Heavy metal accumulation characteristics and physiological response of Sabina chinensis and Platycladus orientalis to atmospheric pollution.

Heavy metal (Cu, Mn, Zn, Pb, and Cd) concentrations were measured in the leaves of Sabina chinensis and Platycladus orientalis collected from urban, suburban, and rural sites in Tianjin, China. Photosynthetic pigment contents, reactive oxygen species content, malondialdehyde (MDA) content and antioxidant enzyme activity were investigated, providing physiological response parameters. Our comparison of the sites revealed that urbanization significantly influenced the heavy metal concentrations in both plant leaves. At the rural site, both plant leaves exhibited the lowest heavy metal accumulation. The highest Cu, Mn, and Zn concentrations were found in S. chinensis leaves from the urban site; the highest Pb and Cd concentrations were found in P. orientalis leaves from the urban site. These results indicate that the urban site contained larger heavy metal concentrations in the plant leaves that may reflect the anthropogenic emission gradient. It is also found that S. chinensis may be used to monitor airborne heavy metal pollution because it is highly quick response to heavy metals, while P. orientalis may be used for mitigation due to its high resistance. The results of this study can contribute to the development of monitoring and environmental management plans by providing information on sensitive and resistant tree species for city greening in North China.

Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments.

Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification.

Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60ß (60-kDa chaperonin ß-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

Impacts of elevated ozone on growth and photosynthesis of Metasequoia glyptostroboides Hu et Cheng.

One-year-old Metasequoia glyptostroboides seedlings were exposed to non-filtered ambient air (NF) and elevated ozone (E-O3, NF+60 ppb) in open-top chambers for two years. E-O3 accelerated leaf senescence, as indicated by significant decreases in photosynthetic pigment contents with the elongation of O3 exposure. E-O3 significantly affected gas exchange and carboxylation, inducing reductions in light-saturated photosynthesis (Asat), the maximum activity of Rubisco (Vc,max) and the maximum electron transport rate (Jmax). Chl a/b, Vc,max/Jmax and stomatal limitation (l) were not affected. Stomatal conductance (gs) was significantly decreased by E-O3 in the first year, but remained unchanged in the second year. It can be inferred that the decrease in Asat by E-O3 was mainly attributed to the changes in non-stomatal factors. After two years' exposure, E-O3 caused significant decreases in canopy photosynthesis and leaf mass per area, and a significant increase in the number of branches, but induced slight, not significant decreases in growth and biomass. Therefore, it can be concluded that the carbon accumulation of the species M. glyptostroboides could be negatively affected after long-term exposure to high O3 concentration.

Effects of water stress, organic amendment and mycorrhizal inoculation on soil microbial community structure and activity during the establishment of two heavy metal-tolerant native plant species.

Our aim was to examine the effect of water stress on plant growth and development of two native plant species (Tetraclinis articulata and Crithmum maritimum) and on microbial community composition and activity in the rhizosphere soil, following the addition of an organic amendment, namely sugar beet residue (SBR), and/or the inoculation with an arbuscular mycorrhizal (AM) fungus, namely Glomus mosseae, in a non-sterile heavy metal-polluted soil. The AM inoculation did not have any significant effect on plant growth of both species. In T. articulata, SBR increased shoot growth, foliar P, total phospholipid fatty acids (PLFA), fungi-related PLFA, AM fungi-related neutral lipid fatty acid, bacterial gram-positive/gram-negative PLFA ratio and the ß-glucosidase and dehydrogenase activities. SBR and AM inoculation increased phosphatase activity in T. articulata plants grown under drought conditions. In both plants, there was a synergistic effect between AM inoculation and SBR on mycorrhizal colonisation under drought conditions. In C. maritimum, the increase produced by the SBR on total amounts of PLFA, bacterial gram-positive-related PLFA and bacterial gram-negative-related PLFA was considerably higher under drought conditions. Our results suggest that the effectiveness of the amendment with regard to stimulating microbial communities and plant growth was largely limited by drought, particularly for plant species with a low degree of mycorrhizal colonisation.

Antifungal properties of ethanolic extract and its active compounds from Calocedrus macrolepis var. formosana (Florin) heartwood.

The ethanolic extract of Calocedrus macrolepis var. formosana heartwood was screened for antifungal compounds by agar dilution assay and liquid chromatography. Two compounds, beta-thujaplicin and gamma-thujaplicin, responsible for the antifungal property of C. macrolepis var. formosana heartwood were isolated by high performance liquid chromatography (HPLC), and identified by 1H NMR and 13C NMR. The antifungal activities of these two compounds were further evaluated against total 15 fungi, including wood decay fungi, tree pathogenic fungi and molds. The hexane soluble fraction showed the strongest antifungal activities among all fractions. beta-Thujaplicin and gamma-thujaplicin exhibited not only very strong antifungal activity, but also broad antifungal spectrum. The MIC values of beta-thujaplicin and gamma-thujaplicin were in the range of 5.0-50.0 microg/ml. In addition, scanning electron microscopy (SEM) was carried out to study the structural change of fungal hyphae induced by beta-thujaplicin. Strong cell wall shrinkage indicated the fungicidal effect could be attributed to the combined actions of metal chelating and cytoplasm leakage. It also suggests that the role of metal chelating is indispensable in the design of environmental-friendly fungicides.

Sensitivity of Mediterranean woody seedlings to copper, nickel and zinc.

The restoration of heavy metal contaminated areas requires information on the response of native plant species to these contaminants. The sensitivity of most Mediterranean woody species to heavy metals has not been established, and little is known about phytotoxic thresholds and environmental risks. We have evaluated the response of four plant species commonly used in ecological restoration, Pinus halepensis, Pistacia lentiscus, Juniperus oxycedrus, and Rhamnus alaternus, grown in nutrient solutions containing a range of copper, nickel and zinc concentrations. Seedlings of these species were exposed to 0.048, 1 and 4 microM of Cu; 0, 25 and 50 microM of Ni; and 0.073, 25 and 100 microM of Zn in a hydroponic silica sand culture for 12 weeks. For all four species, the heavy metal concentration increased in plants as the solution concentration increased and was always higher in roots than in shoots. Pinus halepensis and P. lentiscus showed a higher capacity to accumulate metals in roots than J. oxycedrus and R. alaternus, while the allocation to shoots was considerably higher in the latter two. Intermediate heavy-metal doses enhanced biomass accumulation, whereas the highest doses resulted in reductions in biomass. Decreases in shoot biomass occurred at internal concentrations ranging from 25 to 128 microg g-1 of Zn, and 1.7 to 4.1 microg g( -1) of Cu. Nickel phytoxicity could not be established within the range of doses used. Rhamnus alaternus and J. oxycedrus showed higher sensitivity to Cu and Zn than P. halepensis and, especially, P. lentiscus. Contrasted responses to heavy metals must be taken into account when using Mediterranean woody species for the restoration of heavy metal contaminated sites.