Comparative proteomic analysis on fruit ripening processes in two varieties of tropical mango (Mangifera indica)

Mango (Mangifera indica L.) is an economically important fruit. However, the marketability of mango is affected by the perishable nature and short shelf-life of the fruit. Therefore, a better understanding of the mango ripening process is of great importance towards extending its postharvest shelf life. Proteomics is a powerful tool that can be used to elucidate the complex ripening process at the cellular and molecular levels. This study utilized 2-dimensional gel electrophoresis (2D-GE) coupled with MALDI-TOF/TOF to identify differentially abundant proteins during the ripening process of the two varieties of tropical mango, Mangifera indica cv. ‘Chokanan’ and Mangifera indica cv ‘Golden Phoenix’. The comparative analysis between the ripe and unripe stages of mango fruit mesocarp revealed that the differentially abundant proteins identified could be grouped into the three categories namely, ethylene synthesis and aromatic volatiles, cell wall degradation and stress-response proteins. There was an additional category for differential proteins identified from the ‘Chokanan’ variety namely, energy and carbohydrate metabolism. However, of all the differential proteins identified, only methionine gamma-lyase was found in both ‘Chokanan’ and ‘Golden Phoenix’ varieties. Six differential proteins were selected from each variety for validation by analysing their respective transcript expression using reverse transcription-quantitative PCR (RT-qPCR). The results revealed that two genes namely, glutathione S-transferase (GST) and alpha-1,4 glucan phosphorylase (AGP) were found to express in concordant with protein abundant. The findings will provide an insight into the fruit ripening process of different varieties of mango fruits, which is important for postharvest management

Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins