Experimental design and plant growth

Our approach was to use a hydroponic system to expose plants to salt treatments. We then measured the growth, physiological responses and herbivore resistance of a subset of plants. The remaining plants were exposed to herbivores and had half of their leaves removed, or left as controls, to examine the effect of the salt treatment on induced herbivore resistance and tolerance. These plants were grown to maturity and their fitness (seed yield) measured.

Brassica juncea var. cutlass seeds were germinated in Petri plates for one week under fluorescent lights (125–150 μmol s⁻¹ m⁻²). After one week, 5 mL of half-strength modified Hoagland’s nutrient solution (Sabra et al. 2012) was added to each Petri plate and the seedlings were left to grow for another week. Six randomly chosen seedlings were then transferred to each of 15 10-L plastic containers (a total of 90 plants) filled with half-strength modified Hoagland’s solution that was kept aerated using an aquarium pump (Renault et al. 2001). Plants were grown at 25 °C, under a 14:10 h light:dark photoperiod for 2 weeks to allow for their acclimation to hydroponic conditions. When plants were 4 weeks old, each container was randomly assigned to one of three salinity treatments consisting of 0, 50 or 100 mM NaCl solutions prepared in half-strength modified Hoagland’s nutrient solution. Thus, each salinity treatment was replicated five times. To avoid osmotic shock, seedlings in the 100 mM NaCl treatment were exposed to 50 mM NaCl for 6 h prior to increasing the concentration to 100 mM. Conductivity and water levels were monitored daily to keep the salt and nutrient concentrations constant (7.15 dS m⁻¹ for 50 mM NaCl and 12.50 dS m⁻¹ for 100 mM). The hydroponic solutions were replaced weekly to avoid nutrient deficiency.

After 2 weeks in their salinity treatments (at an age of 6 weeks), two randomly selected plants from each treatment (a total of 30 plants) were harvested and used to ascertain the effects of salinity on tissue quality and plant growth. The harvested plants were washed three times with distilled water, and the fresh weights of roots, stems and leaves were determined. Leaf area of fresh leaves was measured using a leaf area meter (LI-COR, Nebraska, USA). Plant parts were lyophylized to obtain their dry weights. Leaf tissue quality was assessed in terms of specific leaf area (SLA, calculated per plant as: total leaf area of plant/total leaf dry weight), chlorophyll, crude protein, proline and water content. Leaf water content was determined from the fresh and dry weights of four leaf disks (0.6 cm²) from each plant. Leaf chlorophyll content was determined by spectrophotometry (650 and 665 nm) of three methanol washes from similar leaf disks (Renault et al., 2001). To determine the crude protein content of the leaves, frozen samples (0.5 g) were ground in liquid nitrogen. Proteins were extracted with 25 ml of cold phosphate buffer (0.05 M; pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM L-ascorbic acid, along with 1% polyvinylpyrrolidone (PVP) (Jones et al. 1989). The homogenate was kept on ice for 20 min. After extraction the homogenate was centrifuged at 4 °C for 20 min at 15 000 g. The supernatant (200 μl) was mixed with 5 ml of Comassie Brilliant Blue G-250 reagent and the absorbance was read at 595 nm (Bradford 1976). Bovine serum albumin was used as a standard. To determine leaf proline content, a modified Bates method (Bates et al. 1973) was used. Proline was extracted with 10 ml of sulphosalicylic acid (3%) for 30 min and centrifuged for 5 min at 4900 g from frozen leaf tissues (0.5 g) previously ground in liquid nitrogen. The supernatant (1 ml) was incubated with 2 ml of a 60% acetic acid and 1% ninhydrin reagent for 1 h at 100 °C. This solution was then cooled on ice, 3 ml of toluene were added and the 2 phases rigorously mixed. After separation of the phases, the organic phase was isolated and its absorbance read at 520 nm. Proline content was determined from a standard curve prepared using standard L-Proline (Sigma-Aldrich). Lyophylized ground tissues were used to determine the nutrient and Na content of the leaves. Samples were analyzed with a CHNOS elemental analyser ‘vario Micro’ (Elementar, Hanau, Germany).

Of the remaining four plants from each replicate of each salt treatment, two were randomly assigned to an herbivory treatment and also used to obtain leaf disks for bioassays to assess constitutive and induced resistance to herbivory. The other two plants were kept without herbivory. For the herbivory treatment, four T. ni larvae were placed on each plant and allowed to feed on its leaves for 4 h. Larvae consumed roughly one third of the leaf area on each plant. Larvae were constrained to feed on the leaves only, and kept away from the flowers. After the larval feeding, we also simulated herbivory on these plants by manually removing half the leaves from one side of the plant.

Two weeks after the herbivory treatments were applied (at an age of 8 weeks and a size too large to be kept in hydroponic growth), all plants were transferred to pots with a 1:2:1 (V:V:V) mix of sand, peat and perlite containing 0, 50 and 100 mM of NaCl. The soil moisture levels were examined daily and distilled water was added accordingly to keep the soil moist. Two weeks after being transplanted (4 weeks after the application of herbivory treatments) transpiration and stomatal conductance was measured on undamaged leaves on all plants. As plants senesced, all mature fruits were collected and air-dried at room temperature; their seeds were counted and weighed. Senescent (dry) plants were harvested and separated into roots, stems and leaves, oven-dried at 62 °C for 3 days and weighed.

Constitutive and induced resistance of plants to herbivores were assessed by means of bioassays using Trichoplusia ni. Eggs of T. ni were obtained from the Canadian Forest Service (Insect Production Services) and reared on the McMurran artificial diet from the same supplier at 21 °C until they reached the late third or early fourth instar (Tucker and Avila-Sakar 2010). Choice assays were conducted using larvae that had been starved for 20 h. Larvae were individually placed in Petri plates and presented with three 0.6 cm² leaf disks, each freshly cut from mature leaves of a plant grown in one of the three salinity treatments (Hoque and Avila-Sakar 2015; Kornelsen and Avila-Sakar 2015). The disk area remaining after 40 min was measured with a portable leaf area analyzer, and used to estimate resistance as:

where R is resistance, A_i is the initial area of the leaf disk and A_f is the disk area remaining after exposure to the larva. Two sets of disks per plant were tested, and the estimates of resistance obtained were then averaged for each plant. For constitutive resistance, leaf disks were cut from plants assigned to the herbivory treatment before larvae were placed on plants. For induced resistance, leaf disks were obtained one day after larvae had fed on plants. We estimated tolerance to herbivory as the difference between the mean life-time seed production of damaged and undamaged plants within a replicate of salinity level: Delta-seeds = S_d − S_u. In this manner, a positive value indicates over-compensation, a value of zero indicates exact compensation and a negative value indicates under-compensation (Strauss and Agrawal 1999).