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).