Exogenous spermidine is enhancing tomato tolerance to salinity–alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism
Li et al. BMC Plant Biology
Exogenous spermidine is enhancing tomato tolerance to salinity-alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism
Jianming Li 0 1 2
Lipan Hu 0 1 2
Li Zhang 1 2
Xiongbo Pan 1 2
Xiaohui Hu 1 2
0 Equal contributors
1 Key Laboratory of Protected Horticultural Engineering in Northwest, Ministry of Agriculture , Yangling 712100Shaanxi , China
2 College of Horticulture, Northwest A&F University , Yangling 712100Shaanxi , China
Background: Salinity-alkalinity stress is known to adversely affect a variety of processes in plants, thus inhibiting growth and decreasing crop yield. Polyamines protect plants against a variety of environmental stresses. However, whether exogenous spermidine increases the tolerance of tomato seedlings via effects on chloroplast antioxidant enzymes and chlorophyll metabolism is unknown. In this study, we examined the effect of exogenous spermidine on chlorophyll synthesis and degradation pathway intermediates and related enzyme activities, as well as chloroplast ultrastructure, gene expression, and antioxidants in salinity-alkalinity-stressed tomato seedlings. Results: Salinity-alkalinity stress disrupted chlorophyll metabolism and hindered uroorphyrinogen III conversion to protoporphyrin IX. These effects were more pronounced in seedlings of cultivar Zhongza No. 9 than cultivar Jinpengchaoguan. Under salinity-alkalinity stress, exogenous spermidine alleviated decreases in the contents of total chlorophyll and chlorophyll a and b in seedlings of both cultivars following 4 days of stress. With extended stress, exogenous spermidine reduced the accumulation of δ-aminolevulinic acid, porphobilinogen, and uroorphyrinogen III and increased the levels of protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide, suggesting that spermidine promotes the conversion of uroorphyrinogen III to protoporphyrin IX. The effect occurred earlier in cultivar Jinpengchaoguan than in cultivar Zhongza No. 9. Exogenous spermidine also alleviated the stress-induced increases in malondialdehyde content, superoxide radical generation rate, chlorophyllase activity, and expression of the chlorophyllase gene and the stress-induced decreases in the activities of antioxidant enzymes, antioxidants, and expression of the porphobilinogen deaminase gene. In addition, exogenous spermidine stabilized the chloroplast ultrastructure in stressed tomato seedlings. Conclusions: The tomato cultivars examined exhibited different capacities for responding to salinity-alkalinity stress. Exogenous spermidine triggers effective protection against damage induced by salinity-alkalinity stress in tomato seedlings, probably by maintaining chloroplast structural integrity and alleviating salinity-alkalinity-induced oxidative damage, most likely through regulation of chlorophyll metabolism and the enzymatic and non-enzymatic antioxidant systems in chloroplast. Exogenous spermidine also exerts positive effects at the transcription level, such as down-regulation of the expression of the chlorophyllase gene and up-regulation of the expression of the porphobilinogen deaminase gene.
Spermidine; Tomato; Salinity-alkalinity stress; Chloroplast; Chlorophyll precursor; Antioxidant system
Tomato (Solanum lycopersicum L.) is one of the most
widely cultivated vegetables in the world. However, tomato
production is negatively impacted by soil salinization and
alkalization, which frequently co–occur in nature and are
some of the most adverse environmental stresses to plants
and tomato in particular [
]. Salinity–alkalinity stress is
known to adversely affect a variety of processes in plants,
such as seed germination, ion uptake, stomata opening,
and photosynthetic rate . Our previous study showed
that salinity–alkalinity stress decreases tomato growth,
nitrogen metabolism [
], polyamine metabolism [
photosynthetic efficiency, which significantly impacts the
growth and development of plants.
Chlorophyll (Chl) receives solar energy in photosynthetic
antenna systems and mediates charge separation and
electron transport within reaction centers [
]. Chl is
essential for light harvesting and energy transduction
in photosynthesis. The Chl content determines
photosynthesis, which in turn determines plant growth and
development. The level of Chl is maintained by a
balance between Chl biosynthesis and degradation [
Previous research has found that salt stress disturbs
the balance between Chl biosynthesis and degradation,
thus altering the Chl content . The Chl synthesis
pathway is mediated by more than 17 enzymes [
Blockade of any step in the chlorophyll biosynthesis
pathway will cause a decline in Chl content.
Chlorophyllase (Chlase) plays an important role in chlorophyll
degradation. Regulation of the levels of Chl and its
derivatives, such as protochlorophyll (Pchl) and
protoporphyrin IX (Proto IX), is extremely important, because
these molecules are strong photosensitizers; that is,
when present in excess, they will generate reactive oxygen
species (ROS) [
]. ROS, in turn, may retard cell growth
or even cause cell death. Therefore, to maintain healthy
growth, plants must exert fine control over the entire
Chl metabolic process. Sun et al. reported that in
spinach cultivars undergoing seawater stress, the levels of
Chl b, Chl a, total Chl decreased significantly [
decreased chlorophyll may attribute to accumulate
much more ROS in chloroplast. ROS hinders the
transformation of porphobilinogen (PBG) to uroorphyrinogen
III (URO III) [
The accumulation of ROS is a general feature of
salinity stress that alters the antioxidation capacity of
cells, leading to oxidative damage [
] as well as ROS
]. Chloroplasts are major sites of ROS
generation under stress conditions [
]. To counteract
the toxicity of ROS, plants have highly efficient
antioxidative systems composed of both nonenzymatic
antioxidants and antioxidant enzymes. The non–enzymatic
antioxidants include ascorbate (AsA), glutathione (GSH),
carotenoids, flavanones, and anthocyanins, whereas
antioxidant enzymes include superoxide dismutase
(SOD), catalase (CAT), ascorbate peroxidase (APX),
monodehydroascorbate reductase (MDHAR),
dehydroascorbate reductase (DHAR), glutathione reductase
(GR), glutathione peroxidase (GPX), and glutathione
]. It has been hypothesized that the
accumulation of ROS in chloroplasts due to
salinity–alkalinity stress can be mitigated by enhancing the
antioxidant capacity [
]. The ascorbate–glutathione cycle
appears to play an important role in maintaining the
redox status in plant cells, especially under abiotic
Polyamines are a class of biogenic amines that exert
multiple in vivo effects on cellular processes in most
]. Considerable research indicates that
polyamines play an important role in protecting plants
against abiotic stress [
]. Compared with other
polyamines (PAs), spermidine (Spd) more effectively
alleviates the adverse effects of salinity–alkalinity stress
. We found that exogenous Spd treatment can regulate
the metabolic status of polyamines caused by
salinity–alkalinity stress, and eventually enhance tolerance of tomato
plants to salinity–alkalinity stress [
]. PAs catabolism is
tightly linked to ROS generation, because amino oxidases
generate hydrogen peroxide (H2O2), which mediates ROS
]. In a previous study, we found that
exogenous Spd can alleviate the decrease of root dry weight
caused by salinity–alkalinity stress [
]. However, whether a
close relationship exists between exogenous Spd and
increased stress tolerance in tomato seedlings due to
induction of antioxidant enzymes and altered chlorophyll
metabolism in chloroplasts is unclear.
In this study, we examined the effects of exogenous
Spd on the antioxidant system in chloroplasts in salinity–
alkalinity–stressed tomato seedlings. We also examined
the effects of exogenous Spd on the Chl synthesis and
degradation pathways to evaluate the role of exogenous Spd
in Chl metabolism. Specifically, we examined the levels of
Chl and related molecules, the activities of various
enzymes, the expression of relevant genes, and changes in
chloroplast ultrastructure. The overall objective of the
present study was to elucidate the mechanism of Spd–
mediated protection of the photochemical pathways
and structures from salinity–alkalinity–induced damage
in tomato seedlings.
We found that exogenous Spd is effective in triggering
protection against cellular and macromolecular damage
in tomato seedlings during salinity–alkalinity stress.
Exogenous Spd showed positive effects on maintaining the
structural integrity of chloroplasts. This may be because
exogenous Spd alleviate salinity–alkalinity–induced
oxidative damage, through regulation of Chl metabolism
and enzymatic and non–enzymatic antioxidant systems
in the chloroplasts.
The impact of Spd on Chl content in salinity–alkalinity–
stressed tomato seedlings
As shown in Fig. 1, the contents of Chl a, Chl b and
total Chl in salinity–alkalinity–stress (S)–treated two
tomato cultivars increased early and decreased later, and
peaked on fourth day, except for Chl b and total Chl
contents in cv. Jinpengchaoguan (cv. JP) peaked on the
second day. Compared with the control, the Chl content
trended upward for 4 days after the initiation of salinity–
alkalinity conditions, but then the levels declined and
became significantly lower compared with CK–treated
plants. During salinity–alkalinity stress, this trend was
suppressed to some extent by salinity–alkalinity plus
Spd (SS) treatment, as after 4 days of SS treatment,
the decreases in Chl a, Chl b, and total Chl content in
stressed seedlings of both cultivars were alleviated
Effect of Spd on Chl precursor content in salinity– alkalinity–stressed tomato seedlings
The level of ALA (δ–aminolevulinic acid) in both
cultivars under CK conditions rose during the early period of
treatment and then decreased, peaking on day 6 and day
4 after treatment in cv. Zhongza No.9 (cv. ZZ) and cv.
JP, respectively. ALA levels in S–treated seedlings were
significantly higher than in CK–treated seedlings in both
cultivars. However, exogenous Spd significantly reduced
the stress–induced increase in ALA level. In addition,
cv. JP had higher ALA levels than cv. ZZ during
treatment days 0 to 4, but after day 4, cv. JP had lower ALA
levels than cv. ZZ (Fig. 2).
The PBG and uroorphyrinogen III (URO III) contents
in both cultivars grown under CK conditions exhibited a
similar but slightly different trend as ALA (Fig. 3). Under
salinity–alkalinity stress, the PBG content significantly
increased and peaked on treatment day 6. The stress–
induced accumulation of PBG was alleviated by
exogenous Spd in cv. ZZ. Stress also caused significant
increase in the URO III content in both cv. ZZ and cv.
JP after treatment day 2, peaking on day 6 (Fig. 3). SS
treatment reduced the stress–induced increase in URO III
content. In addition, cv. JP had higher PBG content and
lower URO III content than cv. ZZ under the same
treatment conditions (Fig. 3).
Under salinity–alkalinity stress, the Proto IX and
Mg–Proto IX contents in both cultivars exhibited
similar changes, rising early but declining later, with
maximum levels occurring on day 4 (Fig. 4). Compared
with S treatment, SS treatment led to a significant increase
in the Proto IX content, except on day 6. SS treatment also
significantly increased the Mg–Proto IX and Pchl levels,
except on day 4 (Fig. 4).
Effect of Spd on Chlase activity in salinity–alkalinity–
stressed tomato seedlings
Under CK conditions, Chlase activity remained relatively
stable and low in both cultivars (Fig. 5). An increase in
Chlase activity was evident on the second day after
exposure to salinity–alkalinity stress. With the exception
of day 4 for cv. ZZ and day 2 for cv. JP, the Chlase activity
in both cultivars was higher with S treatment than with SS
treatment. Throughout the stress period, no obvious
difference was observed in Chlase activity in SS–treated cv.
ZZ and cv. JP seedlings.
Effect of Spd on Malondialdehyde (MDA) content and O2−
generation rate in salinity–alkalinity–stressed tomato
MDA is the final product of lipid peroxidation, and the
MDA level increased in the chloroplasts of both tomato
cultivars under stress conditions compare with CK
treatment, reaching the highest level on day 6 (Fig. 6). Under
salinity–alkalinity stress with application of exogenous
Spd, the MDA content in the chloroplasts was
significantly reduced in both cultivars. 6 days after treatment,
compared with S treatment, MDA content in SS
treatment of plants decreased by 25.01 % (for cv. Zhongza
No.9) and 33.79 % (for cv. Jinpengchaoguan),
respectively (Fig. 6).
ROS levels are indicators of stress in plants. The rate
of O2− generation was higher in the chloroplasts of
stressed tomato seedlings compared with CK–treated
seedlings, and the rate was higher in cv. JP than in cv. ZZ
during the experimental period, except on day 4 (Fig. 6).
However, the O2− generation rate was significantly lower
in the chloroplasts of SS–treated seedlings of both
cultivars subjected to salinity–alkalinity stress. Furthermore,
the amplitude of the change in O2− generation rate was
higher in cv. ZZ than in cv. JP when seedlings were treated
with exogenous Spd under conditions of salinity–alkalinity
stress (Fig. 6).
Effect of Spd on the chloroplast antioxidant system of
salinity–alkalinity–stressed tomato seedlings
The activities of superoxide dismutase (SOD), ascorbate
peroxidase (APX), and glutathione reductase (GR)
increased significantly in chloroplasts of seedlings of the
both tomato cultivars during exposure to
salinity–alkalinity stress, peaking on day 2 in cv. ZZ seedlings and on days
6, 4, and 6, in cv. JP seedlings, respectively (Figs. 7 and 8).
The monodehydroascorbate reductase (MDHAR) activity
in the chloroplasts of stressed tomato seedlings of both
cultivars was significantly higher than that of CK–treated
seedlings (Fig. 8). Compared with CK–treated seedlings,
those subjected to salinity–alkalinity stress exhibited
significantlly reduced dehydroascorbate reductase (DHAR)
activity in cv. ZZ and increased DHAR activity in cv. JP
(Fig. 8). SS treatment resulted in marked increases in
SOD, MDAHR, DHAR, and GR activities in the
chloroplasts of stressed seedlings, and the activity levels were
higher than those in S–stressed plants (Figs. 7 and 8).
Compared with S treatment, SS treatment also increased
the activity of APX in chloroplasts in seedlings of both
tomato cultivars. APX activity increased early and declined
during the later stages of treatment, with the exception of
day 2. This effect was more obvious in cv. JP seedlings
After salinity–alkalinity stress, the ascorbic acid (AsA)
content decreased early and then increased. The AsA
concentration in S treatment was lower than that of
the control in chloroplasts of both cv. ZZ and cv. JP
seedlings (cv. ZZ, 6.21 % versus 47.54 %; cv. JP, 26.86 %
versus 56.07 %; Fig. 9). Compared with CK treatment, cv.
ZZ seedlings subjected to S treament exhibited
significantly lower reduced glutathione (GSH) concent, whereas
no obvious change in GSH content was observed in cv. JP
seedlings (Fig. 9). SS treatment resulted in a marked
increase and similar pattern of change in both the
AsA and GSH contents in the chloroplasts of both
tomato seedlings. In addition, the extent of the increase
in GSH content in cv. ZZ chloroplasts was higher than
that in cv. JP chloroplasts, despite on day 0 and day 6
Effect of Spd on Chloroplast ultrastructure of salinity–
alkalinity–stressed tomato seedlings
Typical spindle chloroplasts were observed in both
tomato seedlings under CK treatment, with intact double
membranes and a regular arrangement of granal and
stromal thylakoids (Fig. 10a–d). Under salinity–alkalinity
stress, the chloroplast structures in cv. ZZ seedlings
were heavily damaged; the chloroplasts were swollen,
the stroma thylakoid stack and grana thylakoid were
blurred, and the lamellar structure was destroyed
(Fig. 10e and f ). The extent of damage to the
chloroplast structures of cv. JP seedlings was less than that
observed in cv. ZZ seedlings, with some stroma and
grana thylakoid structures remaining completely intact
(Fig. 10g and h).
The number of plastoglobuli was increased and the
plastoglobular volume was abnormally large in S–
stressed tomato seedlings of both cultivars, suggesting
that the plants were undergoing significant stress.
Exogenous Spd alleviated the salinity–alkalinity–induced
damage to the chloroplast structure, with a more
normal chloroplast ultrastructure observed in SS–treated
seedlings. Fewer platoglobuli and lower plastoglobular
volume were observed in seedlings subjected to SS
treatment versus those subjected to S treatment
The relative expression of chloroplast genes (rbcL, psbA,
psbC, and psbD) and Chlase was relatively low in
CKtreated plants (Fig. 11). Salinity–alkalinity stress
enhanced the expression of rbcL, psbA, psbC, psbD, and
Chlase, with significantly higher levels of expression of
these genes in both tomato cultivars compared with
the CK. Under salinity–alkalinity stress, SS treatment
resulted in higher levels of rbcL, psbA, psbC, and psbD
expression in S–stressed cv. ZZ seedlings and lower levels of
expression of these genes in S–stressed cv. JP seedlings
(Fig. 11). Under salinity–alkalinity stress, SS treatment
significantly down–regulated expression of the Chlase gene
in both cultivars (Fig. 11e), and the extent of this down–
regulation was greater in cv. ZZ than in cv. JP seedlings. S
treatment also markedly down–regulated expression of
the pbgD in both cultivars (Fig. 11f ), but this change was
partly alleviated by exogenous Spd in comparison to S–
Chl is directly involved in the absorption, transmission,
distribution, and transformation of light energy in
plants, facilitating the synthesis of organic material from
photosynthetic products. In the present study, we found
that the Chl a content in stressed cv. JP tomato seedlings
was higher than that in control plants from days 2 to 8.
The Chl a content in stressed cv. ZZ seedlings and the Chl
b and total Chl content in stressed seedlings of both
tomato cultivars were lower than in controls after 4 days of
stress treatment (Fig. 1). The Chl content increased during
the early stress period (days 0–4) and declined during the
later stress period (days 4–8), consistent with the report of
Romero et al. [
]. These results suggest that transient
salinity–alkalinity stress stimulates the accumulation of Chl,
but as the duration of stress increases, the Chl content
Chl content is affected by the rates of Chl synthesis and
]. The Chl biosynthesis pathway in higher
plants is complex, mediated by more than 17 enzymes
]. The conversion of glutamic acid into Mg–proto IX
occurs in the chloroplast, and the conversion of Mg–proto
IX into Chl b occurs in the thylakoid membrane [
Disruption of any of these reaction steps may result in
significant accumulation of intermediates produced in steps
prior to the point of disruption and a significant decrease
in the amount of products produced in subsequent steps.
Chen et al. found that seawater stress hinders the
transformation of PBG to URO III in spinach [
]. Wang et al.
suggested that UV–B disrupts Chl synthesis at the point
of ALA conversion to PBG [
]. This difference may be
crop– or cultivar–specific [
]. In the present study,
salinity–alkalinity stress induced the over–accumulation of
ALA, PBG, and URO III in seedlings of both tomato
cultivars throughout the experimental period (Figs. 2 and 3).
Salinity–alkalinity stress also caused an increase in the
Proto IX content from days 0–2 in cv. ZZ seedlings and
days 0–4 in cv. JP seedlings and an increase in the
contents of Mg–proto IX and Pchl in both tomato cultivars
from days 0–4, relative to the controls. However, between
days 6 and 8, levels of Proto IX, Mg–proto IX and Pchl
declined and were significantly lower than in controls (Fig. 4).
These results indicated that salinity–alkalinity stress
disrupted Chl synthesis at the step of URO III conversion
into Proto IX, which can be attributed to damage to the
thylakoid membrane [
]. These results also indicated that
salinity–alkalinity stress upset the Chl biosynthesis balance
differently in cv. ZZ and cv. JP seedlings.
An increase in Chl content could also be due to a
decrease in Chl degradation or to an increase in Chl
synthesis. In the present study, stress led to an increase in
Chl content between days 0 and 4 and a decrease in Chl
content thereafter, whereas more severe salinity–alkalinity
stress stimulated the activity of Chlase over time (Fig. 5).
These results indicate that Chlase accelerates the
degradation of Chl in tomato during long–term salinity–alkalinity
stress, which could explain in part why long–term stress
leads to disorganization of chloroplasts followed by
increased contact of Chl with Chlase, in turn leading to
an increase in Chlase activity. Maintenance of the
structural integrity of chloroplasts is necessary for the
conversion of light energy during photosynthesis. Fang et
al. hypothesized that chloroplast degradation is
responsible for the decrease in Chlase activity [
analysis of the ultrastructure of chloroplasts in the present
study indicated that salinity–alkalinity stress induced
destruction of the chloroplast envelope and increased the
number of plastoglobuli and aberrations in the thylakoid
membrane (Fig. 10). These results demonstrate that
although Chl degradation is undoubtedly responsible
at least in part for the decline in Chl content, during
severe stress this process is not dependent on the
activity of Chlase, suggesting that an alternative pathway
must be involved. The decrease in Chl content may be
attributed to molecular–level Chl damage, resulting in
decrease in the efficiency of light energy absorption and
transmission in the chloroplast.
Polyamines exert positive effects on photosynthetic
efficiency under stress conditions due to their acid–
neutralizing and antioxidant properties, as well as their
membrane– and cell wall–stabilizing activity [
with a high net positive charge can stabilize photosystem
II (PSII) proteins such as D1 and D2 under
photo–inhibition conditions [
]. PAs binding to membrane proteins
may stabilize the protein structure during stress and
consequently preserve photosynthetic activity. Exogenous Spd
alleviated the negative effects of salinity–alkalinity stress
on Chl content (Fig. 1) and the damage to the chloroplast
photosynthetic apparatus, resulting in a more normal
chloroplast ultrastructure in Spd–treated plants (Fig. 10).
These results indicate that exogenous Spd may play a
protective role in chloroplasts, ensuring that a sufficient
supply of enzymes are available for conversion of URO III to
Proto IX, thus promoting Chl synthesis and enhancing
Chl a and Chl b levels in tomato seedlings grown under
salinity–alkalinity stress. Under salinity–alkalinity stress,
exogenous Spd reduced the stress–induced increase in
Chlase activity and the ALA, PBG, and URO III levels in
both tomato cultivars; the URO III content in SS–treated
cv. ZZ and cv. JP seedlings declined on days 4 and 2,
respectively (Figs. 2 and 3), suggesting that the effect of Spd
on stress–induced changes in Chl synthesis differs
between cultivars, with the effect of Spd apparent earlier in
the more tolerant cultivar (cv. JP) than in the more
sensitive cultivar (cv. ZZ). Exogenous Spd also attenuated the
increase in Chlase activity after day 4 of the stress period,
maintaining the Chl a, Chl b, and total Chl levels, contrary
Fig. 11 Effect of exogenous Spd on the expression of chlorophyll metabolism enzyme genes. cv. ZZ, cv. Zhongza No. 9; cv. JP, cv.
Jinpengchaoguan; CK, 1/2 Hoagland’s solution; S, 75 mM saline–alkaline solution (NaCl:Na2SO4:NaHCO3:Na2CO3 = 1:9:9:1); SS, sprayed with
0.25 mM Spd and treated with 75 mM saline–alkaline solution. a, c and e represent cv. Zhongza No.9; b, d and f represent cv. Jinpengchaoguan
to the trend observed in stressed plants not treated with
Spd. These results indicate that exogenous Spd decreases
the accumulation of URO III by promoting the conversion
of Proto IX to Chl, thus overcoming the stress–associated
blockade of URO III conversion to Proto IX. These effects
may be attributed to the stabilization of chloroplast
structure by Spd, which ensures that sufficient enzymes
are available for conversion of URO III to Proto IX,
thereby promoting Chl synthesis.
The psbA gene plays a critical role in the de novo
synthesis of D1 protein and the repair of photo–damaged
PSII components [
]. A previous study reported that
salinity stress reduces the transcript levels of several
chloroplastic genes (rbcL, psbA, psbB, and psbE) [
the present study, salinity–alkalinity stress led to
increases in the levels of transcripts of the rbcL, psbA,
psbC, and psbD genes. This increase was more
pronounced in cv. JP than cv. ZZ (Fig. 11). Our results show
that exogenous Spd leads to down–regulation of the
expression of rbcL, psbA, psbC, psbD and Chlase and the
maintenance of near–normal transcript levels in cv. JP,
in agreement with the results of Chattopadhayay et al.
]. However, exogenous Spd up–regulation of the
expression of rbcL, psbA, psbC, psbD and pbgD in cv. ZZ,
which may be one of the reasons for cv. JP was more
tolerant to salinity–alkalinity stress than cv. ZZ. The
expression of pbgD was down–regulated and that of
Chlase was up–regulated under salinity–alkalinity stress.
However, the down–regulation of pbgD and
up–regulation of Chlase was alleviated by exogenous Spd (Fig. 11).
These results provide conclusive evidence that
exogenous Spd has a positive effect in preventing the loss of
Chl in stressed plants by promoting Chl synthesis and
alleviating Chl degradation.
Of all plant organelles, chloroplasts seem to be the
most sensitive to salt stress and are the major source of
ROS. ROS such as O2−, hydroxyl ions (OH−), and H2O2,
may oxidize proteins, lipids, and nucleic acids. This may
result in abnormalities at the cellular level when plants
are exposed to environmental stresses [
]. This may
particularly affect photosystem I through the oxidation of
iron oxide reducing protein [
]. ROS can be generated by
the direct transfer of the excitation energy from Chl to
produce singlet oxygen or by oxygen reduction through the
Mehler reaction in the chloroplasts, which leads to
membrane lipid peroxidation [
]. Environmental stresses such
as high salinity aggravate photo–inhibition and over a long
period may induce photo–oxidization, resulting in
accumulation of ROS in chloroplasts. Over–accumulation of ROS
leads to enzyme inactivation, pigment decolorization,
protein degradation, and lipid peroxidation, ultimately
inhibiting plant growth. In the present study, seedlings of two
tomato cultivars exhibited increased chloroplast ROS
accumulation under salinity–alkalinity stress (Fig. 6). In plants,
antioxidant systems readily scavenge ROS to protect cells
from oxidative damage. However, under stressful
conditions, the production of ROS may overwhelm the capacity
of the antioxidant system, thereby resulting in oxidative
stress symptoms [
]. In an efficiently functioning
antioxidant system, a high level of antioxidant enzyme activity and
high levels of non–enzymatic components are maintained.
In the present study, salinity–alkalinity stress led to
enhanced chloroplast SOD, GR, APX, DHAR, and MDHAR
activities in seedlings of both tomato cultivars (Figs. 7 and
8). An increase in MDHAR activity can provide reducing
equivalents for APX, which can maintain the AsA–GSH
cycle. MDHAR activity was higher than DHAR activity in
our study (Fig. 8), indicating that AsA regeneration may act
through MDHAR reduction to monodehydroascorbate.
However, the ability of DHAR and MDHAR to catalyze
AsA regeneration is limited, resulting in reduced AsA
content. In the present study, AsA regeneration under
salinity–alkalinity stress was primarily driven by APX
in cv. ZZ and by MDHAR in cv. JP. Glutathione acts as
a substrate for glutathione peroxidase and is considered
the critical component of the AsA–GSH cycle for
maintaining intracellular defenses against ROS–induced
oxidative damage [
]. The increase in GR activity directly
promotes conversion of oxidized glutathione to GSH,
which eliminates H2O2 and reduces the accumulation of
ROS in chloroplasts .
PAs are also well known for their positive effects on
photosynthetic efficiency under stress conditions due to
their acid–neutralizing and antioxidant properties. Spd
contains highly protonated amino and imino groups and
may conjugate with other negatively charged organic
molecules such as nucleic acids, proteins, and
phospholipids. Such binding is important for the stabilization of
the thylakoid membranes and prevention of the hydrolysis
of photosynthetic proteins [
]. In the present study,
application of exogenous Spd also resulted in suppression of
physiological damage associated with salinity–alkalinity
stress, as shown by the lower MDA content and O2−
generation rate (Fig. 6), thus confirming previous observations
that exogenous PAs significantly improve the physiological
status of stressed plants [
]. Moreover, the positive
influence of Spd on MDA content and O2− generation rate
differed between cultivars, possibly indicating that Spd has
a more beneficial effect on sensitive cultivars grown under
stress conditions. In the present study, application of
exogenous Spd significantly increased the activities of the
ROS–scavenging enzymes SOD, APX, and GR in the
chloroplasts of salinity–alkalinity–stressed tomato seedlings.
Moreover, exogenous Spd induced the synthesis of
antioxidant metabolites that provide additional capability to
neutralize the toxic effects of ROS generated during salt
]. We observed that Spd increased the contents
of AsA and GSH in chloroplasts, which enhanced the
salinity tolerance of the photosynthetic apparatus. The
contents of antioxidant metabolites and activities of enzymes
in salinity–alkalinity–stressed chloroplasts were enhanced
by Spd application, consistent with the observed effects of
Spd in reducing the O2− generation rate and MDA content
in tomato seedling chloroplasts. These results showed that
Spd alleviates chloroplast membrane injury resulting from
salinity–alkalinity stress through an increase in ROS
scavenging, indicating that Spd may protect PS II from
In conclusion, the two tomato cultivars examined in the
present study exhibited different response capacities to
salinity–alkalinity stress. Exogenous Spd is effective in
triggering protection against cellular and macromolecular
damage in tomato seedlings during salinity–alkalinity stress,
probably by maintaining the structural integrity of
chloroplasts and alleviating salinity–alkalinity–induced oxidative
damage, most likely through regulation of Chl metabolism
and enzymatic and non–enzymatic antioxidant systems in
the chloroplasts. Exogenous Spd alleviates the
down–regulation of pbgD and up–regulation of Chlase expression
under stress conditions, which may promote an increase in
Chl content. Exogenous Spd also exhibits positive effects in
maintaining the expression of the rbcL and psbA genes.
Exogenous Spd decreases the accumulation of URO III and
promotes the conversion of Proto IX to Chl, thus alleviating
the stress–associated blockade of URO III conversion to
Proto IX. This effect was more pronounced in the sensitive
cultivar than the tolerant cultivar and earlier in the more
tolerant cultivar than in the more sensitive cultivar.
Plant culture, salinity–alkalinity stress, and sample
Six true–leaves–old tomato (Solanum lycopersicum L.)
seedlings of cv. JP (tolerant to salinity–alkalinity stress)
and cv. ZZ (sensitive to salinity–alkalinity stress) were
initially grown in one–half–strength Hoagland’s solution
in an environmentally controlled greenhouse, as
described by Zhang et al. [
]. After 7 days of pre–culture
under controlled conditions, the seedlings were treated
with 75 mM salinity–alkalinity solution (molar ratio of
NaCl:Na2SO4:NaHCO3:Na2CO3 = 1:9:9:1) and the
foliage was sprayed with 0.25 mM Spd. The experimental
plots included three treatments: (a) CK, half–strength
Hoagland’s nutrient solution + 0 mM Spd; (b) S, 75 mM
salinity–alkalinity + 0 mM Spd; and (c) SS, 75 mM
salinity–alkalinity + 0.25 mM Spd.
The containers were arranged in completely randomized
blocks, with four replicates per treatment. The nutrient
solutions were renewed every 2 days. After 0, 2, 4, 6, and
8 days of stress treatment at the final concentration, the
second fully expanded leaf from the top of each plant was
used to analyze chlorophyll content, chlorophyll
metabolism, and antioxidant enzymes in the chloroplasts. The
relative expression of genes in the tomato seedlings was
analyzed after 4 days of treatment. Changes in chloroplast
ultrastructure were evaluated after 6 days of treatment.
Determination of Chl precursors
Chl a, Chl b, and Chl (a + b) levels were estimated following
the method of Holden [
]. Proto IX, Mg–proto IX, and
Pchl were extracted using a mixture of acetone:ammonia
(1 %) (4:1) and the contents were determined based on the
absorbance of the extracts at 575, 590, and 628 nm,
]. Fresh leaves were homogenized on ice in
Tris–HCl (pH 7.2), the homogenate was centrifuged at
5000 × g for 15 min at 4 °C, and the URO III content in the
supernatant was determined by the method of Bogorad
]. For PBG determination, leaf samples were
homogenized with Tris–HCl buffer (pH 8.0) containing 100 mM
Tris and 50 mM mercaptoethanol, and the homogenate
was centrifuged at 8000 × g for 15 min at 4 °C. Next, 2 mL
of the supernatant or standard solution was mixed with
2 mL of freshly prepared Ehrlich’s reagent, and after
30 min, the mixture was used to determine the PBG
content at 555 nm according to the method of Bogorad [
For the determination of ALA, fresh leaves were
homogenized in acetic sodium buffer (pH 4.6), the homogenate was
extracted in a boiling water bath for 15 min and then
centrifuged at l0,000 × g for 20 min at 4 °C, and the
ALA content in the resulting supernatant was determined
according to the method of Richard [
]. The ALA
content was based on reference to an ALA–HCl standard
(Sigma–Aldrich, St. Louis, MO, USA).
Assay of Chlase activity
Samples of frozen tomato leaves were ground on ice
with pre–chilled acetone (−20 °C). The homogenate was
centrifuged at 3000 × g for 5 min at 4 °C, and the pellet
was collected. The cold acetone extraction procedures
were repeated three times in the same manner to remove
all traces of Chls and carotenoids. The resulting acetone
powder was dried under nitrogen gas and stored at −20 °C
until use [
]. The acetone powder was homogenized in
5 mL of extraction buffer (50 mM potassium phosphate
[pH 7.0], 50 mM KCl, and 0.24 % Triton X–100) for 1 h at
30 °C in a water bath. After centrifugation at 12,000 × g for
10 min at 4 °C, the supernatant was used for the enzyme
assay. A total of 4 g of spinach fresh mass (FM) were
homogenized in 40 mL of acetone:water (80:20, vol/vol) at
4 °C using an omnimixer. The suspension was centrifuged
at 9000 × g, and 40 mL of petroleum ether was added to
the supernatant to extract the Chls. The ether was then
evaporated under N2, and the extracted Chls were
dissolved in 4–5 mL of acetone. To assay Chlase activity,
1 mL of supernatant, 0.5 mL of reaction buffer (50 mM
sodium phosphate [pH 7.0] and 0.24 % Triton X–100),
and 2 mL of Chl substrate were mixed, incubated for
30 min at 40 °C, and then poured into 5 mL of
hexane:acetone (7:3) pre–cooled in ice water. The resulting
mixture was stirred vigorously until an emulsion formed,
and then centrifuged at 6000 × g for 5 min at 4 °C. The
upper phase of the resulting supernatant contained the
remaining Chl, whereas the lower phase contained the
chlorophyllide. Chlase activity was monitored by
measuring the absorbance of the lower phase at 663 nm.
Enzyme activity was expressed as the increment of optical
density at 663 nm per minute under the test conditions
Transmission electron microscopy of chloroplasts
The second fully expanded leaves from the top of the
plants were randomly selected for electron microscopic
examination. The leaf samples were sectioned and then
examined using a HITACHI HT7700 transmission
electron microscope according to the method described by
Hu et al. [
Isolation of intact chloroplasts, MDA and O2− generation
Intact chloroplasts were isolated using the method
described by Shu et al. [
]. MDA was measured according
to the method of Xu et al. [
]. The O2− generation rate
was determined according to the method of Elstner
and Heupel [
] with a slight modification. The 100 μL
chloroplast supernatants were added into 200 μL of
ice–cold PBS buffer (65 mM, pH 7.8) and 300 μL
hydroxylamine chlorhydrate, placed at 30 °C for 20 min,
extracted by diethyl ether, and then centrifuged at
3000 × g for 5 min at room temperature. Three hundred
microliters of the extract was added to a tube, and 500 μL
17 mM sulfanilamide and 500 μL 7 mM α–naphthylamine
were added. The mixture was then placed at 30 °C for
another 20 min before mixing with 2.25 mL pure ether. The
absorbance was measured at 530 nm and the O2−
generation rate was calculated from a NaNO2 standard curve.
Extraction of chloroplast antioxidant enzymes and
A 3–mL aliquot of Chl–containing supernatant was mixed
with 3 mL of ice–cold HEPES buffer (25 mM, pH 7.8)
containing 0.2 mM ethylene diamine tetraacetic acid and
2 % (w/v) poly vinyl pyrrolidone. The mixture was then
centrifuged at 4 °C at 12,000 × g for 20 min. The resulting
supernatant was used to assay the antioxidant enzyme
activity and determine the content of antioxidants (AsA
Measurement of SOD, APX, GR, MDHAR, and DHAR
SOD activity was assayed by monitoring SOD–mediated
inhibition of the photochemical reduction of nitro blue
tetrazolium (NBT) [
]. One unit of SOD activity was
defined as the amount of enzyme required for 50 %
inhibition of the reduction of NBT, as monitored at
APX activity was assayed using the method of Nakano
and Asada by monitoring the ascorbate oxidation rate at
290 nm [
GR activity was measured by tracking NADPH
oxidation by monitoring the decrease in absorbance at 340 nm
over 3 min [
The activities of MDHAR and DHAR were assayed
according to the method described by Zhang et al., with a
slight modification [
]. MDHAR activity was assayed at
340 nm in a 1–mL sample containing 50 mM HEPES–
KOH (pH 7.6), 25 mM AsA, 1 mM NADH, 0.5 units of
ascorbate oxidase, and 50 μL of enzyme extract. DHAR
activity was assayed at 265 nm in a 2.9–mL sample
containing 100 mM HEPES–KOH (pH 7.6), 25 mM reduced
GSH, 2 mM dehydroascorbate, and 50 μL of enzyme
Protein was determined according to the method of
Bradford, using bovine serum albumin as a standard [
Determination of AsA and GSH content
Ascorbate was determined according to the method of
Shu et al. [
], with a minor modification. The reaction
mixture contained 200 μL of 5 % trichloroacetic acid,
100 μL of 0.4 % H3PO4–ethanol, 100 μL of 0.03 %
FeCl3–ethanol, 200 μL of 0.5 % BP–ethanol, and 300 μL
of extract. The sample was incubated at 40 °C for 1 h,
after which the absorbance was measured at 534 nm.
AsA content was calculated based on an ascorbic acid
GSH content was assayed as described by Li and
]. GSH was determined by subtraction of
oxidized glutathione from total glutathione.
Expression of chlorophyll metabolism enzyme genes
Total RNA was extracted from tomato leaves using an
E.Z.N.A.® Plant RNA Kit (Omega Bio–Tek, Doraville,
GA, USA) according to the manufacturer’s instructions.
The total RNA was then reverse–transcribed using a
PrimeScriptTM RT reagent kit with gDNA Eraser (Takara,
Shiga, Japan) in a 20–μL reaction mixture containing
1 μL of total RNA from each individual sample. Real–time
PCR was performed on a CFX96™ real–time PCR cycler
(Bio–Rad, Hercules, CA, USA) and a SYBR Premix Ex Taq
(TliRNaseH Plus) Kit (Takara). Initial denaturation at 95 °C
for 30 s was followed by 40 cycles of 95 °C for 5 s, 58 °C for
30 s, and a melting curve of 65–95 °C. Primers for the actin
gene were used as an internal control. Primers for psbA
and actin were designed as described by Wu et al. [
Primers for the pbgD and Chlase genes were designed using
Primer3, version 4.0.0 (website software), with the primer
length set at 20 − 24 bp; melting temperature of 58 − 62 °C;
CG content, 30 − 70 %; and product size, 150–250 bp. All
samples were analyzed three times.
All experiments were performed with at least three
replicates. Data represent the mean ± SE. Data were analyzed
with SAS 9.0 software (SAS Institute, Cary, NC, USA)
using Duncan’s multiple range tests, with P < 0.05 defining
significance. Different letters in table indicate significant
differences between means.
ALA: δ–aminolevulinic acid; APX: ascorbateperoxidase; AsA: ascorbic acid;
Chl: chlorophyll; Chlase: chlorophyllase; CK: control; cv. JP: cultivar
Jinpengchaoguan; cv. ZZ: cultivar Zhongza No. 9; DHAR: dehydroascorbate
reductase; GL: grana lamellae; GR: glutathione reductase; GSH: glutathione;
H2O2: hydrogen peroxide; MDA: malondialdehyde;
MDHAR: monodehydroascorbate reductase; Mg–proto IX: Mg–protoporphyrin
IX; O2–⋅: superoxide radical; OH−: hydroxyl ion; P: plastoglobuli; PAs: polyamines;
PBG: porphobilinogen; pbgD: porphobilinogen deaminase;
Pchl: protochlorophyll; Proto IX: protoporphyrin IX; PSII: photosystem II;
ROS: reactive oxygen species; S: salinity–alkalinity–stress treatment; SG: starch
grains; SL: stroma lamellae; SOD: superoxide dismutase; SS: salinity–alkalinity plus
Spd treatment; URO III: uroorphyrinogen III.
The authors declare that they have no competing interests.
XH, JL and LH conceived the study. XH and LH designed the experiments.
LH carried out the molecular genetic studies, participated in the expression
of chlorophyll metabolism enzyme genes and revised the manuscript.
LZ and XP carried out the chloroplast antioxidant enzymes activities and
antioxidant contents and chlorophyll metabolism. XH and JL interpreted the
experimental data. All authors read and approved the final manuscript.
This work was supported by grants from science and technology project of
Shaanxi province (2015KTTSNY03–03; 2015NY102) and the Scientific Research
Special Fund of Northwest Agriculture & Forestry University (QN2013018,
2452015138). The authors are grateful to Yanyan Zhao, PhD (NWSUAF), for
advice regarding laboratory techniques and to Xiaoting Zhou, PhD (NWSUAF),
for data analysis methods.
1. Zhang Y , Hu XH , Shi Y , Zou ZR , Yan F , Zhao YY , et al. Beneficial role of exogenous spermidine on nitrogen metabolism in tomato seedlings exposed to saline-alkaline stress . J Am Soc Horticultural Sci . 2013 ; 138 ( 1 ): 38 - 49 .
2. Hu L , Xiang L , Zhang L , Zhou X , Zou Z , Hu X. The photoprotective role of spermidine in tomato seedlings under salinity-alkalinity stress . PLoS One . 2014 ; 9 ( 10 ): e110855 .
3. Iqbal M , Ashraf M. Changes in hormonal balance: a possible mechanism of pre‐sowing chilling‐induced salt tolerance in spring wheat . J Agronomy Crop Sci . 2010 ; 196 ( 6 ): 440 - 54 .
4. Hu XH , Zhang Y , Shi Y , Zhang Z , Zou ZR , Zhang H , et al. Effect of exogenous spermidine on polyamine content and metabolism in tomato exposed to salinity-alkalinity mixed stress . Plant Physiol Biochem . 2012 ; 57 : 200 - 9 .
5. Tanaka A , Tanaka R . Chlorophyll metabolism . Curr Opin Plant Biol . 2006 ; 9 ( 3 ): 248 - 55 .
6. Pattanayak GK , Biswal AK , Reddy VS , Tripathy BC . Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants . Biochem Biophys Res Commun . 2005 ; 326 ( 2 ): 466 - 71 .
7. Rüdiger W. Chlorophyll metabolism: from outer space down to the molecular level . Phytochemistry . 1997 ; 46 ( 7 ): 1151 - 67 .
8. Sairam R , Srivastava G. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress . Plant Sci . 2002 ; 162 ( 6 ): 897 - 904 .
9. Von Wettstein D , Gough S , Kannangara CG . Chlorophyll biosynthesis . Plant Cell . 1995 ; 7 ( 7 ): 1039 .
10. Sun J , Jia Y , Guo S , Li J , Shu S. Resistance of spinach plants to seawater stress is correlated with higher activity of xanthophyll cycle and better maintenance of chlorophyll metabolism . Photosynthetica . 2010 ; 48 ( 4 ): 567 - 79 .
11. Tanou G , Job C , Rajjou L , Arc E , Belghazi M , Diamantidis G , et al. Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity . Plant J . 2009 ; 60 ( 5 ): 795 - 804 .
12. Foyer CH , Noctor G. Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications . Antioxid Redox Signal . 2009 ; 11 ( 4 ): 861 - 905 .
13. Xu S , Li J , Zhang X , Wei H , Cui L . Effects of heat acclimation pretreatment on changes of membrane lipid peroxidation, antioxidant metabolites, and ultrastructure of chloroplasts in two cool-season turfgrass species under heat stress . Environ Exp Bot . 2006 ; 56 ( 3 ): 274 - 85 .
14. Hasanuzzaman M , Fujita M. Exogenous sodium nitroprusside alleviates arsenic-induced oxidative stress in wheat (Triticum aestivum L.) seedlings by enhancing antioxidant defense and glyoxalase system . Ecotoxicology . 2013 ; 22 ( 3 ): 584 - 96 .
15. Saxena M , Roy SD , Singla-Pareek SL , Sopory SK , Bhalla-Sarin N . Overexpression of the glyoxalase II gene leads to enhanced salinity tolerance in Brassica juncea . Open Plant Sci J . 2011 ; 5 : 23 - 8 .
16. Bouchereau A , Aziz A , Larher F , Martin-Tanguy J . Polyamines and environmental challenges: recent development . Plant Sci . 1999 ; 140 ( 2 ): 103 - 25 .
17. Tanou G , Ziogas V , Belghazi M , Christou A , Filippou P , Job D , et al. Polyamines reprogram oxidative and nitrosative status and the proteome of citrus plants exposed to salinity stress . Plant Cell Environ . 2014 ; 37 ( 4 ): 864 - 85 .
18. Duan J , Li J , Guo S , Kang Y. Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances short-term salinity tolerance . J Plant Physiol . 2008 ; 165 ( 15 ): 1620 - 35 .
19. Shu S , Yuan LY , Guo SR , Sun J , Yuan YH . Effects of exogenous spermine on chlorophyll fluorescence, antioxidant system and ultrastructure of chloroplasts in Cucumis sativus L. under salt stress . Plant Physiol Biochem . 2013 ; 63 : 209 - 16 .
20. Romero-Aranda R , Soria T , Cuartero J . Tomato plant -water uptake and plant-water relationships under saline growth conditions . Plant Sci . 2001 ; 160 ( 2 ): 265 - 72 .
21. Yu M , Hu CXWYH . Effects of molybdenum on the precursors of chlorophyll biosynthesis in winter wheat cultivars under low temperature . Scientia Agricultura Sinica . 2006 ; 39 ( 4 ): 702 - 8 .
22. Porra RJ . Recent progress in porphyrin and chlorophyll biosynthesis . Photochem Photobiol . 1997 ; 65 ( 3 ): 492 - 516 .
23. Chen X , Sun J , Guo S , Gao P , Du J . Chlorophyll metabolism of spinach leaves under seawater stress . Acta Botanica Boreali-Occidentalia Sinica . 2012 ; 09 : 1781 - 7 .
24. Wang X . Research advances about effects of enhanced UV-B radiation on plants and ecosystems . Acta Botanica Boreali-occidentalia Sinica . 2002 ; 03 : 670 - 81 .
25. Santos CV . Regulation of chlorophyll biosynthesis and degradation by salt stress in sunflower leaves . Sci Hortic . 2004 ; 103 ( 1 ): 93 - 9 .
26. Hao SQ , Liu SQ , Zhang ZK , Cui HR , Duan JF , Chen Q. Characteristics of chlorophyll metabolism and chlorophyll fluorescence in the silvered leaf of summer squash . Acta Horticulturae Sinica . 2009 ; 6 : 021 .
27. Fang Z , Bouwkamp JC , Solomos T. Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L. J Exp Bot . 1998 ; 49 ( 320 ): 503 - 10 .
28. Mapelli S , Brambilla I , Radyukina N , Ivanov YV , Kartashov A , Reggiani R , et al. Free and bound polyamines changes in different plants as a consequence of UV-B light irradiation . Gen Appl Plant Physiol . 2008 ; 34 : 55 - 66 .
29. Hamdani S , Gauthier A , Msilini N , Carpentier R . Positive charges of polyamines protect PSII in isolated thylakoid membranes during photoinhibitory conditions . Plant Cell Physiol . 2011 ; 52 ( 5 ): 866 - 73 .
30. Andersson B , Aro EM . Photodamage and D1 protein turnover in photosystem II . In: Regulation of photosynthesis. Springer; 2001 : 377 - 393 .
31. Chattopadhayay MK , Tiwari BS , Chattopadhyay G , Bose A , Sengupta DN , Ghosh B . Protective role of exogenous polyamines on salinity‐stressed rice (Oryza sativa) plants . Physiol Plant . 2002 ; 116 ( 2 ): 192 - 9 .
32. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions . Plant Physiol . 2006 ; 141 ( 2 ): 391 - 6 .
33. Stepien P , Klobus G . Antioxidant defense in the leaves of C3 and C4 plants under salinity stress . Physiol Plant . 2005 ; 125 ( 1 ): 31 - 40 .
34. Alam M , Nahar K , Hasanuzzaman M , Fujita M. Alleviation of osmotic stress in Brassica napus, B. campestris, and B. juncea by ascorbic acid application . Biologia Plantarum . 2014 ; 58 ( 4 ): 697 - 708 .
35. Mullineaux P , Rausch T . Glutathione, photosynthesis and the redox regulation of stress-responsive gene expression . Photosynth Res . 2005 ; 86 ( 3 ): 459 - 74 .
36. Gill SS , Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants . Plant Physiol Biochem . 2010 ; 48 ( 12 ): 909 - 30 .
37. Hasanuzzaman M , Hossain MA , Fujita M. Exogenous selenium pretreatment protects rapeseed seedlings from cadmium-induced oxidative stress by upregulating antioxidant defense and methylglyoxal detoxification systems . Biol Trace Elem Res . 2012 ; 149 ( 2 ): 248 - 61 .
38. Ahmad P , Azooz MM , Prasad MNV . Ecophysiology and responses of plants under salt stress: Springer Science & Business Media . 2012 .
39. Sen G , Eryilmaz IE , Ozakca D. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina . Phytochemistry . 2014 ; 98 : 54 - 9 .
40. Yiu JC , Juang LD , Fang DYT , Liu CW , Wu SJ . Exogenous putrescine reduces flooding-induced oxidative damage by increasing the antioxidant properties of Welsh onion . Sci Hortic . 2009 ; 120 ( 3 ): 306 - 14 .
41. Goodwin TW . Chemistry and biochemistry of plant pigments . London: Academic; 1965 . p. 461 .
42. Hodgins R , Van Huystee R. Rapid simultaneous estimation of protoporphyrin and Mg-porphyrins in higher plants . J Plant Physiol . 1986 ; 125 ( 3 ): 311 - 23 .
43. Bogorad L. Methods in Enzymology, vol. 5 . San Diego, New York, Berkeley, Boston, London, Sydney, Tokyo, Toronto Academic Press; 1962 . p. 885 - 895
44. Morton RA . Biochemical spectroscopy: A. Hilger; London; Bristol 1975 . Vol. 1 .
45. Chen MM , Chao PY , Huang MY , Yang JH , Yang ZW , Lin KH , et al. Chlorophyllase activity in green and non-green tissues of variegated plants . S Afr J Bot . 2012 ; 81 : 44 - 9 .
46. Costa ML , Civello PM , Chaves AR , Martínez GA . Effect of ethephon and 6-benzylaminopurine on chlorophyll degrading enzymes and a peroxidaselinked chlorophyll bleaching during post-harvest senescence of broccoli (Brassica oleracea L .) at 20 C. Postharvest Biol Technol. 2005 ; 35 ( 2 ): 191 - 9 .
47. Xu PL , Guo YK , Bai JG , Shang L , Wang XJ . Effects of long-term chilling on ultrastructure and antioxidant activity in leaves of two cucumber cultivars under low light . Physiol Plant . 2008 ; 132 ( 4 ): 467 - 78 .
48. Elstner EF , Heupel A . Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase . Anal Biochem . 1976 ; 70 ( 2 ): 616 - 20 .
49. Giannopolitis CN , Ries SK . Superoxide dismutases I. Occurrence in higher plants . Plant Physiol . 1977 ; 59 ( 2 ): 309 - 14 .
50. Nakano Y , Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts . Plant Cell Physiol . 1981 ; 22 ( 5 ): 867 - 80 .
51. Gupta AS , Webb RP , Holaday AS , Allen RD . Overexpression of superoxide dismutase protects plants from oxidative stress (induction of ascorbate peroxidase in superoxide dismutase-overexpressing plants) . Plant Physiol . 1993 ; 103 ( 4 ): 1067 - 73 .
52. Zhang J , Niu J , Duan Y , Zhang M , Liu J , Li P , et al. Photoprotection mechanism in the 'Fuji'apple peel at different levels of photooxidative sunburn . Physiol Plant . 2015 ; 154 ( 1 ): 54 - 65 .
53. Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem . 1976 ; 72 ( 1 ): 248 - 54 .
54. Li P , Cheng L. The shaded side of apple fruit becomes more sensitive to photoinhibition with fruit development . Physiol Plant . 2008 ; 134 ( 2 ): 282 - 92 .
55. Wu Q , Su N , Shen W , Cui J . Analyzing photosynthetic activity and growth of Solanum lycopersicum seedlings exposed to different light qualities . Acta Physiologiae Plantarum . 2014 ; 36 ( 6 ): 1411 - 20 .