Molecular markers in medicinal plant biotechnology: past and present

Plant based medicines have gained popularity worldwide due to their almost negligible side effects. In India, the three traditional medicinal systems, namely homeopathy, Ayurveda and Siddha rely heavily on plants for medicinal formulations. To prevent the indiscriminate collection of these valuable medicinal plants and for their proper authentication and conservation, it is imperative to go for sustained efforts towards proper germplasm cataloguing and devising conservation strategies. For this purpose, molecular markers have a significant role, as they provide information ranging from diversity at nucleotide level (single nucleotide polymorphisms) to gene and allele frequencies (genotype information), the extent and distribution of genetic diversity, and population structure. Over the past twenty years, the molecular marker field has completely transformed the meaning of conservation genetics which has emerged from a theory-based field of population biology to a full-fledged pragmatic discipline. In this review, we have explored the transition and transformation of molecular marker technologies throughout these years.     

Transdifferentiation: a cell and molecular reprogramming process

Evidence has emerged recently indicating that differentiation is not entirely a one-way process, and that it is possible to convert one cell type to another, both in vitro and in vivo. This phenomenon is called transdifferentiation, and is generally defined as the stable switch of one cell type to another. Transdifferentiation plays critical roles during development and in regeneration pathways in nature. Although this phenomenon occurs rarely in nature, recent studies have been focused on transdifferentiation and the reprogramming ability of cells to produce specific cells with new phenotypes for use in cell therapy and regenerative medicine. Thus, understanding the principles and the mechanism of this process is important for producing desired cell types. Here some well-documented examples of transdifferentiation, and their significance in development and regeneration are reviewed. In addition, transdifferentiation pathways are considered and their potential molecular mechanisms, especially the role of master switch genes, are considered. Finally, the significance of transdifferentiation in regenerative medicine is discussed.     

Inflorescence and floral ontogeny in Crocus sativus L. (Iridaceae)

Inflorescence and floral ontogeny of the perennial, herbaceous crop Crocus sativus L. were studied using epi-illumination light microscopy. After production of leaves with helical arrangement a determinate inflorescence forms which becomes completely transformed into a single terminal flower. In some cases, bifurcation of the inflorescence meristem yields two or three floral meristems. The order of floral organs initiation is outer tepals – stamens – inner tepals – carpels. Stamens and outer tepals are produced from the lateral bifurcation of three common stamen-tepal primordia. Within each whorl, organs start developing unidirectionally from the adaxial side, except for the stamens which begin to grow from the abaxial side. Specialized features during organ development include interprimordial growth between tepals forming a perianth tube, fusion at the base of stamen filaments, and formation of an inferior ovary with unfused styles.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Clinical response to chemotherapy in locally advanced breast cancer was not associated with several polymorphisms in detoxification enzymes and DNA repair genes

The main aim of the present study was to investigate the association between several genetic polymorphisms (in glutathione S-transferase members and DNA repair genes) and clinical response to chemotherapy in locally advanced breast cancer. A sequential series of 101 patients were prospectively included in this study. Clinical assessment of treatment was accomplished by comparing initial tumor size with preoperative tumor size using revised RECIST guideline (version 1.1). Clinical response was regarded as a response or no response. There was no difference between non-responders and responders for the prevalence of genotypes of the study polymorphisms.     

Development of a thermostable β-glucuronidase-based reporter system for monitoring gene expression in hyperthermophiles

Mesophilic glucuronidases are the most widely used reporters of gene expression in plants, but unsuitable as reporters in (hyper-)thermophiles due their insufficient thermal stability. Here we present the native 66.8 kDa thermostable β-glucuronidase of Sulfolobus solfataricus. The enzyme activity is characterized in a wide temperature range ideal for, but not limited to, in vivo genetic study of hyperthermophiles. As a proof of concept, we demonstrate its use as a reporter of gene expression in Sulfolobus, by monitoring a promoter fusion created with the β-glucuronidase coding gene gusB and a copper-responsive promoter.     

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker^® staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.     

The influence of peroxisome proliferator-activated receptor γ(1) during differentiation of mouse embryonic stem cells to neural cells

Peroxisome proliferator activated receptor γ, belongs to PPARs, which exerts various metabolic functions including differentiation process. To testify the importance of PPARγ in neural differentiation of mouse embryonic stem cells (mESCs), its expression level was assessed. Data revealed an elevation in expression level of PPARγ when neural precursors (NPs) are formed upon retinoic acid treatment. Thus, involvement of PPARγ in two stages of neural differentiation of mESCs, during and post-NPs formation was examined by application of its agonist and antagonist. Our results indicated that PPARγ inactivation via treatment with GW9662 during NPs formation, reduced expression of neural precursor and neural (neuronal and astrocytes) markers. However, PPARγ inactivation by antagonist treatment post-NPs formation stage only decreased the expression of mature astrocyte marker (Gfap) suggesting that inactivation of PPARγ by antagonist decreased astrocyte differentiation. Here, we have demonstrated the stage dependent role of PPARγ modulation on neural differentiation of mESCs by retinoic acid treatment for the first time.