Blossom-end rot is a physiological disorder causing significant losses in the produce industry each year. Accumulation of reactive oxygen species has been established as a key characteristic of blossom-end rot development. An increase in peroxidase activity and lignin precursor content are also associated with blossom-end rot symptoms, leading to the hypothesis that lignification may be occurring during blossom-end rot development. To investigate the potential involvement of lignification, hydrogen peroxide content, catalase activity, and peroxidase activity were measured in the top, bottom, and blossom-end rot affected tissue of blossom-end rot affected fruit. The top and bottom of healthy fruit from the same growing conditions were used as controls. Lignin was assayed using histochemical staining, autofluorescence, and thioglycolic acid degradation methods. Hydrogen peroxide content was increased in blossom-end rot affected and blossom-end rot adjacent tissues compared to healthy fruit and the top of blossom-end rot affected fruit. Catalase activity was significantly reduced and ferulic acid peroxidase was increased in the blossom-end-rot affected and unaffected tissue at the bottom of blossom-end rot affected fruit compared to the bottom of healthy fruit. Toluidine blue O staining, thioglycolic acid lignin determination, and autofluorescence analysis all showed increased lignin content in blossom-end rot affected tissue compared to tissue from the bottom of healthy fruit. These results show that lignification occurs during blossom-end rot development, likely through a peroxidase-mediated pathway.

Materials and methods section from manuscript submitted for publication:

Plant growth and tissue sampling

Solanum lycopersicum L. seeds (var. HM 4885) were obtained from Agseeds Unlimited (Woodland, CA, USA) and sprouted in peat pellets and with double deionized water during the summer of 2018. Approximately 2 weeks after germination, seedlings were transplanted into 7.57 liter pots with a mixture of 1/3 peat, 1/3 sand, and 1/3 rosewood compost, augmented with 1.56kg m^-3 dolomite lime. Plants were irrigated with a solution of 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 until one day prior to the opening of the first flower. One day prior to the opening of the first flower, daily water use (DWU) was determined by weighing individual pots (including plants) after application of the nutrient solution and prior to nutrient solution application the next day. Upon flowering, nutrient solution application was discontinued, and 20 grams of a calcium free slow release fertilizer (Osmocote Plus, The Scotts Company, Marysville, OH, USA) was mixed with the top 1.5cm of soil. This fertilizer contained 15% nitrogen, 9% phosphate, 12% soluble potash, 1.3% magnesium, 6% sulfur, 0.02% boron, 0.05% copper, 0.46% iron, and 0.06% manganese. After the opening of the first flower, deionized water was added at 200% of the DWU daily. The first flower on each cluster was removed, and each subsequent flower was tagged with the date and manually pollinated.

Fruit were harvested 21 days after pollination during September and October of 2018. Each fruit was rated as healthy, moderately affected, or severely affected based on a 0-5 rating scale, where 0 represented healthy fruit, 2 represented moderately-affected fruit, and 3-4 represented severely affected fruit. All skin was removed from the tissue prior to sampling. Approximately 2 grams of sample was taken from the top and bottom pericarp of healthy fruit. In BER-affected fruit, three samples were taken: unaffected top pericarp tissue, unaffected bottom pericarp tissue (adjacent to BER symptoms), and bottom pericarp tissue exhibiting BER symptoms. These tissues were designated Top, Bottom, and BER tissue. Samples from severely affected fruit was frozen in liquid nitrogen, and then stored at -80°C for subsequent use in enzyme activity analysis.

During the spring of 2019, germination and growth were completed as previously described. Pollination and harvest were completed in June and July of 2019. Tissue from healthy and BER affected fruit was sampled as stated previously and immediately frozen for later use in thioglycolic acid lignin determination. Unfrozen samples were used immediately for H₂O₂ determination and toluidine blue staining microscopy, and whole tomatoes were held overnight before UV autofluorescence microscopy.

Hydrogen peroxide content

Hydrogen peroxide was assayed by measuring the oxidation of potassium iodide at A₃₅₀, compared to a standard curve⁹, using a BioTek H1 multimode plate reader (Biotek, Winooski VT, USA).  Tissue (250 mg) was ground in 1mL of reaction solution using a mortar and pestle. Each 1mL of reaction solution consisted of 250μL 100mM phosphate buffer (pH 6), 250μL 0.1% trichloroacetic acid, and 500μL 1M potassium iodide. Grinding fresh samples, rather than frozen, was found to be imperative in obtaining accurate measurements, with frozen samples having little or no H₂O₂ content. Samples were ground with the reaction mixture and centrifuged for 15 min at 12,000g. The supernatant (100 μL) was added to a microplate (Fisherbrand, Pittsburg, PA, USA) and the absorbance at 350nm was measured. Controls for each sample were prepared in the same manner, but with ultrapure water added instead of the potassium iodide solution. Samples were compared to a standard curve of 1μM-5mM H₂O₂ in phosphate buffer and presented on a per gram fresh weight basis.

Enzyme extraction

Tissue samples were ground into a fine white powder with a mortar and pestle with liquid nitrogen. In a 2 mL centrifuge tube, 0.2 grams of sample was vortexed with 1mL of 100mM phosphate buffer, pH 6, and centrifuged for 20 min at 20,000g. The supernatant was frozen in liquid nitrogen for later use as the water-soluble enzyme extract.

Catalase

Catalase was assayed spectrophotometrically using a BioTek H1 multimode plate reader (Biotek, Winooski VT, USA). Soluble enzyme extract (8.33μL) was added to the reaction mixture (241μL) containing 0.036% H₂O₂ in 100mM phosphate buffer pH 6. This was allowed to react for 20 min. The reaction was stopped by adding 500μL of 1M potassium iodide and 250μL 0.1% trichloroacetic acid (TCA), and incubated for 10 min in the dark. Sample blanks were assayed by substituting ultrapure water for potassium iodide. The initial H₂O₂ concentration for each reaction was determined by adding potassium iodide and TCA immediately after adding sample. The absorbance at 350nm was compared to a 1μM to 1mM H₂O₂ standard curve_­­.

Peroxidase activity

Peroxidase activity was assayed spectrophotometrically using two different electron donor substrates. The total reaction volume of 100μL included 70μL of ultrapure water, 10.66μL of 100mM phosphate buffer pH 6, 10.66μL of either pyrogallol or ferulic acid, 3.33μL of the 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 for pyrogallol peroxidase activity. For ferulic acid peroxidase activity, the decrease in absorbance at 310nm was measured at 11 sec intervals for 16 measurements of each well over 2.93 min. The average change in absorbance over 2.93 min was used to calculate enzyme activity on a per minute basis. The extinction coefficients used for the calculations was 12.0 (mg/mL)^-1 cm^-1 for pyrogallol¹⁶ and 8400 M­­^-1 cm^-1 for ferulic acid.

TGA lignin

Healthy, moderately affected, and severely BER-affected fruit were harvested 21 days after pollination, frozen in liquid nitrogen, and stored at -80°C until analysis. Samples were ground to a fine white powder in liquid nitrogen. Samples (0.5g) were combined with 1.666 mL 95% ethanol, vortexed thoroughly, and centrifuged at 4°C, 20,000g, for 20 min. The supernatant was discarded and the samples allowed to dry overnight at room temperature. The next day, 0.025g of dried sample was weighed into 15mL centrifuge tubes, and 2.5 mL 2M hydrochloric acid and 0.25mL thioglycolic acid were added and mixed. Tubes were incubated at 95°C for 4 hours, cooled, and centrifuged at 12,000g for 20 min at 4°C. The supernatant was discarded and the pellet was washed with DI water. The pellet was resuspended in 2.5mL 1M sodium hydroxide and held for 18 hours. The mixture was centrifuged at 12,000g for 20 min at room temperature and the supernatant was transferred to a separate tube. Concentrated hydrochloric acid (0.5mL) was added to each tube and the mixture was allowed to precipitate overnight at room temperature before the samples were centrifuged for 20 min at 12,000g and room temperature. The pellet was suspended in 1M sodium hydroxide and the absorbance read at 280nm with 1M sodium hydroxide used as a blank.

Toluidine blue O staining

 Freehand slices of approximately 500μm thickness were made from the top and bottom of a BER-affected fruit using a razor. Bottom slices spanned healthy and BER-affected tissue. Slices were incubated in 0.1% toluidine blue O for 1 min, then rinsed and mounted on slides with water under a cover slip at 4X magnification. Stained samples were imaged using an Evos FL Auto microscope in the color transmitted light setting.

Lignin autofluorescence

Autofluorescence of fresh tomato tissue was measured using a EVOS FL Auto fluorescence microscope (Thermo Fisher, Waltham, MA, USA). Slices of tomato tissue approximately 5mm by 5mm by 500μm were cut from the top and bottom of fruit with and without BER on the day after harvest. To make each 500μm slice, a subsection of tomato (approximately 1cm x 1cm x 4cm) was placed in a graduated handheld microtome, and the protruding section was removed to create a flat surface. Vertical incisions were made to create a 5mm x 5mm square section that included the skin, perpendicular to the movement of the microtome. Slices of 500μm thickness were made by hand using a razor. Bottom slices from BER-affected fruit were cut at the water-soaking boundary, including both healthy tissue and tissue exhibiting moderate BER symptoms. Slices were mounted on glass slides with a solution containing 50μL 0.2 M mannitol and 1μM Fluo-4 pentasodium salt (Thermo Fisher, Waltham, MA, USA), and a cover slip. Slices were imaged using a EVOS^TM DAPI filter cube (excitation: 357nm with a bandpass of ±22nm, emission: 447nm with a bandpass of ±30nm, Thermo Fisher) at 4X magnification. Fluo-4 impermeant was included for calcium analysis (data not presented here) and did not affect autofluorescence in the fluorescence range analyzed when compared to samples not treated with Fluo-4 impermeant (data not shown). Images were analyzed for mean fluorescence of the pericarp tissue using ImageJ software¹⁷. The analysis excluded the thin layer of smaller cells near the skin surface and the highly autofluorescent cuticle. Two slices were analyzed from each location on four healthy fruit and four BER-affected fruit, with the mean fluorescence of the two slices being averaged into one final value for each fruit.

Statistical analysis

Statistical analyses were completed using SAS On Demand for Academics (SAS Institute Inc., Cary, NC, USA). A general linear model and Tukey’s honest means separation were used to test for significant differences between means at p<0.05."