Abstract
The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.
Highlights
There are only two species of flowering plants in the Antarctic Peninsula, Antarctic hairgrass (Deschampsia antarctica) and Antarctic pearlwort (Colobanthus quitensis)
A single signal intensity corresponding to a region within ~45 kDa) was detected from transformed yeast cells with the p426GPD-DaMDHAR construct (DM), whereas there were no intensity in wild-type (WT) yeast cells with vector alone (Figure 1B)
The spotting assay demonstrated that DaMDHAR-expressing yeast (DM) cells had acquired tolerance to freezing and thawing (FT) stress compared to WT cells, dependent onactqhueirceydclteoloefraFnTcesttoreFsTs fsrtroemss FcoTm1 ptoarFeTd6t,oaWlthTocuelglsh, dsterpeesnsdreensitsotanntchee dcyecclreeoafseFdT wstriethssprogrfersosmioFnTi1nttohFeTF6T, aclythcloeu. gThhsetrreesws aressaisdtainstciendctecdriefafesreednwceitihnpbrootghreDssMionanindtWheTFTcecllysculen. der F1CTTtgh)rhs.eoetrTwrreeeotbswhcseoakicnsniongfanienrdtdmioiciststdiitonwihfncfeaetsssrdeedpinfreefecseresprfueouintrlnetmcsde,teehigdnrer.nrboIeoownrtbhmtthheDaienklMggicnroaonennwtoddictidhtsWi-iowfkTnfieasncrsee(eFltpnliiscegcsureufnarousdersnem1ardCyFee,)dTrD
Summary
There are only two species of flowering plants in the Antarctic Peninsula, Antarctic hairgrass (Deschampsia antarctica) and Antarctic pearlwort (Colobanthus quitensis). During their entire life cycle, Antarctic plants are exposed to multiple abiotic stresses, including extreme temperatures, varying oxygen concentrations, water and nutrient deficiency, extremely short growing seasons, common summer frosts, strong winds, low light quality, and photoperiod changes [1,2,3,4]. Freezing temperatures, can dramatically affect plants from the cellular level to ecosystem scales [5]. Various cellular changes induced by low-temperature lead to the excess accumulation of toxic compounds, reactive oxygen species (ROS) [2,10]. A considerable amount of research has been conducted to explore the correlation between ROS scavenging and plant stress tolerance under extreme temperatures [13,14,15]
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