Molecular characterization of a tomato purple acid phosphatase during seed germination and seedling growth under phosphate stress.

KEY MESSAGE: SlPAP1 is a phosphate starvation responsive purple acid phosphatase during tomato seed germination. Future research on its family members in tomato might improve the phosphate stress tolerance. Phosphate deficiency is a major constraint upon crop growth and yield. In response to phosphate deficiency, plants secrete acid phosphatases (APases) to scavenge organic phosphate from soil. In this study, we investigated the impact of Pi starvation on germination and seedling growth of tomato, and we then cloned and characterized a phosphate starvation responsive purple APase (SlPAP1) that expressed during tomato seedling growth. Our results showed that phosphate deficiency reduced germination and growth rates of tomato, and also increased intracellular and secretory APase activity in a concentration-dependent manner. An in-gel activity assay found that two APases of 50 and 75 kDa were secreted during conditions of phosphate deficiency. SlPAP1 is a single copy gene belonging to a small gene family. It was expressed as a cDNA of approximately 1.5 kbp encoding a secreted glycoprotein of 470 amino acids. Northern blot analysis showed that SlPAP1 was specifically expressed in root tissue during phosphate deficiency. SlPAP1 had high sequence identity (56-89%) with other plant PAPs and contained highly conserved metal-binding residues. SlPAP1 is the first PAP to be cloned and characterized from tomato. This study provides useful information for future research on PAP family members in tomato, leading to better understanding of phosphate deficiency in this important crop plant.

An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells.

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSR N-terminal binding domains (VSRNTs) were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein-protein interaction in vivo.

Lysine-rich rice partially enhanced the growth and development of skeletal system with better skeletal microarchitecture in young rats.

Rice is the primary staple food for half of the world's population but is low in lysine content. Previously, we developed transgenic rice with enhanced free lysine content in rice seeds (lysine-rich rice), which was shown safe for consumption and improved the growth in rats. However, the effects of lysine-rich rice on skeletal growth and development remained unknown. In this study, we hypothesized that lysine-rich rice improved skeletal growth and development in weaning rats. Male weaning Sprague-Dawley rats received lysine-rich rice (HFL) diet, wild-type rice (WT) diet, or wild-type rice with various contents of lysine supplementation diet for 70 days. Bone microarchitectures were examined by microcomputed tomography, bone strength was investigated by mechanical test, and dynamics of bone growth were examined by histomorphometric analysis. In addition, we explored the molecular mechanism of lysine and skeletal growth through biochemical testing of growth hormone, bone turnover marker, and amino acid content of rat serum analysis, as well as in a cell culture system. Results indicated that the HFL diet improved rats' bone growth, strength, and microarchitecture compared with the WT diet group. In addition, the HFL diet increased the serum essential amino acids, growth hormone (insulin-like growth factor-1), and bone formation marker concentrations. The cell culture model showed that lysine deficiency reduced insulin-like growth factor-1 and Osterix expression, Akt/mammalian target of rapamycin signaling, and matrix mineralization, and inhibited osteoblast differentiation associated with bone growth. Our findings showed that lysine-rich rice improved skeletal growth and development in weaning rats. A further increase of rice lysine content is highly desirable to fully optimize bone growth and development.

A dual-targeted purple acid phosphatase in Arabidopsis thaliana moderates carbon metabolism and its overexpression leads to faster plant growth and higher seed yield.

• Overexpression of AtPAP2, a purple acid phosphatase (PAP) with a unique C-terminal hydrophobic motif in Arabidopsis, resulted in earlier bolting and a higher seed yield. Metabolite analysis showed that the shoots of AtPAP2 overexpression lines contained higher levels of sugars and tricarboxylic acid (TCA) metabolites. Enzyme assays showed that sucrose phosphate synthase (SPS) activity was significantly upregulated in the overexpression lines. The higher SPS activity arose from a higher level of SPS protein, and was independent of SnRK1. • AtPAP2 was found to be targeted to both plastids and mitochondria via its C-terminal hydrophobic motif. Ectopic expression of a truncated AtPAP2 without this C-terminal motif in Arabidopsis indicated that the subcellular localization of AtPAP2 is essential for its biological actions. • Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution. AtPAP2 is the first PAP shown to modulate carbon metabolism and the first shown to be dual-targeted to both plastids and mitochondria by a C-terminal targeting signal. • One PAP-like sequence carrying a hydrophobic C-terminal motif could be identified in the genome of the smallest free-living photosynthetic eukaryote, Ostreococcus tauri. This might reflect a common ancestral function of AtPAP2-like sequences in the regulation of carbon metabolism.

A 64 kDa sucrose binding protein is membrane-associated and tonoplast-localized in developing mung bean seeds.

Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

Improved growth performance, food efficiency, and lysine availability in growing rats fed with lysine-biofortified rice.

Rice is an excellent source of protein, and has an adequate balance of amino acids with the exception of the essential amino acid lysine. By using a combined enhancement of lysine synthesis and suppression of its catabolism, we had produced two transgenic rice lines HFL1 and HFL2 (High Free Lysine) containing high concentration of free lysine. In this study, a 70-day rat feeding study was conducted to assess the nutritional value of two transgenic lines as compared with either their wild type (WT) or the WT rice supplemented with different concentrations of L-lysine. The results revealed that animal performance, including body weight, food intake, and food efficiency, was greater in the HFL groups than in the WT group. Moreover, the HFL diets had increased protein apparent digestibility, protein efficiency ratio, and lysine availability than the WT diet. Based on the linear relationship between dietary L-lysine concentrations and animal performance, it indicated that the biological indexes of the HFL groups were similar or better than that of the WT20 group, which was supplemented with L-lysine concentrations similar to those present in the HFL diets. Therefore, lysine-biofortified rice contributed to improved growth performance, food efficiency, and lysine availability in growing rats.

Sclerostin, an emerging therapeutic target for treating osteoporosis and osteoporotic fracture: A general review.

Osteoporosis and its associated fracture risk has become one of the major health burdens in our aging population. Currently, bisphosphonate, one of the most popular antiresorptive drugs, is used widely to treat osteoporosis but so far still no consensus has been reached for its application in treatment of osteoporotic fractures. However, in old patients, boosting new bone formation and its remodelling is essential for bone healing in age-related osteoporosis and osteoporotic fractures. Sclerostin, an inhibitor of the Wnt/ß-catenin signalling pathway that regulates bone growth, has become an attractive therapeutic target for treating osteoporosis. In this review, we summarize the recent findings of sclerostin and its potential as an effective drug target for treating both osteoporosis and osteoporotic fractures.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult Male Rats.

We investigated the systemic effect of sclerostin monoclonal antibody (Scl-Ab) treatment on intact non-operated bones in an open osteotomy male Sprague Dawley (SD) rat model. Six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were injected subcutaneously with vehicle or Scl-Ab (25 mg/kg, 2 times per week) treatment for 9 weeks. Compared with vehicle control, Scl-Ab treatment significantly improved trabecular and cortical bone mass and microarchitecture at L5 vertebrae and left femora by micro-CT at week 6 and 9. Mechanical testing showed that Scl-Ab treatment resulted in significantly higher stiffness, energy to failure and ultimate load at the femora at week 9. Mineral apposition rate, mineralizing surface and bone formation rate on the trabecular bone in the distal femora was significantly increased in Scl-Ab group at week 6 and 9. The administered Scl-Ab was localized in the osteocytes and beta-catenin was strongly expressed in osteoblasts. Scl-Ab treatment significantly increased serum P1NP level and there was no between-group difference in serum level of CTX-1. In conclusion, Scl-Ab treatment could induce rapid and sustained increase in bone formation, bone mass and bone strength in non-operated bones. Sclerostin inhibition might be advantageous to prevent secondary fracture(s).

Sclerostin monoclonal antibody enhanced bone fracture healing in an open osteotomy model in rats.

Sclerostin is a negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment promoted bone healing in various animal models. To further evaluate the healing efficiency of Scl-Ab in osteotomy healing, we investigated the time course effects of systemic administration of Scl-Ab on fracture repair in rat femoral osteotomy model. A total of 120 six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were treated with vehicle or Scl-Ab treatment for 3, 6, or 9 weeks. Fracture healing was evaluated by radiography, micro-CT, micro-CT based angiography, 4-point bending mechanical test and histological assessment. Scl-Ab treatment resulted in significantly higher total mineralized callus volume fraction, BMD and enhanced neovascularization. Histologically, Scl-Ab treatment resulted in a significant reduction in fracture callus cartilage at week 6 and increase in bone volume at week 9, associated with a greater proportion of newly formed bone area at week 6 and 9 by fluorescence microscopy. Mechanical testing showed significantly higher ultimate load in Scl-Ab treatment group at week 6 and 9. This study has demonstrated that Scl-Ab treatment enhanced bone healing in a rat femoral osteotomy model, as reflected in increased bone formation, bone mass and bone strength.

Extracorporeal shockwave enhanced regeneration of fibrocartilage in a delayed tendon-bone insertion repair model.

Fibrous tissue is often formed in delayed healing of tendon bone insertion (TBI) instead of fibrocartilage. Extracorporeal shockwave (ESW) provides mechanical cues and upregulates expression of fibrocartilage-related makers and cytokines. We hypothesized that ESW would accelerate fibrocartilage regeneration at the healing interface in a delayed TBI healing model. Partial patellectomy with shielding at the TBI interface was performed on 32 female New Zealand White Rabbits for establishing this delayed TBI healing model. The rabbits were separated into the control and ESW group for evaluations at postoperative week 8 and 12. Shielding was removed at week 4 and a single ESW treatment was applied at week 6. Fibrocartilage regeneration was evaluated histomorphologically and immunohistochemically. Vickers hardness of the TBI matrix was measured by micro-indentation. ESW group showed higher fibrocartilage area, thickness, and proteoglycan deposition than the control in week 8 and 12. ESW increased expression of SOX9 and collagen II significantly in week 8 and 12, respectively. ESW group showed a gradual transition of hardness from bone to fibrocartilage to tendon, and had a higher Vickers hardness than the control group at week 12. In conclusion, ESW enhanced fibrocartilage regeneration at the healing interface in a delayed TBI healing model.

AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria.

To date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. Using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (OM) of chloroplasts and mitochondria and exposed its N-terminal enzymatic domain to the cytosol. It was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. Mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.   

Extracorporeal shockwave therapy for treatment of delayed tendon-bone insertion healing in a rabbit model: a dose-response study.

BACKGROUND: Tendon-bone insertion (TBI) consists of both hard and soft tissues. TBI injury with delayed repair is not uncommon. High-dose extracorporeal shockwave (ESW) is effective for treating nonunion fracture, whereas low-dose ESW is used for tendinopathy therapy. The dosing effect of ESW on delayed TBI healing is lacking. HYPOTHESIS: Low-dose ESW might have a healing enhancement effect comparable to that of high-dose ESW in treating delayed TBI healing. STUDY DESIGN: Controlled laboratory study. METHODS: Partial patellectomy was adopted to create a delayed TBI healing model by shielding the healing interface between tendon and bone. Ninety-six female New Zealand White rabbits with unilateral delayed TBI healing at the knee joint were divided into 3 groups: controls, low-dose ESW (LD-ESW; 0.06 mJ/mm(2), 4 Hz, 1500 impulses), and high-dose ESW (HD-ESW; 0.43 mJ/mm(2), 4 Hz, 1500 impulses). The TBI shielding was removed at week 4 after partial patellectomy, followed by treatment with control or ESW at week 6. The rabbits were euthanized at week 8 and week 12 for radiological, microarchitectural, histological, and mechanical assessments of healing tissues. RESULTS: Radiologically, both the LD-ESW group and the HD-ESW group showed larger new bone area than the controls at week 8 and week 12. Microarchitectural measurements showed that the LD-ESW and HD-ESW groups had larger new bone volume than the controls at week 12. Histological assessments confirmed osteogenesis enhancement. Both the LD-ESW and HD-ESW groups showed significantly higher failure load at the TBI healing complex than the control group at week 12. No significant difference was detected between the 2 ESW treatment groups at week 8 or week 12. CONCLUSION: Extracorporeal shockwave, a unique noninvasive physical modality, had similar effects between the low and high dose for treating delayed TBI healing. CLINICAL RELEVANCE: Low-dose ESW for TBI delayed healing might be more desirable and have better compliance in clinical applications.