Screening of environmental stimuli for the positive regulation of stomatal aperture in centipedegrass.

Grasslands, the largest carbon pool in China, possess enormous potential for carbon sequestration. Increasing the stomatal aperture to increase the CO2 absorption capacity is a potential method to improve plant photosynthetic efficiency and ultimately enhance the carbon sequestration capacity of grass plants. Research on stomatal aperture regulation has focused mostly on Arabidopsis or crops, while research on grass plants in these areas is scarce, which seriously restricts the implementation of this grassland carbon sequestration strategy. Here, a widely used ecological grass, centipedegrass, was used as the experimental material. First, a convenient method for observing the stomatal aperture was developed. The leaves were floated in a potassium ion-containing open solution (67 mM KCl, pH 6.0) with the adaxial surface rather than the abaxial surface in contact with the solution and were cultivated under light for 1.5 h. Then, nail polish was applied on the adaxial surface, and a large number of open stomata were imprinted. Second, with the help of this improved method, the concentration‒response characteristics of the stomatal aperture to eleven environmental stimuli were tested. The stomatal aperture is dependent on these environmental stimuli in a concentration-dependent manner. The addition of 100 µM brassinolide led to the maximal stomatal aperture. This study provided a technical basis for manipulating stomatal opening to increase the carbon sequestration capacity of centipedegrass.

An appropriate ammonium: nitrate ratio promotes the growth of centipedegrass: insight from physiological and micromorphological analyses.

Reasonable nitrogen fertilizer application is an important strategy to maintain optimal growth of grasslands, thereby enabling them to better fulfil their ecological functions while reducing environmental pollution caused by high nitrogen fertilizer production and application. Optimizing the ammonium (NH4 +):nitrate (NO3 -) ratio is a common approach for growth promotion in crops and vegetables, but research on this topic in grass plants has not received sufficient attention. Centipedegrass, which is widely used in landscaping and ecological protection, was used as the experimental material. Different NH4 +:NO3 - ratios (0: 100, 25:75, 50:50, 75:25, 100:0) were used as the experimental treatments under hydroponic conditions. By monitoring the physiological and morphological changes under each treatment, the appropriate NH4 +:NO3 - ratio for growth and its underlying mechanism were determined. As the proportion of ammonium increased, the growth showed a "bell-shaped" response, with the maximum biomass and total carbon and nitrogen accumulation achieved with the NH4 +:NO3 - ratio of 50:50 treatment. Compared with the situation where nitrate was supplied alone, increasing the ammonium proportion increased the whole plant biomass by 93.2%, 139.7%, 59.0%, and 30.5%, the whole plant nitrogen accumulation by 44.9%, 94.6%, 32.8%, and 54.8%, and the whole plant carbon accumulation by 90.4%, 139.9%, 58.7%, and 26.6% in order. As a gateway for nitrogen input, the roots treated with an NH4 +:NO3 - ratio of 50:50 exhibited the highest ammonium and nitrate uptake rate, which may be related to the maximum total root length, root surface area, average root diameter, root volume, and largest root xylem vessel. As a gateway for carbon input, leaves treated with an NH4 +:NO3 - ratio of 50:50 exhibited the highest stomatal aperture, stomatal conductance, photosynthetic rate, transpiration rate, and photosynthetic products. The NH4 +:NO3 - ratio of 50:50 treatment had the largest stem xylem vessel area. This structure and force caused by transpiration may synergistically facilitate root-to-shoot nutrient translocation. Notably, the change in stomatal opening occurred in the early stage (4 hours) of the NH4 +:NO3 - ratio treatments, indicating that stomates are structures that are involved in the response to changes in the root NH4 +:NO3 - ratio. In summary, we recommend 50:50 as the appropriate NH4 +:NO3 - ratio for the growth of centipedegrass, which not only improves the nitrogen use efficiency but also enhances the carbon sequestration capacity.

Roles of plastid-located phosphate transporters in carotenoid accumulation.

Enhanced carotenoid accumulation in plants is crucial for the nutritional and health demands of the human body since these beneficial substances are acquired through dietary intake. Plastids are the major organelles to accumulate carotenoids in plants and it is reported that manipulation of a single plastid phosphate transporter gene enhances carotenoid accumulation. Amongst all phosphate transport proteins including phosphate transporters (PHTs), plastidial phosphate translocators (pPTs), PHOSPHATE1 (PHO1), vacuolar phosphate efflux transporter (VPE), and Sulfate transporter [SULTR]-like phosphorus distribution transporter (SPDT) in plants, plastidic PHTs (PHT2 & PHT4) are found as the only clade that is plastid located, and manipulation of which affects carotenoid accumulation. Manipulation of a single chromoplast PHT (PHT4;2) enhances carotenoid accumulation, whereas manipulation of a single chloroplast PHT has no impact on carotenoid accumulation. The underlying mechanism is mainly attributed to their different effects on plastid orthophosphate (Pi) concentration. PHT4;2 is the only chromoplast Pi efflux transporter, and manipulating this single chromoplast PHT significantly regulates chromoplast Pi concentration. This variation subsequently modulates the carotenoid accumulation by affecting the supply of glyceraldehyde 3-phosphate, a substrate for carotenoid biosynthesis, by modulating the transcript abundances of carotenoid biosynthesis limited enzyme genes, and by regulating chromoplast biogenesis (facilitating carotenoid storage). However, at least five orthophosphate influx PHTs are identified in the chloroplast, and manipulating one of the five does not substantially modulate the chloroplast Pi concentration in a long term due to their functional redundancy. This stable chloroplast Pi concentration upon one chloroplast PHT absence, therefore, is unable to modulate Pi-involved carotenoid accumulation processes and finally does affect carotenoid accumulation in photosynthetic tissues. Despite these advances, several cases including the precise location of plastid PHTs, the phosphate transport direction mediated by these plastid PHTs, the plastid PHTs participating in carotenoid accumulation signal pathway, the potential roles of these plastid PHTs in leaf carotenoid accumulation, and the roles of these plastid PHTs in other secondary metabolites are waiting for further research. The clarification of the above-mentioned cases is beneficial for breeding high-carotenoid accumulation plants (either in photosynthetic or non-photosynthetic edible parts of plants) through the gene engineering of these transporters.

Bladder reconstruction using autologous smooth muscle cell sheets grafted on a pre-vascularized capsule.

Rationale: Construction of functional vascularized three-dimensional tissues has been a longstanding objective in the field of tissue engineering. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate functional vascularized smooth muscle tissue flaps for bladder reconstruction in a rabbit model was tested. Methods: Skin tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Smooth muscle cells and endothelial progenitor cells were harvested and cocultured to form pre-vascularized smooth muscle cell sheet. Then repeated transplantation of triple-layer cell sheet grafts onto the vascularized capsular tissue was performed at 2-day intervals to prefabricate functional vascularized smooth muscle tissue flaps. Bladder muscular wall defects were created and repaired by six-layer cell sheet graft (sheet only), capsule flap (capsule only) and vascularized capsule prelaminated with smooth muscle cell sheet (sheet plus capsule). The animals were followed for 3 months after implantation and their bladders were explanted serially. Results: Bladder capacity and compliance were maintained in sheet plus capsule group throughout the 3 months. Tissue bath stimulation demonstrated that contractile responses to carbachol and KCl among the three groups revealed a significant difference (p < 0.05). Histologically, inflammation was evident in the capsule only group at 1 month and fibrosis was observed in sheet only group at 3 months. The vessel density in capsule only and sheet plus capsule group were significantly higher than in the sheet only group at each time point (p < 0.05). Comparison of the smooth muscle content among the three groups revealed a significant difference (p < 0.05). Conclusion: These results proved that the capsule may serve as an induced vascular bed for vascularized smooth muscle tissue flap prefabrication. The prefabricated functional vascularized smooth muscle tissue flap has the potential for reliable bladder reconstruction and may create new opportunities for vascularization in 3-D tissue engineering.

Tissue expander capsule as an induced vascular bed to prefabricate an axial vascularized buccal mucosa-lined flap for tubularized posterior urethral reconstruction: preliminary results in an animal model.

Surgical repair of complex posterior urethral disruptions remains one of the most challenging problems in urology. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate axial vascularized buccal mucosa-lined flaps for tubularized posterior urethral reconstruction in a rabbit model was tested. The experiments were performed in three stages. First, silicone tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Next, buccal mucosa grafts were transplanted into the newly formed capsular tissue supplied by axial vessels for buccal mucosa-lined flap prefabrication. Then, circumferential posterior urethral defects were created and repaired with the buccal mucosa graft (Group 1), the capsule flap (Group 2), and the prefabricated capsule buccal mucosa composite flap (Group 3). After surgery, notable contracture of the tubularized buccal mucosa graft was observed in the neourethra, and none of the rabbits in Group 1 maintained a wide urethral caliber. In Group 2, the retrieved neourethra showed little evidence of epithelial lining during the study period, and the lumen caliber was narrowed at the 3-month evaluation. In Group 3, the buccal mucosa formed the lining in the neourethra and maintained a wide urethral caliber for 3 months. The capsule may serve as an induced vascular bed for buccal mucosa-lined flap prefabrication. The prefabricated buccal mucosa-lined flap may serve as a neourethra flap for posterior urethral replacement.

Surgical treatment of pelvic fracture urethral distraction defects in boys: which approach is suitable?

Pelvic fracture urethral distraction defects (PFUDDs) are relatively infrequent in boys, and treatment for PFUDDs presents one of the most difficult problems in urological practice. Anastomotic urethroplasty is considered an ideal surgical procedure for PFUDDs in boys. However, various surgical approaches for anastomotic urethroplasty have been proposed, including a simple transperineal approach, a transperineal intercorporal septal separation approach, a transperineal inferior pubic approach, and a combined transpubic-perineal approach. This study aims to determine which surgical approach is best for PFUDDs in boys. We retrospectively identified 22 boys with PFUDDs aged 2-14 years who underwent anastomotic urethroplasty via different approaches between January 2008 and December 2017. Follow-up was performed in all the 22 patients for 6-123 (mean: 52.0) months. Finally, 20 of the 22 boys (90.9%) were successfully treated, including 1 of 2 patients treated with a simple transperineal approach, 3 of 3 with a transperineal approach with intercorporal septal separation, 14 of 15 with a transperineal inferior pubic approach, and 2 of 2 with a combined transpubic-perineal approach. Two patients had failed outcomes after the operation, and stenosis recurred. Based on the outcome of the 22 patients, we can draw a preliminary conclusion that most boys (20/22) can be treated with a transperineal inferior pubic approach or simpler procedures without the need of completely removing or incising the pubis. The combined transpubic-perineal approach can be used in cases of extremely long urethral distract defects.

Tubularized urethral reconstruction using a prevascularized capsular tissue prelaminated with buccal mucosa graft in a rabbit model.

Tubularized graft urethroplasty fails largely because of inadequate graft take. Prefabrication of buccal mucosa lined flap has theoretical indications for constructing neourethra with an independent blood supply. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate an axial vascularized buccal mucosa-lined flap for tubularized urethral reconstruction in a rabbit model was tested. The experiments were performed in three stages. First, silicone tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Next, buccal mucosa grafts were transplanted to the newly formed capsular tissue supplied by the axial vessel for buccal mucosa-lined flap prefabrication. Then, circumferential urethral defects were created and repaired by buccal mucosa graft (Group 1), capsule flap (Group 2) and prefabricated capsule buccal mucosa composite flap (Group 3). With retrograde urethrography, no rabbits in Group 1 maintained a wide urethral caliber. In Group 2, the discontinued epithelial layer regenerated at 1 month, and the constructed neourethra narrowed even though the lumen surface formed intact urothelial cells at 3 months. In Group 3, buccal mucosa formed the lining in the neourethra and kept a wide urethral caliber for 3 months. The capsule may serve as an induced vascular bed for buccal mucosa-lined flap prefabrication. The prefabricated buccal mucosa-lined flap may serve as a neourethra flap for circumferential urethral replacement.

Association of molecular markers with cold tolerance and green period in zoysiagrass (Zoysia Willd.).

Cold tolerance and the green period are key traits in the breeding of zoysiagrass (Zoysia Willd.). Identification of molecular markers associated with cold tolerance and the green period of zoysiagrass will contribute to efficient selection of elite cultivars. These two traits were measured in 96 zoysiagrass accessions in 2004 and 2005-2006, respectively. The mapping population was screened with 29 pairs of simple sequence repeat (SSR) primers and 54 pairs of sequence-related amplified polymorphism (SRAP) primers. A multi-loci in silico mapping approach implemented with an empirical Bayes method was applied for association mapping of cold tolerance and green period. We detected 254 SSR polymorphic loci and 338 SRAP polymorphic loci, among which three SSR loci (Xgwm131-3B-187, Xgwm469-6D-194 and Xgwm234-5B-244) and one SRAP locus (Me11Em7-406) were significantly associated with cold tolerance with effect values of 57.83%, 38.05%, 36.92% and 37%, respectively. Three SSR loci (Xgwm132-6B-225, Xgwm111-7D-34 and Xgwm102-2D-97) and two SRAP loci (Me19Em5-359 and Me16Em8-483) were significantly associated with the green period with effect values of 79.54%, 62.59%, 99.04%, 49.01% and 82.57%. These markers will be useful for genetic improvement of the cold tolerance and green period of zoysiagrass by marker-assisted breeding.