The Mechanism Of Cell Death Caused By Disruption Of Fumarylacetoacetate Hydrolase In Arabidopsis

The Tyrosine degradation pathway is a very important metabolic pathway in animals,blockage of this pathway may cause serious metabolic disorder.Deficiency of fumarylacetoacetate hydrolase(FAH),the last enzyme in the Tyrosine degradation pathway,causes an inborn lethal disease.But there are few researches on Tyrosine degradation pathway in plant.We isolated and identified an Arabidopsis mutant sscd1(short-day sensitive cell death1)that displays programmed cell death under short-day conditions(8hours light/16 hours dark)by EMS mutagenesis.The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH.This finding first time demonstrates that Tyrosine degradation pathways have biological functions in plants.Further studies revealed that programmed cell death in sscd1 mutant is closely related to accumulation of succinylacetone,an abnormal metabolite of Tyrosine degradation pathway.Since both sugar metabolism and chlorophyll biosynthesis pathways are associated with programmed cell death,and succinylacetone has a similar structure to chlorophyll biosynthesis precursor,?-aminolevulinic acid(ALA),it is possible to competitively inhibit ALA dehydratase(ALAD)activity,thus affects the chlorophyll biosynthesis pathway.Therefore,this paper first studied whether sugar affects cell death in sscd1 mutant,and then focused on the regulation of cell death in sscd1 mutant by chlorophyll biosynthesis pathway.Through a series of physiological and biochemical experiments and related gene expression analysis,the following main results were obtained:(1)The lethal phenotype of the sscd1 seedlings was suppressed by sugars including sucrose,glucose,fructose,and maltose in a dose-dependent manner.The Tyrosine degradation pathway genes homogentisate dioxygenase(HGO)and maleylacetoacetate isomerase(MAAI)was up-regulated in the sscd1 mutant.However,this upregulation could be repressed by sugars including sucrose,glucose,fructose,and maltose.In addition,a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone,an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway.These results indicated that sugar could suppress celldeath in sscd1,which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates,resulting in suppression of cell death.(2)The sucrose metabolism genes cell-wall invertase 1(cw INV1)and alkaline/neutral invertase G(A/N-inv G)was up-regulated in the sscd1 mutant,however,this upregulation could be repressed by sugars including sucrose,glucose,fructose,and maltose.In addition,exogenous succinylacetone treatment of Arabidopsis wild-type seedlings resulted in up-regulation of cwINV1 and A/N-invG gene expression.Similarly,this up-regulation was inhibited by high concentration of sugar.These results suggest that cwINV1 and A/N-invG may be involved in cell death in sscd1 mutants.(3)The activity of ALAD in the sscd1 mutant was decreased,which proved by in vivo and in vitro experiments,resulting in a decrease in the accumulation of the protochlorophyllide(Pchlide),intermediate in the chlorophyll synthesis pathway,in the etiolated sscd1 seedlings;exogenous succinylacetone treatment of wild-type seedlings was able to mimic the decrease in ALAD activity and Pchlide accumulation in the sscd1 mutant.These results indicate that the activity of ALAD in the sscd1 mutant is inhibited by succinylacetone,resulting in reduced accumulation of Pchlide in the etiolated sscd1 seedlings.(4)Cell death in sscd1 requires a certain darkness and light.When 4-d etiolated sscd1 seedlings were transferred to long-day condition(16 hours light/8 hours darkness)for one photoperiod,Pchlide accumulated although the ALAD activity was still inhibited.Upon re-illumination after Pchlide levels increased,the expression of the reactive oxygen species marker genes including APX2,gstF8,OXI1,DIL,ZP and BAP1 genes was significantly up-regulated,followed by cell death.These results suggested that excessive accumulation of Pchlide result in the accumulation of reactive oxygen species in sscd1 mutant upon re-illumination,eventually leading to programmed cell death(5)Chlorophyll biosynthesis genes HEMA1 and CHLH are upregulated during light-dark transformation before Pchlide levels increase,indicating feedback inhibition of chlorophyll synthesis pathway in sscd1 mutant during light-dark transformation was affected,thereby the chlorophyll biosynthesis pathway in the sscd1 mutant may be activated in darkness and result in the increase of Pchlide.This paper firstly found that the Tyrosine degradation pathway in plants is related to sugar metabolism and chlorophyll biosynthesis pathway,revealing the molecular mechanism of cell death cause by loss of FAH in Arabidopsis thaliana.It is of great scientific significance to further study the biological functions of Tyrosine degradation pathways in plants and the regulation mechanism of plant programmed cell death.