Identification of quantitative trait loci that regulate Arabidopsis root system size

Transcription

1 Genetics: Published Articles Ahead of Print, published on September 12, 2005 as /genetics Identification of quantitative trait loci that regulate Arabidopsis root system size and plasticity Jonathan N. Fitz Gerald *,1,2, Melissa D. Lehti-Shiu *,2, Paul A. Ingram *, Karen I. Deak *, Theresa Biesiada *, and Jocelyn E. Malamy * * Molecular Genetics and Cell Biology Department, The University of Chicago, Chicago, IL Present address: Temasek Lifesciences Laboratory, National University of Singapore, Singapore, J. N. F. G. and M. D. L. contributed equally to this work. 1

2 Running head: Genetic regulation of root system size Key words: root system, drought/osmotic stress, natural variation, QTL mapping, developmental plasticity Corresponding Author: Jocelyn E. Malamy Mailing Address: Rm 209 Erman Biology Center University of Chicago 1103 E. 57 th Street Chicago, IL Phone: Fax:

3 ABSTRACT Root system size (RSS) is a complex trait that is greatly influenced by environmental cues. Hence, both intrinsic developmental pathways and environmental response pathways contribute to RSS. To assess the natural variation in both types of pathways, we examined the root systems of the closely related Arabidopsis ecotypes Landsberg erecta (Ler) and Columbia (Col) grown under mild osmotic stress conditions. We found that Ler initiates more lateral root primordia, produces lateral roots from a higher percentage of these primordia, and has an overall larger root system than Col under these conditions. Furthermore, although each of these parameters is reduced by osmotic stress in both ecotypes, Ler shows a decreased sensitivity to osmotica. To understand the genetic basis for these differences, QTL for RSS under mild osmotic stress were mapped in a Ler X Col recombinant inbred population. Two robust quantitative trait loci (QTL) were identified and confirmed in near-isogenic lines (NILs). The NILs also allowed us to define distinct physiological roles for the gene(s) at each locus. This study provides insight into the genetic and physiological complexity that determines RSS, and begins to dissect the molecular basis for naturally occurring differences in morphology and developmental plasticity in the root system. 3

4 INTRODUCTION Most development in plants occurs post-embryonically. Hence, plants have the opportunity to incorporate environmental cues into developmental decisions, allowing each plant to optimize its morphology for its unique microenvironment. This developmental plasticity is exemplified in the root system. Genetically identical plants can produce root systems with extremely divergent morphologies (i.e. size, degree of branching, distribution of lateral roots) when grown on different soil types or under varying conditions of nutrient or water availability (reviewed in LOPEZ-BUCIO et al and MALAMY 2005). Root system plasticity is not unlimited, however, and the range of possible root system morphologies is constrained by the genetic make-up of a particular plant. Therefore, we can predict that two different categories of genes will affect root system morphology: 1) genes that constrain growth and development under any condition and 2) genes that define how a plant perceives and alters its morphology in response to the specific environmental cues it encounters. We refer to these categories of genetic pathways as 1) intrinsic and 2) environmental response pathways, respectively. Plant species/ecotypes that have different root system morphologies under a given growth condition might differ in components of intrinsic or environmental response pathways, or in components of both. The root system is composed of an embryo-derived primary root and a variable number of lateral roots that continually form as the plant grows. Not much is known about the overall regulation of lateral root formation in the root system. However, the physiological events that occur during formation of a single lateral root have been extensively characterized in many plants, including Arabidopsis. Lateral roots initiate 4

5 through anticlinal divisions in a subset of root pericycle cells called founder cells". This is followed by a stereotypic pattern of divisions leading to the development of lateral root primordia (LRP) (MALAMY and BENFEY 1997a,b). Some of these LRP undergo a cell expansion process to emerge through the endodermal, cortical, and epidermal cell layers of the parent root into the soil. After LRP emerge from the primary root, cell divisions commence in the meristem of the new lateral root; meristem activation is the definitive step in the formation of a lateral root from an LRP (MALAMY and BENFEY 1997a,b). Each of the events in lateral root formation is a potential target for intrinsic and environmental-response regulatory pathways. Indeed, different environmental conditions have been shown to regulate different developmental events, and forward genetics screens have identified mutants compromised at various developmental stages. Analysis of these mutants has led to the identification of several genes that regulate lateral root formation and that coordinate lateral root formation with environmental cues (LOPEZ- BUCIO et al. 2005; DEAK and MALAMY 2005; reviewed in MALAMY 2005, MIURA et al. 2005, reviewed in CASIMIRO et al. 2003). Several groups have taken advantage of natural variation in root system morphology to map quantitative trait loci (QTL) controlling root system development in Arabidopsis (KOBAYASHI and KOYAMA 2002; RAUH et al. 2002; HOEKENGA et al. 2003; LOUDET et al. 2005), maize (reviewed in TUBEROSA et al. 2003), rice (reviewed in PRICE et al., 2002; ZHENG et al., 2003; LI et al. 2005) and other plants. QTL mapping has the advantage of identifying genomic regions containing genes with subtle effects and genes whose effects are masked in a particular background and would not be identified by traditional mutagenesis screens. In addition, exploring the polymorphisms underlying 5

6 natural variation can identify the DNA sequence changes that have effectively modified root system architecture in nature. It is now possible to identify genes underlying QTL, even if these genes have subtle effects on phenotype (TAKAHASHI et al. 2001; reviewed in PARAN and ZAMIR 2003). This is especially true in Arabidopsis where well characterized recombinant inbred lines (RILs) allow QTL that control natural variation to be mapped with precision and where the availability of a wide range of genomics tools facilitates gene identification (ALONSO-BLANCO and KOORNNEEF 2000; BOREVITZ and NORDBORG 2003; MALOOF 2003). Only one gene that underlies a root system trait QTL in any plant has been identified to date. MOUCHEL et al. (2004) used variation in primary root length between the UK-1 and Sav-0 Arabidopsis ecotypes to identify a novel transcription factor, BREVIX RADIX, that controls primary root elongation. Therefore, QTL analysis provides a valuable and under-exploited tool for exploring the genetic basis of natural variation in root system morphology and for discovery of root system regulatory genes in Arabidopsis. Root system size (RSS), a major component of root system morphology, is determined by the number and growth rate of the lateral roots that form along the primary root, and along these lateral roots in turn. The number of lateral roots is determined by the number of lateral root primordia that are initiated and the percentage of these primordia that form lateral roots. It is easy to see how variation in these parameters over the lifetime of the root system, as a result of either intrinsic or environmental signaling, could lead to tremendous variation in RSS. RSS is also affected by environmental cues. Previously, we showed that the size of the Arabidopsis thaliana var. Columbia (Col) root system is reduced by mild osmotic stress and demonstrated that the main cause of the 6

7 reduction in RSS was the repression of lateral root formation from LRP (DEAK AND MALAMY 2005). Here we show that there is natural variation in RSS among Arabidopsis ecotypes grown under mild osmotic stress and focus on the fact that Arabidopsis thaliana var. Landsberg erecta (Ler) produces a larger, more highly branched root system than Col. Our data indicate that the distinct phenotypes of the Ler and Col root systems are caused by both intrinsic differences (environment-independent) and differences in the extent of environmental response. We used RILs to identify two robust QTL, called EDG1 and EDG2 (ELICITORS OF DROUGHT GROWTH), that contribute to the natural variation in RSS under mild osmotic stress. Near isogenic lines (NILs) confirmed the predicted effect of each QTL and allowed us to define distinct physiological roles for EDG1 and EDG2 during root system development. The EDG loci have relatively subtle effects; therefore, QTL mapping has uncovered key regulators of root system morphology that might not have been identified in a standard forward genetics screen. Although previous studies have mapped QTL for various root traits in Arabidopsis and other plants, our work dissects the physiological events that contribute to natural variation in RSS and plasticity and demonstrates how the gene(s) in each QTL function in the regulation of these complex traits. MATERIALS AND METHODS Plant growth: Seeds of all ecotypes were obtained from the Arabidopsis Biological Resource Center ( For a complete list of the ecotypes evaluated in this study, see Supplemental Table 1. Seeds were surface sterilized for 3 min in 100% bleach with Tween-20, rinsed 4 times with sterile water, and stratified for 3 7

8 days at 4 C before sowing. Six to ten sterile seeds were planted on plates containing standard Murashige and Skoog media [0.33 g/l CaCl 2.6H 2 O; g/l MgSO 4, 1.7 g/l KH 2 PO 4, 100 ml/l Murashige and Skoog micronutrient solution (10x stock from Sigma #M0529), 0.5 g/l MES (2-(N-morpholino)ethanesulfonic acid)], except that nitrogen salt concentrations were reduced (5mM each NH 4 NO 3 and KNO 3 ) and 45 grams per liter of sucrose was added. The ph was adjusted to 5.7 using 1N KOH, and 7 g/l BRL Ultrapure Agar (Fisher #B-11849) was added before autoclaving. To impose mild osmotic stress, either 60 mm mannitol or 30 mm nitrogen salts (15 mm each NH 4 NO 3 and KNO 3 ) were added to this media before adjusting the ph. For all experiments, plates were placed in either a Percival growth chamber or a Conviron walk-in growth chamber and oriented vertically to allow the roots to grow on the surface of the media. In all cases plants received 16 hrs of light, 8 hrs of dark, and the temperature was maintained at 22 C. Quantification of RSS: Digital images of roots were traced in Image J 1.29j (Rasband W. National Institutes of Health, USA) using the line tool or in Adobe Photoshop with the pencil tool (3 pixel width). Tracings from Photoshop were saved as black and white jpeg images, opened in Image J, and analyzed with the perimeter tool. The resulting text files were imported into StatView (SAS Institute) to extract the descriptive statistics. TOT (total lateral root length) represents the sum of the lengths of all lateral roots per seedling. Microscopic analysis of lateral root initiation and formation of lateral roots from LRP: Seedlings were grown for 12 days as described above, then cleared and visualized as described in MALAMY and BENFEY (1997a). Seedlings were scored for the total number of initiated lateral roots and the developmental stage of all LRP and lateral 8

9 roots. The total number of lateral root initiations is defined as the number of all lateral roots, including LRP. The percentage of lateral root formation from LRP was determined by counting all of the autonomous lateral roots (defined as those roots where there was evidence for an active meristem based on an increase of cell number at the tip of the LRP; MALAMY and BENFEY 1997a) and by dividing this number by the total number of lateral root initiations. Statistical Analysis: Variance increases with mean TOT value. Therefore, all statistical analyses were done with log transformed TOT values. In cases where datasets included values that were zero or less than one, one was added to each sample before log transformation. Variance in TOT among the RILs was partitioned into genetic (V G ) and error (V E ) components using a random effects analysis of variance (ANOVA) with the SAS statistical package (GLM and VARCOMP procedures; SAS Institute 1988). Broad sense heritability (H 2 ) in the RILs was calculated by dividing V G, the among RIL variance component, by the total variance (V G +V E ). TOT, total number of initiations, and the percent LRP that form lateral roots were measured in Col, Ler, and the NILs in at least 3 cohorts grown at separate times. The combined data were analyzed using a mixed model ANOVA using the PROC MIXED procedure in SAS Institute 1988, with the following model: y = Genotype + Mannitol + Genotype X Mannitol + Cohort + Genotype X Cohort + Mannitol X Cohort + error, where y= the measured trait and where genotype (Ler, Col, and NILs), mannitol (indicating presence or absence of mannitol treatment), and the genotype X mannitol interaction are fixed effects. Cohort (experimental replicate), Genotype X Cohort, and Mannitol X Cohort are random effects. Post-hoc pairwise T-tests were conducted among 9

10 the LS means of genotype and mannitol interaction, and significance thresholds were adjusted using a sequential Bonferroni procedure. All p values reported as significant are below the adjusted significance thresholds of p < In cases where p values are significant at the p < 0.05 threshold before sequential Bonferroni adjustment, this is noted in the text. As described above, log transformed TOT values were used for statistical analysis in place of TOT. The untransformed TOT means are presented in the figures, and the back-transformed TOT means and 95% confidence intervals calculated from the log transformed TOT data are provided in Supplemental Table 2. To test for proportional differences in response to osmotic stress, log transformed values were used for all GXE analyses. Linkage map construction: 230 markers were selected from those publicly available ( that had been scored in at least 85% of the RILs, and were also represented on the RIL genetic map (LISTER AND DEAN 1993). Mapmaker/EXP 3.0 (LANDER et al. 1987) was then used to construct a linkage map as described (UNGERER et al. 2002), with the modification that distances between markers were subsequently assigned directly (using the seq function) from the data in the RIL map. QTL mapping: For the establishment of heritability (H 2 ) values and QTL mapping, an average of five plants were scored for each RIL. TOT determination in the Ler X Col RIL population was repeated twice using 12 day old seedlings for experiment 1 and 14 day old seedlings for experiment 2. The first experiment included RILs 1901 through 1950 (42 lines total) and the second experiment included lines (54 lines total). Several lines in each experiment were not analyzed due to poor germination, 10

11 bacterial contamination or lack of genotypic data. The full dataset can be found online at Plants for both experiments were grown in similar Percival growth chambers except that for experiment 1 the chamber was fitted with yellow plexiglass filters to reduce light intensity. Mean TOT and mean log transformed TOT values were used as mapping traits for QTL mapping. Mapping was performed using the composite interval mapping function (ZENG 1993; ZENG 1994) of QTL Cartographer (version 1.16; BASTEN et al. 2002), essentially as described elsewhere (UNGERER et al. 2002). The background markers used to control for the effects of closely linked QTL were identified by forward-backward stepwise regression. All mapping was done with a window size of 10 cm and a walking speed of 0.1 cm. Mapping was done separately for two independent datasets. For each experiment, one hundred permutations of the data were performed to establish the 5% and 1% significance thresholds. The proportion of variance explained by the EDG1 and EDG2 QTLs was determined by partitioning the variance attributable to the marker nearest the peak LOD score and was calculated using the log transformed TOT data (GLM and VARCOMP procedures; SAS Institute 1988). Creation and testing of near-isogenic lines (NILs): NILs designed to confirm the predicted promotive effects of the Ler allele of EDG1 (EDG1 Ler ) and the Col allele of EDG2 (EDG2 Col ) were constructed by introgressing these QTL regions into Col or Ler, respectively. The EDG1 Ler (Col) NILs contain approximately 6.2 Mb of Ler sequence at the bottom of chromosome 4 in an otherwise Col background. The EDG2 Col (Ler) NILs have approximately 9 Mb of Col sequence at the top of Chromosome 3 in an otherwise Ler background. 11

12 To introgress the EDG1 Ler allele into the Col background, RIL 1950 was backcrossed to Col. Using one to two markers per chromosome (I: NCC1, II: RGA and ER, IV: RPS2, V: RCI1B), approximately 170 F2 plants were screened for those that had at least one Col allele at loci that were Ler in RIL 1950 but retained the Ler allele at the EDG1 locus (RPS2). Two BC1F2 plants were selected, and BC1F3 plants derived from these plants were backcrossed to Col. BC2F1 plants were then screened using markers spaced approximately 2 Mb apart in regions that were Ler in RIL EDG1 Ler (Col) NILs were isolated among the BC2F2 progeny. Two NILs that are descended from the two independent BC1F2 parents were used for all the experiments described in this paper. Introgression of the EDG2 Col allele into the Ler background was performed by backcrossing RIL1950 to Ler. Approximately 170 F2 plants were screened using one or more markers per chromosome (I: g17311, II: CD2131, III: CA1, MNSOD, and m255 and BGL1, V: LFY3) to identify plants that retained the Col allele at the EDG2 locus (CA1, MNSOD, and m255) and that were Ler at most other loci. BC1F3 plants derived from two selected BC1F2 plants were genotyped with markers spaced approximately 2 Mb apart in regions that were Col in RIL Selected BC1F3 plants were backcrossed to Ler. BC2F1 plants were screened with chromosome 3 markers approximately 2Mb apart to identify lines that maintained the EDG2 Col allele and were Ler at all other loci. These plants were backcrossed a third time to Ler, and screening was repeated for BC3F1 plants. EDG2 Col (Ler) NILs were identified in the BC3F2 generation. Two NILs that descended from two independent BC1F2 plants were used for all experiments described in this paper. 12

13 The effect of the EDG2 Col allele in the Ler background (EDG2 Col (Ler) NIL) was highly environment-dependent; therefore, for these experiments, the positions of the plates in the growth chamber were rotated daily. RESULTS Arabidopsis ecotypes show variation in the size of the root system: When Col seedlings are grown on media supplemented with 60 mm mannitol or 30 mm nitrogen salts (15 mm KNO 3, 15 mm NH 4 NO 3 ), they produce very few lateral roots and therefore have a small root system (DEAK and MALAMY 2005). Previous work showed that the mild osmotic stress caused by these conditions is sufficient to repress or significantly delay the formation of lateral roots from LRP (DEAK and MALAMY 2005). These mild osmotic stress conditions provide a convenient starting point to assess natural variation in RSS among Arabidopsis ecotypes. Seedlings of 71 ecotypes, chosen at random, were grown for 12 days under mild osmotic stress conditions, using 30 mm nitrogen salts as the osmoticum. To quantify RSS, we measured all of the lateral roots on each plant and added these lengths together to get the total lateral root length (TOT). [Since TOT and primary root length were found to be independent in linear regressions using an RIL population (described in a later section; Figure 3B), we considered these to be separate traits and therefore did not normalize TOT to primary root length.] In this survey of ecotypes, we found that 44 out of 71 ecotypes tested produced few or no lateral roots under mild osmotic stress conditions, resembling Col. However, the remaining ecotypes had higher TOT values under these conditions. TOT values of ten representative ecotypes that differ from Col under mild osmotic stress conditions are shown (Figure 1A; see Supplemental Table 1 for a complete dataset). These data demonstrate that there is 13

14 considerable natural variation in intrinsic and/or environmental response pathways that regulate RSS. The difference between Ler and Col TOT values under mild osmotic stress was particularly fortuitous, as well-characterized RILs have been developed from crosses between these ecotypes. Ler had a visibly larger, more highly branched root system than Col under mild osmotic stress, whether nitrogen salts or mannitol were used as the osmoticum (Figure 1A-C). Ler consistently had a higher TOT value than Col when grown on 60 mm mannitol (p=0.0013; Figure 1C). Therefore, 60 mm mannitol, which avoids the potential for nitrogen-specific effects when using nitrogen salts as the osmoticum, was used to define the genetic basis of RSS variation between Ler and Col ecotypes. Ler and Col ecotypes differ in intrinsic and environmental-response pathways that determine RSS: Differences in RSS under mild osmotic stress conditions could be due to intrinsic developmental differences between Ler and Col and/or due to differences in response to the stress. To distinguish between these possibilities, we grew Ler and Col seedlings on media containing a range of mannitol concentrations. Ler had a significantly higher TOT value than Col on 0, 40, and 60 mm mannitol (p = at each mannitol concentration) (Figure 2A). The fact that Ler had a larger TOT value under all conditions tested suggests that Ler and Col differ in an intrinsic, environment-independent pathway(s) that regulates RSS. In addition to intrinsic differences in RSS regulation, Ler and Col also showed different sensitivities to osmotica (Figure 2B). In Col, the TOT value was reduced 80% by the addition of 40 mm mannitol, with the same level of repression observed with 60 14

15 and 80 mm mannitol (p > 0.46). In contrast, in Ler the TOT value was reduced only 64% and 62% by addition of 40 and 60 mm mannitol, respectively. This percent reduction of Ler TOT values by 40 and 60 mm mannitol is significantly less than observed for Col at 40 mm mannitol (p = and p = 0.025, respectively; Figure 2B). 80 mm mannitol was required to repress Ler root system development to the same extent as Col at 40 mm mannitol (p=0.7829, Figure 2B). These data suggest that Ler and Col differ in their ability to modify root system morphology in response to osmotic stress. Together, these findings suggest that variation in both intrinsic and environmental-response pathways contribute to the differences in Ler and Col RSS under mild osmotic stress. It should be noted that GXE analysis of the log transformed TOT values did not provide support for the model that Ler is less responsive to mannitol than Col (ecotypic response to 40 mm mannitol, p=0.45; ecotypic response to 60 mm mannitol, p=0.95). However, a trend of reduced sensitivity of Ler to osmotic stress was consistently seen over many experiments. The failure to observe statistical significance in the GXE analysis may be due to the high variance in TOT under conditions where root systems are highly branched. Ler x Col RILs exhibit heritable differences in RSS: To identify genetic components responsible for RSS differences under mild osmotic stress conditions, we analyzed Ler x Col RILs in two experiments, using 42 and 54 RILs respectively (LISTER and DEAN 1993). RILs were grown on 60 mm mannitol for days and TOT values were determined. We expected that root system development would be extremely sensitive to experimental and environmental variation. Therefore, we performed the experiments in two different growth chambers and analyzed the datasets independently (see Materials and Methods). 15

16 To first ascertain if TOT values represent a complex trait, the distribution of mean TOT values for the RILs on 60 mm mannitol was investigated (Figure 3A). Data from one of the two experiments is shown as both were qualitatively similar. The majority of the RILs had mean TOT values intermediate to those of Ler and Col (Figure 3A). The continuous distribution of trait means is consistent with multiple genes contributing quantitatively to this trait. In addition, we observed RILs in each experiment that had TOT values greater than one standard deviation above the Ler controls ( mm; Figure 3A). These transgressive trait values suggest that novel combinations of alleles yield lines that have an intrinsically larger RSS and/or are even less repressed by mannitol than Ler. Furthermore, the existence of transgressive phenotypes suggests that even though Ler plants have a larger TOT value than Col, there are some Ler alleles that are less promotive of root system development than the corresponding Col allele. The presence of promotive alleles in Col is interesting given that Col has little lateral root development on 60 mm mannitol. One trivial explanation for increased TOT values could be that longer primary roots produce more lateral roots simply as a function of length. In this scenario, RILs with large TOT values might simply be those that grow the fastest overall. To test this possibility, primary root lengths were measured in each RIL and a linear regression of TOT values to primary root length was done to determine if these traits are correlated (Figure 3B). This analysis demonstrated that TOT values are independent of primary root length (r 2 = 0.008), suggesting that the genetic pathways that determine TOT values are specific to lateral root formation. 16

17 To determine if TOT values represent a heritable trait and thus are amenable to QTL mapping, we calculated the broad sense heritability of TOT in the RILs (see Materials and Methods). Log transformation of TOT values improved normality (data not shown); therefore, log transformed data were used for statistical analysis. TOT values demonstrate a large heritable component, with an H 2 value of 57.6% and 64.4% in experiments 1 and 2, respectively (Table 1). This indicates that a strong genetic component exists that may be amenable to mapping in the RILs. EDG1 and EDG2 are major effect QTL that control variation in RSS: Mean TOT values for each RIL in each of the two experiments were used to generate trait means for mapping. Composite interval mapping of the log transformed TOT values on 60 mm mannitol uncovered two QTL, termed EDG1 and EDG2 (Figure 4, Table 2). EDG2 had a LOD score greater than the p = 0.05 significance level in both experiments. EDG1 had a LOD score greater than the p = 0.05 significance level in experiment 2, and a suggestive QTL localized to a nearby marker in experiment 1. Furthermore, a strong EDG1 QTL peak was identified when untransformed TOT values were used as the mapping trait in experiment 1 (Table 2). Additional major QTL with significant LOD scores were identified in one of the two experiments (Figure 4), and are listed in Table 2. (A complete list of all QTL identified by QTL Cartographer is provided in Supplemental Table 3.) In this paper we have focused on EDG1 and EDG2, the two QTL that were strongly indicated in both replicates, for further analysis. QTL maps were also generated based on primary root lengths of the RILs, but no peaks with LOD scores above the p = 0.05 significance level were found, and no suggestive QTL overlapped with any of the TOT QTL (data not shown). This further 17

18 supports the idea that primary root length and lateral root formation are independent traits. EDG1: The EDG1 QTL is located on the bottom of Chromosome 4. In both experiments, the location of the peak marker was approximately 80 cm (Table 2). The presence of a Col allele at the EDG1 locus is associated with decreases in TOT values; hence, the Col allele is more repressive to root system development than the Ler allele (Table 2). (From here on we will describe allelic effects as repressive or promotive. This refers to the alleles effects relative to each other and does not imply that the function of these alleles is actually to repress or promote root system development.) The log transformed data were used to calculate the percent variance explained by EDG1 (Table 2). The EDG1 QTL explains 25.3 and 31.5 % of the variance in TOT among the RILs in experiments 1 and 2, respectively (Table 2). EDG2: The EDG2 QTL maps to the top of Chromosome 3. In both experiments, the QTL peak mapped to 38.3 cm (Table 2). The presence of a Col allele at the EDG2 locus is associated with increased TOT values, suggesting that the Col allele of a gene(s) in this region promotes root system development (Table 2). The finding that the more repressive EDG2 allele originates from the Ler background could explain some of the transgressive TOT values in the RILs, as the combination of Ler EDG1 with Col EDG2 would be expected to yield higher TOT values than either parental genotype. Indeed, in 3 out of the 4 RILs that showed transgressive trait means in experiments 1 and/or 2, both promotive alleles were present. EDG2 explains 53.2 and 31.4% of the variance in TOT in experiments 1 and 2, respectively (Table 2). 18

19 Gene Interactions: The absolute value of the EDG1 and EDG2 additive effects are very similar. This leads to difficulty in explaining the parental phenotypes. Theoretically, since each ecotype carries a repressive and promotive EDG allele of approximately equivalent effect, Col and Ler plants should appear morphologically similar on 60 mm mannitol. One explanation for this inconsistency could be that the effects of the EDG2 allele, which should promote lateral root formation in Col and repress it in Ler, are masked by other genetic modifiers. One candidate genetic modifier of EDG2 is EDG1. Therefore, an ANOVA was done to test for epistatic interactions between markers at the EDG1 and EDG2 loci, where a significant p value for marker by marker interaction is taken to be evidence for epistasis between loci (GLM procedure; SAS Institute 1988). However, we failed to detect any interaction between these loci based on this test (p > 0.25 in experiments 1 and 2). This suggests that other loci promote lateral root development, potentially by unmasking the effects of the EDG2 Col allele. For example, 3 out of 4 of the additional QTLs detected in experiment 2 on chromosomes 4 and 5 are predicted to contribute to the repression of TOT in Col (Table 2). These QTLs are therefore candidates for genetic modifiers of EDG2, although no evidence for epistasis was detected (not shown). The existence of genetic modifiers of EDG1 and EDG2 is further supported by the fact that one of the four transgressive lines only carries one of the promotive EDG alleles, and that many other RILs that carry both promotive alleles do not show transgressive trait means. Confirmation of QTL effects in NILs: To confirm the effects of EDG1 and EDG2 in a biological context, we created NILs. To do this, the promotive EDG1 Ler allele was introgressed into Col to create EDG1 Ler (Col), and the promotive EDG2 Col allele was 19

20 introgressed into the Ler background to create EDG2 Col (Ler) (Figure 5A). Two independent NILs were generated and tested for each QTL (see Materials and Methods). This controls for the potential presence of small, undetected regions of Ler and Col sequence in the EDG1 Ler (Col) and EDG2 Col (Ler) NILs, respectively, since it is unlikely that independently derived lines would have these regions in common. Each parental ecotype has one promotive and one repressive allele at the EDG loci. Therefore, both NILs were expected to have higher TOT values than either Col or Ler under mild osmotic stress, as they each have a combination of two promotive alleles. Indeed, two independently derived EDG1 Ler (Col) NILs had a significantly higher TOT value than Col (p = for both NILs; Figure 5B). This confirms the promotive effect of the Ler allele of EDG1. The EDG2 Col (Ler) NILs also had the expected phenotype: both NILs had significantly higher TOT values than Ler on 60 mm mannitol (p = for both NILs, Figure 5C). Based on these results, we concluded that the EDG1 and EDG2 loci do in fact contribute to differences in RSS between Ler and Col, as was predicted by QTL analysis. Although we predicted that combining the promotive alleles of EDG1 and EDG2 would create NILs with larger TOT values than either parent, the EDG1 Ler (Col) NILs were not significantly different from Ler (p = and p = for NILs 1 and 2, respectively; Figure 5B), again suggesting the presence of repressive alleles at unknown loci in the Col background. EDG1 Ler and EDG2 Col contribute to intrinsic and environmental response pathways that regulate RSS: We know that Ler and Col show variation in both intrinsic pathways and in environmental response pathways that affect RSS. The EDG 20

21 loci contribute significantly to the differences in TOT, and therefore may be components of either type of genetic pathway. To determine the roles of the EDG loci in determining RSS, TOT values for each NIL and parental line were compared in the presence or absence of osmotic stress. Furthermore, the extent of repression by 60mM mannitol was also compared. EDG1: As on 60 mm mannitol, EDG1 Ler (Col) NILs grown on media containing 0 mm mannitol had significantly higher TOT values than Col (p = for both NILs), although not significantly different from Ler (p = and p = for NILs 1 and 2, respectively). The fact that the EDG1 Ler allele promoted lateral root formation on both conditions suggests that the EDG1 locus contains gene(s) that are components of intrinsic pathway(s) regulating RSS. To test whether EDG1 also plays a role in environmental response pathways that influence RSS, we looked for differences in sensitivity to mannitol. The mean TOT value in the two EDG1 Ler (Col) NILs was reduced 84% and 80% by 60 mm mannitol, compared to 86% for Col. This slight difference in response to mannitol between the EDG1 Ler (Col) NILs and Col was not significant (GXE on log transformed data, p = 0.458). Hence, it appears that EDG1 is a component of an intrinsic pathway(s) that regulates RSS and has no effect on root system sensitivity to osmotica. EDG2: TOT values in the EDG2 Col (Ler) NILs were significantly higher than Ler on media containing 0 mm mannitol (p = for both NILs) as well as media containing 60 mm mannitol, indicating that the EDG2 locus defines a gene(s) that plays a role in intrinsic regulation of RSS. The mean TOT values of the EDG2 Col (Ler) NILs were reduced by only 56% by 60 mm mannitol, while the TOT value in Ler was reduced 21

22 71%. This difference in response was significant based on GXE analysis (GXE on log transformed data, p=0.0017). These data suggest that the promotive EDG2 Col allele reduces sensitivity to osmotica, and therefore that EDG2 has a role in environmental response in addition to a role in an intrinsic regulatory pathway. Ecotypic differences in RSS under mild osmotic stress are due to differences in both lateral root initiation and in formation of lateral roots from LRP: Estimates of RSS based on TOT allowed us to focus on two regions of the genome with large effects on this trait. However, TOT values provide only a very rough picture of the many physiological events that determine RSS and that differ between ecotypes. Indeed, TOT values can even obscure subtle differences in the regulation of root system development, as differences in initiation of LRP, formation of lateral roots from LRP or lateral root growth rates can compensate for each other to create root systems with similar TOT values but very different developmental histories. A final problem with TOT values is that they show extremely high variability, especially in genotypes or conditions where root systems are highly branched. To understand the physiological events that are differentially modulated in Ler and Col at a finer level, we measured two parameters of root system development that are known to be affected by mild osmotic stress: 1) the number of LRP that are initiated in a given period of time; and 2) percentage of these LRP that form lateral roots within the same time period (DEAK AND MALAMY 2005). Seedlings of Ler and Col were grown on 0 and 60 mm mannitol for 12 days and cleared for microscopic examination (Figure 6). The total number of initiation events was assessed by counting LRP of all sizes and autonomous lateral roots. In addition, the percentage of initiation events that resulted in 22

23 autonomous lateral roots in 12 days was calculated. (TOT values were found to be independent of primary root length in an RIL population (Figure 3B); therefore, the number of initiations was not normalized to primary root length.) A significantly larger number of lateral roots were initiated in Ler than in Col on 60 mm mannitol (p = ) and on 0 mm mannitol (p = ) (Figure 6A), suggesting an intrinsic difference in lateral root initiation rates. Lateral root initiation in both ecotypes was reduced by mild osmotic stress to the same degree (28.8% and 28.7% for Ler and Col, respectively; GXE based on ANOVA of log transformed data, p = ); therefore, lateral root initiation does not appear to be differentially affected by mannitol in the two ecotypes. These results suggest that differences in lateral root initiation are caused by variation in intrinsic regulatory pathways in Ler and Col. In Ler, 46% of LRP formed lateral roots on 60 mm mannitol, compared to only 8% in Col (Figure 6B; significant difference between Col and Ler, p = ). On media containing 0 mm mannitol, 66% of LRP formed lateral roots in Ler, compared to 37% in Col (Figure 6B; significant difference between Col and Ler, p = ). These data suggest that intrinsic differences in lateral root formation from LRP may contribute to RSS variation between Ler and Col. More strikingly, in Ler the percentage of LRP that developed into lateral roots was reduced only 30% in 60 mm mannitol as compared to 0 mm mannitol, while in Col the percentage of LRP that formed lateral roots was reduced 80% by 60 mm mannitol (Figure 6B; significant GXE based on ANOVA of log transformed data, p = ). These data suggest that one of the most important differences between the ecotypes is that Col more strongly represses lateral root formation from LRP in response to osmotic stress. 23

24 In summary, characterization of the Ler and Col ecotypes shows that Ler initiates a larger number of lateral roots than Col on both 0 and 60 mm mannitol, and the repression of initiation by mannitol is similar in both Ler and Col. Therefore, the rate of lateral root initiation reflects an intrinsic difference in the regulation of Ler and Col root system development. In contrast, the formation of lateral roots from LRP is higher in Ler under both conditions, and is not repressed by 60 mm mannitol in Ler as strongly as it is in Col. Therefore, differential regulation of lateral root formation from LRP also contributes to ecotypic differences in RSS under mild osmotic stress and reflects ecotypic variation in both intrinsic and environmental-response pathways. EDG1 Ler and EDG2 Col play distinct physiological roles in lateral root formation: The NILs created to confirm the QTLs for TOT also allowed us to dissect the role that gene(s) at these loci play in root system development. To determine the physiological roles of the EDG1 and EDG2 loci, we compared lateral root initiation and the percentage of LRP that formed lateral roots in day old seedlings grown on media containing 0 or 60 mm mannitol. EDG1: The EDG1 Ler (Col) NILs had a slightly higher mean number of initiations than Col on both 0 and 60 mm mannitol (Figure 6A). This difference in mean number of initiations was significant for NIL2 on 0 mm mannitol (p = ), but not on 60 mm mannitol (p = ). After sequential Bonferroni adjustment of significance levels, NIL1 was not significantly different from Col on either 0 or 60 mm mannitol (p = , associated α=0.0064, for 0 mm mannitol and p = for 60 mm mannitol). These data suggest that EDG1 may have an effect on lateral root initiation on 0 mm mannitol, but we cannot say this with confidence. In contrast, the effect of EDG1 on the 24

25 formation of lateral roots from LRP was more clear. A higher percentage of LRP formed lateral roots in the EDG1 Ler (Col) NILs than in Col on both 60 mm mannitol (p = for both NILs; Figure 6B) and 0 mm mannitol (p = for both NILs; Figure 6B). Therefore, EDG1 acts primarily at the level of lateral root formation from LRP, and is a component of an intrinsic pathway that regulates this developmental event under all growth conditions tested. To test whether EDG1 also plays a role in environmental response pathways that influence lateral root formation from LRP, we looked for differences in sensitivity to mannitol. The percentage of LRP that formed lateral roots was reduced 50% by 60 mm mannitol in the EDG1 Ler (Col) NILs compared to 80% in Col (significant GXE based on ANOVA of log transformed data, p=0.0001). Hence, lateral root formation from LRP in the EDG1 Ler (Col) NILs was less sensitive to mild osmotic stress than Col. Together, these data indicate that a gene(s) at the EDG1 locus plays a role in both intrinsic pathways that control lateral root formation from LRP and response pathways that control the sensitivity of this process to osmotic stress. Only the role in intrinsic pathways was predicted by analysis of the effect of EDG1 on TOT values, again emphasizing the higher resolution of the current assays in dissecting the developmental events that determine RSS. Interestingly, the EDG1 Ler (Col) NILs did not have as high a percentage of LRP forming lateral roots as Ler under either condition (p = for both NILs on 0 and 60 mm mannitol) and were more sensitive to repression by mannitol than Ler (p = ). Hence, the EDG1 Ler allele in the Col background is not sufficient to cause plants to have Ler-like development of lateral roots, or a Ler-like root system response to osmotic 25

26 stress, and suggests that additional loci promote lateral root formation in the Ler background. EDG2: Although the mean number of lateral root initiations for both EDG2 Col (Ler) NILs was slightly higher than Ler on both 0 and 60 mm mannitol, these differences were not significant after sequential Bonferroni adjustment of significance levels (Figure 7A; p = and p = 0.031, associated α = , for NIL1 on 0 and 60 mm mannitol, respectively and p = and p = for NIL2 on 0 and 60 mm mannitol, respectively). Therefore, EDG2 does not appear to affect lateral root initiation. In contrast, EDG2 has a strong effect on the formation of lateral roots from LRP (Figure 7B). In the EDG2 Col (Ler) NILs, a significantly higher percentage of LRP formed lateral roots than in Ler on both 0 and 60 mm mannitol (p < for both NILs on 0 and 60 mm mannitol). The percentage of LRP that formed lateral roots was reduced 25% and 17% by 60 mm mannitol in the two EDG2 Col (Ler) NILs compared to 27% in Ler (GXE analysis on log transformed data, p = ), suggesting that EDG2 does not play a role in environmental response. These data indicate that EDG2 functions only in an intrinsic pathway regulating the formation of lateral roots from LRP. Again, this differs from the predictions based on TOT values, which indicated that EDG2 does play a role in environmental response. This suggests that there are additional parameters of root system development that are differentially affected by osmotic stress in Ler and Col. DISCUSSION A quantitative genetic approach uncovers the genetic basis for natural variation in RSS: The size of the root system is a result of many separate physiological 26

27 events, each of which may be under independent control during plant development. In addition, these events are strongly affected by the plant s ability to perceive and respond to growth conditions. Hence, it is not surprising that RSS is a multigenic trait and that it is influenced by genes involved in development and in environmental response pathways (MALAMY 2005). Complex traits such as this can be difficult to dissect using standard forward genetics approaches, as individual gene effects are often subtle or hard to distinguish from environmental effects. In contrast, a quantitative genetics approach allows the simultaneous definition of multiple genomic regions that contribute quantitatively to RSS. We took advantage of natural variation among Arabidopsis ecotypes to identify genomic regions that play a role in RSS and that contribute to root system differences among closely related plants. We used TOT values as a simple measure to characterize RSS in the Ler and Col ecotypes. Plants were grown under mild osmotic stress conditions, as these conditions were previously demonstrated to repress lateral root formation in Col (DEAK AND MALAMY 2005). Comparisons to plants grown in the absence of osmotic stress allowed us to detect ecotypic differences in both intrinsic and environmental response pathways between the Ler and Col ecotypes. Ler produces a larger root system on all conditions tested, and RSS in Ler is also less sensitive to repression by osmotica. To define the genetic basis for differences between Ler and Col, we mapped QTL for TOT on mild osmotic stress conditions in a Ler X Col RIL population. We identified two QTL, EDG1 and EDG2, that contributed to TOT in two separate experiments, indicating that these loci are robust to experimental variation. The effects of the two QTL were confirmed in NILs. In both cases, the alleles predicted to 27

28 promote RSS (TOT) had the expected effect when introgressed into the opposite genetic background. Interestingly, analysis of the effects of Ler and Col alleles of each QTL demonstrated that, even though Col is essentially unbranched under mild osmotic stress conditions and highly sensitive to osmotica, the EDG2 Col allele promotes RSS. The existence of this "hidden" genetic potential explains some of the transgressive phenotypes that segregate in the RILs. Although calculation of the percent variance explained by EDG1 and EDG2 predict that these loci explain all of the heritable variance in TOT observed in the RILs, this is unlikely to be the case. The percent variance explained by QTLs is often an overestimate, especially when mapping is done with a small sample size (DARVASI et al. 1993; BEAVIS 1994; BEAVIS 1998; GÖRING et al. 2001). Analysis of our data further supports the idea that the EDG QTLs are not the only genetic contributors to TOT. First, EDG1 and EDG2 are not sufficient to explain all of the transgressive phenotypes observed. Second, although the EDG1 Ler (Col) NILs and many of the RILs contain two promotive EDG alleles while Ler contains only one, the root systems of these plants are not larger than that of Ler. Third, the EDG2 Col (Ler) NILs have a much larger root system than predicted based on the additive effects of EDG1 Ler and EDG2 Col. Fourth, the promotive effects of EDG2 Col are clearly not observed in the Col background, even in the absence of the repressive EDG1 Col allele. Finally, neither EDG1 nor EDG2 has a strong effect on lateral root initiation despite the clear difference in initiation between Col and Ler. Together, these results indicate that, not surprisingly, there are additional loci that contribute to RSS. Even though we were not able to reproducibly detect these loci in our QTL mapping, our data allows us to predict that genetic modifiers in Col background 28

29 mask the promotive effects of the EDG2 Col allele and contribute to ecotypic differences in lateral root initiation. Identification of EDG1 and EDG2 reveal the advantages of a natural variation approach to understanding RSS. On average, TOT values for Ler are 6 mm greater than for Col on 60 mm mannitol (Figure 1B). This is a relatively small difference compared to that observed between, for example, Col and the lrd2 mutant; lrd2 produces approximately 100 mm more lateral roots than Col on 60 mm mannitol (DEAK and MALAMY 2005). Not surprisingly, NILs demonstrate that the EDG1 Ler and EDG2 Col alleles have relatively subtle effects on root development. This factor along with the large degree of variance in TOT would have made it difficult to identify these genes in a traditional forward genetic screen. In addition, EDG2 would not have been identified in a screen in the Col background because other modifiers in this background mask the promotive effects of this locus (see above). The identification of a QTL whose effects are background-dependent illustrates the importance of using natural variation in combination with mutant analysis to identify genes regulating RSS. In addition, knowledge of these modifiers will help guide future QTL mapping studies and genetic screens. EDG1 and EDG2 have distinct roles in root system development: TOT values provide a representation of the size of the root system and allowed rapid scoring of large numbers of RILs and identification of QTL affecting RSS. However, TOT does not adequately describe all aspects of root system development. For example, we showed that Ler differs from Col in the rate of lateral root initiation, the percentage of LRP that form lateral roots, and the sensitivity of the latter process to osmotica. The NILs provide 29

30 a valuable tool for dissecting out the specific physiological events that are influenced by EDG1 and EDG2. In this study, we found that both loci play a role in regulating the formation of lateral roots from LRP. This is consistent with our previous finding that osmotic stress primarily affects this stage of lateral root formation. This characterization is not complete, however. Discrepancies between the effects of the EDG loci on TOT and on the physiological events measured reveal where additional events need to be assessed. For example, although EDG2 participates in environmental response pathways regulating TOT, we have not yet defined an EDG2-dependent event that is differentially affected by mild osmotic stress. The identity of the genes at the EDG1 and EDG2 loci: The combined 2 LOD support intervals for both EDG1 and EDG2 span a region of approximately 15 cm. We made the introgressed regions in the EDG1 Ler (Col) and EDG2 Col (Ler) NILs fairly large (45-50 and cm respectively) to ensure that the QTL regions remained intact. This interval is currently too large to predict the gene(s) that underlie the QTL. Once fine mapping reduces the interval, we will search for likely candidate genes, such as genes previously shown to regulate root system development (reviewed in CASMIRO et al. 2003; LOPEZ-BUCIO et al. 2005; reviewed in MALAMY 2005) and genes involved in environmental response pathways (reviewed in MALAMY 2005; MIURA et al. 2005). Genes that affect the anatomy or physiology of the root would also be candidates. Hormones have also been implicated in lateral root development (see MALAMY 2005 for review). For example, the hormone auxin is necessary for lateral root formation, and many genes that affect lateral root development also compromise auxin signaling (CASIMIRO et al. 2003; LOPEZ-BÚCIO et al. 2005; MALAMY 2005). The 30

31 hormone ABA appears to regulate both intrinsic (DEAK AND MALAMY 2005) and response pathways (SIGNORA et al. 2001; DEAK and MALAMY 2005). Likely candidates for EDG1 and EDG2 could therefore include auxin and ABA signaling components or biosynthetic genes. We can also gain insight into EDG1 and EDG2 function from other QTL mapping studies. Recently LOUDET et al. mapped QTL for primary root length and for lateral root number, density, and total lateral root length in the Bay-0 X Shahdara RIL population (2005). They identified QTLs for primary root length (PRL3) and lateral root number (LRN2) that map close to EDG1 at the bottom of Chromosome IV. A third QTL for lateral root length (LRL3) mapped to a region further north on Chromosome IV, but its co-localization with PRL3 and LRN2 could not be excluded. Co-localization of PRL3, LRN2, and/or LRL3 with EDG1 would suggest that differences in EDG1 gene sequence contribute to variation in root system development in other accessions. Although we did not identify any contribution of EDG1 to primary root length or lateral root number differences between Col and Ler (data not shown), it is possible that EDG1 alleles in other ecotypes differentially regulate these aspects of root development. A clue to EDG2 function may come from a study on nitrogen use in the Ler X Col RIL population. The authors of this study measured root length and root mass of plants grown on different nitrogen sources (RAUH et al. 2002). They identified a QTL for root length, and root mass that maps to the same marker at the top of chromosome 3 as EDG2 (mi225). The Col allele at this locus decreases root length, and root mass. In addition, for root length, this QTL showed a significant interaction with nitrogen source, indicating the involvement of this locus in nitrogen response. Co-localization of EDG2 and this 31

32 QTL could suggest that EDG2 acts in the nitrogen signaling pathways targeted in these experiments and that the EDG2 Col allele may not always promote lateral root development. However, caution must be taken in comparing QTLs, as QTL regions are very large. Hence, QTL co-localization does not confirm that variation in the same gene is implicated in both situations. Assessing the benefits of RSS and developmental plasticity: Having an intrinsically large and deep root system is thought to be one of the most important factors allowing crop plants to survive drought conditions (PRICE and COURTOIS 1999; BRUCE et al. 2002; TUBEROSA et al. 2003). Other studies have indicated that the ability to regulate root system morphology in response to environmental cues can give plants a selective advantage (reviewed in MALAMY 2005). Our work indicates that Col has a greater ability to modify root system morphology in response to osmotic signals than Ler. This is consistent with a study of drought response in Col and Ler, where Col was found to alter development in order to withstand drought, whereas Ler exhibited drought escape strategies such as early flowering (MEYRE et al. 2001). Col and Ler provide an interesting system to study the potential benefits or disadvantages of having a small and plastic root system vs a large root system that is less sensitive to osmotic signals. It is important to emphasize that all the plants characterized in this study were grown on nutrient media in petri dishes, and therefore that it is difficult to predict how the genes identified in this study influence RSS in soil-grown plants. Studies of root system development and fitness in Col, Ler, and NILs in different water environments along with the identification of the genes encoded by the EDG1 and EDG2 will allow us to gain a 32

33 better understanding of how changes in these genes in nature can lead to potentially adaptive differences in intrinsic and environmental response pathways. ACKNOWLEDGEMENTS The authors are grateful to Justin Borevitz, Jean Greenberg, Arnar Palsson, Michael Purugganan and Mark Ungerer for helpful discussions, advice and comments on the manuscript and Sudeep Agarwala for technical assistance. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number # ( ) to M.D.L. and a Faculty Research Fund award from the University of Chicago to J.E.M. 33

34 LITERATURE CITED Alonso-Blanco, C., and M. Koornneef, 2000 Naturally occurring variation in Arabidopsis: an underexploited resource for plant genetics. Trends in Plant Sci. 5: Basten, C. J., B. S. Weir, and Z. B. Zeng, 2002 QTL Cartographer. Beavis, W. D., 1994 The power and deceit of QTL experiments: lessons from comparative QTL studies. Proc. Corn and Sorghum Industry Res. Conf., Am. Seed Trade Assoc., Washington D.C Beavis, W. D., 1998 QTL analyses: power, precision, and accuracy, pp in Molecular Dissection of Complex Traits, edited by A. H. Paterson. CRC, Boca Raton, Florida. Borevitz, J. O., and M. Nordborg, 2003 The impact of genomics on the study of natural variation in Arabidopsis. Plant Physiol. 132: Bruce, W. B., G. O. Edmeades, and T. C. Barker, 2002 Molecular and physiological approaches to maize improvement for drought tolerance. J. Exp. Bot. 53: Casimiro, I., T. Beeckman, N. Graham, R. Bhalerao, H. Zhang et al., 2003 Dissecting Arabidopsis lateral root development. Trends in Plant Sci. 8: Darvasi, A., A. Weinreb, B. Minke, J. I. Weller, and M. Soller, 1993 Detecting marker- QTL linkage and estimating QTL gene effect and map location using a saturated genetic map. Genetics 134: Deak, K. I. and J. E. Malamy, 2005 Osmotic regulation of root system architecture. Plant J. 43: Göring, H. H. H., J. D. Terwilliger, and J. Blangero, 2001 Large upward bias in 34

35 estimation of locus-specific effects from genomewide scans. Am. J. Hum. Genet. 69: Hoekenga, O. A., T. J. Vision, J. E. Shaff, A. J. Monforte, G. P. Lee, et al., 2003 Identification and characterization of aluminum tolerance loci in Arabidopsis (Landsberg erecta x Columbia) by quantitative trait locus mapping. A physiologically simple but genetically complex trait. Plant Physiol. 132: Kobayashi, Y. and H. Koyama, 2002 QTL analysis of Al tolerance in recombinant inbred lines of Arabidopsis thaliana. Plant Cell Physiol. 43: Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M. J. Daly, et al., 1987 MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: Li, Z., P. Mu, C. Li, H. Zhang, Li Z, et al., 2005 QTL mapping of root traits in a doubled haploid population from a cross between upland and lowland japonica rice in three environments. Theor. Appl. Genet. 110: Lister, C., and C. Dean, 1993 Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana. Plant J. 4: Lopez-Bucio, J., A. Cruz-Ramirez, and L. Herrera-Estrella, 2003 The role of nutrient availability in regulating root architecture. Curr. Opin. Plant Biol. 6: Lopez-Bucio, J., E. Hernandez-Abreu, L. Sanchez-Calderon, A. Perez-Torres, R. A. Rampey, et al., 2005 An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. 35

36 Identification of BIG as a mediator of auxin in pericycle cell activation. Plant Physiol. 137: Loudet, O., V. Gaudon, A. Trubuil, and F. Daniel-Vedele, 2005 Quantitative trait loci controlling root growth and architecture in Arabidopsis thaliana confirmed by heterogeneous inbred family. Theor Appl Genet. 110: Malamy J., 2005 Intrinsic and environmental response pathways that regulate root system architecture. Plant Cell Environ. 28: Malamy, J. and P. Benfey, 1997a Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124: Malamy, J. and P. Benfey, 1997b Down and out in Arabidopsis: the formation of lateral roots. Trends in Plant Sci. 2: Maloof, J. N., 2003 QTL for plant growth and morphology. Curr. Opin. Plant Biol. 6: Meyre, D., A. Leonardi, G. Brisson, N. Vartanian, 2001 Drought-adaptive mechanisms involved in the escape/tolerance strategies of Arabidopsis Landsberg erecta and Columbia ecotypes and their F1 reciprocal progeny. Journal of Plant Physiology 158: Miura, K., A. Rus, A. Sharkhuu, S. Yokoi, A. S. Karthideyan et al., 2005 The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc. Natl. Acad. Sci. USA 102: Mouchel, C. F., G. C. Briggs, and C. S. Hardtke, 2004 Natural genetic variation in Arabidopsis identifies BREVIS RADIX, a novel regulator of cell proliferation and elongation in the root. Genes Dev. 18:

37 Paran, I., and D. Zamir, 2003 Quantitative traits in plants: beyond the QTL. Trends in Genetics 19: Price, A. H. and B. Courtois, 1999 Mapping QTLs associated with drought resistance in rice: progress, problems and prospects. Plant Growth Regul. 29: Price, A. H., J. E. Cairns, P. Horton, H. G. Jones, and H. Griffiths, 2002 Linking droughtresistance mechanisms to drought avoidance in upland rice using a QTL approach: progress and new opportunities to integrate stomatal and mesophyll responses. J. Exp. Bot. 53: Rauh L., C. Basten, and S. Buckler IV, 2002 Quantitative trait loci analysis of growth response to varying nitrogen sources in Arabidopsis thaliana. Theor. Appl. Genet. 104: Signora, L., I. De Smet, C. H. Foyer, and H. Zhang, 2001 ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis. Plant J. 28: Takahashi Y., A. Shomura, T. Sasaki, and M. Yano, 2001 Hd6, a rice quantitative trait locus involved in photoperiod sensivity, encodes the α subunit of protein kinase CK2. Proc. Natl. Acad. Sci. USA 98: Tuberosa, R., S. Salvi, M. C. Sanguineti, M. Maccaferri, S. Giuliani, et. al., 2003 Searching for QTLs controlling root traits in maize: a critical appraisal. Plant and Soil 255: Ungerer M.C., S. S. Halldorsdottir, J. L. Modliszewski, T. F. C. Mackay, and M. D. Purugganan, 2002 Quantitative trait loci for inflorescence development in Arabidopsis thaliana. Genetics 160:

38 Zeng, Z. B., 1993 Theoretical basis of separation of multiple linked gene effects on mapping quantitative trait loci. Proc. Natl. Acad. Sci. USA 90: Zeng, Z. B., 1994 Precision mapping of quantitative trait loci. Genetics 136: Zheng, B. S., L. Yang, W. P. Zhang, C. Z. Mao, Y. R. Wu, et al., 2003 Mapping QTLs and candidate genes for rice root traits under different water supply conditions and comparative analysis across three populations. Theor. Appl. Genet. 107:

39 TABLE 1 Variance components and heritability for TOT a in the Ler X Col RILs Experiment V g b V e c H 2d P value a Variance component and heritability calculations were done with log transformed TOT values. b Component of variance due to among RIL variation c Component of variance due to within RIL variation d Broad sense heritability calculated as the proportion of total variance (V g +V e ) attributable to genotype (V g ). 39

40 TABLE 2 QTL for TOT and logtot mapped in the Ler X Col RILs QTL peak Position LOD 2 LOD Additive %Variance (Chromosome, (cm) score support effect explained by marker) interval (cm) (mm) a QTL b EDG1 Exp 1 TOT 4, CDs logtot 4, m c Exp 2 TOT 4, mi d logtot 4, mi EDG2 Exp 1 TOT 3, mi logtot 3, mi Exp 2 TOT 3, mi logtot 3, mi Additional QTL Exp2 logtot 4, mi , mi , mi , ve a Reflects the effect of the Col allele on the trait. The sign indicates the direction of the effect. 40

41 b Proportion of the total variance in logtot explained by each QTL (model: y=v EDG1 + V EDG2 + V E for experiment 1 and y= V EDG1 + V EDG2 + V mi204 + V mi32 + V mi61 + V ve032 + V E for experiment 2. c not significant d significant at p =

42 FIGURE LEGENDS Figure 1. Ecotypic variation in RSS under mild osmotic stress. (A) Arabidopsis seedlings were grown for 12 days on growth media supplemented with 30 mm nitrogen salts to create a mild osmotic stress. RSS was assessed as the sum total of the lengths of all lateral roots (TOT). While most ecotypes, like Col, form no lateral roots under these growth conditions and therefore have very low TOT values, some ecotypes show higher TOT values; TOTs of 10 representative examples are shown here. Col and Ler are represented in a different graph to indicate that these plants were not grown concurrent with the other ecotypes. In all cases, means for 6 to 9 seedlings are shown + standard error. (B) Ler and Col seedlings were grown for 12 days on growth media supplemented with 60 mm mannitol to create a mild osmotic stress. As in (A), lateral root formation in Col is largely repressed under these conditions; however, lateral roots are visible in Ler. (C) Quantification of the experiment shown in B. TOT values for 12 day old Col (n=130) and Ler (n=123) seedlings over four experiments were pooled. Mean TOT values are shown + standard error. Figure 2. Intrinsic and environment-dependent differences in Ler and Col root system development. (A) Ler and Col were grown for days on growth media containing 0, 40, 60, or 80 mm mannitol, and TOT values were determined. Ler has a significantly higher mean TOT value than Col on media containing 0, 40, and 60 mm mannitol (p = ), suggesting that Ler has an intrinsically higher TOT than Col irrespective of growth conditions. Three replicates of this experiment were pooled and mean TOT values + standard error are shown (n=44 to 50 for each genotype and 42

43 condition). (B) To evaluate relative sensitivity to mannitol, TOT values are plotted as a percentage of the mean on growth media containing 0 mm mannitol. Data from four separate experiments, in which each TOT value at 60 mm mannitol was normalized to the mean TOT value at 0 mm mannitol for that experiment, were pooled. Ler is less sensitive to increasing concentrations of mannitol than Col. Data are shown + standard error. Figure 3. Frequency distribution of TOT values in the Ler X Col RIL population. (A) 55 RILs (6 plants/line) were grown on growth media containing 60 mm mannitol for 14 days and TOT values were obtained. The Col and Ler means + standard deviation are shown in the inset and means are indicated in the histogram by arrows. The continuous distribution suggests that more than one locus controls differences in RSS between Ler and Col. (B) Mean primary root lengths were determined for each RIL. A linear regression was done to determine the dependence of TOT on primary root length. No correlation was observed (r 2 = 0.008). Data are shown from one of two experimental replicates. Figure 4. QTL map of loci affecting TOT values under mild osmotic stress. Shown are the results of mapping the log transformed data from experiments 1 and 2. The dashed black and solid red lines indicate the LOD scores for experiments 1 and 2, respectively. The p = 0.05 thresholds determined by permutations are 3.65 for experiment 1 and 3.32 for experiment 2. The dotted black line indicates the p = 0.05 threshold for experiment 1. The locations of the EDG1 and EDG2 peaks are indicated by 43

44 arrows. A + indicates that the Col allele has a more promotive effect on lateral root development, and a - indicates that the Col allele has a more repressive effect. Figure 5. Near isogenic lines confirm the predicted effects of EDG1 and EDG2. (A) Schematic representation of the EDG1 Ler (Col) and EDG2 Col (Ler) NILs. White indicates the Col genotype, and gray indicates the Ler genotype. Two mm equals 1 Mb. The indicated markers define the boundaries of the introgressed regions. (B) EDG1 Ler NILs have a significantly higher mean TOT value than Col on both 0 and 60 mm mannitol. The results of three independent experiments were pooled, and are shown as means + standard error. (n=97 to 143 for each genotype and condition). C) The EDG2 Col (Ler) NIL have a significantly higher mean TOT value than Ler on both 0 and 60 mm mannitol. For B and C, data are shown for two independently generated NILs. (n=57 to 63 for each genotype and condition). Figure 6. Col and Ler ecotypes differ in the intrinsic regulation of lateral root initiation and lateral root formation from LRP, and the sensitivity of the latter process to mannitol. EDG1 contributes to ecotypic differences in lateral root formation from LRP, but plays little if any role in lateral root initiation. Seedlings were grown for 12 days on growth media containing 0 or 60 mm mannitol and then cleared and examined microscopically to determine the total number of lateral root initiations (A) and the percentage of lateral root primordia (LRP) that developed into lateral roots (B). Data from three experiments were combined and are shown as mean + standard error. (n=34 to 36 for each genotype and condition). 44

45 Figure 7. EDG2 plays a role in intrinsic regulation of lateral root formation from LRP, but has no apparent role in lateral initiation. Seedlings were grown for 12 days on media containing 0 or 60 mm mannitol and cleared for microscopic analysis. Data from three experiments were pooled and means are shown + standard error. (n=30 for each genotype and condition). 45

46 Figure 1 A B TOT (mm) Di-G Hau-0 Kas-1 Mz-0 Nd-1 Rou-0 Sha Ts-1 Wei-0 Col Ler C TOT (mm) Col Col Ler Ler

47 Figure 2 A Ler Col TOT (mm) B mm Mannitol TOT repression by mannitol (% of 0 mm mannitol control) Ler Col mm Mannitol

48 Figure 3 A Number of RILs Col Ler Col: mm Ler: mm 2 B TOT (mm) TOT (mm) R 2 = Primary root length (mm)

49 Figure 4 LOD I Experiment 1 Experiment 2 P=0.05 +, - Direction of the effect of the Col allele 0 15 II 10 LOD III + LOD EDG IV LOD EDG V LOD cm

50 Figure 5 A EDG1 Ler (Col) NIL I II III IV V EDG2 Col (Ler) NIL I II III IV V CA1 EDG2 m255 EDG1 SNP DHS1 B TOT (mm) Col EDG1 Ler (Col) NIL1 EDG1 Ler (Col) NIL2 Ler C TOT (mm) EDG2 Col (Ler) NIL1 EDG2 Col (Ler) NIL2 Ler mm Mannitol 0 mm Mannitol mm Mannitol 0 mm Mannitol

51 Figure 6 A Number of initiations B Percent of LRP that form lateral roots Col EDG1 Ler (Col) NIL1 EDG1 Ler (Col) NIL2 Ler 60 mm Mannitol Col EDG1 Ler (Col) NIL1 EDG1 Ler (Col) NIL2 Ler 60 mm Mannitol 0 mm Mannitol 0 mm Mannitol

52 Figure 7 A Number of initiations EDG2 Col (Ler) NIL1 EDG2 Col (Ler) NIL2 Ler 10 B Percent of LRP that form lateral roots mm Mannitol 0 mm Mannitol EDG2 Col (Ler) NIL1 EDG2 Col (Ler) NIL2 Ler 60 mm Mannitol 0 mm Mannitol