We report for the first time that expression of potato PR10a gene in faba bean causes enhanced tolerance to drought and salinity. Grain legumes such as soybean (Glycine max L. Merrill), pea (Pisum sativum L.) and faba bean (Vicia faba L.) are staple sources of protein for human and animal nutrition. Among grain legumes, faba bean is particularly sensitive to abiotic stress (in particular osmotic stress due to lack of water or enhanced soil salinity) and often suffers from severe yield losses. Many stress responsive genes have been reported with an effect on improving stress tolerance in model plants. Pathogenesis-related proteins are expressed by all plants in response to pathogen infection and, in many cases, in response to abiotic stresses as well. The PR10a gene isolated from the potato cultivar Desiree was selected for this study due to its role in enhancing salt and/or drought tolerance in potato, and transferred into faba bean cultivar Tattoo by Agrobacterium tumefaciens-mediated transformation system based upon direct shoot regeneration after transformation of meristematic cells derived from embryo axes. The transgene was under the control of the constitutive mannopine synthase promoter (p-MAS) in a dicistronic binary vector, which also contained luciferase (Luc) gene as scorable marker linked by internal ribosome entry site elements. Fertile transgenic faba bean plants were recovered. Inheritance and expression of the foreign genes were demonstrated by PCR, RT-PCR, Southern blot and monitoring of Luciferase activity. Under drought condition, after withholding water for 3 weeks, the leaves of transgenic plants were still green, while non-transgenic plants (WT) wilted and turned brown. Twenty-four hours after re-watering, the leaves of transgenic plants remained green, while WT plants did not recover. Moreover, the transgenic lines displayed higher tolerance to NaCl stress. Our results suggested that introducing a novel PR10a gene into faba bean could be a promising approach to improve its drought and salt tolerance ability, and that MAS promoter is not only constitutive, but also wound-, auxin/cytokinin- as well as stress-inducible.
Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.
Bone loss varies according to disease and age and these variations affect bone cells and extracellular matrix. Osteoporosis rat models are widely investigated to assess mechanical and structural properties of bone; however, bone matrix proteins and their discrepant regulation of diseased and aged bone are often overlooked. The current study considered the spine matrix properties of ovariectomized rats (OVX) against control rats (Sham) at 16 months of age. Diseased bone showed less compact structure with inhomogeneous distribution of type 1 collagen (Col1) and changes in osteocyte morphology. Intriguingly, demineralization patches were noticed in the vicinity of blood vessels in the OVX spine. The organic matrix structure was investigated using computational segmentation of collagen fibril properties. In contrast to the aged bone, diseased bone showed longer fibrils and smaller orientation angles. The study shows the potential of quantifying transmission electron microscopy images to predict the mechanical properties of bone tissue.
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