MdBT2 regulates nitrogen-mediated cuticular wax biosynthesis via a MdMYB106-MdCER2L1 signalling pathway in apple.

Cuticular waxes play important roles in plant development and the interaction between plants and their environment. Researches on wax biosynthetic pathways have been reported in several plant species. Also, wax formation is closely related to environmental condition. However, the regulatory mechanism between wax and environmental factors, especially essential mineral elements, is less studied. Here we found that nitrogen (N) played a negative role in the regulation of wax synthesis in apple. We therefore analysed wax content, composition and crystals in BTB-TAZ domain protein 2 (MdBT2) overexpressing and antisense transgenic apple seedlings and found that MdBT2 could downregulate wax biosynthesis. Furthermore, R2R3-MYB transcription factor 16-like protein (MdMYB106) interacted with MdBT2, and MdBT2 mediated its ubiquitination and degradation through the 26S proteasome pathway. Finally, HXXXD-type acyl-transferase ECERIFERUM 2-like1 (MdCER2L1) was confirmed as a downstream target gene of MdMYB106. Our findings reveal an N-mediated apple wax biosynthesis pathway and lay a foundation for further study of the environmental factors associated with wax regulatory networks in apple.

[Effects of potassium levels on translocation of 13C-photoassimilates to fruit in 'Fuji' apple during fruit expanding period].

A field experiment was carried out in a six-year old 'Fuji'3/M26/Malus hupehensis Rehd. apple with the 13C tracer method to examine the changes of chlorophyll fluorescence parameters, photosynthetic characteristics of leaf, sugar transporter gene expression, 13C assimilation capability and the characteristics of translocation and distribution of 13C-photoassimilates to fruit under different levels of potassium addition (K2O 0, 0.5%, 1.0%, 1.5%, 2.0%, expressed by CK, K1, K2, K3, K4, respectively). Potassium aqueous solution smear the leaves within 20 cm around the fruit at fruit enlargement stage. Compared with other treatments, K3 treatment significantly increased Rubisco enzyme activity, net photosynthetic rate, maximal photochemical efficiency of PSII, actual photochemical efficiency of PSII, coefficient of photochemical quenching, sorbitol and sucrose content, sorbitol 6-phosphate dehydrogenase (S6PDH) and sucrose phosphate synthase (SPS) enzyme activities and 13C assimilation capability of leaves. Furthermore, K3 treatment increased gene expression of sorbitol transporter MdSOT1 and MdSOT2 and sucrose transporter MdSUT4, and promoted the unloading of sugar in fruit. The 13C of self retention (self leaves and self branches) was the highest in CK (82.6%) and the lowest in K3 treatment (60.5%). With increasing potassium concentration, the 13C absorption of fruit first increased and then decreased, which was the highest in K3 treatment (1.31 mg·g-1) and the lowest in CK (0.57 mg·g-1). Our results indicated that foliage application of potassium solution improved PSII photochemical efficiency, activities of key enzymes related with carbon assimilation, synthesis ability, and outward transport ability of photosynthates in leaves, and consequently promoted the directional transportation of sugar to fruit. The amount of photoassimilates transported to fruit was the most under 1.5% K2O treatment (K3).

[Effects of potassium supply on the growth, photosynthesis and 15N and 13C absorption and utilization of M9T337 seedling.] / ä¾›é’¾æ°´å¹³å¯¹è‹¹æžœç §æœ¨M9T337å¹¼è‹—ç”Ÿé•¿ã€å ‰åˆç‰¹æ€§ä¸Ž15N、13C吸收利用的影响.

Hydroponic experiment was carried out on M9T337 seedlings using 15N and 13C isotope tracer technology to study the effects of different potassium supply levels (K0, K1, K2, K3 and K4 were equivalent to 0, 3, 6, 9 and 12 mmol·L-1, respectively) on the growth, photosynthetic characteristics and 15N and 13C absorption and utilization of M9T337 seedlings. The results showed that dry mass, root length, root surface area, number of tips and root activity of M9T337 seedlings under the K2 level were significantly higher than those under other levels. The net photosynthetic rate (Pn) of leaves increased at low K+ concentration and then decreased with the increases of potassium supply level, and reached the maximum value at K2 treatment (15.5 µmol CO2·m-2·s-1). At the 30th day after treatment, the activities of nitrate reductase (NR) and carbon metabolism enzyme were highest in K2 treatment, and lowest in K0 treatment. With the increases of potassium application rate, the 13C accumulation of seedlings were first increased and then decreased, with the 13C distribution rate of each organ being the most balanced at K2 treatment. There were significant differences in 15N uptake and utilization rate among treatments. 15N uptake and utilization rates of seedlings under K2 treatment were the highest, which were 16.1 mg and 17.9%, respectively. Therefore, too low or too high potassium supply could inhibit seedling root growth and leaf photosynthesis, which was not conducive to carbon and nitrogen absorption. Appropriate potassium supply could improve root activity and net photosynthetic rate, enhance nitrate reductase (NR) and carbon metabolic enzyme activity, and promote carbon and nitrogen metabolism.

[Effects of nitrogen application levels on translocation and distribution of 13C-photosynthate and 15N to fruit from leaves of apple tree]. / 氮水平对苹果叶片13Cå ‰åˆäº§ç‰©å’Œ15Nå‘æžœå®žè½¬ç§»åˆ†é çš„å½±å“.

A field experiment was carried out in a six-year old 'Fuji3'/M26/Malus hupehensis Rehd. apple with 15N and 13C labeled tracers, to understand 13C assimilation capability and the characteristics of translocation and distribution of 13C-photosynthate and 15N to fruit under different nitrogen application levels (urea 0%, 0.6%, 1.2%, 1.8%, 2.4%, CK, N1, N2, N3, N4, respectively) to smear the leaves within 20 cm around the fruit at late stage of fruit enlargement. The results showed that, with the increases of urea application, the chlorophyll content, nitrogen content, net photosynthetic rate, sorbitol and sucrose content, sorbitol 6-phosphate dehydrogenase (S6PDH) and sucrose phosphate synthase (SPS) activities, 13C assimilation capability of leaves were first increased and then decreased, with the highest value in 1.8% urea smear treatment and the lowest value with the treatment of clear water. The 13C of self retention (self leaves and self branches) was the highest in clear water (81.6%) and the lowest in 1.8% urea smear treatment (63.5%). The 13C was mainly allocated to fruit, followed by unlabeled perennial branch, and the lowest in unlabeled leaves. With the increases of urea application, the 13C absorption of fruit was first increased and then decreased, with the highest value in 1.8% urea smear treatment (1.21 mg·g-1) and the lowest value in clear water (0.51 mg·g-1). The 15N absorption of fruit was enhanced with the increases of urea application. These results indicated that foliage application of urea solution improved translocation and distribution of leaf photosynthate and nitrogen to fruit with varying degrees, which was the highest in 1.8% urea smear treatment and could avoid excessive intake of nitrogen to fruit.

Dynamics of growth and nitrogen accumulation and utilization of young apple trees.

Taking 6-year-old Yanfu3/SH6/Malus hupehensis Rehd. as the test material, the dynamics of plant growth and nitrogen (N) accumulation under seven periods from germination stage to fruit maturity stage were examined by destructive analysis. The absorption, utilization, and distribution of fertilizer N were studied by 15N isotope tracer technique to clarify the N accumulation dynamics of apple trees and the maximum efficiency period of fertilizer N, and to provide theoretical basis for scientific application of N fertilizer. The results showed that the net accumulation of dry matter was 4.51 kg in germination stage (March 25) to fruit maturity stage (210 d after budbreak), with fruit accounting for 66.5%, the leaves and new shoots accounting for 20.2%, and the perennial organs accounting for 13.3%. The dry matter accumulation in 30-60 d after budbreak was the fastest, accounting for 42.9% of the whole treatment period. The fruit dry matter accumulation in 120-180 d after budbreak was the fastest, which accounted for 70% of the whole treatment period. The total N accumulation of the plant was 29.1 g, which increased rapidly in the 30-60 d and 120-180 d after budbreak by 7.2 g and 12.8 g, respectively accounting for 24.7% and 44% of the whole treatment period. The N accumulation of leaves and new shoots was the fastest in 0-60 d after budbreak, which accounted for 69.1% of the whole period. The N accumulation of fruit was the fastest in 120-180 d after budbreak, accounting for 60.8% of the whole period. The N accumulation of the perennial organ decreased first and then increased, and reached the lowest level at 60 d after budbreak. The 15N utilization rate of plant differed significantly in different periods which was at a high level in 30-60 d,120-150 d and 150-180 d after budbreak with 2.3%, 4.1% and 4.0% respectively. The 15N distribution rate in perennial organs in each period showed a high level, that of the new born organ increased first and then decreased which reached the highest level of 38.4% in 30-60 d after budbreak. The fruit reached the highest in 120-150 d and 150-180 d after budbreak by 15.0% and 16.6% respectively. Therefore, the key period of N accumulation in leaves and shoots was 30-60 d after budbreak, and that in fruit was 120-180 d after budbreak. The period with maximum efficiency for fertilizer N was at 30-60 d and 120-180 d after budbreak.

Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits.

OBJECTIVE: To label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits. METHODS: GFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen. RESULTS: The expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen. CONCLUSIONS: The hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

[Effects of BMP-2 gene therapy on vascularization in repairing bone defects].

OBJECTIVE: To study the effects of BMP-2 gene therapy on vascularization in repairing bone defects. METHODS: The isolated rabbit mesenchymal stem cells (rBMSC), after being transfected by adenovirus carrying BMP-2 gene (Ad-BMP-2) and seeded on xenogeneic bone scaffolds, were used to repair 1.5 cm-long radius bone defects. Five methods were in use in the experiments: Ad-BMP-2 infected rBMSC plus antigen-free bovine cancellous bone (BCB, Group A), rBMSC-BCB plus reconstructed hBMP-2 (Group B1), Ad-LacZ infected rBMSC-BCB (Group C), rBMSC-BCB (Group D) and only BCB scaffolds (Group E). After 4, 8, and 12 weeks of the operations, capillary vessel ink infusion, vascular endothelial growth factor ( VEGF) immunohistochemical staining and histological examination were conducted. RESULTS: After 4 weeks of the operations, usually in Group A one newly formed artery was found in every pore between the trabeculae of the BCB. The density of these intraosseous vessels was high in the periphery and decreasing towards the center of the grafts; by transmission electron microscopy, osteoblasts were always next to vascular endothelial cells and gradually developed into osteocytes with the increase of capillary vessel; VEGF expression were apparently enhanced in mesenchymocytes. CONCLUSIONS: BMP-2 gene therapy, by up-regulating VEGF expression, indirectly induces vascularization of grafts and is of great value to the treatment of bone in union and bone defects.

[Research on chondrogenic differentiation and immunologic response of allogeneic mesenchymal stem cells implanted into joint cavity].

OBJECTIVE: To evaluate the cartilage formation ability of allogeneic mesenchymal stem cells implanted into sheep joint cavity without the use of immunosuppressive therapy. METHODS: Allogeneic mesenchymal stem cells (MSCs) loaded onto porous beta-tricalcium phosphate ceramic (beta-TCP) were implanted into normal sheep joint cavity. A complete mismatch between donor stem cells and recipient sheep was confirmed by mixed lymphocyte reaction assays prior to implantation. Eight weeks after implantation, the implants were taken out for histological and immunohistochemical analysis. The histological results were compared with data derived from joint cavity implantation of autologous MSCs-ceramic composites and cell-free ceramics. The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells. RESULTS: For implantation with allogeneic MSCs, no sign of adverse immune response was detected. Histologically, few inflammation cells infiltration occurred and no antibodies against allogeneic cells were detected. Neocartilage formation in implants loaded with either allogeneic or autologous mesenchymal stem cells was revealed by histochemical and immunohistochemical analysis. In implants without stem cells, no cartilage formation was detected. CONCLUSIONS: Allogeneic mesenchymal stem cells are capable of forming cartilage under the effect of joint cavity environment. Without the use of immunosuppressive therapy, allogeneic MSCs do not provoke an adverse immune response in vivo.

The expression of N-cadherin, fibronectin during chondrogenic differentiation of MSC induced by TGF-beta(1).

OBJECTIVE: To investigate the role of N-cadherin and fibronectin during chondrogenesis. METHODS: Immunohistochemical method and autibody induced changes of aggregation of cells were used to assay the expressions of N-cadherin and fibronectin during cell differentiation. RESULTS: The N-cadherin was present in the area of the cell nodular area in the 24 hours group after adding chondrogenic revulsant, then there was a down-regulating trend. Fibronectin was expressed in 48 and 72 hours groups after adding chondrogenic revulsant, and showed to be negative afterward. The antibody against fibronectin or N-cadherin could inhibit the formation of cellular nodule markedly. CONCLUSIONS: Cell adhesion factors play an important role during cell differentiation. TGF-beta(1) stimulates chondrogenesis via transition from an initial N-cadherin-contributing stage to a succedent fibronectin-contributing stage during the process of chondrogenesis in MSCs. Further study is needed to evaluate whether or not it can promote chondrogenesis by transfecting cDNA of CAMs to MSCs.

Labeling of human insulin-like growth factor-I eukaryotic expression vector with green fluorescent protein.

OBJECTIVE: To label human insulin-like growth factor-I (hIGF-I) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects. METHODS: GFP cDNA was inserted into pcDNA(3.1)-hIGF-1 to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine. RESULTS: Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-I cDNA. Expression of hIGF-1 and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy. CONCLUSIONS: hIGF-I eukaryotic expression vector has been successfully labeled with GFP.

Comparative study on seeding methods of human bone marrow stromal cells in bone tissue engineering.

BACKGROUND: In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering. METHODS: DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope (SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated. RESULTS: DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation. Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs. CONCLUSION: dynamic seeding of hBMSCs is a promising technique in bone tissue engineering.

[Experimental study on the prevention of epidural scar adhesion with polycaprolactone/polylactic acid membrane].

OBJECTIVE: To evaluate the ability of a polycaprolactone/polylactic acid (PCL/PLA) membrane to inhibit epidural scar adhesion after laminectomy, and observe the responsive changes of the pain media in the spinal cord. METHODS: L(1), L(3) laminectomies were performed on 96 Wistar rats. The rats were divided into 3 groups: None-implant Control Group (NC), Autologous free fat graft group (AFFG) and PCL/PLA membrane group (PCL/PLAm). The rats were killed at 1, 3, 6, and 12 weeks postoperatively. Epidural scar formation and adhesion were observed grossly and histologically. Reverse transcription polymerase chain reaction (RT-PCR) were used to analyses the expression of Transforming growth factor beta (TGF-beta) in the epidural scar. Immunohistochemistry stain and RT-PCR were performed to evaluate the expression of the substance P and the c-fos gene in the relevant spinal cord, and the results were analyzed statistically. RESULTS: Gross evaluation and histological evaluation showed that in the NC lamina defect site had much scar tissue and had wide and tight adhesions to the dura; in the AFFG, with the fat degrading gradually, the adhesions were increased; whereas in the PCL/PLAm group, there were slightly adhesions to the dura. RT-PCR showed that the expression of the TGF-beta was much less in the PCL/PLAm group than in the NC group. The insertion of the PCL/PLA membrane and the fat patch reduced the expression of the substance P and the c-fos gene in the spinal cord. CONCLUSION: The insertion of the PCL/PLA membrane reduces scar formation and separates fibrosis tissue from the dura, the results indicate that PCL/PLA membrane is an effective way of reducing peridural scar formation and preventing the failed back surgery syndrome.

[Orthopaedic applications for biodegradable and absorbable internal fixation of fractures].

OBJECTIVE: To illustrate the effect and complication of orthopedic applications for biodegradable and absorbable internal fixation of fractures, and to indicate the existent problem and research aspect currently. METHODS: The recent literatures on orthopedic applications and study of biodegradable and absorbable internal fixation for fractures were reviewed. The effect of biodegradable materials on bone healing was summarized. RESULTS: It is good for the stability of fracture fixation and result of treatment. The biodegradable and absorbable internal fixation fractures had no adverse effect on bone healing. CONCLUSION: There will be more widespread application for biodegradable and absorbable materials in orthopedics, but the intensive research should be carried out to prevent its complication.