Tissue specific proteome analysis deciphers regulation of unique set of proteins from varied metabolic pathways during pre-climacteric and climacteric stages in banana

Abstract

Globally, bananas are a major staple food with significant nutritional and commercial potentials and are extremely perishable with a very short shelf life. Understanding the proteins expressed in different tissues during ripening can help in improving postharvest quality and shelf life. A gel-free comparative proteomic analysis was followed to investigate protein alterations in peel and pulp tissues during pre-climacteric and climacteric phases of banana. Total starch content was decreased when ripening progresses. In contrast, total sugar, sucrose, glucose and maltose content was increased multi folds during the climacteric phase, in both peel and pulp tissues. The protein samples were subjected to orbitrap fusion mass spectrometry coupled with nano LC-MS/MS resulted in the identification of 950 and 1300 proteins in pulp and 1416 and 1279 proteins in peel tissues at pre-climacteric and climacteric stages, respectively. Mass-spectrometry identified proteins were categorised and they were involved in starch and sugar metabolism, cell wall modification, hormonal regulation and detoxification of reactive oxygen species (ROS), which were more prevalent in the pulp tissue during the climacteric period as compared to the pre-climacteric stage. Proteins such as α-1,4-glucan (α-gluc) phosphorylase, pectin esterase (PE), β-galactosidase (β-gal), α-mannosidase (α-man), pectate lyase (PEL), xyloglucan endotransglucosylase/hydrolase (XTH), pectin acetylesterase and β-hexoaminidase (β-hex) were found to be more in number in climacteric stage, and could be responsible for pulp softening, de-greening of the peel, alterations in texture and fruit quality. The study found that proteins related to sugar metabolism, such as fructose bisphosphate aldolase and sucrose synthase (SUS), were more abundant during the climacteric than pre-climacteric stage. This suggests that these proteins could contribute to sugar synthesis and the degradation of starch during fruit ripening. Interestingly, some of the proteins that play a crucial role in hormonal regulation were identified in the form of cysteine synthase (CS) (#17), amino methyltransferase (#10), and a more significant number of CS proteins were identified in pulp tissue at the climacteric stage. Venn analysis of the proteins from different tissues and stages indicates the presence of unique set of proteins in tissue specific manner, and could have a specialized role. Further, the protein-protein interaction study confirms that the XTH could be an ideal candidate known to be associated with unique pathways in peel at the climacteric stage. RTqPCR analysis revealed greater transcript levels of XTH4 and SOD in pulp, and PEL in peel tissue at climacteric stage indicates cell wall disintegration and loosening at climacteric stage. The potential candidate proteins identified in this investigation could be of immense help to gain insights of the regulatory mechanism of banana ripening process. Identified proteins can be further validated using genome editing technology to assign individual functional roles.