全 文 ：Overexpression of Proline Transporter Gene Isolated from Halophyte
Confers Salt Tolerance in Arabidopsis
SHEN Yi_Guo1＊ , ZHANG Wan_Ke1＊ , YAN Dong_Qing2 , DU Bao_Xing1 , ZHANG Jin_Song1 , CHEN Shou_Yi1＊＊
(1.Institute of Genetics and Developmental Biology , The Chinese Academy of Sciences , Beijing 100101 , China;
2.School of Life Science , Northwest Agriculture University , Yanglin , Shaanxi 712100 , China)
Abstract: Proline is one of the most important and widespread osmolyte which functions in adaptation to ad-
verse environmental stresses in many organisms.Also it is an important carbon and nitrogen resource in higher
plants.Metabolism of proline has been elucidated in many plant species.However , transport of proline was
poorly characterized although transport system plays an important role in proline distribution in different tis-
sues.We isolated one full_length cDNA encoding proline transporter from the typical halophyte:Atriplex hort-
ensis L.through cDNA library screening and 5′_RACE.The deduced amino acid sequence had eleven trans-
membrane domains , showed 60%-69% similarities to other ProTs and the gene was designated AhProT1 .In
the phylogenetic tree , higher plants ProTs , e.g.AhProT1 , showed more similar to ProP from microorganisms
than ProT from mammalians.AhProT1 gene was transformed into Arabidopsis thaliana under 35S promoter.In
MS medium containing [ U_14C] proline , AhProT1 + plants were able to accumulate much more radiolabeled
proline in the roots than control plants.InMS medium containing different concentrations of NaCl , AhProT1 +
plants could endure 200 mmol/L NaCl and keep development and biomass increase with proline supply ,
whereas control plants died back at 150 mmol/L NaCl.
Key words: Atriplex hortensis;proline transporter;deposition;salt stress
Crops suffered from adverse environmental stresses
such as salinity , drought and freezing.Salinity causes ion
homeostasis and osmotic stress in plant cells , drought de-
creases water content and osmotic pressure in plant cells ,
and freezing injury is thought to result primarily from
membrane lesions caused by cellular dehydration
[ 1] .As
adaptation to these stress conditions , many plants can ac-
cumulate some highly soluble compounds to raise osmotic
pressure in the cytoplasm and stabilize proteins and mem-
branes.These compounds are called osmoprotectants or
osmolytes , including glycine betaine , proline (Pro),
mannitol , etc., and proline is the most widespread os-
molyte existed in all biological kingdoms.In plant tissues
such as meristems and seeds , proline can also serve as
important carbon and nitrogen resource[ 2 ,3] .
After decades of researches , metabolism of Pro in
different organisms now has been well characterized.In
higher plants , Pro is synthesized through two independent
pathways from different precursors , mainly from Glu and
partially from Orn[ 4] .Two enzymes , P5CS and P5CR ,
catalyze the pathway from Glu.Stress conditions such as
salinity can induce the expression of P5CS , P5CR andδ_OAT (Orn pathway), reduce ProDH (Pro decomposi-
tion pathway)transcript , and thus triggers the Pro accu-
mulation in cytoplasm[ 5] .However , not all the tissues ac-
cumulating Pro depend on Pro biosynthesis.In some tis-
sues without chloroplast , such as roots , the restricted pre-
cursor resources of Glu and NADPH result in limitation of
Pro biosynthesis , and in some tissues such as pollen and
seeds that undergo dehydration during their maturation ,
neither the synthesis pathways nor decomposition pathway
contributed to the higher concentrations of Pro(>70%of
free amino acids)[ 6] .Using labeled chemicals , physio-
logical researches pointed that metabolism of Pro in these
tissues has only minor effect , whereas transport of Pro
plays a major role in Pro accumulation[ 7] .It is then nec-
essary to make clear how the Pro transport system con-
tributes to osmotic pressure adjustment in some tissues un-
der salt stress.
For many years it has been a longstanding target of
agricultural biotechnology to improve salt and drought re-
sistance in crops.Plant genetic engineering of osmolytes
had been viewed as the most effective pathway to achieve
stress tolerance in crops , e.g.rice which was an os-
molyte non_accumulating plants.In several reports , intro-
duction of foreign genes encoding biosynthesis enzymes of
osmolytes into transgenic rice had led to modest accumu-
lation of some osmolytes e.g.GlyBet and Pro , apparently
in consequence , limited increase in stress tolerance[ 8 ,9] .
In previous reports , we isolated several genes involved in
osmolyte biosynthesis and performed further genetic engi-
neerings in tobacco , rice and wheat.We also introduced
AhCMO and AhBADH into tobacco and gained salt_toler-
ant transgenic plants[ 8 ,10 ,11] .In Atriplex hortensis , a Pro
and GlyBet accumulating halophyte of Chenopodiaceae ,
Received:2001-12-24 Accepted:2002-02-25
Supported by the Major Basic Research Program of China(G19990117003)and the National Transgenic Plants Program of China(J99_A_004 and J00_A_008_02).
＊ These authors contribute equally to the work.
＊＊ Author for correspondence.E_mail:
Abbreviations:P5CS , Δ1_pyrroline_5_carboxylate synthetase;P5CR, pyrroline_5_carboxylate reductase;δ-OAT , ornithine_δ_aminot ransferase.
植 物 学 报
Acta Botanica Sinica 2002 , 44(8):956-962
biosynthesis of Pro and GlyBet was recently clari-
fied[ 10 ,12 , 13] .However , mechanisms regarding the trans-
port of Pro and GlyBet inside plant cells remained un-
known.As to the osmolyte transporter , functional re-
searches were few.The first Pro transporter (ProT)was
cloned from Arabidopsis thaliana [ 14 ,15] , and recently two
other ProTs were characterized in tomato and rice[ 6 , 16] ,
respectively.All of these ProTs are categorized into the
AAAP (amino acid/auxin permease)family , which in-
cludes over four dozens proteins from eukaryotes[ 17] .
However , only few of them were functionally character-
ized , and till now there is no report characterizing possi-
ble function of Pro transporter in plant osmolyte engineer-
ings.To obtain the knowledge about Pro transport in a
GlyBet natural accumulator under salt stress , we isolated
a new ProT gene , AhProT1 , from Atriplex hortensis , pre-
dicted the arrangement of secondary structures and investi-
gated its phylogenetic tree.Particularly , overexpression of
AhProT1 could obviously promote salt tolerance in trans-
genic Arabidopsis , and our result indicated new pathway
of plant genetic engineering to improve crop resistance to
osmotic stresses.
1 Materials and Methods
1.1 Plant materials and growth condition
Seeds of Atriplex hortensis L.were germinated hy-
droponically at 25 ℃ for 2 d , and then grown in green-
house at 22 ℃ in natural daylight during October and
November.Mature plants were irrigated with solutions
containing 400 mmol/L NaCl for 4 d as salt treatment be-
fore mRNA isolation and cDNA library construction.Ara-
bidopsis plants (Arabidopsis thaliana ecotype Columbia)
were grown in soil or MS plates at 21 ℃with constant
light(100 μmol photons·m-2·s-1)and 60%humidity.
1.2 Construction and screening of a cDNA library
Total RNA was isolated from leaves of A.hortensis
after stressed with 400 mmol/L NaCl for 4 d and 2μg of
poly (A+)RNA were used for cDNA library construc-
tion.mRNA purification and cDNA synthesis (Promega)
followed manufacture s protocol , and pExcell vector and
Ready_To_Go Kit(Amersham)were used for ligation and
packaging.Approximately 250 000 plaques were screened
with AtProT1 cDNA as a probe.Positive plaques were
obtained from the third round screening and then excised
in vivo into pExCell plasmids and the plasmid with the
longest insert was subjected to sequencing analysis.
1.3 Rapid amplification of cDNA end(5′_RACE)
Three gene_specific primers were designed according
to the partial sequences of the positive clone obtained by
screening cDNA library.The SP1 (5′_CTGATAAG-
TACTCGGGATCC_3′)was used to reverse_transcribe the
mRNA into first_strand cDNA , and the second , nested
primer SP2(5′_GTGAACTGAAAGTACAGGCC_3′)locat-
ed upstream of SP1 was used in combination with the
adaptor primer for the PCR amplification of the 5′termi-
nus of this gene following the manufacturer s instruction(Roche).The PCR product was ligated into pGEM_T
easy vector (Promega)and subjected to transformation.
Positive clones were identified by PCR with gene_specific
primers SP2 and SP3(5′_AAACTTCATGAGTATGGTGG_
3′)and subjected to sequencing.Then a pair of primers(5′_TGAAACCCCTCTTCTTCCTCC_3′, 5′_GGGAAAAAT_
AATATTCTTCC_3′)were designed based on the 5′_ and
3′_termini , respectively , and used to amplify the full_
length cDNA.
1.4 Data analysis
The nucleotide and amino acid sequences were com-
pared with those released in GenBank databases by using
the GAPPED BLAST analysis program.The full_length
sequence of AhProT1 has been deposited in GenBank
databases under the accession number AF274032.The
alignment and phylogenetic tree reports were produced by
software DNASTAR.
1.5 Arabidopsis transformation and cultivation
A Sma Ⅰ/ Sac Ⅰ fragment encoding the full_length
AtProT1 was inserted downstream of the 35S promotor in
the plant expression vector pBI121 (Clontech)and intro-
duced into Arabidopsis plants by the vacuum infiltration
technique.Nine independent homozygous transgenic lines
were obtained after selection of T3 progeny on MS media
containing 50 mg/L kanamycin and expression of trans-
gene was confirmed by Northern blotting.Ten_day_old
seedlings of nine transgenic progenies and wild type plants
are cultured onMS medium and used for further analysis.
1.6 L_[ U_14 C] proline uptake and salt stress test
with exogenous osmolytes
Seedlings of wild type and transgenic lines were
moved into MS bottles containing 0.01 μCi·mL-1 L_[ U_
14
C] proline and cultured for 3 d.Radiolabeled plants
were then washed carefully and dried on filter paper for
fifteen minutes at room temperature , and analyzed by
STORM 840 phosphor imager (Molecular Dynamics ,
USA)after 24 h exposure to a 14C_sensitive screen.
To test salt tolerance , 0mmol/L , 150 mmol/L , 175
mmol/L and 200 mmol/L NaCl was used , and seedlings
of wild type and transgenic lines were moved into these
MS medium containing 5 mmol/L proline.Photos were
taken after cultured for four weeks.
2 Results
2.1 Isolation of AhProT1 gene from Atriplex hort-
ensis
A cDNA library was constructed using mRNA from
salt_treated A. hortensis plants and screened with
AtProT1 cDNA.Four 5′_truncated cDNA clones with dif-
ferent lengths were isolated.A 3′_truncated fragment of
583 bp was successfully obtained by 5′_RACE_PCR ,
which contains a 63 bp 5′_untranslated region and 240 bp
coding region overlapped with the previous clone obtained
from cDNA library.Then the full_length cDNA of 1 684
bp was generated by Reverse Transcription_PCR and veri-
fied by sequencing.Analysis of this cDNA sequence re-
vealed an ORF of 1 362 bp in length , which comprises
453 amino acid residues with a calculated molecular
SHEN Yi_Guo et al:Overexpression of Proline Transporter Gene Isolated from Halophyte Confers Salt Tolerance in Arabidopsis 957
Fig.1. Nucleotide and deduced amino acid sequence of AhProT1.
Dashed line marks the segment of low compositional complexity.Solid lines indicate transmembrane segments.Secondary structure of AhProT1
was predicted by SMART program on EMBL website(http:// smart.embl_heidelberg.de), and the full_length sequence of AhProT1 has been
deposited in GenBank databases under the accession number AF274032.
weight of 49.7 kD(Fig.1).The deduced amino acid se-
quence was 69% identical to the Arabidopsis ProT1 pro-
tein , and it also exhibits significant sequence similarities
to other characterized ProTs.Thus the gene corresponding
to this cDNA was designated AhProT1.Secondary struc-
ture analysis of AhProT1 predicted totally eleven possible
transmembrane domains and categorized AhProT1 into the
AAAP (amino acid/auxin permease) family , which
958 植物学报 Acta Botanica Sinica Vol.44 No.8 2002
Fig.2. Phylogenetic analysis of AhProT1 with other Pro transporters and GlyBet transporters from microorganisms , higher plants and mam-
malians.
Genes and corresponding accession numbers are EcProP(U75904), CgProP(Y12537), RnProT1(P28573), HsProT1 (Q99884), RnBGT1
(P48056), CfBGT1 (P27799), HsBGT1 (S68236), AtProT1 (X95737), AtProT2 (X95738), AtProT3 (AC006919), LeProT1(AF014808), LeProT2(AF014809), LeProT3(AF014810)and OsProT1(AB022783).
Fig.3. Uptake of [ U_14 C] proline in AhProT1 + and control
plants.
Numbers of 1 , 2 and 3 indicate three independent transgenic T3
lines , CK means wild type plant.Seedlings were cultured for 3 d ,
then washed carefully and dried on filter paper 15 min at room tem-
perature , and analy sis by STORM 840 phosphor imager after 24 h
exposure to a 14C_sensitive screen.A.Four dried seedlings.B.
The exposure image.
consists of many eukaryote proteins with similar trans_
membrane domains.
2.2 Phylogenetic analysis of ProTs
We performed phylogenetic analysis based on amino
acid sequence alignment.Totally eight deduced ProTs
from plants , two ProPs from microorganisms , two ProTs
and two BGTs (Betain/GABA Transporters)from mam-
malians were analyzed.Figure 2 shows that ProTs in
plants and ProPs in microorganisms were more similar to
each other than ProTs in mammalians in which proline
and Betaine were transported by two independent protein
systems.And Fig.2 also shows that AhProT1 , LeProT
and AtProT in dicots were divergent from OsProT1 in
monocots.
2.3 L_[ U_14C] proline uptake
To characterize the proline uptake of AhProT1 in
plants , AhProT1 was inserted into binary vector under
35S promoter and transformed into Arabidopsis thaliana.
After homozygous T3 progenies were identified , nine in-
dependent T3 lines were obtained for the following study.
Expression of AhProT1 in the nine seedlings was detected
by Northern blotting (Data not shown).After the
seedlings were cultured on MS medium containing 0.01
μCi·mL-1 L-[ U_14C] proline for 3 d , the labeled exoge-
nous proline was transported into the whole plants from
roots to apices (Fig.3).Deposition of proline in Ah-
ProT1 + plants and in control plants is different in roots
SHEN Yi_Guo et al:Overexpression of Proline Transporter Gene Isolated from Halophyte Confers Salt Tolerance in Arabidopsis 959
but similar in stems or leaves.It has not yet been identi-
fied whether AhProT1 can increase similar deposition in
flower organs as that in roots due to the limitation of our
methods.
2.4 Salt stress on AhProT1+ plants
In order to figure out the effect of active foreign ProT
in crops genetic engineering , we use Arabidopsis as a
simple model.Wild type Arabidopsis plants respond to 50
mmol/L NaCl on molecular level , grow slowly in 100
mmol/L NaCl , wilt and die away in 150 mmol/L and
higher NaCl.We thus set up salt gradient of 0 , 150 , 175
and 200 mmol/L NaCl , each with additional 5 mmol/L
Pro supply.After transfer to salt medium for 4 weeks ,
AhProT1 + seedlings showed an obvious tolerance to salt
stress compared with wild type seedlings not only in sur-
vival but also in biomass increase.Figure 4 shows the
phenotype of AhProT1 +and wild type seedlings on differ-
ent salt medium , and Fig.5 listed the biomass of Ah-
ProT1 + and wild type seedlings after four_week_stress on
MS medium containing salt.These data indicate that ,
with exogenous Pro , AhProT1+ seedlings kept growth at
175 mmol/L NaCl , even endured 200 mmol/L NaCl ,
whereas exogenous Pro had no obvious effect on wild type
seedlings.
Fig.4. Salt tolerance of seedlings of AhProT1 transgenic plants
and control plants grown onMS medium containing different concen-
trations of salt and additional osmolytes.
Photos were taken after culture for 4 weeks.A.5 mmol/ L proline
and 0 mmol/ L NaCl.B.5 mmol/ L proline and 150 mmol/ L NaCl.
C.5 mmol/ L proline and 175 mmol/ L NaCl.D.5 mmol/ L proline
and 200 mmol/L NaCl.
3 Discussion
As one important amino acid , Pro exists in all bio-
logical kingdoms from microorganisms to higher plants and
mammalians.Metabolism and transport of Pro were well
characterized in microorganisms and mammalians.In mi-
croorganisms , Pro is transported by two systems of ProP
and ProU , which also transport another important osmolyte
GlyBet at high affinity
[ 18] .In higher plants , Pro and
Fig.5. The biomass (including roots)of seedlings of AhProT1 +
and control plants.
After culture for four weeks on salt medium as described in Materi-
als and Methods , seedlings were weighed.Biomass of AhProT1 +
seedlings(black)is obviously higher than control seedlings(white)
on 150 , 175 and 200 mmol/ L NaCl medium.
GlyBet transporter kept unknown till recently genes en-
coding ProTs were isolated from Arabidopsis and tomato.
LeProT1 can transport both Pro and GlyBet[ 6] , and At-
ProT2 can also transport Pro and GABA which was an im-
portant neurotransmitter in mammalians
[ 15] .However , in
mammalian cells , Pro transporters is quite different from
GlyBet/GABA transporters , indicating an evolution diver-
gence of Pro transporter[ 19 , 20] .In present study , we iso-
lated the AhProT1 from a halophyte which accumulated
both Pro and GlyBet as osmolytes , and concluded the
phylogenetic tree of Pro transporters as well as GlyBet
transporters.Figure 2 shows that ProTs in plants are more
similar to ProP/ProU in microorganism not only in precur-
sor affinity but also in evolution relationship.In fact ,
some genes e.g.P5CR1 of tomato were found to be a
protoplast homolog[ 21] .Among the ProTs in plants ,
AhProT1 and the three ProTs in dicots were obvious diver-
gent from OsProT1 although the sequence divergence was
little.
Plants accumulate Pro as carbon and nitrogen re-
source during development and as critical response to os-
motic stress.In higher plants , two systems of Pro
metabolism and transport contribute to the Pro deposition
in different tissues , especially in tissues without redun-
dant supply of Glu and NADPH.For example , in pollen
cells that undergo dehydration process during matura-
tion[ 6] , or in root tip cells under lowψw[ 7] , osmotic pres-
sure was adjusted by increasing high concentration Pro in
cytoplasm which was achieved through active transport of
exogenous Pro instead of native Pro biosynthesis.We thus
performed a radiolabeled experiment to investigate the ex-
ogenous Pro deposit when foreign Pro transporter
960 植物学报 Acta Botanica Sinica Vol.44 No.8 2002
(AhProT1)was constitutively active not only in root but
also in other tissues.Figure 3 shows that when cultured
under non_stressed condition , root has no advantage in
exogenous Pro deposit in wild type Arabidopsis , but in
transgenic Arabidopsis overexpressed AhProT1 , root obvi-
ously obtained more exogenous Pro than other tissues al-
though AhProT1 was constitutively expressed in most tis-
sues under 35S promoter.Our result strongly suggested
that Pro transporter itself could not afford the specific Pro
accumulation in root tips , and Pro transport system con-
sisting of ProT and other unknown proteins might be the
reason for the Pro deposit in root , although it is complex
to verify this system in such experiment.In addition ,
current study of ProTs hadmade it clear that ProTs had no
tissue specificity in root as well as in leaves or
stems[ 6 ,14] .
Chenopodiaceae plants such as spinach , A.horten-
sis can endure adverse salt stress by accumulating high
concentration of osmolytes like proline and glycine betaine
in cytoplasm[ 13 , 22] .In plant metabolic engineering , trans-
genic plants overexpressing P5CS has a phenotype of salt
tolerance , which indicates that strengthened Pro biosyn-
thesis has obvious effect on salt tolerance in plants[ 9] ;on
the other hand , suppressed P5CS with antisense P5CS
under ubiquitin promoter caused death in barley seedlings(Preben B.Holm , personal communication), which sug-
gests the necessity of Pro accumulation in reproductive tis-
sues of plants.It is then of great interest to investigate the
effect of active foreign Pro transporter on plant salt toler-
ance.In present report , Arabidopsis overexpressing
AhProT1 could endure as high as 200 mmol/L NaCl , and
kept growth and increased biomass in a salt gradient con-
sisted of 150 , 175 and 200 mmol/L NaCl.In contrast ,
wild type Arabidopsis grew slowly in MS containing
100 mmol/L NaCl , died away in 150 mmol/L NaCl , and
perished in 175 or 200 mmol/L NaCl.Our data demon-
strated that active AhProT1 could increase Pro content in
root tips and consequently promoted salt_tolerance in
transgenic plants obviously.Because Pro is widespread in
nature , crops could obtain Pro of different forms from hu-
mus , fertilizer or soil.Our result firstly threw light on
such new method to achieve salt_tolerance in crops instead
of constructing a foreign biosynthesis pathway in plants.
Acknowledgements:We are thankful to Miss RAN Yan_
Chao , Miss WANG Chun_Mei , Mrs.YU Jia_Ning for
technical help , we also thank Dr.Zi_Yin LI (School of
Pharmacy , University of California , San Francisco)for
critical reading.
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榆钱菠菜脯氨酸转运蛋白基因的克隆
及转基因拟南芥的耐盐性
沈义国1＊ 张万科1＊ 阎冬青2 杜保兴1 张劲松1 陈受宜1＊＊
(1.中国科学院遗传与发育生物学研究所 , 北京 100101;2.西北农林科技大学生命科学学院 , 陕西杨凌 712100)
摘要: 脯氨酸是自然界中分布最广泛 , 作用最重要的渗透保护剂之一 , 同时又是高等植物中一类重要的碳源和氮
源物质。为了解环境胁迫下脯氨酸的转运调节 ,从一个典型的盐生植物榆钱菠菜(Atriplex hortensis L.)中通过 cDNA
文库筛选和 5′_RACE的方法获得了一个全长的 cDNA(AhProT1), 其编码蛋白与脯氨酸转运蛋白有 60%～ 69%的同
源性 ,含有 11 个跨膜结构域。聚类分析表明 , 微生物和高等植物的脯氨酸转运蛋白同源程度相对高于哺乳动物。
为进一步分析脯氨酸转运蛋白在植物中的功能 ,将 AhProT1 置于 35S 启动子下转入拟南芥(Arabidopsis thaliana)。通
过同位素示踪法发现 , 与对照植物相比 , 转基因植物在根中积累更多的脯氨酸;在一系列不同浓度的盐胁迫试验
中 ,转基因植株最高可耐受 200 mmol/ L NaCl , 并可持续生长 ,而对照植株在 150 mmol/L NaCl下即已死亡。
关键词: 榆钱菠菜;脯氨酸转运蛋白;沉积作用;盐胁迫
中图分类号:Q943.2 文献标识码:A 文章编号:0577-7496(2002)08-0956-07
收稿日期:2001-12-24 接收日期:2002-02-25
基金项目:国家重大基础研究项目(G19990117003);国家转基因植物研究与产业化项目(J99_A_004, J00_A_008_02).
＊并列第一作者。
＊＊通讯作者。E_mai l:
(责任编辑:谢 巍)
962 植物学报 Acta Botanica Sinica Vol.44 No.8 2002