Metabolomic, enzymatic, and histochemical analyzes of cassava roots during postharvest physiological deterioration

Selection of cassava cultivars and on-farm trials

Four cultivars were selected for this study as follows: SCS 253 Sangão (hereinafter
SAN), Branco (hereinafter BRA, a landrace), IAC576-70 (hereinafter IAC, a commercial
variety), and Oriental (hereinafter ORI, a landrace). On-farm trials were carried
out at the Ressacada Experimental Farm (Plant Science Center, Federal University of
Santa Catarina, Florianópolis, SC, Brazil—27°35?48? S, 48°32?57? W), using the four
cassava cultivars, as noted above, provided by Santa Catarina State Agricultural Research
and Rural Extension Agency (EPAGRI) at Urussanga county (southern Brazil), the official
state agriculture agency.

Postharvest physiological deterioration (PPD)

Cassava root samples (12 months old) were collected for analysis of non-stored samples
and for induction of physiological deterioration under controlled conditions in the
laboratory. Immediately after harvest, the roots were washed, proximal and distal
parts of the root were removed, and cross sections were made (0.5–1 cm) over the remaining
root, followed by storage at room temperature (66–76 % humidity, 25 °C). Induction
of PPD was performed for 11 days. Monitoring the evolution of PPD and associated metabolic
disturbances were performed daily after induction of PPD. Non-stored samples and those
at 3, 5, 8, and 11 days after PPD induction were collected at each time point, dried
(35–40 °C/48 h) in an oven, milled with a coffee grinder (Model DGC-20N series), and
kept for analysis. For enzymatic analysis, fresh samples were collected and stored
(?80 °C) until analysis.

Postharvest physiological deterioration scoring (PPD scoring)

Seven independent experiments of PPD were carried out in which a randomized sampling
of 3 sliced roots from each plant variety was scored (from 1–10 % of PPD to 10–100 %
of PPD) over the 11-day experimental period. The information was imaged through a
digital camera (OLYMPUS FE-4020, 14 megapixel) and the results were analyzed by visual
inspection of the images.

Metabolomic, enzymatic, and histochemical analyzes

The dried and powdered cassava material (1 g per batch) was mixed with 10 mL ethanol
80 % and extracted using water bath at 55 °C, during 30 min. The mixture was centrifuged
(4000 rpm/5 min), filtered on Whatman No. 2 filter paper, ethanol was removed using
rotatory evaporator at 65 °C, and dried extract diluted to 3 mL with ethanol 6].

The total phenolic contents of the cassava extracts during PPD were determined through the Folin–Ciocalteau
(FCR) method. For a 2.0 mL total volume, 200 µL of extract were first mixed with 100
µL FCR reagent after adding 1.40 mL distilled water and the contents were kept at
room temperature for 10 min. Later, 300 µL Na
₂
CO
₃
aqueous solution (20 %, w.v
^?
) were added and incubated for 1 h. The absorbance was measured at 765 nm through
a UV–visible spectrophotometer (Spectrumlab D180). Total phenolics content was expressed
as µg of gallic acid equivalents/g of dry extract (µg GAE/g) using a standard curve
(0–1000 ?g/mL) of gallic acid 7].

Carotenoid content was determined according to the described method 8]. Briefly, 1 g of flour samples was added to 2 mL of cold acetone. After 10 min, 2 mL
petroleum ether were added and mixed using ultraturrax for 1 min. Samples were then
centrifuged (3000 rpm/10 min), supernatant collected, 2 mL sodium chloride 0.1 M were
added, the solution centrifuged again (3000 rpm/7 min), dried in rotatory evaporator
(55 °C), and the dried extract dissolved with 3 mL petroleum ether. Absorbance was
read at 450 nm in a UV–visible spectrophotometer using the absorption coefficient
of ?-carotene in petroleum ether (2592 L/mol cm).

For Anthocyanins, 1 g of flour sample, 5 mL methanol acidified with 1 N HCl (85:15 v/v) were added
and pH adjusted to 1. The solution was centrifuged (4000 rpm/15 min), the supernatant
collected and dried in a rotatory evaporator (55 °C). The dried extract was reconstituted
with 2 mL methanol and filtered (0.45 µm). Two dilutions were made, one to pH 1.0
buffer by using 3 M potassium chloride and other to pH 4.5 using 3 M sodium acetate
buffer. Samples were diluted tenfold to a final volume of 2 mL and the absorbance
read after 30 min of incubation at 520 and 700 nm (Spectrumlab D180 spectrophotometer)
9], 10].

Total flavonoid content of plant extract was determined using aluminum chloride colorimetric method
11], 12] and standard solutions (0–1000 µg/mL of quercetin in 80 % methanol). For that, 1 mL
of extract solution was mixed with 0.5 mL 95 % ethanol (v/v), 0.1 mL 1 M potassium
acetate, 0.1 mL aluminum chloride solution (10 % AlCl
₃
), and 0.8 mL distilled water to a total volume of 2.5 mL. The mixture was well mixed
and incubated at room temperature for 30 min, versus reagent blank containing water
instead of sample. Quercetin was used as the standard (y = 0.0006x, r
²
 = 0.98) for the quantification of total flavonoid.

To determine total cyanide, the method reported by Bradbury 13] with some modifications was used. Briefly, 1 g flour samples during PPD were weighed
out into plastic bottles; 10 mL 1 M phosphate buffer pH 7.0 and buffer paper were
added. A picrate paper was also added; the bottle was closed with a lid and was left
16 h at 30 °C. The picrate paper was removed, eluted with 0.5 mL water, incubated
during 30 min, and absorbance measured at 510. Acetone cyanohydrin was determined
on the same flour samples as described for total cyanide, but by adding also 0.5 mL
0.1 M HCl.

For the measurement of enzyme activity, flour samples (1 g) from different days of PPD (0, 3, 5, 8, and 11) were homogenized
in 5 mL 10 mM potassium phosphate buffer (pH 7.0) containing 4 % (w/v) PVP (Mr 25,000).
The homogenate was centrifuged (4000 rpm/30 min) and the supernatant used as enzyme
extract 14]. Catalase (CAT) activity was measured directly by the decomposition of H
₂
O
₂
at 240 nm in a spectrophotometer (y = 2.1247x, r
²
 = 0.97) and expressed in units (U) per milligram (U mg
^?1
, 1U = 1 mM of H
₂
O
₂
reduced per minutes × milligrams of protein) 15]. The reaction mixture contained 1 mL 50 mM potassium phosphate buffer (pH 7.0), 1 mL
10 mM H
₂
O
₂
, and 1 mL of the extract. Hydrogen peroxide was determined according to Velikova
16]. 1 g flour sample was homogenized in ice bath with 5 mL 0.1 % (w/v) trichloroacetic
acid (TCA). The homogenate was centrifuged (4000 rpm for 5 min), the supernatant collected
(1 mL) and added of 50 mM 1 mL potassium phosphate buffer (pH 7.0) and 2 mL 1 M KI.
The reaction mixture was read at 390 nm in a spectrophotometer and the content of
hydrogen peroxide calculated through a standard curve (y = 2.1247x, r
²
 = 0.97).

SOD family of enzymes analysis was carried out according to Fridovich 17]. Briefly, 1 g flour sample was homogenized with 10 mL 50 mM potassium phosphate buffer
(pH 7.0), centrifuged (4000 rpm/30 min) and the supernatant containing the crude enzyme
extract for assay recovered. For total superoxide dismutase enzyme (Total SOD), 1 mL
0.05 M sodium carbonate buffer (pH 10.2) was added to 1 mL of enzyme extract and 0.5 mL
4 × 10
^?4
 M of epinephrine. The rate of epinephrine auto-oxidation was determined by monitoring
spectrophotometrically the absorbance in samples in a starting point of reaction and
2.5 min later. The MnSOD was assayed using the same method as above, except with the
addition of sodium cyanide (NaCN), an inorganic compound with high affinity for metals
to inhibit Cu/ZnSOD activity. The enzyme activity of Cu/ZnSOD was then determined
as difference of total SOD and MnSOD

The linamarin solution was assayed in triplicate by adding 100 µL of the pink solution (previously
described by Uarrota 18]) and 0.5 mL water to a small plastic bottle, followed by a 2.1 cm diameter filter
paper disc previously loaded with phosphate buffer 0.1 M at pH 6 (3 mL) and 3 mL linamarase.
A picrate paper was placed in the bottle, which was closed with a screw cap and left
at 30 °C overnight. The brownish picrate paper was removed from the bottle and immersed
in 5.0 mL water for 30 min and the absorbance of the solution measured at 510 nm (Spectrumlab
D180 spectrophotometer). Linamarase assay was carried out by using 1.5 mL of the homogenate,
0.5 mL 5 mM linamarin in 50 mM of Na-citrate, pH 6.0 at 37 °C 19]. After 15 min, the reaction was stopped by boiling the reaction mixture for 2 min
and the glucose released was measured using glucose oxidase method using glucose-oxidase
kit (Glucose-PAP, LAB TEST diagonostica). Briefly, 3 mL kit reagent was added of 0.3 mL
of sample, followed by mixing and incubation at 37 °C during 15 min and absorbance
read at 520 nm (Spectrumlab D180 spectrophotometer). The glucose released in (mg/dL)
was quantified and converted to mmol/L.

For Polyphenol oxidase (PPO) analysis, 2 g of fresh tissue were homogenized with 0.6 g of PVPP and 8 ml 50 mM
(pH 7) phosphate buffer, recovering the supernatant by filtration and centrifugation
(4000 rpm, 4 °C, 15 min, 18 cm of rotor radius) and this constituted a enzymatic extract.
PPO activity was measured using 2.85 ml 0.2 mM (pH 7) phosphate buffer, 50 µl catechol
(60 mM) as substrate, and 100 ?l enzymatic extract, at 25 °C. Changes in absorbance
(420 nm) were recorded over a 5 min period in a UV–visible spectrophotometer (Spectrumlab
D180, BEL Photonics, Brazil—20]).

Ascorbic acid (AsA) content was assayed as described previously with slight modifications 21]. The extract was prepared by grinding 1 g of sample with 5 ml 10 % TCA, centrifuged
(3500 rpm, 20 min), re-extracted twice, and the supernatant made up to 10 ml and this
constituted the extract. The reaction medium was done by 1.0 ml of extract, 1 ml DTC
reagent (2, 4-dinitro phenyl hydrazine–thiourea–CuSO
₄
), incubated (37 °C, 3 h) and 0.75 ml ice-cold 65 % H
₂
SO
₄
(v/v) added after incubation, allowed to stand for 30 min, at 30 °C. The resulting
color was read at 520 nm in the spectrophotometer (Spectrumlab D180, China). The AsA
content was determined using a standard curve build with AsA (y = 0.0361x, r
²
 = 0.99, 0–1000 mg mL
^?1
) and the results were expressed in µg g
^?1
(ppm) of fresh weight.

Protein content was determined in the cassava root samples (non-stored and 3, 5, 8, and 11 days postharvest)
using Coomassie brilliant blue G-250 22] reagent, with bovine serum albumin as standard (y = 0.0159x, r
²
 = 0.98). For enzymatic activities, cassava root samples (1 g, grated samples) were
collected directly into liquid nitrogen in a mortar, with 2 % PVPP, 1 mM PMSF, 10 mM
DTT, and 0.1 mM EDTA (MW: 292.2 g mol
^?1
) in 50 mM Na-P buffer, pH 7.5. For analysis of ascorbate peroxidase (APX), the extraction
buffer also contained 2 mM ascorbate (MW: 176.13 g mol
^?1
). The suspension was centrifuged (4000 rpm, 30 min, 4 °C) and the supernatant used
for enzyme assay.

Ascorbate peroxidase (APX) activity was measured by monitoring the decline in absorbance at 290 nm, as
ascorbate (? = 2.8 mM
^?1
cm
^?1
) was oxidized, for 3 min 23]. The assay medium consisted of 1200µL 50 mM potassium phosphate buffer (pH 7.0),
200 µL EDTA, 200 µL ascorbate, 200 µL of sample, and 200 µl 0.1 mM H
₂
O
₂
to start the reaction. APX activity was expressed in mM ascorbate min
^?1
 mg
^?1
of proteins.

Guaiacol Peroxidase (GPX) activity was measured using a reaction medium containing 50 mM phosphate buffer
(pH 7), 9 mM guaiacol, and 19 mM H
₂
O
₂24]. The kinetic evolution of absorbance at 470 nm was measured during 1 min. Peroxidase
activity was calculated using the extinction coefficient (26.6 mM
^?1
cm
^?1
, at 470 nm). One unit of peroxidase was defined as the amount of enzyme that caused
the formation of 1 mM tetraguaiacol per minute.

Tocopherol (?-TOC or vitamin E) activity was assayed as described by Backer 25] with small modifications. Briefly, 1 g of cassava sample was homogenized with 5 ml
of a mixture of petroleum ether and ethanol (2: 1.6, v/v), the extract was centrifuged
(4000 rpm, 30 min, 4 °C), and the supernatant was used to estimate ?-TOC content.
To one milliliter of extract, 3 ml 2 % 2, 2-dipyridyl in ethanol were added, mixed
thoroughly, and kept in dark for 5 min. The resulting red color was diluted with 4 ml
distilled water and mixed well. The resulting color in the aqueous layer was measured
at 530 nm. The ?-TOC content was calculated using a standard curve (y = 0.1115x, r
²
 = 0.96) of ?-TOC (0–100 mg mL
^?1
) and expressed in mg g
^?1
of fresh weight (FW).

Sugars and organic acids were extracted from 0.5 g of cassava root flour samples in 10 ml mobile phase (H
₂
SO
₄
, 5 mM) and determined accordingly 26]. Briefly, the suspension was homogenized using an ultra-turrax apparatus and mixed
slowly using a horizontal shaker (Microplate shaker, 330 rpm), for 30 min. The suspension
was centrifuged (8000 rpm, 10 min), filtered through a 0.22 ?m disposable syringe
membrane filter and the supernatant collected. Sugars and organic acids were analyzed
by HPLC using a Biorad Aminex HPX 87H column, equipped with a UV detector (MWDG 1365D,
for organic acids), connected in series with a refractive index detector (RID G 1362A,
for sugars) and an injection valve fitted with a 15 ?L loop. The samples were separated
isocratically at 0.6 ml min
^?1
at 30 °C.

Retention times and standard curves were prepared for sugars and organic acids (see
Additional file 1 : Table S1). Three consecutive injections (10 ?L) were performed. Sugars and organic
acids were expressed (mg g
^?1
) as mean ± standard deviation.

For scopoletin analysis, cassava root flour samples (1 g) were placed in 50 mL falcon tubes containing 2 mL
98 % ethanol (JT Baker, USA) and homogenized with an ultraturrax (IKA T18 basic, IKA,
China) for 30 s. The suspension was vortexed (1 min), incubated (microplate shaker,
600 rpm, 30 min), and centrifuged (7000 rpm, 5 min). The extract was filtered on a
Whatman # 1 paper and through a 0.22 ?m nylon membrane. Samples were transferred to
1.5 mL vials for HPLC (Agilent Technologies 1200 series, Waldbronn, Germany) analysis
27]. For that, samples (50 µL) were injected into an HPLC (Agilent Technologies 1200
series, Waldbronn, Germany), equipped with a reverse-phase column (Techsphere BDS
C18, 250 mm × 4.6 mm, 5 ?m) and a diode array detector. The column was kept at 25 °C
and acetonitrile and 0.5 % phosphoric acid (v/v) in aqueous solution were used as
mobile phase. The gradient profile was 60–1 % for 30 min with a 0.5 mL min
^?1
flow and 50 ?L injection volume. Scopoletin was detected at 215, 280, and 350 nm and
according to its retention time, using a standard compound sample (Sigma–Aldrich:
scopoletin ?99 %—no. S2500). Scopoletin quantification was determined through a calibration
standard curve (y = 158159.59x, r
²
 = 0.99, 1–75 mg L
^?1
). Three consecutive injections (10 ?L) were performed and quantifications were made
on a dry weight basis, and data represented in nmol g
^?1
, as mean ± standard deviation.

For histochemical analysis, cassava root samples (non-stored and 3, 5, 8, and 11 days of PPD) were collected
and small pieces were made (0.5 × 0.5 cm
²
) for subsequent fixation in paraformaldehyde. Samples of cassava roots were fixed
in 2.5 % paraformaldehyde in 0.1 M (pH 7.2) phosphate buffer (72 h). Subsequently,
the samples were dehydrated in increasing series of ethanol aqueous solutions 28], 29]. After dehydration, the samples were infiltrated with historesin (Leica Historesin,
Heidelberg, Germany). Sections (5 ?m length) were stained with different histochemical
techniques and investigated with an Epifluorescent (Olympus BX 41) microscope equipped
with Image Q Capture Pro 5.1 software (Qimaging Corporation, Austin, TX, USA). LM
sections were stained as follows: Periodic Acid-Schiff (PAS) used to identify neutral
polysaccharides 18], Toluidine Blue (TB-O) 0.5 %, pH 3.0 (Merck Darmstadt, Germany) used for acid polysaccharides
through a metachromatic reaction 28], and Coomassie Brilliant Blue (CBB) 0.02 % (w/v) in Clarke’s solution (Serva, Heidelberg,
Germany) used for protein identification 30].

Statistical analysis

All statistical analyzes and graphics were implemented in R language (R core team-2014,
version 3.1.2) 31]. Data are represented as mean ± standard deviation of a minimum of three repetitions
(n = 3). Two-way ANOVA using randomized complete design was applied. Ordinary least
square (OLS) regression models and decision regression trees were applied for predictive
models (see Additional files 2, 3, 4, 5, 6, 7, 8 and 9). Histochemical micrographs were performed in Photoshop, version 7. Raw data in csv
format—Additional files 2, 3 and 4, R software report (html format—Additional file 5) and data as R objects (RData format—Additional files 6, 7, 8 and 9) are also provided.