Poplar Callus Research Articles

Silicon Actuates Poplar Calli Tolerance after Longer Exposure to Antimony.

The presence of antimony (Sb) in high concentrations in the environment is recognized as an emerging problem worldwide. The toxicity of Sb in plant tissues is known; however, new methods of plant tolerance improvement must be addressed. Here, poplar callus (Populus alba L. var. pyramidallis) exposed to Sb(III) in 0.2 mM concentration and/or to silicon (Si) in 5 mM concentration was cultivated in vitro to determine the impact of Sb/Si interaction in the tissue. The Sb and Si uptake, growth, the activity of superoxide dismutase (SOD), catalase (CAT), guaiacol-peroxidase (G-POX), nutrient concentrations, and the concentrations of photosynthetic pigments were investigated. To elucidate the action of Si during the Sb-induced stress, the impact of short and long cultivations was determined. Silicon decreased the accumulation of Sb in the calli, regardless of the length of the cultivation (by approx. 34%). Antimony lowered the callus biomass (by approx. 37%) and decreased the concentrations of photosynthetic pigments (up to 78.5%) and nutrients in the tissue (up to 21.7%). Silicon supported the plant tolerance to Sb via the modification of antioxidant enzyme activity, which resulted in higher biomass production (increased by approx. 35%) and a higher uptake of nutrients from the media (increased by approx. 10%). Silicon aided the development of Sb-tolerance over the longer cultivation period. These results are key in understanding the action of Si-developed tolerance against metalloids.

Silicon alleviates the negative effects of arsenic in poplar callus in relation to its nutrient concentrations

The contamination of water and soil by arsenic (As) is a global environmental, health, and agricultural issue. The study of As toxicity in plants and its interaction with nutrients requires special attention. We investigated the impact of low concentrations of As on poplar callus and the effectiveness of silicon (Si) in the alleviation of As stress. We focused on the effects of As and/or Si on the concentration of nutrients and its relationship with other parameters studied: callus growth, the concentration of photosynthetic pigments, and the accumulation of As. Using a correlation matrix heatmap we revealed the associations between the above-mentioned parameters. The principal component analysis (PCA) was used to show the variability between the treatments (control, Si, As, As + Si) and the time stages (short and long cultivation). The concentrations of photosynthetic pigments were impacted extremely by both the As and the As + Si treatments. The nutrient balance was heavily disrupted by As but considerably improved by Si. The PCA revealed a strong correlation among growth, photosynthesis pigments, K, Zn, and Mn. We analysed the data from the literature that specified the changes in the concentrations of nutrients and growth induced by As, and compared them with our results. This heatmap analysis revealed the similarity between poplar callus and sensitive plants. The principal component analysis showed that the variability between the short and long cultivation in the As and/or Si treatment was high in callus growth, nutrient, and chlorophyll concentrations.

The cadmium tolerance development of poplar callus is influenced by silicon.

Cadmium-tolerant plants were studied for their possible usage in phytoremediation techniques. However, their response to cadmium cations at a cellular level has not been properly studied. Silicon is a beneficial element that seems to change the plant's response to the Cd2+ presence. The aim of the present study was to investigate the Cd2+ tolerance patterns of poplar callus cells exposed to Cd+2 and/or Si over short and long cultivation periods. We determined the growth parameters of the callus, the growth dynamics, cell vitality, photosynthetic pigment concentrations and the activity of antioxidant enzymes. The effects were studied over short (21 days) and long (63 days) cultivation periods. The most important result proved that the poplar callus tissue is able to build up a tolerance to Cd2+ after a longer cultivation period. On the 63rd day of the cultivation, Cd2+ stressed calli showed improvement in studied parameters and the callus cells accumulated Cd2+ more efficiently than on the 21st day. Supplementation with Si in higher concentrations (2.5 mM and 5 mM) heightened the Cd-tolerance potential of the tissue. The treatment of Cd2+, and Si in a 2.5 mM concentration was the most efficient variant for Cd2+ removal from medium. The activity of antioxidant enzymes showed that poplar callus cells effectively develop tolerance against Cd2+ after a longer cultivation period.

Osmotic adjustments support growth of poplar cultured cells under high concentrations of carbohydrates.

Poplar callus maintained a specific difference in osmotic potential with respect to media when supplemented with different carbohydrate concentrations. This balance in osmotic potential guaranteed the growth capacity. Osmotic stress is caused by several abiotic factors such as drought, salinity, or freezing. However, the threshold of osmotic potential that allows the growth under stress conditions has not been thoroughly studied. In this study, different levels of osmotic stress in Populus alba (L.) callus have been induced with the addition of mannitol or sorbitol in the medium (from 0 to 500mM). The key factor for preserving the growth was observed to be the restoration of a constant difference in osmotic potential between callus and medium for all the tested conditions. The osmotic adjustments were primarily achieved with the uptake of mannitol or sorbitol from the media considering their chemical properties instead of their biological functions. The decrease in water content (from -1 to -10% after 21days) and mineral elements, such as potassium, calcium, and magnesium, together with the alterations in cell morphology, did not show negative effects on growth. The activity of sorbitol dehydrogenase was detected for the first time in poplar (+ 4.7 U l-1 in callus treated with sorbitol compared to control callus). This finding suggested the importance of choosing carefully the molecules used to exert osmotic stress for separating the dual function of carbohydrates in osmotic adjustments and cell metabolism.

The modulation of ion homeostasis by silicon in cadmium treated poplar callus cells.

One of the major reasons why cadmium is toxic in plants is because it disturbs their nutrient balance. The aim of this work is to investigate the effects of cadmium (Cd) and/or silicon (Si) on the nutrient status of poplar callus cells after 3 and after 9 weeks of Cd exposure and to study its possible relationship with the changes in the fresh and dry mass, the plasma membrane integrity, and cadmium tolerance patterns. A principal component analysis (PCA) was performed to reveal the associations among the elements, and the variability between both treatments, and between the 3- and 9-week stages. Cadmium reduced the fresh and dry mass, the plasma membrane integrity, and the concentration of all nutrients except for P. After 9 weeks of exposure, the Cd concentration in callus cells had almost doubled, in spite of an improvement in all studied parameters. These changes may be due to the callus acclimatizing to the Cd stress. In the Cd + Si treatment, the fresh and dry mass, the plasma membrane integrity, and the concentration of nutrients, as well as the growth tolerance index, increased in comparison with the Cd treatment. We assumed that the enhancement in the plasma membrane integrity mediated by Si under Cd stress had caused the improvement in the uptake of nutrients and, consequently, the fresh and dry mass of callus cells had increased. The reduction in Cd concentration due to the Si impact also contributed to the increase in fresh and dry mass.

Cadmium impact, accumulation and detection in poplar callus cells.

Effect of cadmium cations and their interaction with silicon cations was determined in poplar calli and expressed as changes in callus growth, cell viability and cadmium cation accumulation. Cell viability throughout culture versus cadmium cation accumulation in cells is discussed. At the same time, the study sought appropriate methods for cadmium cation detection in callus cells and also in experiments with low plant material (e.g. protoplasts). Cadmium cations were determined by atomic absorption spectroscopy and using fluorescence microscopy with a specific cadmium cation fluorescent dye. The detection of cadmium cations in callus cells by the latter method appears suitable because the callus cells are surrounded by primary cell walls without auto-fluorescence and these values fit well with atomic absorption spectroscopy quantification. However, the visualisation method has some limits discussed below.

Putative Cationic Cell-Wall-Bound Peroxidase Homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, Are Involved in Lignification

The final step of lignin biosynthesis, which is catalyzed by a plant peroxidase, is the oxidative coupling of the monolignols to growing lignin polymers. Cationic cell-wall-bound peroxidase (CWPO-C) from poplar callus is a unique enzyme that has oxidative activity for both monolignols and synthetic lignin polymers. This study shows that putative CWPO-C homologues in Arabidopsis , AtPrx2, AtPrx25, and AtPrx71, are involved in lignin biosynthesis. Analysis of stem tissue using the acetyl bromide method and derivatization followed by the reductive cleavage method revealed a significant decrease in the total lignin content of ATPRX2 and ATPRX25 deficient mutants and altered lignin structures in ATPRX2, ATPRX25, and ATPRX71 deficient mutants. Among Arabidopsis peroxidases, AtPrx2 and AtPrx25 conserve a tyrosine residue on the protein surface, and this tyrosine may act as a substrate oxidation site as in the case of CWPO-C. AtPrx71 has the highest amino acid identity with CWPO-C. The results suggest a role for CWPO-C and CWPO-C-like peroxidases in the lignification of vascular plant cell walls.

Role of Tyr residues on the protein surface of cationic cell-wall-peroxidase (CWPO-C) from poplar: Potential oxidation sites for oxidative polymerization of lignin

It was previously reported that an unique peroxidase isoenzyme, cationic cell-wall-bound peroxidase (CWPO-C), from poplar callus oxidizes sinapyl alcohol, ferrocytochrome c and synthetic lignin polymers, unlike other plant peroxidases. Here, the catalytic mechanism of CWPO-C was investigated using chemical modification and homology modeling. The simulated CWPO-C structure predicts that the entrance to the heme pocket of CWPO-C is the same size as those of other plant peroxidases, suggesting that ferrocytochrome c and synthetic lignin polymers cannot interact with the heme of CWPO-C. Since Trp and Tyr residues are redox-active, such residues located on the protein surface were predicted to be active sites for CWPO-C. Modification of CWPO-C Trp residues did not suppress its oxidation activities toward guaiacol and syringaldazine. On the other hand, modification of CWPO-C Tyr residues using tetranitromethane strongly suppressed its oxidation activities toward syringaldazine and 2,6-dimethoxyphenol by 90%, respectively, and also suppressed its guaiacol oxidation activity to a lesser extent. Ferrocytochrome c was not oxidized by Tyr-modified CWPO-C. These results indicate that the Tyr residues in CWPO-C mediate its oxidation of syringyl compounds and high-molecular-weight substrates. Homology modeling indicates that Tyr-177 and Tyr-74 are located near the heme and exposed on the protein surface of CWPO-C. These results suggest that Tyr residues on the protein surface are considered to be important for the oxidation activities of CWPO-C with a wide range of substrates, and potentially unique oxidation sites for the plant peroxidase family.

The cationic cell-wall-peroxidase having oxidation ability for polymeric substrate participates in the late stage of lignification of Populus alba L

Previously we reported that purified Cell Wall Peroxidase-Cationic (CWPO-C) from poplar callus (Populus alba L.) oxidizes sinapyl alcohol and polymeric substrate unlike other plant peroxidases and proposed that this isoenzyme is a conceivable lignification specific peroxidase. In this study, we cloned full-length cDNA of CWPO-C and investigated the transcription of CWPO-C gene in various organs and the localization of CWPO-C protein in the differentiating xylem of poplar stem.Real-time PCR analyses indicated that CWPO-C gene is constitutively expressed in the developing xylem, leaf, and shoot but not affected by many stress treatments. Immunohistochemical analysis showed that CWPO-C locates in the middle lamellae, cell corners, and secondary cell walls of the fiber cells during the lignification. The intensity of the CWPO-C labeling increased gradually from the cell wall thickening stage to mature stage of fiber cells, which is very consistent with the increase of lignin content in the developing xylem. These results strongly support that CWPO-C is responsible for the lignification of the secondary xylem. Interestingly, immuno-labeling of CWPO-C was also observed inside of the ray parenchyma cells instead no signals were detected within the developing fiber cells. This suggests that CWPO-C is biosynthesized in the parenchyma cells and provided to the middle lamellae, the cell corners, and the cell walls to achieve lignin polymerization.

Stable haploid poplar callus lines from immature pollen culture.

Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids (Populus nigra L. x hybrid 'Aue1' and 'Aue2'). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.

Lignin dehydrogenative polymerization mechanism: a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in β-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO-C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO-C was capable of oxidizing high-molecular-weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO-C characteristics are important to produce polymer lignin. The results suggest that CWPO-C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.

Treatment of poplar callus with ferulic and sinapic acids I: incorporation and enhancement of lignin biosynthesis

Ferulic acid (FA), tetradeuteroferulic acid (DFA), sinapic acid (SA), or heptadeuterosinapic acid (DSA) was exogenously supplied to poplar (Populus alba L.) callus. Administration of FA or SA increased the lignin content of the callus to about twice that of the control callus. Gas chromatographic analysis of the alkali hydrolysate of the cell wall residue revealed that only a trace amount of SA was bound to the cell wall, and the amount of FA was less than 2% of the total callus lignin. Thioacidolysis of the DFA-treated callus indicated that DFA is effectively converted to both coniferyl and sinapyl alcohols and then incorporated into the corresponding lignin. Incorporation of DSA into syringyl lignin or guaiacyl lignin was not observed, but yields of syringyl lignin thioacidolysis products were markedly increased by DSA treatment of the callus. These results suggest that SA may not be a precursor of sinapyl alcohol and syringyl lignin per se, but it may induce or enhance the biosynthesis of syringyl lignin in poplar callus.

Treatment of poplar callus with ferulic and sinapic acids II: effects on related monolignol biosynthetic enzyme activities

In a previous study we reported that administered ferulic acid (FA) was incorporated into guaiacyl and syringyl lignin in poplar callus (Populus alba L.) but administered sinapic acid (SA) was not. It is possible that SA may induce or enhance the biosynthesis of syringyl lignin. In this study, enzyme activities in lignin biosynthesis were assayed in FA- or SA-treated callus. The activity of 4-coumarate:coenzyme A ligase (4CL) toward FA in FA-treated callus was not affected, and its activity toward caffeic and sinapic acids was quite low. In addition, phenylalanine ammonia-lyase (PAL) activity, cinnamate:4-hydroxylase (C4H) transcription in the upstream steps of lignin biosynthesis, also was not affected. 4CL from SA-treated callus exhibited no detectable activity toward SA, suggesting that SA is not a precursor for syringyl lignin. However, all upstream steps of lignin biosynthesis (PAL activity, C4H transcription, 4CL activity toward FA) exhibited increased activity. These results suggest that SA may induce or enhance enzyme activities involved in syringyl lignin biosynthesis in poplar callus.

Sinapyl alcohol-specific peroxidase isoenzyme catalyzes the formation of the dehydrogenative polymer from sinapyl alcohol

Two peroxidases, CWPO-A and CWPO-C, were isolated from the cell walls of poplar (Populus alba L.) callus culture. The cationic CWPO-C showed a strong preference for sinapyl alcohol over coniferyl alcohol as substrate. Thus, the monolignol utilization of CWPO-C is unique compared with other peroxidases, including anionic CWPO-A and horseradish peroxidase (HRP). CWPO-C polymerized oligomeric sinapyl alcohol (S-oligo) and sinapyl alcohol, producing a polymer of greater molecular weight. In contrast, HRP, which is specific to coniferyl alcohol, produced sinapyl alcohol dimers, rather than catalyzing polymerization. Adding coniferyl alcohol as a radical mediator in the HRP-mediated reaction did not result in S-oligo polymerization. This report shows that CWPO-C is an isoenzyme specific to sinapyl alcohol that polymerizes oligomeric lignols. Its catalytic activity toward oligomeric lignols may be related to the lignification of angiosperm woody plant cell walls.

Frost Tolerance Estimation in Callus Derived From Poplar and Winter Rape Plants Using Three Different Methods

Summary Callus derived from poplar (Populus nigra L.) or from winter rape (Brassica napus L. var. oleifera) plants was exposed to freezing temperature in a range of -2°C to -12°C. The frost-induced injuries were estimated either by an electro-conductivity method, by the TTC reduction test or by determination of the ability of the tissue to regrow on a fresh medium after freezing. Comparison of the results obtained indicates that poplar callus has a higher frost resistance than rape callus. In both kinds of tissue, membrane permeability alterations higher than 20% (as detected with the conductivity method) seem to be deleterious for the further functioning and regrowth abilities of callus.

Phytotoxicity and fungitoxicity tests for tree wound paints

SUMMARYTo assess quickly the wide range of potential treatments for pruning wounds phytotoxicity and fungitoxicity tests were devised. The phytotoxicity test was based on the growth of poplar callus and the fungitoxicity test on the inhibition of fungal growth on wood strips. Results of field tests were in broad agreement with the laboratory phytotoxicity tests.

Control of root and shoot formation and production of trees from poplar callus

The technique of trees production from the undifferentiated poplar callus tissue is described. The best root formation was observed on the modifiedWolter andSkoog medium when NAA in concentration 0.2 to 0.4 mg l−1 was used as an auxin and cytokinins were omitted. The induction of leafy shoots from the undifferentiated callus was the most effective on the modifiedLinsmaier andSkoog medium in the absence of auxin and with 0.15 to 0.70 mg l−1 of BAP. The best development of roots at the basal end of excised shoots was achieved when shoots were transferred into the sterile mixture of perlit and sand (3: l, v/v) containing a modifiedWolter andSkoog medium.