Rice OsGL1-1 Is Involved in Leaf Cuticular Wax and Cuticle Membrane

Molecular Plant?Volume4?Number6?Pages985–995?November2011RESEARCH ARTICLE Rice OsGL1-1Is Involved in Leaf Cuticular Wax and Cuticle Membrane

Bao-Xiang Qin a,2,Ding Tang a,2,Jian Huang a,Ming Li a,Xin-Ru Wu a,Li-Li Lu b,Ke-Jian Wang a,Heng-Xiu Yu b, Jian-Min Chen b,Ming-Hong Gu b and Zhu-Kuan Cheng a,1

a State Key Laboratory of Plant Genomics and Center for Plant Gene Research,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences, Beijing100101,China

b Key Laboratory of Plant Functional Genomics of Ministry of Education,Yangzhou University,Yangzhou,Jiangsu225009,China

ABSTRACT Cuticular wax forms a hydrophobic barrier on aerial plant organs;it plays an important role in protecting a plant from damage caused by many forms of environmental stress.In the present study,we characterized a rice leaf wax-de?cient mutant osgl1-1derived from a spontaneous mutation,which exhibited a wax-de?cient and highly hydro-philic leaf phenotype.We cloned the OsGL1-1gene by the map-based cloning method and performed a complementation test to con?rm the function of the candidate gene.Molecular studies revealed that OsGL1-1was a member of the OsGL1 family,and contained regions that were homologous to some regions in sterol desaturases and short-chain dehydro-genases/https://www.360docs.net/doc/1417918541.html,pared to the wild-type,the osgl1-1mutant showed decreased cuticular wax deposition,thinner cuticular membrane,decreased chlorophyll leaching,increased rate of water loss,and enhanced sensitivity to drought. OsGL1-1is expressed ubiquitously in rice.The transient expression of OsGL1-1–green?uorescent protein fusion protein indicated that OsGL1-1is localized in the cytoplasm,plasma membrane,and nucleus.

Key words:Cuticular wax;cuticular membrane;OsGL1-1;rice.

INTRODUCTION

Leaves of higher plants are covered by a cuticle consisting of intracuticular waxes embedded in a polymer matrix of cutin and epicuticular waxes deposited on the outer surface of the cutin layer.The waxes are synthesized and secreted by epi-dermal cells and confer essential ecophysiological functions, including the prevention of uncontrolled nonstomatal water loss and protection of the plant against UV radiation as well as against bacterial and fungal pathogens(Reicosky and Hanover,1978;Jenks et al.,1994;Suh et al.,2005).In addition, waxes in the tryphine-containing layer of pollen grains are essential for proper pollen-stigma signaling required for fertilization(Preuss et al.,1993).

Cutin and wax are derived from fatty acid precursors.Cutin is composed of C16and C18hydroxy and epoxy-hydroxy fatty acid monomers(Heredia,2003),while waxes predominantly contain alcohols,aldehydes,ketones,alkanes,and esters de-rived from very long-chain fatty acids(VLCFAs),with chain lengths ranging from C20to C34(Kunst and Samuels,2003). Some researches show that synthesis and transport of wax and cutin are closely coordinated(Suh et al.,2005).Neverthe-less,how the deposition of waxes is coordinated with cutin for-mation is still completely unknown.

Epicuticular waxes in?uence light refraction.In many species, the epicuticular waxes form microscopic crystals that scatter light and give the surface a whitish(glaucous)appearance

(Clark and Lister,1975);mutations in genes involved in wax ac-cumulation are commonly characterized by a glossy phenotype

that can be detected visually.On the basis of this difference,

mutants have now been isolated from a number of plant spe-

cies,including barley,Arabidopsis thaliana,Zea mays,and Bras-

sica napus.In addition,epicuticular waxes strongly in?uence

leaf wettability.The leaves of wax-de?cient mutants are more hydrophilic and water droplets on these leaves do not run off

easily.These wetting characteristics of the hydrophilic leaf sur-

faces are relatively easy to detect and facilitate the identi?ca-

tion of rice leaf wax-de?cient mutant lines,whose leaf

cuticle is not smooth and which do not display a glossy pheno-

type without water adhesion.

In recent years,the molecular identi?cation of wax-related

genes has been achieved mainly in Arabidopsis and maize by

1To whom correspondence should be addressed.E-mail zkcheng@genetics.

https://www.360docs.net/doc/1417918541.html,.

2These authors contributed equally to this work.

aThe Author2011.Published by the Molecular Plant Shanghai Editorial

Of?ce in association with Oxford University Press on behalf of CSPB and

IPPE,SIBS,CAS.

doi:10.1093/mp/ssr028,Advance Access publication21April2011

Received1September2010;accepted13March2011

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using either forward or reverse genetic approaches;further,the function of the corresponding proteins has been proposed on the basis of the phenotype of the corresponding mutants.Among these genes,some encode wax biosynthetic enzymes,such as FATB ,FAE1,KCS1,KCS2,CUT1,HIC ,FDH ,CER10,CER4,CER8,WSD1,MAH1,LACS2,GL8A ,and GL8B (James et al.,1995;Millar et al.,1999;Todd et al.,1999;Yephremov et al.,1999;Gray et al.,2000;Pruitt et al.,2000;Bonaventure et al.,2003;Schnurr et al.,2004;Dietrich et al.,2005;Zheng et al.,2005;Rowland et al.,2006;Greer et al.,2007;Li et al.,2008;Lee et al.,2009;Lu et al.,2009).Some genes are involved in wax secretion,such as LTPG ,CER5,and WBC11(Pighin et al.,2004;Bird et al.,2007;Debono et al.,2009).Some genes en-code regulatory proteins;overexpression of WIN1/SHN1,which are AP2/EREBP-type transcription factors,can cause an increase in cuticular wax levels (Aharoni et al.,2004;Broun et al.,2004).CER7is a core exosome subunit that controls the expression of CER3/WAX2/YRE/FLP (Hooker et al.,2007).Although several genes have been isolated,their precise bio-logical function remains poorly characterized thus far;these genes include members of the GL1homologous gene family,such as GL1,CER1,and WAX2(WAX2is an allele of CER3/YRE/FLP )(Aarts et al.,1995;Ariizumi et al.,2003;Chen et al.,2003;Kurata et al.,2003;Sturaro et al.,2005;Rowland et al.,2007).Although these genes are involved in similar processes across different plant species,mutations in these homologous genes result in different phenotypic changes in the cuticular wax compositions of Arabidopsis and maize.The cer1mutant is proportionally de?cient only in alkanes (and related metabo-lites)and has elevated levels of aldehydes (Aarts et al.,1995);the wax2mutant is de?cient in both aldehydes and alkanes (and related metabolites)(Chen et al.,2003),while the gl1mu-tant is de?cient in aldehydes and has high levels of alkanes (Sturaro et al.,2005).Hence,the biochemical function of the corresponding proteins cannot be deduced on the basis of changes in wax composition alone;most studies related to these genes were generally limited to the identi?cation of their roles in plant growth and development.

Since cuticular wax is the outermost barrier against nonsto-matal water loss and against damages caused by many forms of environmental stress,the deposition and composition of cu-ticular wax are closely correlated to drought tolerance in plants (Jenks et al.,2001;Cameron et al.,2006;Kosma et al.,2009).The wax2mutant exhibited elevated epidermal perme-ability and reduced cuticular wax loading and stomatal index on adaxial and abaxial leaf surfaces (Chen et al.,2003).Over-expression of WXP1led to increased cuticular wax loading on the leaf surfaces,reduced water loss,and enhanced drought tolerance in transgenic alfalfa (Zhang et al.,2005).Overexpres-sion of WIN1/SHN1induced wax production and enhanced drought tolerance (Kannangara et al.,2007).

Rice (Oryza sativa L.)is one of the three staple crops world-wide,but rice production requires large amounts of water.Drought has become the largest factor limiting rice production in water-limited areas;therefore,rice production can be stabi-

lized by screening for drought-resistant germplasm and devel-oping drought-tolerant rice cultivars.Tremendous efforts have been devoted to identifying key regulators in rice drought re-sponse and exploring for their application in the improvement of drought tolerance,and many regulators have been identi-?ed.However,only a small portion of these regulators have been explored as potential candidate genes for their applica-tion in the improvement of drought tolerance in rice.Based on biological functions,these genes can be classi?ed into the fol-lowing three categories:(1)stress-responsive transcriptional regulation;(2)post-transcriptional RNA or protein modi?ca-tions such as phosphorylation/dephosphorylation;and (3)oso-moprotectant metabolism or molecular chaperones (Yang et al.,2010).Among these genes,compared to CBF3,SOS2,NCED2,NPK1,and NHX1,LOS5and ZAT10have been showed to display relatively better effect in improving drought resis-tance of transgenic rice under ?eld conditions (Xiao et al.,2009).While continuing down the path to discovery of new genes involved in drought tolerance,serious efforts are also focused on plant leaf cuticular wax because its accumulation is closely correlated with drought tolerance.However,very few leaf wax-de?cient mutants associated with drought sensitivity have been characterized in rice thus far.Recently,WSL1,an ortholog of Arabidopsis KCS ,was isolated and characterized as being involved in wax deposition.wsl1displayed sparse wax crystals and enhanced sensitivity to drought (Yu et al.,2008).Moreover,the GL1homologous genes in rice have been systematically analyzed for sequence and expression diversi?-cation.The results suggested that 11putative GL1-like genes were found in the rice genome;these genes were designed OsGL1-1;OsGL1-11.Overexpression of one of these genes (OsGL1-2)increased cuticular wax accumulation and perhaps cause enhanced drought tolerance (Islam et al.,2009).

In this study,we cloned the OsGL1-1gene by adopting a map-based cloning strategy;OsGL1-1was shown to be a member of the OsGL1family,corresponding to OsGL1-1in OsGL1-1;OsGL1-11(Islam et al.,2009).Among its orthologs from Oryza sativa ,Arabidopsis thaliana ,Zea mays ,and Med-icago truncatula ,OsGL1-1had highest sequence similarities to GL1in Zea mays and contained certain regions that were homologous to some regions in sterol desaturases and short-chain dehydrogenases/reductases.The OsGL1-1gene is universally expressed and the OsGL1-1-encoded protein is localized in the nuclei,cytoplasm,and plasma membrane.

RESULTS

Characterization of the osgl1-1Mutant

Morphologically,the osgl1-1mutant showed a normal pheno-type.Nevertheless,osgl1-1could be distinguished from wild-type (WT)plants based on the wetting characteristics of leaf surfaces by water (Figure 1A).In WT plants,due to the presence of epi-cuticular wax bloom,the wetting of the superhydrophobic leaf cuticle by water is minimized,water molecules tend to cluster

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together to form a spherical water bead,and a slight tilting of the leaf will cause the water bead to run off.In the osgl1-1mutant,the leaf cuticle is highly hydrophilic,so water molecules spread out on the whole leaf surface and confer a wettish,glossy appearance to leaf surface (Figure 1B).

Positional Cloning of the OsGL1-1Gene

We demonstrated that OsGL1-1was controlled genetically by a single locus based on the segregation ratio that revealed that 127out of 410F 2plants derived from a cross between osgl1-1and Zhonghua 11showed the mutant phenotype ( 2=0.488,P .0.05).Using this population,the locus was mapped between STS markers,P1and P6,on the long arm of rice chromosome 9(Figure 2A).We further generated a larger F 3mapping popula-tion with 1842homozygous recessive plants and ?ne-mapped the locus between STS markers,P3and P4.Between the two markers,we developed three additional STS markers,and map-ped the OsGL1-1gene within an interval of 35kb between two STS markers:P7and P9.No recombinant near the STS marker P8was found (Figure 2B).Within this 35-kb region,there was only one annotated gene (09g25850)(Figure 2C);the candidate gene 09g25850showed high similarity with GL1from Zea mays and was designed OsGL1-1in the OsGL1family (Islam et al.,2009).The mutants related to the GL1gene displayed a reduction in cuticular wax deposition on young leaves of Zea mays .Thus,this candidate gene in the osgl1-1mutant was chosen to be ampli?ed and sequenced.OsGL1-1was found to be mutated through a sub-stitution mutation in nucleotide 1729(CAC /GAC)in the tenth exon of the OsGL1-1gene,which substitutes aspartic acid for his-tidine (Figure 2D).

The candidate gene was further con?rmed by a comple-mentation test.The plasmid pCGL,containing the entire open reading frame (ORF),a 2899-bp upstream sequence,and a 1436-bp downstream sequence (Figure 2E),was trans-

formed into osgl1-1mutant embryogenic callus via Agrobac-terium tumefaciens -mediated transformation.Twenty-three transgenic plants transformed with pCGL were identi?ed,and they showed a complementation of the osgl1-1pheno-type (Figure 1C and 1D).

Characterization of the Predicted OsGL1-1Protein

The OsGL1-1cDNA (accession AK060786)was 1857nucleotides long with an ORF that encoded a polypeptide of 619amino acids.The N-terminal portion of OsGL1-1protein contains a fatty acid hydroxylase/sterol desaturase domain (gray lines in Figure 3),whereas the C-terminus of OsGL1-1comprises a short-chain dehydrogenase/reductase domain.The TMHMM analysis shows that OsGL1-1may be an integral membrane protein with ?ve transmembrane helices at the N-terminus as well as a cluster of water-soluble and globular domains at the C-terminus.The three histidine-rich motifs (HX 3H,HX 2HH,and HX 2HH,where X stands for any amino acid)

were

Figure 1.Phenotype of the osgl1-1Mutant.

(A)Phenotypic comparison between WT (left)and osgl1-1(right).(B)Comparison of wetting characteristics of leaf surfaces by water between WT (left)and osgl1-1(right).

(C)Transgenic plant generated by complementation.

(D)Wetting characteristics of leaf surfaces of transgenic plant gen-erated by complementation by water.Bars:(B,D),5

mm.

Figure 2.Fine Mapping and Positional Cloning of the OsGL1-1Gene.

(A)The OsGL1-1locus was mapped on the chromosome 9between markers P1and P6.

(B)Fine mapping of OsGL1-1was positioned on BAC3(BAC OSJNBb0057D06)within a 35-kb region ?anked by the markers P7and P9.

(C)Only one candidate gene existed within the 35-kb restricted region.

(D)Mutation position in the OsGL1-1coding region that is sepa-rated by nine introns.

(E)Complementation constructs.The construct pCGL contains the entire OsGL1-1gene,plus a 2899-bp upstream region and 1436-bp downstream region.

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conserved in OsGL1-1and its orthologs (darker gray lines in Figure 3).Taton et al.(2000)have demonstrated by site-directed mutagenesis that the His residues in His-rich motifs forming a divalent metal ions-binding site are essential for the catalytic activity of sterol desaturases.

Orthologs of OsGL1-1from Oryza sativa ,Arabidopsis thali-ana ,Zea mays ,and Medicago truncatula were found in the NCBI database.OsGL1-1has 37–85%amino acid sequence identity with the ?ve characterized orthologs,including GL1,OsGL1-2,WAX2,CER1,and WDA1.Among these,OsGL1-1exhibited maximum sequence similarity with GL1in Zea mays ,with 85%amino acid identity.Phylogenetic anal-ysis indicated that 14OsGL1-1-related proteins could be di-vided into two subgroups (Figure 4):OsGL1-1groups with Maize GL1,Arabidopsis WAX2,and two rice proteins (OsGL1-2and LOC_Os06g44300),whereas the WDA1,CER1,and CER1homologous proteins formed the other group.Ara-bidopsis CER1functions in the biosynthesis of stem wax and pollen coat tryphine alkanes (Aarts et al.,1995),WDA1is involved in wax production in rice anther walls (Jung et al.,2006),whereas maize GL1,Arabidopsis WAX2,and rice OsGL1-2are required for cuticle membrane and wax produc-tion (Chen et al.,2003;Sturaro et al.,2005;Islam et al.,2009).Based on the similar role of these genes within different

plant

Figure 3.Multiple Alignments of Plant Proteins Showing High Similarity with the OsGL1-1Protein.

OsGL1-1(top line)was aligned with three rice proteins (OsGL1-2,LOC_Os06g44300,and WDA1),two Arabidopsis proteins (CER1and WAX2),and one maize protein (GL1).Identical residues are boxed in black and similar residues in gray.The conserved desaturase/hydrox-ylase domain is marked above the OsGL1-1sequence with a gray line.Black segments identify conserved His residues within the desaturase/hydroxylase domain.Putative transmembrane domains (TM1–TM5)of OsGL1-1are marked above the OsGL1-1sequence with a darker gray line.The identity and similarity of the aligned proteins for OsGL1-1are shown at the end of the alignments.

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species,we deduced that OsGL1-1may also function in reg-ulating cuticular wax deposition during leaf development in rice,and the loss of function of OsGL1-1is consistent with the osgl1-1mutant phenotype.

OsGL1-1Expression Patterns

To investigate the expression patterns of the OsGL1-1gene at the organ level,experiments using GUS fusion constructs and RT–PCR were performed.The results of RT–PCR showed that OsGL1-1is expressed in all organs of WT plants(Figure5).Com-pared to WT,the transcripts of the osgl1-1mutants were down-regulated,indicating that mutations in the OsGL1-1 lead to a decreased level of OsGL1-1mRNA.

When expressed under the control of the native OsGL1-1 promoter in a WT background,GUS protein activity was found to be ubiquitously present in aerial parts and roots of seedlings (Figure6A).In glumous?owers,GUS activity was detected in palea,lemma,stigma,anthers,?lament,and ovary(Figure6B and6C).When seeds were differentially stained for GUS activ-ity,younger seeds exhibited intense GUS staining,whereas older seeds showed faint GUS expression(Figure6D and6E). Analyses of a transverse section of the leaf indicated that the GUS activity was localized mainly in mesophyll cells and vascular bundles(Figure6G).In addition,GUS staining was also observed in leaf epidermal cells(Figure6G).In older stems, faint GUS staining was observed in vascular bundles(Figure 6F and6H).In roots,the exodermal cells,endodermal cells,cor-tical cells,and vascular cylinder were stained blue(Figure6I).

Altered Cuticular Wax and Cuticle Membrane in the

osgl1-1Mutant

To further investigate the effect of OsGL1-1disruption on wax accumulation and cuticle structure,SEM and TEM were used for a detailed comparison between the leaf surfaces and the ultra-structure of WT and the osgl1-1mutant.In WT,the adaxial and abaxial leaf surfaces were densely covered by platelet-type wax crystals including the unevenly distributed cuticular papillae (Figure7A and7B),whereas the adaxial and abaxial leaf surfa-ces of the osgl1-1mutant generally exhibit only a small number of wax crystals and the conical cuticular papillae were nearly naked(Figure7D and7E).Ultrastructural analysis of the leaf cuticle by TEM indicated that the WT cuticle membrane appears to be divided into an outermost translucent layer and an inner-most opaque layer(Figure7C).In the osgl1-1mutant,cuticle membrane thickness is obviously reduced,and the outermost translucent layer appears almost absent(Figure7F).

Altered Cuticular Permeability and Drought Sensitivity in the osgl1-1Mutant

Several studies indicated that the change in accumulation of waxes is generally associated with the change in cuticular permeability and whole-plant drought sensitivity.To investi-gate whether cuticular permeability and whole-plant drought sensitivity of the osgl1-1mutant is altered,chlorophyll leaching assays and whole-plant drought-sensitivity assays were con-ducted.Chlorophyll leaching assays showed that chlorophyll leaching from the osgl1-1mutant leaves was slower than that from the WT plant leaves(Figure8A),thus indicating a decrease ofcuticularpermeabilityinthe osgl1-1mutant.Thewhole-plant drought-sensitivity assays revealed that most WT plants recovered from the stress treatment,whereas only a few osgl1-1mutant plants survived(Figure9).As an indicator of drought sensitivity at the whole-plant level,water-loss assay of detached leaves was also performed to compare the water retention capacity of the osgl1-1mutant and the WT plant. As shown in Figure8B,rate of water loss of detached leaves from the osgl1-1mutant was signi?cantly higher than that of the WT plant at various time points.These results show the osgl1-1mutant is more susceptible to drought than the WT plant,andOsGL1-1maybeinvolved inprotectionfromdrought.

Sub-Cellular Localization of OsGL1-1

Secondary structure prediction of OsGL1-1suggests that it may be an integral membrane protein.To determine the localization of OsGL1-1,a full-length OsGL1-1CDS was intro-duced into the N-terminal of the expression vector pJIT163–hGFP,and the fusion gene was transformed into onion skin epidermal cells.Meanwhile,GFP alone was used as a

control.

Figure5.RT–PCR Analysis of OsGL1-1in Seedlings,Root,Culm,

Leaf,and Panicle of WT,the Leaf of WT,and OsGL1-1.

UBQ gene was used as a control.

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Unexpectedly,localization of OsGL1-1–GFP fusion protein was identical to that of GFP alone;their ?uorescence was observed in the nucleus,cytoplasm,and plasma membrane (Figure 10).

DISCUSSION

In this study,we cloned the OsGL1-1gene by way of a map-based cloning strategy,using single-recessive mutants exhibit-ing wetted leaves.Nucleotide sequencing results revealed that the osgl1-1mutant had a single nucleotide substitution in the

tenth exon of the OsGL1-1gene,which resulted in the failure to produce a functional OsGL1-1.The complementation of the osgl1-1phenotype indicated that the mutant phenotype is due to the single nucleotide substitution in the OsGL1-1gene.Some studies show that deposition of waxes may be co-regulated with cutin biosynthesis (Suh et al.,2005).Our studies demonstrated that the osgl1-1mutant alters both wax produc-tion and cutin https://www.360docs.net/doc/1417918541.html,pared to WT,the osgl1-1mutant displays a reduction in cuticular membrane thickness and cuticular wax deposition.Moreover,the defects in

the

Figure 6.Histochemical Staining of GUS Activity in P OsGL1-1:GUS Transgenic Rice Plants.

GUS activity was detected in aerial parts and roots of seedlings (A),glumes (B),stigma,anthers,?lament,ovary (C),young seed (D),older seed (E),internode of older stem (F),transversal section of the leaf (G),transversal section of older stem (H),and root (I).Bars:(A,B),2mm;(C),0.5mm;(D–F),1mm;(G,I),10l m;(H),80l

m.

Figure 7.Electron Microscopic Analysis of WT and osgl1-1.

(A)Adaxial surface of WT.(B)Abaxial surface of WT.

(C)TEM analysis of WT cuticle mem-branes.

(D)Adaxial surface of osgl1-1mutant.(E)Abaxial surface of osgl1-1mutant.(F)TEM analysis of osgl1-1mutant cuti-cle membranes.

P ,papillae;CW,cell wall;TL,translucent layer;OL,opaque layer.Bars:(A,B,D,E),2.5m m;(C,F),200nm.

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cuticle of osgl1-1mutant are further demonstrated by the chlo-rophyll leaching and water-loss https://www.360docs.net/doc/1417918541.html,pared to WT,the osgl1-1mutant shows decreased chlorophyll leaching,in-creased rate of water loss,and enhanced sensitivity to drought.Wax-de?cient mutants in Arabidopsis and rice generally display increased chlorophyll leaching,rate of water loss,and sensitivity to drought (Chen et al.,2003;Islam et al.,2009).However,in transgenic Arabidopsis lines overexpressing the SHN1gene,although both chlorophyll ef?ux and rate of water loss were increased,the plants had increased wax accumulation and

enhanced drought tolerance (Aharoni et al.,2004).The overex-pression of WXP2in Arabidopsis results not only in increased chlorophyll leaching and decreased water loss,but also in increased wax accumulation and enhanced drought tolerance (Zhang et al.,2007).These ?ndings show that cuticular perme-ability may not be correlated with the rate of water loss,wax accumulation,and whole-plant drought sensitivity.The changes in the cuticle properties of the osgl1-1mutant may re?ect changes in cuticle structures.The increased rate of water loss in the osgl1-1mutant may result from the changes in chemical composition of cuticular wax.Some investigations demonstrated that the chemical composition of cuticular wax was an important factor to determine the degree of resistance to water evaporation.The thickness of leaf wax deposits have little in?uence on cuticular water transpiration (Riederer and Schreiber,2001;Oliveira et al.,2003).

In contrast to no transcription of WAX2and CER1in roots,GL1in root and silk OsGL1-1is universally expressed,similar to WSL1,a KCS gene in rice (Yu et al.,2008).Therefore,OsGL1-1may be involved in a variety of developmental pro-cesses as well as cuticle formation.However,its reduced ex-pression causes phenotypic alterations only in the leaf and not in other organs in which it is expressed.It may be due to the presence of OsGL1-1homologs showing functional re-dundancy in the rice genome;o ther homologous genes might compensate for mutations in the OsGL1-1gene in these organs.

Secondary structure prediction of OsGL1-1suggests that it may be an integral membrane protein with

?ve

Figure 8.Altered Cuticular Permeability in the osgl1-1Mutant.(A)Chlorophyll leaching assays with matured leaves of WT and osgl1-1mutant,immersed in 80%ethanol for different time inter-vals.Data are shown by mean 6SE with three replicates.

(B)Water-loss rate of detached leaves of WT and osgl1-1mutant;x -axis is the scale for different time points and the y -axis is percentage of free water loss from leaves.Data are shown by mean 6SE with three

replicates.

Figure 9.Effects of Drought Stress Comparison between WT and osgl1-1Mutant.

(A)Six-week-old WT (left)and osgl1-1mutant (right)before drought stress.

(B)WT (left)and osgl1-1mutant (right)after re-watering for 14

d.

Figure 10.Sub-Cellular Localization of OsGL1-1–GFP in Onion Skin Epidermal Cells.

An onion skin epidermal cell expressing GFP alone (A),its DIC image (B),and merge (C),showing ?uorescent signals in nucleus,mem-brane,and cytoplasm.An onion skin epidermal cell expressing OsGL1-1–GFP alone (D),its DIC image (E),and merge (F),showing a similar signal pattern.

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transmembrane helices at the N-terminus and a cluster of wa-ter-soluble and globular domains at the C-terminus,which are predicted to reside in the cytoplasm.It is also demonstrated that CER1and WAX2are localized in the ER (Kamigaki et al.,2009).However,in our study,we found that OsGL1-1is localized in the nucleus,cytoplasm,and plasma membrane,which is not consistent with previous reports.

Eleven members of the GL1homologous gene family have been annotated in rice genome (Islam et al.,2009);the 11putative GL1-like genes were designed OsGL1-1;OsGL1-11.Among these,the most homologous gene to Zea mays GL1is OsGL1-1(85%identity at the protein level).YRE (YRE is an allele of CER3/WAX2/FLP )and CER1in Arabidopsis shared 64%and 37%amino acid similarity with OsGL1-1,respectively.CER1and YRE are known to play essential roles in trichome development in addition to cuticular wax https://www.360docs.net/doc/1417918541.html,bined loss-of-function mutations in CER1and YRE result in a much stronger phenotype than either single mutant.Either cer1or yre mutants alone have very limited or no morphological alteration of trichomes;however,trichomes in the double yre cer1mutants were heavily deformed (Kurata et al.,2003).In the present study,we found that the loss of function of OsGL1-1does not result in changes in trichomes;osgl1-1displays a reduction in cuticular wax deposition and cuticle membrane thickness—a phenotype similar to but less severe than that observed in Arabidopsis mutants cer1and yre .These ?ndings suggest that OsGL1-1functions similarly to its homologs,with some differences during rice leaf wax biosynthesis and trichome formation.

In Arabidopsis ,PEX10is essential for expression of CER1and WAX2.The suppression of the PEX10gene expression results in the down-regulation of CER1and WAX2gene expression and mislocalization of the gene products (Kamigaki et al.,2009).CER7protein is a regulator of wax biosynthesis that acts by degrading a speci?c mRNA species encoding a negative regulator of WAX2transcription;cer7mutant exhibits reduced cuticular wax accumulation and contains considerably lower levels of WAX2transcripts (Hooker et al.,2007).However,these genes have not yet been characterized in rice.The relationships among CER7,PEX10,and OsGL1-1in rice leaf development remain to be

elucidated.The availability of knowledge vis-a

`-vis OsGL1-1will facilitate an understanding of the mechanism of rice leaf wax accumulation.

METHODS

Plant Material

The rice leaf wax-de?cient spontaneous mutant,osgl1-1,was derived from an indica rice variety,Zhongxian 3037,which was used as WT in this study.To generate a large F 2population for gene mapping and cloning,osgl1-1was crossed with a japonica variety,Zhonghua 11.The WT and the osgl1-1plants for

drought-sensitivity experiments were grown in the pot under normal growth conditions.In addition,all other rice plants were grown in paddy ?elds under normal growth conditions.

Molecular Cloning of the OsGL1-1Gene

A total of 1842leaf wax-de?cient plants selected from the F 2generation were used for ?ne-mapping of OsGL1-1.Genomic DNA was extracted from rice leaves using the CTA

B method.STS markers were developed based on the diversity between the genomic DNA sequence of Nipponebare and 9311in the region spanning the OsGL1-1locus,and the sequences of the STS markers (Table 1)were designed using the DNAS-TAR-Lasergene v6software.A contig was constructed accord-ing to the results of ?ne mapping.

Gene prediction was performed using the Rice Genome Automated Annotation system (RiceGAAS,http://rice-gaas.dna.affrc.go.jp/),and intron/exon structures were ver-i?ed by alignment of the cDNA sequence of rice with the genomic DNA sequence.The genomic DNA fragments of candidate genes from the mutant and WT plants were am-pli?ed and sequenced.The sequencing reaction was per-formed by Sinogenomax Co.,Ltd.

Complementation of the osgl1-1Mutant

An 8.26-kb genomic DNA fragment isolated from the Xba l-digested BAC clone,AP005568(OSJNBb0057D06),containing the entire OsGL1-1gene,the 2899-bp upstream sequence,and the 1436-bp downstream sequence,was inserted into the binary vector pCAMBIA1300.Then,the resulting plasmid was introduced into the osgl1-1mutant for complementation testing using an Agrobacterium tumefaciens -mediated transformation method.Transgenic plants were selected on hygromycin medium,and further veri?ed by PCR ampli?cation using genome-speci?c primers.The genome-speci?c primers were 5#-AGAGATTCCA-GAAGATACAG-3#and 5#-GGGCCCTATGTAAAATCTTA-3#.

RT–PCR Analysis

Total RNA was extracted from leaves of different lines or differ-ent tissues of WT plants using the TRIzol RNA isolation reagent (Invitrogen).A total of 3l g of total RNA was treated with RNase-free DNase,and ?rst strand of cDNA was synthesized through reverse transcription by an oligo (dT)primer (TaKaRa).The pri-mers for OsGL1-1RT–PCR were 5#-ACCCCAACGTACCACACGAT-3#and 5#-ACCCCTGCGCTGGTTTTCTT-3#;the primers for the control gene UBQ were 5#-CAAGATGATCTGCCGCAAATGC-3#and 5#-TTTAACCAGTCCATGAACCCG-3#.Both sets of primers were designed to span an intron in order to speci?cally amplify target fragments.

OsGL1-1:GUS Construct and Histological GUS Assay

The 3003-bp region immediately upstream of the start codon of OsGL1-1was ampli?ed and inserted in front of the GUS gene in pCAMBIA1301.The resulting plasmid containing the OsGL1-1:GUS reporter gene was introduced into the WT background by Agrobacterium -mediated transformation.

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Transgenic plants were selected on hygromycin medium and T 1transgenic plants were used for analysis of GUS activity.

GUS staining was performed using a standard protocol (Jefferson et al.,1987).Transgenic plant samples were incu-bated in X-gluc buffer overnight at 37°C,then the staining so-lution was removed and the samples were cleared of chlorophyll through dehydration with graded ethanol.The stained tissues were observed and photographed using a ste-reomicroscope with a digital camera.

Scanning and Transmission Electron Microscopy

For scanning electron microscopy (SEM),samples were ?xed overnight at 4°C with 2.5%glutaraldehyde in 0.1M phosphate buffer (pH 7.4).After dehydration in a graded ethanol series and substitution with isoamyl acetate,the samples were criti-cal-point dried,sputter coated with gold,and observed under a QUANTA 200scanning electron microscope (FEI,Eindhoven,The Netherlands)at 10kV.For transmission electron microscopy (TEM)analysis,samples were ?xed overnight at 4°C with 2.5%glutaraldehyde in 0.1M phosphate buffer (pH 7.4).After wash-ing in phosphate buffer,samples were post?xed overnight in 2%(v/v)OsO 4in phosphate buffer.After being en bloc stained and dehydrated in a gradient alcohol series,the samples were then in?ltrated with LR White resin and polymerized for 48h.Ultrathin cross-sections were prepared using a Leica UC6ultra-microtome.Sections were inspected with a JEM-1400TEM.

Analysis of Cuticular Property of osgl1-1Mutant

For chlorophyll leaching assay,the third leaf from the top was sampled from each tiller at the heading stage of plants and the leaf was cut into segments (about 3cm)and immersed in 30ml of 80%ethanol at room temperature (gently agitating in the dark).At 1,2,3,4,5,6,9,and 12h,3ml of aliquot was taken out for chlorophyll quanti?cation,and the aliquot was poured back to the same tube after measurement.Measurements were performed in a dark room with very weak light.The chlo-rophyll concentration was quanti?ed using a DU800UV/Vis spectrophotometer at wavelengths of 663.2and 646.8nm

by using the standard method (Lolle et al.,1997).Chlorophyll ef?ux at each time point was expressed as a percentage of total chlorophyll extracted after 13h.

For drought-sensitivity experiments,the WT and the osgl1-1plants were grown in the same pot with three individual rep-licate experiments.After being grown under normal conditions for 6weeks,the whole plants were subjected to drought stress by stopping watering.Twelve days after inducing stress,the plants were rehydrated and observed for recovery.

For water-loss measurements,the third leaf from the top was detached from each tiller at the heading stage of plants.The detached leaves were weighed at 0.5,1,1.5,2,3,4,5,6,7,and 8h.Measurements were performed in a dark room with very weak light.The percentage loss of fresh weight was cal-culated based on the initial weight of the samples.

Cellular Localization Assays with Green Fluorescent Protein Fusion Protein

The OsGL1-1–green ?uorescent protein (GFP )fusion gene was constructed by fusing the GFP ORF to the C-terminal end of the OsGL1-1ORF (ampli?ed from WT cDNA with the follow-ing primers:5#-AAGCTTATGGGTGCCGCATTCTT GTC-3#and 5#-GGATCCGACAGGCCGGAGGCCGTGCC-3#)and cloning into the vector pJIT163.Both the OsGL1-1–GFP fusion and GFP alone were transfected into onion skin epidermal cells with the Bio-Rad PDS-1000/He device (Bio-Rad,Hercules,CA,USA).After bombardment,tissues were incubated for 14h at 25°C in the dark.Images were captured under the Olympus BX61?uorescence microscope conjunct with a microCCD camera.Grayscale images were captured for each color chan-nel and then merged using the software of IPlab.

FUNDING

This work was supported by grants from the Ministry of Sciences and Technology of China (2009ZX08009-068B)and the National Natural Science Foundation of China (30900885and 31000695).No con?ict of interest declared.

Table 1.List of STS Markers Developed in this Study.Markers Primer pairs

a

Fragment size b Zhongxian 3037Zhonghua 11P1F 5’-TAAAAAAGATGGCACCAGTG-3’;R 5’-CTACATTTAGTTTGCTGCCA-3’136166P2F 5’-TGATATCGAAGTAATGGCGA-3’;R 5’-AAGTTTCATGTTGACAGCAC-3’106119P3F 5’-CATTTCCTCACTCAATCAAC-3’;R 5’-AGATCGATTTTCGCTACAAC-3’276258P4F 5’-ACCGATAAAACTGCTCAAGA-3’;R 5’-CAAAGAAAGAACTTGGTTGC-3’7459P5F 5’-AACACCAGATCGACCTCTAC-3’;R 5’-CAGCCATGTCAACGAAGGT-3’98113P6F 5’-TGGCCACAGCATGGTAAAAA-3’;R 3’-CCCACATGTCAGTATGCGTA-3’11091P7F 5’-TGTCACATCTCGTTCTAAGT-3’;R 5’-AATGGTGGTGGCTATGATTT-3’90115P8F 5’-AATGAGCGTATCGGGTATTT-3’;R 5’-GATATCAGCTCGTCATTTAC-3’137150P9

F 5’-ATTGATATACTCCTTCCGTC-3’;R 5’-CATACTACTTTCTCTGTCTC-3’

157

139

a F ,forward primer;R,Reverse primer

b Numbers indicate the size (in bp)of ampli?ed fragments.

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