Stress can be remembered by plants in a form of stress legacy that can alter future phenotypes of previously stressed plants and even phenotypes of their offspring. DNA methylation belongs among the mechanisms mediating the stress legacy. It is however not known for how long the stress legacy is carried by plants. If the legacy is long lasting, it can become maladaptive in situations when parental-offspring environments do not match. We investigated for how long after the last exposure of a parental plant to drought can the phenotype of its clonal offspring be altered. We grew parental plants of three genotypes of Trifolium repens for five months either in control conditions or in control conditions that were interrupted with intense drought periods applied for two months in four different time-slots. We also treated half of the parental plants with a demethylating agent (5-azaC) to test for the potential role of DNA methylation in the stress legacy. Then, we transplanted parental cuttings (ramets) individually to control environment and allowed them to produce offspring ramets for two months. The drought stress experienced by parents affected phenotypes of offspring ramets. The stress legacy resulted in enhanced number of offspring ramets originating from parents that experienced drought stress even 8 weeks before their transplantation to the control environment. 5-azaC altered transgenerational effects on offspring ramets. We confirmed that drought stress can trigger transgenerational effect in T. repens that is very likely mediated by DNA methylation. Most importantly, the stress legacy in parental plants persisted for at least 8 weeks suggesting that the stress legacy can persist in a clonal plant Trifolium repens for relatively long period. We suggest that the stress legacy should be considered in future ecological studies on clonal plants.

Stress can be remembered by plants in a form of ‘stress memory’ that can alter future phenotypes of previously stressed plants and even phenotypes of their offspring. It was shown that DNA methylation is among the mechanisms mediating the memory. It is not known for how long the memory is kept by plants. If the memory is long lasting, it can become maladaptive in situations when parental-offspring environment differ. We investigated for how long can a parental plant “remember” that it experienced a stress and pass the memory to its clonal offspring. We grew parental plants of three genotypes of Trifolium repens for five months either in control conditions or in control conditions that were interrupted with drought pulses applied for two months in four different time-slots. We also treated half of the parental plants with 5-azacytidine (5-azaC) to test for the potential role of DNA methylation in the stress memory. Then, we transplanted parental cuttings (ramets) individually to control environment and allowed them to produce offspring ramets for two months. The drought stress experienced by parents affected phenotypes of offspring ramets. Such a memory resulted in enhanced number of offspring side branches originating from plants that experienced drought stress maximally 6 weeks before their transplantation to control environment. We did not find any transgenerational memory in offspring of plants that experienced drought stress later than 6 weeks before their transplantation. 5-azaC also reduced the effect of transgenerational memory on offspring ramets. We confirmed that drought stress can trigger transgenerational memory in T. repens that is very likely mediated by DNA methylation. Most importantly, the memory was time limited and was gradually erased. We conclude that the time limited memory on environmental stress can be adaptive as climate tends to be variable and parental-offspring environmental conditions often do not match.