全 文 ：Effects of Adaptation to Elevated Salinity on Some Enzymes Salt_tolerance
in Vitro and Physiological Changes of Eelgrass
YE Chun_Jiang , ZHAO Ke_Fu＊
(Institute of Plant Stress , Shandong Normal University , Jinan 250014 , China)
Abstract: The eelgrass(Zostera marina L.)was treated with artificial seawater (ASW)of different salini-
ties(100%, 150% and 200% seawater)for 5 d.The activities of two enzymes extracted from the plant leaves
were determined under a salinity grade in vitro.So were the photosynthesis rates of the plants from the three
treatments in the media with different salinities(100%, 150%, 200%, 300%ASW)and some physiological
data.The data showed that under increased salinities(concentrated seawater), Na+ , Cl- , MDA(malon di-
aldehyde)and glucose contents and the osmotic potentials (absolute value)in the leaves increased with the
salinity elevation in the medium (ASW), but both K+ and free amino acid (mainly proline)contents de-
creased.Malate dehydrogenase (MDH)from the plant leaves under a salinity grade showed its activities (A)
as follows:A100%ASW>A150%ASW>A200%ASW.Phosphoenolpyruvate carboxylase (PEPC)extracted from the
100%ASW_ and 200%ASW_treated plants showed similar activities(both insensitive to salinities)under the
salinity grade in vitro , but the activities of PEPC from plants treated with 150%ASW were dependent on
salinity.Whether the plant is stressed at 150%ASW and can stand higher salinity than seawater needs to be
studied further.Meantime , the data do not agree with the opinion that the adaptation of the eelgrass to seawa-
ter salinity is partly fulfilled by its insensitiveness to salinities in some metabolic enzymes.It can be inferred
that the lack of transpiration may be an important aspect of the plant s tolerance to seawater salinity.
Key words: eelgrass;salinity adaptation;enzyme salt_tolerance;PEPC;MDH;MDA
The eelgrass(Zostera marina L.), one of the most
typical seagrasses in the north hemispheres , plays an im-
portant role in the shallow sea ecology[ 1] .In recent
decades , the plant has attracted much attention from
botanists and ecologists mainly for its rapid disappearance
from its former habitats for uncertain reasons.Also inter-
esting is its salt_tolerance mechanism to seawater salinity
for scientists engaged in illuminating the salt_tolerance
strategies in halophytes , although little work has been
done.Compared with the studies on terrestrial halo-
phytes , our knowledge about how seagrasses adapt to sea-
water salinity (3.3%)has been scarce.About the salt
tolerance mechanisms of eelgrass , several hypotheses have
been presented:1)invaginated plasma membrane of epi-
dermal cells in mature leaves may be important for its salt
tolerance;it is found that there are two types (spherical
and non_spherical)of protoplasts released from eelgrass
leaves by enzymatic methods.Spherical protoplasts are
obtained from epidermal cells of immature leaves (which
are enclosed by sheathes from seawater)and other types
of cells , and non_spherical protoplasts are from epidermal
cells of mature leaves and sheathes , both of which are ex-
posed to seawater directly.Moreover , the non_spherical
protoplasts are highly resistant to detergents and osmosis
stress , for their plasma membrane is coated(invaginated)
with certain thick substances(its chemical nature still not
determined), which also accounts for their non_spherical
shapes and makes the cells have a transfer_cell_like ap-
pearance.So it comes easily to connect the existence of
the invaginated plasma membrane with the plant salt toler-
ance[ 2] ;2)P_type H+_ATPase may be the most impor-
tant pump for salt exclusion.With cytochemical tech-
nique , P_type H+_ATPase was found to be far more ac-
tive in epidermal cells of the mature leaves than in those
of immature leaves[ 3] , and a gene for putative P_type
H+_ATPase in the eelgrass was cloned from the plant
leaves(highly homologous with the one from tobacco),
whose expression is tissue_specific , i.e.expressedmainly
in epidermal cells of mature leaves (exposed to seawa-
ter), but not in those of immature leaves (isolated from
seawater)[ 4] ;3)The mechanism of the cooperation be-
tween a proton pump and Na+/H+ antiporter on the plas-
ma membrane for Na+ exclusion;fusicoccin (FC), an
inhibitor of Na+/H+ antiporter , can make the plasma
membrane super polarized , indicating the existence of
Na+/H+ antiporter[ 5] ;4)Salt tolerance in the metabolic
enzymes and ion “compartmentalization” between different
kinds of cells.Beer et al[ 6] reported that under high
salinity activities of Rubisco from leaves of the eelgrass
decreased but still remained high enough , and the ion
compartmentalization in the plant occurred not only within
cells (between cytoplasma and vacuoles), but also be-
tween different types of cells (epidermal cells of leaves ,
Received:2001-09-29 Accepted:2002-01-15
Supported by the State Key Basic Research and Development Plan of China(G1999011700).
＊Author for correspondence.E_mail:
Abbreviations:ASW , artificial seawater;MDA , malon dialdehyde;MDH , malate dehydraogenase;PEPC , phosphoenolpyruvate carboxylase;Rubisco , ribulose
1 , 5 biphosphate carboxylase.
植 物 学 报
Acta Botanica Sinica 2002 , 44(7):788-794
i.e.photosynthesis cells and mesophyll cells)①.
In the present study , we examined the effects of
adaptation to elevated salinity of the eelgrass on the salt
tolerance of MDH and PEPC , both of which were the key
enzymes in the plant metabolisms in vitro , and some
physiological changes such as photosynthesis rate under a
series of salinity grades and the changes in inorganic ions
(Na+ , K+ , Cl-), MDA , free amino acids and dis-
solved sugars contents in leaves after being treated with
ASW of different salinities.
The aims of the study are:1)to determine whether
the salt_tolerance of metabolic enzymes is common in sea-
grasses for their adaptation to sea water;2)to show the
effects of adaptation to increased salinities on the salt_tol-
erance of key metabolic enzymes in vitro;3)to prove how
the plant responds to increased salinities (150%ASW ,
200%ASW)in ions (Na+ , K+ , Cl-), free amino
acids(including proline)and glucose and sucrose con-
tents in leaves;4)to present a factor which may be im-
portant for the adaptation of the eelgrass to sea water
salinity;5)to indicate whether the salinity increase is
important for the plant to fare well in sea water.
1 Materials and Methods
1.1 Plant material and cultivation
The eelgrass(Zostera marina L.)was collected in
May 2001 from Changdao Island , Shandong Province ,
China , where there was no apparent evidence for the de-
grade of eelgrass communities (It still could be easily
found in shallow sea waters).The plants were transferred
to laboratory in natural seawater(NSW)within 10 h and
kept in three 30 L aquariums with artificial sand beds in a
medium of ASW (Made from Sude Instant Seawater
Essence , Qingdao Ocean University product which was
proved an effective substitution for the natural seawater).
After 3 d adaptation in ASW , the media in the aquariums
were adjusted to 150% and 200%ASW respectively , at
a 50%step with the same seawater essence.The sea wa-
ter was changed every 3 d and the temperature was kept at
25 ℃ by an air conditioner.The cultivation room was
kept dim , and the aquariums were bubbled 30 min every
hour with an auto_switched air pump.After 5 d treat-
ment , various assays were carried out.
1.2 Measurements of MDH and PEPC activities in
vitro under a salinity grade
Crude extracts were prepared by homogenizing 1 g
fresh leaves with 100 mmol/L maleic_KOH , pH 6.8[ 7] .
The homogenate was centrifuged at 5 000 r/min for 30
min , and the supernatant was assayed forMDH and PEPC
activities in series of media with different salinities.
Then , the measurements of MDH and PEPC activities
were carried out as descried by Soussi et al[ 8] .All steps
were carried out at 4 ℃.Triplicates were performed for
each assay of enzyme activity.
1.3 Measurements of photosynthesis rates under a
series of salinity grades
The determination of photosynthesis rates of eelgrass
leaves was made according to Major s[ 9] method with
slight modification.Leaves of varying maturities were cut
into small blades , and an appropriate number of blades
were put into 250mL flasks , which were filled with ASW
of different salinities (100%, 150%, 200%, 300%
ASW)in each experiment.The flasks were incubated for
2 h in a shaking bed at 30 ℃.The light was from fluo-
rescent tubes (light intensity:about 500 μmol·m-2·
s-1).After the incubation , KI_NaOH and MnSO4 were
introduced into each bottle with syringe and titration of
100 mL aliquots against sodium thiosulphate , which was
standardized for each set of experiment , and should be
finished as soon as possible.Variations in salinity of the
reaction medium were realized by adding appropriate
amount of the same sea water essence (Sude).For each
salinity medium(ASW), blank experiments were carried
out as control.The blades were weighted before being put
into the flasks.
1.4 Measurements of ion contents in leaves and
ASW
Na
+ , K+ contents in leaves andASW of each salin-
ity were determined by atomic absorption spectrometry
(Hitachi Z_8000)[ 10] and Cl- by titration against
AgNO3
[ 11] .
1.5 Measurements of MDA content in leaves
Nought point four gram fresh leaves were homoge-
nized in 0.1% trichloro acetic acid (TCA)and extracts
were incubated with 0.5% thibarbituric acid solution in a
boiling water bath for 10 min(timing from bubbling on).
Then , absorbencies at 532 nm and 600 nm were record-
ed.The MDA content was calculated as described by
Lin[ 12] .
1.6 Measurements of osmotic potentials and water
content in leaves
Leaves were put into liquid nitrogen after being col-
lected from the plants , and 15 min later cut into small
pieces.The leaf sap was squeezed into EP tubes with a
syringe.Then , the saps were centrifuged at 3 000 r/min
for 10 min , and osmotic potentials were determined with
an osmometer (Osmomat 030).For water content , the
leaves were blotted dry and weighted , then dried at 80 ℃
for 24 h to get their dry weight.
1.7 Measurements of free amino acids (including
proline), glucose and sucrose contents in eelgrass
leaves
For free amino acid measurement , 3 g leaves were
homogenized in 80%ethanol.Then , the homogenate was
diluted to 100 mL with distilled water.After infiltration ,
20 mL aliquots were dried under reduced air pressure un-
til solid appeared.Then the solid was redissolved in dis-
tilled water.The method was as described by Rosen[ 13] .
For proline content measurement , 2 g fresh leaves
were ground in 10 mL 5% acetic acid , and the
①Ye C_J , Zhao K_F.Osmotically active compounds and their subcel lular lo-
calization in the marine halophyto eelgrass.Biol Plantarum , 2002.(in
press)
YE Chun_Jiang et al:Effects of Adaptation to Elevated Salinity on Some Enzymes Salt_tolerance and Physiological Changes of Eelgrass 789
homogenate was diluted to 50 mL with distilled water.
Proline concentration was determined as described by
Trolly and Lindaloy[ 14] .
For measurement of glucose and sucrose , 1 g of dry
leaves were homogenized in 80% ethanol , then trans-
ferred into 250 mL flasks which then were kept in 70 ℃
water bath for 3 h.The method followed Somogyi s[ 15] .
2 Results
Figure 1A indicated that MDH activities showed a
similar trend for the three treatments (100%, 150%,
200%ASW).That is , under different salinities(0 , 50 ,
100 , 200 , 300 mmol/L NaCl), MDH was activated by
low salinity but inhibited under high salinity , which was
common for most of enzymes in plants either glycophytes
or halophytes
[ 16] .Figure 1A showed that increased salini-
ties had negative effects on MDH activities.At each
salinity , MDH activities (A)were differently higher in
the sequence:A100%ASW>A150%ASW>A200%ASW.Mean-
time , for 150%ASW treatment , MDH activity was stim-
ulated by 100 mmol/L NaCl that had an inhibiting effect
on the MDH activities for 100% and 200%ASW treat-
ments.
PEPC activities had a similar change trend for 200%
ASW and 100%ASW treatments.Namely , as the salini-
ty increased PEPC activities showed a slight decrease , but
relatively stable.With 300 mmol/L NaCl its activity was
still high enough (only 11.3% and 9.2% decrease for
100% and 200%ASW treatments against controls , i.e.
Fig.1. MDH (A)and PEPC(B)(from eelgrass leaves of three
treatments)activities under different salinities in vitro.
0 mmol/L NaCl , respectively).For plants treated with
150%ASW , PEPC was best activated by 100 mmol/L
NaCl(almost 2fold those for 200% ASW and 100%
ASW treatments).It seemed that PEPC activities from the
eelgrass being treated with 150%ASW were dependent
on salinity (activated by <100 mmol/L NaCl)and had a
72% increase in activity under 300 mmol/L NaCl com-
pared with the control(0 mmol/L)(Fig.1B).
Z .marina treated with 100% ASW and 200%
ASW showed a similar trend in photosynthesis rates under
varying salinities in the assay medium.But the case for
150%ASW treatment was distinct from 100%and 200%
ASW treatments(Fig.2).Compared with PEPC activi-
ties , interestingly , the changes in photosynthesis rates
were highly consistent with the changes in PEPC activities(when salinity is more than 50 mmol/L NaCl in vitro).
Although the plant appeared to be a C3 plant , PEPC
might play a more important role than Rubisco in photo-
synthesis under increased salinity.Additionally , at 300%
sea water salinity the plant s respiration was stronger than
the photosynthesis for the 100% and 200%ASW treat-
ments as indicated by the negative amount of released O2.
But for the 150% treatment , the plant photosynthesis
rates were still high enough , almost equal to those of the
plants from the other two treatments at 100% sea water
salinity.
The MDA content , which represented the superoxi-
dation degree of the plasma membrane , increased with the
salinity in the cultivation medium (Fig.3A , B).Both
Na+ and Cl- contents in the leaves increased with the el-
evated salinities in the ASW(Fig.3C ,D).But the case
is opposite for K+(Fig.3C).
There were no apparent changes in water contents of
eelgrass leaves among the three treatments(Fig.4A).But
as the osmotic potential increased in ASW , osmotic po-
tentials of leaf sap also increased significantly (Fig.4B).
Between the treatments , there was a big difference in total
free amino acids(especially proline), which was the im-
portant organic osmoticum for the plant osmotic adjust-
ment①(Fig.5).The glucose content increased with the
salinity elevation but sucrose not(Fig.5B).
Fig.2. Photosynthesis rates of the eelgrass (Zostera marina L.)
from three treatments under a salinity grade.
790 植物学报 Acta Botanica Sinica Vol.44 No.7 2002
Fig.3. Na+(A), Cl-(B)and K+(C)contents in the ASW with various salinities and eelgrass leaves from different treatments and MDH(D)contents in eelgrass leaves from different treatments.
Fig.4. Water contents(A)and osmotic potentials (B)of eelgrass leaves from different treatments.
Fig.5. Nitrogen in the form of amino acids , proline(A)and sugars(glucose and sucrose)contents(B)in eelgrass leaves under different
salinities.
YE Chun_Jiang et al:Effects of Adaptation to Elevated Salinity on Some Enzymes Salt_tolerance and Physiological Changes of Eelgrass 791
3 Discussion
3.1 What is the maximum salinity ?
Eelgrass can thrive in a wide range of salinities(0.5%-3.5%).And some studies have shown that the
decrease in photosynthesis rates of the eelgrass in reduced
salinity (diluted sea water)is mainly due to the decrease
of the inorganic carbon content in sea water[ 17] .All of
these indicates little influence of simple NaCl factor on the
survival of the plant.But the effects of salinity increase
on eelgrass survival have not yet been determined.The
present study shows that under increased salinity the MDA
content (C)in eelgrass leaves increases significantly ,
i.e.C200%ASW =2.5C150%ASW =4.5C100%ASW(Fig.
3D).MDA is one of the products from superoxidation of
plasma membrane , and its content can represent the dam-
age degree of the plasma membrane[ 12] .Additionally ,
Na+ , Cl- and glucose contents in eelgrass leaves also in-
crease steeply with their increase in the ASW medium ,
which is also necessary for the plant osmotic adjustment
under higher osmotic potential stress.But the K+ content
decreases sharply , whose absorption is competitively in-
hibited by Na
+(Fig.3A -C).At the same time , the
contents of free amino acids , which are the most important
organic osmotica for the eelgrass osmotic adjustment① ,
decrease significantly with the salinity increase in the
medium(ASW), which may be caused by the broken
synthesis process of these organic osmotica(Fig.4 A ,B).
It seems that the plant is under salt stress once the ASW
is concentrated.However , 150%ASW_treated eelgrass
has a higher photosynthesis rate at each salinity than 1
ASW_treated plants.And under 300%sea water salinity ,
eelgrass photosynthesis of plants from 100% and 200%
treatments are inhibited completely , but plants treated
with 150%ASW seems fare well under this salinity (a
high enough photosynthesis rate)(Fig.2).For 100%
ASW and 150%ASW treatments , the difference in activ-
ity of MDH is not significant , especially under the higher
salinity in vitro , and for PEPC , its activity is higher for
150% ASW treatment than for 100% ASW under the
higher salinity (>50 mmol/L NaCl)(Fig.1A , B).
Therefore , the plant may fare well under this salinity by
“ compartmentalizing” Na+ , Cl- into parenchymatous
cells and a more active P_type H+_ATPase to exclude
such toxic ions.
For the question whether eelgrass can adapt to 150%
ASW and fare well at this salinity to be resolved , we must
first know what are the most effective indicators for the
plant stress level , which is still an open question.In ad-
dition , 5 days may not be long enough for the plant to
adapt to the sharp salinity increase;therefore a longer ex-
periment period under natural conditions should be adopt-
ed to resolve the question , and more indicators need to be
introduced to illuminate the stress level suffered by the
plant under increased salinity , especially ion X_ray mi-
croanalysis through which we can know if the “ compart-
mentalization” status of toxic ions among the different
kinds of cells is destroyed.Therefore , the question is still
open and needs to be studied further.
3.2 Tips for salt_tolerance mechanisms
Except for some archibacteria , the metabolic en-
zymes of higher plants(either glycophytes or halophytes)
are sensitive to salt in vitro[ 16] .But some researchers
have found that the case may be different for seagrass-
es
[ 18] .In the present study , PEPC from eelgrass leaves is
not sensitive to salinity in vitro , but MDH is sensitive to
salinity and under each salinity (in the salinity grade in
vitro) the activity (A) sequence is:A100%ASW >
A150%ASW>A200%ASW , indicating that not only in vitro
higher salinity has an inhibiting effect on MDH activities
but also the salt level in vivo of photosynthesis cells for
150%ASW and 200%ASW has already imposed an in-
hibiting effect on its activity (at 0 mmol/L NaCl the dif-
ference in activities among the treatments still exists)(Fig.1A).The case is different for PEPC , and only that
from 150%ASW_treated eelgrass shows a similar trend in
activity change to MDH under the salinity grade(first ac-
tivated , then inhibited), which may indicate that PEPC
from the 150% ASW_treated eelgrass functions in the
presence of NaCl at certain concentration in cells that
have no inhibiting but activating effects , and its better
performance is dependent on salinity(Fig.1B).Although
for 100%ASW and 200%ASW treatments , PEPC has
the similar activity change trend , they may represent dif-
ferent situations as far as Na+ and Cl- concentrations in
the photosynthesis cells are concerned:for 100%ASW ,
Na
+
and Cl
-
are effectively excluded or compartmental-
ized into parenchymatous cells from the photosynthesis
cells , so with a low salt concentration in the cytoplasm① ,
but for 200%ASW treatment the plant s ability to deal
with the high Na+ , Cl- concentrations in the medium(sea water)may not be effectively enough.As a result ,
the salt concentration in the cytoplasm is so high that its
effect on the PEPC is inhibited.
From the above , the results do not agree with the ar-
gument that the salt_tolerance of eelgrass is realized partly
through its metabolic enzymes insensitiveness to salini-
ty[ 18] for the plant s metabolic maintenance needs all of
the enzymes to perform well in cooperation and only one
enzyme s insensitiveness to salinity (such as PEPC in
eelgrass)in vitro should not be regarded as important for
plant s salt tolerance.Salt tolerance through “ compart-
mentalization” among different kinds of cells and salt ex-
clusion is more practical for the eelgrass than through its
metabolic enzymes salt tolerance[ 19] (which would re-
quire all the metabolic enzymes resistance to salinity and
is not the case as demonstrated by MDH sensitiveness to
salinity in vitro).
In addition , although the plant appear to be a C3
plant , we have found that the plants photosynthesis rates
are highly consistent with their PEPC activities in the
change trend for the three treatments under the salinity
grade(for PEPC activity , salinity >50 mmol/L NaCl ,
which may be the case in the photosynthesis cells of the
plant)(Fig.1B).So it is possible that PEPC plays a
792 植物学报 Acta Botanica Sinica Vol.44 No.7 2002
more important role in photosynthesis under increased
salinities(leading to a relatively higher salinity within the
cells), and such a transition in carbon metabolisms has
been found in terrestrial halophytes and exonphytes under
salt stress and water stress respectively[ 20] .Such a transi-
tion might also exist in the eelgrass under salt stress.
As important osmolytes for xenophytes and terrestrial
halophytes under water stress
[ 19] , free amino acids (in-
cluding proline)and sugars (including sucrose)contents
decrease at varying degrees.Maybe at increased salini-
ties , such organic osmotica are replaced partly by inor-
ganic osmotica such as Na
+ , Cl- ions and it is more eco-
nomic for the plant to accumulate inorganic ions than to
synthesis organic osmolytes as far as the energy cost is
concerned.
3.3 A possible important factor for eelgrass salt_tol-
erance
About 30 years ago , Oertli[ 21] pointed out that for
terrestrial halophytes , failure to accumulate salt ions into
the protoplast would result in the development of a low
apoplastic solute potential with resultant cellular dehydra-
tion.The drive forNa+ , Cl- ions to enter the plant body
through root is mainly transpiration.Even when the exter-
nal salt concentration is low , still high salt concentration
can be accumulated in the apoplastic space of leaves
mainly for the strong transpiration and the little volume of
the apoplastic space in leaves[ 22] .But for the eelgrasses ,
which grow in sea water submerged completely , transpira-
tion does not exist unless they are exposed to air , which is
fatal to the plant even for a short time (author , unshown
data).So the drive for external ions to enter the plant is
mainly the diffusion of ions , which is much easier to deal
with by a delicate couple (or balance)between the plant
growth and amount of ions that enter into the plant body
as well as the “compartmentalization” of these ions from
the photosynthesis cells into parenchymatous cells①.
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YE Chun_Jiang et al:Effects of Adaptation to Elevated Salinity on Some Enzymes Salt_tolerance and Physiological Changes of Eelgrass 793
盐分胁迫对大叶藻某些胞内酶耐盐性
及其生理功能的影响
叶春江 赵可夫＊
(山东师范大学逆境植物研究所 , 济南 250014)
摘要: 分别以 3 个盐度(100%、150%和 200%人工海水)处理大叶藻(Zostera marina L.)植株 5 d ,然后在体外测定
了大叶藻叶片中两个胞内酶(PEP 羧化酶和苹果酸脱氢酶)的耐盐性和在不同盐度介质中(100%、150%、200%、
300%人工海水)经不同盐度(100%、150%和 200%人工海水)海水预处理的大叶藻叶片光合速率以及一些生理指
标的改变。结果表明在高于海水的盐度下 ,大叶藻叶片中丙二醛(MDA)、还原性糖和Na+ , Cl-含量及渗透势(绝对
值)均随海水盐度的提高而增加 , 但 K+和游离氨基酸的含量均降低。从处理 5 d 的植株中提取的苹果酸脱氢酶在
体外每一盐度下 ,其活性(A)大小依次为:A100%ASW>A150%ASW>A200%ASW(artificial seawater),并且其活性对盐度敏感
(低盐度激活高盐度抑制);但从 100%及 200%人工海水(ASW)处理的植株中提取的 PEP羧化酶的活性在体外对盐
度不敏感 ,而从 150%人工海水处理的植株中提取的 PEP 羧化酶的活性对盐度具有很高的抗性。实验结果显示由
150%人工海水处理的植株 ,在每一特定的盐度下其光合速率均高于另外两组处理(100% ASW 和 200% ASW), 并
且 3个处理的光合速率均与其 PEPC 在不同盐度下的活性表现相一致。但是 , 经 150%ASW 处理的大叶藻植株是
否受到盐害还有待于进一步探讨。实验表明“大叶藻耐盐性在一定程度上是由于其代谢酶对盐度的耐性造成的”
这一说法是不合适的;不存在蒸腾作用可能是大叶藻耐盐机理的重要组成部分。
关键词: 大叶藻;盐度适应;酶的耐盐性;PEP羧化酶;苹果酸脱氢酶;丙二醛
中图分类号:Q945 文献标识码:A 文章编号:0577-7496(2002)07-0788-07
收稿日期:2001-09-29 接收日期:2002-01-15
基金项目:国家重点基础研究发展规划项目(G199011700)。
＊通讯作者。E_mai l:
(责任编辑:贺 萍)
794 植物学报 Acta Botanica Sinica Vol.44 No.7 2002