All life on earth depends on the process of pho-tosynthesis， which occurs in the chloroplasts. Photo-synthesis is responsible for not only CO2 fixation butalso other essential plant functions such as the pro-duction of carbon skeletons， fatty acids， and pig-ments and the synthesis of amino acids from inorganicnitrogen ［1］. Exploration of the functions of genes involved in chloroplast development and photosynthesisis a topic of importance in plant science.

Mutants are powerful tools for the examination ofgene function and plant development， and leaf colormutants are usually associated with chloroplast devel-opment. Thus far， many mutants that interfere with

chloroplast development have been identified in manyplant species such as maize， tobacco， Arabidopsis，and rice ［2-6］. According to the changes in leaf colorthat they induce， mutants are classified into 3 class-es: albino， pale green and variegation. Due to muta-tions in Alb3， Cla1 and Pac， the leaves of Ara-bidopsis plants are albino ［7-11 ］. When App1， Cao，

一个新的水稻斑马叶突变（zebra leaf 1）对叶绿体发育的影响（简报）张道允 1，2 巩子英 1 叶建伟 1，2 吴彦霞 1，2 米华玲 1 陈根云 1*

（1中国科学院上海生命科学研究院植物生理生态研究所， 上海 200032； 2中国科学院研究生院， 北京 100049）

摘要 通过 γ射线诱变， 在水稻粳稻栽培品种 9522 中得到一个斑马叶突变体 zebra leaf 1。 为了研究 zl1 的功能， 我们对突变体进行了形态学和细胞学的分析， 同时也对此基因突变以后对叶绿体发育和光合作用的影响作了评价。 突变体叶片上绿色和枯白色条纹相间， 叶绿素含量显著的下降。电镜显示叶绿体类囊体的排列被打乱， 变得杂乱无章。 这表明 zl1 突变体在叶绿体发育过程中出现障碍。 zl1基因的突变使得净光合速率显著的下降。 参与光合作用的一些关键蛋白， 比如核酮糖1， 5-二磷酸羧化酶/加氧酶 （Rubisco）、 Rubisco 活化酶、 D1 蛋白、 CF1β 亚基的表达量也显著的下调。 但是， zl1突变体对外界环境非常敏感， 有时会没有表型。

关键词： 水稻 叶绿体发育 光合作用

**Supported by the Chinese Academy of Sciences （CAS，KSCX2-YW-N-059），the Ministry of Science and Technology，China（2005cb121106）.

**Corresponding author，Tel：021-54924235； Fax：021-54924015；Email: chenggy@sippe.ac.cn

第 41 卷 第 5 期 Ｖｏｌ． 41, Ｎｏ．5October ２００8２００8年 10 月

分 子 细 胞 生 物 学 报Journal of Molecular Cell Biology

本文 2008 年 4 月 25 日收到。 2008 年 7 月 20 日接受。**基金项目：科学院院部基金项目（CAS，KSCX2-YW-N-059），973 项目（2005cb121106）。**通讯作者，Tel： 021-54924235； Fax：021-54924015； Email: chenggy@sippe.ac.cn

Ｋｅｙ ｗｏｒｄｓ： Rice. Chloroplast development. Photosynthesis

EFFECTS OF ZEBRA LEAF 1-A NEW VARIEGATIONMUTATION-ON CHLOROPLAST DEVELOPMENT IN RICE*

ZHANG Dao Yun1，2 GONG Zi Ying1 YE Jian Wei1，2

WU Yan Xia1，2 MI Hua Ling1 CHEN Gen Yun1，**

（1Institute of Plant Physiology and Ecology， Shanghai Institutes for Biological Sciences， Chinese Academy ofSciences， Shanghai 200032， China； 2Graduate School of the Chinese Academy of Sciences， Beijing 100049， China）

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41卷张道允 巩子英 叶建伟 吴彦霞 米华玲 陈根云

and VIPP1 were mutated， the chlorophyll content ofthe mutants was significantly decreased， and theleaves were pale green ［12-14］. It has been shown thatmutations in Chm， Im， Var1， and Var2 are correlatedwith leaf variegation ［15-18］. Leaf variegation has longbeen known as a recessive genetic trait in higherplants. Variegation results from a defect that destabi-lizes chloroplast development; it does not terminatechloroplast development， as indicated by the fact thatat least a part of the plant tissues produces normalchloroplasts. Although leaf -variegated mutants havecontributed to the information on maternal inheritanceand have been used as genetic markers， these muta-tions remain poorly understood at the molecular level.

In this paper， we reported a new leaf variegationmutant-zebra leaf 1 （zl1） -in rice. We studied theeffect of this mutation on chloroplast development andphotosynthesis.

The variegation mutant zl1 generated by γ -rayirradiation was gifted to us by Professor Da -BingZhang （ Shanghai Jiaotong University）. All plantswere grown in a paddy field at Shanghai Academy ofAgriculture Sciences. For physiological and biochemi-cal analyses， the zl1 mutant and wild -type plantswere cultivated in a phytotron （photon intensity: ap-proximately 200-300 μmol m-2 s-1； 12h light at 25°Cand 12h dark at 20°C）. Fluorescence images of chlor-ophyll were acquired with the confocal microscope as

described by Vermaas et al. ［19］. Transmission electronmicroscopy analysis was made as described by Peng etal ［20］. Chlorophyll was separately extracted with 80%acetone from 8 fresh leaves， and its content was de-termined according to the method of Arnon ［21］. Theleaf net photosynthetic rate （Pn） of the zl1 mutantand the 9522 cultivar grown in the phytotron wereperformed as described by Chen et al ［22］. Blades ofrice leaves were ground in liquid N2 with a disintegra-tor and extracted with an extraction buffer （50 mmol/L Tris-Cl pH 7.6， 50 mmol/L NaCl， 1% SDS）. Im-munodetection was conducted according to the methodof Hong［23］.

Characterization of zl1 mutantsThe leaves of the zl1 mutants resembled the

coat of a zebra. Stripes of green and chlorotic areaswere observed along the leaf vein and the leaf sheath（Fig.1A）. The zl1 mutant was sensitive to variationsin the environment. For 2 years， no phenotype wasobserved for the zl1 mutants in the fields in Shanghai.However， the phenotype of the zl1 mutant was alwaysobserved in fields in Hainan and phytotrons in Shang-hai.

The intensity of chlorophyll fluorescence was sig-nificantly lower in the case of the zl1 mutants than inthe case of the wild-type plants（Fig.1B，C），indicatingthat chlorophyll synthesis or chloroplast developmentwas disturbed in the zl1 mutants.

Fig.1 Phenotypes of the zl1 mutant and wild-type plantsA. Leaves of the zl1 mutant and wild-type plants

B，C. Chlorophyll fluorescence images of each leaf of the zl1 mutant and wild-type plants.

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There was no difference in the size of the chloro-plasts between the zl1 mutants and wild-type plants（Fig.2）. However， the thylakoid membrane systemsin the zl1 chloroplasts were disturbed， and the mem-brane spacing was not as distinct（Fig.2）.

Effects of the zl1 mutation on the chlorophyllcontent， leaf Pn and some proteins involved inphotosynthesis

The chlorophyll content in the zl1 mutant wasapproximately 30% less than that in the 9522 ricecultivar （wild type）（Fig.3）. The chlorophyll a and bcontents were also significantly lower in the zl1 mu-tant （Fig.4）. The Pn value of the zl1 mutant was sub-

stantially lower than that of the wild -type cultivar（Fig.4）. This finding indicated that the zl1 mutationaffected photosynthesis to a considerable extent.

In order to identify the components of the photo-synthetic system that are affected by the zl1 gene，immunodetection was used to probe some key proteinsinvolved in photosynthesis. The amount of Rubisco，the key enzyme responsible for CO2 carboxylation，was significantly decreased in the zl1 mutant （Fig.5）.The amount of Rubisco Activase （Rca）， which pro-motes Rubisco activation， was also significantly de-creased （Fig.5）. Further， we also found that the lev-els of D1 and CF1β were significantly reduced in the

Fig.2 Electron micrographs of chloroplasts from the leaves of the zl1 mutant and wild-type plantsA. Chloroplast of wild type plants； B. Chloroplast from the chlorotic areas of the leaves of zl1 mutant.

Chloroph

yllc

ontent（ m

g/dm

2 ）

3.2

2.4

1.6

0.8

0.0Chl（a+b） Chla Chlb

WTzl1

Pn（ μ

molCO

2/m

2 s）

16

12

8

4

0WT zl1

Fig.3 Comparison of the chlorophyll content in theleaves of the wild-type plants and the zl1 mutant

Each value is the mean of 6 leaves， and SE is expressedas a vertical bar. Asterisks （*） represent very significant（P<0.05） differences between the wild-type plants and the zl1mutants.

Fig.4 Effect of the zl1 mutation on the net photosyntheticrate（Pn）

Each value is the mean of 8 leaves, and SE is expressedas a vertical bar. Two asterisks （**） represent very significant（P<0.01） differences between the wild-type plants and the zl1mutant.

Zl1WT

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41卷张道允 巩子英 叶建伟 吴彦霞 米华玲 陈根云

Fig.5 Immunodetection of chloroplast proteins involvedin photosynthesis from zl1 and wild-type rice

The proteins were separated by SDS-PAGE， and theblots were probed with specific antibodies to RbcL， Rca， D1，and CF1β.

WT zl1

RbcL

Rca

D1

CF1β

zl1 mutant （Fig.5）.The function of zl1 gene on chloroplast devel-

opmentLeaf variegation would result from defects that

destabilize chloroplast development. Mutations insome genes such as Chm， Im， Var1， and Var2cause the variegation phenotype［15-18］. Chm shares highhomology with yeast MSH1， which is involved in mi-tochondrial genome maintenance［15］. Chm may be as-sociated with mismatch repair and the maintenance ofmtDNA［15］. Im encodes a chloroplast protein similar tomitochondrial alternative oxidase. The variegationcaused by mutations in Im is probably due to thephotooxidation of chloroplasts， directly or indirectlyby the impairment of the carotenoid biosynthetic path-ways ［16］. Var1 and Var2 encode similar chloroplastFtsH proteases ［17，18］. These proteases are the majorcomponents of the FtsH complex， which is involvedin the repair of photodamaged proteins in the thy-lakoid membranes［18］. Mutations in all the abovemen-tioned genes were identified in Arabidopsis. However，they were rarely identified in the other more importantmodel plant， i.e.， rice; the exception to this is that amutation in pyl-v， which encodes a chloroplast pro-tease， was identified in rice［24］.

In this study， we mapped another new variega-tion mutant-zl1-which was different from the pyl -vmutant in the following aspects. First， the phenotype

of zl1 was found to be sensitive to variations in theenvironment， while that of the pyl-v mutant was sta-ble irrespective of variations in the environment ［24］.Second， the variegation observed in the zl1 mutantwas not as severe as that observed in the pyl-v mu-tant. The leaves of the zl1 mutant were greener thanthose of the pyl-v mutant （Fig.1）. Third， the zl1 al-lele was found to be located on chromosome 1 （datanot shown）. In contrast， the pyl-v allele was mappedbetween the SSR markers RM251 and RM282 onchromosome 3［24］.

Because the zl1 mutant exhibited significantlyreduced chlorophyll content and defective chloroplastdevelopment （Fig.1B， Fig.2， Fig.3）， we presumedthat this mutation affects photosynthesis. The signifi-cant decrease in the Pn in zl1 mutant leaves con-firmed our hypothesis （Fig.4）. It is well known thatfor photosynthesis to be successful， coordination be-tween its 2 phases -the light reaction and carbon -linked reactions-is required. O2， NADPH， and ATPare produced in the light reaction through coordina-tion among PSII， PSI， and ATPase， and they areused for carbon fixation. The D1 protein and CF1βsubunit are the core proteins of PSII and ATPase，respectively. The significant decrease in the levels ofthese proteins in the zl1 mutants indicated that theassimilatory power （ATP and NADPH） in these mu-tants was severely reduced （Fig.5）. Rubisco catalyzesCO2 fixation and plays a critical role in chloroplastbiochemistry. Rca promotes Rubisco activation. Theexpression levels of these 2 proteins were found to besignificantly reduced in the mutants （Fig.5）， indicat-ing that the carboxylation capacity was decreased.These results demonstrate that the zl1 mutation affectsboth the assimilatory power and carboxylation. There-fore， it is possible that zl1 functions during the earlystage of chloroplast development.

AcknowledgementThis project was supported by the Chinese A-

cademy of Sciences （CAS， KSCX2-YW-N-059） ，the Ministry of Science and Technology， China（2005cb121106）. We thank Dr. Da-Bin Zhang forthe gift of the zl1 mutant.

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