全 文 ：Expression of Ferric Chelate Reductase Gene in Citrus junos
and Poncirus trifoliata Tissues
LI Ling , FAN Yan_Hua , LUO Xiao_Ying , PEI Yan , ZHOU Ze_Yang＊
(Biotechnology Research Center , Southwest Agricultural University , Chongqing 400716 , China)
Abstract:　It has been hypothesized that under iron stress high ferric chelate reductase (FCR)activity in the
absorptive root of plants tolerant to iron_deficiency will be induced and result in subsequent Fe2+ transport
across the plasmalemma.The activity of FCR and expression of FCR gene (FRO2)in Citrus junos Sieb.ex
Tanaka tolerant to iron_deficiency and Poncirus trifoliata (L.)Raf.susceptible to iron_deficiency were deter-
mined to elucidate the physiological difference which causes the different tolerance of the two citrus rootstocks
to iron stress.The activity of FCR was detectable in excised roots and was stimulated about 20_times in C.
junos and only about 3_times in P .trifoliata under iron deficiency for four weeks.The FRO2 of Arabidopsis
was used as a probe , the tissue print technique was used to ascertain the expression of the FCR gene in C.
junos and P.trifoliata under iron stress.High_level transcripts were observed in the absorptive root , young
green stem as well as new leaf of C.junos under iron stress for two weeks , and the transcripts were accumu-
lated only slightly in P .trifoliata at the same time.The results showed that the obvious increase of FCR ac-
tivity was an important reason for the tolerance of C.junos to iron_deficiency , and the regulation of FCR activ-
ity seemed to be at the transcriptional level , and the expression of FRO2 occurred in the root , stem and leaf.
Key words:　Citrus junos;Poncirus trifoliata;iron stress;ferric chelate_reductase;tissue print;FRO2
gene;Northern hybridization
　　Iron deficiency is a widespread agricultural problem
for many crops grown in alkaline and calcareous soils in
the world.Iron in these soils , although abundant , is of-
ten insoluble and therefore unavailable for plants[ 1] .In
calcareous soils , many crops , such as grape[ 2] , apple ,
pear , citrus , rose[ 3] , soybean[ 4] and many other species ,
appear to be sensitive to iron stress.Losses of fruit har-
vest would happen when Fe_deficiency caused serious
chlorosis and withering.Rom[ 5] indicated that lime in-
duced chlorosis is a major problem in peach production in
44% of the countries surveyed.Iron deficiency also has
afflicted more than three billion people worldwide[ 6] , and
plants are the principal source of iron in most diets.Low
availability of iron often limits plant growth because iron
forms insoluble ferric oxides , leaving only very little solu-
ble form as organic complex (10-17 mol/L)in soil solu-
tion , a value far below the amount required for the opti-
mal growth of plants(10-6 mmol/L)[ 7] .The importance
of a ferric_chelate reductase in Fe absorption by plant
roots has been recognized since the original work by
Chaney et al[ 8] , which showed the existence of an obliga-
tory Fe reduction step from Fe (Ⅲ)to Fe (Ⅱ)prior to
its uptake by the roots of dicotyledonous plants.The in-
crease in the capacity to reduce Fe (Ⅲ)in Fe_efficient
plants is thought to be caused by one or several plasma
membrane_bound ferric chelate reductase (FCR) en-
zymes[ 9 ,10] .The enzyme FCR is required by most plants
to take up soluble iron.Robinson et al[ 11] isolated the
FRO2 gene , which is expressed in iron_efficient root of
Arabidopsis , then was transferred into Arabidopsis mutant
which can not induce FCR activity , an equivalent restora-
tion of FCR activity was observed in the transformed
lines.
C.junos was found to be tolerant to iron chlorosis
in field and laboratory experiment , and it was able to take
up iron from soils of low iron availability , but the bio-
chemical mechanism for the iron efficiency remained un-
clear.The purpose of this paper was to study the toler-
ance mechanism of C.junos by comparing the FCR activ-
ity of C.junos and P .trifoliata under iron stress.The
spatial expression pattern of FRO2 in C.junos and P.
trifoliata under iron stress was investigated by tissue
printing.
1　Materials and Methods
One citrus species , Citrus junos Sieb.ex Tanaka(tolerant , from Chinese Citrus Research Institute)and
one poncirus species , Poncirus trifoliata (L.) Raf.(susceptible , from Horticulture Department , Southwest
Agricultural University)were used.Seedlings of C.
junos and P .trifoliata were obtained by direct germina-
tion of seeds in the soil.The seedlings with three leaves
were transferred into Hoagland solution.Iron was supplied(80μmol/L and 0μmol/L)as Fe(Ⅱ)EDTA.All solu-
tions were made with double distilled de_ionized water ,
pH adjusted to 5.8.The plants were grown in greenhouse
at 30 ℃day/25 ℃ night (±5 ℃)and 45% to 85%
Received:2001-10-16　Accepted:2002-01-04
Supported by the Project of Plant Transformation Research and Application(grant of the Science and Technology Ministry of China(Joo_A_008_07)).
＊Author for Correspondence.E_mai l:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

Acta Botanica Sinica　2002 , 44(7):771-774
relative humidity (RH).
The FCR activity of roots was assayed by measuring
the formation of Fe (Ⅱ)BPDS complex from Fe (Ⅲ)-
EDTA(535 nm with an extinction coefficient of 22.14
mmol·L-1·cm-1[ 12]).Roots , about 2.5 cm long , were
excised , rinsed briefly in water , and collected and im-
mersed in 4 mL of Fe3+ reductase assay solution contain-
ing 5 mmol/L Mes(pH 5.5), 0.5 mmol/L CaSO4 , 0.1
mmol/L Fe3+ EDTA , and 0.3 mmol/L BPDS (Sigma).
Samples were incubated in the dark in a shaker.Mea-
surements were also made in the absence of roots to cor-
rect for any nonspecific Fe reduction.All measurements
were performed at room temperature with a Shimadzu
spectrophotometer(Kyoto , Japan)model UV_120_02.
The FRO2 gene in pELC203 was kindly provided by
Nigel J.Robinson (Newcastle University , UK).The
plasmid DNA was introduced into an E .coli line (XL1_
Blue)and isolated with High Pure Kit.The FRO2 frag-
ment was obtained from Xba Ⅰ digested DNA and labeled
with DIG (Boehringer Mannheim)Kit.
The tissue printing was conducted as described by
Maliga et al[ 13] and McClure and Guibfoyle[ 14] .The ny-
lon filter was rinsed with 0.2×SSC , 1%SDS at 65 ℃,
4 h to remove compounds released from broken cells ,
e.g.pigment , etc.Prehybridization and hybridization
were performed according to standard protocol(Boehringer
Mannheim).Immunological reaction was carried out as
described by the suppliers(Boehringer Mannheim)for 15
h.
2　Results and Discussion
2.1 　Time course for the development of ferric_
chelate reductase activity
The FCR enzyme activity in roots of C.junos was
induced under iron deficiency and increased about twenty-
fold during the four weeks , but P.trifoliata was stimu-
lated to increase only about threefold at the same time un-
der the same condition (Fig.1).This result showed that
the obvious increase of FCR enzyme activity under iron
stress was an important reason for the tolerance of C.
junos to iron_deficiency .
Fig.1.　Time course of activity of ferric chelate reductase under
iron deficiency.
2.2　Spatial expression pattern of ferric_chelate re-
ductase gene
As shown in Fig.2(a , b and c), strong hybridiza-
tion signals were observed in C.junos under iron_defi-
cient condition , and the FRO2 transcripts were expressed
in roots and stems as well as in leaves.The brief spatial
expression pattern of FRO2 in the tissue print of various
organs was examined in tissue printing Northern hybridiza-
tion.The transcripts were detected in the epidermis and
exodermis , primary xylem layers of the absorptive root , in
the collenchyma and primary xylem , and in the primary
phloem layers of the young green stem , but the transcripts
in cortex parenchyma and pith parenchyma were hardly
detectable in the young green stem.FRO2 transcripts
were expressed not only in the vein vascular bundle but
also in all mesophyll palisade tissue and spongy tissue
layers in the new leaves.By contrast , the transcripts were
accumulated slightly in the same organs of C.junos un-
der iron_sufficient conditions (Fig.2 , d , e and f).The
transcripts were also accumulated slightly in all tissue lay-
ers of P .trifoliata at both iron_deficiency and iron suffi-
ciency (Fig.2 , g , h , k , l , m and n).Such results re-
vealed that high_level FRO2 transcripts were presented in
C.junos under iron_deficient conditions , and the tran-
scripts accumulated in response to iron deficiency.This
suggests that the regulation of FCR activity occurs at tran-
scriptional level.
It has been reported that FCR exists on root cell
PM
[ 10 ,15] .In recent years , this point of view has been
developed with some new methods applied to isolated leaf
protoplasts[ 16] and submerged leaf discs[ 17] , because Fe
acquisition from the soil is not the only limiting step in Fe
utilization by plants.After being taken up by the plant
root , Fe must be transported to its sites of utilization all
over the plant.Since most(up to 80%)of the leaf Fe is
located in the chloroplast , most of the Fe must cross sev-
eral biological membrane systems to arrive at its final des-
tination
[ 17] .The present research clearly revealed that
high level of FRO2 transcripts can be found in new leaf of
iron efficient C.junos under iron stress , and can provide
enough capacity of reduction to make Fe(Ⅲ)change into
Fe (Ⅱ)in order to cross mesophyll cell membranes and
meet the need of chlorophyll synthesis.But in sensitive
plants , for instance , P.trifoliata , we have observed
that the transcripts were expressed slightly in the new
leaves either under iron sufficiency or under iron deficien-
cy.This verified that part of the Fe acquired from the soil
by FCR could be immobilized and accumulated in inactive
forms somewhere in the leaf , because the Fe (Ⅲ)could
not cross the mesophyll cell membranes without enough
reducing capacity , so chlorosis symptom appeared in the
new leaves of sensitive P .trifoliata due to less synthesis
of chlorophyll.
Few data are available in the literature about long_
distance transport of Fe in the xylem and phloem of the
stem.In our experiment , the transcripts could be ob-
served in the collenchyma of new stem cortex of C.
junos.It explains that there exist many chloroplasts in
collenchyma cells and Fe reducing is involved to cross the
cell membrane.It should be further studied why the tran-
scripts were also detected in primary xylem and phloem of
the stem vascular cylinder.Iron is commonly assumed to
be transported in the xylem as Fe(Ⅲ)_citrate complexes
or Fe(Ⅲ)nicotinamide complexes that play a role in the
772　 植物学报　Acta Botanica Sinica　Vol.44　No.7　2002
Fig.2.　Ferric chelate reductase mRNA detection in growing Citrus junos and Poncirus trifoliata by tissue printing Northern hybridization.
a, b , c.Root , stem , leaf of Citrus junos under iron deficiency.d , e , f.Root , stem , leaf of C.junos under iron sufficiency.g , h , k.
Root , stem , leaf of Poncirus trifoliata under iron deficiency.l , m , n.Root , stem , leaf of P.trifoliata under iron sufficiency.
long_distance transport in xylem or phloem[ 18 ,19] .Al-
though IRTI gene has been isolated , it is expressed in the
roots of Arabidopsis and it was proposed that IRTI should
be an Fe (Ⅱ)transporter that takes up iron from the
soil[ 20] .The hybridization signals of the stem vascular
cylinder may provide some information for understanding
some questions related to long_distance transport system of
Fe.
Although some authors have studied reduction of
Fe(Ⅲ)_chelates in mesophyll of leaf disks using the
methods of vacuum infiltration or with isolated protoplast ,
none of these could avoid the effects of the compounds re-
leased from the mesophyll cells and broken cells at the
disk edge , or in unreal physiological environments.The
tissue print technique and RNA Northern hybridization
could provide a pure expression of FCR and avert the
above_mentioned effects.Although in situ hybridization
studies can be used to define the exact cell types that ex-
press the FRO2 gene in iron stress , tissue printing can
provide a rapid and reliable method to research the un-
complicated spatial and temporal expression pattern of
FRO2 and other genes in wooden plants , to ascertain the
correlation between the dynamic expression of genes under
iron stress and physiological phenomena traditionally
known as iron stress responses.
Acknowledgements:We thank Prof.N J Robinson(Newcastle University , UK)for kindly providing FRO2
gene and Prof.LI Dao_Gao (Southwest Agricultural Uni-
versity , China)andMr.DENG Lie(Citrus Research In-
stitute , China), Prof.HUANG De_Hui(Southwest Agri-
cultural University , China)for kindly providing seeds of
C.junos and P.trifoliata , Prof.WANG Yi_Min , Prof.
HUANG Xiao_Ming , Dr.XIAO Yue_Hua and Mr.NIU
Guo_Qing for their comments on this manuscript.
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三价铁螯合物还原酶在香橙和枳中的表达
李　凌　范艳华　罗小英　裴　炎　周泽扬＊
(西南农业大学生物技术中心 , 重庆 400716)
摘要:　用耐缺铁的香橙(Citrus junos Sieb.ex Tanaka)和极不耐缺铁的枳(Poncirus trifoliata (L.)Raf.), 在铁胁迫条件
下对根的三价铁螯合物还原酶活性变化和酶基因的表达情况进行了研究。离体根的酶活性测定表明 , 在铁胁迫 4
周时 ,香橙根的酶活性增强约 20 倍 ,枳仅增强约3 倍。用拟南芥的三价铁螯合物还原酶基因作探针进行组织印迹
的Northern 杂交检测香橙和枳三价铁螯合物还原酶的 mRNA ,在铁胁迫 2周时 , 香橙吸收根 、幼茎和新叶中均检测到
强烈的表达信号 ,而枳相同器官的表达信号则极其微弱。实验结果表明 , 三价铁螯合物还原酶活性在缺铁胁迫下
被诱导强烈增加是香橙耐缺铁的重要原因 ,该酶活性的调控发生在转录水平上 ,而且该酶基因在诱导条件下在根 、
茎和叶中均有表达。
关键词:　香橙;枳;缺铁胁迫;三价铁螯合还原酶;组织印迹;FRO2 基因
中图分类号:Q781　　　文献标识码:A　　　文章编号:0577-7496(2002)07-0771-04
收稿日期:2001-10-16　接收日期:2002-01-04
基金项目:国家转基因植物研究与产业化专项课题(Joo_A_008_07)。
＊通讯作者。E_mai l:。
(责任编辑:谢　巍)
774　 植物学报　Acta Botanica Sinica　Vol.44　No.7　2002