Supplement to Marius C. Codrea, Marja Hakala-Yatkin, Anna Kårlund-Marttila, Ladislav Nedbal, Tero Aittokallio, Olli S. Nevalainen and Esa Tyystjärvi: Mahalanobis Distance Screening of Arabidopsis Mutants with Chlorophyll Fluorescence, published in Photosynthesis Research (2010)

Contents:

Table S1

Practical application of the method

(Three R scripts are provided as separate text files)

Table S1. Effects of the different parameter selections on the percentage of plants of different strains detected as outliers. ALL 46 = all parameters; 240 µE = parameters only from the highest PPFD of the treatment of the plants; NINE = nine selected parameters; NO NPQ = all other parameters except NPQ values; PHYSIOL = only physiologically relevant parameters. See Table 2 for a detailed description of the parameter sets.

Strain / Parameters used for detection of outliers / Description of the strain
Number of plants / ALL 46 / 240 uE / NINE / NO NPQ / PHYSIOL
N3772 / 9 / 100.0 / 0.0 / 0.0 / 44.4 / 88.9 / Zeaxanthin epoxidase deficient (At5G67030)
N17 / 12 / 100.0 / 91.7 / 83.3 / 91.7 / 100.0 / Digalactocylglycerol deficient (At3G11670)
N155 / 2 / 100.0 / 0.0 / 0.0 / 100.0 / 100.0 / ABA deficient (At5G67030)
N156 / 9 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / ABA deficient (At1G52340)
N6169 / 5 / 100.0 / 100.0 / 100.0 / 100.0 / 100.0 / Lighter color and slower growth; chlorotic in high CO2
npq4-1 / 34 / 85.3 / 88.2 / 82.4 / 85.3 / 2.9 / Lacks a functional PsbS protein (At1G44575)
N595156 / 9 / 88.9 / 90.0 / 90.0 / 88.9 / 10.0 / T-DNA insert in the At1G44575 gene coding for the psbS protein
N89530 / 3 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Single M3 TILLING line; lesion in the At1G44575 gene coding for the psbS protein
npq1-2 / 12 / 33.3 / 33.3 / 0.0 / 8.3 / 8.3 / Violaxanthin de-epoxidase deficient (At1G08550)
rwt46 / 21 / 66.7 / 45.5 / 18.2 / 40.9 / 66.7 / Only largeisoform of Rubisco activase
rwt43 / 15 / 46.7 / 53.3 / 35.7 / 35.7 / 28.6 / Only small isoform of Rubisco activase
Rca / 14 / 92.9 / 64.3 / 57.1 / 71.4 / 85.7 / Rubisco activase deficient
R100 / 9 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Rubisco activase antisense line (30-40% of WT); ecotype RLD
N3819 / 4 / 75.0 / 0.0 / 0.0 / 0.0 / 50.0 / UV-hypersensitive (At5G41150)
N6217 / 9 / 66.7 / 33.3 / 33.3 / 22.2 / 66.7 / Phytochrome B deficient (At2G18790)
N2355 / 3 / 33.3 / 0.0 / 0.0 / 0.0 / 33.3 / Reduced nitrate reductase activity (At1G37130)
N8326 / 11 / 9.1 / 0.0 / 0.0 / 0.0 / 0.0 / Ascorbic acid deficient (At2G39770)
N8156 / 4 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Chlorotic leaves (At2G20610)
N3889 / 5 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Lhca2 antisense line
N3892 / 4 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Lhca3 antisense line
N6362 / 12 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Lhcb2 antisense line
N6363 / 6 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Lhcb2.1 antisense line
N3354 / 4 / 0.0 / 0.0 / 25.0 / 0.0 / 0.0 / Chl b deficient (At1G44446)
N6204 / 7 / 0.0 / 0.0 / 0.0 / 0.0 / 28.6 / CAB expression affected (At3G02260)
N6147 / 5 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / Glucose insensitive (At1G52340)
N3418 / 4 / 100.0 / 100.0 / 100.0 / 100.0 / 100.0 / Chlorotic spots in leaves
N3530 / 1 / 100.0 / 100.0 / 0.0 / 100.0 / 100.0 / Thiamine requiring (At1G22940)
N6128 / 5 / 60.0 / 20.0 / 20.0 / 40.0 / 60.0 / Increased accumulation of anthocyanins in the cotyledons and reduced chlorophyll content (At3G26790)
N89558 / 5 / 60.0 / 40.0 / 40.0 / 40.0 / 40.0 / Single M3 TILLING line; lesion in the At1G02280 gene coding for a translocon at the outer chloroplast envelope membrane
N559206 / 12 / 41.7 / 25.0 / 9.1 / 5.9 / 16.7 / T-DNA insertion in At5G05000 gene coding for a translocon at the outer chloroplast envelope membrane
N562094 / 1 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / T-DNA insertion in At3G46740 gene coding for a translocon at the outer chloroplast envelope membrane
N8507 / 2 / 50.0 / 50.0 / 50.0 / 50.0 / 50.0 / Severe phosphorus deficiency in leaves; dark green leaves with purple petioles (At3G23430)
N509166 / 5 / 20.0 / 20.0 / 20.0 / 20.0 / 20.0 / T-DNA insertion in gene coding for the SigC sigma factor of chloroplast RNA polymerase (At3G53920)
N524720 / 11 / 9.1 / 0.0 / 0.0 / 0.0 / 9.1 / T-DNA insertion in gene coding for the PSII OEC33 protein (At3G50820)
N3733 / 17 / 5.9 / 0.0 / 0.0 / 0.0 / 5.9 / Sensitive to mechanical stress in a developmentally controlled manner; susceptible to opportunistic pathogens and exhibits decreased growth inhibition of a heterologous pathogen of bean (At3G44880)
N562136 / 9 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0 / T-DNA insertion in the At3G17040 gene; the plants have a high fluorescence phenotype.
N515134 / 7 / 14.3 / 0.0 / 0.0 / 0.0 / 16.7 / T-DNA insertion in a nuclear copy of the chloroplast gene coding for the large subunit of Rubisco (AtCG00490)
N533879 / 18 / 22.2 / 5.6 / 5.6 / 5.6 / 27.8 / T-DNA insertion in a nuclear copy of the chloroplast gene coding for cytochrome f apoprotein (genes At2G27990andAtCG00540)
RLD / 15 / 6.7 / 0.0 / 0.0 / 0.0 / 0.0 / An arabidopsis ecotype
WT (incl. RLD) / 365 / 86.8 / 96.5 / 98.2 / 98.2 / 90.6 / Both ecotypes (Columbia and RLD)

Practical Application of the Method

The method can be used with the free R software (R Development Core Team 2009). The supporting information contains three R scripts. All scripts assume that the user has a set of fluorescence curves and each curve is located in (x,y) plane. Script "fit.txt" contains functions for calculating a linear and an exponential fit using a time series of fluorescence values as input data. The two other scripts use feature files as input data; a feature file is defined as a file containing, on each line, x and y coordinates followed by the values of features. Each line must contain the same number of features. (x,y) values of background pixels should not be included. Script "make_wt_feature_file.txt" uses three separate feature files containing wild-type data and implements the leave-one-out procedure described above to write a file containing the final wild-type distribution. The maximum SD value which determines the validity of a wild-type feature vector, is adjustable, and the default is 1.96. Script "make_score_pictures.txt" produces a score picture from each feature file. The parameter steepness can be determined freely (default = 0.0005, see Fig. 4a). Instead of specifying the threshold value directly, the user can specify a threshold-percentage value (default = 0.5) and the script calculates the corresponding threshold value so that the threshold-percentage of the MD values of the final wild-type distribution exceed this MD (in the default condition, 0.5 % of wild-type feature vectors have MD higher than threshold).