Mechanisms And Functions Of ATG6-dependent Autophagy In Nitrogen Stress Of Tomato

Nitrogen(N)is one of the most important mineral elements in plant growth and development.The deficiency of N and the low-efficiency of N affect the yield and quality of crops and thus recognized as a widespread problem across the world.Autophagy has been demonstrated to facilitate nutrient recycling and remobilization through delivering intracellular materials to the vacuole for degradation in plants under nutrient starvation.However,the role of autophagy in N transportation and utilization and its upstream and downstream regulatory networks are unknown.Here,we focus on the key autophagy-related gene ATG6 in tomato(Solanum lycopersicum),explore its upstream and downstream regulation mechanisms,and clarify the role of ATG6-dependent autophagy in low-N(LN)stress of tomato.The main results are as follows:1.We have found that autophagy played an important role under LN stress in tomato.Tomato atg6,atg10 and atg18a mutants displayed hypersensitivity to N deficiency with increased leaf chlorosis,decreased the plant biomass and chlorophyll content compared with wild-type(WT)seedlings.The N content was decreased more in atg6,atg10 and atg18a seedlings compared with WT under LN conditions.Compared with stronger induction of autophagy in WT leaves and roots,the autophagic activity was inhibited in atg6,atg10 and atg18a mutants under LN stress.These results suggest that the autophagy in tomato leaves and roots could be induced by LN stress,and autophagy is essential for N uptake and seedling growth in tomato under LN stress.2.We have found ATG6-dependent autophagy was involved in the N absorption and assimilation under LN stress in tomato.Total biomass and N accumulation were exacerbated decrease in atg6 mutants but were alleviated in ATG6OE plants under LN stress.Moreover,the expression of NRT1.1 and NRT2.1 were up-regulated in ATG6OE roots promoting an increase of the nitrogen content in ATG6OE under LN stress.Meanwhile,ATG6-dependent autophagy enhanced nitrogen assimilation efficiency and protein production in tomato leaves.The activity and expression of nitrate reductase(NR)and nitrite reductase(Ni R)were severe blocked in atg6mutants but were lightly compromised in ATG6OE plants under LN stress.These results indicate ATG6-dependent autophagy promotes N absorption in tomato roots and enhances N assimilation in tomato leaves under LN stress.3.We have found ATG6-dependent autophagy regulated photosynthetic CO₂assimilation in response to LN stress.Total C content,photosynthetic rates and photosynthesis-related proteins were greatly decreased in atg6 mutants,but these parameters were alleviated in ATG6OE plants under LN stress.Through grafting experiments,we further verified that photosynthetic CO₂ assimilation efficiency was inhibited in grafted plants using atg6 as rootstock or scion.These results suggest ATG6-dependent autophagy regulates N uptake and assimilation together with carbon assimilation,besides nutrient recycling and remobilization in tomato plants facing LN stress.4.We have demonstrated that BZR1 mediated brassinosteroid(BR)-induced autophagy and N starvation in tomato.The formation of autophagosomes and the degradation of ubiquitinated proteins were triggered under N starvation by exogenous application of BR.And the BR-activated transcription factor brassinazole-resistant 1(BZR1)was involved in BR-induced autophagy.The accumulation of the dephosphorylated BZR1 was induced in BZR1OE plants by exogenous application of BR.Exogenous BR also promoted autophagy formation and ATGs transcription in BZR1OE plants,while these impacts were compromised in BZR1-silenced plants.Moreover,BZR1 could directly bind to the promoters of ATG2 and ATG6.In ATG2-and ATG6-silenced plants,the BR-induced autophagy formation was decreased.In additon,the chlorosis and chlorophyll degradation were deteriorated in both BZR1-silenced plants and the BR biosynthetic mutant d^{^im} but were relieved in BZR1OE and bzr1-1DOE plants under N starvation.Taken together,these results suggest that BR BR activates BZR1-mediated downstream signal transduction,leading to the dephosphorylation of BZR1 into the nucleus,thereby regulating the expression of ATGs and inducing autophagy.Thus BZR1-dependent BR signaling pathway induces the formation of autophagosomen and plays a crucial role in response to N starvation in tomato.5.We have studied the critical role of SnRK1.1 in response to LN stress for tomato.We identified interactions between tomato SnRK1.1 and ATGs,and confirmed that tomato SnRK1.1 phosphorylated ATG6 in vitro.Total biomass decerase and chlorophyll degradation were exacerbated in snrk1.1 mutants but were alleviated in SnRK1.1OE plants under LN stress.Compared with stronger induction of autophagy in WT leaves,the autophagic activity was inhibited in snrk1.1 mutants under LN stress,but accelerated in SnRK1.1OE plants.In addition,SnRK1.1 mediated the formation of ATG6-dependent autophagy under LN stress,which was inhibited in the ATG6-silenced in SnRK1.1OE.These results suggest tomato SnRK1.1 interacts with ATG6 and phosphorylates ATG6,SnRK1.1 mediated the formation of ATG6-dependent autophagy and alleviated tomato LN stress.