A standardized method for analysis of Medicago truncatula phenotypic development.

Medicago truncatula has become a model system to study legume biology. It is imperative that detailed growth characteristics of the most commonly used cultivar, line A17 cv Jemalong, be documented. Such analysis creates a basis to analyze phenotypic alterations due to genetic lesions or environmental stress and is essential to characterize gene function and its relationship to morphological development. We have documented morphological development of M. truncatula to characterize its temporal developmental growth pattern; developed a numerical nomenclature coding system that identifies stages in morphological development; tested the coding system to identify phenotypic differences under phosphorus (P) and nitrogen (N) deprivation; and created visual models using the L-system formalism. The numerical nomenclature coding system, based on a series of defined growth units, represents incremental steps in morphological development. Included is a decimal component dividing growth units into nine substages. A measurement component helps distinguish alterations that may be missed by the coding system. Growth under N and P deprivation produced morphological alterations that were distinguishable using the coding system and its measurement component. N and P deprivation resulted in delayed leaf development and expansion, delayed axillary shoot emergence and elongation, decreased leaf and shoot size, and altered root growth. Timing and frequency of flower emergence in P-deprived plants was affected. This numerical coding system may be used as a standardized method to analyze phenotypic variation in M. truncatula due to nutrient stress, genetic lesions, or other factors and should allow valid growth comparisons across geographically distant laboratories.


INTRODUCTION
Medicago truncatula has become a model plant for the study of legume biology (Cook, 1999). With the anticipated sequencing of its genome by 2007, its large EST database, and availability of the Medicago Affymetrix GeneChip®, essential resources are available to genetically dissect this organism. However, to fully utilize genomic approaches the Medicago research community requires standardized methods for growth and phenotypic analysis that can be utilized worldwide. A standardized system, similar to that adopted by the Arabidopsis research community, should allow researchers from geographically separate regions to easily and clearly monitor morphological and developmental changes resulting from mutations, biotic and abiotic stress, and other factors, thereby facilitating the standardization of experimental results across laboratories.
The establishment of a standardized baseline for the temporal developmental growth pattern of M. truncatula should provide a uniform approach to compare growth of genetic mutants or ecotypes having a developmentally impaired phenotype. The development of a numerical nomenclature coding system provides a means to define specific growth stages in plant development thereby making it possible to discern and document specifically where changes occur for phenotypically distinct plants. Additionally, a nomenclature coding system may be used to clearly communicate developmental stages without extensive descriptions. It also provides a basis to standardize tissue collection for analysis. A coding system may also be incorporated into a component of the M. truncatula plant ontology database, (Lancashire et al., 1991). Nomenclature coding systems have been extensively used for describing growth stages in model animal systems as well (Browder, 1980;Wood, 1988;Bate and Arias, 1993).
We have used nutrient stress to simulate changes in whole plant development to assess the effectiveness of a standardized coding system to reproducibly detect morphological changes in plant growth. We have followed shoot and root development of M. truncatula from cotyledon to early pod formation when grown under nitrogen (N) and phosphorus (P) deficient conditions. These particular stress conditions were chosen because of their major role in plant growth and their implication in nutrient signaling. Phosphorus, an important plant macronutrient, is immobile in the soil and is a limiting nutrient in many agricultural systems (see reviews by Raghothama, 1999;Vance et al., 2003). Nitrogen, besides being an important plant macronutrient, plays an integral role in the nodulation process, a key aspect of legume biology.
Additionally, these two nutrients have been implicated in nutrient signaling involving long-distance communication between the root and the shoot (Forde and Lorenzo, 2001;Liu et al., 2005). Documenting the morphological development of M. truncatula under nutrient stress conditions should lead to a better understanding of the mechanisms behind plant development associated with these nutrients and in combination with physiological, biochemical and genetic data should lead to a better understanding of the molecular mechanisms controlling nutrient perception, root-shoot communication and nutrient signaling.
The overall goal of this work was to develop a standardized method to developing metameric units sequentially along the main and axillary axes of growth and designates position of flower emergence. The nomenclature coding system starts with the metamer associated with the unifoliate leaf as metamer 1 (m1). The units developing above this are numbered in ascending order with m2 associated with the first trifoliate, etc. The metamers forming axillary shoots developing off of the main axis are coded first according to their main shoot metamer of origin then numbered sequentially starting at the base with 1 (e.g. The axillary shoot associated with m1 that contains 1 unit of growth is designated as m1-1). Floral location is indicated with an "F" preceding the number of its associated metamer of emergence. Flowers emerging from the axils of the main shoot are designated according to the metamer of origin followed by F0 (e.g., floral emergence from metamer 6 is designated as m6-F0). When considering a flower developing along an axillary shoot it is coded according to the main shoot metamer of origin followed by the axillary shoot metamer of emergence preceded by "F" (e.g. The flower emerging from the axillary shoot associated with m1 and from the axil of metamer 3 of the m1 axillary shoot is coded as m1-F3). The numerical coding system identifying units of M. truncatula growth and their location is illustrated in Figure 1.
Additionally, the nomenclature coding system defines leaf and reproductive organ development into nine sub-stages (Table I) depicted by a decimal added as an extension to the numerical code. These sub-stages were chosen because they are visually identifiable and provide a continuum of growth and development for the organ that they specify. Therefore, to indicate that the m1-1 leaf is fully developed a code of m1-1.9 is used. A measurement component is also associated with the coding system. Various parameters were measured that could distinguish growth alterations that may be missed by the coding system alone. Plant growth parameters measured for this study included shoot and root fresh and dry weights, shoot and root total lengths, leaf size, and internode lengths. There are numerous other parameters that may have been measured throughout development. The above parameters were chosen because they are nondestructive and augment the morphological focus of this study.
By following plant growth under nutrient stress conditions, specifically N and P stress, we have simulated potential changes in whole plant development that may occur due to genetic lesions or other factors. This allowed us to demonstrate the use of the coding system and its measurement component to document plant morphological development. Moreover, by using these treatments, we were able to assess the effectiveness of this system to distinguish differences in phenotypic alterations. When shoot tissue of P-deficient and control plants were analyzed for %P, plants grown under P-deficient conditions had a lower P content relative to the controls (compare 0.194 %P, SE + 0.014, n = 3, for P-deficient plants and 0.462 %P, SE + 0.042, n = 3, for control plants).
The low P tissue content in P-deprived plants indicates that the morphological alterations reported in this study can be attributed to the P stress imposed.
The results from this study were also used to construct an empirical model of M. truncatula growth and development under these stress conditions.
Animations showing model development can be viewed online in supplemental

Chronology and Morphological Development of the Main Shoot for M. truncatula Grown Under Nutrient Sufficient and Deficient Conditions
A simple way to orient oneself to the developing plant is to use the unifoliate leaf as a reference marker. The unifoliate leaf, associated with metamer one (m1), emerges from the developing apical meristem between the two cotyledons ( Fig. 2A). The second leaf (associated with m2) emerges 180 degrees opposite to the unifoliate. The developing leaves from m1 and m2, plus the cotyledons, produce a cross type of appearance to the developing plant ( Fig.   2, A-C). The third leaf (associated with m3) develops 140 degrees relative to the m2 leaf, adjacent to the m1 leaf. The fourth leaf (associated with m4) develops on the same side of the cotyledons as the m2 leaf and approximately 165 degrees from the m3 leaf (Fig. 2D).  (Table I).
By 14 dap plants grown under both nutrient sufficient and P-deficient conditions had m1 at full development or near full development (m1.9 and m1.8, respectively) and m2 at a mean developmental stage of m2.7 (Fig. 2, A  Furthermore, N-deficient plants were slightly chlorotic, including the cotyledons. Additionally, N-deficient leaf petiole angles, relative to the main axis of growth, were quite acute compared to leaves of the other treatments, compare 36 o and The cotyledons also began to accumulate anthocyanin on their slightly chlorotic surface. Although P-deficient plants showed no significant differences in development relative to nutrient sufficient plants some subtle differences were noted. Specifically, P-deficient plants began to develop patches of anthocyanin on the cotyledon surface and plants began to appear diminished in size (Fig. 2B).
By 21 dap the leaf of m3 control plants had a mean developmental stage of m3.9 ( Fig. 2D; Table II). Under P-deficient conditions this leaf was at a mean developmental stage of m3.7 ( Fig. 2E; Table II). This is the first sign of a significant developmental difference between nutrient sufficient and P-deficient By 28 dap the leaf associated with m5 was at a mean developmental stage of m5.9 for plants grown under nutrient sufficient conditions and at m5.7 for P-deficient plants (Fig. 2 By 32 dap the leaf associated with m6 for control plants was at a mean developmental stage of m6.9 and for P-deficient plants it was at a developmental stage of m6.7. The main shoot developed subsequent metamers between 32 and 40 dap. And by 40 dap, a total of 10-11 metamers were produced along the main shoot of both nutrient sufficient and P-deprived plants. In contrast, the N-deficient plants produced only 5 metamers ( Fig. 3; supplemental Fig. 1).

Chronology and Morphological Development of the Axillary Shoots for M.
truncatula Grown Under Nutrient Sufficient and Deficient Conditions.
The first axillary bud to break and initiate growth was associated with m1 ( Fig. 3; Table II). The m1 axillary bud break was initiated between 18 and 21 dap in plants grown under nutrient sufficient conditions ( Fig. 3; Fig. 2D) and was correlated with the development of m4 along the main shoot. The m1 axillary bud break of P-deficient plants occurred between 21 and 25 dap and was correlated with the development of m5 along the main shoot (Fig. 3). By 40 dap the axillary shoot of m1 was a mean developmental stage of m1-6.4 and m1-5.5 for plants grown under nutrient sufficient and P-deficient conditions, respectively. Nitrogendeficient plants failed to develop any axillary shoots. It should be noted that axillary shoot elongation began to occur when the third leaf emerged from its growing point.
The second axillary bud to break and initiate growth was associated with m2 ( Fig. 3; Table II along the main shoot ( Fig. 3; Table II). The m3 axillary bud break of P-deficient plants occurred between 28 and 32 dap and was correlated with the development of m7 along the main shoot (Fig. 3). By 40 dap the m3 axillary shoot was at a mean developmental stage of m3-5.4 and m3-4.4 for nutrient sufficient and P-starved plants, respectively.
Subsequently, the m4 axillary shoot developed between 28 and 32 dap for plants under nutrient sufficient conditions and between 32 and 40 dap for Pdeficient plants ( Fig. 3; Table II). Concurrently, m8 and m9 developed along the main shoot. By 40 dap the m4 axillary shoot was at a developmental stage of m4-4.4 and m4-3.4 for nutrient sufficient and P-deficient plants, respectively. Axillary bud break from m5 was initiated by 32 dap in control plants and between 32-40 dap in P-deficient plants ( Fig. 3; Table II). Axillary bud break from m5 was correlated with the development of m9 and m10 along the main shoot.
By 40 dap the m5 axillary shoot was at the m5-3.4 and m5-2.3 developmental stage for nutrient sufficient and P-deficient plants, respectively. Axillary buds from m6 and m7 initiated growth just prior to 40 dap for both nutrient conditions (data not shown). By 40 dap the m6 and m7 axillary shoots contained 1 leaf.

Flower Emergence Along the Main and Axillary Shoots
Flowers began to emerge along the main shoot by 32 dap in both nutrient sufficient and P-deficient plants ( Fig. 3; Table II). However, P-deficient plants had approximately 3-fold fewer flowers that initially emerge at 32 dap and these flowers were at a significantly younger developmental stage relative to plants grown under nutrient sufficient conditions (compare mean flower developmental stage of 0.1 to 0.3, respectively; n = 11; t-test, P< 0.05) ( Fig. 4A; Table II).
Notably, by 40 dap no differences in number of emerging flowers or their developmental stage was evident for both treatments ( Fig. 4A; Table II). The differences in flower number between the nutrient sufficient and P-deficient plants may be attributed to the delayed development of P-stressed plants since the timing of flower emergence and not their position is affected between the two treatments. The initial location of flower emergence for both nutrient sufficient and P-deficient plants was from the axils of m5, m6 and m7. As the main shoot continued to elongate, flowers emerged from axils above but not below these metamers. N-deprived plants failed to produce any visible reproductive structures.
On elongated axillary shoots, flowers were visible at 32 dap on control plants and at 40 dap on P-deficient plants (Table II). Flowers emerged from elongated axillary shoots that originated from m1, m2, m3, and m4 for both nutrient sufficient and P-deficient plants. However, plants grown under nutrient sufficient conditions had nearly 2-fold more flowers that developed from the elongated axillary shoots relative to P-stressed plants (Fig. 4B). Similar to flower emergence along the main shoot, differences in the number of emerged flowers on the elongating axillary shoots may be attributed to the delayed developmental stage of axillary shoots in P-stressed plants relative to the controls. The location of flower emergence along the axillary shoots occurred at a specific metamer from the main shoot metamer of origin and was different for the m1 axillary relative to the other axillary shoots. The first flower to have emerged for the m1 axillary was from the m1-3 position. Whereas, the first flower to have emerged from the other axillary shoots is from the m2-2, m3-2 and m4-2 position (Table II).
Differences in flower developmental stage between control and P-deficient plants was evident for flowers that emerged from the axillary metamer closest to the main shoot where flowers from the control plants were at a slightly advanced developmental stage relative to the P-deficient plants (Table II). However, such a delay in flower emergence may also be attributed to a delay in axillary shoot development for P-deficient plants. Subsequently, it was difficult to assess whether the delay in flower emergence along the main and axillary shoots for Pdeficient plants could be attributed solely to a delay in whole plant development or if a physiological response to P-stress could also contribute to such a response.
Growth parameters for total shoot length, internode length of main and axillary shoots, shoot and root fresh weights and leaf dimensions were measured every 4-7 days beginning at 7 dap. These measurements were used to augment the nomenclature coding system making the system more sensitive to the identification of phenotypic differences associated with variable treatments.
Main shoot total length and internode lengths were measured at 40 dap for plants grown under nutrient sufficient and P-deficient conditions. The Ndeficient plants failed to produce measurable shoot length therefore were not included in this analysis. The results showed no significant differences for total length of the main shoot for plants grown under nutrient sufficient and P-deficient conditions (t-test, P< 0.05). The mean shoot length for plants grown under nutrient sufficient conditions was 68.7 mm (SE + 13.1, n = 8) and for P-deficient condition was 52.6 mm (SE + 11.6, n = 9). Consistent with total shoot length data, no differences were detected for internode lengths of all measurable metamers. Morphologically, a unique pattern of internode elongation emerged along the main shoot regardless of nutrient treatment. Internodes of m1, m2, m3, and m4 were short and immeasurable. However, once the leaf associated with m4 became fully developed internodes along the main shoot, above m4, began to elongate. Notably, the internode associated with m5 was consistently short (2.2 mm, SE + 1.11, n = 9) relative to other internodes produced above this metamer (e.g. m6 = 15.5 mm, SE + 3.6, n = 9).
Measurement of axillary shoot total length at 40 dap showed that axillary shoots were significantly longer in plants grown under nutrient sufficient relative to P-deficient conditions (t-test, P< 0.05; Fig. 5A). Axillary shoot internode lengths also appeared to be longer for plants grown under nutrient sufficient relative to Pdeficient conditions (Fig. 5 B). However, because of the high variation in internode lengths between plants statistical differences could not be verified.
These results indicate that P-deficiency had a greater affect on axillary shoot elongation relative to main shoot elongation. Such a response resulted in sufficient conditions. The effect of P-deficiency on axillary shoot elongation was not evident until 28 dap and does not affect internodes produced before this time.
Therefore, prior to 28 dap M. truncatula appeared to contain sufficient P to sustain growth comparable to control plants. Whether the lack of difference in main shoot total length would be significant at later stages remains to be established. Perhaps the main shoot was more tolerant of P-stress relative to the axillary shoots.

Leaf Dimensions along the Main Shoot
Leaf petiole length, blade length (the length of the middle leaflet, including its petiolule) and blade width (distance across the two lateral leaflets including their petiolules) were measured for fully developed leaves associated with m1 through m5 along the main shoot (Fig. 6, A and B).
Leaf petiole lengths were significantly different between the three treatments (F-test, P< 0.05; Fig. 6A). Differences in petiole length between plants grown under nutrient sufficient and N deficient conditions were evident at the m1.9 stage of development. By comparison, differences in petiole length of nutrient sufficient and P-deficient plants were evident at the m2.9 stage. Petiole length progressively increased as leaves were produced along the main shoot for nutrient sufficient and P-deficient plants. In contrast, petiole length for N-deficient plants did not vary and remained short relative to the other treatments (Fig. 6A).
Significant differences in blade length were also detected between the three nutrient treatments (F-test, P< 0.05; Fig. 6B). Plants grown under nutrient sufficient conditions consistently had longer leaf blades relative to plants grown under P-and N-deficient conditions. Blade length differences between nutrient sufficient and N-deficient plants were first evident at the m2 position, whereas for P-deficient plants differences were first detected at the m3 position.

Sufficient and Deficient Conditions
Root architectural development of M. truncatula grown under nutrient sufficient, P-and N-deficient conditions was analyzed over a 28 day period.
Plants were harvested at 4, 7, 14, 21 and 28 dap. Roots were excised and scanned on a flatbed scanner to evaluate root architectural development.
Measurements for total length of primary root, first-order laterals, second-and higher-order laterals were calculated. Additionally, total root number for first-order second-and higher-order laterals was also determined. Table III Table III). However, no differences were detectable for primary root length and first-order lateral root total length and total root number, at 14 dap, for all three treatments.
By 21 dap no differences were detected in first-, second-and higher-order lateral root total length and number between nutrient sufficient and P-deficient plants ( Fig. 7; Table III). However, for N-deficient plants first-order lateral root length was significantly shorter and total root number was significantly lower relative to the other two nutrient treatments (F-test, P< 0.05; Fig. 7  Root hair characteristics were compared between the three nutrient treatments (Fig. 8). Differences in root hair appearance, relative to control plants, were first visible at 14 dap for N-deficient and at 21 dap for P-deficient plants.
Root hairs of plants grown under P-deficient conditions (Fig. 8B) were longer relative to their nutrient sufficient counterparts (Fig. 8A). Additionally, in Pdeficient plants, root hair development occurred closer to the root tip in many instances. Root hairs of N-deficient plants were similar in appearance to those of control plants (Fig. 8C). However, the frequency of appearance along the roots varied between the two treatments. Roots of N-deficient plants had a glabrous appearance with root hairs either lacking or patchy in distribution.

DISCUSSION
The M. truncatula growth analysis and model presented here provide a standardized method to evaluate phenotypic development. In this report we have: (1) defined a detail reproducible baseline description of the temporal growth and developmental pattern of M. truncatula from cotyledon to early pod formation; (2) developed a standardized numerical nomenclature coding system that uses easily identifiable developmental growth stages to define plant growth; (3) identified plant morphological differences resulting from growth under nutrient stress conditions thereby illustrating how the nomenclature coding system can be used to discern phenotypic alterations; and (4)  documented timing of metamer production over a 40 day period thereby allowing assessment of alterations in developmental timing. We have included a decimal component to the system that divides metamer growth into nine sub-stages based on leaf or reproductive organ development (Table I). Additionally, a measurement component included in this system helps to distinguish growth alterations that may be overlooked by the coding system alone. The key to detection of subtle changes in growth is to be familiar with whole plant development and its plasticity, compare developmental timing of growth, then monitor and measure growth using various parameters. Regardless of which parameters are monitored, the examination of multiple parameters enhances the potential to detect true phenotypic differences resulting from mutations versus phenotypic plasticity (Coleman et al., 1994;Boyes et al., 2001). This coding system can also be used to standardize tissue collection. Documenting the detailed position from which tissue is harvested and analyzed provides a developmental reference point for comparative analyses across laboratories.
We have validated the effect of the coding system and its measurement component in detecting morphological changes in growth due explicitly to treatment effects over morphological plasticity. Using nutrient stress to modify whole plant development we simulated changes that may occur due to genetic lesions or other factors. Growth under N and P deprivation produced a number of morphological alterations that could clearly be documented using both the coding system and measurement component. Specifically, N and P deprived plants showed a delay in leaf development and expansion along the main and axillary axes of growth (Figs. 2, 3, 6; Table II); a delay in axillary shoot emergence and elongation (Figs 2, 3, 5; Table II); a decrease in leaf and shoot size and an alteration in root growth (Figs. 5,6,7; Table III). Furthermore, the timing and frequency of flower emergence in P-deprived plants were also affected ( Fig. 4; Table II). Both nutrient stress conditions produced similar growth effects, however, the timing, extent and location of these alterations differed depending upon the nutrient stress imposed.
Most of our results were compatible with and could be integrated into previously published studies that addressed the physiological, biochemical and genetic mechanisms of plant responses to nutrient stress. When the description of morphological development is integrated with the mechanistic data and viewed in a comprehensive manner, the relationship between function and morphological development can be better realized. For example, the sequential timing of shoot, root and reproductive structure development of M. truncatula grown under Pdeprivation was similar with reports on P and carbon (C) cycling, and remobilization in a variety of plant systems including Glycine max, Stylosanthes hamata, Phaseolus vulgaris, Triticum aestivum and Oryza sativa (Fredeen et al., 1989;Smith et al., 1990;Marschner et al., 1996;Snapp and Lynch, 1996;Peng and Li, 2005;Wissuwa et al., 2005). In our experiments with M. truncatula grown under P-deficient conditions, decreased shoot growth and development, detected by 14 dap, were among the first responses observed. The reduction in shoot size was consistent with previous published reports on the effect of P deprivation on plant development. The developing roots become a sink for P and photoassimilated C resulting in these nutrients being partitioned from the shoot to the root thereby compromising shoot development (Smith et al., 1990;Marschner et al., 1996;Wissuwa et al., 2005). Later in plant development, when flower initiation commences, the flowers/fruit become the major P and C sinks thereby shifting nutrient allocation from the shoot and root into flower/fruit development (Peng and Li, 2005). In our experiments, at 28 dap, just prior to flower emergence at 32 dap, there was a noticeable decline in root growth. The decreased root growth was consistent with reports that documented flower/fruit development as a new sink thereby causing the re-allocation of nutrients to these developing organs and compromising root growth prior to reproductive structure development (Peng and Li, 2005).
However, not all of our results were consistent with the existing literature.
For example, root architectural analysis of M. truncatula under P-deficiency deviated from that reported for Arabidopsis or P. vulgaris where low P conditions initially increased lateral root length (Lynch and Brown, 2001;Williamson et al., 2001;Al-Ghazi et al., 2003). When we grew M. truncatula under P-deficient conditions there were no detectable differences in root architecture between Pdeficient and control plants until 28 dap when P-deficient plants showed a decline in lateral root total length and number relative to control plants. Notably, the increase in lateral root growth reported in other systems was associated with the development of fine feeder-type of roots that are also vulnerable to damage upon removal from the growing medium (Hodge, 2004). In our study, some of these fine roots may have been damaged during the process of harvesting. Therefore, a more detailed study of M. truncatula root growth using non-invasive techniques and following growth over a shorter period of time would be useful in uncovering subtle spatial and temporal changes in root development due to P-stress.
Timing of development in this study has been described in terms of days after planting, with measurements taken at 4-7 day intervals. This means that our models can not reflect continuous growth of plant parts in detail, as is possible if more frequent measurements are made (Mundermann et al., 2005), but are interpolated linearly based on initiation time, maximum size, and growth duration.

MATERIALS AND METHODS
Medicago truncatula seeds of line A17 of cv. Jemalong were chemically scarified with concentrated sulfuric acid for 8 minutes and surface sterilized for 3 min with commercial grade bleach (5.25% sodium hypochlorite). Seeds were given a 2 day germination period at 4 o C then transferred to petri plates with moistened filter paper and given a 14 d vernalization period at 4 o C. Vernalized seeds with a radical length of 1-1.5 cm were planted in pots containing quartz sand. Pot size was 10cm x 10cm x 35cm. Plants were grown at 24-26 o C and a 16h photoperiod (http://www.isv.cnrs-gif.fr/embo01/manuels/pdf/module1.pdf) with a light intensity at the sand surface of 430 -490 µmol . m -2 sec -1 . Plants were fertilized 3x per week (150 ml of nutrient solution per fertilizer application) with the following  , 7, 11, 14, 18, 21, 25, 28, 32, and 40 days after planting (dap). Five seedlings for each treatment were selected for continuous monitoring of shoot growth. These five plants were photographed and various parameters were measured throughout the 40 day period. Plant growth parameters measured included shoot and root fresh and dry weights, shoot and root total lengths, leaf size, and internode lengths. An additional set of plants was grown in parallel for collection and processing of tissue. Total P analysis was done on dried shoot tissue of P-deficient and control plants harvested at 40 dap.
Tissue P content was analyzed by optical emission spectroscopy (OES-ICP).
Three replicated experiments were performed. Images were taken with a Sony DSC-D770 digital camera. Growth angles were calculated from the digital images. All other parameters were measured using a handheld ruler.
An empirical model of M. truncatula development capturing differences in plant structure due to the different nutrient treatments was developed using the L-system-based L-studio software from the University of Calgary (Prusinkiewicz et al., 2000). L-systems are a formalism for describing plant development (Lindenmayer 1968) that can support a wide range of empirical and mechanistic modeling approaches (Prusinkiewicz, 2004;Mundermann et al., 2005). In an Lsystem, plant components are represented by an alphabet of symbols with associated parameters. These are arranged in a string that represents the plant structure, with branching topology imposed by a hierarchy of brackets. Growth and development are captured by applying production rules, which describe the changes in components on a daily basis in this case, to all symbols in the current string to produce a new string representing the plant on the next day. The application of these rules allows development to be visualized in schematic form ( Fig. 1)  To statistically examine developmental scale differences between the treatments, the Levene's test was first performed to satisfy the parametric ANOVA (Analysis of Variance) test assumption of homogeneity of variance and normality. A single-factor ANOVA was used to analyze developmental scale treatment differences. If a significant F-test statistic was obtained, then comparisons between each treatment and the control were performed using a Dunnett's test. Additionally, t-tests for unequal replication and equal or unequal variances were used for the analysis of leaf and flower developmental scale differences and shoot and internode length differences. For most analyses differences were considered to be significant at p< 0.05, but in some instances p< 0.10 was considered appropriate for distinguishing treatment differences.
Data represent the mean of three independent experiments + standard error (SE).    color-coded as shown in the legend. Time of axillary shoot appearance is depicted by an asterisk ( * ), color-coded according to its metamer of origin. Time of flower appearance is indicated by a yellow oval. The nomenclature code of most metamers, at completion of growth, is labeled and indicated with an arrow.     conditions. Roots stained with basic fuschin. Magnification is 20x. Scale bar = 1mm.