A Low Glutathione Redox State Couples with a Decreased
Ascorbate Redox Ratio to Accelerate Flowering in
Oncidium Orchid
Institute of Plant Biology, National Taiwan University, Taipei, Taiwan. No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan
*Corresponding author: E-mail, [email protected]; Fax, +886-2-23622703.
(Received August 20, 2015; Accepted December 18, 2015)
Glutathione (GSH) plays multiple roles in plants, including
stress defense and regulation of growth/development.
Previous studies have demonstrated that the ascorbate
(AsA) redox state is involved in flowering initiation in
Oncidium orchid. In this study, we discovered that a significantly decreased GSH content and GSH redox ratio are
correlated with a decline in the AsA redox state during
flowering initiation and high ambient temperatureinduced flowering. At the same time, the expression level
and enzymatic activity of GSH redox-regulated genes, glutathione reductase (GR1), and the GSH biosynthesis genes gglutamylcysteine synthetase (GSH1) and glutathione
synthase (GSH2), are down-regulated. Elevating dehydroascorbate (DHA) content in Oncidium by artificial addition of
DHA resulted in a decreased AsA and GSH redox ratio, and
enhanced dehydroascorbate reductase (DHAR) activity.
This demonstrated that the lower GSH redox state could
be influenced by the lower AsA redox ratio. Moreover, exogenous application of buthionine sulfoximine (BSO), to
inhibit GSH biosynthesis, and glutathione disulfide
(GSSG), to decrease the GSH redox ratio, also caused
early flowering. However, spraying plants with GSH
increased the GSH redox ratio and delayed flowering.
Furthermore, transgenic Arabidopsis overexpressing
Oncidium GSH1, GSH2 and GR1 displayed a high GSH
redox ratio as well as delayed flowering under high ambient
temperature treatment, while pad2, cad2 and gr1 mutants
exhibited early flowering and a low GSH redox ratio. In
conclusion, our results provide evidence that the decreased
GSH redox state is linked to the decline in the AsA redox
ratio and mediated by down-regulated expression of GSH
metabolism-related genes to affect flowering time in
Oncidium orchid.
Keywords: AsA–GSH cycle Flowering High-ambient temperature Oncidium Redox regulation.
Abbreviations: AP1, APETALA 1; APX, ascorbate peroxidase;
AsA, ascorbate; BSO, buthionine sulfoximine; CO, CONSTANS;
cytAPX1, cytosolic ascorbate peroxidase 1; DHA, dehydroascorbate;
DHAR,
dehydroascorbate
reductase;
FLC,
FLOWERING LOCUS C; FT, FLOWERING LOCUS T; GI,
GIGANTEA; GR, glutathione reductase; GSH, glutathione;
H2O2, hydrogen peroxide; LFY, LEAFY; OE, overexpression;
ROS, reactive oxygen species; SD, short day; WT, wild type.
The nucleotide sequences reported in this paper have been
submitted to GenBank with the following accession numbers:
OgDHAR1 (KR149279), OgGSH1 (KR140025), OgGSH2
(KR140026) and OgGR1 (KR140024).
Introduction
Plants throughout their life cycle, and particularly in unfavorable environmental conditions, are affected by accumulation
of reactive oxygen species (ROS). An appropriate increase in
hydrogen peroxide (H2O2), a ROS, triggers redox signaling
pathways that regulate several biological processes, including
development, growth and various defense mechanisms
against environmental stress (Foyer and Noctor 2005, Spoel
and van Ooijen 2014). However, it may accumulate to toxic
levels, leading to severe damage or plant cell death (Baxter
et al. 2014).
Ascorbate (AsA) and glutathione (GSH) are the two major
antioxidant compounds found in plant cells and are known to
play a central role in redox regulation via the AsA–GSH cycle,
which maintains cellular redox homeostasis (Foyer and Noctor
2011). The oxidation of AsA to dehydroascorbate (DHA) by
ascorbate peroxidase (APX; EC 1.11.1.11) is a key reaction to
remove H2O2 and eliminate oxidation damage in the cell. GSH
is used as an electron donor by dehydroascorbate reductase
(DHAR; EC 1.8.5.1) to reconvert DHA to the reduced form of
AsA. The oxidized form of GSH, glutathione disulfide (GSSG),
can be recycled to GSH by glutathione reductase (GR; EC
1.6.4.2) using reduced NADPH as the electron donor. The
AsA–GSH cycle includes three interdependent redox couples:
AsA/DHA, GSH/GSSG and NADPH/NADP; the biochemical
and physiological functions of these have been intensively studied (Noctor 2006). In addition to the effect of abiotic and
biotic environmental factors on H2O2 and the antioxidation
system, these are also influenced by the developmental stage
of the plants (Considine and Foyer 2014).
GSH is a multifunctional metabolite that interacts with several molecules through thiol–disulfide exchange. Changes in
the GSH redox ratio regulate several transcription factors and
Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206, Advance Access publication on 6 January 2016,
available online at www.pcp.oxfordjournals.org
! The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Regular Paper
Dan-Chu Chin, Chia-Chi Hsieh, Hsin-Yi Lin and Kai-Wun Yeh*
D.-C. Chin et al. | AsA–GSH cycle and floral initiation
protein activity, and influence cellular redox potential, which
has been identified as a stress indicator (Noctor et al. 2012,
Dietz 2014). Previous studies indicated that GSH and its
redox state are involved in floral initiation in Arabidopsis,
Eustoma grandiflorum and wheat. The Arabidopsis fca
mutant has a high GSH level and late-flowering phenotype,
with a high expression level of FLOWERING LOCUS C (FLC),
which is a flowering repressor. However, the flowering time
could be accelerated by treatment with the GSH biosynthesis
inhibitor buthionine sulfoximine (BSO), in both the wild type
(WT) and fca mutant (Ogawa et al. 2001, Ogawa 2005).
Conversely, low levels of GSH were associated with a vernalization-related flowering pathway and resulted in delayed flowering in E. grandiflorum (Yanagida et al. 2004). Overexpression of
the GSH1 gene in Arabidopsis with a high content of the oxidized form GSH strongly suggests that GSSG is a key regulator in
the response to low temperature and flowering. Moreover, the
level of the flowering repressor FLC in GSH1 overexpression lines
was higher than in the WT, without seed vernalization treatment, indicating that GSH metabolism is involved in the FLCregulated flowering pathway. Thus, the higher GSH level in the
35S-GSH1-overexpressing Arabidopsis line was associated with
delayed flowering (Hatano-Iwasaki and Ogawa 2012). Plants of
spring and winter wheat varieties were treated with various
antioxidants, redox-related chemicals and osmotic stress to
alter the GSH content and redox state. The results revealed
that the changes in the GSH redox state influenced the transcriptional level of freezing tolerance-related genes and the
floral gene, ZCCT2, which is involved in the vernalization pathway (Gulyás et al. 2014). These reports demonstrate the coordination of GSH and its redox state in response to environmental factors to regulate flowering in plants. It raises a central
question of how the environment fluctuations adjust endogenous GSH and which redox state, determined by both the biosynthesis and recycling of the oxidized form of GSH, triggers the
induction of flowering.
In our previous study, we found that the AsA redox ratio
(AsA/DHA) is dramatically decreased in the bolting period
during Oncidium growth (Chin et al. 2014). In addition, a
prolonged high ambient temperature treatment (30 C) of
Oncidium plants can promote early bolting. Evidence revealed that the decreased AsA redox ratio occurring at the
high ambient temperature was a critical factor for the transition from vegetative to bolting stage. The lower AsA redox
ratio was mainly due to the elevated activity of cytosolic
ascorbate peroxidase 1 (cytAPX1), which was activated to
scavenge ROS at the high ambient temperature. Our results
demonstrate that Oncidium responds to high ambient temperature by elevation of the ROS level and a decrease in the
AsA redox ratio. The decreased AsA redox ratio, in turn, acts
as a signal to initiate flowering. In the present study,
we investigated the correlation between AsA/DHA and
GSH/GSSG redox states, as well as flowering initiation of
Oncidium orchid. We suggested that the change in GSH
redox state associated with flowering initiation in
Oncidium is co-ordinated with the AsA redox state through
the AsA–GSH cycle.
424
Results
A low glutathione redox state (GSH/GSSG) occurs
during phase transition in Oncidium
Oncidium orchids grown under regular greenhouse conditions
were employed to study the change in GSH associated with
flowering initiation. A previous report revealed that a decreased
AsA level, high DHA content and lower AsA redox ratio
occurred at the natural bolting stage (Shen et al. 2009). Here,
the GSH and GSSG content and the GSH redox ratio were
measured at various growth stages, including the mature
pseudobulb (vegetative growth; V), bolting (B), flowering (F)
and next generation (NG) stage (Fig. 1A). As shown in Fig. 1B,
oxidation and reduction of AsA and GSH alternate to regulate
the cell redox balance and comprise the AsA–GSH pathway in
plants. The content of GSH was much less in B and F stages,
compared with V and NG stages; however, the oxidized form of
GSH, GSSG, was not significantly different in the various developmental stages (Fig. 1C). The GSH redox ratio at the B and F
stages was one-third that at the V and NG stages (Fig. 1D).
Furthermore, a higher H2O2 content was measured in B and F
stages compared with V and NG stages, suggesting that an
increased H2O2 level and low GSH redox ratio were in parallel
with aging-dependent flowering process (Fig. 1E).
Further investigation of the expression of genes involved in
redox in the AsA–GSH cycle and GSH biosynthesis was carried
out. Data revealed that both DHAR1 and GR1 displayed a lower
transcription level and lower enzymatic activity in the B and F
stage, compared with the V and NG stage (Fig. 1F, G). Likewise,
the GSH biosynthesis genes, i.e. g-EC synthetase (GSH1) and
glutathione synthase (GSH2), also showed down-regulation at
the B and F stage (Fig. 1H). The results confirmed that the
down-regulated gene expression level and enzymatic activity
of the relevant genes in AsA–GSH and GSH biosynthesis corresponded to a lower GSH content and GSH redox ratio in the
bolting and flowering stages.
A low GSH redox ratio (GSH/GSSG) is associated
with a decreased AsA redox ratio (AsA/DHA)
during the high ambient temperature-induced
flowering process in Oncidium
In our previous study, we established that 14 d prolonged high
ambient temperature (30 C) exposure promoted Oncidium
early flowering through the activation of cytAPX1 to oxidize
AsA for scavenging of H2O2. A striking reduction in the ascorbate level and a decreased AsA redox ratio were detected
during flowering initiation after the 14 d 30 C treatment
(Chin et al. 2014). In the present study, we analyzed both
AsA and GSH levels and redox states in Oncidium treated
with high ambient temperature (30 C). The AsA level and
AsA redox ratio (AsA/DHA) were significantly decreased from
4 h to 14 d, compared with levels at 20 C (Fig. 2A, E).
Concurrently, the level of DHA, the oxidized form of AsA,
increased (Fig. 2C). The changes in GSH and GSSG content
and the GSH redox ratio at 30 C from 4 h to 14 d were similar
to the changes in the AsA and DHA level, and redox state,
Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206
Fig. 1 Correlation of glutathione metabolism during developmental stages of Oncidium. (A) Life cycle of Oncidium. Growth stage of (i) young
plantlet (seedling); (ii) unsheathing stage, (iii) mature pseudobulb; (iv) bolting stage with floral bud emerging from the base of the pseudobulb;
(v) flowering stage with an inflorescence developing at the base of the pseudobulb; (vi) next generation stage with an adventitious bud emerging
(continued)
425
D.-C. Chin et al. | AsA–GSH cycle and floral initiation
respectively (Fig. 2B, D, F). Furthermore, the content of H2O2
accumulated in 30 C, suggesting that the decreased redox
ratios of AsA and GSH occurred simultaneously and in conjunction under the high ambient temperature-mediated oxidative stress, and acted synergistically to affect Oncidium
flowering time (Fig. 2G).
Assays of the expression pattern of the genes and enzyme activities involved in the AsA–GSH cycle revealed that
DHAR activity was higher in the 14 d 30 C high ambient
temperature treatment (Fig. 3A), due to the higher metabolic
activity present at higher temperature. In contrast, another key
gene involved in the AsA–GSH cycle, GR1, as well as the GSH
biosynthetic genes, GSH1 and GSH2, were down-regulated under
the 30 C high ambient temperature condition (Fig. 3B, C). The
low expression of GR1, GSH1 and GSH2 suggested that downregulation of GSH biosynthesis and the co-ordinated regeneration led to decreased GSH production and a decreased GSH
redox ratio under the 30 C condition.
The GSH redox state is linked to the AsA redox
state
We have previously shown that treatment with 4 mM DHA
cause a decreased AsA redox ratio and early flowering in
Oncidium (Chin et al. 2014). To validate the finding that the
GSH redox state is tightly linked to the upstream AsA redox
state, Oncidium orchid plants were exogenously sprayed with
AsA (100 mM) or DHA (4 mM) for 28 d. A noticeable decrease
over time in GSH content was observed after DHA treatment,
and a significant increase in GSSG content at day 14, compared
with mock and AsA treatments (Fig. 4A, B). In addition, the
GSH redox ratio (GSH/GSSG) with DHA treatment was much
lower than the redox ratio with AsA or mock treatment at day
14, and the activity of GSH-dependent DHAR significantly
increased with DHA treatment (Fig. 4C, D). This indicates
that endogenous GSH is consumed as a reducer when excess
DHA is reduced to AsA by DHAR (Fig. 1B).
A low GSH redox state is the consequence of
down-regulation of GSH biosynthesis and
regeneration
To establish further the relationship between the GSH redox
state and flowering time, the GSH redox status was altered by
exogenous application of BSO (a GSH biosynthetic inhibitor),
GSSG or GSH. Oncidium plants at the unsheathing growth stage
were treated for 28 d and then grown for a further 60 d. The
plants showed significantly earlier bolting with either GSSG or
BSO treatment (21.11 ± 1.6% and 64.44 ± 12.6% bolting, respectively) at the beginning of day 14, compared with mock
or GSH treatment (Table 1). On day 28, 51.55 ± 6.3% and
73.33 ± 9.4% of the plants were observed to be bolting in the
GSSG and BSO treatment groups, respectively. However, there
were no bolting plants in the GSH treatment group, and
12.04 ± 2.4% bolting was observed in the mock treatment
group. On the final 60th day, the GSSG treatment group
showed 78.88 ± 12.6% bolting, and nearly 100% (93.33 ± 5.4%)
bolting was observed in the BSO treatment group (Table 1;
Fig. 5A). In contrast, a much longer period of time was required
for the mock and GSH treatments to induce bolting. The quality of cut flowers in the BSO and GSH treatment groups showed
no significant difference compared with the mock group.
However, GSSG treatment causes a reduction in the length of
floral stalks (Supplementary Fig. S1).
The exogenous application of BSO and GSSG, which reduce
the endogenous GSH level and GSH redox ratio (GSH/GSSG),
validated the hypothesis that a lower GSH redox state is effective in reducing the time to flowering. A higher level of the
reduced form of GSH was present in the GSH treatment, and
the GSH content in the BSO treatment was lowest in the first
14 d. During 14–28 d of treatment, the GSH content in the BSO
and GSSG treatment groups was markedly decreased and was
significantly lower than in the mock and GSH treatment
groups. In contrast, the GSH level remained high in the GSH
treatment group throughout the treatment period (Fig. 5B).
The oxidized form of GSH, GSSG, significantly increased in the
GSSG treatment group at day 14. At day 28, however, there
were no significant differences in GSSG content between all
treatments, even though GSSG levels gradually increased
during the 28 d period of treatment (Fig. 5C). The GSH level
was highest in the GSH treatment group and lowest in the BSO
treatment group. In addition, a higher GSSG level was observed
in the GSSG treatment group. This led to the highest GSH redox
ratio in the GSH treatment group and lower redox ratios in the
GSSG and BSO treatment groups, compared with the mock
teatment (Fig. 5D). Moreover, compared with the mock treatment, the level of AsA and the AsA redox ratio (AsA/DHA)
were lower in Oncidium treated for 14 d with BSO and GSSG,
where DHA content were the most predominantly high. In
contrast, an increased level of AsA and a decreased content
of DHA resulting in a higher AsA redox ratio were observed in
GSH-treated Oncidium (Fig. 5E, F).
Fig. 1 Continued
from the opposite side of the base of the pseudobulb; and (vii) a new plantlet developed from the adventitious bud at the base of the pseudobulb. (B)
Model of the AsA–GSH cycle and GSH biosynthesis pathway. The associated enzymes and antioxidants involved in the AsA–GSH cycle and GSH
biosynthesis are indicated. Ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) are the key enzymes that
link the redox state of the antioxidants in the AsA–GSH cycle. g-Glutamylcysteine synthetase (g-ECS) and glutathione synthase (Glus), encoded by GSH1
and GSH2, play crucial roles in glutathione biosynthesis. g-EC: g-glutamylcysteine. (C, D and E) The GSH and GSSG content varied with the GSH redox
ratio (GSH/GSSG) and the H2O2 content at different developmental stages, i.e. vegetative growth (V), bolting (B), flowering (F) and next generation
growth (NG), respectively. (F and G) The expression (gray bar) and enzymatic activity (white bar) of DHAR and GR during various developmental stages.
(H) The expression of GSH biosynthesis genes, GSH1 and GSH2, from vegetative to reproductive growth stages. Transcript levels were calculated and
normalized with respect to Oncidium ACTIN mRNA. Bars represent the SEM (n = 6).
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Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206
Fig. 2 Effect of temperature on the content of AsA/GSH and DHA/GSSG, and the AsA/GSH redox ratio in Oncidium. Oncidium plants were
grown at 20 and 30 C for 14 d, and pseudobulb tissue was sampled at 0, 4 and 24 h, and 3, 7 and 14 d to determine changes in the following: (A
and B) total AsA and GSH content; (C and D) DHA and GSSG content; (E and F) AsA and GSH redox ratio (AsA/DHA; GSH/GSSG). (G) H2O2
content. Bars indicate the SEM (n = 6).
427
D.-C. Chin et al. | AsA–GSH cycle and floral initiation
Analysis of flowering-related genes, including photoperiodic
floral genes, GIGANTEA (GI), CONSTANS (CO) and FLOWERING
LOCUS T (FT), as well as floral identity genes, APETALA 1 (AP1)
and LEAFY (LFY), in relation to the time of bolting in each
treatment demonstrated that GI, FT, LFY and AP1 were significantly up-regulated under GSSG and BSO treatment (>2.5fold) compared with mock and GSH treatment (Fig. 5G, H).
GSH biosynthesis genes (GSH1 and GSH2) and
glutathione reductase (GR1) contribute to the
regulation of GSH redox homeostasis in the
AsA–GSH cycle and affect flowering time
To verify whether GSH redox changes associated with the time
of flowering are mediated by products of glutathione synthase
(GSH1 and GSH2) and glutathione reductase (GR1) genes in
the AsA–GSH pathway (Figs. 1G, H, 3B, C), we isolated the
full length of GSH1, GSH2 and GR1 from Oncidium (OgGSH1,
OgGSH2 and OgGR1, respectively). The subcellular localization
analysis indicated that OgGSH1, OgGSH2 and OgGR1 were
cytosolic form enzymes (Supplementary Fig. S2). In parallel,
transgenic Arabidopsis plants ectopically overexpressing
OgGSH1,
OgGSH2
and
OgGR1
were
generated
(Supplementary Fig. S3). Transgenic Arabidopsis lines with
high expression of the ectopically overexpressed genes were
assayed. When the independent Arabidopsis transgenic lines
were grown under short day (SD) conditions (8/16 h photoperiod) at 22 C for 6 weeks, and then transferred to 30 C for
14 d, OgGSH1-overexpression (OE), OgGSH2-OE and OgGR1-OE
lines displayed delayed flowering, with an increased number of
rosette leaves, compared with the WT (Col-0) grown under the
same conditions (Fig. 6A–F). However, no significant difference
in flowering time was observed between the transgenic
Arabidopsis and WT plants grown at 22 C (Supplementary
Fig. S4A–F).
Two independent GSH1-deficient Arabidopsis mutants,
pad2 and cad2, as well as the GR-deficient mutant, gr1, were
grown at 22 C under SD conditions (8/16 h photoperiod) for 6
weeks and then transferred to 30 C. An early flowering phenotype was observed in all mutant plants compared with the WT
(Fig. 6G, H). However, the early flowering phenotype was not
observed when these mutant lines were grown continuously at
22 C (Supplementary Fig. S4G, H). These data suggested that
GSH1 and GR1 accelerate Arabidopsis flowering under high
ambient temperature.
In an attempt to unravel the correlation between GSH redox
state and flowering initiation, analysis of the content of GSH
and GSSG, and the GSH redox ratio (GSH/GSSG) in Arabidopsis
WT, homozygous lines OgGSH1-OE, OgGSH2-OE and OgGR1OE, as well as the deficient mutants cad2, pad2 and gr1 was
performed. The data showed that a significantly higher GSH
content, lower level of GSSG and higher GSH redox ratio were
present in OgGSH1-OE, OgGSH2-OE and OgGR1-OE Arabidopsis
plants under high ambient temperature, i.e. 30 C (Fig.7A–F).
The mutant lines pad2 and cad2, however, displayed a significantly opposite effect, with a lower level of GSH, higher content
of GSSG and low GSH redox ratio (Fig. 7G, H). In contrast, there
428
Fig. 3 Effect of temperature on the transcription level and enzymatic
activity of DHAR and GR, and the GSH1 and GSH2 expression level in
Oncidium. Oncidium plants at the ‘unsheathing’ stage were grown at
20 C for 1 week and then placed at 20 C continuously or moved to
30 C for 14 d. The following analyses were carried out. (A and B) The
expression profile (gray bar) and enzymatic activity (white bar) of
DHAR and GR at 20 and 30 C. (C) Comparison of gene expression
levels of GSH biosynthesis genes, GSH1 and GSH2, at 20 and 30 C.
Transcript levels were calculated and normalized with respect to
Oncidium ACTIN mRNA. Bars indicate the SEM (n = 6).
is no significant difference in GSH content in the gr1 mutant
compared with the WT, but it did show a high level of GSSG
with a low GSH/GSSG redox ratio at 30 C. However, it is
noticeable that there were no significant differences in the
GSH/GSSG redox ratio or GSSG content among pad2 and
cad2 mutant and WT plants at 22 C (Supplementary Fig.
S5), and these lines all flowered at the same time at 22 C
(Supplementary Fig. S4). These data confirm that GSH1,
Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206
GSH2 and GR1 contributed to the balance of the GSH level and
redox status and this was associated with a change in flowering
time.
Discussion
Fig. 4 Effect of changes in the AsA redox ratio by AsA/ DHA treatment on the GSH redox state and enzymatic activity of DHAR
in Oncidium. Plants at the ‘unsheathing’ stage were first grown at
20 C for 1 week, and then were continuously sprayed with 100 mM
AsA or 4 mM DHA in buffer or were subjected to mock treatment
(buffer only) for 28 d to examine: (A) GSH content, (B) GSSG content,
(C) GSH redox ratio (GSH/GSSG) and (D) DHAR activity. Bars indicate
the SEM (n = 6).
The present study examined the effect of the redox state of the
GSH pool (GSH + GSSG) on induction of flowering in Oncidium
and the mechanisms involved in the AsA–GSH cycle. The GSH
content, as well as the GSH redox ratio (GSH/GSSG), in pseudobulb tissue was significantly decreased at bolting (B) and flowering (F) stages, but was relatively high in the vegetative (V) and
axillary bud developmental stages (NG) (Fig. 1C, D). The alternation of the GSH level is associated with the transition from
vegetative to reproductive phases. Moreover, the changes in
GSH redox ratio are strongly linked with the upstream decrease
in AsA content and its redox ratio (Wang et al. 2008, Shen et al.
2009).
Several pathways, involving both environmental and endogenous factors, regulate flowering in plants, such as photoperiod, gibberellin, vernalization, ambient temperature,
autonomous factors and the aging process, which recently
has been proposed to be associated with redox signaling transduction (Greenup et al. 2009, Kocsy et al. 2013, Khan et al.
2014). The process of aging was reported to be related to the
redox changes of AsA and GSH through the AsA–GSH cycle
(Chen and Gallie 2006, Loscos et al. 2008). Three key enzymes
participating in reducing/oxidizing AsA and GSH in the AsA–
GSH cycle are APX, DHAR and GR. In this study, the gene expression level and enzymatic activity of DHAR and GR
decreased markedly during the transition from vegetative to
reproductive phases, but were relatively high at the axillary
bud developmental stage (Fig. 1F, G). In contrast, cytAPX1
activity increased during the transition to reproduction (Chin
et al. 2014). This suggests that the regulation of the AsA level/
AsA redox ratio and GSH level/GSH redox ratio leading to
flower initiation is a fine-tuned mechanism operating through
the AsA–GSH pathway (Figs. 1B, 4).
Ambient temperature is a strong non-stressful environmental factor that affects flowering and the circadian clock; however, the identity of the sensors of ambient temperature is
currently a challenging question (Wigge 2013). The diurnal
and seasonal redox changes are non-stressful and influence
plant growth and development, and involve interaction of
ROS/reactive nitrogen specis (RNS) with several environmental
factors such as temperature and light (Kocsy et al. 2013).
Recently, it has been reported that circadian clock genes not
only play a crucial role in the photoperiod-dependent flowering
pathway, but also regulate redox homeostasis (Lai et al. 2012,
Shim and Imaizumi 2014). This implies the possibility of crosstalk between ambient temperature and redox changes in the
flowering pathway. The ambient temperature range for
Oncidium is about 20–30 C, and prolonged high temperature
(30 C) exposure is an effective environmental factor to induce
flowering in Oncidium, which has been shown to result in a
decreased AsA level and AsA redox ratio, due to the H2O2 level
429
D.-C. Chin et al. | AsA–GSH cycle and floral initiation
Fig. 5 Effect of changes in the GSH redox state by GSH, GSSG and BSO treatment on flowering time and the expression of floral genes in
Oncidium. Plants at the ‘unsheathing’ stage were first grown at 20 C for 1 week, and then were continuously sprayed with 10–3 M GSH, 10–3 M
GSSG, 10–4 M BSO or water (mock treatment) for 28 d for further determinations. (A) The flowering phenotype was recorded at 30 d after the
28 d treatment period. Bolting plants were defined as those in which the inflorescence bud length was >3 cm. Each test group consisted of 12
(continued)
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Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206
rising and elevated expression of cytAPX1 (Chin et al. 2014). In
the present work, a lower AsA redox ratio, lower GSH redox
ratio and flowering initiation occurred synchronously during
the 14 d high ambient temperature (30 C) exposure of
Oncidium (Fig. 2). Also, the exogenous application of DHA
(2 mM) resulted in low GSH content, high GSSG content and
low GSH redox ratio, leading to rising DHAR activity (Fig. 4). By
combining these data, it can be firmly concluded that the GSH
redox state couples with the AsA redox ratio to determine
flowering initiation in the AsA–GSH pathway.
DHAR plays a physiological role in conversion of the oxidized form of DHA to the reduced form of AsA in the AsA–GSH
cycle (Fig. 1B). It is reasonable to conclude that a low AsA level
is associated with down-regulated expression and activity of
DHAR in the AsA oxidative cycle. As shown in Fig. 1F, DHAR
declined in bolting and flowering stages compared with vegetative and NG stages, when orchid plants were grown under
standard glasshouse conditions. This corresponds to the aging
process and our previous data showing that a low AsA redox
ratio affects flowering initiation (Chen and Gallie 2006, Shen
et al. 2009). On the other hand, when orchid plants were grown
under the high ambient temperature (30 C), high expression
and activity of DHAR is possible owing to the higher metabolism at the higher temperature compared with 20 C (Fig. 3A;
Gallie 2013, Sgobba et al. 2015). High DHAR activity accompanied by lower GR activity at 30 C effectively facilitates the resulting lower GSH redox ratio (Fig. 3B). In parallel, the relatively
lower expression of GSH1 and GSH2 enhances both the low
GSH accumulation and low GSH redox ratio. In consequence,
the synergistic effects lead to flowering initiation.
Table 1 Bolting of Oncidium treated with 103M GSH, 103M
GSSG and 104M BSO for 28 d, and then grown for a further 60 d
No. of days of
treatment
Bolted plants (%)
Mock
GSH
GSSG
0
0
0
14
0
0
21.11 ± 1.6
64.44 ± 12.6
0
51.55 ± 6.3
73.33 ± 9.4
78.88 ± 12.6
93.33 ± 5.4
28
12.04 ± 2.4
60
32.08 ± 7.7
30.02 ± 4.7
0
BSO
0
Bolting was recorded at 0, 14, 28 and 60 d after treatment. Each test group
contains 12 individual plants. Bolted plants were defined as those in which the
inflorescence bud length was >3 cm.
Data are the mean ± SE and are the averages of three independent experiments
(n = 36).
The exogenous application of various redox-altering compounds, such as BSO, GSH and GSSG, to orchid plants revealed
that BSO induced flowering initiation at day 14 after treatment,
and GSSG induced flowering induction at day 28, while GSH
treatment delayed flowering initiation compared with mock
treatment (Table 1). The inhibition of GSH1 activity by BSO
treatment directly reduced the generation of GSH and effectively caused a reduction in GSH content, leading to a lower GSH
and AsA redox ratio (Fig. 5B–F). In consequence, the early
flowering phenotype occurred (Fig. 5A; Table 1). GSSG has
been considered the key factor in the regulation of flowering
(Kocsy et al. 2013). The application of GSSG to Oncidium
caused an increase in GSSG and decline of the GSH redox
ratio, and led to early flowering (Fig. 5A, C, D). In parallel, a
high level of DHA with low AsA redox ratio was also observed
with GSSG treatment (Fig. 5E, F). In contrast, GSH application
elevated the GSH level and the GSH redox ratio, and delayed
flowering (Fig. 5A, B, D). Taken together, the results strongly
validate the hypothesis that a low GSH content and GSH redox
ratio were associated with the AsA redox state (Fig. 5E, F; de
Pinto et al. 1999, Stasolla et al. 2008), and further resulted in
early flowering in Oncidium orchid and Arabidopsis (Figs. 5–7;
Ogawa et al. 2001).
By using a transgenic approach to identify the function of
glutathione metabolism genes, transgenic Arabidopsis lines,
OgGSH1-OE, OgGSH2-OE and OgGR1-OE, with a high GSH
redox ratio, were found to display delayed flowering (Figs.
6A–F, 7A–F). On the other hand, Arabidopsis mutants pad2,
cad2 and gr1, with a lower GSH redox ratio, displayed early
flowering (Figs. 6G, H, 7G, H). cad2 and gr1 were reported to
be more oxidized than the WT (Col-0) and have a decreased
buffer capacity to respond to ROS (Meyer et al. 2007, Marty
et al. 2009), implying that GSH biosynthetic and GR genes function via regulation of the GSH content and GSH redox ratio,
altering the redox potential to affect flowering. Notably, the
early or delayed flowering phenotypes of transgenic
Arabidopsis and mutants were only observed in high ambient
temperature (30 C) conditions and not at 22 C
(Supplementary Fig. S4), suggesting that the changes in GSH
level and GSH redox ratio affecting flowering are also controlled
by H2O2 resulting from environmental factors, such as vernalization, light intensity and high ambient temperature (Ogawa
et al. 2004, Hatano-Iwasaki and Ogawa 2012, Chin et al. 2014), in
addition to the function of GSH metabolism genes.
Recently, the redox homeostasis-associated flowering pathway has attracted increased attention. The redox changes,
Fig. 5 Continued
individual plants, and three independent experiments were carried out (n = 36). Similar results were obtained in all duplicates. (B–D) The influence of
GSH, BSO and GSSG treatment for 28 d on GSH content, GSSG level and the GSH redox ratio (GSH/GSSG), respectively. (E and F) The contents of AsA
and DHA, and the AsA redox ratio (AsA/DHA) after GSH, BSO and GSSG application for 14 d. (G) Transcriptional levels of the photoperiodic flowering
pathway genes, GI, CO and FT, under the various treatments. Leaves (for measurement of CO expression) and pseudobulbs (for measurement of GI and FT
expression) were harvested at 1 h before dusk. (H) Effect of the treatments on the expression patterns of the meristem identity genes, LFY and AP1, in
meristem and floral bud tissues. Transcript levels were calculated and normalized with respect to Oncidium ACTIN mRNA. Bars indicate the SEM (n = 6).
Significant differences in comparison with mock treatment are indicated with asterisks: *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.
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D.-C. Chin et al. | AsA–GSH cycle and floral initiation
Fig. 6 Flowering time of OgGSH1-OE, OgGSH2-OE and OgGR1-OE (transgenic Arabidopsis ectopically expressing OgGSH1, OgGSH2 and OgGR1),
gr1 (glutathione reductase 1-deficient Arabidopsis mutants) and cad2 and pad2 (GSH1-deficient Arabidopsis mutants) growing at 30 C. (A), (C)
and (E) OgGSH1-OE, OgGSH2-OE and OgGR1-OE Arabidopsis lines exhibited delayed flowering compared with the WT (Col-0) Arabidopsis. (B),
(D) and (F) Comparison of flowering time in the WT and OgGSH1-OE, OgGSH2-OE and OgGR1-OE Arabidopsis lines as represented by the rosette
leaf number. (G) Flowering time of the WT, gr1, cad2 and pad2. (H) Comparison of flowering time in the WT, gr1, cad2 and pad2 lines as
represented by the rosette leaf number. The experiment was repeated twice using 20 plants of each genotype. The number of rosette leaves was
determined when inflorescences were 1 cm in length. Plants were grown at 22 C under SD conditions (8/16 h photoperiod) for 6 weeks and then
transferred to 30 C under SD conditions for 1 d. After the 14 d 30 C treatment, plants were placed at 22 C under SD conditions for recovery to
determine the number of rosette leaves at flowering. Bars indicate the SEM (n = 40). Significant differences in comparison with the WT (Col-0)
are indicated with asterisks: *P < 0.05, **P < 0.01, by Student’s t-test.
432
Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206
Fig. 7 GSH, GSSG and the GSH redox ratio (GSH/GSSG) in WT, OgGSH1-OE, OgGSH2-OE and OgGR1-OE, and GSH1- and GR1-deficient mutants
of Arabidopsis, pad2, cad2 and gr1, growing at 30 C. (A), (C) and (E) The content of GSH and GSSG in OgGSH1-OE, OgGSH2-OE and OgGR1-OE
plants of transgenic Arabidopsis, respectively, compared with the content in the WT (Col-0). (B), (D) and (F) GSH redox ratio (GSH/GSSG) in
OgGSH1-OE, OgGSH2-OE and OgGR1-OE plants of transgenic Arabidopsis, respectively, compared with the content in the WT (Col-0). (G and H)
The content of GSH and GSSG, and the GSH redox ratio (GSH/GSSG) in pad2, cad2and gr1 mutants, compared with the levels and redox ratio in
the WT (Col-0). Plants were grown at 22 C under SD conditions (8/16 h photoperiod) for 6 weeks, and then transferred to 30 C for 14 d. Ten
plants of each genotype were used in the experiment. Bars indicate the SEM (n = 10). Two independent experiments were conducted and similar
results were obtained each time. Significant differences in comparison with the WT (Col-0) are indicated with asterisks: *P < 0.05, **P < 0.01,
***P < 0.001 by Student’s t-test.
which are triggered by photosynthesis and environmental factors, and determined genetically, are capable of controlling several developmental processes and growth in plants, including
floral induction and floral organ development (Meyer et al.
2005, Considine and Foyer 2014, Dietz 2014). The water–
water cycle and AsA–GSH cycle are the two major systems to
metabolize H2O2 and maintain redox homeostasis in the
chloroplast (Miyake 2010). Thylakoid ascorbate peroxidase
(tylAPX) is a key enzyme involved in the water–water cycle
(Maruta et al. 2010, Awad et al. 2015), and it is reported that
its single and double mutants in Arabidopsis displayed a significant difference of flowering time compared with the WT
(Miller et al. 2007). These studies suggest that the redox homeostasis in the water–water cycle may play a role in redox-regulated flowering. Our previous study suggested that the
expression level of tylAPX displayed less correlation with flowering during the Oncidium developmental process (Chin et al.
2014). It could be assumed that the pseudobulb tissue in the
Oncidium response to redox-associated flowering is a sink tissue
without abundant chloroplasts and an active photosynthesis
system. Hence, we consider that all the compounds and enzymes involved in the cytosolic AsA–GSH cycle are important
and specific for the regulatory metabolism controlling growth
and development in Oncidium (Supplementary Fig. S2).
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D.-C. Chin et al. | AsA–GSH cycle and floral initiation
In conclusion, both aging and high ambient temperature
decrease the AsA/DHA and GSH/GSSG redox ratio in the
AsA–GSH cycle (Fig. 8). In addition, the transcriptional
levels of GR1 and the GSH biosynthesis genes GSH1 and
GSH2 were down-regulated, leading to low production of
GSH and altering redox potential (Meyer et al. 2007). On the
other hand, a low GSH redox state also influences the AsA
redox ratio in the AsA–GSH cycle (Fig. 5E, F). The redox signaling is thus generated to trigger the flowering pathways.
Future work to identify the downstream target proteins that
are involved in gene regulation of flowering routes will advance
our understanding of the potential function of the redox
homeostasis network.
Materials and Methods
Plant material and growth conditions
Oncidium ‘Gower Ramsey’ plants were grown in a greenhouse at 20–25 C and
exposed to the natural daily photoperiod for investigation of developmental
stages. Oncidium plants at the ‘unsheathing’ stage (Fig. 1A) were selected and
grown at 20 C for 1 week; groups of plants were then maintained at 20 C or
transferred to 30 C for temperature treatment. The AsA and DHA treatment of
Oncidium followed conditions from Shen et al. (2009) and Chin et al. (2014).
Plants at the ‘unsheathing’ stage were first grown at 20 C for 1 week, and were
then continuously sprayed with 100 mM AsA and 4 mM DHA dissolved in
buffer (30 mM KCl, 10 mM MES–KOH pH 6.0). The GSH, GSSG and BSO treatments of Oncidium were carried out as in Ogawa et al. (2001). The plants were
treated for 28 d either with water (mock treatment), or with GSH, GSSG or BSO,
which were dissolved in water at a concentration of 10–4, 10–3 and 10–3 M,
respectively.
Plants of Arabidopsis thaliana Col-0, SALK lines and plants ectopically overexpressing the related Oncidium genes (OgGSH1-OE, OgGSH2-OE and OgGR1OE) were grown at 22 C under SD conditions (8/16 h photoperiod) for 6 weeks.
Plants were subsequently maintained at 22 C or transferred to 30 C to conduct
biochemical assays and to determine the number of leaves prior to floral
initiation.
Fig. 8 Proposed mechanism of the AsA–GSH cycle and GSH biosynthesis in Oncidium flowering. Hydrogen peroxide (H2O2) is generated
with aging and at high ambient temperature. Cytosolic ascorbate
peroxidase 1 (cytAPX1) is up-regulated to produce a high level of
dehydroascorbate (DHA) and causes a decrease in the AsA/DHA
redox state. A low AsA/DHA redox state results in an increased
level of GSSG and a low GSH/GSSG ratio by activating dehydroascorbate reductase (DHAR) activity. Concurrently, transcriptional levels of
Glutathione reductase (GR1) and the GSH biosynthesis genes, GSH1
and GSH2, are down-regulated by aging and high ambient temperature, causing low production of GSH. This results in a decrease in the
GSH/GSSG redox ratio. Overall, the redox signaling triggers Oncidium
flowering. Glu, glutamate; Cys, cysteine; g-EC, g-glutamylcysteine; gECS, g-glutamylcysteine synthetase; Gly, glycine; Glus, glutathione
synthase.
Estimation of GSH/GSSG and AsA/DHA
The measurement of the content of GSH and GSSG was carried out as
described (Rahman et al. 2006) with the following modifications. A 0.1 g aliquot of tissue was homogenized in 0.6% 5-sulfosalicylic acid solution (Sigma)
and centrifuged at 13,000 r.p.m. for 10 min at 2–4 C. The clear supernatant
was collected and used for the total GSH assay in 96-well microtiter plates as
follows. In each well, 20 ml of KPE buffer (0.1 M potassium phosphate buffer
with 5 mM EDTA disodium salt, pH 7.5) was added together with 20 ml of the
sample. Next, equal volumes of freshly prepared 0.06% DTNB [5,50 -dithiobis
(2-nitrobenzoic acid); Sigma] in KPE and 3 U of GR (Sigma) in KPE were mixed
and 120 ml of this solution was added to each well. While keeping the tube
containing the DTNB–GR mixture covered in aluminum foil to avoid direct
exposure to light, the conversion of GSSG to GSH was allowed to proceed for
30 s, and then 60 ml of b-NADPH (Sigma) was added. The reaction produced a
yellow product. The absorbance at 412 nm was read immediately in a microplate reader and further measurements taken every 30 s for 2 min (five readings in total from 0 to 120 s). The rate of 2-nitro-5-thiobenzoic acid formation
(change in absorbance min–1) was calculated. For GSSG assay, 2 ml of 2-vinylpyridine (Sigma) was added to 100 ml of cell extract and mixed well to produce GSH. The reaction was allowed to proceed for 1 h at room temperature
in a fume hood. The derivatized samples were assayed by the same method as
described for total GSH. The total GSH and GSSG concentrations in samples
were determined by using linear regression to calculate values obtained from
a standard curve of GSH and GSSG (containing 2 ml of 2-vinylpyridine)
(Sigma).
434
Total AsA, reduced AsA and total DHA were measured following the
method of Gillespie and Ainsworth (2007). Briefly, 40 mg of tissue was extracted
with 6% trichloroacetic acid (TCA) followed by the addition of 10 mM dithiothreitol (DTT) to reduce the pool of oxidized AsA. The total AsA content was
measured at OD525. Total AsA (DTT added) and reduced AsA levels (DTT not
added) were obtained using this method. Total DHA content was obtained by
subtracting the reduced AsA from total AsA.
Enzyme assays
The DHAR assay was carried out using the method of Hossain et al. (1984), with
the following modification. A 0.1 g aliquot of homogenized tissue in 1 ml of
buffer containing 50 mM Tris–HCl (pH7.4), 100 mM NaCl, 2 mM EDTA and
1 mM MgCl2 was centrifuged at 13,000 r.p.m. for 15 min at 2–4 C. A 50 ml
aliquot of the clear supernatant was mixed with 800 ml of reaction solution
[100 mM KPE (pH 6.6), 1 mM DHA, 1 mM GSH and 150 ml of water]. The mixture was incubated for 1 min and absorbance was monitored at 290 nm. DHAR
activity was determined from the change in absorbance at 265 nm (with an
absorbance coefficient of 14 mM–1cm–1).
Assay of GR activity was performed according to the method of Mannervik
et al. (1999). A 0.1 g aliquot of homogenized tissue in 1 ml of buffer containing
50 mM Tris–HCl (pH7.8), 0.1 mM EDTA, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM DTT was centrifuged at
13,000 r.p.m. for 15 min at 2–4 C. Clear supernatant (20 ml) was mixed with
Plant Cell Physiol. 57(2): 423–436 (2016) doi:10.1093/pcp/pcv206
180 ml of reaction solution [25 mM KPE (pH 7.8), 5 mM GSSG, 1.2 mM NADPH
(Sigma)]. The mixture was incubated for 1 min and absorbance was monitored
at 340 nm. GR activity was determined from the decrease in NADPH by
the change in absorbance at 265 nm (with an absorbance coefficient of
6.2 mM–1cm–1).
RNA extraction, cDNA synthesis and gene
expression analysis
Total RNA was isolated from the pseudobulb, leaves and meristem using the
method of Chang et al. (1993). Total RNA (5 mg) was used to synthesize cDNA
using the First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions. Synthesized cDNA was utilized for quantitative reverse
transcription–PCR (qRT–PCR) using the specific primers listed in
Supplementary Table S1. qRT–PCR was performed using a KAPA SYBR
FAST qPCR Kit and an ABI 7500 Fast Real-Time PCR System. The Oncidium
ACTIN gene was used as a reference.
Gene cloning by RACE
Sequencing of the full-length OgGSH1, OgGSH2 and OgGR1 genes was completed using the rapid amplification of cDNA ends (RACE) method (GeneRacer
TM
RLM-RACE kit, Invitrogen ). Sequences were identified, confirmed and deposited in GenBank under the assigned accession numbers.
Transformation of Arabidopsis
The full-length target gene was ligated to the pCAMBIA 1300 binary vector in between the 35 S Cauliflower mosaic virus (CaMV) promoter and
NOS terminator. The constructions were transformed into Arabidopsis
by using Agrobacterium (GV3101) and the floral dipping transformation
method (Zhang et al. 2006). The seeds were plated on half-strength
Murashige and Skoog (MS) medium and 1% sucrose. Seedlings selected
on 25 mg l–1 hygromycin were subsequently transferred to the growth
chamber.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Ministry of Science &
Technology, Taiwan. [MOST-103-2812-8-002-00 to K.W.];
Academia Sinica Innovative Translational Agricultural
Research Program, Taiwan. [2014CP02s5 to K.W.].
Disclosures
The authors have no conflicts of interest to declare.
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