Defense proteins
To investigate the interactive effect of salt stress and insect herbivory on tomato defense responses, the activity of two jasmonic acid (JA)-inducible plant defense proteins: polyphenol oxidase (PPO: mOD/min/mg tissue) and trypsin proteinase inhibitor (TPI: % inhibition/mg protein) were measured (Tan et al. , 2018).
PPO and TPI activity levels were measured in three experiments: salt addition alone over 1, 3, and 5 days, insect herbivory for 3 hours, followed by salt addition, and short term (6 hours, 24 hours) and long term (3 days, 7 days) salt priming followed by insect herbivory.
A two-factorial assay with salt (0 mM, 200 mM) and herbivory (no herbivory, herbivory) was conducted. A single 5thinstar H. zea caterpillar was allowed to feed inside a clip cage (3.15 cm2 ­­ leaf area) on the fourth fully expanded leaf of the plant as part of the herbivory treatment. Empty clip cages were placed on plants in the no herbivory treatment. Forty-eight hours after tomato plants were subjected to their respective treatments, 50 mg of leaf tissue was collected in liquid nitrogen and stored at -80°C until further analysis. PPO and TPI levels were analysed using a spectrophotometric method (Acevedo et al. , 2017). Leaf total protein content was estimated using a Bradford protein assay using a bovine serum albumin standard curve (Bradford, 1976).