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Mutation of the Rice Narrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport 1[OA] Jing Qi 2 , Qian Qian 2 , Qingyun Bu, Shuyu Li, Qian Chen, Jiaqiang Sun, Wenxing Liang, Yihua Zhou, Chengcai Chu, Xugang Li, Fugang Ren, Klaus Palme, Bingran Zhao, Jinfeng Chen, Mingsheng Chen, and Chuanyou Li* State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (J.Q., Q.B., Q.C., J.S., W.L., S.L., Y.Z., C.C., J.C., M.C., C.L.); State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China (Q.Q.); Graduate School of Chinese Academy of Sciences, Beijing 100049, China (J.Q., Q.C., W.L., S.L.); Institut fu ¨ r Biologie II und Zentrum fu ¨r Angewandte Biowissenschaften, Universita ¨t Freiburg, D–79104 Freiburg, Germany (X.L., F.R., K.P.); and China National Hybrid Rice Research and Development Center, Changsha 410125, China (B.Z.) The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth. The plant vascular system, a continuous network of vascular bundles that interconnects all major plant organs, enables long-distance transport of photoassim- ilates and soil nutrients and provides mechanical support to the plant body (Esau, 1965; Dengler, 2001; Aloni, 2004; Scarpella and Meijer, 2004). Anatomical studies revealed that all vascular cells are differenti- ated from a vascular meristematic tissue, the procam- bium (Foster, 1952; Esau, 1965). Procambial cells are characteristically arranged in continuous strands and acquire their narrow shape through coordinated, ori- ented divisions, parallel to the axis of the emerging strand (Nelson and Dengler, 1997; Dengler, 2001; Ye, 2002; Scarpella and Meijer, 2004). Despite extensive descriptions of different aspects of vascular develop- ment and patterning, the molecular mechanisms gov- erning these strictly regulated processes remain to be elucidated. Among the various plant hormones that influence vascular development and patterning, the role of auxin is unique (Dengler, 2001; Aloni, 2004; Fukuda, 2004; Scarpella and Meijer, 2004; Teale et al., 2006). A long history of elegant physiological experiments established that the polar cell-to-cell transport of auxin plays a crucial role in vascular tissue differentiation and vascular patterning (Sachs, 1989; Aloni, 2004; Fukuda, 2004; Blakeslee et al., 2005; Sauer et al., 2006; Teale et al., 2006). The classical auxin application studies and polar auxin transport (PAT) inhibition studies also led to the formulation of the ‘‘auxin-flow canalization hypothesis,’’ which suggests that auxin flow, starting initially by diffusion, induces the forma- tion of a polar auxin transport system and, as a con- sequence, promotes auxin transport and leads to canalization of the auxin flow along a narrow file of cells. This continuous polar transport of auxin through cells finally results in the differentiation of vascular strands (Sachs, 1981, 2000; Aloni, 2004; Fukuda, 2004). Molecular genetic studies, primarily using the di- cotyledon Arabidopsis (Arabidopsis thaliana) as a model system, have boosted our understanding of 1 This work was supported by grants from the Ministry of Science and Technology of China (grant nos. 2005CB120801 and 2006AA10A101) and the National Natural Science Foundation of China (grant nos. 30700054 and 90717007). 2 These authors contributed equally to the article. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Chuanyou Li ([email protected]). [OA] Open Access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.118778 Plant Physiology, August 2008, Vol. 147, pp. 1947–1959, www.plantphysiol.org Ó 2008 American Society of Plant Biologists 1947 www.plantphysiol.org on March 10, 2020 - Published by Downloaded from Copyright © 2008 American Society of Plant Biologists. All rights reserved.

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Page 1: Mutation of the Rice Narrow leaf1 Gene, Which …...Mutation of the RiceNarrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport1[OA] Jing

Mutation of the Rice Narrow leaf1 Gene, Which Encodesa Novel Protein, Affects Vein Patterning and PolarAuxin Transport1[OA]

Jing Qi2, Qian Qian2, Qingyun Bu, Shuyu Li, Qian Chen, Jiaqiang Sun, Wenxing Liang, Yihua Zhou,Chengcai Chu, Xugang Li, Fugang Ren, Klaus Palme, Bingran Zhao, Jinfeng Chen,Mingsheng Chen, and Chuanyou Li*

State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beijing 100101, China (J.Q., Q.B., Q.C., J.S., W.L., S.L.,Y.Z., C.C., J.C., M.C., C.L.); State Key Laboratory of Rice Biology, China National Rice Research Institute,Chinese Academy of Agricultural Sciences, Hangzhou 310006, China (Q.Q.); Graduate School of ChineseAcademy of Sciences, Beijing 100049, China (J.Q., Q.C., W.L., S.L.); Institut fur Biologie II und Zentrum furAngewandte Biowissenschaften, Universitat Freiburg, D–79104 Freiburg, Germany (X.L., F.R., K.P.); andChina National Hybrid Rice Research and Development Center, Changsha 410125, China (B.Z.)

The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plantleaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristicphenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number oflongitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in whichthe number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analysesdemonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showingthat Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxintransport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plantsand plays an important role in the control of lateral leaf growth.

The plant vascular system, a continuous network ofvascular bundles that interconnects all major plantorgans, enables long-distance transport of photoassim-ilates and soil nutrients and provides mechanicalsupport to the plant body (Esau, 1965; Dengler, 2001;Aloni, 2004; Scarpella and Meijer, 2004). Anatomicalstudies revealed that all vascular cells are differenti-ated from a vascular meristematic tissue, the procam-bium (Foster, 1952; Esau, 1965). Procambial cells arecharacteristically arranged in continuous strands andacquire their narrow shape through coordinated, ori-ented divisions, parallel to the axis of the emergingstrand (Nelson and Dengler, 1997; Dengler, 2001; Ye,2002; Scarpella and Meijer, 2004). Despite extensive

descriptions of different aspects of vascular develop-ment and patterning, the molecular mechanisms gov-erning these strictly regulated processes remain to beelucidated.

Among the various plant hormones that influencevascular development and patterning, the role ofauxin is unique (Dengler, 2001; Aloni, 2004; Fukuda,2004; Scarpella and Meijer, 2004; Teale et al., 2006). Along history of elegant physiological experimentsestablished that the polar cell-to-cell transport of auxinplays a crucial role in vascular tissue differentiationand vascular patterning (Sachs, 1989; Aloni, 2004;Fukuda, 2004; Blakeslee et al., 2005; Sauer et al., 2006;Teale et al., 2006). The classical auxin applicationstudies and polar auxin transport (PAT) inhibitionstudies also led to the formulation of the ‘‘auxin-flowcanalization hypothesis,’’ which suggests that auxinflow, starting initially by diffusion, induces the forma-tion of a polar auxin transport system and, as a con-sequence, promotes auxin transport and leads tocanalization of the auxin flow along a narrow file ofcells. This continuous polar transport of auxin throughcells finally results in the differentiation of vascularstrands (Sachs, 1981, 2000; Aloni, 2004; Fukuda, 2004).

Molecular genetic studies, primarily using the di-cotyledon Arabidopsis (Arabidopsis thaliana) as amodel system, have boosted our understanding of

1 This work was supported by grants from the Ministry of Scienceand Technology of China (grant nos. 2005CB120801 and2006AA10A101) and the National Natural Science Foundation ofChina (grant nos. 30700054 and 90717007).

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Chuanyou Li ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.118778

Plant Physiology, August 2008, Vol. 147, pp. 1947–1959, www.plantphysiol.org � 2008 American Society of Plant Biologists 1947 www.plantphysiol.orgon March 10, 2020 - Published by Downloaded from

Copyright © 2008 American Society of Plant Biologists. All rights reserved.

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the molecular basis of PAT, PAT-mediated auxin dis-tribution, and their association with vascular differen-tiation and patterning (Palme and Galweiler, 1999;Dengler, 2001; Friml and Palme, 2002; Ye, 2002; Aloni,2004; Fukuda, 2004; Blakeslee et al., 2005; Paponovet al., 2005; Teale et al., 2006). In-depth characterizationof Arabidopsis mutants defective in PAT had enabledthe identification of the putative auxin influx as well asefflux carriers, two of the major components of the PATsystem. For example, the pin-formed1 (pin1) mutantshowed irregular vascular patterns and strongly re-duced basipetal auxin transport, which resembledplants treated with inhibitors of auxin efflux (Okadaet al., 1991; Galweiler et al., 1998). Gene cloning andprotein localization analyses demonstrated that AtPIN1was a member of a family of transmembrane proteinsthat asymmetrically distributed at the basal end ofPAT-competent cells, supporting the hypothesis thatAtPIN1 as well as other PIN proteins were compo-nents of the auxin efflux machinery (Galweiler et al.,1998; Palme and Galweiler, 1999; Friml, 2003; Aloni,2004; Blakeslee et al., 2005; Leyser, 2005; Paponov et al.,2005; Petrasek et al., 2006). In addition, mutations inAtPGP1 and AtPGP19, two genes encoding putativeP-glycoprotein ABC transporters, resulted in the dis-ruption of PAT and pleiotropic growth phenotypes,including stunted stature and curled leaves (Noh et al.,2001, 2003; Luschnig, 2002). Recently, heterologousexpression studies demonstrated that both AtPGP1and AtPGP19 exhibited auxin efflux transport activity(Geisler et al., 2005).

Through monitoring the dynamic patterns of thepolar AtPIN1 expression during critical stages of theformation of various defined veins, recent studiesrevealed a directing influence of auxin transport inthe epidermis on vein patterning (Scarpella et al., 2003;Sauer et al., 2006). These works refined the overallpicture of the canalization hypothesis for PAT-mediatedvascular patterning (Scheres and Xu, 2006).

Although a large number of studies have estab-lished an instrumental role of PAT and PAT-mediatedauxin distribution for vascular patterning in Arabi-dopsis, much less is known about whether the sameparadigm also underlies vascular patterning in rice(Oryza sativa), a model system of monocot plants.While dicot plants show a reticulate pattern of highlybranched veins, most monocot plants, such as rice,show parallel (striate) vascular patterns (Nelson andDengler, 1997; Scarpella et al., 2003; Scarpella andMeijer, 2004). The auxin-inducible homeobox geneOshox1 in rice was shown recently to be a molecularmarker of a stage in vascular tissue development atwhich procambial cell fate is specified but not yetstably determined (Scarpella et al., 2000, 2005). Over-expression of Oshox1 led to reduced sensitivity to PATinhibitors, indicating a role of PAT in vascular tissuedifferentiation in monocot species (Scarpella et al.,2002). An association between PAT and venation wasdemonstrated by characterization of the maize (Zeamays) rough shealth2 mutant, which shows aberrant leaf

development (Tsiantis et al., 1999; Scanlon et al., 2002).Stronger evidence supporting the notion that PAT isinvolved in vascular patterning in monocot plantscame from the characterization of the classic brachytic2(br2) mutant in maize, which shows irregular vascularpatterns in stalks (Multani et al., 2003). br2 carriedmutation in a gene that is homologous to the above-mentioned Arabidopsis P-glycoproteins AtPGP1 andAtPGP19. Similar to the Arabidopsis case, PAT wassignificantly reduced in br2 mutants. The radicleless1(ral1) mutant of rice showed distinctive embryonic andpostembryonic vascular defects, including alterednumber and spacing of parallel veins, and was defec-tive in auxin response (Scarpella et al., 2003). However,the gene responsible for the ral1 phenotype had yet tobe identified.

Here, we report the in-depth characterization of apreviously described rice mutant named narrow leaf1(nal1; Dong et al., 1994; Zeng et al., 2003). The nal1mutant exhibits altered vascular patterns in leaves andculms. Nal1 encodes a plant-specific protein with un-known biochemical function. We provide evidenceshowing that Nal1 is expressed preferentially in vas-cular tissues and that mutation of its function leads toreduced PAT capacity. These results support the hy-pothesis that Nal1 plays important roles in regulatingPAT as well as vascular patterning of rice.

RESULTS

The nal1 Mutant Shows Reduced Leaf Width

One of the most characteristic phenotypes of the ricenal1 mutant was reduced leaf width (Dong et al., 1994;Zeng et al., 2003). The leaves of nal1 plants appearednormal in shape but were generally smaller than thoseof wild-type plants (Table I; Fig. 1C). Measurement ofleaf growth indicated that the nal1 mutation signifi-cantly affected the width of the leaf blade. Comparedwith width, the length of the mutant leaf blades wasless affected. For example, the mature second leaves(from the top of the plant) of soil-grown nal1 plantsusually attained approximately 63% of the width ofthe wild-type counterparts (Table I), while the lengthof the nal1 leaves was about 87% of that of wild-typeplants (Table I).

The nal1 Mutant Exhibits an Altered InternodeElongation Pattern

In addition to narrow leaves, the nal1 mutant alsoshowed an altered internode elongation pattern (Fig. 1,A, B, and D). At maturity, the height of the mutantreached approximately 71% of the wild-type height(Table I). We compared the internode elongation pat-terns between nal1 and wild-type plants. As shown inFigure 1D and Table I, while the nal1 mutation hadlittle effect, if any, on the length of the panicle and theuppermost two internodes (designated internodes Iand II, respectively), it significantly shortened the

Qi et al.

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length of the third, the fourth, and the fifth internodes(designated internodes III, IV, and V, respectively).

To determine whether the dwarf phenotype of thenal1 culm results from defective cell division and/orcell elongation, longitudinal sections of the severelyshortened internode IV of the mature mutant culmwere compared with their wild-type counterparts. Asshown in Figure 1D and Table I, the total cell numberin y axes in internode IV of nal1 was approximately60% less than that of wild-type plants, while nosignificant change in cell length was observed. Theseobservations suggested that the Nal1 gene may play arole in cell division rather than cell elongation.

The nal1 Mutant Shows Defective Vascular Patterns inLeaves and Culms

Wild-type rice leaves show a typical parallel vena-tion pattern, in which three orders of major longitudi-nal veins (i.e. the midveins, large veins, and smallveins) lie parallel along the distal axis of the leaf(Nelson and Dengler, 1997; Scarpella et al., 2003). Awealth of anatomical studies revealed a constant rela-tionship between leaf blade width and longitudinalvein quantity in monocots (Russell and Evert, 1985;Dannenhoffer et al., 1990; McHale, 1993; Nelson andDengler, 1997). A comparison of mature leaves re-vealed that the total numbers of large veins and smallveins were all reduced in the nal1 mutant (Table I).Notably, the average number of small veins betweentwo large veins was reduced from approximately fivein the wild type to approximately three in the mutant

(Fig. 1, E and F; Table I). These observations indicatedthat the nal1 mutation affected the formation of leafveins, which might account for the narrow leaf phe-notype of the mutant.

The finding that the nal1 mutation affected leafvenation prompted us to examine the vascular systemin culms. Transverse sections of internode IV, thelength of which was significantly reduced in nal1(Fig. 1D; Table I), were compared between wild-typeand mutant plants. In cross sections of the wild-typeinternode IV, vascular bundles of inner ring and outerring were uniformly arranged and formed a typicalradial organization, with outer small vascular bundlesarranged concentrically below the epidermis (Fig. 1G,left). In contrast, the distribution pattern of vascularbundles in the nal1 mutant culm was severely dis-rupted (Fig. 1G, right). In addition to the altered dis-tribution pattern, the mutant culm also containedimmature vascular bundles and an overall increasednumber of vascular bundles, especially in the outerring (Table I; Fig. 1G, right). Furthermore, increasednumbers of parenchyma cells in the region betweenthe epidermis and the outer ring of small vascularbundles could be observed (Fig. 1G, right).

Positional Cloning of Nal1

The genetic locus defined by the nal1 mutant waspreviously located on the long arm of rice chromo-some 4 (Dong et al., 1994; Zeng et al., 2003). For finemapping, the nal1 mutant in the genetic background ofZhefu802 (an indica cultivar) was crossed to Nip-

Table I. Morphometric analysis of wild-type and nal1 plants

Leaf morphometric analyses were done on mature leaves of 3-month-old plants. Blade width andvascular pattern parameters were measured through the middle region of the leaf blade. Vascular patternparameters were measured in dark-field microscopic digital images of cleared leaf preparations. Numbersof stem vascular bundles in the outer and inner rings were determined in digital microscopic images oftransverse sections through the middle part of the fourth internode of plants at the ripened inflorescencestage. The internode IV morphometric analyses and plant height measurement were done at the maturestage of 3-month-old plants. Results represent means 6 SD of populations of the size indicated inparentheses. Asterisks indicate the significance of differences between wild-type and nal1 plants asdetermined by Student’s t test: * 0.01 # P , 0.05, ** 0.001 # P , 0.01, *** P , 0.001.

Organ Wild Type nal1

LeafBlade width (cm) 1.9 6 0.2 (15) 1.2 6 0.1 (15)***Blade length (cm) 32.1 6 5.5 (15) 27.7 6 1.7 (15)**Number of large veins 8.1 6 0.8 (33) 7.2 6 1.1 (42)***Total number of small veins 39.4 6 2.8 (30) 22.2 6 3.5 (30)***Number of small veins between

two adjacent large veins4.9 6 0.3 (30) 3.0 6 0.4 (30)***

StemMature plant height (cm) 88.1 6 2.9 (20) 62.8 6 1.9 (20)***Internode IV length (cm) 4.5 6 1.4 (20) 1.2 6 0.2 (20)***Number of vascular bundles in the outer ring 23.6 6 2.7 (23) 25.2 6 2.5 (21)*Number of vascular bundles in the inner ring 25.5 6 1.99 (23) 26.5 6 4.36 (21)Total cell number of

internode IV in y axis530.3 6 100.6 (16) 159.1 6 69.1 (16)***

Nal1 Affects Venation and Polar Auxin Transport

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ponbare (a japonica cultivar) to generate a large F2population. Genetic analysis with 2,000 segregantsshowing the nal1 mutant phenotype initially placedthe Nal1 gene between simple sequence repeatmarkers RM5503 and RM3836 (Fig. 2A). Within thisregion, we developed four PCR-based molecularmarkers (Table II) and further delimited the targetgene to a 29-kb region defined by markers M3 and M4on a single bacterial artificial chromosome (BAC)clone, AL662950 (Fig. 2A). Within this 29-kb interval,four open reading frames (ORFs), which were desig-nated ORF1 to ORF4, were predicted (Fig. 2A). Ge-nomic DNAs corresponding to the four putative ORFswere PCR amplified from the nal1 mutant and se-quenced. Sequence comparison revealed a 30-bp de-letion in the nal1-derived ORF1 (Fig. 2B), while no

sequence difference was found in ORF2 to ORF4.ORF1, therefore, was considered a strong candidatefor the Nal1 gene.

To test the possibility that the 30-bp deletion in nal1-derived ORF1 is responsible for the mutant pheno-type, we performed genetic complementation analysis.A 12.4-kb genomic DNA fragment containing theentire Nal1 coding region, the 2,673-bp upstream se-quence, and the 1,685-bp downstream sequence wasintroduced into the nal1 background by Agrobacteriumtumefaciens-mediated transformation (Hiei et al., 1994).The 30-bp deletion in the nal1-derived ORF1 led to thedevelopment of a PCR-based marker to determine themutant background in complementation tests (Fig. 2C,bottom). Among the 24 transgenic plants confirmed bygenomic PCR, 17 primary lines (T0) showed comple-

Figure 1. The nal1 mutant shows a defective vascular system in leaves and culms. A, Gross morphology of 1-month-old wild-type (left) and nal1 (right) plants. B, Gross morphology of wild-type (left) and nal1 (right) plants at the heading stage. C, Leafmorphology of wild-type (left) and nal1 (right) plants. Shown are the second leaf blades (from the top of the plant) of 3-month-oldplants. D, Phenotypic exhibition of the components of rice plant height in representative wild-type (left) and nal1 (right) plants atthe mature stage. P, Panicle. I to V indicate the corresponding internodes from top to bottom. E, Whole-mount clearing of themature fifth leaves of wild-type (WT) and nal1 plants. Shown are regions between two large veins in the middle of mature leafblades. lv, Large vein; sv, small vein. Bars 5 150 mm. F, Transverse sections of the mature fifth leaves in the middle part of wild-type and nal1 plants. Bars 5 20 mm. G, Transverse sections of the middle part of internode IV of wild-type (left) and nal1 (right)plants at the mature stage. ep, Epidermis; lvb, large vascular bundles; pc, parenchyma cells; svb, small vascular bundles. Thesingle red arrow indicates an immature vascular bundle, and the double red arrows indicate parenchyma cell layers in the regionbetween the epidermis and the outer ring of small vascular bundles. Bars 5 20 mm.

Qi et al.

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mentation of the nal1 phenotype (Fig. 2C; data notshown). Further characterization of three of these linesshowed that the complemented phenotype was inher-ited in the T1 generation (data not shown). These resultsdemonstrated that ORF1 is equivalent to the Nal1 gene.

Nal1 Encodes a Plant-Specific Protein with Unknown

Biochemical Function

Sequence comparison between genomic DNA and

cDNA revealed that the Nal1 gene was composed

Figure 2. Nal1 encodes a plant-specific protein with unknown biochemical function. A, Fine genetic and physical mapping ofNal1. The target gene was initially mapped to a genetic interval between markers RM5503 and RM3836 on rice chromosome 4.Analysis of an F2 mapping population of 2,000 plants delimited the gene to a 29-kb region between markers M3 and M4 on BACclone AL662950. Markers and the numbers of recombination events identified are indicated. B, Schematic representation of theNal1 gene structure. The nal1 allele contains a 30-bp deletion in the fourth exon. Black boxes indicate the coding sequence, whiteboxes indicate the 5# and 3# untranslated regions, and lines between boxes indicate introns. The start codon (ATG) and the stopcodon (TGA) are indicated. C, Genetic complementation of nal1 (top) and molecular identification of transgenic plants (bottom).The wild type (left), nal1 containing an empty vector (middle), and a transgenic plant containing the 12.4-kb DNA fragment ofORF1 in the genetic background of nal1 (right) are shown. The 30-bp deletion in the nal1 was used as a PCR-based marker todistinguish the three plants. D, Deduced amino acid sequence of Nal1. The deleted 10 amino acids are underlined, and the redletters indicate the putative nuclear localization signal. E, Phylogeny of the Nal1 protein family. A neighbor-joining tree was built byMEGA3 using a Poisson correction model with gaps to complete deletion. Topological robustness was assessed by bootstrapanalysis with 500 replicates. The bar is an indicator of genetic distance based on branch length. Nal1 and AK120320 are from rice;BT014552 and BT013672 are from tomato; AC191706, AC191543, AC205276, and AC208803 are from maize; and AK248497and AK252329 are from barley. F, Confocal image (left), transmitted light image (middle), and merged image (right) of Arabidopsiscell culture protoplasts transfected with free GFP. Bar 5 10 mm. G, Confocal image (left), transmitted light image (middle), andmerged image (right) of Arabidopsis cell culture protoplasts transfected with Nal1-GFP fusion proteins. Bar 5 10 mm. H, Steady-state nuclear protein body patterns of 35S:Nal1-GFP in root cells from a 5-d-old transgenic Arabidopsis seedling. Confocal image(left), transmitted light image (middle), and merged image (right) of root cortex cells. Bar 5 20 mm.

Nal1 Affects Venation and Polar Auxin Transport

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of five exons and four introns (Fig. 2B) that encodeda protein with 582 amino acids (Fig. 2D). In nal1,the 30-bp deletion, which existed in exon 4 of themutated Nal1 gene, resulted in the in-frame absenceof 10 amino acids (Fig. 2, B and D).

A survey of the published rice genome database(http://www.gramene.org/) identified the existenceof another gene named Nal1-like (AK120820), whichwas predicted to encode a protein that shares greaterthan 70% amino acid sequence identity with Nal1 (Fig.2E). BLAST searches of the DNA and protein data-bases also identified Nal1-like genes from other plantspecies, including monocots and dicots (Fig. 2E). No-tably, three putative homologous genes, At2g35155(AY059774), At5g45030 (AY099637), and At3g12950(BT012224), which shared around 60% amino acidsequence identity with the Nal1 protein, were identi-fied in the Arabidopsis genome (Fig. 2E). Putative Nal1homologous genes were also identified from the ge-nomes of tomato (Solanum lycopersicum), maize, andbarley (Hordeum vulgare; Fig. 2E). Database searchesfailed to identify yeast or animal proteins showingsignificant similarity to Nal1. The existence of Nal1-likegenes in different plant species might suggest con-served biochemical function of the Nal1 protein family.However, the Nal1 sequence did not match any proteinof known biochemical function in the public databases.

Subcellular Localization of Nal1

Using the WoLF PSORT programs (http://wolfpsort.org/), it was predicted that the Nal1 protein contains aputative nuclear localization signal (PKRLRSD, aminoacid residues 567–573; Fig. 2D). To determine thesubcellular localization of Nal1, GFP was fused tothe C terminus of Nal1 under the control of the 35Spromoter and the fusion gene was transformed intoArabidopsis protoplast (Meskiene et al., 2003). TheNal1-GFP chimeric protein was localized to the nucleo-plasm and cytoplasm and concentrated in discretestructures (Fig. 2G), which appeared similar to nuclearprotein bodies. However, nuclear protein body forma-tion was not detected in control cells, which expressedGFP alone under the control of the 35S promoter (Fig.2F). Similarly, our parallel experiment indicated that

At3g12950, one of the Arabidopsis homologs of Nal1,also can form nuclear protein bodies in Arabidopsisprotoplast (data not shown). To substantiate theseobservations, we generated transgenic Arabidopsisplants containing the 35S:Nal1-GFP fusion gene. Con-focal observation of the root segments of the trans-genic seedlings indicated that the Nal1-GFP fusionprotein form nuclear protein bodies and that thenumber, size, and brightness of the nuclear proteinbodies show high variation in different cells (Fig. 2H;data not shown).

Expression Pattern of Nal1

Reverse transcription (RT)-PCR analysis indicatedthat Nal1 was expressed in organs such as leaf sheaths,leaves, culms, and young panicles (Fig. 3A). The nal1mutation led to irregular venation patterns in leavesand culms, suggesting that the Nal1 gene might act invascular tissues. To test this, we generated transgenicrice plants expressing a 1,918-bp fragment of the Nal1promoter fused with the GUS reporter gene. GUSstaining of transgenic plants indicated that Nal1 waspreferentially expressed in vascular tissues of etiolatedcoleoptiles (Fig. 3, B and C). In a cross section of culmat heading stage, strong GUS staining was found in thephloem of vascular bundles (Fig. 3D). RNA in situhybridization with cross sections of 1-month-old seed-lings provided further evidence that Nal1 was richlyexpressed in vascular tissues (Fig. 3, E–I). The findingthat Nal1 was preferentially expressed in vascular tis-sues together with the fact that the nal1 mutant showeddefective vascular patterns in leaves and culms impliedthat Nal1 might be involved in rice vascular patterning.

The nal1 Mutant Shows Reduced Polar AuxinTransport Activity

The phytohormone auxin plays a pivotal role indifferent aspects of vascular tissue development andpatterning (Ye, 2002; Aloni, 2004; Fukuda, 2004; Scarpellaand Meijer, 2004; Teale et al., 2006). Our finding thatthe nal1 mutation led to defective vascular develop-ment in leaves and culms raised the interesting pos-

Table II. PCR-based molecular markers used for genetic mapping

Marker Primer Pairsa Fragment Sizeb Restriction Enzyme

M1 F, 5#-ACAGTACCTTCATTCAGCCTCTTC-3#;R, 5#-GATGCGTTCGTCTCACCAGTT-3#

368 FokI

M2 F, 5#-CAACTAAGGCTGGGGATGAAAAG-3#;R, 5#-ACCGAGTCTGGCGTCTACACAA-3#

911 PstI

M3(STS) F, 5#-TGGGGGTGGAGGACAGGT-3#;R, 5#-TTTTTTGCGGGGAGATGGAG-3#

433

M4 F, 5#-GGACAGCCAAAAGTAGTAGTAGTAG-3#;R, 5#-GGTAAATAATAGGGGGAAATA-3#

313 NsiI

aF, Forward primer; R, reverse primer. bNumbers indicate the size (in base pairs) of amplifiedfragments.

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sibility that the polar auxin flow in the nal1 mutantmay be altered. Given that the etiolated cereal coleop-tile is used as a model system to study auxin-mediatedgrowth and development (Haga et al., 2005; Li et al.,2007), together with our anatomical investigationsshowing that the overall structures and vasculaturesof etiolated coleoptiles between the wild type and nal1were similar (Fig. 4, D–G), we compared the PATactivities in etiolated coleoptiles of wild-type andmutant plants. We first examined the uptake of tritium-labeled indole-3-acetic acid ([3H]IAA) using 2-mmcoleoptile fragments and found that the uptake capac-ity of [3H]IAA in nal1 was comparable to that of thewild type (Fig. 4A). To measure basipetal transport of

IAA, [3H]IAA was applied to the apical tips of dark-grown coleoptile fragments, and the basipetal auxintransport activity in nal1 coleoptiles was reduced toapproximately 33% of that of wild-type plants (Fig.4B). As a control, acropetal transport was also mea-sured through feeding [3H]IAA to the basal tips ofcoleoptile fragments and found to be much lower thanbasipetal transport, but it was similar between nal1and the wild type (Fig. 4B). The PAT activities of nal1and wild-type plants were also examined in the pres-ence of N-1-naphthylphthalamic acid (NPA), an inhib-itor of PAT (Morris et al., 2004). As shown in Figure 4B,in the presence of 20 mM NPA, the PAT activity in wild-type coleoptiles was reduced to approximately 36% of

Figure 3. Nal1 expression pattern. A, RT-PCR analysis of Nal1 expression. TotalRNA was isolated from leaf sheaths (S),leaves (L), culms (C), and panicles (P) ofwild-type plants. Amplification of the riceACTIN1 gene was used as a control. B to D,Nal1 expression revealed by GUS stainingin Nal1 promoter-GUS transgenic riceplants. B, Nal1 promoter-GUS expressionpatterns in 5-d-old dark-grown coleoptilesshowing intense GUS staining in vasculartissues. C, Transverse section of the middlepart of the coleoptile in B showing intenseexpression of GUS in the phloem. D, Trans-verse section of a fourth internode at theheading stage showing the expression ofNal1 promoter-GUS in the phloem of vas-cular bundles. ph, Phloem; v, vascularbundle. Bars 5 400 mm in B, 20 mm in C,and 100 mm in D. E to I, Nal1 expressionpatterns revealed by RNA in situ hybridi-zation. E, Transverse section of a 1-month-old seedling showing intense expression ofNal1 in vascular tissues of leaves and leafsheaths. F, Magnified image of the boxedregion in E showing the strong expressionof Nal1 in developing vascular bundles ofleaf sheath. G, Transverse section of ayoung culm. H, Magnified image of theboxed region in G showing the strongexpression of Nal1 in vascular bundles. I,Background control. No hybridization sig-nal was observed with the sense probe. X,Xylem. Bars 5 200 mm in B, 130 mm in E,G, and I, 70 mm in F and H, 50 mm in C,and 20 mm in D.

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Figure 4. The nal1 mutant shows defective PATactivity. A, Comparison of auxin uptake between wild-type (WT) and nal1 plantsin dark-grown coleoptiles. Values represent means 6 SD of six independent assays. B, Comparison of auxin transport betweenwild-type and nal1 plants in dark-grown coleoptiles. Values represent means 6 SD of six independent assays. The asteriskindicates that the difference between the wild type and nal1 is significant (Student’s t test, P , 0.001). C, IAA efflux of dark-growncoleoptile segments. Five coleoptile segments were used in each assay, and values are given as means 6 SD of three independentassays. The difference between the wild type and nal1 is significant (Student’s t test, P , 0.05). D and E, Cross sections of dark-grown wild-type (D) and nal1 (E) shoots showing that the vasculature of nal1 coleoptile is similar to that of the wild type. Bars 5

50 mm. F, Magnified image of the boxed region in D. Bar 5 50 mm. G, Magnified image of the boxed region in E. Bar 5 50 mm. Hto K, Immunohistochemical analysis of etiolated wild-type and nal1 seedlings using the anti-AtPIN1 antibodies as probe. H,Transverse section through the middle part of a 5-d-old dark-grown wild-type seedling. I, Bright-field image of K. J, Transversesection through the middle part of a 5-d-old dark-grown nal1 seedling. K, Bright-field image of J. Bars 5 50 mm. L, Protein gel-blot analysis using the anti-AtPIN1 antibodies as probe. Membrane protein extracts from wild-type and nal1 coleoptiles were

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that of buffer control. In the case of nal1, the PATactivity was already low and application of NPA led toonly a little reduction in PAT. In order to compare theefflux rates of auxin transport between the wild typeand mutant, coleoptile segments were incubated sequen-tially in the [3H]IAA-containing solution and in auxin-free buffer, and then the amount of auxin retained inthe coleopitle segments was measured. As shown inFigure 4C, in the absence of NPA, the [3H]IAA retainedin nal1 coleoptiles was approximately 32% higher thanthat of the wild type. In the presence of 2 mM NPA, theamount of auxin retained in wild-type coleoptiles wasincreased by 140%, whereas that in nal1 coleoptileswas only increased by 24%. Together, these resultsindicated that mutation of Nal1 led to defective basip-etal PAT activity and rendered the nal1 mutant lesssensitive than the wild type to NPA-mediated inhibi-tion of polar auxin flow.

The nal1 Mutation Might Affect the Abundance of a

Deduced Rice Homolog of AtPIN1, an Auxin EffluxFacilitator in Arabidopsis

The findings that nal1 shows reduced PAT activity inthe shoot and decreased sensitivity to NPA-mediatedPAT inhibition raised the interesting possibility thatthe nal1 mutation might affect auxin efflux. Consider-ing the established roles of the Arabidopsis PIN familyproteins in the regulation of auxin efflux, together withthe existence of highly conserved PIN proteins in rice(Galweiler et al., 1998; Palme and Galweiler, 1999;Paponov et al., 2005; Xu et al., 2005), we conductedimmunohistochemical analysis in 5-d-old dark-grownseedlings using antibodies raised against the AtPIN1protein, a well-characterized auxin efflux carrier inArabidopsis. As shown in Figure 4, H to K, the AtPIN1antibodies detected signals in the vascular tissues ofboth mutant and wild-type seedlings and, interest-ingly, the signals detected in nal1 was weaker thanthose in wild-type plants. To validate this observation,membrane proteins were extracted from both nal1 andwild-type coleoptiles and subjected to protein gel-blotanalysis using the same AtPIN1 antibodies as probe.As shown in Figure 4L, a specific band with compa-rable size could be detected in both nal1 and the wildtype. However, the abundance of the signal in nal1 wassignificantly reduced over that in the wild type (Fig.4L). These results suggested that the nal1 mutationaffected the abundance of a deduced rice homolog ofAtPIN1, an auxin efflux facilitator in Arabidopsis.Among the three PIN1 family genes in rice, OsPIN1

(AF056027) shows the highest sequence similarity toAtPIN1 (Xu et al., 2005). Our quantitative real-time RT-PCR assays indicated that the OsPIN1 expressionlevels in dark-grown coleoptiles of nal1 were significantlyreduced compared with those in the wild type (Fig. 4M),suggesting that the nal1 mutation affects the OsPIN1abundance and activities at the transcription level.

DISCUSSION

Our results demonstrated that the nal1 mutationaffects vascular patterns in leaves and culms of rice.Compared with their wild-type counterparts, matureleaves of the nal1 mutants showed distinctive vasculardefects, including a reduced number of parallel veins,especially small veins (Fig. 1, E and F). On the otherhand, transverse sections of the shortened internodesin the nal1 mutant also exhibited disturbed vascularpatterns, in which the number and arrangement of thevascular bundles were altered (Fig. 1G). Gene cloningstudies indicated that Nal1 encodes a novel proteinthat was preferentially expressed in vascular tissues(Fig. 3, B–I).

It had been established that auxin, especially itscharacteristic polar transport, plays a key role duringpattern formation of the vascular system in both dicotand monocot plants (Tsiantis et al., 1999; Dengler, 2001;Scanlon et al., 2002; Aloni, 2004; Fukuda, 2004; Scarpellaand Meijer, 2004; Teale et al., 2006). Our findings thatthe nal1 mutant showed abnormal vascular patterns inleaves and culms suggested that the nal1 phenotypesmight be associated with auxin-dependent deficiencies.A series of analyses indicated that the nal1 mutant isdefective in polar auxin transport. Supporting evidencefor this notion came from measurements of [3H]IAA inetiolated coleoptiles, showing that the basipetal PATactivity in nal1 was significantly decreased (Fig. 4B)whereas the auxin retention in nal1 was significantlyincreased (Fig. 4C). Given that nal1 plants show abnor-mal vascular patterns in leaves and culms, the reducedPAT activity in nal1 could be due to defective vasculardevelopment in coleoptiles. Our investigation of 5-d-old dark-grown coleoptiles indicated that the morphol-ogy, vascular bundle number, and vascular structurebetween nal1 and the wild type were similar (Fig. 4,D–G). These observations rule out the possibility thatthe observed difference in IAA transport arises fromstructure differences between nal1 and the wild type.

Our auxin transport assay also indicated that thenal1 mutant was less sensitive than the wild type to the

Figure 4. (Continued.)probed with anti-AtPIN1 antibodies. The signal (arrowhead) abundance detected in nal1 was significantly reduced from that inthe wild type. The numbers at left denote the molecular masses of marker proteins in kilodaltons. Coomassie blue staining of theprotein samples was used as a loading control (bottom gel). M, Quantitative real-time RT-PCR analysis comparing OsPIN1relative expression levels in 5-d-old coleoptiles of the wild type and nal1. Values represent means 6 SD of three independentassays. The difference between the wild type and nal1 is significant (Student’s t test, P , 0.05).

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inhibitory effect of NPA on basipetal PAT (Fig. 4, B andC). It was proposed that the action of NPA to inhibitauxin transport was achieved through interacting withthe so-called NPA-binding protein, which has longbeen considered part of the auxin efflux carrier com-plex (Dixon et al., 1996; Ruegger et al., 1997; Blakesleeet al., 2005). On the other hand, a characteristic featureof NPA action is that auxin accumulates in treatedcells, and the effect of NPA on PAT was generallyinterpreted as an inhibition of auxin efflux (Morriset al., 2004). In this scenario, the findings that the nal1mutant exhibited decreased PAT activity and alteredresponses to NPA led us to the hypothesis that the nal1mutation might affect, directly or indirectly, auxinefflux. To test this, we looked at Arabidopsis, a modelsystem in which the auxin efflux apparatus is rela-tively well understood. Among the molecular compo-nents involved in polar auxin transport in Arabidopsisidentified to date, the best characterized AtPIN1 pro-tein was believed to play an important role in thecontrol of auxin efflux (Palme and Galweiler, 1999).The existence of a family of highly conserved PINproteins in the rice genome (Paponov et al., 2005),together with recent experimental evidence showingthat one member of the rice PIN protein family, namedOsPIN1, plays a role in auxin-dependent development(Xu et al., 2005), suggested that the function of the PINproteins in regulating auxin efflux is conserved be-tween these two species (Paponov et al., 2005; Xu et al.,2005). Therefore, available antibodies raised againstthe auxin efflux carrier AtPIN1 were used as probes toconduct immunohistochemical and protein gel-blotanalyses in nal1 and wild-type plants. In both assays,the AtPIN1 antibodies could detect specific signals invascular tissues (Fig. 4, H–K). Furthermore, signalsdetected by the AtPIN1 antibodies in the nal1 mutantwere shown to be significantly weaker than those inwild-type plants (Fig. 4L). Gene expression analysissuggested that the nal1 mutation might affect theabundance and activities of OsPIN1 (Fig. 4M). To-gether, these results led us to the following specula-tions that need to be explored in our ongoing andfuture studies. First, the signals detected by the AtPIN1antibodies might represent the rice homolog(s) of theAtPIN1 protein, which presumably acts as an auxinefflux carrier in rice. Second, reduced abundance ofthe rice homolog(s) of AtPIN1 might account for thePAT deficiency in the nal1 mutant. The results pre-sented here demonstrated that mutation of the Nal1function affects both auxin transport and vascularpatterns; however, further studies are required toaddress whether the nal1 aberrations in vascular pat-terns are the result of its deficient auxin transport.

Another significant aspect of the nal1 phenotype isdefective cell division. For example, the total cellnumber of the longitudinal sections of internode IVin nal1 mutants decreased to around one-third of thatof wild-type plants (Fig. 1D; Table I). Given theestablished role of auxin in the control of cell division(Meijer and Murray, 2001; Morris et al., 2004; Woodward

and Bartel, 2005), it is reasonable to generally associatethe reduced cell number of the nal1 internode IV withits defective PAT-mediated auxin distribution. How-ever, the detailed molecular mechanism by whichNal1 exerts its action on the cell cycle needs to beexplored in future studies.

Subcellular localization studies using Arabidopsisprotoplast or transgenic plants indicated that the Nal1-GFP fusion protein appears to form discrete nuclearprotein bodies (Fig. 2, G and H). Accumulating evi-dence in Arabidopsis indicated that nuclear proteinbodies are the sites of regulated protein degradationmediated by the ubiquitin/26S proteasome. For ex-ample, a Rac GTPase stimulates the recruitment ofGFP-labeled AUXIN/IAAs from the nucleoplasm intonuclear protein bodies to be degraded by the ubiqui-tin/26S proteasome apparatus (Tao et al., 2005). AFP, anegative regulator of abscisic acid signaling, colocal-izes with ABI5, a positive regulator of abscisic acidsignaling, in nuclear protein bodies (Lopez-Molinaet al., 2003). Abscisic acid also induces a heteroge-neous nuclear ribonucleoprotein-type protein namedAKIP1 to form nuclear protein bodies (Ng et al., 2004).Phytochrome B moves to the nucleus and forms nu-clear protein bodies in response to different lightconditions (Chen et al., 2003). Similarly, the E3 ligaseCOP1 has been observed to form nuclear proteinbodies with several regulators of light signaling, in-cluding HY5 (Saijo et al., 2003), LAF1 (Seo et al., 2003),and HFR1 (Jang et al., 2005; Yang et al., 2005). In thiscontext, our results showing that Nal1-GFP formsnuclear protein bodies link the Nal1 protein to theproteolysis process. As a first step to elucidate thebiochemical and molecular mechanisms underlyingthe Nal1 actions, we are conducting experiments todetermine whether Nal1 is involved in the degrada-tion of other proteins or Nal1 itself is subjected todegradation by the 26S proteasome.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

A rice (Oryza sativa) mutant line showing a characteristic phenotype of

narrow leaves was kindly provided by Dr. G.S. Khush at the International Rice

Research Institute in the Philippines. The original mutant was back-crossed to

the indica cultivar Zhefu802 for 10 successive generations, and the resulting

mutant line was renamed nal1 (Dong et al., 1994; Zeng et al., 2003). nal1 and

Zhefu802 were therefore considered to be a pair of nearly isogenic lines, and

Zhefu802 was used as the wild-type line for all experiments involving

comparisons of mutant and wild-type plants in this study. Rice plants were

cultivated in the experimental field at the China National Rice Research

Institute in the natural growing season.

Phenotypic Characterization of the nal1 Mutant

To investigate the morphology of the vascular bundles of the leaf blade and

the internode, tissues at indicated stages were fixed in 5% formaldehyde, 5%

glacial acetic acid, and 63% ethanol and dehydrated in a graded ethanol series.

The samples were embedded in Paraplast Plus chips (Sigma). Microtome

sections (8 mm thick) were affixed to poly-Lys-coated slides (Sigma), dewaxed

in xylene, rehydrated through a graded ethanol series, and dried overnight

before staining with toluidine blue for light microscopy.

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Map-Based Cloning of Nal1

To identify the Nal1 gene using a positional cloning approach, nal1 was

crossed to Nipponbare, a japonica cultivar, to generate a large F2 mapping

population. For fine mapping, PCR-based markers were developed based on

the sequence difference between japonica variety Nipponbare and indica variety

9311 (http://www.gramene.org/resources/). The primer sequences of the

molecular markers used are listed in Table II. For RT-PCR, we used total

RNA isolated from young seedlings as template. The full-length cDNA of Nal1

was amplified using the primer pair 5#-CGCTTTCGGCATTCGTTATCTACC-3#and 5#-GGGATCCGGCAATGGTGTATATCA-3#, sequenced, and cloned into

pBluescript SK1 vector (Stratagene).

Complementation Test

A 12.4-kb genomic DNA fragment containing the entire Nal1 coding

region, the 2,673-bp upstream sequence, and the 1,685-bp downstream se-

quence was cut from BAC AL662950 with the restriction enzyme XbaI. This

fragment was cloned into the XbaI sites of the binary vector pCAMBIA2300 to

generate the transformation plasmid for the complementation test. The

resulting transformation plasmid, as well as the empty pCAMBIA2300 vector

as control, was introduced into the nal1 mutant by Agrobacterium tumefaciens-

mediated transformation according to a published protocol (Hiei et al., 1994).

The 30-bp deletion of the putative Nal1 allele in the mutant, which was

confirmed in both cDNA and genomic sequences between nal1 and wild-type

plants, could be detected with PCR using the primer pair 5#-GATGACTTT-

GACATTTCCACCGT-3# and 5#-GAGTGATTCATTGGTAATGATAA-3#.

Phylogenetic Analysis

Homologous sequences of Nal1 were obtained through National Center for

Biotechnology Information BLAST search. Maize (Zea mays) coding sequences

were predicted by Fgenesh using homologous BAC clone sequences as input.

Others were directly from a homologous cDNA clone. The collected protein

sequences were then aligned by DIALIGN 2. Note that the program DIALIGN

2 employs the BLOSUM62 amino acid substitution matrix but does not use

gap penalty. Instead, it assembles global alignments from gap-free local pair-

wise alignments. The alignment was revised critically and used as input for

MEGA3 to construct a phylogenetic tree. A neighbor-joining tree was built by

MEGA3 using a Poisson correction model with gaps to complete deletions.

Topological robustness was assessed by bootstrap analysis with 500 replicates.

Gene Expression Analysis

For RT-PCR analysis, total RNA was extracted from various tissues using

TRIZOL reagent (Invitrogen). After RNase-free DNase (Takara) treatment, 2.5

mg of RNA was reverse transcribed using oligo(dT) primer and SuperScript III

(Invitrogen). An appropriate amount of the products was further applied for

a 20-mL PCR amplification using the following gene-specific primer pairs:

5#-GATGACTTTGACATTTCCACCGT-3# and 5#-GAGTGATTCATTGGTAA-

TGATAA-3# for Nal1 (40 cycles of 15 s at 94�C, 15 s at 60�C, and 30 s at 72�C)

and 5#-CATCTTGGCATCTCTCAGCAC-3# and 5#-AACTTTGTCCACGC-

TAATGAA-3# for ACTIN1 (25 cycles of 15 s at 94�C, 15 s at 60�C, and 30 s at 72�C).

For quantitative real-time RT-PCR, first-strand cDNAs were synthesized

by reverse transcription from 3 mg of total RNA. The cDNAs were then used as

templates in real-time PCR using the SYBR Green PCR Master Mix (Applied

Biosystems) according to the manufacturer’s instructions. The amplification

of the target genes was analyzed using the ABI Prism 7000 sequence detection

system and software (PE Applied Biosystems). The relative expression levels

of each transcript were obtained by normalization to the UBIQUITIN5 (UBQ5)

gene. The following gene-specific primers were used for real-time PCR

analysis: 5#-CGCCGTGCCGCTGCTGTCGTTCC-3# and 5#-TCCCCCGGCGG-

CTGAGGTG-3# for OsPIN1 and 5#-ACCACTTCGACCGCCACTACT-3# and

5#-ACGCCTAAGCCTGCTGGTT-3# for UBQ5. Three independent RNA iso-

lations were used for cDNA synthesis, and each cDNA sample was subjected

to real-time PCR analysis in triplicate.

For promoter-GUS assay, a 1,918-bp genomic fragment upstream of the

Nal1 gene translation start codon was PCR amplified with the primers

5#-AAGTCGACTCCGAACCAAACACCAACACAC-3# and 5#-AAGAATTC-

ACAGTTTGCGAACCTATTATA-3#. The DNA fragment was cloned into the

SalI and EcoRI sites of the vector pCAMBIA1391Z. The resulting plasmid was

transformed into rice, and the resulted transgenic plants were analyzed by

GUS staining assay as described (Scarpella et al., 2003).

RNA in situ hybridization was performed as described by Schwarzacher

and Heslop-Harrison (2000). The 3# end of Nal1 was amplified from wild-type

plants with the following primers: 5#-CACCAACCTGAACAACCCCT-3# and

5#-GGTCTTTTGATCTACTACTA-3#. The PCR product was subcloned into

pGEM-T Easy vector (Promega) and used as a template to generate RNA

probes. Transverse sections (8 mm thick) were probed with digoxigenin-

labeled antisense probes (DIG Northern Starter Kit; Roche). The slides were

observed with a microscope (Leica) and photographed using a 3CCD color

video camera (Apogee Instruments).

Subcellular Localization of Nal1

The Nal1 cDNA was amplified from wild-type rice plants with the primers

5#-AAGTCGACATGAAGCCTTCGGACGATAA-3# and 5#-AGCCATGGTCT-

CCAGGTCAAGGCTTGAT-3# and cloned into the SalI/NcoI sites of the

CaMV35S:GFP (S65T)-NOS-30 cassette vector (Niwa et al., 1999; Li et al.,

2003). The resulting 35S:Nal1-GFP construct was introduced into Arabidopsis

(Arabidopsis thaliana) protoplast as described (Meskiene et al., 2003).

The 35S:Nal1-GFP construct was digested with HindIII/EcoRI and cloned

into the binary vector pCAMBIA1300 (http://www.cambia.org.au) to trans-

form Arabidopsis plants using the floral dip method (Clough and Bent, 1998).

Progeny of an identified T3 transgenic plant homozygous for the transgene

were used to investigate the Nal1-GFP localization. The root cells of the

5-d-old transgenic seedlings grown on Murashige and Skoog (1962) agar

plates were analyzed. Confocal images were collected using a Zeiss LSM 510

Meta confocal laser scanning microscope. GFP fluorescence was excited

with a 488-nm argon laser, and images were collected in the 500- to 530-nm

range. In all cases, transmissible light images were collected simultaneously.

At least 20 seedlings were observed, and a representative image is

presented.

Auxin Influx and Efflux Assays

Auxin uptake assays were conducted according to a method described

previously (Dai et al., 2006) with some modifications. The upper ends (0.5 cm

away from the tip) of 3-d-old dark-grown coleoptiles were harvested and

allowed to equilibrate in a 1.5-mL microcentrifuge tubes containing 1/2 MS

buffer (5 mM MES and 1% [w/v] Suc, pH 5.8) for 2 h. The coleoptile fragments

(0.2 cm in length) were then incubated in 1/2 MS buffer containing 100 nM

[3H]IAA (25 Ci/mmol; Amersham) for 10 min. Samples were then transferred

into a new 1.5-mL microcentrifuge tube and rinsed in 1 mL of ice-cold 1/2 MS

buffer three times. After rinsing, the samples were incubated in scintillation

liquid for 18 h and the radioactivity was counted with a liquid scintillation

counter (1450 MicroBeta TriLux; Perkin-Elmer). For each assay, five coleoptiles

were measured.

The polar auxin transport assays were performed using coleoptiles of 5-d-

old dark-grown rice seedlings as described previously (Okada et al., 1991; Dai

et al., 2006; Li et al., 2007) with minor modifications. In each assay, five apical

coleoptile segments each (0.2 cm away from the tip) of 2.0-cm length were

preincubated in 1/2 MS medium with shaking at 100 rpm for 2 h. The

coleoptile segments were then placed in a 1.5-mL microcentrifuge tube with

one end submerged in 10 mL of 1/2 MS liquid medium containing 500 nM

[3H]IAA plus 500 nM free IAA in the dark at room temperature for 2 h. NPA

(Chem Service) was added to the medium as indicated. The unsubmerged

ends of the segments (0.5 cm in length) were excised and washed three times

in 1/2 MS liquid medium. After rinsing, the segments were incubated in 2.0

mL of universal scintillation fluid for 18 h and the radioactivity was counted

with a liquid scintillation counter (1450 MicroBeta TriLux; Perkin-Elmer).

Based on the orientation of the coleoptile segments submerged in the buffer,

basipetal or acropetal auxin transport was measured, with the acropetal auxin

transport as control.

IAA efflux of dark-grown coleoptile segments was assayed as described

previously (Dai et al., 2006). In each assay, five apical dark-grown coleoptile

segments (2.0 mm in length) were put into 50 mL of 1/2 MS medium buffer

containing 500 nM [3H]IAA plus 500 nM free IAA and incubated at room

temperature for 2 h with shaking at 100 rpm. NPA (2 mM) was added to the

medium as indicated. The segments were then rinsed and incubated for

another 2 h in the same buffer without IAA to allow the IAA to be

transported out. After washing twice, the segments were then incubated in

2.0 mL of universal scintillation fluid for 18 h, and the radioactivity was

counted with a liquid scintillation counter (1450 MicroBeta TriLux; Perkin-

Elmer).

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Immunohistochemical Localization and Protein

Gel-Blot Analysis

Immunohistochemical localizations and microscopy were conducted as

described previously (Noh et al., 2003). The antibody used was anti-AtPIN1

(Galweiler et al., 1998) at a final dilution of 1:200. Fluorescein isothiocyanate-

conjugated antibody was used as a secondary antibody. A confocal laser

scanning microscope (Zeiss LSM 510 Meta) was used for immunofluorescence

imaging.

One gram of coleoptiles from 5-d-old dark-grown seedlings was used

for membrane protein extraction using the ProteoExtract kit (M-PEK;

Calbiochem). Membrane proteins were separated by 10% SDS-PAGE and

probed with anti-AtPIN1 (1:200) antibodies.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under the following accession numbers: Nal1 (EU093963), putative

OsPIN1 (AF056027).

ACKNOWLEDGMENTS

We thank Dr. Jiayang Li (Institute of Genetics and Developmental Biology,

Chinese Academy of Sciences) for critical reading of the manuscript. We

thank Dr. Gurdev Singh Khush (International Rice Research Institute) for

providing the original nal1 mutant, Dr. Bin Han (National Center for Gene

Research, China) for providing the BAC clone AL662950, and Dr. Kang

Chong (Institute of Botany, Chinese Academy of Sciences) for assistance in

RNA in situ hybridization.

Received March 6, 2008; accepted June 10, 2008; published June 18, 2008.

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Nal1 Affects Venation and Polar Auxin Transport

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