Comparative Physio-biochemical And Molecular Analysis Of Interaction Between Herbicides And Salinity In Rice And Barnyard Grass

Plants are often subjected to various abiotic and biotic stresses,such as extreme temperatures,drought,salinity,cold,pathogen infection,herbivore attack,herbicides,toxic metal,nutrient deficiency and exposure to other chemicals throughout their life cycles.Combined or simultaneous occurrence of these stresses like salinity and herbicides have shown multifaceted plant responses,because stress responses under combined stress conditions are governed by different signaling pathways that may interact and inhibits one another.Modulation of antioxidant defense system,osmolyte synthesis,photosynthetic boost-up,hormonal interaction and transcription factors are the main metabolic and signaling cascade involved in the response of plants to stress combination.The herbicide butachlor is widely used in paddy field,while 2,4-dichlorophenoxyacetic acid(2,4-D)is an old and also widely used herbicide in cropping system.In present research investigations,we tried to evaluate how rice plant response under widely applied herbicides and how commonly used practice of herbicide application interact with abiotic stresses like salinity in rice and Echinochloa crusgalli(barnyard grass).1.We have investigated how butachlor-induced toxic effects in two rice cultivars,Zhejing 88 and Xiushui 134.We found significant decrease in growth rate,pigment concentration,and chlorophyll fluorescence and alternations in chloroplast structures were observed in both rice cultivars at higher treatment levels.The malfunctioning of antioxidant enzymes,the higher uptake of butachlor,and the downregulation of the putative fatty acid elongase gene suggested severe oxidative damage,which was also indicated by enhanced reactive oxygen species formation(H2O2,O2-),lipid peroxidation,and electrolyte leakage and reduced total solubleprotein levels in cultivar ZJ 88 than in cultivar XS 134.However,higher proline accumulation and pyrroline-5-carboxylate synthase and GABA-transaminase transcript levels in ZJ 88 suggested their ameliorative role under butachlor-induced oxidative stress in the sensitive cultivar.In contrast,the stimulated activation of antioxidant enzymes,glutathione,soluble sugars,and phenolics played a vital role in protection against butachlor stress in cultivar XS 134.Thus,butachlor-induced oxidative stress tolerance in the resistant cultivar(XS 134)is related to the enhanced activation of different isoforms of antioxidant enzymes and a reduced uptake of butachlor compared with the sensitive cultivar(ZJ 88).The present work provides valuable insights into butachlor-induced toxicity in rice plants.We also study the interaction of salinity with butachlor,which showed additive stress responses.2.Effects of individual and combined stress of herbicide 2,4-D and salinity(NaCI)on Oryza sativa cultivars(ZJ 88 and XS 134)were investigated.Both herbicide and saline stress affected the plant growth differentially and produced oxidative stress in rice cultivars.Interestingly,the combination of herbicide and salinity showed a significant protection to both rice cultivars by reducing ROS(H2O2,O2-)and lipid peroxidation through modulation of enzymatic(SOD,POD,CAT and APX)and non-enzymatic(TSP,sugars,phenolic and proline)antioxidants.In addition,active regulation of transcript levels of genes encoding Na+ and K+ transporter proteins reduced sodium and enhanced potassium accumulation under combined stress,resulted a better growth and ionic homeostasis in both rice cultivars.The production of ABA and IAA was significantly higher in cultivar XS 134 compared with cultivar ZJ 88 under control conditions.However,combined herbicide and saline stress enhanced the accumulation of phytohormones(IAA and ABA)and transcription of ethylene in cultivar ZJ 88,which might be one of the factors responsible for poor salt tolerance in sensitive cultivar.These findings indicated that herbicide application under saline stress confers tolerance to salinity in rice cultivars,likely by reducing oxidative damage,modulating mineral absorption,upgradation of antioxidant defense and by dynamic regulation of key genes involved in Na+ and K+ homeostasis in plants.3.We have investigated the effects of recommended dose of 2,4-D on growth,oxidative damage,antioxidant defense,regulation of cation transporter genes and anatomical changes in the roots of rice cultivars XS 134(salt resistant)and ZJ 88(salt sensitive)were investigated under different levels of saline stress.Individual treatments of saline stress and 2,4-D application induced oxidative damage as evidenced by decreased root growth,enhanced ROS production,more membrane damage and Na+ accumulation in sensitive cultivar compared with the tolerant cultivar.Conversely,combined treatments of 2,4-D and saline stress significantly alleviated the growth inhibition and oxidative stress in roots of rice cultivars by modulating lignin and callose deposition,redox states of AsA,GSH,and related enzyme activities involved in the antioxidant defense system.The expression analysis of nine cation transporter genes showed altered and differential gene expression in salt-stressed roots of sensitive and resistant cultivars.Together,these results suggest that 2,4-D differentially regulates the Na+ and K+ levels,ROS production,antioxidant defense,anatomical changes and cation transporters/genes in roots of rice cultivars.4.E.crusgalli is a most noxious weed that affects rice and many other crops globally.Sometime the efficacy/performance of herbicide is compromised due to the environmental factors.We have explored the interaction of salinity and herbicide 2,4-D on weed control because effects of saline stress on weed growth and herbicide activity are important for sustainable weed management.Previously,salinity was reported to enhance plant' s tolerance to several other stresses.The aim of the present work was to investigate whether saline stress enhances the herbicide tolerance in the E.crusgalli by evaluating the individual and combined interactive effect of salinity(4 dS m-1 and 8 dS m-1)and recommended dose of 2,4-dichlorophenoxyacetic acid(2,4-D)on growth,ROS production,antioxidant activities,chloroplast ultrastructure,hormonal regulation and Na+and K+ accumulation in E.crusgalli plants.The results showed that individual treatment of 2,4-D reduced the chlorophyll contents,photosynthetic capacity,enhanced MDA,electrolyte leakage,ROS production(H2O2,O2-)and inhibited the activities of ROS scavenging enzymes.Severe saline stress(8 dS m-1)followed by mild saline stress treatment(4 dS m-1)also reduces the E.crusgalli growth,but have less impact as compared with the 2,4-D treatment alone.Surprisingly,under combined treatments,the phytotoxic effect of 2,4-D was reduced on saline stressed E.crusgalli plants,especially under mild saline + 2,4-D treatment.This stimulated growth of E.crusgalli is related to the higher activities of enzymatic and non-enzymatic antioxidants and dynamic regulation of IAA,ABA in combined stressed plants.This shows that 4-D efficacy was affected by salinity in a stress intensity-dependent manner,and suggesting that the presence of salt tolerant E.crusgalli in paddy fields requires special weeds control measures for sustainable crop production under saline environment.5.To explore the transcriptional responses of rice cultivars to individual and combined stresses,we performed transcriptome sequencing of seedlings under normal conditions or subjected to herbicide treatment,saline stress treatment,or their combination.We found that under herbicide treatment,63%of transcripts were upregulated in resistant cultivar,while 56%of transcripts were upregulated in sensitive cultivar.A strong induction in Cytochrome P450 and Glutathione transcripts upregulation was observed in resistant cultivar under the herbicide 2,4-D treatment.Under saline stress conditions,Genes related to several cellular functions such as membrane transport,osmoprotection,molecular chaperoning,redox metabolism or protein synthesis were differentially expressed in both cultivars,which may explain their tolerance against salinity.Under combined stress conditions,it was observed that 2,4-D effects the regulation of ion transporters,IAA,GA,ABA and brassinosteriods signal transduction proteins differentially in resistant and sensitive cultivars.The RNA sequencing data revealed that dynamic regulation of underling process in resistant cultivar under both sensitive and resistant cultivar provides new insights into the molecular action of salinity and 2,4-D signaling to enhance plant tolerance to combined stresses,and elucidates the transcriptional network of different adaptations strategies under individual and combined stress treatments in contrasting rice cultivars.