LTP2 hypomorphs show genotype-by-environment interaction in early seedling traits in Arabidopsis thaliana

Abstract

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LTP2 greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs, and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

Keywords: LTP2; cuticle integrity; gene-by-environment interaction; hypocotyl; hypomorph; lipid transfer protein; non-genetic variation; phenotypic robustness; phenotypic variation; stochasticity.

Figure 1.. LTP2 hypomorphs increase phenotypic variation in skotomorphogenesis traits.

A. Approximate location of the T-DNA insertion (LB) relative to the LTP2 gene in ltp2-1 and ltp2-2 lines. B. LTP2 relative expression in the shoots of wild-type (Col-0), ltp2-1 and ltp2-2 seedlings grown in the dark for 7 days (d) after stratification. In parenthesis are the % of Col-0 LTP2 transcript levels detected in the ltp2 lines. C. Representative image of Col-0 (top), ltp2-1 (middle) and ltp2-2 (bottom) dark-grown seedlings at 7 days after stratification; the roots were trimmed. D. Density lines of the distributions of hypocotyl length scored at 7 days post-stratification in Col-0 and ltp2 seedlings grown in MS media with 1% sucrose; each line represents the merged distribution of 5 biological replicates (n=78-210 each) per genotype. E. Coefficient of variation (sd/mean) in hypocotyl length, a measure of variation, for (1) 5 biological replicates (open circles) and (2) merged for all 5 replicates (filled circle, same data as in D). F. Hypocotyl negative gravitropism measured as the deviation from vertical (in degrees) of the hypocotyl apex in 7d dark-grown seedlings grown on MS+1% sucrose. The density distribution of measured angles for all genotypes is plotted. G. Comparison between the hypocotyl length of individual ltp2 parents (5 long and 5 short) and the mean hypocotyl length of their offspring; all lengths measured at 7d post-stratification on seedlings grown on MS+1% sucrose. H. Beanplots of hypocotyl (left side) and root (right side) length from the merged dataset used in D and E. I. Percentage of dark-grown seedlings with an open cotyledon phenotype, in wild-type (Col-0), ltp2-1 and ltp2-2 genotypes, at 7 days after stratification on MS+ 1% sucrose. Two replicates are shown (n=79-208).

Figure 2.. Sucrose-dependent loss of cuticle integrity in ltp2 is associated with short hypocotyls.

A. Density lines of the distribution of hypocotyl length for seedlings grown on plates containing MS medium, MS+1% glucose or MS+1% sucrose for 7 days post-stratification. Shown are the merged distributions of two biological replicates per condition/per genotype. B. Coefficient of variation in hypocotyl length for the distributions shown in A. C. Representative shoots of 7d dark-grown seedlings grown on plates containing MS medium, MS+1% glucose or MS+1% sucrose. Hypocotyl negative gravitropism is largely rescued in ltp2 seedlings grown without sucrose. D. Boxplots comparing the hypocotyl/root ratio for every seedling in A across different growth media. E. Magnified images of the epidermal surface of Col-0 and ltp2-1 hypocotyls at 7d post-stratification when grown with (left) or without (right) 1% sucrose. F. Comparing the effects of 1% sucrose on hypocotyl cuticle permeability: dark-grown seedlings of Col-0 and ltp2-1 were stained with toluidine blue after growing for 5 days on MS+1% sucrose (left) or 7 days on MS alone (right). G. Relationship between hypocotyl length at 7 days after stratification on MS+1% sucrose and toluidine blue staining coverage expressed in % of hypocotyl length. Filled dots represent seedlings with the wild-type developmental pattern of hypocotyl length > root length.

Figure 3.. Hypocotyl length is associated with many transcriptional differences in ltp2 but not Col-0 seedlings, mostly related to stress, defense and growth.

A. Experimental setup for collecting RNA-seq samples. For Col-0 and ltp2-1, the first boxplot shows the distribution of hypocotyl length from one out of seven hypocotyl assays done per replicate; the second set of four boxplots (two replicates with short and two replicates with long hypocotyls) shows the actual distribution of hypocotyl lengths (n=70) from the selected individuals used to generate RNA-seq libraries. B. Biplot of Principal Component Analysis showing the first two PCs. The percentage of the total variance explained by PC1 and PC2 is indicated on the top right corner. C. Correlation between mean hypocotyl length for the eight RNA-seq samples and PC1 loadings; shown on the bottom left corner is the Spearman rho. D. Shortlisted GO enrichments (fold-enrichment >=2 and adjusted p-value < 0.05) for the set of 584 differentially expressed genes between ltp2-L and ltp2-S, split by down- and upregulated genes. Shown are the log10(fold-enrichment) and the number of DEGs per enriched term. E-F Row-scaled heatmap visualizations of a subset of DEGs downregulated (E) or upregulated (F) in ltp2-S relative to ltp2-L. Pink dots indicate whether each gene was also a DEG on other pairwise-comparisons.

Figure 4.. The ltp2 phenotype depends on the expression ratio of LTP2 and its close paralog LTP1

A. Row-scaled heatmap visualization of cuticle-related genes differentially expressed between Col-0 and ltp2 samples. B. Detail of a tree depicting amino acid sequence similarity among PR14/Type I LTP proteins (full tree in Figure S5A). C. Relative expression of LTP1 and LTP2 in the shoots of light or dark-grown (gray box) Col-0 and ltp2 seedlings grown for 7 days after stratification on MS+ 1% sucrose. D. Comparison of the LTP1/LTP2 expression ratio between Col-0, ltp2-1 and an ltp1ltp2 double mutant. E-G Hypocotyl length distribution (E), coefficient of variation of hypocotyl length (F) and (G) hypocotyl/root ratio for Col-0, ltp2-1 and an ltp1 ltp2 double knockdown; all seedlings were grown in the dark for 7d after stratification on MS+ 1% sucrose. H. Representative images of toluidine blue stained etiolated hypocotyls from Col-0, ltp2-1 and an ltp1 ltp2 double mutant grown for 7 days after stratification on MS+ 1% sucrose