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Abstract The 3’ end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plantsArabidopsis thalianaandZea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators. 

Summary 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 inArabidopsis thaliana, we found that hypomorphs of the cuticle‐related geneLIPID TRANSFER PROTEIN 2(LTP2) greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Manyltp2hypocotyls were significantly shorter than wild‐type hypocotyls while others resembled the wild‐type.Differences in epidermal properties and gene expression betweenltp2seedlings 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 inltp2hypomorphs and found that increased expression of its closest paralogLTP1is necessary forltp2phenotypes.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.