BENT UPPERMOST INTERNODE1Encodes the Class II Formin FH5Crucial for Actin Organization and Rice Development
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Weibing Yang,a,1Sulin Ren,b,1Xiaoming Zhang,c,1Mingjun Gao,a Shenghai Ye,c Yongbin Qi,c Yiyan Zheng,b Juan Wang,b Longjun Zeng,a Qun Li,a Shanjin Huang,b,2and Zuhua He a,2,3
a National
Key Laboratory of Plant Molecular Genetics,Institute of Plant Physiology and Ecology,Chine Academy of Sciences,
王的部首
Shanghai 200032,China
b Key Laboratory of Photosynthesis and Environmental Molecular Physiology,Institute of Botany,Chine Academy of Sciences,Beijing 100093,China
c State Key Laboratory Breeding Ba for Zhejiang Sustainable Pest an
d Dias
e Control,Zhejiang Academy o
f Agricultural Sciences,Hangzhou 310021,China
The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants.However,the molecular mechanisms linking the actin cytoskeleton to the process remain largely unknown.Here,we report the functional analysis of rice (Oryza sativa )FH5/BENT UPPERMOST INTERNODE1(BUI1),which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice.The bui1mutant displayed pleiotropic phenotypes,including bent uppermost internode,dwarfism,wavy panicle rachis,and enhanced gravitropic respon.Cytological obrvation indicated that the growth defects of bui1were caud mainly by inhibition of cell expansion.Map-bad cloning revealed that BUI1encodes the class II formin FH5.FH5contains a phosphata tensin-like domain at its amino terminus and two highly conrved formin-homology domains,FH1and FH2.In vitro biochemical analys indicated that FH5is capable of nucleating actin asmbly from free or profilin-bound monomeric actin.FH5also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end.Interestingly,the FH2domain of FH5could bundle actin filaments directly and stabilize actin filaments in vitro.Consistent with the in vitro biochemical activities of FH5/BUI1,the a
mount of filamentous actin decread,and the longitudinal actin cables almost disappeared in bui1cells.The FH2or FH1FH2domains of FH5could also bind to and bundle microtubules in vitro.Thus,our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments,which are important for proper cell expansion and rice morphogenesis.
INTRODUCTION
Rice (Oryza sativa )is a major food resource for nearly half of the world human population.Rice productivity is highly associated with its architectural pattern,including plant height,which is attributable mainly to stem internode elongation (Sasaki et al.,2002;Wang and Li,2008).The uppermost internode is of particular importance for rice productivity,since the elongation of the uppermost internode promotes panicle emergence (Zhu et al.,2006).The phytohormones gibberellins (GAs)and brassinosteroids are the two major factors that affect rice internode length by modulating cell expansion (Wang and Li,2008).The cytoskeleton,including microtubules and actin
microfilaments,is also esntial for plant development and morphogenesis by modulation of cell expansion.For example,loss of function of DWARF AND GLADIUS LEAF1,which encodes an ATPas
e katanin-like protein in rice,caud disor-ganization of microtubule arrays and inhibited cell elongation,resulting in a dwarf phenotype (Komorisono et al.,2005).However,the information about the functions of the actin cyto-skeleton in cell elongation and rice morphogenesis is rather limited.
Pharmacological perturbation of actin organization indicates that the actin cytoskeleton is a major regulator of cell elongation in Arabidopsis thaliana and other plant species (Baluska et al.,2001;Collings et al.,2006).Simultaneous downregulation of ACTIN2and ACTIN7reduced cell elongation in Arabidopsis hypocotyls (Kandasamy et al.,2009).Mixpression of actin regulatory proteins,such as profilin and actin-depolymerizing factors,also perturbs cell elongation (Ramachandran et al.,2000;Dong et al.,2001;Kandasamy et al.,2009).In addition,the actin cytoskeleton plays pivotal roles in polar cell expansion and the establishment of cell division planes by governing cytoplasmic streaming,organelle movement,and vesicle trans-port (Martin et al.,2001;Staiger and Blanchoin,2006).However,
1The authors contributed equally to this work.2The authors contributed equally to this work.
3Address correspondence to zhhe@sibs.ac.
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the molecular mechanisms by which actin regulates the phys-iological process remain poorly understood.
The function of the actin cytoskeleton is tightly coupled with its dynamic properties(Traas et al.,1987).Actin dynamics include maintenance of the monomeric actin(G-actin)pool,nucleation, actinfilament asmbly and disasmbly,actin bundle forma-tion,and actin cable construction,which are modulated by a preci orchestration of the activities and functions of a plethora of actin binding proteins(Staiger and Blanchoin,2006;Higaki et al.,2007).Nucleation is the rate-limiting step during sponta-neous actinfilament asmbly(Pollard and Borisy,2003).To date,veral actin nucleation factors have been identified, including the Actin-Related Protein2/3complex,formins,Spire, Cordon-bleu,Leiomodin,and Junction-Mediating and Regula-tory protein,which allow the cell to determine when and where to polymerize actinfilaments(Baum and Kunda,2005;Quinlan et al.,2005;Ahuja et al.,2007;Chereau et al.,2008;Zuchero et al.,2009).
Formins,originally identified from a mou limb deformity mutant,have been found to exist in many eukaryotic organisms, including animals,fungi,and plants,and are involved in many fundamental cellular process,including cytokinesis,cell mo-tility,and polarity(Woychik et al.,1990;Goode and Eck,2007). Formins are multidomain-containing proteins,characterized by two highly conrved formin-homology domains,FH1and FH2. Some formins in fungi and animals also share additional con-rved domains such as the FH3domain,the Rho binding domain,the Diaphanous-autoregulator
y domain,and the Diaph-anous-inhibitory domain,which confer functional regulation of the formins(Goode and Eck,2007).The FH1domain,con-sisting of veral concutive polyproline stretches,binds pro-filin or profilin/actin complexes to induce actin polymerization from the barbed end(Pruyne et al.,2002;Kovar et al.,2006).The number of polyproline stretches differs among formin proteins. The FH2domain contains actin binding sites and acts as a dimer to nucleate new actinfilaments(Pruyne et al.,2002;Xu et al., 2004;Otomo et al.,2005).General activities of formins include nucleating actin asmbly and interacting with the barbed end of actinfilaments(Kovar,2006;Goode and Eck,2007).Some formins also have vering and bundling activities(Harris et al., 2004;Michelot et al.,2005;Moley and Goode,2005;Harris et al.,2006).Along with their functions in regulating actin cyto-skeleton organization,veral animal formins,including mDia1, mDia2,Cappuccino(Capu),and Inverted Formin1(INF1),have been shown to bind directly to microtubules,thus regulating their dynamic properties(Palazzo et al.,2001;Rosales-Nieves et al., 2006;Bartolini et al.,2008;Young et al.,2008;Miki et al.,2009). The plant formin At FH4also associates with microtubules via the unique group Ie domain,which is located between the trans-membrane domain and the FH1domain(Deeks et al.,2010), suggesting that the formins may function in the crosstalk between microtubule and actin cytoskeleton systems.
Plant genomes encode a large family of formins that do not share recognizable autoregulatory domains found in animal and yeast formins(Grunt et al.,2008).Bad on quence similarity and conrvation,plant formins are divided into two class, referred to as class I and class II.Class I formins have an N-terminal membrane-anchoring domain followed by a transmembrane region and C-terminal FH1and FH2domains,whereas class II formins carry an N-terminal phosphata and tensin-related (PTEN)-like domain besides the conrved FH1and FH2do-mains(Deeks et al.,2002;Cvrckova´et al.,2004;Grunt et al., 2008).Functional analysis of class I formins has shown that they are involved in many fundamental physiological process.For instance,a loss-of-function mutation of At FH5leads to defective cytokinesis in ed endosperm(Ingouff et al.,2005).At FH5was recently shown to stimulate actin asmbly from the subapical domain of pollen tubes to facilitate pollen tube growth,and downregulation of FH5inhibits tip growth of pollen tubes in Nicotiana tabacum(Cheung et al.,2010).Downregulation of AFH3caus depolarized pollen tube growth,whereas over-expression of AFH1arrests pollen tube tip growth,and over-expression of At FH8disturbs root hair tip growth(Cheung and Wu,2004;Yi et al.,2005;Ye et al.,2009).By contrast,functional studies of class II formins are relatively limited.Recent results from the moss Physcomitrella patens demonstrated that class II formins could elongate actinfilaments at an amazingly rapid rate and are esntial for polar cell expansion(Vidali et al.,2009). Most recently,a cla
ss II formin,AFH14,was reported to bind both microtubules and microfilaments and was shown to play important roles in cell division and microspore formation in Arabidopsis(Li et al.,2010).However,it remains a major chal-lenge to explore the physiological functions of each formin protein in plants,particularly in the model cereal rice,for which no single formin has been functionally characterized.
We previously reported the identification of the rice ELON-GATED UPPERMOST INTERNODE(EUI)gene encoding a P450 that deactivates bioactive GAs.Mutation of Eui led to incread bioactive GA levels and an extremely elongated uppermost internode(Zhu et al.,2006).To understand rice internode devel-opment better,we characterized a rice mutant with a bent uppermost internode,designated as bent uppermost internode1 (bui1).The bui1mutant displayed multiple defects in plant architecture,including a bent uppermost internode,short culms, high tillering,wavy panicle rachis,and enhanced gravitropism. Map-bad cloning revealed that BUI1encodes the class II formin FH5.Cytological and biochemical analys demonstrated that FH5/BUI1plays esntial roles in diffu cell expansion and rice morphogenesis.
RESULTS
Phenotypic Characterization of the bui1Mutant
To understand better the cellular mechanism underlying rice internode development,we isolated a nonclassic dwarfed mu-tant from a g-ray–induced mutation pool(Zhu et al.,2006).The mutant was named bui1bad on its bent uppermost internode phenotype(e below).Compared with wild-type plants,bui1 had more tillers but significantly reduced plant height(Figure1A; e Supplemental Figures1A and1B online).During the early heading stage,the bui1uppermost internodes were much shorter than tho of the wild-type plants,and young panicles of bui1could hardly break out of theflag leaf sheath(Figure1B). Longitudinal ction analysis revealed that the cells of the bui1
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uppermost internode were markedly shorter and swelled com-pared with the wild-type cells (Figures 1C and 1D;e Supple-mental Figure 1C online).The results indicate that cell elongation was inhibited by the bui1mutation.
Although the bui1panicles could eventually protrude from the leaf sheath,they were small and loo,exhibiting a cockscomb shape (Figure 1B).The uppermost internode of bui1did not grow upward;instead,it was bent almost perpendicularly to the vertical axis (Figure 1E).To understand fully the cellular basis underlying the bending phenotype,we analyzed cell morphology in the middle part
of the uppermost internode,where shoot bending was most vere.Longitudinal ction analysis revealed that,in addition to the shorter and slightly swollen cell shapes,bui1also displayed other cellular phenotypes.In the wild-type plants,cell margins were even and connected tightly with each other (Figure 1F),whereas cell boundaries in bui1were rough,with obvious protuberances along the margins and ruptures in cell-to-cell boundaries (Figure 1G).Compared with the rectan-gular cells in the wild type,most bui1cells were slanted,and cell
files were not aligned properly (Figure 1H).Taken together,the results indicate that polar cell expansion was also disrupted in bui1.
The bui1mutant displayed other morphological defects be-sides the bent uppermost internode.All primary and condary rachis of bui1were wavy,and the number of rachis branches and spikelets were reduced compared with the wild-type plants (Figure 1I).The eds of bui1were smaller with irregular shapes and were not well filled (Figure 1J).
Mutation of BUI1Affects Seedling Development and Gravitropism
Wild-type young edlings display erect upward growth due to negative gravitropism,whereas bui1edlings bend in all direc-tions (Figure 2A),suggesting an altered gravitropic respon in bui1.T
he bending of bui1under normal growth conditions makes it difficult to analyze shoot gravitropism.We thus analyzed the gravitropic respon of the roots instead.The kinetics
of
Figure 1.Phenotypes of Wild-Type and bui1Mature Plants.
微波炉可以做什么美食(A)Wild-type (Zhejing 22;left)and bui1(right)plants.Bar =2cm.
(B)Panicle exrtion of wild-type (left three)and bui1(right three)plants.Bar =2cm.
(C)and (D)Longitudinal ctions of the uppermost internodes of the wild type (C)and bui1(D)at heading stage.The regions ud for analysis are indicated with squares in (B).Bars =100m m.
(E)Panicles of the wild type (left)and bui1(right)at the mature stage.Bar =2cm.
(F)to (H)Longitudinal ctions of the uppermost internode regions (indicated with squares in [E])of the wild type (F)and bui1(G).Severely slanted cells of the same bui1internode region are shown in (H).Bars =50m m.
(I)Panicle rachis of the wild type (left)and bui1(right).Note the wavy rachis in bui1.Bar =2cm.(J)Grains of the wild type (top)and bui1(bottom).Bar =0.5cm.
Rice FH5Regulates Actin Organization 3of 20
gravity-induced root bending of light-grown edlings revealed that gravitropism in bui1was strongly promoted,with an accel-erated respon to the gravitropic stimulus and an incread bending degree (e Supplemental Figure 2online).
The shoots of bui1edlings were shorter than tho of wild-type plants (Figure 2B;e Supplemental Figure 3A online).Longitudinal ction analysis revealed that cell elongation was inhibited in the bui1shoot (Figures 2C and 2D;e Supplemental Figure 3B online).Interestingly,when cultured in half-strength Murashige and Skoog medium,bui1roots displayed a strong wavy phenotype,with root length only slightly reduced (Figure 2E;e Supplemental Figure 3C online).To gain insight into the cellular mechanism for the wavy-root phenotype,we analyzed the internal structure of bui1and wild-type roots.The analys revealed that cells in the bui1roots were slanted with reduced cell length but little change in cell width (Figures 2F and 2G;e Supplemental Figure 3D online).Cell files corresponding to the wavy region also exhibited a wavy manner,but they still
remained parallel to each other (Figure 2G),suggesting that this wavy growth behavior is different from the twisted growth in the Arabidopsis microtubule-related mutants (Ishida et al.,2007).We speculated that bui1might be defective in normal root thigmot-ropism,as obrved in the Arabidopsis mildew resistance locus o4mutant (Chen et al.,2009).Bad on the obrvations,we concluded tha
t cell growth was inhibited in bui1but to a lesr extent at the edling stage.
BUI1Encodes the Class II Formin FH5
To understand fully the cellular mechanism by which the bui1mutation inhibits cellular growth,we isolated the BUI1gene using a map-bad cloning strategy.An F2population of ;10,000plants was generated from a cross between bui1and Zhenshan 97(an indica cultivar)and was ud for PCR-bad mapping.The BUI1locus was delimited to a genomic region on chromo-some 7between two simple quence repeat (SSR)markers,RM1132and RM505(Figure 3A).Six inrtion/deletion markers were developed between the two SSR markers,and BUI1was further narrowed down to a 60-kb DNA region between 7WB8and 7WB16on a single BAC,AP004275.There are three open reading frames (ORFs)in this region (Figure 3B).Sequencing analysis revealed that bui1had a substitution of A to G in the sixth intron of the gene FH5/Os07g0596300(Figure 3C),changing the highly conrved 39-intron end AG to GG.We suspected that this mutation interferes with the correct splicing of the sixth intron.Indeed,RT-PCR analysis revealed the prence of the 79-bp intron fragment in the predominantly amplified cDNA fragment of FH5/Os07g0596300(Figure 3D).
RNA gel blot analysis was performed to determine the tran-script length of FH5.Using a probe specifi
c to its 39region,as supported by an isolated cDNA clone (GenBank accession number AK120222),we detected an ;5-kb-long transcript (Figure 3E),suggesting that the previously predicted full-length mRNA quence (GenBank accession number NM_001066706)was a truncated one.The detected transcript size is consistent with the predicted protein of the FH5locus (Q84ZL0,isoform 1,annotated by the UniProtKB/Swiss-Prot website),which is 1627amino acids long.The 59region of the FH5transcript was verified by RT-PCR using two ts of primers (Figures 3C and 3F)and quencing.We thus generated a cDNA clone containing the entire ORF for the annotated FH5polypeptide.This ORF and the native FH5promoter were then cloned into a binary vector,and the resulting transgene was introduced into bui1plants.Figure 3G shows that the FH5minigene successfully complemented the mutant phenotypes.We thus concluded that the A-to-G substi-tution in the FH5locus is responsible for the obrved bui1mutant phenotypes.
Plant formins are named on the basis of quence similarity and conrvation by phylogenetic analysis,and BUI1corre-sponds to FH5,a class II formin (Cvrckova
´et al.,2004;Grunt et al.,2008).FH5is predicted (hits.isb-sib.ch/cgi-bin/PFSCAN)to contain a PTEN-like domain (amino acids 198–336)at its N terminus,followed by a typical FH1domain (amino acids 825–1170)and an FH2domain (amino acids 1188–1588;e Supplemental Figure 4A online).Th
e FH1domain consists of 21polyproline stretches (e Supplemental Figure 5online),
and
Figure 2.Comparison of Wild-Type and bui1Seedlings.
(A)Wild-type (WT)and bui1edlings (7d old).Note that the bui1shoots are bent.Bar =2cm.
开发部(B)to (D)Longitudinal ctions of edling shoots of the wild type (C)and bui1(D).(B)shows 9-d-old edlings of the wild type (left)and bui1(right).Bar in (B)=2cm;bars in (C)and (D)=100m m.
(E)Roots of the wild type (left)and bui1(right)grown in half-strength medium.Bar =1cm.
(F)and (G)Cell morphology of wild-type (F)and bui1(G)roots by propidium iodide staining.The regions ud for analysis are indicated with squares in (E).Bars =50m m.
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the FH2domain shares 31,31,29,30,61,and 24%amino acid similarity with tho of the formins AFH1,AFH3,At FH5,At FH8,For2A,and Bni1p,respectively (e Supplemental Figure 4B online).Importantly,the key residues (Ile-1431and Lys-1601)of Bni1p involved in actin nucleation and barbed end capping are also conrved in FH5FH2(e Supplemental Figure 4B online;Xu et al.,200
4),implying that FH5may perform conrved actin regulatory actions.Inclusion of the sixth intron possibly intro-duced a premature stop codon and could result in a truncated protein that contains the PTEN domain,the FH1domain,and part of the FH2domain in bui1,suggesting that a complete FH2domain is esntial for FH5function.Becau the mutation in the FH5/BUI1gene is fully recessive,we propod that the predicted truncated protein,which could accumulate predominantly in
bui1,could interfere with the function of the less produced full-length protein.
RNA gel blot analysis revealed that FH5is expresd ubiqui-tously in all tissues (Figure 3H).RT-PCR analysis demonstrated that most rice formins are expresd ubiquitously in all the examined tissues,with the exception of FH18and FH12,who transcripts could be detected only in the young panicles (e Supplemental Figure 6online).Actin Filament Organization in bui1
Becau formins are known for their abilities to regulate actin cytoskeleton asmbly and organization (Goode and Eck,2007),we sought to determine the effect of bui1mutation on
鬼吹灯结局actin
Figure 3.Map-Bad Cloning of BUI1.
(A)The BUI1locus was mapped on chromosome 7between two SSR markers,RM1132and RM505.
(B)Fine-mapping of BUI1.BUI1was narrowed to a 60-kb region between two makers,7WB8and 7WB16,on a single BAC (AP004275),which contains three predicted genes.
(C)Sequence comparison revealed a substitution of A to G in one intron of the gene FH5/Os07g0596300in bui1.WT,wild type.(D)Detection of altered splicing in bui1by RT-PCR analysis.Rice UBI1was ud as an internal control.
(E)RNA gel blot analysis to confirm the length of the FH5/BUI1transcript.Total RNA (10m g)extracted from the wild type and bui1was ud for the analysis (shown below the blot).M,RNA ladder.
(F)Confirmation of the full-length FH5/BUI1transcript by RT-PCR.The primer pairs (P1and P2)ud for RT are indicated in (C).(G)Complementation test of the FH5/BUI1gene.One reprentative line (1300-BUI1)of complementation is shown.Bar =2cm.
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(H)Expression pattern of FH5revealed by RNA gel blot.YS,Young edling;FL,flower;RA,rachis;LF,leaf;LS,leaf sheath;ND,node;IN,internode;RT,root.
Rice FH5Regulates Actin Organization 5of 20
organization.We examined the actin cytoskeleton organization in the wild type and bui1directly by staining root cells with AlexaFluor488–phalloidin,which has been widely ud for stain-ing actin cytoskeleton in plants.We found that the overall fluorescence signal of the bui1cells was much weaker than that of the wild-type cells under identical staining conditions and confocal ttings(Figures4A and4B).Since phalloidin specifi-cally binds to actinfilaments(F-actin),the amount of F-actin is proportional to thefluorescence intensity.Therefore,we decided to compare the F-actin levels between the wild-type and bui1 cells by quantifyingfluorescence intensity.As shown in Figure 4C,the averagefluorescence intensity was reduced by nearly threefold in bui1.The results suggest that FH5/BUI1plays an important role in maintaining the level of F-actin.
When the detection ttings for bui1were incread so that both the wild-type and bui1cells gave clearfluorescence signals, we found that actinfilaments were verely disorganized in the bui1cells.In wild-type plants,cells in the root elongation zone displayed a clearly organized actin cyto
skeleton structure. Prominent actin cables oriented longitudinally through the whole cell cortex and were cross-linked by transverly or longitudi-nally orientedfine actinfilaments(Figure4D).However,longitu-dinal actin cables were barely detected in the bui1cells.Although some thick actin cables were obrved in bui1,they were quite short.In addition,thefine actinfilaments were also shorter and arranged randomly in bui1cells compared with tho in the wild-type cells(Figure4E).Measurement offluorescence intensity revealed peaks in the wild-type cells,which correspond to
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Figure4.F-Actin Organization in Wild-Type and bui1Cells.
F-actin organization was visualized by AlexaFluor488–phalloidin staining.Each image is a maximum projection of thefluorescence signals.
红楼梦全部歌曲(A)and(B)F-actin organization in the cortex cells of the root elongation regions of the wild type(A)and bui1(B).Bars=20m m.人体最大的器官
(C)Quantitative analysis of F-actin levels in wild-type(WT)and bui1cells as detected in(A)and(B).Data shown are means6SE offluorescence intensity of144cells in the wild type and bui1.P<0.01,by t test.
(D)and(E)F-actin organization in the root elongation region cells of the wild type(D)and bui1(E).Confocal ttings for bui1were incread to give clear signals.Bars=20m m.
(G)and(H)F-actin organization in the root transition region cells of the wild type(G)and bui1(H).Confocal ttings for bui1were incread to give clear signals.Bars=20m m.
(F)and(I)Fluorescence intensities corresponding to the regions marked in(D)/(E)and(G)/(H),respectively.
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