Agave sisalana, a hard succulent CAM (crassulacean acid metabolism) plant along with other land plants like cactus, pineapple and vanilla orchids, has evolved over millions of years to drive a different kind of photosynthesis that allows the plants to survive in semiarid environments where water isn‟t always readily available. This process is known as CAM and it is the main area of interest for the scientists all over the world to develop drought resistant plants. Modern genomic approaches like gene identification, their characterization and expression under various abiotic stresses lead to the crafting of genetically resistant crops. The genomes of a number of different CAM plants have been sequenced in the past two years but Agave sisalana L. has received limited attention by many researchers around the globe as far its ability to survive under drought conditions is concerned. Therefore present study was aimed to develop drought stressed cDNA library and to find out potentially up-regulated genes expressed under drought in leaf epidermal tissue of Agave sisalana. Agave sisalana plant selection under drought stressed conditions was done on the basis of their physio-biochemical and water related attributes. All the physiological parameters including photosynthetic and transpiration rate, stomatal conductance and water use efficiency showed decreasing trend with increasing drought stressed conditions. Biochemical analyses of control and drought stressed Agave sisalana plants also played an important role in the adaptation of plants to adverse environment conditions. Plant under drought stress produces certain osmolytes like proline and products of reactive oxygen species which triggers the regulatory signaling pathways involved in the plant defense mechanism. In the present study the correlation coefficients (r) among various physiological, biochemical and water related factors under drought stress conditions also indicated that decreasing photosynthesis, transpiration, total chlorophyll content and other parameters are well monitored by the accumulation of osmolytes produced in response to abiotic traumas. In case of water related measuring parameters, the values of relative water content decreases with the drought stress compared to control where it remain significantly high. Microscopic examination of epidermal tissues of leaves of Agave sisalana showed partially and fully closed stomata upon the induction of drought stress. Stomatal conductance also decreased and showed accordance with the closed stomata under drought stress. Instantaneous water use efficiency showed contradictory results of showing decreasing trend with abiotic stress. It has been reported in many cases that it varies with different plant species as has been explained in the discussion part of the dissertation. All the biochemical assays performed in Agave plants confirmed the accumulation of macro molecules that could lead to support the idea of searching drought tolerant functional EST in leaves of Agave sisalana L. A cDNA library from drought stressed epidermal tissue of leaves of Agave sisalana L. has been constructed. Ten thousands clones were randomly picked, replicated and PCR amplified. The inserts size was found in a range of 100-1000bp. One hundred and five (105) clones (submitted to NCBI GenBank (JZ892707 - JZ892811) were sequenced and annotated. As there were no reported sequences on Agave sisalana L. under drought stress so the homology search was done in comparison with already reported sequences in land plants. Blast ( EST, nucleotide and protein) database in NCBI GenBank was used for homology search. Approximately 4% clone sequences didn‟t show homology at selection criteria (e < 1.0) whereas maximum homology was found in Elaeis guineensis (African palm 16%) followed by Phoenix dactylifera (date palm 8%), Musa acuminate (Banana 5%), Solanum species (5%), Medicago tranculata (5%), Populus trichochorpa (desert poplar 4%), Vitis vinifera (Grapes 4%), Nicotiana species (3%) and Agave species (3%) respectively. The clones (amplified PCR products) were printed in duplicate at an expected ratio of 9,408 spots per microarray chip. The labeled cDNAs were prepared from total RNAs of control and drought stressed leaves of Agave sisalana L. These labeled cDNAs were hybridized to cDNA chips, scanned and data were analyzed. Ten (10) clones were found to be differentially expressed on cDNA microarray platform. The microarray results were validated by real time PCR. EST sequences of potential candidates for drought stressed genes were analyzed through BLAST2GO programe. Out of ten (10) ESTs, maximum homology was found in Phoenix dactylifera plant. Six EST‟s which gives the best homology with drought tolerant genes in other land plants on nucleotide BLAST (nr) and protein BLAST (p) were further evaluated with real time PCR along with bioinformatics studies. Real time data analysis showed highest expression in EST‟s (JZ892752 and JZ892726) whereas EST (JZ892743, JZ892761 and JZ892787) showed low expression of drought tolerance in Agave sisalana L. The differentially expressed EST JZ892778 showed no expression or upregulated activity in stressed leaves of Agave sisalana L. Similar expression of two up-regulated ESTs (JZ892726 and JZ892752) was observed in expression behavior and fold change when compared with K means in cluster analysis of microarray. This new EST collection for the first time in Agave sisalana L. indicated an important step towards the identification of many molecular markers that aided the selective hybridization and biotechnological approaches to further improve Agave sisalana L. as drought resistant plant. Furthermore, the public availability of the novel up regulated cDNA clone sequences generated in this study will enable testing of the biological function of the genes represented and the development of transgenic plants in future.
The Islamic laws prevent us from the evil deeds. As well as, according to Shariah, it is permissible and sometimes even compulsory to save the believers from the actions that may lead them towards the banned activities. Therefore, the ruling of prohibition from these types of activities is called Sadd-e-Zaree’a. This is the principle extracted from the Quran and Sunnah. As Almighty Allah forbade the believers to say ‘Ra’ina’ because this word was used by Jews knowingly in a wrong manner with evil intentions, whereas, Muslims presented their requests by this same word in the highest court of The Holy Prophet (peace and blessings be upon him) for seeking easiness and relaxation in their concerned matters. As in Quran: O People who Believe, do not say (to the Prophet Mohammed- peace and blessings be upon him), " Ra’ina (Be considerate towards us)" but say, " Unzurna (Look mercifully upon us)", and listen attentively in the first place. [Baqarah 2: 104]. (To disrespect the Holy Prophet – peace and blessings be upon him – is blasphemy.) Another example by Hadith, in this regard, it is also evidenced by the good character of The Holy Prophet (peace and blessings be upon him) that sometimes He stopped himself intentionally from the permissible activities lest the common masses may involve in undesired activities on the basis of The Holy Prophet’s act. Concisely, keen consideration is required on rational basis when explaining the Shariah rulings of any matter. Otherwise, the beauty andbenefits of the Shariah may be lost. Consequently, to prevent from the future turmoil is actually the spirit of Sadd-e-Zaree’ah. As this is the actually basic need and prerequisite of Shariah. It saves and prevents its believers form expected turmoil. Therefore, if the risk of turmoil would trigger with committing the permissible activity it must be abandoned due to the risk of turmoil.
Embryonic stem cells are the pluripotent cells that act as a root of embryonic development to engender the specialized cells of the body. This property can be captured in vitro to cultivate indefinitely, providing a remarkable model to study early development and diseases. In vitro, mouse pluripotent embryonic stem cells (mESCs) exist in two different states, naïve and primed. The naïve state mimics in vivo inner cell mass of blastocyst with the pluripotency genes expressed more uniformly and have more developmental potential than the primed state. The primed state corresponds to a later embryonic developmental stage than the naïve state. Both states of cells express similar level of some key pluripotency genes but the signaling pathways that support their self-renewal and pluripotency are strikingly different. Mitochondrial metabolism, among others, is a major difference between the two states. The primed state has more mature mitochondria but doesn’t use them for ATP generation, which is generated solely through glycolysis. The naïve state however, has a bivalent metabolic state, i.e. uses both glycolysis and mitochondria to generate ATP. What role mitochondria play in these two states remains a very interesting and relevant question. Here in this study, we investigated the function of mitochondrial respiration in naïve mESCs by blocking the electron transport chain (ETC) with specific pharmacological inhibitors, Rotenone and Antimycin, and genetically with shRNAs against mitochondrial specific transcription factor A (TFAM) and a helicase, Twinkle. Both inhibitors and shRNAs resulted in blockade of cell proliferation and when released, the cells resumed self-renewal without affecting the pluripotency. The paused phenotype by ETC inhibition also appeared in the blastocysts cultured in vitro, which naturally die within 2-3 days of the development while Rotenone treatment extends survival of blastocysts for 3 more days. Moreover, the treated blastocysts were able to give live pups when injected back into surrogate mother, confirming a normal developmental potential. Upon ETC inhibition with either compounds or shTFAM, the mitochondrial mass and membrane potential increased and level of reactive oxygen species (ROS) decreased. The shRNAs against TFAM and Twinkle greatly reduced the mitochondrial DNA (mtDNA) copy number and expression of mtDNA encoded mRNA, leading to complete blockade of mitochondrial respiration. Paused mESCs stopped oxygen consumption and enhanced glycolysis instead to maintain a steady level of ATP generation, which is essential for the cells to be alive. Mechanistically, ETC inhibition induced pause is distinct from the pause induced through either mTOR or Myc inhibition. Neither pyruvate, aspartate nor nucleosides supplementation could rescue the self-renewal of mESCs which were shown to have the ability in other cell types upon ETC inhibition. The total non-targeted metabolomics showed that the ETC regulates the carbohydrates, proteins, lipid and nucleic acid metabolism. Specifically, lysosomal-related pathways are found to be significantly and commonly changed from both metabolomics and proteomic analyses. We could confirm the blockade of autophagy and lysosomal pathways in ETC inhibition-induced paused cells. These analyses suggest that an intimate link exists between mitochondria and lysosome in mESCs and highlights the potential important function of lysosomes for mESCs self-renewal. This work reported a new type of pause by ETC inhibition in mESCs and identified some unique metabolic roles of mitochondrial ETC for mESCs self-renewal. Furthermore this study first time shows the effect of Ursolic acid on mouse embryonic stem cells.