Cloning and Characterization of 1-Aminocyclopropane-1-carboxylate Oxidase cDNAs from Guava（Pisdium guajava L.） Varieties with Different Ripening-behaviour
Date Issued
2010
Date
2010
Author(s)
Liu, Te-Chun
Abstract
Guava (Psidium guajava L.) is a tropical evergreen fruit crop of Myrtaceae, and its fruit is rich in vitamins and minerals and has antioxidant capacity. The guava cultivars in Taiwan are divided into 2 groups, climacteric and non-climacteric, based on their ripening behavior. Five of the common non-climacteric guava cultivars in Taiwan, ‘Jen-Ju Bar’, ‘Shuei-Jin Bar’, ‘Di-Wang Bar’, ‘Er-Shr-Shr-Ji Bar’ and ‘Hung-Shin Bar’, and one of the climacteric guava cultivar, ‘Li-Tzy Bar’, were used to investigated the postharvest physiology. The ethylene production of non-climacteric fruits remained under 0.20 μL‧kg-1‧L-1 during 204 hours storage at 20℃, while that of climacteic fruits could reach 71.02 μL‧kg-1‧L-1. Treatment of exogenous propylene induced the ripening related response of non-climacteric cultivar guava, indicating that the ripening mechanism in cultivars of this group is closely linked with ethylene synthesis instead of ethylene perception.
The two ACO cDNA clones in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ tissues were isolated by RT-PCR (reverse transcriptase- polymerase chain reaction). The numbers of base pairs, molecular weights of the derived amino acid sequence, and the pI value of these two cDNA clones were 1,259 and 1,241, 36.21 and 36.28 kDa, and 5.39 and 5.78, respectively. It was estimated that both of the cDNA clones were able to translate 321 amino acids, in which with 12 ACO activity related conserved residues and 4 leucine zipper sites. Fusion proteins of the two cDNA clones obtained by using E. coli heterologous expression system demonstrated ACO enzymatic activity, which confirm that the two cDNA clones are guava ACO cDNA clones, and thus named as Pg-ACO1and Pg-ACO2.The result of Southern blotting suggested that the copy number of ACO genes in guava genome is approximately 4 or 5.
The results of northern blotting and RT-PCR assay on Pg-ACO1and Pg-ACO2 showed that there was accumulation of transcripts of Pg-ACO1and Pg-ACO2 in guava fruits and shoots but not significant in flowers and leaves. Pg-ACO1 in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ constitutively expressed from 60 and 120 days after anthesis, respectively, to 12 days during postharvest. Pg-ACO2 transcripts in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ were detectable in the 15th day after anthesis, and then decreased until the 60th and 120th day after anthesis, respectively. Transcripts of Pg-ACO1and Pg-ACO2 in ‘Li-Tzy Bar’ fruit significantly increased during ripening, and that in ‘Jen-Ju Bar’ constitutively expressed during storage. The results of 100 μL‧L-1 ethylene and 10 mg‧L-1 1-MCP treatments showed that the transcripts of Pg-ACO1 and Pg-ACO2 in mature green ‘Li-Tzy Bar’ were both increased by ethylene elicitation and suppressed by 1-MCP. Therefore, it was inferred that two ACO cDNA clones possess the characteristics of “system Ⅱ” ethylene synthesis. However, in ‘Jen-Ju Bar’ fruits, the two ACO transcripts were detected from 60th day after anthesis to 12th day postharvest suggesting that both ACO clones might be involved in ethylene biosynthesis in both fruit development and ripening stage and that the two ACO transcripts possess characteristics of both “system Ⅰ” and “system Ⅱ”. In addition, the ACO activity of ‘Jen-Ju Bar’ was significantly higher than that of ‘Li-Tzy Bar’ during the first 10 days postharvest.
The results shown above indicate that the difference in the ripening behaviors between ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ is neither caused by silence of Pg-ACO1and Pg-ACO2 nor by the lack of ACO activity. Furthermore, according the ethylene and 1-MCP experiment on ACO transcripts in ‘Jen-Ju Bar’ fruits, it is hard to conclude which system of ethylene synthesis that both of Pg-ACO1 and Pg-ACO2 belong to.
Subjects
guava
ethylene
ACC oxidase
ripening
climacteric
cloning
Type
thesis
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