Regulation Mechanism Of Melatonin And Nitric Oxide On Abiotic Stress Tolerance And Branch Growth In Tomato

Tomato(Solanum lycopersicum L.)is one of the mostly consumed vegetable crops in the world.In tomato production,various abiotic stresses,abuse of pesticides,and pesticide residues seriously limit the yield and quality of tomato fruits.Braching is considered as one of determinant factors affecting plant architecture.Appropriate plant architecture can coordinate plant growth and development,and thus benefit the final yield.Therefore,it is of great significance to study the regulation mechanism underlying plant architecture and abiotic stress tolerance of tomato,as well as the related cultivation technologies.The effects of melatonin(MEL)and nitric oxide(NO)on abiotic stress tolerance and plant architecture of tomato were investigated.The results were as follows:1.Enhancement of tomato tolerance to saline-alkali stress through DREB1?-IAA3 singaliing cascade by MEL.When MEL was applied to tomato seedlings under saline-alkali stress,stress-related parameters were significantly reduced.RNA-seq was performed for tomato roots under MEL,NaHCO₃,and MEL+NaHCO₃ stresses,respectively.Six transcription factors were identified to respond to both MEL and saline-alkali stresses simultaneously.Using virus-induced gene silencing(VIGS)system,we observed that knockdown of DREB1? and IAA3 expression significantly decreased MEL-induced salinity tolerance in tomato,while silencing of the other four transcription factors had no similar effects.Interaction analysis showed that the decreased IAA3 expression did not affect the expression of DREB1?,but inhibition of DREB1?significantly reduced the expression of IAA3.DREB1?responded to saline-alkali stress earlier than IAA3.Yeast one hybridization showed that DREB1?bound to the promoter region of IAA3 and activated IAA3 expression.The physiological mechnisem was furher explored for MEL-induced enhancement of saline-alkali tolerance via DREB1? and IAA3 in tomato plants.We found that the lowered expression of DREB1? and IAA3 significantly reduced the expression of ion transport genes that were induced by MEL,the accumulation of organic acids,the stomatal movement,the water retention,and the expression of antioxidant enzymes in roots.This evidence suggests that DREB1?and IAA3 might be key downstream genes in MEL-inviolved tomato tolerance to saline-alkali stress,and the MEL-DREB1?-IAA3signalling cascade plays multiple roles in balancing plant growth and stress tolerance.2.MEL reduces the toxicity and residue of carbendazim in tomatoThe application of 1 m M MBC interfered with photosynthetic electron transport(F_v/F_m),decreased chlorophyll content and net photosynthetic rate(P_N),and increased the content of malonaldehyde(MDA)in tomato plants.The damages of MBC to tomato leaves were significantly relieved by 0.1 to 1.5?M MEL,and the best was observed at 0.5?M,possibly duo to the better degradation of MBC under 0.5?M MEL treatment than other concentration levels.Compared with WT,overexpression of the MEL biosynthesis gene caffeic acid O-methyltransferase 1(COMT1)significantly increased the content of MEL and reduced the pesticide residues in tomato.MEL significantly increased the activities of SOD and other antioxidant enzymes in tomato plants under MBC stress.In addition,MEL application promoted the accumulation of total ascorbic acid,the transition from DHA to ASA and the accumulation of GSH and GSSG,and enhanced the synthesis and recycling of endogenous GSH in MBC-stressed tomato.The activities of peroxidase(POD)and glutathione S-transferase(GST)were significantly increased,and more GSH was allocated into GST-mediated heterotoxic degradation.Grafting expreiment showed that,on one hand,the MEL content was increased,and on the other hand,the toxicity and residue of MBC were lowered in WT plants,when the COMT1-overexpressing tomato(COMT1-OE)was used as rootstock.3.MEL coordinates NO and ROS to confer broad-spectrum tolerance in tomatoExogenous application of MEL at 1,10,100 and 1000?M was able to lessen the detrimental effects of drought,high and low temperature stresses on tomato seedlings,and the best alleviatory performance was observed for MEL treatment at 100?M.Upon 100?M MEL treatment,the expression of CDPKs,MAPKs,WRKYs and other stress response genes were induced,the content of SNOS and NO was reduced,and the activity of NADPH oxidase(RBOH)and ROS content were increased in tomato plants.Correlation analysis revealed that MEL content was negatively correlated with SNOs content,while was positively correlated with RBOH activity.We further found that RBOH denitrosylation was stimulated by MEL treatment.Inhibition of RBOH activity or reduction of ROS level by pharmacological and genetic approaches significantly inhibited MEL-induced tomato tolerance to abiotic stresses,and reduced the expression of stress-responsive genes and the activities of antioxidant enzymes.Altogether MEL promoted the denitrosylation of RBOH and enhanced the RBOH activity and intercellular ROS content via elimination of intracellular NO in tomato plants,,thus conferring the broad-spectrum tolerance.4.NO promotes branch growth of tomato via balancing IAA and CKsGSNOR functioned as a negative regulator for endogenous NO synthesis and branch growth in tomato.Inhibition of GSNOR expression reduced the expressions of FZY,AUX1and PIN1 in apical buds,promoted the expressions of AUX1 and PIN1 in lateral branches,and diminished the accumulation of IAA in apical buds and lateral branches.Also the decresed GSNOR expression stimulated the expression of genes related to CK synthesis in roots.Five grafting combinations,WT/WT/WT,RNAi/RNAi/RNAi,RNAi/WT/WT,WT/RNAi/WT and WT/WT/RNAi,were obtained by grafting RNAi materials as apical buds,middle stem segments,and roots,respectively.The growth performace of lateral branches was ordered as RNAi/RNAi/RNAi>WT/RNAi/WT>RNAi/WT/WT>WT/WT/RNAi>WT/WT/WT.Considering both the content of IAA/CKs and the expression of genes related to their synthesis and signalling,we speculate that the decreased GSNOR expression could generate different outcomes in a tisuues-dependent manner:CK synthesis in roots,and IAA outflow and CK activation in lateral branches are promoted,while the downward transport of IAA in apical buds is inhibited,thus stimulating the growth of lateral branches in tomato plants.These results were consistent with the previous observations that exogenous application of 6-benzylaminopurine(6-BA)or apical bud removal can promote lateral branch growth,possibly due to the enhanced expression of ARR5 and CYCD3.1,as well as the decreaseed expression of DRM1,AD1 and BRC1.At the same time,we investigated the NO signal transduction pathway during lateral branch growth,and found that nitrate reductase(NR)acted as a NO-producing enzyme at the early stage of lateral branch growth.The generation of NO inhibited GSNOR activity,and further benefited NO accumulation and CK signal transduction,thus promoting lateral branch growth.