Chemical Engineering of Plants for Salt Tolerance

Salinity is one of the serious threats to global agriculture that threatens human food security. To tackle the problem, there were and still are quite several scientific efforts in place. Hence, in this dissertation, we used chemical and genetic engineering approaches to unravel the sophisticated knot of the problem using a Trojan peptoid called plant PeptoQ and overexpression of OsOPR7 gene in non-transformed WT tobacco BY-2 cells. First, plant PeptoQ that can be used to target a functional cargo (a rhodamine-labelled semiquinone peptoid as mimetic of coenzyme Q10) into mitochondria of tobacco BY-2 cells was characterized with regard to its cellular uptake and potential cytotoxicity. We found that the uptake is specific for mitochondria, rapid, dose-dependent, and requires both clathrin-mediated and clathrin-independent endocytosis, as well as actin filaments, while microtubules seem to be dispensable. Viability of the treated cells was not affected, and they showed better survival under salt stress, a condition that perturbs oxidative homeostasis in mitochondria. Using double labelling with appropriate fluorescent markers, we showed that targeting of this Trojan Peptoid to the mitochondria is not based on a passage through the plasma membrane (as thought hitherto), but on import via endocytotic vesicles and subsequent accumulation of the positively charged side chains at the negatively charged inner mitochondrial membrane. ... mehrSecond, the effects of pretreatment with plant PeptoQ and OsOPR7 overexpression, on salt stress induced detrimental effects in WT BY-2 cells were investigated. In general, both pretreatment with plant PeptoQ and overexpression of OsOPR7 in WT BY-2 cells, mitigated salt stress induced deleterious effects more or less in a similar manner. Cell expansion and cell viability were fully and partially compensated at moderate (75 mM NaCl) and high (150 mM NaCl) salt stress respectively by peptoid treatment and OsOPR7 overexpression. However, even if, the detrimental effects of salt stress on cell division (proliferation) were mitigated by both approaches, it was more sensitive as compared to cell expansion and viability. Furthermore, they significantly ameliorated doubling time, and effectively suppressed salt stress induced increase in MDA and superoxide levels in WT BY-2 cells. However, both approaches had no effect on hydrogen peroxide level. Plant PeptoQ pretreatment and OsOPR7 overexpression lead to increased SOD activity but decreased Mn-SOD transcript induction. However, they had no effect on catalase (CAT) activity. Except SOS1, NAC and OPR7 genes , other salt-related genes such as ion channels (NHX1 and SKOR), regulators for ion channels (SOS3 and SLT1) and jasmonate related gene (JAZ3) did not show strong transcript modulation in response to salinity, plant PeptoQ treatment and OsOPR7 overexpression. Similarly, even if, ionic balance was strongly perturbed by salt stress, both plant PeptoQ treatment and OsOPR7 overexpression had no mitigatory role at all. On the other hand, pretreatment of salt stressed WT and OsOPR7 overexpressor (OE) BY-2 cells with plant PeptoQ, caused increased OPDA level; however, it had no significant effect on JA-Ile level. It lead to a significant shift of the biosynthetic pathway from JA-Ile to OPDA, and this channeling of the pathway towards OPDA was significantly more accentuated for moderate salt stress (75 mM NaCl) but it faded as it proceeds to high salt stress (150 mM NaCl). Both plant PeptoQ pretreatment and OsOPR7 overexpression conferred salt tolerance to the non-transformed WT BY-2 cells by mitigating the salt stress induced detrimental effects effectively and efficiently.