Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1.

BACKGROUND: Magnolia is a woody ornamental plant, which is widely used in urban landscaping. However, its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes. RESULTS: We developed an efficient protoplast isolation and transient expression system for Magnolia denudata × Magnolia acuminata 'Yellow River'. The highest yield of protoplasts was obtained from young leaves digested in 3% Cellulase R10, 0.8% Macerozyme R10, 0.04% pectinase and 0.4 M mannitol enzymolysis solution for 6 h. For transfection of protoplasts, 20% PEG4000 for 5 min was optimal. To verify the protoplast system and begin to understand heat tolerance in Magnolia, a heat shock transcription factor MdeHSF1 was cloned from 'Yellow River', which belongs to the HSF subfamily A and has significant homology with AtHSFA1A. Subcellular localization analysis indicated that MdeHSF1 was expressed in the cell nucleus. Furthermore, qPCR analysis of the MdeHSF1 transcript level in response to high temperature stress suggested that MdeHSF1 might be involved in regulating heat stress tolerance in 'Yellow River'. CONCLUSION: The described protocol provides a simple and straightforward method for isolating protoplast and exploring gene subcellular localization of MdeHSF1 in Magnolia. This expands the new research of protoplast isolation and transfection in Magnolia.

Effect of biochar on the extractability of heavy metals (Cd, Cu, Pb, and Zn) and enzyme activity in soil.

Biochar is a carbon-rich solid material derived from the pyrolysis of agricultural and forest residual biomass. Previous studies have shown that biochar is suitable as an adsorbent for soil contaminants such as heavy metals and consequently reduces their bioavailability. However, the long-term effect of different biochars on metal extractability or soil health has not been assessed. Therefore, a 1-year incubation experiment was carried out to investigate the effect of biochar produced from bamboo and rice straw (at temperatures ≥500 °C) on the heavy metal (cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn)) extractability and enzyme activity (urease, catalase, and acid phosphatase) in a contaminated sandy loam paddy soil. Three rates (0, 1, and 5%) and two mesh sizes (<0.25 and <1 mm) of biochar applications were investigated. After incubation, the physicochemical properties, extractable heavy metals, available phosphorus, and enzyme activity of soil samples were analyzed. The results demonstrated that rice straw biochar significantly (P < 0.05) increased the pH, electrical conductivity, and cation exchange capacity of the soil, especially at the 5% application rate. Both bamboo and rice straw biochar significantly (P < 0.05) decreased the concentration of CaCl2-extractable heavy metals as biochar application rate increased. The heavy metal extractability was significantly (P < 0.01) correlated with pH, water-soluble organic carbon, and available phosphorus in soil. The 5% application rate of fine rice straw biochar resulted in the greatest reductions of extractable Cu and Zn, 97.3 and 62.2%, respectively. Both bamboo and rice straw biochar were more effective at decreasing extractable Cu and Pb than removing extractable Cd and Zn from the soil. Urease activity increased by 143 and 107% after the addition of 5% coarse and fine rice straw biochars, respectively. Both bamboo and rice straw biochars significantly (P < 0.05) increased catalase activity but had no significant impact on acid phosphatase activity. In conclusion, the rice straw biochar had greater potential as an amendment for reducing the bioavailability of heavy metals in soil than that of the bamboo biochar. The impact of biochar treatment on heavy metal extractability and enzyme activity varied with the biochar type, application rate, and particle size.

Subcellular distribution of metals within Brassica chinensis L. in response to elevated lead and Chromium Stress.

Differential centrifugation and synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) microprobe were used to study the distribution of the elements in tissue cross sections of pakchoi ( Brassica chinensis L.) under stress of elevated Pb and Cr. Subcellular fractionation of the different tissues grown in a nutrient solution containing 200 mg L(-1) Pb or 5 mg L(-1) Cr showed that 86.7 and 76.3% of the Pb that accumulated in the roots and shoots, respectively, was contained in the cell wall and vacuoles in those areas. Whereas 75.0% of the Cr that accumulated in the root was contained in the cell wall, 63.1% of the Cr that accumulated in the shoot was found in the vacuoles and cell wall. SRXRF analysis revealed that, when pakchoi seedlings were placed under excess Pb stress, the Pb, Ca, Cu, and Zn were concentrated in the cortex and vascular bundle of the root and mixed Fe-Mn plaques were seen on the surface of the pakchoi root. In the Cr treatment group, Cr, Ca, Mn, and Zn were mainly located in the cortex of the root, whereas in the stem, only Ca, Cu, and Zn were detected at higher levels in the cortex area. Thus, this study provides evidence that, in response to Pb and Cr stress, pakchoi uses cell walls and vacuoles to reduce the transport of these heavy metals through the plant, as well as restrict transport from the root to the stem.