery. In contrast, the ability of juice extract from a local cultivar of parsley to cleave heteroduplex DNA substrates was clearly more than that of celery juice extract, whereas the level of CEL I gene expression in parsley was obviously lower than in celery. We concluded that there are multiple mismatch-cleaving enzymes collaborating in digestion of heteroduplex DNA substrates by plant juice extracts.     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and coppper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H₂O₂). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and copper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H2O2). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.