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Plant Direct. 2020;4:1–13.
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1wileyonlinelibrary.com/journal/pld3
Received: 22 July 2019
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Revised: 13 December 2 019
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Accepted: 21 January 2020
DOI: 10.1002 /pld3.202
ORIGINAL RESEARCH
A metabolomics study of ascorbic acid-induced in situ freezing
tolerance in spinach (Spinacia oleracea L.)
Kyungwon Min1 | Keting Chen2 | Rajeev Arora1
This is an op en access arti cle under the ter ms of the Creative Commons Attribution L icense, which pe rmits use, dis tribu tion and reprod uction in any med ium,
provide d the original wor k is properly cited.
© 2020 The Authors. Plant Direct pu blishe d by Amer ican So ciety of Plant Biologists and the Society for E xperimenta l Biology and Jo hn Wiley & Sons Ltd
This man uscript was pre viousl y deposited as a pr eprint at http s ://doi.org/10.1101/202 0.01. 23.916973.
1Depar tment of Horticulture, Iowa St ate
University, Ames, IA, USA
2Depar tment of Genetic, Development, and
Cell Biology, Iowa St ate Universit y, Ames,
IA, USA
Correspondence
Rajeev A rora, D epar tment of H orticulture,
Iowa State University, Ames, IA 50 011, USA.
Email: rarora@iast ate.edu
Funding information
Hatch Act and State of Iowa Funds
Abstract
Freeze–thaw stress is one of the major environmental constraints that limit plant
growth and reduce productivity and quality. Plants exhibit a variety of cellular dys-
functions following freeze–thaw stress, including accumulation of reactive oxygen
species (ROS). This means that enhancement of antioxidant capacity by exogenous
application of antioxidants could potentially be one of the strategies for improving
freezing tolerance (FT) of plants. Exogenous application of ascorbic acid (AsA), as an
antioxidant, has been shown to improve plant tolerance against abiotic stresses but
its effect on FT has not been investigated. We evaluated the effect of AsA-feeding
on FT of spinach (Spinacia oleracea L.) at whole plant and excised-leaf level, and con-
ducted metabolite profiling of leaves before and after AsA treatment to explore
metabolic explanation for change in FT. AsA application did not impede leaf growth,
instead slightly promoted it. Temperature-controlled freeze–thaw tests revealed
AsA-fed plants were more freezing tolerant as indicated by: (a) less visual damage/
mortality; (b) lower ion leakage; and (c) less oxidative injury, lower abundance of free
radicals (
O⋅−
2
and H2O2). Comparative leaf metabolite profiling revealed clear separa-
tion of metabolic phenotypes for control versus AsA-fed leaves. Specifically, AsA-fed
leaves had greater abundance of antioxidants (AsA, glutathione, alpha- & gamma-
tocopherol) and compatible solutes (proline, galactinol, and myo-inositol). AsA-fed
leaves also had higher activity of antioxidant enzymes (superoxide dismutase, ascor-
bate peroxidase, and catalase). These changes, together, may improve FT via allevi-
ating freeze-induced oxidative stress as well as protecting membranes from freeze
desiccation. Additionally, improved FT by AsA-feeding may potentially include en-
hanced cell wall/lignin augmentation and bolstered secondary metabolism as indi-
cated by diminished level of phenylalanine and increased abundance of branched
amino acids, respectively.
KEYWORDS
alpha-tocopherol, freeze–thaw injury, glutathione, in situ freezing test, proline, reactive
oxygen species
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MIN et al.
1 | INTRODUCTION
Sub-freezing temperatures are the major environmental constraint
affecting crop performance and limiting plant distribution. This
provides ample incentive to improve plants' freezing tolerance
(FT). Freeze–thaw-injured tissues undergo various cellular dys-
functions. Thus far, two of the most studied loci of such injury are
(a) leakage of cellular solutes, that is. physico-molecular perturba-
tions in cell membranes, and (b) oxidative injury to macromolecules
due to cellular accumulation of reactive oxygen species (ROS; e.g.,
superoxide, singlet ox ygen, etc.) (Arora, 2018; Kendall & McKersie,
1989; Min, Chen, & Arora, 2014; Mittler, 2002). Hence, detoxifi-
cation of excess ROS is believed to be one of the major strategies
of frost survival (McKersie, Bowley, & Jones, 1999; McKersie et
al., 1993).
Certain plants from temperate region have an ability to in-
crease their FT, via a process called cold acclimation, when ex-
posed to cold temperature (Thomashow, 2010). This involves a
myriad of adjustments at physiological, biochemical, and meta-
bolic levels, including an upregulation or accumulation of enzy-
matic and/or non-enzymatic antioxidants (Thomashow, 2010; Xin
& Browse, 2000). This suggests enhancement of antioxidant ca-
pacity by exogenous application of antioxidants could potentially
be an intervention strategy to increase plants' FT. Ascorbic acid
(AsA) is a well-known water-soluble antioxidant involved in ascor-
bate–glutathione cycle, espe cially as a substr ate for ascorbate per-
oxidase (APX) which is responsible for conver ting H2O2 into H2O
(Foyer & Noctor, 2011; Smirnof f, 2000). Research has shown ex-
ogenous AsA to improve plant tolerance against salt, drought, and
chilling (Ahmad, Basra, & Wahid, 2014; Akram, Shafiq, & Ashraf,
2017; Amin, Mahleghah, Mahmood, & Hossein, 2009; Azzedine,
Gherroucha, & Baka , 2011). But no s tudy, to our knowledge, exis ts
on the ef fect of AsA on FT of whole plants. Moreover, a compre-
hensive study of metabolome changes induced by AsA-feeding of
tissues could provide additional insight into biochemical mecha-
nis m and in vivo role of AsA- in du ce d str es s to leran ce , in cl uding F T.
These studies may also lead to identification of beneficial metabo-
lites vis-à-vis FT enhancement.
In the present study, our main goals are twofold to: (1) in-
vestigate the effect of AsA-feeding on FT of spinach seedlings
at the whole plant as well as excised-leaf level, and (2) explore
metabolome changes induced by AsA treatment using gas chro-
matography–mass spectrometry (GC-MS). We used spinach as
a model because of its moderate constitutive FT allowing suf-
ficient range of freezing treatment temperatures for the pres-
ent study and our previous experience with this system (Chen &
Arora, 2014; Min, Showman, Perera, & Arora, 2018; Shin, Min,
& Arora, 2018). Visual estimation and ion leakage test were
used to evaluate AsA-induced FT following in situ freezing test.
Other physiological parameters, that is, histochemical detection
of ROS, activit y of antioxidant enzymes, and leaf content of glu-
tathione (GSH), were also determined for untreated control and
AsA-fed tissues.
2 | MATERIALS AND METHODS
2.1 | Plant materials
Spinach seedlings were grown as described previously (Min et al.,
2018). Briefly, seeds of “Reflect,” a F1 hybrid cultivar (Johnny's se-
lected seeds, Inc), were sown in plug flats filled with Sunshine LC-1
mix (Seba Beach) and placed in growth chambers at 15/15°C (D/N)
with 12-hr photoperiod under average PAR of ~300 µmol m−2 s
−1
at plant height provided by incandescent and fluorescent lamps.
Seedlings were watered as needed via sub-irrigation (approximately,
5-day interval). After two weeks from the sowing, temperature in
chambers was elevated to 20/18°C (D/N), and seedlings were sub-
fertigated only once with either 300 ppm EXCEL (Everris NA Inc)
nutrient solution (hereafter referred to as F-control) or with 0.5 and
1.0 mM AsA treatment made with 300 ppm EXCEL as solvent. About
24-day-old spinach seedlings, that is 10 day after fertigation treat-
ments, were used for experiments as described below.
2.2 | Growth measurement
Leaf growth was evaluated by measuring fresh weight (FW), dry
weight (DW ), and leaf area of F-control and AsA-fed leaves. Briefly,
7 to12 pairs of leaves (total 14 to 24 leaves) per treatment were first
used to measure leaf area using LI-3100 Area Meter (LI-COR , Inc),
quickly followed by the measurement of FW on the same leaves.
DW was measured following oven-drying leaves at 75 ± 1°C for 72-
hr. Water content was calculated on FW basis. Data of leaf growth
across five biological replications (14 to 24 leaves per biological rep-
licate) were pooled to c alculate the representative treatment means
with standard errors. Mean differences between treatments were
analyzed by least significant difference (L SD) test .
2.3 | Freezing tolerance measurement
2.3.1 | In situ freezing test
Temperature-controlled, whole-plant freezing protocol was used, as
described by Min et al. (2018), to compere FT between F-control and
AsA-fed seedlings. Three plug flats—one of F-control and the other
two wit h 0. 5 or 1.0 mM As A-fe d pl ants—were tr an sferred to a fr ee z-
ing chamber (E41L1LT, Percival Scientific, Inc) kept at 0°C; other
such three plug flats were transferred to another identical freezing
chamber. The two freezing chambers, respectively, were used for
freezing treatments of −5.5 or −6. 5°C , and subseq uent thawing. The
two test temperature treatments (−5.5 and −6.5°C) used in the pre-
sent study were selected based on our previous data of leaf-freezing
response curve for “Reflect” leaves, and LT50 (lethal temperature
for 50% injur y) of ~−6.0°C (Shin et al., 2018). These two test tem-
peratures represent relatively moderate (−5.5°C) and severe (−6.5°C)
stress bordering LT50, and are, therefore, physiologically relevant.
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MIN et al .
After 2-hr at 0°C, temperature in freezing chambers was lowered
at 1°C/hr up to −2°C at which ice nucleation was conducted by
quickly misting pre-chilled (0°C) ddH2O onto leaves, and held at
this temperature for 1-hr. Plants were then frozen to −5.5 or −6.5°C
at −0.5°C/30 min. Plants kept at each targeted temperature for
30 min were allowed to thaw at 0°C overnight (~13-hr). Unfrozen
control (UFC) seedlings of each treatment were kept at 0°C in an-
other identical chamber throughout the freeze–thaw cycle. Gradual
thaw continued by subjecting plants, including UFC, to 5°C for 2-hr.
Entire freezing and thawing were performed in dark. All the plants
were transferred from chambers to the laboratory bench (~20°C)
under dim light (~15 μmol m−2 s−1, cool white fluorescent) for ~1 day.
Freeze–thaw injury to seedlings was then evaluated visually and
photographed. Additional assessment of freeze injury/tolerance for
F-control versus AsA-fed plants was made by measuring ion leakage
on leaves excised from plants that had been exposed to whole-plant
freezing. Percent injury was calculated using percent ion leakage
data as described by Lim, Arora, and Townsend (1998).
Whole-plant freezing test along with visual estimation and ion
leakage measurement were repeated thrice, each with 14 to 16
plants per temperature per treatment (2 leaves per plant replicate).
Injury percent data across three independent experiments were
pooled to calculate the representative treatment means with stan-
dard errors. Mean differences were analyzed by LSD test.
2.3.2 | Excised-leaf freezing test (Bath freezing)
Excised leaves from F-control and AsA-fed seedlings were subjected
to a temperature-controlled freeze–thaw protocol as described by
Chen and Arora (2014), using a glycol bath (Isotemp 3028; Fisher
Scientific) (hereon referred as to ‘bath freezing’). Briefly, a pair of
petiolate leaves (rinsed with ddH2O and blotted on paper towel) was
placed in a 2.5 × 20 cm test tube containing 150 μl ddH2O and slowly
cooled down at −0.5°C/30 min to four different test temperatures
(i.e., −4.5, −5.5, −6.5, and −7.5°C) following ice nucleation at −1°C.
Samples were kept for 30 min at each selected temperature and
thawed on ice overnight. UFC leaves of each treatment were main-
tained at 0°C throughout the freeze–thaw c ycle. The next morning,
samples were kept at 4°C for 1-hr followed by 1-hr at room tem-
perature (~20°C) before measuring ion leakage. Bath freezing was
independently repeated thrice, each with 5 technical replicates per
temperature per treatment (2 leaves per technical replicate). Injur y
percent data (calculated from percent ion leakage) from 3 biologi-
cal replications were pooled to calculate the treatment means with
standard errors. Mean differences were analyzed by LSD test.
2.4 | ROS staining
Superoxide (
O⋅−
2
) and hydrogen peroxide (H2O2) distribution were
visualized by nitroblue tetrazolium (NBT) and 3,3’-diaminobenzidine
(DAB) staining, respectively, using the protocol previously used in
our laboratory for spinach (Chen & Arora, 2014; Min et al., 2014).
Staining intensities were visually evaluated between F-control and
AsA-fed leaves that were subjected to bath freezing at −5.5, −6.5,
and −7.5°C. This experiment was independently repeated twice,
each with 2 to 3 replications (2 leaves/ replicate) per temperature
per treatment. A representative picture showing staining intensities
is presented in this study.
2.5 | Measurement of antioxidant enzyme activity
The activity of three antioxidant enzymes, that is, SOD, CAT, and
APX, was measured using a protocol as described by Chen and Arora
(2011, 2014). Essentially, ground frozen leaf tissue (150 mg) was ho-
mogen ized with 1 ml of 100 mM pot assium phosphate buf fer (p H 7.0).
The samples were then centrifuged at 10,000 g for 25 min at 4°C,
and supernat ants were use d as th e enzy me ext ra ct for SOD, C AT, and
APX. Enzyme activity was calculated as described by Chen and Arora
(2014). This experiment was independently repeated four times, each
with 3 to 4 technical replicates per treatment. Mean difference was
analyzed as per Student's t test.
2.6 | Measurement of glutathione (GSH)
GSH level was determined using high-performance liquid chroma-
tography as described by Zheng et al. (2018) with slight modifica-
tions. Ground frozen leaf tissues (~0.2 g) were mixed with extraction
buffer containing 0.1% trifluoroacetic acid and 200 mM dithiothrei-
tol to extract GSH. The homogenate was centrifuged at 15,300 g
for 10 min. The supernatant (0.5 ml) was transferred to a spin filter
and centrifuged for 5 min. The filtrate was injected into Spherisorb
5 μm ODS column (250 mm × 4.6 mm) for HPLC (model 1,260) cou-
pled to 1, 200 series evaporative light scattering detector (Agilent
Technologies). This analysis was conducted twice independently
with 2 to 3 technical replications each. Mean difference was ana-
lyzed as per Student 's t test.
2.7 | Sample extraction for metabolite profiling
Frozen leaf tissues were ground and used for metabolite profiling.
Sample extraction was conducted as detailed by Min et al. (2018);
each treatment from F-control and AsA-fed leaves consisted of 4
biological replications, each with 3 technical replications.
2. 7.1 | Metabolite identification and quantification
Metabolite identification was performed based on compounds’
chromatographic retention time indices following deconvolution of
ra w GC-MS ch rom at ogr a ms us ing AM DIS softw a re, as des c rib ed by
Min et al. (2018). Each identified metabolite was quantified based
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MIN et al.
on internal standards and dr y weight; missing dat a were replaced
by a number (i.e., the smallest peak area /2) for further statistical
anal ysis as re po r ted by Xia , Psy ch ogi os , Youn g, an d Wis har t (2009).
2. 7. 2 | Statistical analysis for metabolite profiling
Principal component analysis (PCA) was conducted with R (version
3.2.2, The R Foundation for Statistical Computing, ISBN 3-900051-
07-0) on log10 transformed relative metabolite concentration between
two treatments (F-control vs. 1.0 mM AsA-fed tissues). Mean differ-
ence in th e ab undance of each me ta bo li te between tr ea tm ents was de-
termined via Student's t test (Table S1). A volcano plot was generated
using log2-scaled mean difference in each metabolite concentration
and log10 -transformed p-values between two treatments; only those
metabolites were numbered on a volcano plot for which the abundance
between the two treatments was significantly different (p < .05).
3 | RESULTS
3.1 | Effect of exogenous AsA on growth
Water content was slightly higher in 0. 5 and 1.0 mM AsA-fed leaves
compared with F-control, (Table 1). Leaf area of seedlings treated
with 0.5 or 1.0 mM AsA was larger than the F-control by 7.0% or
15.9%, respectively. DW/leaf area of F-control and 0.5 mM AsA-
fed leaves was similar but slightly smaller than 1.0 mM AsA-fed
seedlings.
3.2 | Freezing tolerance and leaf AsA
A representative pic ture of seedlings exposed to freeze–thaw stress
is shown in Figure 1a where either 0.5 mM- or 1.0 mM AsA-fed
seedlings are visually more freeze-tolerant than F-control at both
−5.5 and −6.5°C stress. The beneficial effec t of AsA on FT was
especially more pronounced at the moderate stress level (−5.5°C).
AsA (1.0 mM)-fed leaves accumulated ~2.4-fold AsA compared with
F-control (Figure 1b); leaf AsA of 0. 5 mM AsA-fed leaves was not
determined.
Relatively less freeze–thaw injur y in A sA-fed tissues was also ev-
ident by the ion leakage from the leaves excised from seedlings that
had been subjected to in situ freeze–thaw (Figure 2a). Seedlings fed
with 0.5 mM A sA had ~52% and ~13% less injur y at −5.5 and −6.5°C ,
respectively, compared with F-control whereas those treated with
1.0 mM AsA had ~69% and ~41% less injury at both stress levels rel-
ative to F-control. Bath freezing tests using excised leaves (not whole
seedlings) from three different treatments also exhibited lower freez-
ing injury in AsA-fed tissues compared with F-control (Figure 2b).
3.3 | Histochemical detection of ROS (
O⋅−
2
and H2O2)
A representative image of the quantitative estimate of
O⋅−
2
and H2O2
(as indicated by the color intensity) in the leaves from three treat-
ments (F-control, 0.5 mM, and 1.0 mM AsA) af ter having been ex-
posed to bath freezing at −5.5, −6.5, and −7.5°C, and that of unfrozen
control (UFC) is shown in Figure 3. The two ROS accumulated at
higher abundance in F-control than 0.5 and 1.0 mM AsA-fed leaves
after free zi ng at −5. 5 and −6. 5°C, wit h 1.0 mM As A tr eatment show-
ing the lowest accumulation. Little to no protection was apparent by
AsA application at −7.5°C stress level.
3.4 | Biochemical analysis
FT data indicated 1.0 mM AsA treatment to be more protective
than 0.5 mM. Therefore, F-control was hereon compared only with
1.0 mM AsA treatment for all biochemical analyses (below).
3.5 | Antioxidant enzyme activities and leaf
glutathione (GSH)
Quantification of antioxidant enzyme activities and GSH was ex-
pressed on DW basis, since water content of F-control versus AsA-
fed leaves was different (Table 1).
SOD (Figure 4a), CAT (Figure 4b), and APX (Figure 4c) activities in
1.0 mM AsA-fed leaves, respectively, were 1.1-, 2.4-, and 2.7-fold of
F-control. GSH in 1.0 mM AsA-fed leaves was ~1.3-fold of F-control
(Figure 4d).
3.6 | Principal component analysis (PCA)
In total, 46 metabolites were identified following GC-MS analysis
and clustered into 6 groups—17 amino acids, 8 carbohydrates, 2 fatty
acids, 4 TCA intermediates, 3 antioxidant s, and 12 others (Table S1).
TABLE 1 Leaf growth parameters of spinach (Spinacia oleracea L.
cv. Reflect) seedlings sub-fer tigated with fer tilizer alone (F-control),
fertilizer +0.5 mM ascorbic acid (0.5 mM AsA), or fertilizer +1.0 mM
ascorbic acid (1.0 mM AsA). DW, dry weight
Growth
parameters
Treatment
F-control 0.5 mM AsA 1.0 mM AsA
Water content (%)a91.9 ± 0.14 b 92.5 ± 0.08 a 92.3 ± 0.05 a
Leaf area (cm2)a6.30 ± 0.28 b 6 .74 ± 0.19 ab 7.30 ± 0.29 a
DW/Leaf area
(mg/c m2)a
1.89 ± 0.03 b 1.86 ± 0.03 b 2.06 ± 0.02 a
Note: Value s with the same letter within the same row are not different
(LSD test, p < .05).
aPooled means ± SE from f ive biological replications, each including
7–12 plants. Two leaves/plant were used resulting in a total of 14–24
leaves/biological replication.
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MIN et al .
PCA was performed to explore whether metabolite phenotype
between F-control and 1.0 mM AsA was different, and to deter-
mine which metabolites affected such differences the most. Data
indicated clear separation between the two treatments wherein
two components (PC1 and PC2) explained 55.6% of the total vari-
ance (Figure 5a). The first component (PC1) accounts for 37.4% of
the variance separating AsA-feeding versus F-control. The second
component (PC2) accounting for 18.2% of the variance primarily
indicates different abundance of metabolites across technical repli-
cations within each treatme nt (Figure 5a). Loading values for metab-
olites separated by the two PCs are shown in Table S2. For example,
urea and GABA with most positive or negative loading values, re-
spectively, contribute most for the separation of F-control against
AsA-fed treatment on PC1.
FIGURE 1 (a) Visual estimation of in situ whole-plant freezing (−5.5 and −6.5°C) response of spinach (Spinacia oleracea L. cv. Reflect)
seedlings sub-fertigated with fertilizer alone (F-control), fertilizer +0.5 mM ascorbic acid (0.5 mM A sA), and fertilizer + 1.0 mM ascorbic
acid (1.0 mM AsA); UFC, unfrozen control. (b) Ascorbic acid content (mean ± SE) in F-control and 1.0 mM AsA; different letters indicate
significant differences between treatments at p < .05 as per LSD test
FIGURE 2 Freeze–thaw injury assessed by ion leakage of spinach (Spinacia oleracea L. cv. Reflec t) leaves: (a) excised from the seedlings
subjected to in situ whole-plant freezing at −5.5 and −6.5 ˚C; values represent the average ± SE from three independent experiments, each
with 14–16 plants per temperature per treatment and (b) exposed to bath freezing at −4.5, −5.5, −6.5, and −7.5°C; values represent the
average ± SE from three independent experiments, each with 5 plants per temperature per treatment. Different letters indicate significant
differences bet ween treatments at p < .05 as per LSD test. UFC , unfrozen control; F-control, seedlings sub-fertigated with fertilizer alone;
0.5 mM AsA, seedlings sub-fertigated with fertilizer +0.5 mM ascorbic acid; 1.0 mM AsA, seedlings sub-fertigated with fertilizer +1.0 mM
ascorbic acid
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MIN et al.
3.7 | Comparative metabolite profiles of 1.0 mM
AsA versus F-control
Mean abundance of total 46 identified metabolites was pair-wise
compared (1.0 mM AsA vs. F-control) using log2-scaled fold change
and −log10 scaled p-values. Data indicate that 23 out of 46 metabo-
lites marked as red in Figure 5b exhibited major changes in abun-
dance (signific antly different at p < .05) between F-control and
AsA-fed treatment. Numerical fold change (log2 scaled) for these
metabolites in AsA-fed versus F-control is shown in Table 2, where
th e spo t nu mber fo r ea c h metab oli t e cor res p o n ds to th e num b e r as-
signed in Figure 5b. These 23 metabolites are also identified for sig-
nificance level (t test) with asterisk notations in Table S1. Nineteen
of these metabolites, that is cysteine, glycine, glutamine, glutamic
acid, leucine, methionine, proline, threonine, galactinol, myo-inosi-
tol, citric acid, malic acid, α-tocopherol, γ-tocopherol, AsA, ferulic
acid, gl ycer ic acid, phytol, and ure a, were mor e abund an t in 1.0 mM
AsA-fed leaves as indicated by a positive value (1.0 mM AsA /F-
control ratio) (Table 2). Four metabolites, phenylalanine, fructose,
GABA, and phosphoric acid, were less abundant (minus sign) rela-
tive to F-control (Table 2). These 23 metabolites were placed in five
categor ie s (n ot 6, as in Table S1) because abu nd ance in “fa tty aci ds”
was not found to be significantly dif ferent.
4 | DISCUSSION
In recent years, exogenous application of beneficial chemicals has
received some attention as potential means for improving plant
tolerance against various abiotic stresses (Savvides, Ali, Tester, &
Fotopoulos, 2016). While AsA application has been a subject of such
efforts in the context of salt, drought, and chilling stresses, its effect
on FT remains unknown. In the present study, we have evaluated
the effect of AsA fer tigation on FT at whole plant as well as excised
tissue level determined through various parameters of freeze–thaw
injury, and conducted metabolite profiling of leaves before and after
AsA treatment to explore metabolic explanation for AsA-mediated
change in FT.
FIGURE 3 Distribution of superoxide (
O⋅−
2
) (a-c) and hydrogen peroxide (H2O2) (d-f) in unfrozen controls (UFC) and freeze–thaw-injured
spinach (Spinacia oleracea L. cv. Reflect) leaves that were sub-fertigated with fertilizer alone (F-control), fertilizer +0.5 mM ascorbic acid
(0.5 mM AsA), and fertilizer +1.0 mM ascorbic acid (1.0 mM AsA) before exposure to bath freezing at −5.5, −6.5, and −7.5˚C
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MIN et al .
FIGURE 4 The activity of SOD, CAT,
and APX (a-c) in spinach (Spinacia oleracea
L. cv. Reflect) leaves sub-fertigated with
fertilizer alone (F-control), and fertilizer
+1.0 mM ascorbic acid (1.0 mM AsA).
One unit of SOD activity is defined as
the amount of enzyme required for
50% inhibition of formazan formation
at 560 nm; one unit of CAT activity is
defined as the degradation of 1 μM H2O2
in 1 min at 240 nm; one unit of APX
activity is defined as the degradation of
1 μM AsA into monodehydroascorbate
in 1 min at 290 nm; values represent the
average ± S.E from four independent
experiments, each with 3–4 replications
per treatment. (d) Glutathione
concentration of spinach leaves in
F-control and 1.0 mM AsA; values
represent the average ± SE from two
independent experiments, each with 2–3
replications per treatment. *indicates
significant difference at p < .05 (t test) for
all four panels
FIGURE 5 (a) Metabolic phenotype clustering for spinach (Spinacia oleracea L. cv. Reflect) leaves through principal component analysis
(PCA) of log10-scaled 46 metabolite data from a total of 12 replications (triplicates from 4 biological replications) each originating from two
treatments (i.e., F-control and 1.0 mM AsA); 1.0 mM AsA: treated with fertilizer +1.0 mM AsA; F-control: treated with fer tilizer. Principal
component 1 (PC1) indicates differential response to AsA application. Principal component 2 (PC2) indicates variation of metabolite
concentration among replications. F-control (black) and 1.0 mM AsA (red) are shown in 2D plot. (b) Volcano plot of comparative abundance
of metabolites in 1.0 mM AsA versus F-control. Each dot represents a metabolite with the -log10 (p value) as a function of abundance
difference between two biological conditions (log2 fold change on the abscissa). Metabolites are numbered and colored (red) if significantly
different at corrected p-value < .05. The two vertical lines on either side of the central vertical line indicate range of twofold cutoff in
abundance whereas the horizontal line represents a threshold of −log10 = 0.05
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MIN et al.
4.1 | AsA fertigation and leaf growth
The effect of AsA on plant growth as well as stress tolerance is de-
pendent upon the mode of ap plication and concentration (Ak ram et al. ,
2017). Henc e, we fir s t te ste d fou r AsA con cen t rat ion s (i .e. , 0. 5, 1.0 , 2.0 ,
and 4.0 mM) as sub-fertigation treatments. Seedlings fed with 2.0 and
4.0 mM AsA showed somewhat stunted growth relative to F-control,
whereas 0.5 and 1.0 mM AsA-feeding did not show any detrimental
effect. Hence, 2.0 and 4.0 mM AsA were not used for subsequent ex-
periments (data not shown for these comparisons). Higher leaf AsA
concentration in AsA-fed leaves than F-control (Figure 1b) indicates
seedlings effec tively absorbed and assimilated AsA.
Research shows that exogenous application of AsA improved
plant growth in wheat (Athar, Khan, & Ashraf, 2008) and millet
(Hussein & Alva, 2014). This supports our observation of 7%–16%
higher leaf area in AsA-fed leaves (Table 1). Specific mechanism of
AsA-induced growth is beyond the scope of this study but increase in
AsA level has been associated with enhanced cell division (Smirnoff,
1996) and expansion (De Cabo, González-Reyes, Córdoba, & Navas,
1996). Moreover, repression of L-galactono-1,4-lactone dehydroge-
nase, an enzyme involved in the biosynthesis of AsA, in tobacco BY-2
cell lines caused a decline in cellular AsA content as well as in cell
division and growth (Horemans, Pot ters, Wilde, & C aubergs, 2003).
In contrast, cell division in maize root treated with AsA was higher
than control (Kerk & Feldman, 1995). AsA may promote cell division
by inducing G1 to S prog ression (Galie, 2013), and AsA-indu ced plant
growth could also involve auxin regulation via interac tion between
ascorbate oxidase and auxin (Esaka, Fujisawa, Goto, & Kisu, 1992;
Key, 1962; Pignocchi, Fletcher, Wilkinson, Barnes, & Foyer, 2003;
Smirnof f, 2000).
4.2 | AsA-feeding improves freezing tolerance
Visual evaluation of injured seedlings (whole-plant freezing) and cor-
responding percent injury based on ion leakage from leaves excised
fr om th ese seed lin gs in di c at e s AsA-fed plan t s to be mor e fr e eze-tol er-
ant than F-control (F igures 1a and 2a), and that 1.0 mM As A was mor e
effective than 0.5 mM AsA. “Bath freezing” tests with excised spin-
ach leaves further supported this observation (Figure 2b). Induction of
freezing tolerance (as in cold acclimation) typically involves decrease
in cellular hydration status (Xin & Browse, 2000). Therefore, it is some-
what intriguing that AsA-fed leaves, which are more hydrated, though
marg ina ll y, tha n F-co nt rol (Tab le 1), had gr ea ter FT. Co nce iv abl y, ot her
physiological and biochemical changes induced by AsA-feeding (as
discussed below) override this apparent contradiction.
4.3 | Higher antioxidant enzyme activity in AsA-
fed leaves
Evidence abounds that plant tissues subjected to freeze–thaw
accumulate excess
O⋅−
2
and H2O2 (Kendall & McKersie, 1989; Min
et al., 2014; Shin et al., 2018). Our data of visual detection of
ROS (Figure 3) are consistent with these findings and show that
more severe freezing stress (−6.5˚C) resulted in higher ROS ac-
cumulation. More importantly, less accumulation of
O⋅−
2
and H2O2
in AsA-fed leaves compared with F-control indicates alleviation of
oxidative stress by AsA application. Relatively higher scavenging
of H2O2 in 1.0 mM AsA-feeding compared with 0.5 mM (compare
Fi gur e 3e an d f ) indi c ate s the for m er tre a tme n t was mo r e effe c t ive
scavenger of free radicals. Our results also indicate a close corre-
spondence between antioxidant enzyme activities and ROS abun-
dance. For instance, SOD activity in AsA-fed leaves was higher
than F-control (Figure 4a), which might be responsible for less
accumulation of
O⋅−
2
. Likewise, CAT and APX activities in AsA-fed
leaves were higher than in F-control (Figure 4b,c), suggesting more
TABLE 2 Significant changes in the concentrations of leaf
metabolites bet ween 1.0 mM AsA versus F-control
Group
Spot
number Metabolite
log2 (1.0 mM
AsA/F-control)
Amino acids 1aCysteine 0.79b
2Glutamine 0.64
3Glutamic acid 0.50
4Glycine 0.94
5Leucine 1.62
6Methionine 1.33
7Phenylalanine −0.33
8Proline 1.47
9Threonine 0.64
Carbohydrates 10 Fr uctose −0.72
11 Galactinol 1.15
12 Myo-inositol 0.83
TCA
intermediates
13 Citric acid 0.60
14 Malic acid 0.58
Antioxidants 15 α-tocopherol 0.99
16 γ-tocopherol 0.36
17 Ascorbic acid 1.24
Others 18 Phytol 0.64
19 Ferulic acid 0.56
20 GABA −0 .61
21 Glyceric acid 1.00
22 Phosphoric acid −0.44
23 Urea 1.46
aSpot number indicates a metabolite that is signific antly different
(p < .05).
bFold changes in the concentrations of each metabolites betwe en
two groups (12 replic ations per treat ment) were calculated using
the formula log2 (1.0 mM AsA /F-control); spot numbers and the
numerical value of metabolites in this table are illustrated in Figure
5b. Metabolites are classified into 5 groups, that is amino acids,
carbohydrates, TCA intermediates, antioxidant s, and others. 1.0 mM
AsA: seedlings sub-fer tigated with fertilizer +1.0 mM ascorbic acid;
F-control: seedlings sub-fertigated with fertilizer alone.
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9
MIN et al .
efficient scavenging of H2O2. A higher GSH content in 1.0 mM
AsA-fed leaves than F-control (Figure 4d) further supports higher
APX activit y in these tissues since GSH works together with APX
in ascorbate–glutathione cycle (Foyer & Noctor, 2011). Several
studies have also noted enhanced activity of antioxidant enz ymes
by exogenous application of AsA, especially when tissues are ex-
posed to abiotic stresses (Alam, Nahar, Hasanuzzaman, & Fujita,
2014; Athar et al., 2008; Kumar et al., 2011).
4.4 | AsA-feeding alters leaf metabolome
PC1, accounting for 37.4% of total variance, clearly separated
F-control from AsA-fed treatment (Figure 5a). In contrast, PC2,
explaining 18.2% of total variance, may reflect differences of me-
tabolite concentration across replications. Discussion under several
sections (below) further highlights specific differences in metabo-
lism between two treatments.
Figure 5b illustrates comparative metabolite abundance for AsA-
fed versus F-control tissues. Ensuing metabolite-specific sections
discuss their putative roles in relation to FT.
4.4.1 | Amino acids
AsA-fed leaves had significantly higher levels of cysteine, methio-
nine, proline, glutamine, glutamic acid, glycine, threonine, and leu-
cine but lower level of phenylalanine relative to F-control (spots
1–9; Figure 5b; Table 2). Higher cysteine, glutamic acid, and glycine
in AsA-fed tissues support our results of a higher activity of APX
as wel l as high er GS H in th ese tissue s (Figu re 4c ,d). Cyst ei ne, a sul-
fur-containing amino acid, is known as a key component for GSH
biosynthesis (Noctor et al., 2012), which consist s of two steps: (a)
formation of γ-glutamyl-cysteine, catalyzed by glutamate-cysteine
ligase, and (b) addition of glycine (or, β-alanine, serine and glutamic
acid) to γ-glutamyl-cysteine, catalyzed by glutathione synthase.
GSH is involved in AsA-GSH cycle as a substrate for dehydroascor-
bate reductase which reduces dehydroascorbate to ascorbate
(Foyer & Noctor, 2011; Smirnoff, 200 0). Others have also noted a
higher accumulation of AsA and GSH induced by exogenous AsA
under heat (Kumar et al., 2011) and salt stress (Billah, Rohman,
Hossain, & Uddin, 2017).
Methionine is an indispensable building block for protein syn-
thesis. Higher methionine in AsA-fed leaves may be useful for the
synthesis of various stress proteins associated with FT induction
(Chen et al., 2015; Espevig, Xu, Aamlid, DaCosta, & Huang, 2012).
Arrigoni, Arrigoni-Liso, and Calabrese (1977) reported that AsA was
necessary to synthesize hydroxyproline-containing proteins, a cell
wall structural entity important for cell expansion/ growth (Cleland,
1968; Kavi Kishor, Hima Kumari, Sunlta, & Sreenivasulu, 2015; Ridge
& Osborne, 1971). Conceivably, higher methionine in AsA-fed leaves
may also be associated with small but significantly better leaf growth
of AsA-fed seedlings (Table 1). Methionine also serves as a substrate
for the synthesis of polyamines (Alcázar et al., 2011). Accumulation
of polyamines has been implicated in stress tolerance, including
freezing (Alcázar et al., 2011).
Phenylalanine, an aromatic amino acid, serves as a precursor
for a wide range of imp or tant secon da ry meta bolites (Tzin & Ga lili,
2010). One such metabolite, lignin, a strengthening component
of cell wall, is synthesized via phenylpropanoid/lignin biosyn-
thetic pathway (Vanholme, Demedts, Morreel, Ralph, & Boerjan,
2010). In the present study, AsA-fed leaves had lower levels of
phenylalanine. This may be due to either decreased synthesis or
increased consumption of phenylalanine, the latter presumably
for lignin biosynthesis. Lignin content was not measured in this
study. However, higher lignin content has been widely linked with
increased FT (Huner, Palta, Li, & Carter, 1981; Stefanowska, Kuras,
Kubacka-zebalska, & Kacperska, 1999). Cold acclimation-induced
upregulation of C3H gene (a key enzyme for lignin biosynthesis)
has also been repor ted for Rhododendron leaves (Wei et al., 2006).
Higher lignin content is also expec ted with greater tissue growth
as well as higher leaf DW; higher leaf area and DW/leaf area for
AsA-fed tissues in this study are in line with this notion. Future
study of lignin biosynthesis and content in AsA-fed tissues is war-
ranted to test above stated notion.
AsA-fed leaves had ~2.8-fold proline relative to F-control (spot
8; Fi gure 5b ; Table S1) . Proli n e, a com p atib l e solut e , has be en wi d ely
known to accumulate under stress conditions with roles in cellu-
lar osmotic adjustment, and membrane and protein stabilization
(Hay at et al. , 201 2). Its accu mulation has also bee n widel y reported
in cold-acclimated plants including spinach (Kaplan et al., 2004; Min
et al., 2018; Shin et al., 2018). Concordantly, AsA-fed plants were
also more freeze-tolerant in the present study. How AsA-feeding
causes proline accumulation is not known. However, a relatively
higher amount of glutamine and glutamic acid in AsA-fed leaves
compared with F-control (spot 2, 3; Figure 5b; Table 2) suggest s
stimulation of proline biosynthesis since glutamine is converted
into glutamic acid, a primary precursor of proline biosynthesis
(Forde & Lea, 2007; Hayat et al., 2012). Indeed, AsA-induced pro-
line accumulation has been reported in okra under drought (Amin
et al., 2009) and wheat under salt stress (Azzedine et al., 2011).
On the other hand, Hoque et al. (2007) reported that activity of
enzymes involved in AsA-GSH cycle, including APX, was stimulated
by exogenous proline in tobacco cultures under salt stress.
Leucine, a branched amino acid, was significantly higher in AsA-
fed leaves (spot 5; Figure 5b; Table 2). Although not significantly,
other branched amino acids, isoleucine and valine, also were higher
in these tissues compared with F-control (Table S1). Branched amino
acids serve as precursors for the biosynthesis of secondar y metab-
olites involved in various plant defenses (Bennett & Wallsgrove,
1994; Dixon, 2001). Also, upregulation of genes involved in second-
ary metabolism has been well correlated with improved FT (Hannah
et al., 2006). Hence, higher abundance of branched amino acids in
AsA-fed leaves may indicate higher level of secondary metabolites
specifically contributing to higher FT. However, this notion deserves
further confirmation.
10
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MIN et al.
Threonine (spot 9; Figure 5b; Table 2) was higher in AsA-fed
leaves relative to F-control, but no explanation is available at this
time for their role in F T induction.
4.4.2 | Carbohydrates
Fructose (as well as glucose) was less abundant in AsA-fed leaves
compared with F-control (spot 10; Figure 5b; Table 2). Reason for
this is unclear but may have resulted from decreased breakdown of
sucrose which accumulated at higher levels in AsA-fed (Table S1).
Higher sucrose in AsA-fed leaves may be associated with increased
FT due to its well established role as a compatible solute under des-
iccation stress (Bocian et al., 2015; Kaplan et al., 20 04).
AsA-fed leaves had higher abundance of galactinol and myo-ino-
sitol (spot 11, 12; Figure 5b; Table 2). Galactinol and myo-inositol
are involved in the biosynthesis of raffinose family oligosaccha-
rides (RFOs) (Kannan et al., 2016; Sengupta, Mukherjee, Basak, &
Majumder, 2015). RFOs also serve as compatible solutes under stress
conditions (Bocian et al., 2015; Kaplan et al., 20 04). Hincha, Zuther,
and Heyer (2003) noted that RFOs stabilized cellular membrane
under desiccation stress via sugar–membrane interaction. Although
RFOs were not detected in the present study due to technical lim-
itations (oligosaccharides were undetectable by GC-MS used here),
it may be reasonable that A sA-fed leaves have higher abundance of
RFOs which may contribute, in par t, to enhanced FT.
4.4.3 | Antioxidants
Alpha-tocopherol and gamma-tocopherol were ~2.0- and ~1.3-fold
of F-control, respectively (spot 15, 16; Figure 5b; Table S1), indeed
a substantially high accumulation. Alpha-tocopherol is a potent an-
tioxidant protecting membranes by scavenging singlet oxygen and
reacting with lipid peroxyl radicals, that is reducing lipid peroxida-
tion (Munné-Bosch, 2005; Sattler, Gilliland, Magallanes-Lundback,
FIGURE 6 Illustrative summary of the
effect of exogenous application of AsA
on leaf metabolome vis-à-vis improved
freezing tolerance (F T) of spinach (Spinacia
oleracea L. cv. Reflect); for explanation,
refer to “Conclusions.” AsA, ascorbic
acid; GSH, glutathione; SOD, superoxide
dismutase; CAT, catalase; APX, ascorbate
peroxidase; ROS, reactive ox ygen species;
Leu, leucine; Ile, isoleucine; Val, valine;
Phe, phenylalanine
|
11
MIN et al .
Pollard, & DellaPenna, 200 4), which may contribute to amelioration
of freeze injury. The exact mechanism of how A sA-feeding induces
accumulation of alpha-tocopherol is unclear. However, data from the
present study could provide tentative explanation as follows: to-
copherol biosynthesis requires phytyl-diphosphate which is derived
by phosphor ylation of free phytol (Soll & Schultz, 1981; Vom Dorp et
al., 2015). AsA-fed leaves, in the present study, had higher free phy-
tol levels than F-control (spot 18; Figure 5b; Table 2). Moreover, it
has been noted that tocopheroxyl radical, that is oxidized form of to-
copherol, is reduced by ascorbic acid and therefore, tocopherols and
AsA work collaboratively in controlling ROS levels (Munné-Bosch,
2005; Szarka, Tomasskovics, & Bánhegyi, 2012). AsA and GSH (dis-
cussed earlier) in conjunction with alpha-tocopherol constitute a
robust antioxidant machinery in AsA-fed leaves enabling greater re-
sistance to freeze-induced oxidative stress.
4.4.4 | TCA intermediates and other metabolites
Citric acid and malic acid were more abundant in AsA-fed leaves
compared with F-control (spots 13, 14; Figure 5b; Table 2); al-
though not significantly different, two other TCA intermediates,
fumaric acid and succinic acid, were also more abundant in AsA-
fed leaves (Table S1). TCA cycle is pivotal in producing energ y for
various biochemical processes and delivering carbon skeleton and
reducing equivalents (Meyer et al., 2007). Hence, this bigger pool
size of TCA metabolites in AsA-fed leaves may be associated with
accumulation of many useful metabolites which contribute to im-
proved FT.
GABA was higher in F-control compared with AsA-fed leaves
(spot 20; Figure 5b; Table 2). GABA is a four-carbon non-proteino-
genic amino acid requiring glutamic acid as a precursor for its syn-
thesis (Shelp, Bown, & McLean, 1999). In the present study, AsA-fed
leaves had substantially higher proline, which too requires glutamic
acid for its biosynthesis. Could it be that lower level of GABA in AsA-
fed ti ss ues res ulted from the lack of suff i ci ent pre cur so r? Thi s hy poth-
esis warrants fur ther confi rmation. Phosph oric acid was also higher in
F-control than A sA-fed leaves (spot 22; Figure 5b; Table 2) whereas
ferulic acid, glyceric acid, and urea were more abundant in AsA-fed
leaves than F-control (spots 19, 21, 23; Figure 5b; Table 2). No expla-
nation is available at this time for their specific role, if any, in FT.
5 | CONCLUSION
Su mmariz ed co n c l u s i ons ar e ill ustrate d in Figure 6 . As A-f e e d i n g of sp in-
ach seedlings enhanced activity of SOD, CAT, and APX, and bolstered
the accumulation of antioxidants (alpha- and gamma-tocopherol, AsA,
glutathione) and compatible solutes/osmolytes (proline, galactinol, and
myo-inositol). These changes may synergistically enhance FT via alle-
viating freezing-induced oxidative stress as well as protecting mem-
branes from freeze desiccation. Additional components of improved
FT of A sA-fed leaves can be enhanced secondary metabolite system
and lignin/ cell wall augmentation (as indicated by dashed arrows);
these two presumed changes are supported by increase in branched
amino acids (leucine, isoleucine, valine) and possibly higher consump-
tion of phenylalanine, respectively. Lastly, AsA-feeding induced small
but significant increase in leaf grow th is possibly a result of enhanced
expansion and/or division.
ACKNOWLEDGEMENTS
This journal paper of the Iowa Agriculture and Home Economics
Experiment Station, Ames, Iowa, Project no. 3601 was supported by
Hatch Act and State of Iowa Funds. Technical assistance by Mr. Peter
Lawlor (Manager, Horticulture Greenhouses) and Drs. Ann Perera
and Lucas Showman (W.M. Keck Metabolomics Laboratory), Iowa
State University is gratefully acknowledged.
CONFLICT OF INTEREST
The authors declare no conflict of interest associated with the work
described in this manuscript.
AUTHOR CONTRIBUTIONS
R.A. and K.M. jointly conceived the idea and designed experiments.
K.M. performed the experiment s and analyzed the data with help
from K.C. K.M., and R. A. jointly wrote the paper. R.A. provided all
financial support for this research.
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How to cite this article: Min K, Chen K, Arora R. A
metabolomics study of ascorbic acid-induced in situ freezing
tolerance in spinach (Spinacia oleracea L.). Plant Direct.
2020;4:1–13. https ://doi.org/10.1002/pld3.202
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