Abstract Background Blossom-end rot in tomatoes is often used as a model system to study fruit calcium deficiency. The study of blossom-end rot development in tomatoes has been greatly impeded by the difficulty of directly studying and applying treatments to the affected cells. This manuscript presents a novel method for studying blossom-end rot development after harvest in immature whole fruit and in pericarp discs. Results Pericarp discs removed from the bottom pericarp of immature healthy fruit developed blossom-end rot like symptoms, corresponding to a decrease in L* value and an increase in a* value. Symptoms also developed in columella tissue, but not in stem-end pericarp tissue, similar to patterns observed during blossom-end rot development on the plant. Ascorbate oxidase and peroxidase activity, which are elevated in blossom-end rot affected fruit compared to healthy fruit, were both correlated with colorimetric measures of tissue darkening in discs. Respiration rate was higher in discs that later developed blossom-end rot symptoms, with increased respiration in asymptomatic discs on day 1 of storage being associated with symptom development on day 2. Calcium chloride and ascorbic acid treatments inhibited symptom development, demonstrating the potential of this method to provide causal evidence. Conclusions Results indicate that symptom development in this system is consistent with blossom-end rot development with regards to location, color change, and the activity of key enzymes. This system has the potential to be used to elucidate the cause of fruit calcium deficiency and improve knowledge of the biological basis for calcium’s diverse effects on fruit.

Materials and methods

Solanum lycopersicum L. (var. HM 4885) seeds were obtained from Agseeds Unlimited (Woodland, CA, USA) and Solanum lycopersicum L. (var. Rutgers) seeds were obtained from the Tomato Genetics Resource Center (University of California, Davis, CA, USA). Seeds were sprouted in peat pellets using double deionized water (ddH₂O). Approximately 2 weeks after germination, seedlings were moved to a greenhouse and transplanted into 7.6 liter pots with a mixture of 1/3 peat, 1/3 sand, and 1/3 rosewood compost, augmented with 1.56kg m^-1 dolomite lime. Plants were irrigated daily until pots were dripping with a solution containing 150ppm nitrogen, 50ppm phosphorus, 200ppm potassium, 175ppm calcium, 55ppm magnesium, 120ppm sulfur, 2.5ppm iron, 0.02ppm copper, 0.5ppm boron, 0.50ppm manganese, 0.01ppm molybdenum, 0.05ppm zinc, and 0.02ppm nickel. Flowers were manually pollinated and tagged. HM fruit used for whole fruit experiments were harvested 9 days after pollination. HM and Rutgers fruit used for pericarp disc experiments were harvested 21 days after pollination.

BER development in harvested whole fruit

Whole fruit were stored blossom-end up in a closed container under a constant flow of humidified air to reduce the accumulation of ethylene and carbon dioxide. Fruit were photographed daily.

Disc Preparation

Discs were prepared from the stem-end pericarp, blossom-end pericarp, and columella tissue on the day of harvest of 21-day-old tomato fruit. Fruit were surface sterilized in approximately 150mL of 1% sodium hypochlorite for 1 minute, then thoroughly rinsed in double deionized water. A 15mm cork borer was used to excise cylindrical tissue samples. For discs made from pericarp tissue, locular and endocarp tissue was removed using a razor blade. Discs were rinsed in ddH₂O and blotted dry on sterilized filter paper. Discs were weighed and placed skin side down in 24 well tissue culture plates (Cellstar, Greiner Bio-one, Kremsmünster, Austria), noting the original fruit and tissue location.

For discs made from columella tissue, a 15mm cylinder from the stem scar to the center of the blossom-end of the fruit was prepared. The blossom-end pericarp and stem-scar were removed, producing a cylinder containing columella tissue with small amounts of locular and seed tissue. This cylinder was cut into discs approximately 2mm in height, and these were rinsed in ddH2O and blotted dry using sterile filter paper. The discs were placed in a 24 well tissue culture plate with the disc locations from the stem-end to the blossom-end preserved. Tissue culture plates were stored without lids in a closed container under a constant flow of humidified air to reduce the accumulation of ethylene and carbon dioxide.

Symptom rating, color, and weight loss

After approximately 3.5 days, discs were scored based on their symptom development on a 0 to 5 scale, with 0 being no symptom development and 5 being complete deterioration and blackening of the tissue. Discs were turned upside down and disc color was measured by inverting a Minolta colorimeter (Konica Minolta Inc. Tokyo, Japan) and measuring the color of the pericarp through the bottom of the plate. Disc color was measured through the bottom of the plate rather than from the top of the well to decrease effects of ambient light. In respiration and treatment testing experiments, disc color was measured through the top to reduce the mechanical damage and potential contamination associated with flipping discs pericarp side down for color measurement from the bottom. Measuring from the top is recommended for future studies. Discs were then weighed, frozen in liquid nitrogen, and stored at -80°C for subsequent enzyme analysis. To determine weight loss trends over time, 6 Rutgers discs from the stem-end pericarp, side pericarp, and blossom-end pericarp were weighed on Day 0, Day 2, and Day 3.5 in a separate experiment.

To compare color changes in discs to the color change of fruit on the plant, 4 fruit exhibiting BER symptoms were harvested. A thin layer of skin was removed from one spot within the BER affected tissue and two spots on the stem end to expose the pericarp tissue for comparable readings with the discs. Color was measured at these spots as stated above, and the two measurements from the stem end were averaged for each fruit to create 4 stem-end measurements and 4 blossom-end measurements.

Enzyme extraction and activity measurement

Frozen discs from HM fruit were ground with a pestle as they thawed in a room temperature mortar with 100mM phosphate buffer, pH 6. The resulting homogeneous mixture was then centrifuged for 20min at 20,000g and 4°C, and the supernatant was used as a crude enzyme extract. Enzyme extracts for ascorbate oxidase activity on whole fruit were prepared using the same method.

Peroxidase activity was assayed spectrophotometrically using a BioTek H1 multimode plate reader (Biotek, Winooski VT, USA). The total reaction volume of 100μL included 70μL of ultrapure water, 3.33μL of 100mM phosphate buffer, 10.66μL of pyrogallol, 10.66μL of enzyme extract, and 5.33 μL 0.5% H₂O₂. H₂O₂ was added last to start the reaction. The increase in absorbance at 420nm, associated with purpurogallin formation, was monitored over 3 minutes. The slope of the regression line for the plot of absorbance over time was used to calculate enzyme activity on a min^-1 g^-1 fresh weight basis. The extinction coefficient used for the calculations was 12.0 (mg/mL)^-1 cm^-1.

Ascorbate oxidase was assayed using a total reaction volume of 100μL including 75.33 μL of ultrapure water, 3.33μL of 100mM phosphate buffer, 10.66μL of 250µM ascorbic acid, and 10.66μL of enzyme extract. The change in absorbance at 290nm over 2 minutes was measured, and activity on a min^-1 g^-1 fresh weight basis was calculated using 2.8 mM^-1 cm^-1 as the extinction coefficient.

Respiration measurement

Respiration rate was measured in HM discs by sealing each of the wells in the 24 well plate with an adhesive PCR plate cover (MicroAmp, Applied Biosystems, Foster City, California, USA) and attaching an adhesive septum (Bridge Analyzers Inc. Alameda, California, USA) to the top. Samples (1mL) were collected by inserting a syringe into the airspace above the discs. Respiration rate was measured 1, 2, and 3 days after harvest. Wells were sealed for 5-11 minutes. CO₂ production (μl CO₂ min^-1 g^-1 fresh weight) was measured using an infrared CO₂ analyzer (Horiba, Irvine, CA).

Calcium and ascorbic acid treatments

Calcium chloride and ascorbic acid treatments were applied to discs as 15-minute dips. Discs were prepared from the blossom-end pericarp tissue of 4 HM fruit as stated above. After rinsing and blotting, 2 discs from each fruit (8 total) were placed in 15mL of either 10g/L (90.1mM) CaCl₂ or 500mM ascorbic acid and shaken gently on a rotating platform for 15 minutes. Controls included 15 minute treatments in ddH2O (pH 5.58) or ddH2O adjusted to pH 2 on 8 discs each from the same fruit. The discs were blotted dry on sterile filter paper and placed in a 24 well tissue culture plate. The plates were stored in a humidified container as stated above. After 3.5 days the discs were photographed and their color measured. For color measurement of the treated discs the discs were left with the pericarp tissue facing up and the colorimeter was placed directly above each well.

Statistical analysis

Statistical analysis was carried out using SAS On Demand for Academics (SAS Institute Inc., Cary, NC, USA). Pearson’s correlation for enzyme analysis was completed on a total of 48 discs, 24 from stem-end pericarp and 24 from blossom-end pericarp. A general linear model procedure was used to determine significant differences in weight loss and a Tukey’s honest means separation was completed. Pearson’s correlation comparing respiration on day 1 with color measurement on day 2 was completed on 24 discs, comprised of 12 stem-end discs and 12 blossom-end discs.