DOI: 10.25338/b8dp7r
title: Validation and demonstration of a pericarp disc system for studying blossom-end rot of tomatoes

description: 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.
description: <b>Materials and
 methods</b> <i>Solanum
 lycopersicum</i> L. (var. HM 4885) seeds were obtained from Agseeds
 Unlimited (Woodland, CA, USA) and <i>Solanum lycopersicum</i>
 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 (ddH<sub>2</sub>O).
 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<sup>-1</sup> 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. <i>BER development in
 harvested whole fruit</i> 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. <i>Disc
 Preparation</i> 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 ddH<sub>2</sub>O 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. <i>Symptom rating, color,
 and weight loss</i> 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. <i>Enzyme extraction and
 activity measurement</i> 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%
 H<sub>2</sub>O<sub>2</sub>.
 H<sub>2</sub>O<sub>2</sub> 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<sup>-1</sup> g<sup>-1
 </sup>fresh weight basis. The extinction coefficient used for the
 calculations was 12.0 (mg/mL)<sup>-1
 </sup>cm<sup>-1</sup>. 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<sup>-1</sup>
 g<sup>-1 </sup>fresh weight basis was calculated using 2.8
 mM<sup>-1</sup> cm<sup>-1</sup> as the extinction
 coefficient. <i>Respiration
 measurement</i> 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. CO<sub>2</sub> production (μl
 CO<sub>2</sub> min<sup>-1</sup>
 g<sup>-1</sup> fresh weight) was measured using an infrared
 CO<sub>2</sub> analyzer (Horiba, Irvine, CA). <i>Calcium and ascorbic
 acid treatments </i> 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) CaCl<sub>2</sub> 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. <i>Statistical analysis
 </i> 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.