全 文 ：磷脂酶 D啄对拟南芥叶片衰老过程中内源 ROS
和激素含量的影响*
贾艳霞1,2, 陶发清1, 李唯奇1**
(1 中国科学院昆明植物研究所中国西南野生生物种质资源库, 云南 昆明摇 650201;
2 中国科学院大学, 北京摇 100039)
摘要: 活性氧 (ROS) 和植物激素是植物衰老过程中重要的内在或者外在的调控因子。 我们发现, 相对于
离体诱导的衰老过程, 在脱落酸 (ABA) 和乙烯 (ethylene) 促进的衰老过程中有较多的活性氧积累; 在
对拟南芥磷脂酶 D啄 (PLD啄) 缺失型突变体的研究中发现, 与野生型相比, 突变体在衰老过程中产生较少
的活性氧。 我们比较了上述两种基因型的离体叶片在离体、 ABA和 ethylene三种衰老处理下内源的 ABA、
茉莉酸甲酯 (MeJA)、 玉米素核苷 (Zeatin Riboside, ZR) 和吲哚乙酸 (IAA) 的含量变化, 发现每一种激
素对上述三种衰老处理的响应模式都很相似。 在离体诱导的衰老中, 两种基因型拟南芥的内源激素含量没
有差异; 而在 ABA促进的衰老过程中, PLD啄缺失型突变体叶片中的MeJA的含量较低, ZR和 IAA含量较
高; 在乙烯促进的衰老过程中, 突变体中的 ABA和 MeJA的含量较低, ZR 和 IAA 含量较高。 上述内源激
素的这种变化可能有助于延缓突变体的衰老。
关键词: 磷脂酶 D啄; ROS; 乙烯; 脱落酸; 衰老
中图分类号: Q 945摇 摇 摇 摇 摇 摇 文献标识码: A摇 摇 摇 摇 摇 摇 摇 文章编号: 2095-0845(2013)02-149-09
The Effects of Phospholipase D啄 Suppression on the Responses
of ROS and Hormones to Artificial Leaf Senescence
in Arabidopsis thanliana
JIA Yan鄄Xia1,2, TAO Fa鄄Qing1, LI Wei鄄Qi1**
(1 The Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China;
2 University of Chinese Academy of Sciences, Beijing 100039, China)
Abstract: The reactive oxygen species (ROS) and hormones can act as an important internal or external factor in鄄
fluencing plant senescence. In the present study, we found that suppression of phospholipase D啄 (PLD啄) attenuated
ROS production during abscisic acid (ABA)鄄 and ethylene鄄promoted leaf senescence. We also comparatively ana鄄
lyzed the content of endogenous hormones, ABA, methyl jasmonate (MeJA), indole鄄3鄄acetic acid (IAA), and total
zeatin in detachment induced鄄senescence leaves, exogenous ABA and ethylene鄄 promoted senescence in wild type
and PLD啄鄄knockout (PLD啄鄄KO) Arabidopsis leaves. We found that the response patterns of the four endogenous
hormones to the three senescence treatments were identical. In comparison with wild type, PLD啄鄄KO plants showed
higher ZR and IAA levels and lower MeJA content under ABA and higher ZR and IAA levels and lower ABA and
MeJA content under ethylene. The results suggested that these hormones might contribute to retarding ABA鄄 and eth鄄
ylene鄄promoted senescence in PLD啄鄄knockout Arabidopsis.
植 物 分 类 与 资 源 学 报摇 2013, 35 (2): 149 ~ 157
Plant Diversity and Resources摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 DOI: 10. 7677 / ynzwyj201312102
*
**
Foundation items: Grants from the National Basic Research Program of China (31070262), Fund of State Key Laboratory of Phytochemistry
and Plant Resources in West China (O97C0211Z1)
Author for correspondence; E鄄mail: weiqili@ mail. kib. ac. cn
Received date: 2012-08-06, Accepted date: 2012-10-09
作者简介: 贾艳霞 (1982-) 女, 博士, 工程师, 主要从事植物逆境分子生理学研究。 E鄄mail: jiayanxia@ mail. kib. ac. cn
Key words: Phospholipase D啄; ROS; Ethylene; Abscisic acid; Leaf senescence
Abbreviations: abscisic acid, ABA; trichloroacetic acid, TCA; ethephon, ETH; thiobarbituric acid, TBA; jas鄄
monic acid, JA; methyl jasmonate (MeJA); indole鄄3鄄acetic acid, IAA; phospholipase D, PLD; reactive oxygen
species, ROS; malonyldialdehyde, MDA; Enzyme鄄linked Immunosorbent Assays (ELISAs)
摇 Leaf senescence is the final phase from matura鄄
tion to attrition in the leaf life history (Wang et al.,
2000). The senescence process takes place in a
highly regulated manner and the cell constituents are
dismantled in an ordered progression (Quirino et al.,
2000). Senescence is associated with an increased
production of ROS such as hydrogen peroxide (H2O2),
superoxide and its more toxic derivative hydroxyl
radical (del Rio et al., 1998). Chlorophyll degra鄄
dation and a marked decline in photosynthesis will
result in excessive excitation energy in leaves and
then the production of ROS will increase (Khanna鄄
Chopra, 2012). After the ROS reach certain thresh鄄
old levels, it will in turn damage the photosynthetic
apparatus, oxidize proteins, lipids and DNA. These
result in degradation of cellular structures and en鄄
zymes, lipid peroxidation and membrane leakiness
(Thompson et al., 1987; del Rio et al., 1998).
Leaf senescence can be induced by both develo鄄
pmental signals and environmental stress, and plant
hormone and related growth substance as internal
factor play important roles in regulating senescence
(Guo and Gan, 2005). Hormonal controls singly or
in combination are essential for overall control of
growth, development and senescence in plants (Gan,
2010). The five major classes of hormones, namely
auxins, cytokinins ( CKs ), gibberellins ( GAs ),
ABA and ethylene, and other plant growth regulators
such as jasmonic acid ( JA), have been implicated
in the regulation of leaf senescence (Chin and Beev鄄
ers, 1970; Bleecker and Patterson, 1997). The ex鄄
ogenous application of ABA, ethylene and MeJA
strongly promotes senescence in detached leaves
(Smart, 1994; Gan and Amasino, 1996), whereas
exogenous application of the remainders inhibit the
process. Knowledge of the endogenous hormonal reg鄄
ulating senescence, gained from leaf senescence
studies in Arabidopsis and tomato, has been used to
manipulate post鄄harvest senescence in crops such as
broccoli and lettuce (Gan and Amasino, 1995; Mc鄄
Cabe et al., 2001 ). Using the SAG12 promoter
fused to the Agrobacterium ipt gene as described a鄄
bove to increase the endogenous cytokinin content
(Gan and Amasino, 1995), delayed yellowing has
been shown in lettuce both before and after harvest
(Chen et al., 2001; McCabe et al., 2001). Mean鄄
while, changes in endogenous hormone levels of de鄄
tachment鄄induced senescing leaves in some plant
species also have been examined. For example,
changes in the levels of endogenous ABA are meas鄄
ured during the senescence of detached lettuce leaves
(Aharoni and Richmond, 1978 ), tobacco leaves
(Even鄄Chen and Itai, 1975), and nasturtium leaves
(Chin and Beevers, 1970), whereas changes in the
levels of endogenous auxin are measured in detached
tobacco leaves (Evenchen et al., 1978), rape and
pumpkin leaves, and excised bean leaves (Sheldrake
and Nobthcote, 1968). However, the correlations
between hormone levels and senescence appear to be
differ among plant species ( Sheldrake and Nobth鄄
cote, 1968). These differences underscore the need
for deeper understanding of the response of endoge鄄
nous hormone levels to detachment鄄induced leaf se鄄
nescence in Arabidopsis. The correlation between
hormone levels and senescence remains undocument鄄
ed in relation to leaf senescence that is promoted by
ABA or ethylene.
The PLD family comprises 12 members, which
are classified into six types, PLD琢 (3), 茁 (2), 酌
(3), 啄, 着, and 灼 (2) (Qin and Wang, 2002),
ABA鄄and ethylene鄄promoted senescence can be re鄄
tarded by the suppression of phospholipase D琢1
(PLD琢1) (Fan et al., 1997). We found that the
retardation of senescence in PLD琢1鄄AS plants was
051摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
associated with changes in ABA, MeJA, ZR and
IAA levels ( our unpublished date ). Most plant
PLDs have distinctive molecular and biochemical
properties that are associated with diverse cellular
and physiological roles (Qin and Wang, 2002). As
one of most abundant of members of the PLD family
in Arabidopsis, PLD啄 regulates plant responses to
abiotic stresses, such as drought, freezing, high sa鄄
linity, and UV stress, through influencing the sig鄄
nalling and / or structural roles of its product phos鄄
phatidic ( PA) ( Bargmann and Munnik, 2006 ).
Our previous results indicated that suppression of
PLD啄 retarded ABA鄄 and ethylene鄄promoted leaf se鄄
nescence through attenuating degradation of mem鄄
brane lipids ( our unpublished data). However, if
and how endogenous hormones respond to senes鄄
cence retarded by suppression of PLD啄 is unknown.
To address these questions, we used histochemi鄄
cal staining to detect the ROS accumulation and
used enzyme鄄linked immunosorbent assays ( ELI鄄
SAs) to measure the changes in the endogenous con鄄
tent of ABA, MeJA, ZR and auxin (indole鄄3鄄acetic
acid, IAA) during detachment鄄induced and ABA鄄
or ethylene鄄promoted senescence in wild鄄type Arabi鄄
dopsis and PLD啄鄄knockout mutant. Our results sug鄄
gested that higher ROS level in ABA鄄 and ethylene鄄
promoted senescence compared to detachment鄄in鄄
duced senescence, and suppression of PLD啄 attenu鄄
ation of ROS accumulation. We also provided the re鄄
lationship between endogenous phytohormones (Me鄄
JA, ABA, ZR, IAA) and the major factor that in鄄
duce senescence. The association of PLD啄 with
these hormones in regulating endogenous hormones
during the three senescence treatments was also in鄄
vestigated.
1摇 Material and Methods
1. 1摇 Plant materials and growth conditions
Arabidopsis thaliana, ecotype Wassilewskija e鄄
cotype (wild鄄type, WS) and PLD啄鄄knockout mutant
(PLD啄鄄KO) generated from Wassilewskija ecotype,
in which PLD啄 is constitutively attenuated by knock鄄
out (Li et al., 2008), were used. The plants were
grown in a controlled growth chamber at 23 益
(day) and 19 益 (night) and 60% relative humidi鄄
ty under a 12鄄hr photoperiod fluorescent lighting of
120 滋mol·m-2·s-1 .
1. 2摇 Hormone treatments
Ethephon and ABA as mixed isomer were pur鄄
chased form Sigma. Ethephon is water soluble and
releases ethylene in the cell, which has the same
function as ethylene but more feasible to control.
The detached leaves for treatment with several senes鄄
cence鄄affecting factors were obtained by cutting at
the approximately middle part of the petioles of the
third of fourth foliar leaves with sharp scalpel to min鄄
imize wound effect. The detached leaves were floa鄄
ted on deionized water containing 50 滋M abscisic
acid, ethephon in Petri dishes with the abaxial side
up (Fan et al., 1997). The leaves were incubated
at 23 益 under a 12鄄hr photoperiod and light of 120
滋mol·m-2·sec-1 .
1. 3摇 Measurement of endogenous hormones
Plant sample extraction and preparation were
modified according to Lei et al. (2007). The de鄄
tached leaves (0. 5-1 g) at sampling time were ho鄄
mogenized in 2 mL of 80% methanol with 1 滋m buty鄄
lated hydroxytoluene and this procedure was repeated
twice by rinsing the mortar. After extracted and cen鄄
trifuged, the extracts were dried under nitrogen gas.
The endogenous hormones in leaves incubated in
ABA, ethephon and water were determined with En鄄
zyme鄄linked Immunosorbent Assays (ELISAs).
1. 4摇 Histochemical staining for ROS detection
Production of ROS in detached leaves was
measured by staining plants with 2忆, 7忆鄄Dichloroflu鄄
orescin diacetate (H2DCFDA) as described with mi鄄
nor modification (McInnis, 2006). Briefly, leaves
were stained for 10 min with 10 滋g·mL-1 H2DCFDA
(sigma), fluorescence intensity of the dye was ob鄄
served using the Olympus LSCM FV1000 (488 nm
excitation, 520 nm emission). H2 DCFDA fluores鄄
cence increases as the dye is oxidized by ROS to di鄄
chlorofluorescein (DCF).
1512 期摇 摇 JIA Yan鄄Xia et al. : The Effects of Phospholipase D啄 Suppression on the Responses of ROS and Hormones …摇 摇
1. 5摇 Detection of lipid peroxidation
The extent of lipid peroxidation in leaves was
estimated by measuring the amount of malonyldialde鄄
hyde (MDA), a decomposition product of the oxida鄄
tion of polyunsatured fatty acids, as described (Ha鄄
vaux et al., 2003). Briefly, about 0. 5 - 1 g leaf
segments were homogenized in 4 mL of chilled rea鄄
gent 10% [W / V] trichloroacetic acid (TCA), and
centrifuged at 12 000 g for 10 min. After that, 2 mL
0. 6% [W / V] thiobarbituric acid (TBA) in 10%
TCA was added to an aliquot of 2 mL from the super鄄
natant. The mixture was heated in boiling water for
30 min, and then quickly cooled in an ice bath. Af鄄
ter centrifugation at 10 000 g for 10 min, the absor鄄
bance of the supernatant at 450, 532 and 600 nm
was determined. The MDA content was calculated
according to Hodges et al. (1999).
1. 6摇 Data analysis
For all quantitative measurements in present
study, five replicates from each sampling time were
analysed. The data were subjected to one鄄way analy鄄
sis of variance (ANOVA) with SPSS 16. 0. Statisti鄄
cal significance was tested by Fisher爷s least signifi鄄
cant difference (LSD) method.
2摇 Results
2. 1 摇 ROS and oxidative stress were attenuated
in PLD啄鄄KO leaves during the ABA鄄 and ethyl鄄
ene鄄promoted senescence
Leaf senescence occurs in detached / harvested
leaves. In our experiment, three senescence proces鄄
ses were conducted. The first, detachment鄄induced
senescence was measured by floating detached Arabi鄄
dopsis leaves on water. In the second and the third,
detachment鄄induced senescence was accelerated by
ABA and ethylene, respectively. ABA鄄and ethylene鄄
promoted senescence was retarded in PLD啄鄄KO
plants as previous found (our unpublished data).
The level of MDA, a product of lipid oxidation,
increased in the three senescence treatments, and
the leaves incubated with ABA and ethylene under鄄
went more severe oxidative stress. There was no
difference in the MDA content between the two geno鄄
types Arabidopsis detached leaves incubated within
water (Fig. 1A, top). With ABA or ethylene treat鄄
ment, the MDA content of mutant leaves was only
70% and 61. 7% compared to wild type on day 3,
respectively ( Fig. 1A). Increased ROS level is a
common factor in different stress responses as well as
in senescence. The accumulation of ROS indicated
oxidative damage in the form of protein and lipid oxi鄄
dative which we had examined. More ROS were de鄄
tected in the ABA and ethylene鄄promoted senescence
leaves, and suppression of PLD啄 attenuated ROS
production (Fig. 1B). These results indicated that
much higher ROS levels produced in ABA鄄 and eth鄄
ylene鄄promoted senescence than that in detachment鄄
induced senescence, and PLD啄 might be involved
in ROS accumulation in detached leaves during
ABA鄄and ethylene鄄promoted senescence and sup鄄
pression of PLD啄 protected the leaves from oxidative
stress.
2. 2 摇 Endogenous ABA levels increased at the
onset of senescence and dropped during the late
stage of senescence
We measured endogenous ABA levels to see
how ABA respond to leaf senescence and if PLD啄
has effect on its levels. Detachment itself could in鄄
duce leaf to senesce in a reasonable rate accompa鄄
nied with many senescence symptoms (Weaver et
al., 1998), and the ABA levels of detached leaves
incubated in water constantly increased at all the
sampling time, and increased to 3鄄fold at 5 days.
The sudden increase in endogenous ABA after the
exogenous application of ABA (Fig. 2, middle pan鄄
el) may be caused by the diffusion and / or uptake of
ABA from outside. Nonetheless, even though the
ABA level increased to its highest point within 1 day
after ABA treatment, it then began to drop (Fig. 2,
middle panel) . Within 3 days of treatment with eth鄄
ylene, ABA levels of the two genotypes Arabidopsis
leaves reached a maximum (3鄄fold and 2 fold com鄄
pared to each initial level), and the levels in PLD啄鄄
KO mutant leaves were much lower than that of wild
251摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
Fig. 1摇 Comparison of ROS levels in WS and PLD啄鄄knockout, analysis of lipid oxidation in senescing leaves of two genotypes Arabidopsis
A. MDA content was calculated based on the absorbance at 600 nm, WS and PLD啄鄄knockout mutant. FW, fresh weight. Values are
mean 依SD (n=5); B. DCF Fluorescent probes reports changes in ROS abundance within water, ABA, ethylene treatment leaves
type WS, 62% and 55% at day 2 and 3, respec鄄
tively ( Fig. 2, bottom panel) . Given above evi鄄
dences, it was concluded that endogenous ABA rap鄄
idly increased to respond to ABA鄄and ethylene鄄pro鄄
moted senescence. Furthermore, suppression of
PLD啄 attenuated endogenous ABA accumulation
which contributed to delaying in ethylene鄄promoted
senescence.
2. 3摇 Suppression of PLD啄 attenuated MeJA ac鄄
cumulation during ABA鄄 and ethylene鄄promoted
senescence
MeJA, which occurs widely in plants, can pro鄄
mote chlorophyll loss and accelerated leaf senes鄄
cence (Ueda and Kato, 1980). In this study, Me鄄
JA levels were measured in the three senescence
treatments. In water treatment, MeJA levels of the
detached leaves increased a little and began to drop
slowly and no difference between WS and PLD啄鄄KO
mutant Arabidopsis (Fig. 3, top panel) . MeJA lev鄄
els of detached WS leaves incubated in ABA and
ethylene increased sharply, which increased to 4. 6鄄
fold and 1. 7鄄fold compared to control at day 3, re鄄
spectively. However, MeJA levels in PLD啄鄄KO
leaves were significantly lower than those in WS
leaves (Fig. 3, middle and bottom panels) . These
results indicated that lower MeJA levels in PLD啄鄄KO
leaves might contribute to retardation of ABA鄄and
ethylene鄄promoted senescence, that is regulated by
suppression of PLD啄 activity.
2. 4摇 ZR levels decreased during detachment鄄in鄄
duced and ABA鄄promoted senescence, and were
higher in PLD啄鄄KO leaves than that in WS leav鄄
es under ABA treatments
Cytokinins (CKs) regulate cell division and va鄄
rious metabolic and developmental processes, inclu鄄
ding senescence ( Smart et al., 1991; Gan and
3512 期摇 摇 JIA Yan鄄Xia et al. : The Effects of Phospholipase D啄 Suppression on the Responses of ROS and Hormones …摇 摇
Amasino, 1995). We measured the ZR content dur鄄
ing the three senescence processes defined above. In
the water treatment, ZR levels dropped by 35. 5%
as the leaves detached from the whole plant for just
one day and kept decreasing during the treatment
period, and at 5 days, it had decreased by 64. 5%
(Fig. 4). Although the ZR levels of WS leaves de鄄
creased to 32. 3% and 22. 6% at 1 day after ABA
and ethylene treatment, respectively, for the remain鄄
der of experiment the values increased, and then de鄄
creased again (Fig. 4). However, deficient of PLD啄
made the mutant leaves have higher ZR content than
that of WS in certain sampling time ( after ABA
treatment for 1, 2, 3 and 5 days; after ethylene
treatment for 5 days) (Fig. 4). The significantly de鄄
crease of ZR levels in senescing leaves of the three
treatments indicated that senescence in detached
leaves associated with decrease in ZR abundance.
The significant difference of ZR levels between WS
and PLD啄鄄KO leaves suggested that ZR might con鄄
tribute to attenuation of PLD啄鄄KO during ABA鄄 and
ethylene鄄promoted senescence.
2. 5 摇 IAA levels decreased during senescence,
and IAA levels in PLD啄鄄KO leaves were higher
than that in WS
In order to make clear how the auxin responded
to the three senescence鄄accelerators and whether
PLD啄 played any role in these processes, we meas鄄
ured content of IAA (a kind of well known auxin)
content. During detachment鄄induced senescence,
Fig. 2摇 Variation in levels of endogenous ABA in leaves from WS and
PLD啄鄄KO plants during three different treatments. Detached leaves
were treated with deionized water ( top), 50 滋M ABA (centre), or
50 滋M ethephon (ETH; bottom) and sampled at the indicated time
points. FW, fresh weight. Values are mean依SD (n=5). Values with
different letters are significantly different (P<0. 05) . “*冶 indicates
that the value is significantly different from that of the WS under the
same condition (P<0. 05)
Fig. 3摇 Variation in levels of endogenous MeJA in leaves from WS and
PLD啄鄄KO plants during three different treatments. Detached leaves
were treated with deionized water ( top), 50 滋M ABA (centre), or
50 滋M ethephon (ETH; bottom) and sampled at the indicated time
points. FW, fresh weight. Values are mean依SD (n=5). Values with
different letters are significantly different (P<0. 05) . “*冶 indicates
that the value is significantly different from that of the WS under the
same condition (P<0. 05)
451摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
IAA levels did not change at the first two days but
dropped to 50% of their initial levels at day 3. We
found no difference in IAA levels between leaves
from WS and PLD啄鄄KO plants in the detachment鄄in鄄
duced senescence. During ABA鄄accelerated senes鄄
cence, IAA levels kept decreasing in the incubation
course and decreased to 60% of the initial levels at
day 5 (Fig. 5, middle and bottom panels) . IAA lev鄄
els of WS leaves incubated in ethylene increased a
litter at day 1 and then decreased constantly in the
following treatment days. Moreover, there was obvi鄄
ous difference about the IAA content detected when
PLD啄 was absent after ABA treated for 1 and 3days
and ethylene treated for 2 and 5 days ( IAA content
of mutant leaves was higher than that of WS leaves)
(Fig. 5). These results indicated that IAA was asso鄄
ciated with senescence and involved in the attenua鄄
tion of ABA鄄 and ethylene鄄promoted senescence in
PLD啄鄄KO plants.
3摇 Discussion
There have been a number of reports indicating
that ROS levels increase during senescence, and the
increase is likely to be associated with macromole鄄
cule degradation, in particular lipid degradation.
We detected less accumulation of ROS and MDA
levels during the ABA and ethylene鄄promoted senes鄄
cence in PLD啄鄄KO plants leaves, in comparison
with WS (Fig. 1A, B). ROS have been implicated
as a second messenger in several plant hormone re鄄
sponses (Lam, 2004; Tamaoki, 2008). ABA and
ethylene signaling have important roles in regulating
Fig. 4摇 Variation in levels of endogenous ZR in leaves from WS and
PLD啄鄄KO plants during three different treatments. Detached leaves
were treated with deionized water ( top), 50 滋M ABA (centre), or
50 滋M ethephon (ETH; bottom) and sampled at the indicated time
points. FW, fresh weight. Values are mean 依 SD ( n = 5). Values
with different letters are significantly different (P<0. 05) . “*冶 indi鄄
cates that the value is significantly different from that of the WS under
the same condition (P<0. 05)
Fig. 5摇 Variation in levels of endogenous IAA in leaves from WS and
PLD啄鄄KO plants during three different treatments. Detached leaves
were treated with deionized water ( top), 50 滋M ABA (centre), or
50 滋M ethephon (ETH; bottom) and sampled at the indicated time
points. FW, fresh weight. Values are mean 依 SD ( n = 5). Values
with different letters are significantly different (P<0. 05) . “*冶 indi鄄
cates that the value is significantly different from that of the WS under
the same condition (P<0. 05)
5512 期摇 摇 JIA Yan鄄Xia et al. : The Effects of Phospholipase D啄 Suppression on the Responses of ROS and Hormones …摇 摇
ROS production (Kwak et al., 2006). Transcrip鄄
tional express of PLD啄 are higher in senescent than
in young tissue and also increases in response to se鄄
vere dehydration and high salt (Wang and Wang,
2001). ABA stimulation of PLD acts upstream in
the transduction pathway leading to RAB18 expres鄄
sion (Hallouin et al., 2002). All these evidences
suggest that the PLD啄 has roles in retardation of
ABA鄄 and ethylene鄄promoted senescence through
ROS accumulation.
Senescence is a very important aspect of post鄄
harvest physiology. Enormous literatures concern the
effects of hormone treatments on maintenance and
survival of plant materials in storage, especially for
fruits and flowers, for example, fruit ripening or
flower opening. Hormones play critical roles in these
processes, although much remains to be learned a鄄
bout correlations between hormone levels and senes鄄
cence, particularly during hormone鄄promoted senes鄄
cence. To the best of our knowledge, this is the first
report on the change patterns of endogenous hor鄄
mones in PLD啄鄄KO plants during detachment in鄄
duced鄄, ABA鄄 and ethylene鄄promoted senescence.
For the senescence of detached leaves, our data in鄄
dicated that each type of endogenous hormone re鄄
spond to the three different senescence inducer (de鄄
tachment, ABA and ethylene) in the same pattern
(Figs. 2-5). The increase of MeJA and ABA and
the decrease of ZR at the initial stage of senescence
might be resulted from the detachment from the
whole plant. However, at the senescence late stage,
there were some differences in the responses of the
four hormones to the three types of senescence. Lev鄄
els of ZR decreased during leaf detachment and ABA
incubation, but sharply increase at day 2 and then
began to drop during ethylene treatment ( Fig. 4).
Therefore, we proposed that, during the initial
stage, the endogenous hormone respond to deficient
in energy and nutrients supplies results from detach鄄
ment, whereas during the late stage, they were re鄄
sponses to ABA and ethylene鄄mediated effects. Mo鄄
reover, higher ZR and IAA content and lower ABA
and MeJA content in PLD啄鄄KO mutant Arabidopsis
were not just the consequence but the regulator to
ABA鄄 and ethylene鄄promoted senescence.
The complexity of leaf senescence is mainly due
to the involvement of multiple components that ex鄄
hibit overlapping effects. This is particularly true for
the action of ROS and hormones. Our results demon鄄
strate the association of ROS and hormones ZR,
IAA, ABA, and MeJA in retardation of senescence
in PLD啄鄄KO plants, which also provides clues for
further investigate the roles of PLD啄 in senescence.
Given the availability of various microarrays and the
complexity of the senescence process, a systems bi鄄
ology approach should be taken for deciphering the
molecular regulatory mechanisms of leaf senescence
by plant hormones and other external and internal
factors. Once senescence is fully understood, it
should allow us to devise ways to manipulate leaf se鄄
nescence for agriculture improvement.
Acknowledgements: We thank Dr. Hongyin Chen for her
critical reading of the paper.
References:
Aharoni N, Richmond AE, 1978. Endogenous gibberellin and ab鄄
scisic acid content as related to senescence of detached Lettuce
leaves [J] . Plant Physiology, 62 (2): 224—228
Bargmann BO, Munnik T, 2006. The role of phospholipase D in plant
stress responses [J] . Current Opinion in Plant Biology, 9 (5):
515—522
Bleecker AB, Patterson SE, 1997. Last exit: senescence, abscis鄄
sion, and meristem arrest in Arabidopsis [ J] . The Plant Celln鄄
line, 9 (7): 1169—1179
Chen LFO, Hwang JY, Charng YY et al., 2001. Transformation of
broccoli (Brassica oleracea var. italica) with isopentenyltrans鄄
ferase gene via Agrobacterium tumefaciens for post鄄harvest yello鄄
wing retardation [J] . Molecular Breeding, 7 (3): 243—257
Chin TY, Beevers L, 1970. Changes in endogenous growth regulators
in nasturtium leaves during senescence [ J] . Planta, 92 (2):
178—188
del Rio LA, Pastori GM, Palma JM et al., 1998. The activated oxy鄄
gen role of peroxisomes in senescence [ J] . Plant Physiology,
116 (4): 1195—1200
Even鄄Chen Z, Itai C, 1975. The role of abscisic acid in senescence
of detached Tobacco leaves [ J] . Physiologia Plantarum, 34
(2): 97—100
651摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 35 卷
Evenchen Z, Atsmon D, Itai C, 1978. Hormonal aspects of senes鄄
cence in detached Tobacco鄄leaves [J] . Physiologia Plantarum,
44 (4): 377—382
Fan L, Zheng S, Wang X, 1997. Antisense suppression of phospho鄄
lipase D alpha retards abscisic acid鄄 and ethylene鄄promoted se鄄
nescence of postharvest Arabidopsis leaves [ J] . The Plant Cell,
9 (12): 2183—2196
Gan SS, 2010. The hormonal regulation of senescence [A]. In Plant
Hormones [C]. In Plant Hormones, 597—617
Gan SS, Amasino RM, 1995. Inhibition of leaf senescence by auto鄄
regulated production of cytokinin [ J] . Science, 270 (5244):
1986—1988
Gan SS, Amasino RM, 1996. Cytokinins in plant senescence: From
spray and pray to clone and play [ J] . BioEssays, 18 (7 ):
557—565
Guo YF, Gan SS, 2005. Leaf senescence: signals, execution, and
regulation [ J] . Current Topics in Developmental Biology, 71:
83—112
Hallouin M, Ghelis T, Brault M et al., 2002. Plasmalemma abscisic
acid perception leads to RAB18 expression via phospholipase D
activation in Arabidopsis suspension cells [J] . Plant Physiology,
130 (1): 265—272
Havaux M, Lutz C, Grimm B, 2003. Chloroplast membrane photosta鄄
bility in chlP transgenic tobacco plants deficient in tocopherols
[J] . Plant Physiology, 132 (1): 300—310
Hodges DM, DeLong JM, Forney CF et al., 1999. Improving the
thiobarbituric acid鄄reactive鄄substances assay for estimating lipid
peroxidation in plant tissues containing anthocyanin and other in鄄
terfering compounds [J] . Planta, 207 (4): 604—611
Khanna鄄Chopra R, 2012. Leaf senescence and abiotic stresses share
reactive oxygen species鄄mediated chloroplast degradation [ J] .
Protoplasma, 249 (3): 469—481
Kwak JM, Nguyen V, Schroeder JI, 2006. The role of reactive oxygen
species in hormonal responses [ J] . Plant Physiology, 141
(2): 323—329
Lam E, 2004. Controlled cell death, plant survival and development
[J] . Nature Reviews Molecular Cell Biology, 5 (4): 305—
315
Lei Y, Korpelainen H, Li C, 2007. Physiological and biochemical re鄄
sponses to high Mn concentrations in two contrasting Populus
cathayana populations [J] . Chemosphere, 68 (4): 686—694
Li W, Wang R, Li M et al., 2008. Differential degradation of extra鄄
plastidic and plastidic lipids during freezing and post鄄freezing re鄄
covery in Arabidopsis thaliana [ J] . The Journal of Biological
Chemistry, 283 (1): 461—468
McCabe MS, Garratt LC, Schepers F et al., 2001. Effects of PSAG12 鄄
IPT gene expression on development and senescence in transgen鄄
ic Lettuce [J] . Plant Physiology, 127 (2): 505—516
McInnis SM, Desikan R, Hancock JT et al., 2006. Production of re鄄
active oxygen species and reactive nitrogen species by angiosperm
stigmas and pollen: potential signalling crosstalk? [ J] . New
Phytologist, 172 (2): 221—228
Qin C, Wang X, 2002. The Arabidopsis phospholipase D family.
Characterization of a calcium鄄independent and phosphatidylcho鄄
line鄄selective PLD zeta 1 with distinct regulatory domains [ J] .
Plant Physiology, 128 (3): 1057—1068
Quirino BF, Noh YS, Himelblau E et al., 2000. Molecular aspects
of leaf senescence [J] . Trends in Plant Science, 5 (7): 278—
282
Sheldrake AR, Nobthcote DH, 1968. Production of auxin by de鄄
tached leaves [J] . Nature, 217 (5124): 195—195
Smart CM, 1994. Gene expression during leaf senescence [J] . New
Phytologist, 126 (3): 419—448
Smart CM, Scofield SR, Bevan MW et al., 1991. Delayed leaf senes鄄
cence in tobacco plants transformed with tmr, a gene for cytokin鄄
in production in Agrobacterium [ J] . The Plant Cell, 3 (7):
647—656
Tamaoki M, 2008. The role of phytohormone signaling in ozone鄄in鄄
duced cell death in plants [J] . Plant Signaling and Behavior,
3 (3): 166—174
Thompson JE, Legge RL, Barber RF, 1987. The role of free鄄radicals
in senescence and wounding [ J] . New Phytologist, 105 (3):
317—344
Ueda J, Kato J, 1980. Isolation and identification of a senescence鄄
promoting substance from wormwood (Artemisia absinthium L. )
[J] . Plant Physiology, 66 (2): 246—249
Wang C, Wang X, 2001. A novel phospholipase D of Arabidopsis that
is activated by oleic acid and associated with the plasma mem鄄
brane [J] . Plant Physiology, 127 (3): 1102—1112
Wang C, Zien CA, Afitlhile M et al., 2000. Involvement of phospho鄄
lipase D in wound鄄induced accumulation of jasmonic acid in Ar鄄
abidopsis [J] . The Plant Cell, 12 (11): 2237—2246
Weaver LM, Gan S, Quirino B et al., 1998. A comparison of the ex鄄
pression patterns of several senescence鄄associated genes in re鄄
sponse to stress and hormone treatment [ J] . Plant Molecular
Biology, 37 (3): 455—469
7512 期摇 摇 JIA Yan鄄Xia et al. : The Effects of Phospholipase D啄 Suppression on the Responses of ROS and Hormones …摇 摇