Natural Genetic Variation in is Identifies BREVIS RADIX, A Novel Regulator of Cell Proliferation and Elongation in the Root

8/2/2019 Natural Genetic Variation in is Identifies BREVIS RADIX, A Novel Regulator of Cell Proliferation and Elongation in the

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10.1101/gad.1187704Access the most recent version at doi:2004 18: 700-714Genes Dev.

Cline F. Mouchel, Georgette C. Briggs and Christian S. Hardtkenovel regulator of cell proliferation and elongation in the root

, aBREVIS RADIXidentifiesArabidopsisNatural genetic variation in

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Natural genetic variation in Arabidopsisidentifies BREVIS RADIX, a novel

regulator of cell proliferationand elongation in the root

Cline F. Mouchel, Georgette C. Briggs, and Christian S. Hardtke1

Biology Department, McGill University, Montreal, Quebec H3A 1B1, Canada

Mutant analysis has been tremendously successful in deciphering the genetics of plant development. However,less is known about the molecular basis of morphological variation within species, which is caused bynaturally occurring alleles. In this study, we succeeded in isolating a novel regulator of root growth by

exploiting natural genetic variation in the model plant Arabidopsis. Quantitative trait locus analysis of across between isogenized accessions revealed that a single locus is responsible for 80% of the variance of theobserved difference in root length. This gene, named BREVIS RADIX (BRX), controls the extent of cellproliferation and elongation in the growth zone of the root tip. We isolated BRX by positional cloning. BRX isa member of a small group of highly conserved genes, the BRX gene family, which is only found inmulticellular plants. Analyses of Arabidopsis single and double mutants suggest that BRX is the only gene ofthis family with a role in root development. The BRX protein is nuclear localized and activates transcriptionin a heterologous yeast system, indicating that BRX family proteins represent a novel class of transcriptionfactors. Thus, we have identified a novel regulatory factor controlling quantitative aspects of root growth.

[Keywords: Arabidopsis; BRX; root growth; root meristem; natural variation; gene family]

Received January 20, 2004; revised version accepted February 6, 2004.

The last several years have witnessed tremendous ad-vances in the genetic analysis of plant development,thanks to the rigorous application of mutagenesis ap-proaches. However, much less is known about the mo-lecular basis for the variation observed within species.This variation is the result of natural genetic hetero-geneity, which is the result of selection pressures thatare created by environmental conditions. For sessile ter-restrial plants, adaptation to local conditions is espe-cially important and has been observed on a temporallyand geographically very small scale (Linhart and Grant1996). Such natural variation can be exploited to isolatenovel genes or alleles involved in plant physiology anddevelopment, for instance by analysis of isogenized ac-cessions of the model plant Arabidopsis thaliana(Alonso-Blanco and Koornneef 2000). This approach hasbeen successful in isolating both novel genes (Johansonet al. 2000) and novel alleles of known genes (El-DinEl-Assal et al. 2001; Maloof et al. 2001). A distinct ad-vantage of exploiting natural genetic variation is its abil-ity to detect alleles that have been subjected to selection

in the wild. This approach, in essence, counter-selectsagainst alleles that are detrimental to plant survival andcan thus complement the more common mutagenesisapproaches, which often target genes that are essentialfor the trait of interest. Here we have exploited naturalvariation in Arabidopsis to isolate a novel regulator ofroot growth.

The root system plays a pivotal role in the survival ofhigher plants. Roots provide the plant with physical sup-port as well as essential nutrients and water, which theytake up from the soil. Arabidopsis thaliana is a dicoty-ledonous plant and has a typical allorhiz root system.Initially, growth is restricted to a primary root, which isformed during embryogenesis. Later in development, theroot system expands by forming lateral roots, whichoriginate from the pericycle, an inner cell layer of theprimary root. Eventually, adventitious roots might alsobe formed at the hypocotylsroot junction. At the cellu-lar level, Arabidopsis roots have a simple organization,consisting of concentric layers of epidermis, cortex, andendodermis, surrounding the stele that contains the vas-cular tissues (Dolan et al. 1993). These tissue layers areformed through the action of a growth zone at the distaltip of the root, the apical root meristem. Within thismeristem, signals emanating from a quiescent center ofslowly dividing cells organize a region of stem cells,

1Corresponding author.E-MAIL [email protected]; FAX (514) 398-5069.Article published online ahead of print. Article and publication date areat http://www.genesdev.org/cgi/doi/10.1101/gad.1187704.

700 GENES & DEVELOPMENT 18:700714 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org

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which give rise to the cell files of the tissue layers bystereotypic divisions in a reiterative fashion (van denBerg et al. 1997; Sabatini et al. 2003). The daughter cellscontinue to divide several times in the distal me-ristematic zone before entering a zone of rapid cell elon-gation and differentiating to maturity.

Genetic analysis has provided evidence that plant hor-mone signaling pathways are fundamentally importantfor root development. An intact auxin signaling path-way, for example, is required for proper root growth(Davies 1995; Sabatini et al. 1999), a growth-promotingeffect that is mediated via signaling through anotherplant hormone, gibberellic acid (Fu and Harberd 2003). Inaddition, root patterning requires correctly localizedpeaks of auxin concentration gradients (Sabatini et al.1999) as well as the action of two transcription factors,SCARECROW (SCR) and SHORT ROOT (SHR). The lat-ter are needed for the asymmetric division of initials thatgive rise to the cortex and endodermis cell layers, as wellas for the differentiation of these tissues (Di Laurenzio et

al. 1996; Helariutta et al. 2000; Nakajima et al. 2001).Interestingly, SCR and SHR also have a fundamental rolein the maintenance of the quiescent center and, thereby,the stem cell population (Sabatini et al. 2003).

The ontogenesis of the root system is highly plasticand sensitive to changes in environmental conditions. Inparticular, the availability of rate-limiting nutrients forplant growth, such as phosphate and nitrate, results inprofound changes in root system architecture. Root sys-tems can react to localized supplies of these nutrients byadjusting their rate and direction of growth, as well astheir extent of branching and their extent of root hairformation (Zhang and Forde 1998; Malamy and Ryan2001; Linkohr et al. 2002; Lopez-Bucio et al. 2002). Theselocalized growth responses are mediated by pathwaysthat appear to be coordinated with phytohormone signal-ing, allowing for their coordination with the cell elonga-tion and proliferation events that underlie all growthphenomena (Lopez-Bucio et al. 2003).

Although environmental inputs have an important in-fluence on root system architecture, it is conceivablethat root growth is limited by inherent genetic bound-aries. Such boundaries are, for instance, set by the cel-lular mechanisms controlling cell elongation and pro-liferation (Beemster et al. 2003). For instance, cell pro-liferation is a particularly important factor in thedetermination of root growth rate, as transgenic interfer-ence with cell cycle progression has profound effects ongrowth rate and sometimes also on meristem organiza-tion of the root (Doerner et al. 1996; Cockcroft et al.2000; De Veylder et al. 2001). Furthermore, cell produc-tion is an important component of root growth rate innatural accessions of Arabidopsis (Beemster et al. 2002).To a significant degree, the effect of plant hormones onroot growth also appears to be mediated by modulationof cell cycle duration (Beemster and Baskin 2000; Stalsand Inze 2001; Werner et al. 2003). At the organ level, theoutputs of the cellular mechanisms that control the sizeof the root meristem, the rate of cell proliferation, andthe extent of cell elongation, are integrated to determine

the overall rate of growth. However, whether or to whatdegree these mechanisms are acting independently fromone another is not clear (Beemster et al. 2003).

The aim of this study was to isolate novel regulators ofquantitative aspects of root growth that are responsiblefor the intraspecific variation of root system morphology

in Arabidopsis. Therefore, we exploited natural geneticvariation rather than mutagenesis of a particular wild-type background. This strategy also avoids the isolationof alleles that affect basic properties of the root system,such as the formation of certain tissue layers or physi-ological responses to nutrient availability. We were suc-cessful in isolating a novel gene that regulates the extentof cell proliferation and elongation in the root. It repre-sents a member of a novel, plant-specific gene family andencodes a novel type of nuclear protein that appears to beinvolved in transcriptional regulation.

Results

Root growth parameters vary among isogenizedArabidopsis wild-type lines

To determine natural genetic variation of root systemmorphology, we compared 44 arbitrarily chosen Arabi-dopsis accessions in tissue culture experiments. Asample of 20 seedlings of each line was grown underconstant illumination on solid medium containing basicmacro- and micronutrients and agar. Nine days after ger-mination (dag), the length of the primary root, the num-ber of lateral roots, and the number of adventitious rootswere recorded. An overall two- to threefold variation inprimary root length and lateral root number was ob-

served between accessions. Adventitious roots were veryrare in all accessions; however, they were observed morefrequently in Umkirch-1 (Uk-1). This accession also de-veloped a significantly shorter primary root than average(Fig. 1A) and a generally more branched root system atlater stages. Because of its clearly distinct root systemphenotype, we chose to analyze this line in further de-tail.

The short primary root of Uk-1 seedlings is largelycaused by a single locus

To test whether the alleles conferring the root phenotypeof Uk-1 are of a dominant or recessive nature, we crossedUk-1 into Slavice-0 (Sav-0), an accession with an averageroot system as compared with other accessions in ourassays. In the F2 generation of our cross we noticed thatthe short primary root phenotype of Uk-1 segregated as arecessive in a ratio close to 3:1. Root development ishighly plastic, and although the average primary rootlengths of the Uk-1 and Sav-0 lines are clearly distinct(Fig. 1A), their ranges of root length in individuals over-lap. By analysis of the F3 progeny, however, it was pos-sible to unequivocally determine the phenotype of theparental F2 plants, confirming the suspected 3:1 ratio.Thus, the short-root phenotype of Uk-1 appears to be

BRX in Arabidopsis root growth

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largely caused by a single locus, which we namedBREVIS RADIX (BRX), latin for short root.

Starting from the F2 progeny of two different F1 plants,we also established a recombinant inbred line populationof 206 lines by repeated selfing for six generations. Theprimary root length of these lines was measured, andeach line was genotyped for a set of simple sequencelength polymorphism markers spread over the Arabidop-sis genome (Table 1). The data were then subjected toquantitative trait locus (QTL) analysis. The results indi-cate that a major QTL for primary root length is located

on the upper arm of chromosome I and identical withBRX (see Fig. 5, below).

The Uk-1 short-root phenotype does not dependon shoot-derived signals

Morphological differences between accessions were ob-served not only in the root system but also in the shootsystem. Because it has been shown that communicationbetween shoot and root tissues can significantly influ-ence each others growth rate and branching pattern

Figure 1. Natural variation in root system morphology among Arabidopsis accessions. (A) Primary root length of Arabidopsis

seedlings at 9 dag, grown in 8 h dark16 h light cycle on 0.5 MS medium. n 10. (B) Representative seedlings of the Uk-1 and Sav-0accessions, and a seedling resulting from introgression of the Uk-1 short-root phenotype into an Sav-0 background ( brxS), 9 dag grownin constant light on 0.5 MS medium containing 0.3% sucrose. Bar, 1 cm. (C) Primary root length of plants of the three genotypesgrown in constant light on 0.5 MS medium containing 1.0% sucrose, 21 dag. n = 6. (D, top) Representative rosette phenotypes of thethree genotypes at 24 dag, grown on soil under constant illumination. ( Bottom) Root system belonging to the shoots shown in the toppanel, dug out from the soil and cleaned. Bar, 1 cm. ( E) Approximate primary root length of plants of the three genotypes grown on soilunder constant illumination, 24 dag; n 7. (F) Transverse cryosection through the mature part of a primary root of a 7-day-old Uk-1seedling. (ep) epidermis; (co) cortex; (en) endodermis. (G) Relative response of Col, Sav-0, Uk-1, and brxS seedlings to differentexogenous plant hormone applications, 6 dag. Seedlings were grown in constant light on 0.5 MS medium containing 2.0% sucroseplus indicated hormone supplement. (IAA) indole acetic acid; (NAA) naphtalene acetic acid; (GA) gibberellic acid; (BA) benzylamino-purine. Error bars are standard error.

Mouchel et al.

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(Turnbull et al. 2002; Sorefan et al. 2003), we wanted todetermine whether the Uk-1 root phenotype is autono-mous from shoot-derived signals. To this end, we intro-gressed the short primary root phenotype into a Sav-0background, whose shoot morphology is very differentfrom Uk-1. Sav-0 plants flower early, approximately afterthe sixth true leaf (under constant illumination), andform multiple shoots. By contrast, in the same condi-tions Uk-1 plants flower late (approximately after the24th true leaf) and form a single shoot. From a sample ofthe F2 generation resulting from our Uk-1 Sav-0 cross,we selected the seedling with the shortest primary root.This plant was then back-crossed into the parental Sav-0

line, a scheme that was in total repeated four times.From this introgression we derived plants whose genomeconsists of 97% of Sav-0 DNA and only 3% of Uk-1DNA. In the following we refer to individuals with ashort-root phenotype that have been derived from thisintrogression into an Sav-0 background as brxS.

The roots of brxS seedlings are as short as those ofUk-1 seedlings, both when grown in the light (Fig. 1B) orin darkness (data not shown). In the adult root system ofbrxS plants, the primary root is slightly longer and theroot system is less branched than in Uk-1. This is truefor root systems grown in tissue culture (Fig. 1C) as wellas for soil-grown roots (Fig. 1D,E). In contrast to the root

Table 1. Analysis of a recombinant inbred line (RIL) population derived from a cross between the Sav-0 and Uk-1 accessions

Columns indicate the line number, average primary root length determined from a sample of 1620 seedlings in the S6 generation, andthe genotype at simple sequence length polymorphism markers distributed throughout the Arabidopsis genome. Genotypes: (A) Uk-1allele; (B) Sav-0 allele; (H) heterozygous; (U) unknown.



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system, the shoot system morphology and floweringtime of brxS plants resembles the Sav-0 shoot system(Fig. 1D). Moreover, grafts between Sav-0 shoots andUk-1 roots, and vice versa, do not influence the respec-tive root system morphologies (data not shown). There-fore, the short-root phenotype conferred by the Uk-1 al-

lele of the BRXlocus is independent from shoot-derivedsignals.

Physiological responses of the root system are intactin brxS plants

Because the influence of patterning genes, plant hor-mones, and environmental stimuli on root growth arewell documented, we checked whether brxS plants areimpaired in any of the corresponding pathways. Trans-verse sections of Uk-1 roots indicate that the cortexand endodermis cell layers are present (Fig. 1F), rulingout defects in the SCR or SHR genes. In addition, brxS

seedlings respond to exogenous application of plant hor-

mones, such as auxins, gibberellins, or cytokinins, inroughly the same proportional range as the parentalSav-0 line (e.g., Fig. 1G). Notably, the application ofplant hormones was in no instance able to rescue theshort-root phenotype (Fig. 1G), even when very low con-centrations were applied (data not shown). Finally, wealso tested the response of brxS seedlings to differentnutrient conditions, as nutrient availability has beendemonstrated to affect root system architecture (Lopez-Bucio et al. 2003). However, we did not observe any ap-parent defects in the numerous assays that we con-ducted, including examination of the responses to low orhigh nitrate or phosphate levels or to different ratios ofnitrogen to carbon source. Again, brxS seedlings re-sponded in proportional ranges similar to those of theparental Sav-0 line (data not shown). In summary, theshort-root phenotype of brxS plants is not the result of amajor defect in basic hormone or physiological responsepathways.

brxS seedlings have shorter and fewer root cells

To characterize the brxS phenotype in further detail, weanalyzed the primary roots of brxS seedlings at the cel-lular level. In principal, the brxS short-root phenotypecould be caused by one of two phenomena: either shortercells or fewer cells. To distinguish between these twopossibilities, we microscopically analyzed mature epi-dermal cell files (i.e., the root hair-bearing region distalto the meristem). Analysis of the size and number ofepidermal cells revealed that brxS roots are composed ofshorter (Fig. 2A) as well as fewer (Fig. 2B) cells. Theseparameters remained relatively constant throughout theperiod of observation (38 dag). In Sav-0, the productionrate of mature epidermal cells was 1924 cells per day,and their average length was 110117 m, whereas inbrxS, 1113 cells per day with a length of 7687 m wereproduced. Because the root growth rate in both geno-types remained roughly the same up to 21 dag, it is rea-sonable to assume that these parameters did not change

throughout development. In line with the observationsin epidermal cell files, confocal microscopy revealed thatthe more evenly sized cortical cells are also shorter inbrxS roots (Fig. 2C,D). In summary, both cell elongation

and cell production rate are decreased in brxS

seedlings,contributing approximately one-third and two-thirds, re-spectively, to the overall difference in root length ascompared with Sav-0 seedlings.

The BRX locus affects cell proliferation in the apicalroot meristem

To visualize the meristematic region of the root, wecrossed a transgenic reporter of cell proliferation, a fu-sion protein between cyclin B1;1 (CYCB1;1) and -gluc-uronidase (GUS) expressed under control of the CYCB1;1promoter (de Almeida Engler et al. 1999), into the brxS

and Sav-0 lines. GUS staining of roots of these seedlingsrevealed that the root meristems of brxS seedlings aresmaller than Sav-0 meristems (Fig. 3A). When investi-gated by confocal microscopy, the organization of brxS

root meristems appears normal (Fig. 3B). However, com-pared with Sav-0 meristems, cells in the meristematiczone in brxS appear to increase in size earlier, and thenumber of cells undergoing division appears to be re-duced (Fig. 3C). This phenotype (shown for 0.5% sucroseconcentration in Fig. 3B,C) becomes more pronounced ingrowth-promoting conditions. In our physiological as-says we noticed that the difference in root length be-tween Sav-0 and brxS seedlings increased when root

Figure 2. Mature cell size and number in the primary roots ofSav-0 and brxS seedlings. (A) Mature epidermal cell length at 3,6, and 8 dag. For each genotype, three seedlings were measured

per time point. The number of cells measured in each seedlingwas 13 at 3 dag, 46 at 6 dag, and 34 at 8 dag. (B) Number ofmature epidermal cells in a cell file of the root at 3, 6, and 8 dag.For each genotype, three seedlings were counted per time point.(C) Mature cortical cell length at 6 dag. For each genotype, threeseedlings were measured. The number of cells measured was50. (D) Confocal microscopy images of the mature region ofSav-0 and brxS roots. Asterisks mark cortical cells. (e) epider-mis; (c) cortex. Error bars are standard error.

Mouchel et al.


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growth rate was stimulated by increasing the amount ofsucrose in the medium (Fig. 3D; Benfey et al. 1993). Thiscorrelates with a further size reduction of brxS me-ristems at a higher growth rate (e.g., 2% sucrose; Fig. 3E).In these conditions, they are composed of fewer cellsthat are less organized and not as isodiametric (Fig. 3F).

To quantify our observations, we measured the size ofthe meristematic and elongation zones of Sav-0 and brxS

seedlings that were grown on 2% sucrose at 6 dag byanalyzing cell files. We took the number of cortical cells,counted from the initial cell up to the first rapidly elon-gating cell, as an indicator of root meristem size(Casamitjana-Martinez et al. 2003). By this measure,brxS root meristems consist of 25% of the number ofcells in Sav-0 meristems (Fig. 3G). We also took the num-ber of cortical cells, counted from the first rapidly elon-gating cell up to the first cell of mature size, as an indi-cator of elongation zone size. By this measure, brxS elon-gation zones consist of 40% of the number of cells inSav-0 elongation zones (Fig. 3G). Therefore, the ratio be-tween the number of cells in the meristematic zone andthe number of cells in the elongation zone is shifted toclose to 1.0 in brxS from 1.7 in Sav-0. Thus, the size ofboth the meristematic and elongation zones of the roottips of brxS seedlings are decreased, but the meristematiczone is affected more severely.

Isolation of the BRX gene by positional cloning

To identify the BRXgene at the molecular level, we fol-lowed a positional cloning approach. To this end,genomic DNA was isolated from 860 individuals of theF2 population from the Uk-1 Sav-0 cross and geno-typed with molecular markers that showed polymor-phism between the two accessions. The root phenotypeof the F2 plants was unequivocally scored by analysisof the F3 progeny. Recombination mapping placed theBRX locus on the upper arm of chromosome I. Subse-quently, novel markers were generated from PCR-ampli-fied DNA fragments arbitrarily chosen from the Arabi-dopsis genome sequence. This strategy allowed us to lo-cate the BRX gene in a zero-recombination interval of45 kb, flanked by proximal and distal markers indicat-ing three and one recombination events, respectively(Fig. 4A).

Crosses of Uk-1 with the Arabidopsis reference acces-sion Columbia (Col) result in segregation of a recessiveshort-root phenotype as well. Thus, we tested five of the10 candidate genes in the 45-kb interval by analyzingrespective T-DNA insertion mutants in Col backgroundthat were available (Alonso et al. 2003). A short-root phe-notype was not observed in any of these mutants (Fig.4B). We also analyzed 8 of the 10 BRXcandidate genes by

Figure 3. Root meristem morphology and size in the primary roots of Sav-0 and brxS seedlings. (A) Activity of aCYCB1;1CYCB1;1:GUS reporter gene in the meristems of brxS and Sav-0 seedlings, detected by GUS staining. Brackets indicate themeristematic region as defined by the GUS signal. (B) Confocal images of root meristems grown on 0.5 MS medium containing 0.5%sucrose. (C) Magnification of cortical cell files (marked by white dots), starting from the initial cell, shown in B. (D) Response of rootgrowth of Sav-0 and brxS seedlings to increasing amounts of sucrose (given in percentages) in the medium, scored 7 dag. n 8. (E)Confocal images of root meristems grown on 0.5 MS medium containing 2.0% sucrose. (F) Magnification of cortical cell files (markedby white dots), starting from the initial cell, shown in E. (G) Number of cells in cortical cell files of the root meristematic andelongation zones as defined in the text, grown on 0.5 MS medium containing 2.0% sucrose and scored 6 dag. n 10. Error bars arestandard error.



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Figure 4. Positional cloning of the BRXgene. (A) Schematic representation of recombination mapping of the BRXlocus to an 45-kbinterval on chromosome I of Arabidopsis. Solid bars indicate predicted genes, numbers indicate their unicode. (B) Summary of thegenetic and sequence analysis of the genes in the region of interest. (n.a.) Not available; (n.d.) not determined. (C) Schematic presen-tation of the intronexon structure of the BRX gene. Boxes represent exons, lines represent introns, and their sizes are given innucleotides below. The shaded boxes indicate the open reading frame. The position of the mutation resulting in a premature stopcodon in the Uk-1 accession is shown. ( D) Representative Uk-1, Sav-0, and brxS seedlings and brxS seedlings carrying a 35SBRXtransgene, 9 dag grown in constant light on 0.5 MS medium containing 0.3% sucrose. ( E) Number of cells in cortical cell files of theroot meristematic and elongation zones as defined in the text, grown on 0.5 MS medium containing 2.0% sucrose and scored 6 dag.

n 10. (F) Primary root length of seedlings grown in constant light on 0.5 MS medium containing 1.0% sucrose, 9 dag. n 15. (G)RTPCR of BRX and the control gene actin4 (ACT4) from RNA isolated from different sources. Control reactions for BRX in whichthe reverse transcriptase was lacking (BRX-RT) are shown as well. (M) DNA size marker. (H) Primary root length of seedlings grownin constant light on 0.5 MS medium containing 1.0% sucrose, 7 dag. n 15. Error bars are standard error.

Mouchel et al.


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comparing the sequence of the Uk-1 alleles with the cor-responding Col alleles. We found no Uk-1 alleles withobvious implications for gene functionality (Fig. 4B),with the exception of the gene represented by unicodeAt1g31880. This gene contains a base pair change in thefourth exon, which results in a premature stop codon in

the open reading frame and, therefore, a truncated pro-tein missing approximately two-thirds of the C terminus(Fig. 4C). This stop codon is not present in the respectivealleles of other accessions with long primary roots (de-termined for accessions Sav-0, Wassilewskaja, Landsbergerecta, Freiburg-1, Eilenburg-0, Loch Ness-0, Chisdra-0,Goettingen-0, and Kindalville-0). Moreover, the stopcodon is also missing from the sequence of the acces-sions Uk-2, Uk-3, and Uk-4, whose BRX alleles arenearly identical to the Col allele apart from very fewsilent polymorphisms or one conserved substitution.These three accessions have long primary roots andwere collected in the immediate vicinity of Uk-1(The Arabidopsis Information Resourse, TAIR, http://

www.arabidopsis.org). Introduction of a transgenic con-struct expressing the open reading frame of At1g31880 un-der control of the 35S cauliflower mosaic virus gene pro-moter (35S) into brxS seedlings largely rescues the short-root phenotype (Fig. 4D) and restores the meristem size toSav-0 dimensions (Fig. 4E). Finally, this is also true for atransgene expressing a BRXopen reading frame in its na-tive start codon context (i.e., including the untranslatedexons and introns up to the ATG; Fig. 4C) under controlof a 1.9-kb fragment of the BRX promoter (data notshown). Thus, the combined evidence demonstrates thatAt1g31880 and BRXare identical.

BRX is expressed in the root at very low levels

From the brxS phenotype it can be expected that BRX isexpressed in the root. To determine whether this is the

case, we analyzed whole seedlings, shoots, and roots byRTPCR. In these experiments, BRX expression can bedetected in all three samples (Fig. 4G). To visualize BRXexpression at spatiotemporal resolution, we also con-structed transgenic plants expressing the green fluores-cent protein (GFP) or a fusion protein of BRX and GFP

under control of the BRX promoter (constructsBRXGFP and BRXBRX:GFP, respectively). Impor-tantly, the BRXBRX:GFP transgene rescues the brxS

root phenotype, demonstrating expression and function-ality of the BRX:GFP fusion protein (Fig. 4H). However,in (confocal) fluorescence microscopy, neither theBRX:GFP fusion protein nor native GFP could be de-tected. In line with these observations, Western analysisof the transgenic lines using an anti-GFP antibody yieldsa very faint signal, and only does so if an excess amountof protein extract is loaded, whereas GFP produced in a35SGFP line is readily detectable in very little extract(data not shown). Therefore, in summary our results in-dicate that BRXis expressed in the shoot and root, albeit

at very low levels.

BRX explains most of the variance in primary rootlength between Uk-1 and Sav-0

We observed rescue of the short-root phenotype of brxS

seedlings in several transgenic lines derived from inde-pendent primary transformants. However, we noticedthat rescue was not complete in any of these lines (e.g.,Fig. 4F,H). This finding is consistent with the idea thatBRX represents the major QTL for primary root lengthpredicted on chromosome I from regression analysis ofour recombinant inbred line population. The creation of

a BseGI restriction enzyme polymorphism by the basepair change in the Uk-1 allele of BRX allowed us to di-rectly score the BRXgenotype in the recombinant inbred

Figure 5. Quantitative trait locus (QTL) analysis of the RIL population. (A) Results from the regression analysis of the data presentedin Table 1 plus the genotypes at the BRX(At1g31880) locus, with respect to primary root length. (B) Graphical presentation of the datashown in A. The different chromosomes and the relative position of the scored simple sequence length polymorphism markers areindicated, along with the likelihood statistics for the positions of QTLs.



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lines and to include this information in the regressionanalysis. The results indicate that the BRX locus ex-plains 80% of the observed variance in primary rootlength in the population (Fig. 5).

BRX is a member of a novel, plant-specific gene familyAt the time of its identification, the BRX gene was notcorrectly annotated in public databases, with most of theopen reading frame predicted to be fused with the neigh-boring gene and consequently considered a novel type ofaquaporin (Johanson et al. 2001). However, the annota-tion of a related gene, which we named BRX-like 1(BRXL1; unicode At2g35600), enabled us to determinethe correct intronexon structure of BRXby comparison,including two noncoding exons representing 5 untrans-lated regions (Fig. 4C). Based on the gene structure ofBRX and BRXL1, we were able to identify and annotatethree more genes of this type in the Arabidopsis ge-nome, BRXL2, BRXL3, and BRXL4 (unicode or fusion ofparts of unicodes At3g14000, At1g54180At1g54190,

and At5g20530At5g20540, respectively). Subsequently,full-length cDNA clones became available for four out ofthe five genes and confirmed the predicted gene models.The BRX family genes and the proteins they encode arehighly conserved (Fig. 6A) and are found in all higherplants for which data are available, but are absent from

unicellular organisms or animals. Therefore, this genefamily appears to be specific to multicellular plants.

To test whether other BRX-like genes act partially re-dundant with BRX in root growth, we obtained pre-sumed null mutants for BRXL1, BRXL2, and BRXL3from the SALK T-DNA insertion mutagenesis project(Alonso et al. 2003). Insertions in the BRXL4 gene couldnot be confirmed. Interestingly, none of these mutantsdisplay a brxroot phenotype. However, partial and asym-metric redundancy has been observed in other cases andmight only become apparent in a brx mutant back-ground. Thus, we created double mutants between theUk-1 brx allele, twice introgressed into a Col back-ground (we refer to these plants as brxC), and the oth-

er brxl mutants. In our analysis, we focused on the

Figure 6. The BRXfamily of genes. (A) Sequence alignment of the predicted sequences of BRX family of proteins. Asterisks indicateidentity, two dots indicate conserved substitutions, and one dot indicates substitutions with similar basic characteristics. A highlyconserved domain, occurring twice in each protein, is highlighted. (B) Unrooted phylogenetic tree based on the amino acid sequencesshown in A. (C) Analysis of brxC;brxl1 double mutants. Representative seedlings of the indicated genotypes, grown in constant lighton 0.5 MS medium containing 1.0% sucrose, are shown at 8 dag. ( D) Primary root length of seedlings grown in constant light on 0.5MS medium containing 1.0% sucrose, scored 8 dag. n 9. Error bars are standard error.

Mouchel et al.


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brxC;brxl1 double mutant, because of the high similarityof BRXL1 to BRXboth in gene structure (only these twoBRX-like genes possess the untranslated exons) andamino acid sequence (Fig. 6A,B). In this double mutant,we did not observe any abnormalities in the root systemthat would indicate an enhancement of the brxC pheno-

type (Fig. 6C,D). Similar results were obtained for thebrxC;brxl2 and brxC;brxl3 double mutants (data notshown). Therefore, BRX likely is the only gene in thisfamily with a role in root development.

The BRX protein is nuclear localized and can activatetranscription in yeast

The BRX protein does not contain any previously char-acterized motifs that would indicate its biochemicalfunction. However, sequence alignment of the BRX fam-ily proteins reveals that all five of them contain threehighly conserved domains (Fig. 6A). One domain is lo-cated at the N terminus, between amino acids 28 and 45

of BRX, whereas two more domains that are highly simi-lar to each other are located between amino acids 169and 182 and between 320 and 334, respectively. Interest-ingly, in secondary structure predictions, these three do-mains all have a high probability of forming -helicalsecondary structures (Fig. 7A).-Helices are characteristic for transcription factor

proteins and are often found in DNA binding and proteininteraction domains (Luscombe et al. 2000). Transcrip-tion factors are nuclear proteins, and therefore we testedwhether BRX accumulates in the nucleus. To this end, afusion between GFP and BRX was transiently expressedin epidermal onion cells and its subcellular localizationwas monitored by fluorescence microscopy. In this as-

say, the GFP:BRX fusion protein is found primarily inthe nucleus (Fig. 7B), unlike GFP by itself, indicatingthat BRX is actively transported into the nucleus.

We also tested whether the BRX protein can activatetranscription in a heterologous yeast system. To thisend, we cloned the BRX open reading frame into a yeast

expression vector, in frame with the lexA DNA bindingdomain of Escherichia coli. Expression of this fusion pro-tein in the presence of a -galactosidase reporter genecontrolled by lexA promoter binding sites results instrong reporter activity (Fig. 7C). This is not the case if acontrol fusion protein between the Arabidopsis tran-scription factor HY5, which lacks transactivation poten-tial (Ang et al. 1998), and lexA is expressed instead. Thetransactivation potential is largely reduced in a trun-cated BRX protein comprising the 100 N-terminal aminoacids. Thus, the data indicate that BRX contains a tran-scription activation domain.

Discussion

Natural genetic variation in root system morphologyofArabidopsis

The goal of our study was to isolate novel regulators ofroot growth that are responsible for the intraspecificvariation in root system morphology. Such genes shouldnot be essential for root development per se, based on theassumption that alleles that are selected in the wild arenot detrimental to basic plant development and that evo-lution preferentially acts on genes controlling nonessen-tial aspects of growth. Because of the well-developed ge-netic resources and the ease of manipulation, we choseto analyze natural genetic variation in isogenized acces-sions of the model plant Arabidopsis.

Figure 7. Analysis of the BRX protein. (A) Secondary structure prediction for the BRX protein. The domains highly conserved betweenBRX-like proteins are indicated in green. (H) Regions with a high probability of forming -helical structures are indicated; (E) regionswith high probability of forming extended sheets. (B) Nuclear localization of BRX. Fluorescent microscopy of transiently transformedepidermal onion cells expressing a BRXGFP fusion protein or GFP alone. (C) Reporter gene activity in yeast expressing the indicatedlexA fusion proteins. n = 8. Error bars are standard error.



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Mutagenesis approaches in Arabidopsis have been tre-mendously successful in isolating genes involved in dif-ferent aspects of root development, such as pattern for-mation, growth rate, or cell shape (e.g., Benfey et al.1993; Hauser et al. 1995). Although the analysis of thesegenes has greatly enhanced our knowledge of root devel-

opment, less is known about the factors that specificallycontrol quantitative aspects of root biology, such as therate of growth. Although it is clear that control of cellproliferation has an important role in root growth (Beem-ster et al. 2003), to our knowledge, loss-of-function mu-tants that are specific to cell proliferation in the rootmeristem have not been isolated to date. Rather, experi-mental evidence for pathways controlling root growthhas been gathered from transgenic gain-of-function ap-proaches, which usually involve the ectopic or overex-pression of candidate genes. By these means, for ex-ample, the control of cell cycle progression (Doerner etal. 1996; Cockcroft et al. 2000; De Veylder et al. 2001)and CLAVATA-type pathways (Casamitjana-Martinez et

al. 2003; Hobe et al. 2003) have been implicated in thecontrol of root growth or meristem size.

Notably, it has been observed that there is detectablevariation in root growth between Arabidopsis accessionsand that this is, to a significant degree, the result of dif-ferences in mature cell size or the rate of cell prolifera-tion (Beemster et al. 2002), supporting the notion thatgenetic analysis of natural variation can identify factorscontrolling these processes. Consistent with this previ-ous report, we observed an average two- to threefoldvariation in root growth parameters of Arabidopsis ac-cessions. The reduction of growth in the Uk-1 line ascompared to average was, however, remarkable. The oc-currence of this phenotype in the wild might be relatedto the fact that the Uk-1 accession has been reportedlycollected from a river embankment (TAIR, http://www.arabidopsis.org). Thus, water availability mightnot be as limiting a growth factor in the natural environ-ment of this line, and this might have permitted theevolution of a shorter root as compared with accessionsthat grow in more arid environments.

The characterization of QTLs by genetic mapping is awell-established procedure; however, isolation of a genecorresponding to a QTL of interest is still an arduoustask. Our success in isolating the BRX gene was greatlyaided by two factors. First, the effect of the Uk-1 allele ofBRX on root growth is a strong one and is, therefore,easily detectable. Second, the unmatched genomic re-sources for Arabidopsis enable fine mapping within areasonable time frame (Borevitz and Nordborg 2003).Nevertheless, it can be expected that increased availabil-ity of molecular markers and automatization of mappingprocedures will soon enable the routine isolation ofsmall effect QTLs in Arabidopsis (Borevitz et al. 2003;Schmid et al. 2003; Torjek et al. 2003).

Specificity of the brx phenotype

By introgression of the Uk-1 allele of BRXinto the Sav-0background, we have demonstrated that the brxS pheno-

type does not depend on shoot-derived signals. More-over, in our phenotypic analysis we could not detect anyabnormalities in the shoot system of brxS plants. ThusBRXactivity is specifically needed in the root. Althoughmany genes influencing root growth have been isolatedby mutagenesis approaches, such specificity is still rare.

Notably, the majority of root growth mutants isolated todate are involved in hormone signaling pathways. In gen-eral, they also display conspicuous defects outside theroot system. For instance, root growth is affected in the

gai and rga mutants, which disrupt gibberellic acid sig-naling (Fu and Harberd 2003). However, these genes alsohave a central role in the growth of stems. The issue isfurther complicated in mutants affecting the auxin sig-naling pathway, which include several gain-of-functionmutants that might occasionally represent neomorphicphenotypes (Leyser 2002). An auxin signaling gene thatappears to be required only in the root is SHY2 (Tian andReed 1999). Although shy2 gain-of-function mutantshave shoot and root phenotypes, corresponding loss-of-

function mutants only display a root growth phenotype.In addition, shy2 loss-of-function results in reduced rootgrowth only in light-grown conditions, and this reduc-tion can be rescued by exogenous application of auxin(Tian and Reed 1999). By contrast, the phenotype of brxS

seedlings is not conditional and cannot be rescued byplant hormone application. It also has to be stressed thatunder all growth conditions tested, brxS roots alwaysgrow at a rate that is two- to threefold lower than in rootsof seedlings carrying the functional Sav-0 allele. Further,the reduction in meristem size in brxS can be observedearly in development, does not change as the roots be-come older, and does not result in growth arrest. Thisdiffers significantly from other studies (Casamitjana-Martinez et al. 2003; Hobe et al. 2003), where the rootmeristem has normal size in early stages and becomesconsumed over time, eventually resulting in the shut-down of growth. In summary, compared to other rootgrowth mutants the phenotype of brxS seedlings isunique in many aspects, and BRX appears to be a verybasic factor, required for an optimal rate of root growthin any condition.

The brx phenotype: cell proliferationversus cell elongation

The slow primary root growth of brxS seedlings is theresult of a reduction in mature cell size as well as cellproliferation. The reduced cell proliferation quantita-tively contributes more to the brxS phenotype than tothe reduced cell size. It has to be noted, however, that inour introgression we always selected the seedlings withthe shortest primary root, thereby likely introducing allthe genetic factors affecting root growth in Uk-1 intobrxS seedlings. Because transgenic expression of BRX inbrxS seedlings restores mature cortical cell size to wild-type dimensions but does not rescue total root length to100%, we must assume that indeed additional, smallereffect QTLs have been introgressed and would have to be

Mouchel et al.


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complemented to fully restore the cell proliferation rateto Sav-0 levels.

The different contributions of cell proliferation andcell elongation to overall root growth have been difficultto dissect. To date, it is not clear whether these processesare controlled independently (Beemster and Baskin 1998;

De Veylder et al. 2001). This issue is also complicated bythe fact that cells still divide, although at a much lowerfrequency, in the elongation zone (Beemster et al. 2003).It is, however, conceivable that a reduction of cell pro-liferation in the meristematic region results in a de-creased supply of cells to the elongation zone, thus de-creasing its size. It also has been suggested that it is thetime a cell spends as part of the elongation zone ratherthan elongation zone size per se that determines finalmature cell length (Beemster and Baskin 1998, 2000).Because decreased cell proliferation in the meristemwould also result in slower displacement of cells fromthe elongation zone, the time they spend elongating con-sequently might not change dramatically, even if the

elongation zone is physically smaller. This explanationaccounts for the observation that interference with cellproliferation in the root meristem, resulting in reducedsize of the meristematic region, always results in a re-duction of elongation zone size, whereas mature cell sizeis usually not affected to the same degree (Beemster andBaskin 2000; De Veylder et al. 2001; Casamitjana-Mar-tinez et al. 2003; Werner et al. 2003).

Genetically, we cannot separate the roles of BRX incell proliferation and elongation. However, several argu-ments support the notion that the reduced mature cellsize might be a secondary consequence of reduced cellproliferation. In brxS seedlings, the growth zone of theroot is reduced in size. This phenotype is enhanced if cellproliferation is stimulated by increased sucrose concen-tration of the medium. Compared with Sav-0, the cellnumber in the meristematic zone is affected to a greaterextent in brxS seedlings than the cell number in the elon-gation zone. Interestingly, this phenotype shows signifi-cant similarity to root tips of seedlings in which cellproliferation has been slowed down; for instance, by cy-tokinin treatment (Beemster and Baskin 2000) or byoverexpression of inhibitors of cell cycle progression (DeVeylder et al. 2001). Finally, previous analyses suggestthat the rate of root growth is primarily controlled at thestep of cell proliferation (Beemster and Baskin 1998;Beemster et al. 2002, 2003). Thus, the primary cause ofthe brxS phenotype might be the reduction of cell prolif-eration in the root meristem.

Implications from the low expression level of BRX

Our expression analyses determined that BRX is ex-pressed in the root as well as the shoot of young seed-lings. Thus, BRX might also have a yet-unknown func-tion in the shoot, which could be masked by redundantlyacting BRX-like genes in brxS plants.

We could not detect GFP fluorescence in our reporterlines in situ. In this context, it is important to note thatwe demonstrate that the BRXBRX:GFP transgene can

substitute for native BRX. The transgenic proteins, thatis, BRX:GFP or native GFP, are also barely detectable inWestern blots, supporting our conclusion that the BRXexpression level is very low. This result is corroboratedindependently by the very rare occurrence of BRXcDNAs in public databases (two hits at time of publica-

tion) and BRX signatures in MPSS experiments (http://mpss.udel.edu/at/java.html). Finally, based on the lowexpression level of BRXand the transgenic rescue of brxS

seedlings with a 35SBRX construct, it can be con-cluded that overexpression of BRX does not stimulateroot growth beyond the rate observed in Sav-0, thereforeindicating that BRX is one of several factors that deter-mine the rate of root growth.

The BRX gene family of Arabidopsis: a novel classof transcription factors?

The BRX family proteins are remarkably well conservedin Arabidopsis (64%93% similarity at amino acid

level), indicating that most of their structure is impor-tant for their function. However, with the possible ex-ception of BRXL4, for which we could not confirm aT-DNA insertion mutant, only BRX appears to have arole in root growth, as demonstrated by the analysis ofthe single and double mutants with brxl1, brxl2, andbrxl3. This could indicate that, despite the similaritybetween these genes, there are functional differences inthe activity of the encoded proteins, or that these genesact only partially redundantly because of differential ex-pression patterns. W

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Natural Genetic Variation in is Identifies BREVIS RADIX, A Novel Regulator of Cell Proliferation and Elongation in the Root