| Abiotic stresses can adversely affect the growth and development of crops.Among the abiotic stresses,drought is one of the main factors limiting crop quantity and quality.It results in abnormal metabolism,reduced growth,and ultimately plant death.The impact of drought stress on crops can be minimized and bring tolerance by applying various techniques,including breeding techniques,application of exogenous phytohormones,various chemical compounds,and osmoprotectants to plants or seeds,and drought-hardening.Drought-hardening involves the utilization of reduced or partial irrigation of seedlings to pre-condition the seedlings to the drought stress.Drought-hardening is a feasible and widely used method in tobacco seedlings cultivation.It has gained extensive interests due to its role in improving drought tolerance.In this present study,the floating breeding system was used for the tobacco seedlings under control conditions in a growth room.Three tobacco varieties NC55(N),Honghuadajinyuan(H),and Yunyan-100(Y),were selected to study their physiological,anatomical,biochemical,and molecular responses under drought stress to evaluate their differential responses and drought tolerance and adaptation mechanisms.On this basis,H and Y cultivars were selected,and different drought-hardening treatments were applied.The physiological,biochemical,and key gene expression analyses were used to explore the drought tolerance mechanisms due to drought-hardening.The main analysis parameters include plant biomass,leaf water potential,chlorophyll content,chlorophyll fluorescence and multicolor fluorescence parameters,antioxidant enzyme activities,osmoregulation substance contents,analysis of important metabolic pathways,and expression analysis of key genes.The main results are as follows:1.Biomass and chlorophyll content: H maintained higher growth and less reduction in fresh plant weight,dry weight,and chlorophyll content as compared with N and Y.The fresh plant weight of N,H,and Y drought-stressed seedlings was reduced by 29%,18%,and 23% and dry weight by 23%,7%,and 10%,respectively,compared with their controls.Similarly,21%,19%,and 21% reduction were observed in the chlorophyll content of N,H,and Y,respectively,in response to drought stress.2.Chlorophyll fluorescence parameter: Drought stress seriously affects the photosynthetic efficiency.The chlorophyll fluorescence parameters showed that the PSII quantum yield decreased 13%,13%,and 17% in N,H,and Y under drought stress.In comparison,the steady-state non-photochemical quenching was increased by 24%,19%,and 40%,respectively,at 72 hours-time points,which represent damage to the photosynthetic machinery.So H was the least affected one among the varieties.Similarly,the multicolor fluorescence parameters,blue and green fluorescence(BF and GF),were significantly increased after 72 hours of drought stress in N,H,and Y with a percent increase of 55% and 57%,42% and 40%,35%,and 33%,respectively.This increment in BF and GF might occur due to the higher content of phenolic compounds and accumulation of intermediary compounds during chlorophyll breakdown,which was an adaptation mechanism of the photosynthetic apparatus to drought stress.3.The antioxidant enzyme activity and osmoregulation substance contents: The three varieties showed differential adaptive responses triggered by drought stress.N accumulated 191% proline content,while H and Y accumulated 331% and 228% proline content upon 72 hours of drought stress in comparison to well-watered seedlings,respectively.Likewise,10%,13%,and 25% increment was observed in the soluble sugars content of N,H,and Y,respectively,in response to 72 hours of drought stress.Drought stress upon 72 hours' time point enhanced the activities of peroxide dismutase(POD)by 21%,60%,and 57%,ascorbate peroxidase(APX)by 29%,40%,and 17%,and glutathione reductase(GR)by 9%,20%,and 5% in N,H,and Y varieties,respectively.Similarly,the superoxide dismutase(SOD)and catalase(CAT)activity were elevated by 55% and 11%,31%,and 41% in N and H,respectively,by 72 hours of drought stress.Finally,the non-enzymatic antioxidants like ascorbic acid(AsA)and reduced glutathione(GSH)were also enhanced by drought stress for 72 hours by 19%,7%,13% and 45%,23%,47% in N,H,and Y,accordingly.This resulted in mitigating the negative impacts of reactive oxygen species and malondialdehyde and ultimately helped in bringing tolerance and adaptation to drought stress.It was concluded that H was the least affected one among three varieties.4.Analysis of the transcriptomics and metabolic pathways: The transcriptomic analysis showed that H resulted in more number of differentially expressed genes(DEG)expressed,followed by Y and N in response to drought stress.Overall,1020 DEGs were expressed among the three varieties,while 567,1061,and 727 DEGs were expressed individually in N,H,and Y during drought stress,respectively.The differential genes expression among N,H,and Y were enriched in the functions of(1)Plant hormone signal transduction pathway,i.e.,PYL,PP2 C,SnRK2,AHP,and A-ARR were the key and most enriched genes.(2)Starch and sucrose metabolism pathways,i.e.,UGP,SUS,BAM,HXK,and glgc were the key upregulated and mostly enriched genes.(3)Arginine and proline metabolism pathways,i.e.,P5 CS,PDH,OAT,and ARG1,were the most enriched and key genes.Compared to N and Y,the differentially expressed genes of H displayed enhanced expression in the corresponding pathways under drought stress,which enhanced the understanding of the molecular mechanisms through the networks of various metabolic pathways mediating drought stress tolerance and adaptation in tobacco seedlings.5.Differential response mechanism to drought-hardening for different varieties: In the second experiment,the results revealed and presented a complete framework of drought tolerance due to drought-hardening at physiological,biochemical,and genes expression levels of the two tobacco varieties in response to drought stress.The results showed that drought-hardening treatments reduced the growth of the seedlings as plant fresh weight(FW)and dry weight(DW).The FW of H was reduced by 11% and 16%,the DW was reduced by 16% and 24% for T1 and T2 compared to CK,respectively,similarly,the FW and DW of Y were reduced by 26% and 16%,and 36% and 24% for T1 and T2,respectively,in response to drought stress.Drought-hardening improved water uptake by decreasing leaf water potential(LWP),the LWP was decreased 24% in HT1,11% in HT2 under drought stress.Similarly,the LWP was also decreased by 12% for YT1 and 10% for YT2 in response to drought stress when compared to their respective controls,which are the adaptive mechanisms.6.The drought-hardening showed differential effects on different varieties in PSII quantum yield(QY_Lss)and the ratio of fluorescence decline(Rfd_Lss): Drought-hardening caused no significant changes in PSII quantum yield(QY_Lss)and the ratio of fluorescence decline(Rfd_Lss)of H variety.The significant changes were observed in Y variety under drought stress.The droughthardening treatments T1 and T3 showed 7% and 3% decline in QY_Lss of Y,respectively.In this way 9% and 6% reduction was observed in Rfd_Lss of Y,respectively,in comparison to control.It was also observed that drought-hardening,especially T1,showed a significant rise in BF under drought stress in H seedlings in comparison with control.In contrast,drought-hardening had no effect on BF in Y seedlings.The rise in BF in H might be due to the breakdown of the chlorophyll into intermediary compounds and the accumulation of phenolic compounds.Thus,the phenolic compounds had a role in mitigating the adverse impacts of drought stress and helped in bringing drought tolerance and adaptation.7.Drought-hardening affected the enzymatic antioxidant defense system and osmoregulation substance contents,but showed differential responses between H and Y: The activity of SOD was enhanced by 16% in HT2.The activity of SOD was also enhanced in Y as 16%,12%,and 38% increment in T1,T2,and T3,respectively,in comparison with control under drought stress.The activity of POD also showed a 22% and 13% increase in HT1 and HT2,accordingly,compared with the control.21%,4%,and 11% increment was also witnessed in YT1,YT2,and YT3,respectively,in comparison with control under drought stress.Similarly,the APX activity was also enhanced by drought-hardening in H as 136%,32%,and 26% increment in T1,T2,and T3,respectively,in comparison with control under drought stress.In Y cultivar,only 37% increment was observed in T1,while T2 and T3 were found similar to control.The AsA and GSH contents were increased in T1 and T2 of H in comparison to control under drought stress.Similarly,the AsA content was enhanced in YT1 by 16% and YT2 by 43%.While a decline was observed in the GSH content in YT1 by 21% and YT2 by 3% in comparison with control under drought stress.Proline content was augmented by 77% and 23% in HT1 and HT2,accordingly,in comparison with control under drought stress,while only 32% of increment was observed only in YT1 in comparison to control under drought stress.Soluble sugars(SS)contents were increased by 69% and 107% in HT1 and HT2,while 37% and 125% in YT1 and YT2,respectively,in comparison to control under drought stress.It was concluded that drought-hardening improved the antioxidant plant defense system and elevated the amounts of proline and SS,which helped in mitigating the negative impacts of oxidative damage and brought drought tolerance and adaptation to both H and Y varieties in response to drought stress.8.Molecular mechanism of Drought-resistance: The drought-hardening also induced drought tolerance in both varieties via the expression of various stress-responsive genes by triggering(1)the biosynthesis pathways of proline(P5CS1)and polyamines(ADC2);(2)ABA-dependent(SnRK2,AREB1)and independent(DREB2B)signaling pathways;and(3)higher expression of antioxidant defense-related genes(CAT,APX1,GR2)in response to drought stress.Drought-hardening improved the expression of the various categorized genes in both varieties,H and Y,in response to drought stress,thus confer stress in bringing drought tolerance and adaptation. |
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