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Comparative Study
. 2010 Apr;152(4):2142-57.
doi: 10.1104/pp.109.148338. Epub 2010 Mar 3.

Probing the Reproducibility of Leaf Growth and Molecular Phenotypes: A Comparison of Three Arabidopsis Accessions Cultivated in Ten Laboratories

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Free PMC article
Comparative Study

Probing the Reproducibility of Leaf Growth and Molecular Phenotypes: A Comparison of Three Arabidopsis Accessions Cultivated in Ten Laboratories

Catherine Massonnet et al. Plant Physiol. .
Free PMC article

Abstract

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype x environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

Figures

Figure 1.
Figure 1.
Macroscopic kinematic leaf growth phenotypes. A, Changes with time of RA. B, Changes with time of rosette LN. C, Individual final leaf area according to leaf nodal position in the rosette. The increase of RA and rosette LN is described by y = A/[1 + e (−(XX0)/B)]. The inset in A shows mean and sd values of maximal rate and duration of rosette leaf expansion (Rmax in mm2 d−1 and duration in d), and the inset in B shows mean and sd values of maximal rate and duration of leaf production (Rmax in leaf no. d−1 and duration in d). Lowercase letters indicate significant differences (P < 0.05). ns, No significant difference.
Figure 2.
Figure 2.
Microscopic kinematic leaf growth phenotypes. A, Epidermal CD in leaf 6. B, Epidermal CN in leaf 6. C, Mean epidermal CA in leaf 6. All data were collected from the sixth leaf of the rosette. The decline of epidermal CD is described by y = D + [(AD)/(1 + 10(X−log(C))B)], and the increases of epidermal CN and CA are shown by y = A/[1 + e (−(XX0)/B)]. The inset in B shows mean and sd values of maximal rate and duration of cell production (Rmax in cell no. d−1 and duration in d), and the inset in C shows mean and sd values of maximal rate and duration of cell expansion (Rmax in μm2 d−1 and duration in d). ns, No difference.
Figure 3.
Figure 3.
Leaf growth phenotypes of three genotypes across nine independent laboratories. Data are represented as box plots with a representation of the first, median, and third quartiles in the box. A to C, RA. D to F, Sixth leaf area. G to I, Epidermal CD in the sixth leaf. Laboratories (L1–L9) with significantly different phenotypes are noted with asterisks for each genotype and each variable (ANOVA and Tukey's posthoc test results). Mean and sd values of homogenous groups are represented on each plot by dashed and dotted lines, respectively. A core group of four laboratories with similar results for all genotypes and at the different organizational levels was identified: L2, L3, L8, and L9.
Figure 4.
Figure 4.
Phenotype ranking per genotype across laboratories. Mean values of RA (A), sixth leaf area (B), and epidermal CD in the sixth leaf (C) calculated per genotype and per laboratory are shown. L1, White circles; L2, black circles; L3, black squares; L4, white squares; L5, white diamonds; L6, white lower triangles; L7, white upper triangles; L8, black upper triangles; L9 black lower triangles. The core laboratories (L2, L3, L8, and L9) are represented by black symbols and straight lines and the others with white symbols and dashed lines. For each variable, ANOVA was performed either on the data including all nine laboratories (italics) or the core laboratories (boldface) to evaluate laboratory (L), genotype (G), and interaction (L×G) effects. Asterisks indicate significant differences (*** P < 0.001, * P < 0.05), and ns indicates the absence of a significant difference.
Figure 5.
Figure 5.
PCA of leaf growth variables (rosette LN, RA, AL6, epidermal CD in leaf 6, and mean epidermal CA in leaf 6; A–C) and metabolic profiles (D–F). Graphs represent data averaged from all laboratories (A and D) or individual replicates from L5 (B and E) and L2 (C and F). Colors indicate the genotype: Col, black; Ler, red; Ws, green. Numbers identify laboratories.
Figure 6.
Figure 6.
Histograms of P values from an ANOVA of all metabolites with the factors genotype (A) and laboratory (B).
Figure 7.
Figure 7.
Box plots of genome-wide correlation coefficients for gene expression values in replicate samples. A, Intralaboratory correlation coefficients. B, Interlaboratory correlation coefficients.
Figure 8.
Figure 8.
PCA of gene expression data based on Col samples only (A), based on Col, Ler, and Ws samples (B), and based on Col, Ler, and Ws samples averaged per laboratory (C). Colors indicate the genotype: Col, black; Ler, red; Ws, green. Numbers identify laboratories.

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