| ATP-citrate lyase(ACL) is an important cytosolic enzyme, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. Citric acid content is one of the important factors which affect citrus fruit quality. Therefore, study the role of ACL in citrus fruit citrate use has an important significance. In this study, we identified ACL genes from the current citrus genome databases and investigated the role of ACL in citrus fruit by analyzing the citric acid content, ACL activity, and gene expression in different citrus cultivar fruits and/or in fruits under different treatments. The main results were as follows: 1. Data mining and molecular characterization of ACL genesWe identified three ACL genes, named Cit ACLα1, Cit ACLα2, and Cit ACLβ1, in the citrus genome database. Both Cit ACLα1 and Cit ACLα2 encode putative Cit ACLα subunits whereas Cit ACLβ1 encodes a putative ACL β subunit. Moreover Cit AClα1 and Cit AClα2 have 82.5 % amino acid identity. Gene structure analysis showed that Cit ACLα1 and Cit ACLα2 had 12 exons and 11 introns, and Cit ACLβ1 had 16 exons and 15 introns. A region of about 60 amino acids between Cit ACLα and Cit ACLβ subunits was absent relative to H. sapiens ACL. 2. Organ-/tissue-specifi expressions of ACL genesThe result shows that Cit ACLα1 was predominantly expressed in flower, and the lowest transcript level of Cit ACLα1 was observed in JS. Cit ACLα2 was predominantly expressed in stem and fibrous roots, and the transcript levels were similar in the other three tissues. Similar to Cit ACLα1, Cit ACLβ1 was predominantly expressed in flower, and the transcript level in ML and JS was similar to that in FR. 3. Citrate content and ACL genes expression in ripening fruits of ‘Anliu’ orange and ‘Hong Anliu’ orangeThe citrate content of ‘Anliu’ orange fruit increased at first, and it is highest in the 134 d after flower fruit, then it decreased. However, the citrate content of ‘Hong Anliu’ orange fruit was always zero in four stages. Analysis the expression of three ACL genes indicated that the transcript level of Cit ACLα1, Cit ACLα2, and Cit ACLβ1 of ‘Anliu’ orange fruit and Cit ACLβ1of ‘Hong Anliu’ orange fruit were similar, the transcript level increased at first, and then decreased. In contrast, the transcript level of Cit ACLα1 and Cit ACLα2 in ‘Hong Anliu’ orange fruit decreased at first, and then increased. Based on the present result, we could conclude that ACL is related to citrate use in ‘Anliu’ orange fruit, during fruit ripening. 4. Citrate content, ACL activity, and ACL gene expression in ripening fruits of different cultivarsAs fruits ripen, the transcript levels of Cit ACLα1, Cit ACLβ1, and/or Cit ACLα2 in cultivars ‘Niuher’ and ‘Owari’ increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in ‘Egan No. 1’ and ‘Iyokan’, although citrate content also decreased. In ‘HB pummelo’, in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of Cit ACLα2, Cit ACLβ1, and/or Cit ACLα1 increased. ACL activity of ‘Niuher’, ‘Owari’, ‘HB pummelo’, and acid-free pummelo significantly increased, however ‘Egan No. 1’ and ‘Iyokan’ significantly decreased. These data indicate that the effect of ACL on citrate use during fruit ripening depends on the cultivar. 5. Responses of citrate content, ACL activity, and ACL gene expression to mild drought, ABA injection, and(-)-hydroxycitrate injectionMoreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only Cit ACLβ1 was significantly reduced by ABA treatment. The ACL activity of mild drought stress and ABA treatment significantly decreased. These results indicate that the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.HCA injection significantly increased citrate contents in fruits, and significantly decreased the ACL activity. The results showed that to a certain extent, the ACL play a role on citrate use in the fruits. |
