Physiological Mechanisms Of Heat Shock-induced Adaptation To Aluminum Toxicity In Wheat(Triticum Aestivum L.)

Acid soils cover 30-40%of the world's arable lands and around 50%of potentially arable land.In acid soils,the major factor limiting crop production is aluminum(Al)toxicity.Since more than 60%of the acid soils are located in subtropical and tropical area in the world,where plants are also exposed to heat stress.For plants,heat shock(HS,non-lethal temperature for short periods of time)can ultimately result in acquired thermotolerance and also improve its defenses against other abiotic stresses.While the effect of HS on other tresses has been well demonstrated,little is konown about the response of plant to HS and Al stress.Wheat is an important cereal crop and a staple food source globally,and about 5%of the wheat production worldwide is on acidic soils,and the late planting in the cropping system or early sowing in order to prolong the winter growth time of wheat results in wheat seedlings or wheat's reproductive stage coincides with high temperatures.In the present study,we use Al-tolerant wheat gentype Jian-864 and Al-sensitive wheat gentype Yangmai-5 to investigated the Al tolerance induced by the response of pior heat shock,the role of ascorbic acid(As A)and glutathione(GSH)in Al tolerance strategies,H₂O₂and NO mediated HS-induced Al tolerance,and cross talk between H₂O₂and NO to heat shock and Al stress.The main resluts are described as below:(1)Using two wheat genotypes differening in Al resistance,we focused on the effect of HS on Al tolerance.HS(45°C,3 h)significantly alleviated the inhibition of root elongation under 30?M Al stress,with the sensitive genotype Yangmai-5 having greater root elongation.Exposure to 3 h HS decreased levels of H₂O₂,O₂.^-,MDA and Evans blue uptake under Al stress in the root tips of both Yangmai-5 and Jian-864.Pretreatment with HS can reduce the ROS level by increasing antioxidant enymes(APX,CAT,SOD,POD)activity and expression and antioxidants level(As A and GSH).These resluts indicated that HS increased the activities of antioxidant enymes and antioxidants levels,scavenged the excessive ROS is improtant for wheat root cross tolerance to Al stress.(2)The effects of exogenous ascorbic acid(As A),the precursor of As A synthesis,L-Gal,and As A synthesis enzyme inhibitor lycorine were used to study the response of As A in HS and the role of HS-induced As A to Al stress.The content of As A increased in root tips after 1 h HS,and Jian-864 maintained higher As A level during 3 h HS.Pretreatment with As A and L-Gal significantly increased Al-induced As A and GSH level,antioxidant enymes and As A-GSH cycle related enyme activities,reduced ROS level;whereas,during HS,Lycorine counteracted the HS-induced As A production and protection against subsequent Al stress.These results suggest that HS-induced As A accumulation play an important role in protection against Al toxicity in wheat root tips through decresing H₂O₂accumulation and its regulation of the As A-GSH cycle.(3)The role of heat shock-induced glutathione(GSH)in heat shock and Al cross tolerance was investigated,using exogenous GSH and GSH synthesis enzyme inhibitor BSO.Exposure to HS caused GSH accmulation after 1 h HS in both Yangmai-5 and Jian-864.Exogenous application of GSH increased As A/DHA and GSH/GSSG ratio to Al stress,while reduced GSH synthesis by BSO in HS reversed this increament.Exogenous GSH application significantly reduced Al-induced H₂O₂accumulation,increased Al-induced the ratio of As A/DHA and GSH/GSSG and As A-GSH cycle related antioxidant enyme activities;whereas reduced GSH synthesis by BSO in HS promoted the Al-induced H₂O₂ accumulation and protection against subsequent Al stress.These results suggest that HS-induced GSH accumulation play an important role in wheat adapation to Al toxicity.(4)We used hydrogen peroxide(H₂O₂)donor and inhibitors to investigate the possible role of H₂O₂ signal in plant responses to HS and Al cross stress.HS could induce the rapid production of H₂O₂ and activity of APX,CAT,POD,GR enyme in Yangmai-5 and Jian-864 root tips,the Al-tolerant Jian-864 respond in a more sensitive manner,showed an H₂O₂ brust at 45 min after HS,with increased activity of APX and CAT after 2 h HS.Pretreatment with H₂O₂ donor alleviated Al-induced root inhibition,while H₂O₂scavenger(DMTU)or NADPH oxidase inhibitor(DPI)treament in HS exacerbated Al-induced H₂O₂accmulation,reduced SOD,CAT,POD,GR enyme activity,this phenomenon occurs even more greater in Yangmai-5.In this study,quantitative real-time PCR(q RT-PCR)revealed that 3 h HS induced HSP70 and HSP26expression,and Yangmai-5 had higher expression than Jian-864.Results imply that pior HS hastened HSP70 and HSP26 expressioning in the presence of Al stress,while in Jian-864 HSP70 and HSP26 expression was observed ealier than Yangmai-5.However,the HS-induced Al tolerance was counteracted by inhibiting the expression of HSP70in wheat root tip,and induced more H₂O₂accumulation.These results suggest that HS-induced H₂O₂ and HSPs were involved in response to Al torlance.Accumulation of H₂O₂during HS signals the increase in Al-induced antioxidant enymes activities,prevents wheat from Al-caused root inhibiton.(5)To illustrate the corss talk of H₂O₂and nitric oxide(NO)in HS and Al corss tolerance of wheat,we foucused on the effect of donors and inhibitors of H₂O₂ and NO production in HS and Al corss stress.HS induced NO burst in both root tips of Yangmai-5 and Jian-864,while the Al-tolerant genotype Jian-864 showed more rapid NO burst at 2 h after HS.HS priming accelerated the NO production in Al stress,and can be detected in Yangmai-5 after 3 h Al treatment.Exogenous application of NO alleviated the Al-induced root inhibiton,whereas NO scavenger(c PTIO)and inhibitors in HS counteracted it.HS induced the activity of nitrate reductase(NR),whereas showed no significant change on nitric oxide synthase(NOS)activity.Treatment of wheat root with H₂O₂ induced NR activity and early NO burst;while H₂O₂ scavenger(DMTU)or inhibitor(DPI)treament during HS reduced the NO proudction and NR activity.HS-induced H₂O₂can mediated the early NO synthesis through NR pathway.Nitric oxide donor(SNP)in HS counteracted H2O2 scavenger effect on Al-induced root elongation;while H2O2 donor did not block NO effect.Pretreatment with H₂O₂ donor induced antioxidant enzyme activities under Al stress in Yangmai-5 and Jian-864,but suppressed by NO scavenger(c PTIO)or NR inhibitor in HS and Al cross stress.These date indicated that H₂O₂ acts upstream of NO through NR in HS,improves the antioxidant capacity which requires enhanced Al tolerance.In summary,heat shock can alleviate the adverse effects of wheat root under Al stress.Heat shock-induced ASA and GSH improved the activity of As A-GSH cycle related antioxidant enyme and antioxidant capacity,and reduce the accumulation of H₂O₂ in root tips,thus alleviated the root inhibition of aluminum toxicity.HS induced both H₂O₂ and NO in wheat root tips.HS-induced H₂O₂ reduced antioxidant enyme activity,and with HSPs synergistically reduce the accumulation of H₂O₂ in root tips under Al stress.Further results showed that H₂O₂ acts upstream of NO through NR under HS,improves the Al-induced antioxidant capacity which alleviated Al toxicity.