Cloning and Characterization of 1-Aminocyclopropane-1-carboxylate Synthase cDNAs from Different Ripening-behaviour Guava (Pisdium guajava L.) Varieties
Date Issued
2009
Date
2009
Author(s)
Chen, Kuo-En
Abstract
Guava (Psidium guajava L.), a tropical evergreen fruit crop of Myrtaceae, is native to in tropical regions of Mexico and Peru. Because of its great environmental adaptability and vigor characteristics, guava is widespread cultivated in tropical and subtropical areas all over the world, and is also a cash fruit tree in Taiwan. According to fruit ripening behavior, guava varieties are generally classified into two groups, namely climacteric and non-climacteric type. The genetic trail of ripening behavior affects not only the way of postharvest handling and utilization of guava, but also the expansion of industrialized cultivation area in Taiwan. Unfortunately, the mechanism(s) causing the different ripening behaviors among guava varieties is still unknown. Therefore, theobjectives of this thesis are to assess the physiological differences in ripening between climacteric and nonclimacteric guava fruits, as well as to investigate the mechanism(s) rendering various ripening behaviors in guava at molecular level. The respiration and ethylene production rate of ‘Li-Tzy Bar’ and ‘Dar-Dih’ guava fruits are significantly increased after harvested. The maximum respiration rate of ‘Li-Tzy Bar’ and ‘Dar-Dih’ at 20 were 95 and 76 mL CO2/kg/hr, respectively; and the peak of ethylene release of these two cultivars were 33 and 16.9 μL C2H4/kg/hr, respectively. The fruits from both cultivars showed the typical characteristics found in climacteric fruits. ‘Jen-Ju Bar’ guava fruit, in contrast, performed as a nonclimateric fruit, since the respiration rate was steady and fluctuated within the range of 18~21 mL CO2/kg/hr as well as the ethylene production was barely detectable in the same storage condition. Although enhancement of respiration, yellowing in peel color, and softening in fruit texture, some typical physiological responses in climacteric fruit ripening, were obviously observed, there was no significant increase of ethylene release from ‘Jen-Ju Bar’ guava fruits after 100 μL/L propylene treatment for 72 hours. These results implies that the reason that ‘Jen-Ju Bar’ guava fruit displayed a nonclimacteric fruit was due to failure of triggering endogenous ‘System Ⅱ’ ethylene synthesis, but not because of defect of ethylene perception and/or signal transduction. Several lines of evidences have demonstrated that ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) is the key enzyme of ethylene biosynthetic pathway in higher plants. In order to illustrate the difference(s) between climacteric and nonclimacteric type guava at molecular level, two full length ACS cDNA clones, 1830 bp and 1861 bp encoding 491 and 486 amino acids, respectively, were isolated from guava tissues by RT-PCR (reverse transcriptase polymerase chain reaction) strategy and used for the further experiments below. Nucleotide sequencing data revealed that there were 7 conserved regions and 11 unvaried amino acids typically found in ACS genes existed in both cDNA clones. They, therefore, were named Pg-ACS1 and Pg-ACS2, respectively. Both genes were considered as Type I ACS genes since four phosphorylated serine sites were found at the C terminal of their derived amino acid sequence. Results of Southern blot analysis showed that Pg-ACS1 was a single copy gene, and Pg-ACS2 was either a single or two copy gene in ‘Li-Tzy Bar’ or ‘Jen-Ju Bar’ guava genome. Moreover, the fact that gene products of Pg-ACS1 and Pg-ACS2 posed ACS enzyme activity was demonstrated by using JAde 6 Escherichia coli expression system. The growth pattern of ‘Li-Tzy Bar’ or ‘Jen-Ju Bar’ guava fruits was a double sigmoid curve characterized with major fruit enlargement in the first and third phase. The highest respiration and ethylene production rate of developing ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ guava fruit were detected at 5 days after anthesis; and then both gradually declined with fruit development before harvest. The maximum values of respiration rate in developing ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ guava fruit were 195 and 229 mL CO2/kg/hr; and the maximum ethylene production rate of these two cultivars were 0.64 and 0.29 μL C2H4/kg/hr, respectively. In case of ‘Li-Tzy Bar’ guava, the respiration and ethylene production rate elevated again afterward when the fruit entered ripening stage. Pg-ACS1 expression was attributed to the ACS catalyzed the ethylene production of ‘ethylene burst’ in ripening ‘Li-Tzy Bar’ guava, a climacteric cultivar, based on northern blot analysis. The fact that ethylene treatment induced the gene expression, and 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, suppressed the transcript accumulation in green-mature ‘Li-Tzy Bar’ guava fruits implied the ethylene autocatalysis property of Pg-ACS1 expression, a ‘System II’ ACS. The mRNA of Pg-ACS2 was detected by RT-PCR in developing ‘Li-Tyz Bar’ guava prior to green mature stage and all the stages in ‘Jen-Ju Bar’ guava fruit. Opposite to Pg-ACS1, the expression of Pg-ACS2 was influenced neither ethylene nor 1-MCP in ‘Li-Tyz Bar’ guava. Thus, Pg-ACS2 was considered as a ‘System I’ or a constitutive ACS in guava. In conclusion, the major difference between climacteric ‘Li-Tzy Bar’ and nonclimacteric ‘Jen-Ju Bar’ guava fruit is that the latter variety cannot switch on Pg-ACS1, a ‘System II’ ACS, during ripening. Therefore, no enough ACC to support massive ethylene production in green mature ‘Jen-Ju Bar’ guava fruit and the fruit exhibit nonclimacteric characteristics, including delay yellowing in peel and fruit firmness last longer.
Subjects
guava
ACC synthase
ethylene
ripening
SDGs
Type
thesis
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