10.25338/B88S4D
Reitz, Nicholas
0000-0002-0152-7352
University of California, Davis
Lignification of tomato (Solanum lycopersicum) pericarp tissue during
blossom-end rot development
Dryad
dataset
2020
Blossom-end rot
Lignification
Hydrogen peroxide
catalase
peroxidase
2020-10-07T00:00:00Z
2020-10-07T00:00:00Z
en
https://doi.org/10.1016/j.scienta.2020.109759
12967 bytes
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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 EVOSTM 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."