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Tomato is a climacteric fruit, which is characterized by ripening-related increase of respiration and elevated ethylene synthesis. Ethylene is the key hormone in ripening process of climacteric fruits. The objective of this research is to study the expression of three ethylene synthesis genes: LeACO1, LeACS1A, LeACS2, and a housekeeping gene LeGAPDH in ripening tomato fruit. Specific primers have been designed to amplify complementary DNA fragment of LeGAPDH (143 bp), LeACO1 (240 bp), LeACS1A (169 bp), and LeACS2 (148 bp) using polymerase chain reaction. Nucleotide BLAST results of the complementary DNA fragments show high similarity with LeGAPDH (NM_001247874.1), LeACO1 (NM_001247095.1), LeACS1A (NM_001246993.1), LeACS2 (NM_001247249.1), respectively. Expression study showed that LeACO1, LeACS1A, LeACS2, and LeGAPDH genes were expressed in ripening tomato fruit. Isolation methods, reference sequences, and primers used in this study can be used in future experiments to study expression of genes responsible for ethylene synthesis using quantitative polymerase chain reaction and to design better strategy for controlling fruit ripening in agroindustry.

Tomato is a climacteric fruit, which is characterized by ripening-related increase of respiration and elevated ethylene synthesis. Ethylene is the key hormone in ripening process of climacteric fruits. The objective of this research is to study the expression of three ethylene synthesis genes: LeACO1, LeACS1A, LeACS2, and a housekeeping gene LeGAPDH in ripening tomato fruit. Specific primers have been designed to amplify complementary DNA fragment of LeGAPDH (143 bp), LeACO1 (240 bp), LeACS1A (169 bp), and LeACS2 (148 bp) using polymerase chain reaction. Nucleotide BLAST results of the complementary DNA fragments show high similarity with LeGAPDH (NM_001247874.1), LeACO1 (NM_001247095.1), LeACS1A (NM_001246993.1), LeACS2 (NM_001247249.1), respectively. Expression study showed that LeACO1, LeACS1A, LeACS2, and LeGAPDH genes were expressed in ripening tomato fruit. Isolation methods, reference sequences, and primers used in this study can be used in future experiments to study expression of genes responsible for ethylene synthesis using quantitative polymerase chain reaction and to design better strategy for controlling fruit ripening in agroindustry. [ABSTRACT FROM AUTHOR]

Ethylene regulates a myriad physiological and biochemical processes in ripening fruits and is accepted as the ripening hormone for the climacteric fruits. However, its effects on metabolome and resulting fruit quality are not yet fully understood, particularly when some of the ripening-associated biochemical changes are independent of ethylene action. We have generated a homozygous transgenic tomato genotype (2AS-AS) that exhibits reduced ethylene production as a result of impaired expression of 1- aminocyclopropane-1-carboxylate synthase 2 gene by its antisense RNA and had a longer shelf life. Double transgenic hybrid (2AS-AS x 579HO) developed through a genetic cross between 2AS-AS and 579HO (Mehta et al., 2002) lines resulted in significantly higher ethylene production than either the WT or 2AS-AS fruit. To determine the effects of reduced ethylene and introgression of higher polyamines' trait, the metabolic profiles of ripening fruits from WT (556AZ), 2AS-AS, and 2AS-AS x 579HO lines were determined using 1H-NMR spectroscopy. The levels of Glu, Asp, AMP, Adenosine, Nucl1, and Nucl2 increased during ripening of the WT fruit. The increases in Glu, Asp, and AMP levels were attenuated in 2AS-AS fruit but recovered in the double hybrid with higher ethylene and polyamine levels. The ripening-associated decreases in Ala, Tyr, Val, Ile, Phe, malate, and myo-inositol levels in the 2AS-AS linewere not reversed in the double hybrid line suggesting a developmental/ripening regulated accumulation of these metabolites independent of ethylene. Significant increases in the levels of fumarate, formate, choline, Nucl1, and Nucl2 at most stages of ripening fruit were found in the double transgenic line due to introgression with higher-polyamines trait. Taken together these results show that the ripening-associated metabolic changes are both ethylene dependent and independent, and that the fruit metabolome is under the control of multiple regulators, including ethylene and polyamines. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is a powerful tool for characterization of functional genes in tomato. In this study, we improved the syringe infiltration method on detached tomato fruits. With PDS (phytoene desaturase gene) used as a reporter gene, harvested mature green tomato fruits were syringe-infiltrated with agrobacterium strain GV3101 containing pTRV1 and pTRV2- LePDS, which resulted in color changing on the fruit surface. The changes in pigments and transcript abundance of PDS were compared between silencing sectors and control. In addition, factors of environmental conditions, such as temperature and humidity, were optimized for gene silencing, and other factors, including volume of agrobacterial culture, were also analyzed. Furthermore, this silencing method was confirmed by silencing LeRIN and LeACS2, which influenced fruit ripening process as positively regulating genes, and LeCTR1, which took part in ethylene signal transduction as a negatively regulating gene. Transcripts of LeRIN, LeCTR1, and LeACS2 were measured in the following analysis of gene silencing. All results indicated that an infiltration-based method is efficient for gene characterization during development and ripening of tomato fruits. [ABSTRACT FROM AUTHOR]

In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence of tomato, two cDNAs (LeNCED1 and LeNCED2) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, two cDNAs (LeACS2 and LeACS4) which encode 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and one cDNA (LeACO1) which encodes ACC oxidase involved in ethylene biosynthesis were cloned from tomato fruit using a reverse transcription-PCR (RT-PCR) approach. The relationship between ABA and ethylene during ripening was also investigated. Among six sampling times in tomato fruits, the LeNCED1 gene was highly expressed only at the breaker stage when the ABA content becomes high. After this, the LeACS2, LeACS4, and LeACO1 genes were expressed with some delay. The change in pattern of ACO activity was in accordance with ethylene production reaching its peak at the pink stage. The maximum ABA content preceded ethylene production in both the seeds and the flesh. The peak value of ABA, ACC, and ACC oxidase activity, and ethylene production all started to increase earlier in seeds than in flesh tissues, although they occurred at different ripening stages. Exogenous ABA treatment increased the ABA content in both flesh and seed, inducing the expression of both ACS and ACO genes, and promoting ethylene synthesis and fruit ripening, while treatment with fluridone or nordihydroguaiaretic acid (NDGA) inhibited them, delaying fruit ripening and softening. Based on the results obtained in this study, it was concluded that LeNCED1 initiates ABA biosynthesis at the onset of fruit ripening, and might act as an original inducer, and ABA accumulation might play a key role in the regulation of ripeness and senescence of tomato fruit. [ABSTRACT FROM PUBLISHER]