| Tomato(Solanum lycopersicum)is a typical respiratory-climacteric fruit.During the process of maturation,the internal structure of chloroplasts is disintegrated,chlorophyll is rapidly degraded;and carotenoids,especially lycopene,are accumulated in large amounts in chromoplasts,and the fruits become red,orange or yellow.At present,the chlorophyll degradation and carotenoid synthesis pathways of higher plants are relatively clear,but the regulation of the two metabolic pathways and the interrelationships between them are still unclear in the process of fruit ripening and color changing.In this paper,the fruits of three stay green tomato mutants were used to investigate the effect of chlorophyll degradation on the synthesis of lycopene during ripening of tomato fruits.Different mutations were identified in the key gene SGR1(STAY GREEN 1)for these mutants,and the ripening characteristics of these sgr1 mutants,chlorophyll degradation,and carotenoid biosynthesis,and the function of mutant sgr1 are further investigated.The results are as follows:1.The fruits of WT(AC)and 3 stay green mutants were initated to ripen by exogenous ethylene.After 9 d of ethylene treatment,WT becomes red,eg remains green and contains large amounts of chlorophyll and eg does not accumulate lycopene;gf1 contains large amounts of chlorophyll and accumulates a large amount of lycopene,which fruits are brown;gf2 contains part of the chlorophyll and accumulates a lot of lycopene,which fruits are brown.All three SGR1 mutants could be normal softened,which were classified as C-type stay-gree2.The wild-type AC contains 818 bp bases in the coding region of the SGR1 gene and encodes a protein of 271 amino acids.32 bp sequence containing the termination codon TAA inserts into the SGR1 transcript of eg after base 460,leading to early termination of its encoded protein containing only 156 amino acids.The C at base 271 of SGR1 in gf1 was replaced by T,resulting in the appearance of the termination codon TAA,which prematurely terminates the encoded protein,which contains only 90 amino acids.The A at base 429 of SGR1 in gf2 is replaced by T,resulting in a mutation that arginine at position 143 changes to serine in the protein with a total of 271 amino acids.In conclusion,the C-terminus of SGR1 plays an important role in its function,and that the arginine which is substituted in gf2 has an important influence on the function of SGR1.3.Compared with the wild type(AC),the expression of the chlorophyll degradation-related genes such as SGR1 and PPH of the three sgr1 mutants are low during the ripening of the fruit,which affect the degradation of chlorophyll and keep the fruit as green.The expression of genes related to carotenoid synthesis are regulated.The expression levels of PSY1 and GGPS2 genes in the mutant are significantly lower than those in wild type;the expression levels of PSY1 and ZDS in gf1 were significantly higher than those in wild type after 6 d of agroinjection;earlier than the wild-type,PSY1,PDS,ZDS of gf2 have been significantly up-regulated after 3 d of agroinjection.4.The expression levels of chlorophyll degradation-related genes,CLH1,CLH3,and CLH4,are higher during the senescence of mutant leaves than those of wild type,suggesting that mutation of SGR1 loses function and transcription is inhibited and CLH,which is not involved in leaf senescence,is up-regulated to degrades chlorophyll in order to reduce the damage.5.By constructing UBQ-10-SGR1-GFP vector and using fluorescence confocal technology,green fluorescence is observed in tobacco leaves transformed with SGR1 of AC and gf2 overexpression vector,but isn’t observed in eg and gf1,assuming that the protein is prematurely terminated.Tobacco leaves transformed with overexpression vector of SGR1 of AC turned yellow after 48 h of agroinjection,and tobacco transformed with the mutant SGR1 overexpression vector is significantly greener than wild type.Detecting demagnetase chelatase activity by HPLC,chlorophyll degradation products are detected in the wild-type,but are not detected in three SGR1 mutants,assuming that the C-terminus of SGR1 is very important.The premature termination of translation will seriously affect its function,and gf2 will undergo an important amino acid mutation,resulting in a change in the active site or configuration affecting the function of SGR1.In summary,the three stay-green mutants are C-type stay-green,which are good materials for studying the pigment metabolism pathway and regulation of tomato.The SGR1 lack of the C-terminal part in eg and gf1 severely affect the function,and the mutation of arginine to serine in gf2 result in the loss of function.Mutations of SGR1 activates CLH gene in the leaves to degrade chlorophyll in order to reduce the toxicity. |
