overexpression of the arabidopsis gene upright ... - plant...

Overexpression of the Arabidopsis Gene UPRIGHTROSETTE Reveals a Homeostatic Control forIndole-3-Acetic Acid1[C][W]

Yue Sun*, Yang Yang, Zheng Yuan2, Jutta Ludwig Muller, Chen Yu, Yanfei Xu, Xinghua Shao, Xiaofang Li,Eva L. Decker, Ralf Reski, and Hai Huang

School of Life Sciences, East China Normal University, 200062 Shanghai, China (Y.S., Y.Y., C.Y., Y.X., X.S.,X.L.); Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Science, 200032 Shanghai,China (Z.Y., H.H.); Department of Biology, Technische Universitat Dresden, D–01062 Dresden, Germany(J.L.M.); and Plant Biotechnology, Faculty of Biology, and Centre for Biological Signalling Studies, Universityof Freiburg, D–79104 Freiburg, Germany (E.L.D., R.R.)

Auxins are phytohormones that are essential for many aspects of plant growth and development. The main auxin produced byplants is indole-3-acetic acid (IAA). IAA exists in free and conjugated forms, corresponding to the bioactive and storedhormones, respectively. Free IAA levels, which are crucial for various physiological activities, are maintained through acomplex network of environmentally and developmentally responsive pathways including IAA biosynthesis, transport,degradation, conjugation, and conjugate hydrolysis. Among conjugated IAA forms, ester- and amide-type conjugates are themost common. Here we identify a new gene, UPRIGHT ROSETTE (URO), the overexpression of which alters IAA homeostasisin Arabidopsis (Arabidopsis thaliana). We previously identified a semidominant mutant, uro, which had multiple auxin-relatedphenotypes. We show here that compared to wild-type plants, the uro plants contain increased levels of free and ester-conjugated IAA, and decreased levels of amino-conjugated IAA. uro plants carrying the pDR5:b-glucuronidase (GUS) constructhave strong GUS staining in cotyledons and stem, and their cotyledons are able to generate roots on auxin-free medium,further confirming that this mutant contains higher levels of free IAA. The URO gene encodes a C2H2 zinc-finger protein thatbelongs to a plant-specific gene family. The response to URO overexpression is evolutionarily conserved among plants, as GUSactivity that may reflect free IAA levels was increased markedly in transgenic p35S:URO/pGH3:GUS/Physcomitrella patens andpNOS:URO/pGH3:GUS/P. patens plants.

Auxin is an important plant hormone that modu-lates numerous processes throughout plant growthand development. It has roles in tropic responses tolight and gravity, general root and shoot architecture,organ initiation and patterning, and vascular devel-opment (Woodward and Bartel, 2005; Benjamins andScheres, 2008; Vanneste and Friml, 2009). The major

naturally occurring auxin in plants is indole-3-aceticacid (IAA). Local free IAA is bioactive and contributesto the regulation of plant growth and development.Free IAA can be converted into conjugated forms,which lose biological activity, through modification ofthe indole ring or side chain (Ljung et al., 2002; Bajguzand Piotrowska, 2009). Hydrolysis of IAA conjugates,which releases free IAA, is another important aspect inplant IAA homeostatic control. The major IAA conju-gates, ester- and amide-type conjugated IAA, can beboth hydrolyzed to free IAA (Ljung et al., 2002; Bajguzand Piotrowska, 2009).

In higher plants, free IAA concentration is usuallyvery low, often in the nanomolar range (Woodwardand Bartel, 2005). In whole seedlings of Arabidopsis(Arabidopsis thaliana), the amide-type IAA conjugatesconstitute approximately 90% of the IAA pool, whereasthe ester-type conjugates and free IAA account forapproximately 10% and 1%, respectively (Tam et al.,2000). This generally maintained distribution of IAAforms indicates that an appropriate amount of free IAAin local plant tissues is crucial for specific physiologicalactivities and must be strictly maintained.

Recent studies have provided important informa-tion toward the understanding of IAA homeostasis.SUPERROOT1 (SUR1) and SUR2 encode a C-S lyase

1 This work was supported by the National Natural ScienceFoundation of China (grant no. 90717107/30300030/30570159), 863research plan (grant no. 2006AA10Z109), Chinese Academy ofSciences (grant no. KSCX2–YW–N–016), Shanghai Scientific Com-mittee (grant no. 08QA14028/075407067), Shanghai Education Com-mittee (grant no. 09ZZ43), and Alexander-von-Humboldt ResearchFellowship for postdoctoral researchers.

2 Present address: School of Life Sciences and Biotechnology,Shanghai Jiao Tong University, Dongchuan Road 800, 200240Shanghai, P.R. China.

* 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:Yue Sun ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.110.154021

Plant Physiology�, July 2010, Vol. 153, pp. 1311–1320, www.plantphysiol.org � 2010 American Society of Plant Biologists 1311

(Mikkelsen et al., 2004) and a cytochrome P450 protein(Barlier et al., 2000), respectively. Both of these genesare likely to be involved in glucosinolate biosynthesis.Since both glucosinolate and IAA biosyntheses requirecommon precursors, a block in glucosinolate biosyn-thesis diverts the precursor compounds into IAAbiosynthesis. Mutant seedlings of sur1 and sur2 havean elongated hypocotyl, epinastic cotyledons, and anincreased number of lateral roots. The YUCCAs, whichencode flavin monooxygenase-like proteins, constituteanother group of genes involved in IAA biosynthesis(Zhao et al., 2001; Cheng et al., 2006, 2007; Kim et al.,2007). Dominant activation-tagged yuccamutants haveelongated hypocotyls, epinastic leaves, and increasedapical dominance (Zhao et al., 2001; Kim et al., 2007).The loss-of-function sur1 and 2 mutants and the gain-of-function yucca mutants contain increased levels offree IAA at specific developmental stages. The sur1mutant also has increased levels of conjugated IAAon a whole-seedling basis. It has been proposed thatthe increased levels of IAA conjugates in sur1 maybe due to an increased rate of conjugation, inducedby the larger amount of free IAA (Ljung et al., 2002).TAA1 encodes an aminotransferase, which catalyzesthe formation of indole-3-pyruvic acid from L-Trp inanother IAA biosynthetic pathway (Tao et al., 2008).Loss-of-function taa1 mutants have a reduction infree IAA levels, demonstrating the importance of theindole-3-pyruvic acid-dependent IAA biosynthesispathway.

In addition to the enzymes that are involved in IAAbiosynthesis, enzymes involved in auxin that conjugateformation and hydrolysis also affect IAA homeostasis.These include IAA glucosyl-transferase, auxin-conjugatehydrolases, and GH3 IAA-amino acid synthases(Bartel and Fink, 1995; Davies et al., 1999; LeClereet al., 2002; Rosamond et al., 2002; Rampey et al., 2004;Staswick et al., 2005; Ludwig-Muller et al., 2009). TheIAR4 gene, which encodes a putative mitochondrialpyruvate dehydrogenase E1a-subunit, is required formaintenance of amino-type conjugate levels. In theiar4-3 mutant, the IAA amino-conjugate level wassignificantly increased (Quint et al., 2009). Severalgenes in the GH3 family, such as GH3.2, GH3.3,GH3.4, GH3.5, GH3.6, and GH3.17 in Arabidopsis(Staswick et al., 2005) or GH3-like genes in moss(Ludwig-Muller et al., 2009), encode IAA-amidosynthetases that catalyze the formation of auxin-amino acid conjugates. In addition, several amido-hydrolases, such as ILR1, ILL1, ILL2, and IAR3, areinvolved in cleaving IAA amino acid conjugates torelease free IAA in Arabidopsis (Davies et al., 1999;LeClere et al., 2002). The triple mutant ilr1 iar3 ill2 haslower levels of free IAA, and higher levels of amino-type IAA conjugates (Rampey et al., 2004).

The recent research has led to a clear picture show-ing that there are a number of components affectingIAA homeostasis in plants. However, little is knownabout how the coordination of these components isregulated to fulfill each corresponding function. Per-

haps one limitation to this question is that most of theIAA homeostasis-affecting components identified thusfar are metabolic enzymes, not putative regulatoryfactors. We previously reported characterizations of asemidominant Arabidopsis mutant, upright rosette(uro), which displayed multiple auxin-related pheno-types (Sun et al., 2000; Guo et al., 2004; Yuan et al.,2007). Our genetic analyses revealed that the urophenotypes are closely linked to a T-DNA insertionand are caused by a single nuclear gene mutation (Sunet al., 2000). In this study, we report the identificationof theURO gene, which encodes a putative C2H2 zinc-finger protein. We demonstrate that the uro auxin-related phenotypes are caused byURO overexpression.Our data also suggest that a potentially existing plantregulatory system(s) for IAA homeostasis controlresponds to the URO overexpression, as the uro mu-tation resulted in an altered distribution of the free,ester-, and amide-conjugated IAA levels in Arabidop-sis. In addition, we show that the response to UROoverexpression is consistent in Physcomitrella mossplants, suggesting a conserved homeostasis controlof IAA in plants.

RESULTS

The URO Gene Encodes a C2H2 Zinc-Finger Protein andBelongs to a Plant-Specific Gene Family

The uro mutant has multiple abnormal plant phe-notypes that have been observed in several otherauxin-defective mutants, such as lacking apical dom-inance with retarded reproductive development andhaving a very soft stem with the reduced xylem sizeand underdeveloped interfascicular fibers (Guo et al.,2004). To identify the URO gene, we first obtained aDNA fragment containing a part of the left border of aT-DNA sequence and a flanking Arabidopsis sequenceby thermal asymmetric interlaced PCR (Liu et al.,1995). Sequence analysis revealed that the T-DNA leftborder was linked to the upstream sequence of thegene At3g23140, 164 bp from the start codon (Fig. 1A),and the gene At3g23140 was up-regulated in the uromutant (see below). To prove whether At3g23140 is theURO gene and that its up-regulation could cause theuro phenotypes, we transformed wild-type and uroplants with several constructs (Fig. 1A).

First, we transformed uro with p35S:dsAt3g23140, inwhich an At3g23140 double-stranded RNA interfer-ence fusion gene driven by the 35S promoter (Fig. 1A).Compared with wild-type (Fig. 1B), uro/+ (Fig. 1C),and uro (Fig. 1D) plants, 30 out of a total of 51 p35S:dsAt3g23140/uro transgenic plants appeared nearlyrescued, showing elongated inflorescence stems andnormally shaped plant organs, although some plantsin this group exhibited a slightly increased number ofbranches (Fig. 1F). Thirteen plants were weakly res-cued, showing a partially restored apical dominancebut a dwarf stature (Fig. 1E), while the other eight

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transgenic plants were with uro phenotypes (data notshown). Strikingly, the At3g21340 transcript level inthe transgenic plants was correlated to the uro phe-notype severity (Supplemental Fig. S1A). The p35S:dsAt3g23140 construct was also used to transformwild-type plants; however, all 12 transgenic plantsthat we obtained appeared normal (data not show).Next, 27 p35S:At3g23140/Landsberg erecta (Ler) and 76pNOS:At3g23140/Ler transgenic plants were obtained,showing upright cotyledons (Fig. 1G) or rosette leaves(Fig. 1H), and all these plants were arrested at thecotyledon (for p35S:At3g23140/Ler plants) or seedling(for pNOS:At3g23140/Ler plants) stages. To determinethe At3g23140 expression-driven sequence in the uromutant, we transformed wild-type plants with theAt3g23140 coding region, either fused to its 164-bpupstream sequence (pAt3g23140:At3g23140) or to the2,986-bp upstream sequence containing the entireIAAH gene (IAAH-At3g23140; Fig. 1A). While trans-genic plants carrying the first fusion did not yieldplant phenotypic changes (data not shown), 82 out of116 transgenic plants harboring IAAH-At3g23140

showed uro-like phenotypes in varying degrees ofseverity, and 27 out of 82 showed the strong bushyphenotype (Fig. 1I). Finally, wild-type plants weretransformed with the 2,986-bp upstream sequenceonly (IAAH) or with a direct downstream gene (p35S:At3g23145; Fig. 1A), and the resulting eight IAAH/Lerand 10 p35S:At3g23145/Ler transgenic lines did notshow altered plant phenotypes (data not shown).Based on the above results, we reasoned that theAt3g23140 gene is URO.

The URO gene encodes a protein of 173 amino acidsin length and has no predicted intron. A single C2H2type of zinc-finger-like motif is located in the N termi-nus (Fig. 2A). A Leu-rich inhibition domain, whichis present near the C terminus, contains a conservedmotif (L/FDLNL/FXP) known as an ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR-associ-ated amphiphilic repression (EAR) motif (Fig. 2B;Fujimoto et al., 2000; Ohta et al., 2001; Hiratsu et al.,2003). An Arabidopsis genome-scale search revealedthat URO belongs to a gene family containing 28members (Englbrecht et al., 2004). Genes in this family

Figure 1. A T-DNA insertion in theURO locus causes the uro pheno-types. A, Diagram of the predictedstructure in the uro locus andconstructs used in plant transfor-mation. Note that the T-DNA left-border (LB) sequence is linked tothe 164-bp upstream sequencefrom the ATG of the URO gene.chr3, Chromosome 3; RB, rightborder. B to D, Mature wild-type(B), uro/+ (C), and uro (D) plants. Eand F, RNA interference of theURO gene using the double-stranded URO sequence (p35S:dsAt3g23140) resulted in partial(E) or much better rescue (F) ofthe apical dominance defect inthe transgenic uro plants. G andH, p35S:At3g23140/Ler (G) andpNOS:At3g23140/Ler (H) trans-genic plants exhibited severe phe-notypes with upright cotyledons orearly appearing rosette leaves, andall these plants were arrestedat the seedling stages. I, Trans-genic plants, carrying the IAAH-At3g23140 construct (the upstreamT-DNA sequence including IAAH,the entire URO coding region,and a part of downstream geneAt3g23145), showed the uro-likephenotypes. Shown is a 4-week-old IAAH-At3g23140/Ler trans-genic plant (I). Bars = 5 mm (B–F,I), 1 mm (G), and 2 mm (H).

Overexpression of the URO Gene Affects IAA Homeostasis

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are also called SUPERMAN (SUP)-like genes, asSUP was the first member to be cloned (Schultz et al.,1991). This gene family, which is plant specific(Englbrecht et al., 2004), includes members from otherplant species such as rice (Oryza sativa), petunia(Petunia hybrida), and even the moss plant Physcomi-trella (Fig. 2, A and B). The URO transcript levels inwild-type and mutant seedlings were analyzed byquantitative real-time reverse transcription (RT)-PCR.Compared with the wild type, the URO level wasdramatically elevated in the uro/+ and uro seedlings(Fig. 2C). In different tissues analyzed in wild-typeArabidopsis, URO transcripts were only weakly de-tected in inflorescence (Fig. 2D), and these results werereproducible even with increased PCR cycles (Supple-mental Fig. S2). The URO expression was strongly up-regulated in different plant tissues in the uro mutant,especially in stems and inflorescences. These resultssuggest that the uro phenotypes are likely to be causedby the abnormal URO expression.

The uro Mutant Has Alterations in IAA Response

and Homeostasis

To address the auxin-related phenotypes in the uromutant, we first investigated the auxin response byperforming a root elongation inhibition test. Wild-typeand uro seedlings were first grown on a hormone-free

medium for 3 d. Seedlings with similar root lengthswere then transferred to newmedia containing a rangeof IAA concentrations. The length of the primary rootwas measured after 3 d growth on the new media. Atlower concentrations (0.001–0.01 nM), IAA promotedroot elongation in both wild-type and uro mutantseedlings, although the promotion in wild-type seed-lings was more evident (Fig. 3, A and B). At a concen-tration of 0.1 nM, IAA promoted the root elongation ofwild-type seedlings even more strongly, whereas elon-gation of uro roots was inhibited (Fig. 3, A and B).Treatment with 1 nM of IAA still promoted root elon-gation in wild-type seedlings, but severely affected uroroot growth (Fig. 3, A and B). With a further increase inIAA concentration, root elongation was inhibited inboth wild-type and uro seedlings (Fig. 3, A and B).These results suggest that root elongation in uro plantsis hypersensitive to IAA. We also analyzed lateral root,and found that in the uromutant the number of lateralroots was reduced as compared with wild-type plants(Supplemental Fig. S3).

We next measured IAA levels in the uro mutant.Total IAA levels, including free, ester-, and amide-linked IAA, were similar in 12-d-old uro and wild-typeaboveground seedlings (Fig. 3D) and 4-week-oldrosette leaves (Fig. 3F). However, levels of each ofthese three IAA forms differed markedly in uro plantscompared to wild-type plants (Fig. 3, C and E). In

Figure 2. Sequence and expression pattern analyses of the URO gene. A and B, Amino acid sequence alignments show that thezinc-finger domain (A) and L/FDLNL/FXP, called EAR motif (B), of the URO protein share a high similarity to those of other planthomologs. The absolutely conserved residues are highlighted in yellow, highly conserved residues in blue, and conservedresidues in green. C, Different expression levels of 6-d-old uro/+ and uro seedlings were shown by quantitative real-time RT-PCR.All values were normalized against the expression level of the ACTIN genes. Three biological replicates, each with threetechnical repeats, were performed, and the data are shown as the averages6 SE. D, In different plant tissues, RT-PCR was used todetectURO transcript. In 5-week-old wild-type plants,URO transcripts were only weakly detected in inflorescence, whereas thetranscription levels were dramatically elevated in several tissues of the uro mutants.

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particular, free and ester-linked IAA levels were ele-vated, whereas amide-linked IAA levels decreased(Fig. 3, C and E). These results suggest that over-expression of theURO gene affects IAA homeostasis inArabidopsis.

To provide additional evidence that the uro mutantcontains increased levels of free IAA, we introducedthe pDR5:GUS construct (Ulmasov et al., 1997) intouro mutant plants by crosses. Because the originalpDR5:GUS plant is in the Columbia-0 background and

Figure 3. The increasedURO expression resulted in the altered IAA response and homeostasis in the uromutant. A and B, Rootselongation of uro showed a higher sensitivity to the IAA concentration. Data in A represent average 6 SE. Asterisks (* and **)indicate significant statistical differences by t test (P , 0.05 and P , 0.01, respectively) between wild type and uro. Ten to 15seedlings were used in each measurement of the different IAA concentrations, with three biological replicates. C to F,Measurement of IAA contents in 12-d-old whole seedlings (C and D) and 4-week-old rosette leaves of wild-type and uro plants(E and F). The free, IAA-ester, and IAA-amino conjugates were respectively measured using three biological replicates (C and E),while the total IAA contents were calculated using the average of the three biological replicates from C and E, and the calculatedresults are shown in D and F, respectively. [See online article for color version of this figure.]



the uro mutant is in Ler, we examined GUS reporteractivity in progeny siblings with wild-type-like (pDR5:GUS/W), uro/+ (pDR5:GUS/uro/+), or uro (pDR5:GUS/uro) phenotypes. In young pDR5:GUS/W seed-lings, GUS signals were weakly present in the root tipand distal parts of leaves (Fig. 4A). In comparison,GUS staining was observed throughout all above-ground tissues of pDR5:GUS/uro seedlings, and washighly accumulated in the vascular tissue (Fig. 4B).We then transformed pDR5:GUS/W plants with thepNOS:URO construct (Fig. 1A). In the resulting pNOS:URO/pDR5:GUS/W plants, the increased URO ex-pression resulted in an even stronger accumulationof GUS staining throughout the entire seedling, withthe plant apex and root tip both being highly stained(Fig. 4C). GUS signals in 5-week-old pDR5:GUS/uro/+(Fig. 4, E and G) or pDR5:GUS/uro (Fig. 4H) plantswere also strong, as compared to the pDR5:GUS/Wplants (Fig. 4, D and F), especially in the stem.

It was previously reported that explants from sur1,sur2, and yucca-D mutants, which contain higher con-centrations of free IAA, are able to generate roots onauxin-free medium (Delarue et al., 1998; Zhao et al.,2001). To determine whether uro tissue could generateroots on hormone-free medium, uro cotyledons wereused as explants. As expected, after 7 d growth onhormone-free media, root initiation was observedfrom uro cotyledons (Fig. 4J), but not from wild-typecotyledons (Fig. 4I). The pDR5:GUS staining andthe root initiation experiments further support theidea that certain uro tissues contain a high level of freeIAA.

Overexpression of the URO Gene Affects Auxin

Homeostasis in Physcomitrella

The moss Physcomitrella belongs to the bryophytes,the evolutionary oldest group of land plants. A searchof a genome sequencing database (www.cosmoss.org)revealed that Physcomitrella contains at least four pu-tative URO homologs. To investigate whether theeffect of URO overexpression on IAA homeostasis isevolutionarily conserved in other plant species, weanalyzed p35S:URO and pNOS:URO transgenic Phys-comitrella lines, whose motherline originally carriedthe pGH3:GUS construct (Bierfreund et al., 2003). As inthe pDR5:GUS/Ler transgenic plants, free auxin is ableto induce GUS expression in the pGH3:GUS line, andstrength of GUS activity was correlated to auxin con-centrations in the plants (Bierfreund et al., 2003). ThepGH3:GUS motherline usually produced very weakGUS signals in the absence of IAA treatment (Fig. 5, Aand D). Six independent p35S:URO/pGH3:GUS andfive pNOS:URO/pGH3:GUS transgenic lines were ob-tained from a moss transformation experiment. Thegametophytes of all transgenic lines had much stron-ger GUS signals than those of the motherline on IAA-free media (Fig. 5, B, C, E, and F). This indicates thatthe effect of URO overexpression on auxin homeosta-sis is evolutionarily conserved.

DISCUSSION

The Cause of the uro Phenotypes

We previously reported isolation and characteriza-tion of the uro mutant, and proposed that the mutantphenotypes may be caused by defective auxin biology(Sun et al., 2000; Guo et al., 2004; Yuan et al., 2007).However, in previous studies of the URO gene, twoimportant questions were unanswered. First, whichgene corresponds to the uro abnormalities? And sec-ond, what is the molecular basis by which uro muta-tion affects plant development? In this work, we report

Figure 4. Auxin reporter expression and adventitious root formation inuromutants. A and B, GUS staining of a 7-d-old wild-type (A) or uro (B)seedling carrying the pDR5:GUS construct. C, A 7-d-old pNOS:URO/pDR5:GUS/W transgenic plant. D to H, GUS staining of a 5-week-oldwild-type (D and F), uro/+ (E and G), or uro/uro (H) plant carrying thepDR5:GUS construct. The strongest DR5::GUS expression is in stem inboth uro/uro and uro/+ plants. I and J, When cotyledon explants weregrown on IAA-free medium containing one-half Murashige and Skoogfor 7 d, the adventitious roots were seen only in the uro cotyledon (J),not in the wild-type cotyledon (I). Bars = 2 mm (A–G), 1 mm (H), and0.5 mm (I–J).

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the identification of the URO gene, which encodes aSUP-like zinc-finger protein. Overexpression of theURO gene results in the abnormal uro phenotypes. Wealso show that the uro auxin-related phenotypes arelikely to be due to the disruption of auxin homeostasis.In answer to our first question, we have providedseveral lines of evidence to prove that the geneAt3g23140 is URO. Transgenic plants harboring anAt3g23140-containing genomic fragment phenocopiedthe uromutant, and RNA interference of theAt3g23140gene in the uromutant resulted in the rescue of the urophenotypes. In addition, transgenic plants containingp35S:At3g23140 and pNOS:At3g23140 both partiallymimicked uro. In answer to our second question, wedemonstrated altered IAA homeostasis in uro by mea-suring free, ester-, and amino-linked IAA levels. Wealso showed indications for elevated IAA levels by aroot regeneration experiment using uro cotyledon ex-plants and increased GUS staining in uro lines con-taining the auxin-responsive pDR5::GUS reporter.To understand the endogenous function of URO in

plant development, we have searched several Arabi-dopsis databases for a loss-of-function uromutant, buthave not yet found one. The p35S:dsURO/uro con-struct restored the uro phenotypes, but p35S:dsURO/Ler transgenic plants did not have visible phenotypicchanges. In addition, the uro mutant has multipleabnormal phenotypes in different plant tissues andorgans, whereas the URO transcripts in wild-typeplants could be detected only in the inflorescence.These suggest that theURO endogenous function maynot correspond directly to the phenotype of the uromutant. However, our data suggest that a regulatorysystem(s) for the IAA homeostasis control exists in

plant, which respond to expression changes ofURO orURO-like genes. The Arabidopsis genome has 28members in this URO/SUP gene family, and it ispossible that URO function is highly redundant.Arabidopsis and Physcomitrella both respond to UROoverexpression by increasing IAA response, stronglysuggesting the existence of an evolutionarily con-served molecular mechanism in which IAA homeo-stasis is affected by changes in the expression ofcertain regulatory genes. We noted that althoughboth Arabidopsis and Physcomitrella respond to UROoverexpression, phenotypic changes in the p35S:URO/pGH3:GUS and pNOS:URO/pGH3:GUS transgenicPhyscomitrella lines were not observed. It is possiblethat different plant species may differ in sensitivity tothe free IAA concentrations, which would causechanges in plant phenotypes.

Different Possibilities for Auxin Homeostatic Control

It has been proposed that one of the major functionsof IAA conjugates is to act as a storage form of free IAA(Woodward and Bartel, 2005; Bajguz and Piotrowska,2009). Plants can keep free IAA concentrations at acertain level, to meet their developmental require-ments or to respond to their environment. This isaccomplished, at least in one respect, as one importantpathway by interconversion of free IAA and IAAconjugates. Levels of free IAA and its conjugates arecontrolled precisely in plants, with different specieshaving distinct IAA conjugate profiles (Woodwardand Bartel, 2005; Bajguz and Piotrowska, 2009). Thebalance between free and bound IAA could also be auseful, highly efficient way to regulate local free IAA

Figure 5. Overexpression of the URO gene inmoss Physcomitrella promoted the pGH3:GUSexpression. A and D, Two-month-old Physcomi-trella gametophores were analyzed. The mother-line carrying pGH3:GUS showed weak GUSsignals (A and D). B, C, E, and F, The motherlinecarrying either p35S:URO (B and E) or pNOS:GUS (C and F) resulted in strong GUS accumu-lations in gametophores. D to F are the magnifi-cation of the plants that were pointed by a blackarrow in A, B, and C, respectively. Bar = 2 mm(A–C) and 0.5 mm (D–F).



concentrations. When needed, overaccumulated freeIAA could result in an increased level of IAA conju-gates. For example, the sur1 and sur2 mutants containincreased levels of both free and conjugated IAA(Boerjan et al., 1995). In both sur1 and sur2 cases,although the concentrations of free IAA and IAAconjugates were increased, the balance between freeIAA and IAA conjugates was not obviously changed.The situation in sur1 and sur2 plants is quite differentfrom that in uro plants. Although the level of free IAAis increased in the uro mutant, which is similar to sur1and sur2, the level of the total IAA pool in uro doesnot seem to be markedly changed. In addition, re-markable changes occurred between different typesof IAA: Increased levels of free and ester-conjugatedIAA were apparently accompanied by a decrease inamide-conjugated IAA.

Enzymes that regulate homeostasis between freeIAA and IAA conjugates have recently been identified.These include the IAA amidohydrolases, ILR1, ILL1,ILL2, and IAR3 (Bartel and Fink, 1995; Davies et al.,1999; LeClere et al., 2002; Staswick et al., 2005); severalGH3 family gene-encoded IAA amino acid conjugatesynthases (Tanaka et al., 2002; Staswick et al., 2005; Jainet al., 2006; Hakkinen et al., 2007; Park et al., 2007;Ludwig-Muller et al., 2009; Zhang et al., 2009); and anArabidopsis enzyme that catalyzes a reaction to formIAA-Glc (Rosamond et al., 2002). In contrast to theseenzyme-encoding genes, URO encodes a putativetranscription factor. To understand whether UROoverexpression could cause changes in the expressionof these enzyme-encoding genes, we previously per-formed a genome-wide analysis of altered gene expres-sion in uro seedlings using the Affymetrix GeneChip,which contains approximately 24,000 Arabidopsis genes(Yuan et al., 2007). Among the many genes withchanges in expression, the YUCCA1 (At4g32540) tran-script level was elevated by a Log2 ratio of 2.2. Al-though it is not clear whether these genes are director indirect targets of URO, it is possible that UROoverexpression could affect transcript levels of genesencoding important enzymes for IAA control, result-ing in the alteration of auxin homeostasis.

Phenotypic Differences between uro and Other Mutantswith Increased Free IAA Levels

Although uro and other mutants such as sur1, sur2,and yucca-D all contain increased free IAA levels thanwild-type plants, their phenotypes vary. For example,sur1, sur2, and yucca-D have epinastic cotyledons,whereas cotyledons of uro are normal. However, therosette leaves of uro seedling and cotyledon of p35S:URO transgenic plants grow vertically similar to thecotyledon petiole of YUCCA6-OX8 (YUCCA6 over-expression transgenic plant; Kim et al., 2007). Cotyle-dons of both p35S:URO/pDR5:GUS/W (SupplementalFig. S4) and dark-grown yucca-D/pDR5:GUS (Zhaoet al., 2001) seedlings exhibit extremely strong GUSstaining, suggesting that the upright cotyledon phe-

notype may be closely related to the highly accumu-lated free IAA levels in the cotyledons.

Interestingly, although uro and yucca-D plants con-tain high free IAA levels, they have opposing apicaldominance phenotypes. In yucca-D plants, apical dom-inance is increased, with an apparently reduced num-ber of lateral branches (Zhao et al., 2001; Kim et al.,2007). However, the uromutant displays a bushy plantstature, indicative of a loss of apical dominance (Guoet al., 2004). The stems of uro, but not yucca-D, showstrong GUS staining in pDR5:GUS plants, reflecting ahigh level of free IAA. This may indicate a reducedefficiency in the basipetal IAA transport, which isgenerally thought to be necessary for the establish-ment of apical dominance (Prusinkiewicz et al., 2009;Shimizu-Sato et al., 2009).

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) mutant uro, which is in the Ler

genetic background, was generated previously from a T-DNA mutagenesis

experiment (Sun et al., 2000). The medium for plant growth contained one-half

Murashige and Skoog salt and 0.8% agar. Seeds on media were kept in 4�C for

3 d and then moved to a growth chamber at 22�C. For growing plants in soil,

seeds were sowed in a 6:1 mixture of vermiculite and dark soil containing

plant nutrition solution (Estelle and Somerville, 1987). The seeds were

incubated in 4�C for 24 h and then moved to greenhouse at 20�C to 24�C.All plants were grown under continuous fluorescent illumination.

RT-PCR

PCR after RT of RNA and real-time RT-PCR analyses were performed

according to our previous method (Xu et al., 2003; Fu et al., 2007). The gene-

specific primers were used in the RT-PCR analysis: 5#-GGTACCATGAAC-

CACCGGGACAAAC-3# and 5#-GAATTCTTAATGATGACGATGACCGC-3#for URO; and 5#-TGGCATCA(T/C)ACTTTCTACAA-3# and 5#-CCACCACT

(G/A/T)AGCACAATGTT-3# for ACTIN.

Constructs for Plant Transformation

For each construct used in plant transformation, DNA fragments were first

PCR amplified and verified by sequencing. The primers are listed as follows:

5#-GTTTATTTCGGCGTGTAGGACATG-3# and 5#-GCATATTTTGTGAAGC-

TAGTTCGG-3# for IAAH-At3g23140, 5#-ATTGATAAAACAATTTAGCCC-3#and 5#-GCATATTTTGTGAAGCTAGTTCGG-3# for pAt3g23140-At3g23140,

5#-GTTTATTTCGGCGTGTAGGACATG-3# and 5#-GTGTGTGAACGGACA-

CTAATTAG-3# for IAAH, 5#-GTGTGTGAACGGACACTAATTAG-3# and

5#-CCGCTGCAAGCTTAATG-3# for p35S:At3g23140 and pNOS:At3g23140,

and 5#-GTTCTTGTTGTCTGAAGTTGGGT-3# and 5#-CTTGGAAGAATC-

CATGGAGTAG-3# for p35S:At3g23145. Vectors pCAMBIA1301 (for IAAH-

At3g23140 or IAAH, pAt3g23140-At3g23140, and pNOS:At3g23140) and

pMON530 (for p35S:At3g23140 or p35S:At3g23145) were used in plant trans-

formation. For 35S:dsAt3g23140, the amplified At3g23140 fragment was first

subcloned into pFGC5941. The resulting construct was then subcloned into

pFGC1008 for plant transformation.

IAA Determination

Extraction and measurement of endogenous IAA through gas chromatog-

raphy-mass spectrometry (GC-MS) were performed according to a recently

reported method (Ludwig-Muller et al., 2009) with slight modification, using

aboveground parts of 12-d-old seedlings (grown on medium), or the second

pair of rosette leaves from 4-week-old plants (grown in vermiculite). Briefly,

Sun et al.


approximately 100 mg fresh weight per sample was extracted with iso-

propanol:acetic acid (95:5, v/v) and 100 ng 13C6-IAA (Cambridge Isotope

Laboratories) per sample was added as internal standard. For each sample

three independent extractions were performed. The samples were incubated

under continuous shaking (500 rpm) for 2 h at 4�C. The samples were then

centrifuged for 10 min at 10,000g, the supernatant removed and the isopro-

panol evaporated under a stream of N2. The samples were diluted 1:5 with

water and further evaporated with N2 until no acetic acid was present. For the

determination of free IAA the aqueous residue was brought to pH 3.5 with 1 N

HCl and extracted twice with equal volumes of ethyl acetate. The ethyl acetate

phases were combined, centrifuged again for 10 min at 10,000g, the superna-

tant removed and placed in a glass vial. The ethyl acetate was evaporated

under a stream of N2 and the samples were suspended in 50 mL ethyl acetate.

Methylation of samples was carried out by adding 950 mL freshly prepared

diazomethane (Cohen, 1984). For GC-MS analysis the methylated samples

were suspended in 30 mL ethyl acetate. Ester conjugates were hydrolyzed with

1 N NaOH for 1 h at room temperature and amide conjugates with 7 N NaOH

for 3 h at 100�C under N2. The latter samples were cooled on ice. The

hydrolysate was filtered, the pH brought to 2.5, and the auxins were extracted

twice with equal volumes of ethyl acetate and methylated as described for free

IAA. GC-MS analysis was carried out on a Varian Saturn 2100 ion-trap mass

spectrometer using electron impact ionization at 70 eV, connected to a

Varian CP-3900 gas chromatograph equipped with a CP-8400 autosampler

(Varian). For the analysis 1 mL of the methylated sample was injected in the

splitless mode (splitter opening 1:100 after 1 min) onto a Phenomenex ZB-5

column, 30 m3 0.25 mm3 0.25 mm (Phenomenex), using helium carrier gas

at 1 mL min21. Injector temperature was 250�C and the temperature

program was 70�C for 1 min, followed by an increase of 20�C min21 to

280�C, then 5 min isothermically at 280�C. For higher sensitivity, the mSIS

mode (Wells and Huston, 1995) was used. The endogenous concentrations

of IAA were calculated according to the principles of isotope dilution

(Cohen et al., 1986) monitoring the quinolinium ions at mass-to-charge ratio

130/136 (ions deriving from endogenous and 13C6-IAA, respectively).

Conjugated IAA was calculated by subtraction of the amount of free IAA

from the amount obtained after hydrolysis with 7 M NaOH. Ester conjugates

were calculated likewise by subtracting free IAA levels and amide conju-

gates were obtained after subtracting the ester-bound fraction from total

conjugates.

Physcomitrella Culture and Transformation

Physcomitrella patens plants were cultured in liquid Knop medium or on

solid Knop plates under a 16-h-light/8-h-dark regime (Bierfreund et al., 2003).

Liquid cultures were mechanically disrupted every week to maintain the

plants in the protonema stage. Gametophore development was induced by

transferring protonema to the solid Knop medium. The Arabidopsis URO

gene under the control of the NOS or 35S promoter was subcloned into a

vector containing the hygromycin B phosphotransferase (hpt) selective marker.

The p35S:URO-HPT or pNOS:URO-HPT fragment was then introduced into

Physcomitrella carrying pGH3::GUS (Bierfreund et al., 2003) by a polyethylene

glycol-mediated transformation method (Strepp et al., 1998). The transgenic

Physcomitrella moss plants were verified by RT-PCR using URO-specific

sequences as primers.

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

libraries under accession numbers ACB88838 (URO), BAD07404 (LIF),

NP_187540 (TAC1), AAY17042 (RAMOSA1), NP_568161 (RBE), CAB78784

(SAZ), AAY78753 (SUP), and ACU12847 (DST).

Supplemental Data

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

Supplemental Figure S1. Levels of the URO gene in different transgenic

plants.

Supplemental Figure S2. URO expression in different tissues of wild-type

plants.

Supplemental Figure S3. uro mutant has reduced number of lateral roots

during early stage growing.

Supplemental Figure S4. The p35S:URO/pDR5:GUS/W transgenic plants

showed strong GUS staining in cotyledons and apex.

ACKNOWLEDGMENTS

We thank Tom J. Guilfoyle for providing pDR5:GUS seeds and Jian Xu for

useful discussion.

Received February 1, 2010; accepted May 11, 2010; published May 13, 2010.

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