Investigation of physiological and molecular mechanisms for quality assurance in post-harvest management of African nightshade (Solanum scabrum Mill.) and cowpea (Vigna unguiculata L. Walp)

Abstract

African indigenous leafy vegetables (ALVs) have a great potential in improving livelihood, offering sustainable food security and solving the malnutrition crisis in Sub-Saharan Africa (SSA). African nightshade (Solanum scabrum Mill.) and cowpea (Vigna unguiculata L.Walp.) are among the major indigenous leafy vegetables utilized in SSA. Currently, farmers suffer over 50 % qualitative and quantitative losses along the field to consumer chain, due to poor production conditions, unknown maturity indices, poor harvesting methods and post-harvest storage conditions. The research aimed at investigating the harvesting methods in terms of yield and post-harvest nutrient contents, determining development stages with optimal phytonutrients, evaluating the dynamics of phytonutrients at different post-harvest storage conditions, evaluating endogenous ethylene production in African nightshade and their responses to exogenous ethylene application, and finally analyzing ethylene-related gene expression during growth and storage of African nightshade. African nightshade and cowpea leaves harvested from middle parts of the plants, and from field experiments at Jomo Kenyatta University in Kenya 30, 60, 90 and 120 days after planting (dap), were used for phytonutrient analysis during development and at storage (5 °C and room temperature (RT). Enzymatic and photometric methods were used to quantify glucose, fructose, sucrose (GFS) and starch. The gallic acid equivalents (GAE) phenolic contents were analyzed using the Folin-Ciocalteu method (FC). The aluminum complexation reaction assay was used for quantifying catechin and quercetin equivalent flavonoids, while trolox equivalent antioxidant capacity (TEAC) assays were conducted for antioxidants. Furthermore, African nightshade leaves (2, 4, 6, 8 weeks old) from greenhouse experiments were used in testing ethylene production and sensitivity at RT for 72 hours. Ethylene was quantified with gas chromatography (GC-FID) method and gene expression analysis was studied using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results indicated that in cowpea the total carbohydrates were highest at 90 dap and at 60 dap in African nightshade. The total phenolics (gallic acid equivalents), catechin equivalent flavonoids, and trolox equivalent antioxidants (TEA) were highest in concentration at 60 dap in cowpea and 90 dap in African nightshade. Chlorophyll content was optimal at 60 dap in both crops. Storage of cowpea and African nightshade leaves at room temperature (RT; 50-55 % relative humidity) led to a strong decline of the analyzed phytonutrients after 4 days, but mostly they remained stable at cold storage (5 °C). The middle leaves aged 4 to 6 weeks (wks) had higher carbohydrate and chlorophyll contents, were less sensitive to exogenous ethylene (5 ppm) application, and emitted less ethylene during storage compared to younger (2 wks) and older (8 wks) leaves. The secondary metabolites were highest in the young leaves (2 wks) compared to other leaves. For RT-qPCR assays, results indicated that the Adenine phosphoribosyltransferase (APRT), Actin, and Glyceraldehyde-3-phosphate dehydrogenase (GAPGH), were stable and acceptable for use as reference genes in the normalization of gene expression data. Gene expression for ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylic acid oxidase (ACO 1 and ACO 4), and ethylene sensitivity (ETR; ethylene receptor) in African nightshade leaves during leaf development, age of the plant and in storage at RT with or without ethylene was investigated. The ACO 1 and 4 were upregulated in older leaves and older plants (8 wks and 90 to 120 dap) compared to younger leaves and plants, while the ETR was highly expressed in both young leaves and plants (2 wks and 30 dap) than in older leaves. Results of this study highlight the importance of developmental stages at harvest (60 to 90 dap), storage conditions and duration (low temperatures 5 °C up to 4 days) for optimal availability of phytonutrients in freshly consumed leaves and for post-harvest quality management. Furthermore, considering leaf position and age (4 to 6 wks old) during harvesting of African nightshade would offer optimal quality benefits and reduced sensitivity to ethylene. Adoption of these research findings by the farmers will be a great milestone in solving post-harvest losses and ensuring quality of cowpea and African nightshade