Loose Plant Architecture1, an INDETERMINATE DOMAIN Protein ... · Loose Plant Architecture1, an INDETERMINATE DOMAIN Protein Involved in Shoot Gravitropism, Regulates Plant Architecture

Loose Plant Architecture1, an INDETERMINATEDOMAIN Protein Involved in Shoot Gravitropism,Regulates Plant Architecture in Rice1[W]

Xinru Wu2, Ding Tang2, Ming Li2, Kejian Wang, and Zhukuan Cheng*

State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Geneticsand Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Tiller angle and leaf angle are two important components of rice (Oryza sativa) plant architecture that play a crucial role indetermining grain yield. Here, we report the cloning and characterization of the Loose Plant Architecture1 (LPA1) gene in rice, thefunctional ortholog of the AtIDD15/SHOOT GRAVITROPISM5 (SGR5) gene in Arabidopsis (Arabidopsis thaliana). LPA1regulates tiller angle and leaf angle by controlling the adaxial growth of tiller node and lamina joint. LPA1 was also found toaffect shoot gravitropism. Expression pattern analysis suggested that LPA1 influences plant architecture by affecting thegravitropism of leaf sheath pulvinus and lamina joint. However, LPA1 only influences gravity perception or signaltransduction in coleoptile gravitropism by regulating the sedimentation rate of amyloplasts, distinct from the actions of LAZY1.LPA1 encodes a plant-specific INDETERMINATE DOMAIN protein and defines a novel subfamily of 28 INDETERMINATEDOMAIN proteins with several unique conserved features. LPA1 is localized in the nucleus and functions as an activetranscriptional repressor, an activity mainly conferred by a conserved ethylene response factor-associated amphiphilicrepression-like motif. Further analysis suggests that LPA1 participates in a complicated transcriptional and proteininteraction network and has evolved novel functions distinct from SGR5. This study not only facilitates the understanding ofgravitropism mechanisms but also generates a useful genetic material for rice breeding.

Rice (Oryza sativa) is the staple food for more thanhalf of the world’s population. In the past 50 years, thegreen revolution and the use of heterosis have greatlyimproved rice yields (Peng et al., 2008). However,because of the increasing demand for rice production,food security can still be a serious problem. Thus, newelite varieties that can produce much higher grainyields need to be developed, and ideal plant architec-ture (ideotype) breeding has been shown to be themost promising strategy in tropical areas (Khush,2005). Rice plant architecture is mainly determined byplant height and tiller and panicle morphology, ofwhich tiller angle and leaf angle are two importantagronomic traits.

Tiller angle, defined as the angle between the mainculm and its side tillers, has long attracted the atten-tion of breeders due to the significant contribution ofthis trait to plant architecture (Wang and Li, 2008). In

cultivated rice, neither a spread-out nor a compacttype of plant architecture is beneficial to grain pro-duction (Xu et al., 1998). Plants with the spread-outarchitecture can escape some diseases that are in-creased by high humidity, but they occupy too muchspace and increase shading and lodging, consequentlydecreasing photosynthetic efficiency and grain yieldper unit area. On the other hand, compact plants areless efficient in capturing light and are more suscep-tible to insects and pathogens transmitted by contact.Thus, an appropriate tiller angle is very important torice production. In determining tiller angle, auxintransport plays an important role. Loss of function ofLAZY1 enhanced polar auxin transport (PAT) andimpaired lateral auxin transport, resulting in reducedshoot gravitropism and a tiller-spreading phenotype(Li et al., 2007). Suppressing the expression of rice PIN-FORMED1 or enhancing that of rice PIN-FORMED2,two auxin efflux transporters, both altered PAT andincreased tiller angles (Xu et al., 2005; Chen et al., 2012).Additionally, many quantitative trait loci (QTLs) affect-ing tiller angle have been identified (Li et al., 1999). Amutation in the major QTL Tiller Angle Control1 (TAC1)decreased its expression level, leading to the compacttiller in japonica rice, in contrast to the generally widertiller angle in indica rice (Yu et al., 2007). Tiller angle isalso associated with rice domestication. Selection for thePROSTRATE GROWTH1 (PROG1) mutant caused thecritical transition from the prostrate tillers of wild rice(Oryza rufipogen) to the erect tillers of cultivated rice (Jinet al., 2008; Tan et al., 2008). However, the molecular

1 This work was supported by the Ministry of Sciences and Tech-nology of China (grant nos. 2012AA10A301 and 2011ZX08009–003),and the National Natural Science Foundation of China (grant nos.31160223 and 31000695).

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 policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Zhukuan Cheng ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.112.208496

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mechanisms underlying the actions of both genes havenot been clarified.

Leaf angle is the inclination between leaf blade andculm. An erect leaf trait can contribute to rice grainyield by both enhancing photosynthetic efficiency andallowing for increased planting density (Sinclair andSheehy, 1999; Morinaka et al., 2006; Sakamoto et al.,2006). Leaf angle is formed when the leaf blade bendsaway from the leaf sheath, during which time celldevelopment on the adaxial side of the lamina jointplays a pivotal role (Duan et al., 2006; Zhang et al.,2009). At present, many related genes have beenidentified, most of which are involved in the biosyn-thesis or signaling of the phytohormone brassinoste-roids (BRs; Yamamuro et al., 2000; Hong et al., 2002,2003, 2005; Tanabe et al., 2005; Bai et al., 2007; Tonget al., 2009). Other phytohormones, including auxin,ethylene, and gibberellin, also affect leaf angle throughtheir synergistic effects with BRs (Cao and Chen, 1995;Shimada et al., 2006; Song et al., 2009). However, newevidence indicates that novel mechanisms also partic-ipate in the determination of leaf angle in rice. Forexample, Leaf Inclination2 controls leaf angle by inhib-iting adaxial cell division but not cell elongation of thelamina joint, distinct from the BR-dependent pathway(Zhao et al., 2010). Recently, another BR-unrelatedgene, Increased Leaf Angle1, was reported to affect leafangle by regulating mechanical tissue formation in thelamina joint (Ning et al., 2011). These findings dem-onstrate the complicated nature of the rice leaf angleregulatory mechanism.

Most studies of rice plant architecture have only fo-cused on either tiller angle or leaf angle. In practice,the two traits are closely related to one another, andmany genes can simultaneously affect both, such asLeaf and Tiller Angle Increased Controller, BrassinosteroidUp-Regulated1, and even LAZY1 and TAC1 (Li et al.,1999, 2007; Wang et al., 2008; Tanaka et al., 2009). In thispaper, we described the rice mutant loose plant archi-tecture1 (lpa1), which displays a relatively larger tillerangle and leaf angle due to reduced shoot gravitropism.LPA1 encodes a novel INDETERMINATE DOMAIN(IDD) protein with transcriptional repression and showsa specific expression pattern. Our analyses suggest thatLPA1 may be involved in a complicated regulatorymechanism. This study can aid in our general under-standing of the gravitropism mechanism and the im-portant role it plays in rice plant architecture formation.

RESULTS

The lpa1 Mutant Displays Loose Plant Architecture

The lpa1 mutant was a naturally occurring mutantisolated from an indica variety, Zhongxian3037. Duringboth the vegetative and reproductive stages, lpa1 al-ways exhibited loose plant architecture with largertiller angle and leaf angle than those of the wild type(Fig. 1, A and B). We measured the tiller angle at

heading date and found that the maximum angle was17.3° in lpa1 but only 9.8° in the wild type (Fig. 1C).Careful observation showed that the large tiller angleof lpa1 was caused by the more symmetrical growth ofthe tiller node compared with the wild type (Fig. 1D).We also measured leaf angles: each angle was larger inlpa1 than in the wild type, and this difference wasmore obvious in older leaves, where the maximumangle of the fourth leaf could reach up to 61.2° in lpa1but only 27.4° in the wild type (Fig. 1E). This differencewas further confirmed by the dynamic change ob-served in the newly developing leaf (Fig. 1F). Detailedexamination revealed that the large leaf angle of lpa1was caused by a more rapid elongation on the adaxialside of the lamina joint (Fig. 1, G and H). We furtheranalyzed the differences in the lamina joint betweenthe wild type and lpa1 at the cellular level. Longitu-dinal sections of the adaxial epidermis in the laminajoint showed that the larger lamina joint of the mutantwas mainly due to the enhanced cell elongation (Fig. 1,I and J).

In addition, we also observed distinct changes inother characteristics. Compared with the wild type,each internode of the lpa1 mutant became shorter butthicker (Supplemental Fig. S1A). Similarly, grains and

Figure 1. Morphological comparison between the wild type and lpa1.A and B, Phenotype of wild-type (WT) Zhongxian3037 and the mutantlpa1 in the tillering stage (A) and the grain-filling stage (B). C, Maxi-mum tiller angle of the wild type and lpa1. Values are means 6 SD. D,Magnified tiller base of the wild type and lpa1. Bar = 1 cm. E, Leafangle of the wild type and lpa1. Values are means 6 SD. Numbersindicate the positions of leaves. F, Kinetic comparison of leaf anglebetween the wild type and lpa1. Values are means 6 SD. G and H,Magnified lamina joint of the wild type (G) and lpa1 (H). Bars = 0.5cm. I and J, Longitudinal section of the adaxial epidermis of a laminajoint of the wild type (I) and lpa1 (J). 30˚ indicates the degree of leafangle. Bars = 0.5 cm.

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leaves became shorter and wider (Supplemental Fig.S1, B and C). Microscopic observation showed that thewall of culm was thickened (Supplemental Fig. S1, Dand E) but that cell size did not change (SupplementalFig. S1, F–I). The structure of vascular bundles alsoshowed complicated changes (Supplemental Fig. S1, Jand K).

The lpa1 Mutant Displays Reduced Shoot Gravitropism

In rice, the lazy1 mutant exhibits a tiller-spreadingphenotype resulting from reduced shoot gravitropism(Li et al., 2007). To examine whether lpa1 was alsoinvolved in the same process, we analyzed the gravityresponse of young seedlings. The result revealed thatboth light- and dark-grown mutant seedlings had areduced gravity response and could not grow uprighteventually (Fig. 2, A–C). However, lpa1 roots showed anormal gravity response (Fig. 2D). These results indi-cated that LPA1 is only involved in shoot gravitropismin rice.We also tested the gravity response of coleoptiles

using 1-cm-long young shoots grown in the dark. Theresult showed that lpa1 could grow upright com-pletely, but the time was delayed about 4 h whencompared with the wild type (Fig. 2, E–G). This indi-cated that the mutant coleoptile gravitropism is aber-rant in gravity perception or signal transduction butnormal in organ bending, according to the gravity re-sponse process.In higher plants, the starch-statolith hypothesis,

postulating that the amyloplast sedimentation alongthe direction of gravity in statocytes is responsible forgravity perception, has been widely accepted (Sack,1997). We observed amyloplast sedimentation in thebasal region of coleoptile where shoot bending occurs.Under gravistimulation by turning young shoots up-side down, most wild-type amyloplasts had moved tothe basal ends of the coleoptile parenchyma cells alongthe direction of gravity within 10 min (Fig. 2, H and J).In contrast, the amyloplasts of lpa1 were only locatedin the middle parts of cells within the same time pe-riod, but they reached to the bottom after 20 min (Fig.2, I and J). This result suggested that LPA1 may affectgravity perception by regulating the sedimentationrate of gravity-sensing amyloplasts in rice coleoptiles.

Map-Based Cloning and Functional Verification

To map LPA1, we generated an F2 population de-rived from the cross between lpa1 and 02428 (a wild-type japonica variety with compact plant architecture).LPA1 was roughly mapped on the short arm ofchromosome 3, linked to sequence-tagged-site (STS)markers S1 and S2. Because the effects of lpa1 are notsignificantly distinct from natural variation betweenindica and japonica types, we developed a series of re-combinant inbred lines containing the heterozygous

LPA1/lpa1 allele from the F2 population, and fourlines showing obvious segregation of phenotype wereidentified in the F5 generation. Using 80 recessive in-dividuals from these lines, we restricted LPA1 to a 570-kb interval between STS markers S6 and S7 and furthernarrowed the interval to 40 kb between cleaved-amplifiedpolymorphic sequence marker C1 and STS marker S7,using 963 recessive individuals from the F6 generation(Fig. 3A).

There are three putative genes within the 40-kb re-gion. Genomic sequencing revealed an insertion in thethird exon of LOC_Os03g13400. We isolated this in-sertion and confirmed its existence at the transcrip-tional level (Fig. 3, B and C). Nucleotide BLASTshowed that the insertion is a Ty1-copia group retro-transposon (Supplemental Fig. S2), which results in apremature transcriptional termination and a deducedtruncated protein with a 203-amino acid C-terminaldeletion in the mutant (Fig. 3D).

To investigate the effects of LOC_Os03g13400, anRNA interference (RNAi) vector and an overexpression(OE) vector driven by the cauliflower mosaic virus(CaMV) 35S promoter were constructed and trans-formed into Yandao8 (a wild-type japonica variety withcompact plant architecture). Most RNAi and OEtransgenic plants showed loose and compact plantarchitectures with differing degrees, respectively,compared with Yandao8 (Fig. 4A), from which onetypical RNAi plant and one typical OE plant wereselected for detailed analysis.

Following two generations of self-pollination, theRNAi and OE transgenic plants showed stable phe-notypes. Detailed observation showed that both tillerangle and leaf angle increased in the RNAi plant butdecreased in the OE plant (Fig. 4, B and C). Real-timePCR analysis showed that the expression level ofLOC_Os03g13400 was down-regulated nearly 4-foldin the RNAi plant but up-regulated more than 20-foldin the OE plant (Fig. 4, D and E). Furthermore, thegravity response was also reduced in the RNAi seedlings(Fig. 4F), and multiple independent RNAi lines providedmore solid evidence (Supplemental Fig. S3, A–C). Theseresults strongly confirmed that LOC_Os03g13400 isLPA1 and also showed that the transcription level ofLPA1 is closely associated with rice plant architecture.

LPA1 Exhibits a Specific Expression Pattern

We performed 59 and 39 RACE, and the resultsrevealed that the full-length complementary DNA(cDNA) of LPA1 is 1,806 bp long with a 1,317-bp openreading frame, a 273-bp 59 untranslated region, and a216-bp 39 untranslated region. Sequence comparisonbetween genomic DNA and cDNA revealed thatLPA1 is composed of three exons and two introns(Supplemental Fig. S4).

To define the temporal and spatial expression pat-tern of LPA1, we performed an analysis of LPA1 tran-scription using RNA samples from different tissues.

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Real-time PCR revealed that LPA1 was highly ex-pressed in the lamina joint and internodes, especiallyin young tissues. However, older tiller base also showeda high expression level equivalent to the young secondinternode (Fig. 5A). LPA1 was also moderatelyexpressed in coleoptile, root, seedling, and paniclebut was barely detectable in leaf blade and leaf sheath(Fig. 5A). The higher expression levels of LPA1 in thelamina joint and tiller node correspond well with themain phenotypes of the mutant.

In grasses, the leaf sheath pulvinus was identified asa specialized organ for gravity response (Kaufmanet al., 1987). We analyzed the expression of LPA1 inthis tissue. To our surprise, LPA1 was exceptionallyabundant in leaf sheath pulvinus, about 35-fold higherthan in the coleoptile, strongly suggesting an impor-tant role for LPA1 in leaf sheath pulvinus gravitropism(Fig. 5B). Additionally, we also found that the ex-pression of LPA1 in dark-grown etiolated seedlingswas 1.5-fold higher than it was in those grown in thelight (Fig. 5C), indicating that light can inhibit the ex-pression of LPA1, consistent with previous reports thatlight affects gravitropism in plants (Correll and Kiss,2002; Kim et al., 2011).

LPA1 Defines a Novel Subfamily of IDD Proteins

LPA1 encodes a predicted 438-amino acid protein.Sequence analysis indicated that LPA1 is a typical Cys-2/His-2 zinc finger protein (Supplemental Fig. S4)belonging to the plant-specific IDD protein family,where it was also known as OsIDD14 (Colasanti et al.,2006). The IDD protein family was first defined by themaize (Zea mays) INDETERMINATE1 (ID1), and allmembers share a conserved IDD that has a putativenuclear localization sequence at the N terminus, fol-lowed by four distinct zinc finger motifs (sequentiallynamed ZF1, ZF2, ZF3, and ZF4; Colasanti et al., 2006).It is interesting that OsIDD12, OsIDD13, and OsIDD14/LPA1 are more similar to one another and divergentfrom the other 12 rice members, like AtIDD14,AtIDD15/SHOOT GRAVITROPISM5 (SGR5), andAtIDD16 among the 16 Arabidopsis (Arabidopsisthaliana) members (Colasanti et al., 2006), suggestingthe specificity of the six proteins in the IDD proteinfamily. Sequence alignment revealed that the six proteinsshared a unique conserved coiled-coil domain at the C

Figure 2. Gravitropism analysis of the wild type and lpa1. A and B,Four-day-old seedlings of the wild type (WT) and lpa1 grown in light(A) and dark (B) after a 24-h gravistimulation. Bars = 1 cm. C, Theultimate curved angle of wild-type and lpa1 seedlings grown in lightand dark after a 3-d gravistimulation. Values are means 6 SD. D, Ki-netic comparison of the curved angle of root between the wild typeand lpa1 under gravistimulation. Values are means 6 SD. E and F,Coleoptiles of the wild type and lpa1 after 4 h (E) and 8 h (F) ofgravistimulation. Bars = 1 cm. G, Kinetic comparison of the curvedangle of coleoptiles between the wild type and lpa1 under

gravistimulation. H and I, Kinetic comparison of the sedimentation ofamyloplasts along the direction of gravity in parenchyma cells of co-leoptiles between the wild type (H) and lpa1 (I). Bars = 25 mm. J,Average position of amyloplasts in gravistimulated wild-type and lpa1parenchyma cells at different time points. Values are means 6 SE. Theschematic cell at right was used to determine the position of amylo-plasts. Each cell was divided into four equal segments along its length,numbered 0 to 3. When coleoptiles were inverted, amyloplasts movedalong the basal-apical axis and their positions were scored. The arrowindicates the direction of gravity (g).


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terminus but have lost two other conserved domains(MSATALLQKAA and TR/LDFLG), one or both ofwhich the other members possess (Fig. 6; Colasantiet al., 2006). This result further indicated that the sixproteins form a distinctive group in the IDD proteinfamily.To identify other homologs of LPA1, a BLASTP

search was performed. The result revealed that thereare 27 proteins showing high sequence similarity withLPA1 in higher plants. Multiple sequence alignmentsshowed that these proteins were highly conserved inthe ID domain and the coiled-coil domain. Further-more, most of these proteins also shared several smallcharacterized motifs, of which the two conservedmotifs, EL1 (ELQLLP) and EL2 (LQLSIG), that aresimilar to ethylene response factor-associated amphi-philic repression (EAR) and EAR-like motifs, were themost remarkable (Supplemental Fig. S5; Kazan, 2006).This result clearly demonstrated that LPA1 defines anovel subfamily of IDD proteins with distinct domains

and motifs. Moreover, phylogenetic analysis indicatedthat the 28 proteins were distinctly classified into twoclades, corresponding to proteins from monocots anddicots, respectively, with the exception of a singleprotein from the bryophyte Physcomitrella patens (Fig.7). However, the four proteins on the LPA1 branch aredistinct from the other monocot proteins (Fig. 7). Someof this divergence may result from their unique con-served spacers between the ZF1 and ZF2 motifs, sim-ilar to that of maize ID1 (Supplemental Fig. S5; Kozakiet al., 2004).

Figure 3. Map-based cloning of LPA1 and gene information. A, Map-based cloning of LPA1. LPA1 was mapped in a 570-kb interval re-stricted by STS markers S6 and S7 using 80 recessive individuals. Theinterval was narrowed to a 40-kb interval on bacterial artificial chro-mosome AC134229 restricted by STS marker S7 and cleaved-amplifiedpolymorphic sequence marker C1 using 963 recessive individuals. Thenumber under the marker indicates the number of recombinant indi-viduals. LPA1 contains three exons (thick black bars) and two introns(thin gray bars). The triangle indicates the insertion of a retro-transposon. B, PCR amplification of the retrotransposon. The arrowsindicate specific bands of the wild type (WT) and lpa1. C, PCR veri-fication of the retrotransposon using cDNA as a template. 59 and 39indicate two primer pairs whose products overlap, partially corre-sponding to the 59 and 39 parts, respectively, of the full-length cDNA ofLPA1. The latter harbors the insertion site of the retrotransposon andcannot get the predicted product in lpa1. The arrows indicate specificbands. D, The insertion of this retrotransposon leads to a prematuretranscriptional termination of LPA1. GTGAG indicates the target siteduplication of the retrotransposon.

Figure 4. Functional verification of LPA1. A, Phenotype of wild-typeYandao8, RNAi, and OE LPA1 transgenic plants. B, Comparison oftiller angle among Yandao8, RNAi, and OE plants by vertical obser-vation. Bar = 5 cm. C, Comparison of the angle of the third leaf amongYandao8, RNAi, and OE plants. Bar = 1 cm. D and E, Relative ex-pression levels of LPA1 in RNAi (Ri; D) and OE (E) transgenic plantscompared with Yandao8 (WT). Values are means 6 SE of three indi-vidual experiments, and the value of Yandao8 is set as 1. F, Four-day-old seedlings of Yandao8 and RNAi transgenic plants grown in lightafter a 24-h gravistimulation. The arrow indicates the direction ofgravity (g). Bar = 1 cm.


Rice LPA1 Cloning





LPA1 Is an Active Transcriptional Repressor

To determine the subcellular localization of LPA1,we transiently expressed GFP alone and the LPA1-GFPfusion protein under the control of the CaMV 35Spromoter in onion (Allium cepa) epidermal cells. Thegreen fluorescence of GFP alone was distributed in thenucleus, cytoplasm, and plasma membrane, whereas

fluorescence from the fusion protein was observed onlyin the nucleus, indicating that LPA1 is a nucleus-localizedprotein (Fig. 8A). The expression of this fusion protein inrice protoplasts also revealed a consistent nuclear locali-zation pattern (Fig. 8B), while OsMADS15/DEP acted asa positive control (Supplemental Fig. S6; Wang et al.,2010).

As LPA1 is a putative transcription factor, we ana-lyzed its transcriptional activity using a dual luciferasereporter (DLR) assay system in Arabidopsis proto-plasts (Fig. 9A). The result showed that LPA1 and its Nterminus (amino acids 1–232) slightly reduced therelative luciferase activity compared with the GAL4binding domain negative control. However, the Cterminus (amino acids 233–438) significantly inhibitedthis activity by more than 50%, indicating the presenceof a strong repression domain (Fig. 9B).

In plants, EAR and EAR-like motifs are well knownfor their transcriptional repression activities, and theywidely modulate various biological processes (Kazan,2006). Our analysis has indicated the presence of twoconserved EAR-like motifs, EL1 and EL2, within thesequence of LPA1. To minimize the C-terminal re-pression domain, we examined the transcriptional ac-tivities of two short polypeptides containing EL1(amino acids 262–271) and EL2 (amino acids 301–312),respectively. The results showed that EL1 stronglyrepressed the expression of the reporter gene by morethan 90%, while EL2 only showed a slight repressioneffect, similar to LPA1 and its N terminus (Fig. 9B). Ineukaryotes, transcriptional repressors can be dividedinto active and passive categories, and active repres-sors contain an intrinsic repression domain (Thielet al., 2004). Thus, our results indicated that LPA1 is an

Figure 5. Expression pattern of LPA1. A, Relative expression levels ofLPA1 in different tissues analyzed by real-time PCR. Values aremeans 6 SE of three independent experiments, and the value of cole-optile is set as 1. B, Relative expression levels of LPA1 in leaf sheathpulvinus. The value of coleoptile is set as 1. C, Relative expressionlevels of LPA1 in seedlings grown in light (set as 1) and dark.

Figure 6. C-terminal sequence alignment of AtIDD14, AtIDD15/SGR5, AtIDD16, OsIDD12, OsIDD13, and OsIDD14/LPA1.Conserved amino acids are highlighted in shades of black and gray: white letters with black background (100% identity), whiteletters with gray background (80% identity), and black letters with gray background (60% identity). The line indicates thecoiled-coil domain of LPA1 predicted by SMART (http://smart.embl-heidelberg.de/).


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active transcriptional repressor and that its main re-pression effect is contributed by an EAR-like motif.

DISCUSSION

The Role of LPA1 Is Distinct from That of LAZY1 in RiceColeoptile Gravitropism

Gravity is an important environmental factor thatcontrols the growth direction of plants. Roots andshoots grow downward and upward, showing posi-tive and negative gravitropism, respectively (Moritaand Tasaka, 2004). This response ensures efficientphotosynthesis and nutrition acquisition, which iscritical for plant survival (Hangarter, 1997).Generally, the gravity response can be divided into

three steps: gravity perception, signal transduction,and organ bending (Haswell, 2003). In studies on themechanism of gravitropism, two hypotheses havebeen proposed. The starch-statolith hypothesis postu-lates that the sedimentation of starch-filled amylo-plasts in the direction of gravity within specific cells(statocytes) is the trigger for gravity perception (Sack,1991), while the Cholodny-Went hypothesis suggeststhat an uneven distribution of auxin between the twosides of a curving organ induced by gravity results indifferential growth and bending (Firn et al., 2000).Genetic evidence from Arabidopsis indicates that bothhypotheses are essentially correct (Blancaflor et al.,

1998; Fukaki et al., 1998; Utsuno et al., 1998; Weise andKiss, 1999; Fujihira et al., 2000; Friml et al., 2002;Ottenschläger et al., 2003).

In this study, we showed that LPA1 is specificallyinvolved in rice shoot gravitropism. The gravitropismof seedlings was impaired partially in the mutant.However, the mutant coleoptile was able to growcompletely upright in the opposite direction of gravity,although the process was delayed, suggesting thatgravity sensing or signal transduction was disruptedin the coleoptile. Further analysis showed that thesedimentation rate of amyloplast in the mutant cole-optile was significantly slower than that of the wildtype. These results indicate that LPA1 may influencegravity perception in the rice coleoptile by regulatingthe sedimentation rate of amyloplasts, supporting thestarch-statolith hypothesis. However, LPA1 may alsoparticipate in signal transduction, because some cyto-plasm components, such as vacuole and actin fila-ments, have been reported to affect the sedimentationof amyloplasts and are likely to be involved in thegravity signal in Arabidopsis shoot gravitropism(Morita et al., 2002; Yano et al., 2003; Silady et al., 2004;Saito et al., 2005; Nakamura et al., 2011).

Our results further suggested that the function ofLPA1 is distinct from that of LAZY1 in rice coleoptilegravitropism. First, the lazy1 coleoptile has partiallylost gravitropism and was unable to grow upright(Yoshihara and Iino, 2007), which is different from

Figure 7. Phylogenetic tree of the 28 IDD proteins ofthe subfamily defined by LPA1. The tree was con-structed using MEGA 4.0 based on the neighbor-joining method. The box indicates the position ofLPA1.


Rice LPA1 Cloning



lpa1. Detailed experiments revealed that enhancedPAT and impaired lateral auxin transport caused anequal distribution of auxin and a reduced gravity re-sponse in lazy1 (Li et al., 2007). This result supports theCholodny-Went hypothesis, which also indicated thatLPA1 may not affect auxin asymmetry. Second, it wasdemonstrated that the sedimentation rate of amylo-plast appeared to be identical between lazy1 and itswild type (Abe et al., 1994b; Godbole et al., 1999),suggesting that LAZY1 is unlikely to participate ingravity perception. Third, the expression of LAZY1 isnot affected by the mutation of LPA1 (SupplementalFig. S7). Given these reasons, we proposed that LPA1and LAZY1 function at different stages of coleoptilegravitropism, and LPA1 may participate in a LAZY1-independent pathway.

LPA1 May Be Involved in a Complicated Transcriptionaland Protein-Protein Interaction Network

IDD proteins are members of a zinc finger proteinfamily of transcription factors in higher plants, and

several members have been well characterized. MaizeID1 is the first described IDD protein, acting as atranscriptional regulator of floral transition (Colasantiet al., 1998). In Arabidopsis, AtIDD3/MAGPIE,AtIDD10/JACKDAW, and AtIDD8/NUTCRACKERare involved in root development with the GRAStranscription factors SHOOT-ROOT and SCARECROW(Levesque et al., 2006; Welch et al., 2007). AtIDD8 alsoregulates photoperiodic flowering by modulating sugartransport and metabolism (Seo et al., 2011b). Recently,AtIDD1/ENHYDROUS was shown to promote germi-nation by regulating light and hormonal signalingduring seed maturation (Feurtado et al., 2011). In rice,three independent studies have demonstrated thatOsID1, the ortholog of maize ID1, plays a pivotal role inrice floral transition (Matsubara et al., 2008; Park et al.,2008; Wu et al., 2008).

In this study, we showed that another rice IDDprotein, LPA1, plays an important role in rice shootgravitropism and defines a novel subfamily of IDDproteins with several unique conserved domains andmotifs, from which a C-terminal EAR-like motif (EL1)was identified because of its typical amphiphilic fea-ture. We further demonstrated that this motif was

Figure 8. Subcellular localization of LPA1. Localization of 35S:GFPand 35S:LPA1-GFP in onion epidermal cells (A) and rice protoplastcells (B) is shown. GFP indicates the green fluorescence of proteins,DIC indicates the differential interference contrast phase, and Mergedindicates the merging of GFP and DIC. Bars = 10 mm.

Figure 9. Transcriptional activity of LPA1. A, Scheme of the constructsused in the Arabidopsis protoplast cotransfection experiment. The re-porter contains five copies of GAL4 binding elements (GAL4), a min-imal TATA box of the CaMV 35S promoter, the firefly luciferase (LUC)gene, and a nopaline synthase (Nos) terminator. Each effector containsa GAL4 DNA binding domain (GAL4 BD) and part of a coding region(D) of LPA1 promoted by CaMV 35S. A translational enhancer se-quence from Tobacco mosaic virus (V) is located upstream of the siteof initiation of translation. B, Relative luciferase activities of Arabi-dopsis protoplasts cotransfected with reporter and different effectors.Schemes of deletion mutants of LPA1 are shown at left. All luciferaseactivities are expressed relative to the value of GAL4 BD alone (set as100). Values are means 6 SD of four independent experiments.


Wu et al.





responsible for the major transcriptional repressionactivity of LPA1. In Arabidopsis, AtIDD8 is a tran-scriptional activator, and its activation domains alsolocate to the C terminus (Seo et al., 2011b). Our resultfor LPA1 not only revealed the diversity of tran-scriptional activities of IDD proteins but also impliedthat such activities are mainly conferred by their Ctermini.Our further analysis suggested that EL1 may have

peculiar features. First, most EAR and EAR-like motifssuppress both intramolecular and intermolecular ac-tivities of other transcriptional activators, such as ViralProtein16 (VP16) from Herpes simplex virus (Ohta et al.,2001; Hiratsu et al., 2002; Tiwari et al., 2004); however,our data demonstrated that EL1 could not significantlyinhibit the activity of VP16 (Supplemental Fig. S8),implying that EL1 may function as a gene-specifictranscriptional repressor (Gaston and Jayaraman, 2003).Second, most full-length repressors containing EAR andEAR-like motifs show strong transcriptional repressionactivities (Hiratsu et al., 2002), but LPA1 only showeda slight repression effect, whereas EL1 by itself hadmuch higher activity. It could be that the flanking se-quence contains an activation domain that counteractsthe effect of EL1. However, no such domain wasfound. Another possibility is that LPA1 may performthe repression effect of EL1 by interacting with otherproteins. As evidence, some EAR repressors were in-deed involved in protein-protein interactions (Chernet al., 2005; Song et al., 2005; Weigel et al., 2005), whilethe conserved coiled-coil domain also suggests thatLPA1 can be a stable part of some protein complexes(Burkhard et al., 2001). However, yeast two-hybridanalyses did not give a meaningful result. Addition-ally, LPA1 also possesses putative DNA-binding ac-tivity, because the highly conserved ID domain hasbeen shown to recognize DNA sequences (Kozakiet al., 2004; Seo et al., 2011b). Therefore, LPA1 may bepart of a complicated transcriptional and protein-protein interaction network that regulates rice shootgravitropism. More studies are required to investigatethis regulatory mechanism further.

LPA1 Is the Functional Ortholog of AtIDD15/SGR5 Butwith Distinct Features

It is intriguing that AtIDD15/SGR5 belongs to theconserved LPA1-defined subfamily, affects the gravityresponse of inflorescence stems by regulating thesedimentation of amyloplasts, and plays an importantrole in the formation of branch angle in Arabidopsis(Tanimoto et al., 2008), quite similar to the function ofLPA1 in rice. Moreover, a previous report revealed thatOsIDD14/LPA1 and its two paralogs, OsIDD12 andOsIDD13, are more closely related to each other but aremore divergent from other members of this family inrice (Colasanti et al., 2006). Furthermore, OsIDD12and OsIDD13 have the same expression patterns asAtIDD14 and AtIDD16, most abundant in panicle or

flower, while LPA1 and SGR5 are mainly expressedin gravitropism-related tissues (Morita et al., 2006;Supplemental Fig. S9), indicating that LPA1 is thefunctional ortholog of SGR5. They may share a con-served biological function in plant shoot gravitropism.

However, LPA1 also has characteristic featuresdistinct from its Arabidopsis counterpart. First, pro-tein sequence alignment revealed that there are sev-eral significant differences between LPA1 and SGR5,such as some amino acid substitutions in the conservedID domain and coiled-coil domain, the insertions/deletions of two minor motifs (EL2 and SYV/MSS),and the most arresting spacer sequence (SupplementalFig. S5), which implies that LPA1 might have otherpeculiar functions in rice. Second, LPA1 affects thedevelopment of internodes, leaves, grains, and vascu-lar bundles as well as shoot gravitropism in rice(Supplemental Fig. S1), but similar effects in Arabi-dopsis were not detected in SGR5 (Morita et al., 2006;Tanimoto et al., 2008). Third, the mutation of SGR5attenuated significantly the shoot circumnutation inArabidopsis (Tanimoto et al., 2008). However, thismovement is only controlled by the LAZY1-dependentpathway in rice (Yoshihara and Iino, 2007), implyingthat LPA1 may not participate in circumnutational reg-ulation. Fourth, it was recently reported that AtIDD14can regulate starch metabolism by means of alternativesplicing under different temperature conditions (Seoet al., 2011a). Although SGR5 also affects starch me-tabolism, its function is obviously divergent from thatof AtIDD14. Additionally, AtIDD16 has a similar ex-pression pattern to AtIDD14 (Morita et al., 2006),suggesting that both genes may have similar functions.These facts further support the hypothesis that SGR5may not function redundantly with its two paralogs inregulating Arabidopsis shoot gravitropism (Moritaet al., 2006; Tanimoto et al., 2008). However, our ex-pression pattern analysis revealed that OsIDD13 wasalso highly expressed in leaf sheath pulvinus(Supplemental Fig. S9). As the loss-of-function mutantfor OsIDD13 has not been described yet, it is unknownwhether OsIDD13 is also involved in the gravitropismof leaf sheath pulvinus. Therefore, whether LPA1 alsohas a similar role to SGR5 in regulating rice shootgravitropism is still in doubt. Further investigationsare required to understand the functions of LPA1 andits paralogs.

LPA1 Could Potentially Be Used in Rice Ideal PlantArchitecture Breeding

In recent years, some genes that affect rice grainyield have been identified, shedding light on some ofthe molecular mechanisms underlying ideal plant ar-chitecture (Ashikari et al., 2005; Song et al., 2007; Xueet al., 2008; Huang et al., 2009; Jiao et al., 2010; Miuraet al., 2010). Gravity is an important regulator of plantarchitecture. In grasses, because of the short-lived co-leoptiles, the leaf sheath pulvini and lamina joints are


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the major gravity-responding organs, which controlthe growth orientations of seedlings, tillers, and leafblades (Maeda, 1965; Kaufman et al., 1987; Abe et al.,1994a). In our study, LPA1 showed a specific expres-sion pattern, and the high expression level in the leafsheath pulvinus and lamina joint suggests that LPA1mainly functions in the gravitropism of these tissues,corresponding to its limited effect on coleoptile gravi-tropism. This analysis demonstrated that LPA1 affectsrice plant architecture by regulating shoot gravitrop-ism in later developmental stages.

There are several genes that control tiller angle inrice. LAZY1 and PROG1 lead to the prostrate growthof tillers, but the major QTL, TAC1, only slightly en-larges the tiller angle in indica rice. Because of theirlarger effects, LAZY1 and PROG1 are not very suitablefor rice breeding, while TAC1 has been extensivelyutilized in rice plant architecture breeding owing to itssmaller effect on tiller angle (Yu et al., 2007). In ourstudy, LPA1 showed an inclined tiller angle of 17.3°,similar to TAC1 (Yu et al., 2007). Although the equiv-alent effect made too many difficulties in map-basedcloning of LPA1, it revealed the potential utilization ofLPA1 in rice plant architecture breeding. Moreover,the transgenic results further demonstrated that riceplant architecture was correlated with the endoge-nous expression level of LPA1. Therefore, LPA1 is auseful gene for plant architecture modification in ricebreeding.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Rice (Oryza sativa) plants were grown in a paddy field in Beijing undernatural conditions. For light or dark treatment, germinated seeds were placedon bottomless 96-well plates floating on water and moved to growth cham-bers (28°C) with or without a 14-h/10-h photoperiod. Seedlings were sampledafter 7 d.

Histological Analysis

Tissues were fixed in 50% ethanol, 5% acetic acid, and 10% formaldehydeovernight, followed by a series of dehydration and infiltration steps, and thenembedded in Technovit 7100 resin (Heraeus Kulzer). Five-micrometer sectionswere sliced with a Leica microtome, stained with 0.25% toluidine blue, andobserved using an Olympus BX51 microscope.

Gravitropism Assay and AmyloplastSedimentation Analysis

The gravity response was measured according to the method describedpreviously (Li et al., 2007). Briefly, rice seeds were grown in half-strengthMurashige and Skoog medium (pH 5.8) after being dehusked and surfacesterilized. Two-centimeter-long roots, 1-cm-long shoots, and 4-d-old seedlingswere used for testing. The gravity response was determined by measuring thecurved angle after reorienting by 90°. For amyloplast staining, a periodic acid-Schiff kit (Sigma-Aldrich) was used according to the manufacturer’s directions.Eight- to 10-mm paraffin sections were deparaffinized and hydrated, immersedin periodic acid solution for 5 min, rinsed with distilled water, and then im-mersed in Schiff’s reagent for 15 min. After dehydrating, clearing, and mounting,sections were observed microscopically. The quantitative analysis of amylo-plast average position was performed as described previously (Tanimotoet al., 2008). For each time point, the average position of amyloplasts in 25

coleoptile parenchyma cells from each tissue section was scored, and four suchsections from different coleoptiles were used.

Primer Design and Retrotransposon Identification

PCR-based molecular markers for map-based cloning were developedbased on sequence differences between indica variety 93-11 and japonica varietyNipponbare according to published data from the National Center for Bio-technology Information (http://www.ncbi.nlm.nih.gov/). Primer sequencesare listed in Supplemental Table S1. Long amplification of the retrotransposonwas performed using LA Taq (TaKaRa) and the ID-6F/ID-1R primer pair, andthe verification of transcriptional level was performed using 59 (C1-F/C1-R)and 39 (C2-F/C2-R) primer pairs. The specific primers are listed in SupplementalTable S2.

Real-Time PCR and RACE Analysis

Total RNA was extracted from various samples using TRIzol reagent(Invitrogen). Two to 3 mg of RNA was treated with RNase-free DNaseI(Invitrogen), and first-strand cDNA was synthesized to using oligo(dT)18primer (TaKaRa) and Moloney murine leukemia virus reverse transcriptase(Invitrogen). The expression levels of LPA1 and other genes were analyzedusing a CFX96 Real Time System (Bio-Rad) and rice Ubiquitin as an internalcontrol. 59 and 39 RACE was performed according to the online protocol fromTaKaRa (http://www.takara-bio.com/). For 59 RACE, cDNA was synthe-sized using the 59 phosphorylated primer 59-RT, treated with RNaseH, andcyclized using T4 RNA ligase, followed by nested PCR with primer pairs59-1F/59-1R and 59-2F/59-1R. For 39 RACE, cDNA was reverse transcribedusing the Oligo-dT-Adaptor, then nested PCR was performed usingprimer pairs 39-1F/Adaptor and 39-2F/Adaptor. The specific primers arelisted in Supplemental Table S2.

Vector Construction and Rice Transformation

To generate the RNAi vector for LPA1, two copies of the 440-bp 59 RACEproduct were repeatedly inserted in opposing orientations into pUCCRNAito form a hairpin, which was then cloned into pCAMBIA1300-35S. Togenerate the OE vector for LPA1, a 1,432-bp fragment containing the entireopen reading frame of LPA1 was amplified using primer pair ZF-C1F/ZF-C2R and cloned into pCAMBIA1300-35S. Both vectors were introduced intoAgrobacterium tumefaciens strain EHA105 through electroporation, and theresulting strains were used to transform the japonica rice variety Yandao8.Rice transformation was performed as described previously (Hiei et al.,1994).

Protein Sequence and Phylogenetic Analysis

Rice and Arabidopsis (Arabidopsis thaliana) protein sequences were obtainedfrom The Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/) andThe Arabidopsis Information Resource (http://www.arabidopsis.org/index.jsp), respectively. Four maize (Zea mays) IDD proteins were obtained through adatabase search from Maize Sequence (http://www.maizesequence.org/index.html) using LPA1 sequence as a query. The other protein sequences were ac-quired by searching GenBank (http://www.ncbi.nlm.nih.gov/genbank/) usingLPA1 sequence as a query. Protein sequences were aligned using ClustalX 1.83,and the conserved residues were displayed by GeneDoc (http://www.nrbsc.org/gfx/genedoc/). A phylogenetic tree was constructed using MEGA 4.0based on the neighbor-joining method. The coiled-coil domain was predicted bySMART (http://smart.embl-heidelberg.de/). The spacers and zinc finger motifswere labeled as described previously (Colasanti et al., 2006).

Subcellular Localization

The coding region of LPA1 was amplified using the primer pair GFP-F/GFP-R (the specific primers are listed in Supplemental Table S2) and clonedinto pJIT163-hGFP, generating the LPA1-GFP fusion under the control of the23 CaMV 35S promoter. GFP and LPA1-GFP plasmids were bombarded intoonion (Allium cepa) epidermal cells using a PDS-1000/He particle gun (Bio-Rad) or transformed into rice protoplasts as described previously (Li et al.,2009). After 20 h of incubation at 28°C, GFP fluorescence was observed with aconfocal laser scanning microscope (Leica TCS SP5).


Wu et al.


http://www.ncbi.nlm.nih.gov/




http://www.takara-bio.com/


http://rapdb.dna.affrc.go.jp/

http://www.arabidopsis.org/index.jsp

http://www.arabidopsis.org/index.jsp

http://www.maizesequence.org/index.html

http://www.maizesequence.org/index.html

http://www.ncbi.nlm.nih.gov/genbank/

http://www.nrbsc.org/gfx/genedoc/

http://www.nrbsc.org/gfx/genedoc/

http://smart.embl-heidelberg.de/



Transcriptional Activity Analysis

The transcriptional activity of LPA1 was analyzed using the DLR assaysystem in Arabidopsis protoplasts (Hao et al., 2010). The firefly luciferasegene driven by the minimal TATA box of the CaMV 35S promoter fol-lowing five copies of the GAL4 binding element was used as a reporter. TheRenilla luciferase gene driven by CaMV 35S was used as an internal control.Different truncated fragments of LPA1 were amplified (the specific primersare listed in Supplemental Table S2) or synthesized and then fused with theyeast GAL4 DNA-binding domain as effectors, driven by CaMV 35S fol-lowed by the translational enhancer V from Tobacco mosaic virus. Arabi-dopsis protoplast preparation and transfection were based on the protocolfor Arabidopsis mesophyll protoplasts (Yoo et al., 2007). For each assay, 6mg of reporter plasmid DNA, 6 mg of effector plasmid DNA, and 1 mg ofinternal control plasmid DNA were cotransfected. After incubating for 16 hat 23°C, the relative luciferase activity was measured using the DLR assaysystem and the GloMax 20-20 luminometer (Promega).

Sequence data from this article can be found in the GenBank databaseunder the following accession numbers: LPA1, JQ681528; Ubiquitin, D12629;OsIDD12, AK119595; OsIDD13, AK288964; LAZY1, AK067664; TAC1,AP005682; PROG1, AP005632.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Characteristics of internode, grain, and leaf of thewild type and lpa1.

Supplemental Figure S2. The sequence of the retrotransposon.

Supplemental Figure S3. Five independent RNAi transgenic lines of LPA1.

Supplemental Figure S4. Full-length cDNA of LPA1 and the deducedamino acid sequence.

Supplemental Figure S5. Sequence alignment of 28 full-length IDD pro-teins of a subfamily defined by LPA1.

Supplemental Figure S6. Subcellular localization of OsMADS15/DEP-GFP in rice protoplast cells.

Supplemental Figure S7. Relative expression levels of LPA1, TAC1, andPROG1 in 7-d-old seedlings grown in different conditions.

Supplemental Figure S8. EL1 cannot significantly inhibit the transcrip-tional activation activity of VP16.

Supplemental Figure S9. Relative expression levels of OsIDD12, OsIDD13,and OsIDD14/LPA1 in different tissues.

Supplemental Table S1. PCR-based molecular markers for map-basedcloning.

Supplemental Table S2. Primer sequences used for functional analysis ofLPA1.

ACKNOWLEDGMENTS

We thank Prof. Shouyi Chen (Institute of Genetics and DevelopmentalBiology, Chinese Academy of Sciences) for kindly providing the DLR assaysystem.

Received October 3, 2012; accepted November 1, 2012; published November 2,2012.

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Rice LPA1 Cloning



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Loose Plant Architecture1, an INDETERMINATE DOMAIN Protein ... · Loose Plant Architecture1, an INDETERMINATE DOMAIN Protein Involved in Shoot Gravitropism, Regulates Plant Architecture