Comparison of Arabidopsis growth analysis using traditional methods versus imaging techniques G.M. Estavillo1, D. Harris-Pascal1, M. Eberius3, R. Furbank2 and B.J. Pogson1 1The ARC Centre of Excellence in Plant Energy Biology, The Australian National University, 2CSIRO Plant Industry , Canberra, Australia, and 3Lemnatec GmbH, Würselen, Germany ABSTRACT Growth analysis using simple primary data can be used to study plant morphology and function. The classical, destructive method involves tissue harvesting and estimation of different plant physical parameters such as leaf area and weight at regular intervals. An alternative method is the use of non invasive techniques based in image recording and software analyses that allows for periodical sampling in a non destructive way. In theory, this approach requires less starting material than the former, and the growth of the same individual can be documented through the life cycle or experimental conditions. It allows also for analysis of many other structural and morphological traits of a large number of plants. Although the intrinsic advantages of the imaging system, the results should be compared with those of traditional techniques to assess its utility in specific cases. Here we analyze and compare the results of plant growth analyses of wild type and mutant Arabidopsis plants performed with the traditional method versus morphological phenotyping with LemnaTec Scanalyzer 3D (Lemnatec). The results suggest that the imaging technique is a powerful tool to identify plants based on morphological parameters and allows for dynamic phenotyping over time. Gonzalo M. Estavillo; 61 2 6125 2663; gonzalo.estavillo@anu.edu.au 22/06/07

(a) (b) (c) Columbia alx8 fry1-1 C24 Figure 1: Imaging of Arabidopsis plants using LemnaTec HTS Bonit Scanalyzer. (a) Digital colored image of 35 day old wild type (Columbia and C24) and mutant (alx8, Rossell et al., 2006, and fry1-1, Xiong, et al., 2001) Arabidopsis plants obtained with Scanalyzer (LemnaTec). (b) Color classified or false colored image. Three different color classes were determined based on visual examination of the live figure using the LemnaTec Bonit HTS software. (c) Quantification of leaf color classes. Scanalyzer allows for rapid and consistent high throughput imaging of Arabidopsis plants once the image capture configurations (i.e., camera, vessel, etc) have been set. The color classification of the different objects performed by the analyzing software provides a summary of morphological features such as tissue color classes, leaf area, etc. Different tissue coloration can be quantified using color classifications. This classification could provide important information when correlated with other measurable parameters such as amount of photosynthetic pigments.

(a) (b) Figure 2: Comparison of leaf area measurements between destructive method and imaging technique. (a) Trays containing 5 individuals of wild type and mutant plants were pictured once a week and total leaf area was measured using the image analysis software Scanalyzer (left). Leaves from the same plants were detached, scanned and the leaf area measured with Leafarea software (http://www.shef.ac.uk/~nuocpe/ucpe/leafarea.html) (right). (b) Correlation of leaf area measurements between non-invasive imaging technique and destructive harvest. Total leaf area for Columbia (left) and alx8 (right) plants obtained using the imaging technique and traditional harvest prior to leaf scanning were plotted against each other. Correlation coefficients (a) close to 1 indicate that the leaf area values obtained with both methods are very similar for both types of plants, despite the apparent leaf overlap present in alx8 mutant.

Figure 3: Comparison of relative growth rates (RGR). (b) Figure 3: Comparison of relative growth rates (RGR). (a) Relative growth rate using leaf are obtained with Scanalyzer. (b) Relative growth rate with leaf are obtained with destructive harvest. Total leaf area of each individual (n=5) for each plant line were used to calculate the relative growth rate (RGR=(Ln Area 2) - (Ln Area 1) /7). RGR values based on leaf area calculated with the imaging technique are lower than the ones obtained with leaf area measurements for detached leaves of 2-week old seedlings. This is probably due to an overestimation of leaf area by the imaging software for small plants as some pixels might not been excluded. This problem is more relevant for younger plants and it can be solved by further improving the imaging process using both color classification and object recognition. The RGR values for weeks 4 and 5 are very similar for both methods.

(a) (c) (b) (d) Figure 4: Grouping of Arabidopsis plants based on the correlation of morphological parameters. Sets of morphological parameters from 5-week old Arabidopsis plants were obtained with LemnaTec Bonit HTS software and plotted against each other. (a) Leaf Area vs Eccentricity. the architecture of mutant plants is more asymmetric (higher eccentricity) than the that of wild type and independent of size. (b) Leaf Area vs Compactness. Compactness refers to the positioning of the leaves relative to the center of the plant, and it is calculated based on the normalized rotational momentum, irrespective of size. Mutant plants group together, showing that their leaves are closer to the center compared to the ones of wild type plants, and this is independent of their size. It is also worth noting that this correlations also segregates Columbia and C24 wild type plants into two groups. (c) Surface Coverage vs Eccentricity: While C24 and Columbia show a high symmetry (low eccentricity) with different surface coverage the mutants combine both relatively high coverage with much lower symmetry (high eccentricity). Reasons for loss of symmetric growth will be interpreted biologically based on dynamic data in the future. (d) Surface Coverage vs Leaf Area: The graph shows that the leaves of mutant and C24 plants cover the surface underneath their growing area (a thought circle around he plant) much more than leaves of Columbia plants.

(a) (c) Columbia alx8 fry1-1 C24 Growth rate: relative growth measured using total leaf area. Eccentricity: degree of deviation of a conic section from being circular. Compactness: positioning of the leaves relative to the centre. Roundness (or “Stockiness”) Surface Coverage (d) (b) Columbia alx8 fry1-1 C24 Figure 5: Phenotyping of Arabidopsis plants based on morphological features. (a) Color classified image of 5 week-old Arabidopsis plants obtained with Scanalyzer. (b) Polar graphs representing five measured parameters of plant growth: RGR, eccentricity, compactness, roundness, and surface Coverage. (c) Features of the polar graph representation. The data was normalized to the highest value in the series (axes scale is 0 to 1). (d) Polar graphs showing the change of morphological features for the four plant lines over a period of three weeks. The combination of the different morphological parameters obtained by the software from recorded images provides important information of changes in phenotype over time. This allows for plant grouping based on quantifiable morphological features that can be used for high throughput screening of plant populations.