Transgenerational decline through insidious effects of drought memory

Climate change increases the occurrence of drought stress, which challenges the sustainability of agroecosystems, forests, and gardens and impairs plant productivity. Nonetheless, repeated drought episodes may train a single plant to better withstand subsequent water deficit (Ding et al., 2012) – a phenomenon referred to as priming, hardening, or acclimation (Gallusci et al., 2023; Harris et al., 2023). There is also evidence that plants may be trained across generations, which requires a transmittable form of stress ‘memory’. This concept has gained biological support with the discovery of molecular mechanisms underlying stress memory, including epigenetic modifications that may be transmitted to the progeny (Crisp et al., 2016). Sexual reproduction resets most of these modifications, and the few examples of transgenerational drought memory show little to no impact on integrated phenotypes such as yield (Liu et al., 2022; Gallusci et al., 2023). Clonal reproduction – the dominant propagation mode in horticulture, arboriculture, and many ecosystems – is more likely to retain epigenetic modifications or any morpho/physiological change involved in stress memory. A pioneering study in poplar showed that clones propagated at distinct nurseries for several decades diverged in their transcriptome and physiological response to drought when grown in a common garden (Raj et al., 2011). In white clover, clonal offspring are slightly less sensitive to drought when the parent has also been exposed (Rendina González et al., 2017). To our knowledge, whether effects of drought memory may accumulate across numerous generations remains largely unexplored. Here, we tested whether training petunia plants to drought, through recurrent exposure to water deficit within and across clonal generations, may provide them with transgenerational and adaptive drought memory. We exposed petunias to recurrent water deficit for > 25 clonal generations, but trained plants did not outperform naive plants under drought and started declining after c. 15 generations, ultimately leading to extinction. Our results emphasize an underappreciated vulnerability of plants to recurrent drought through insidious effects that can be transmitted clonally. Starting from one single plant of petunia (Petunia × hybrida, generation #00), we propagated two clonal lines through cuttings for more than 25 generations. The two lines were grown together in the same growth chamber with all environmental conditions being identical, except for the irrigation regime. We generated a ‘naive line’ exposed to a soil water potential always above −100 hPa (sub-irrigation about once a day), corresponding to well-watered conditions, as well as a ‘trained line’ recurrently exposed to less than −500 hPa (sub-irrigation every 4 d), corresponding to chronic drought conditions around the wilting point (Supporting Information Fig. S1). After c. 1 month in these contrasting conditions, plants were regenerated through cuttings, subjected again to their assigned watering regime and so forth (see the Materials and Methods section). We first assessed the behavior of both lines maintained in their irrigation regime. We observed morphological and physiological effects of water restriction that were consistent across successive generations. Growth was obviously affected by chronic water deficit (Fig. 1a): shoot biomass was reduced on average by 50% in dry weight and in fresh weight across generations #02 to #10 (Fig. 1b; Fig. S2a). Shoot branching, shoot apparent area, flower area, and root development were all negatively affected by water restriction (Fig. S2b–e). Leaves of well-watered petunias showed high water content (94–95%; Fig. 1c). Chronic water deficit significantly reduced leaf water content but only by c. 1–3%, while the plants here had not been irrigated for 3–4 d (Fig. 1c). These results suggest isohydric behavior, with a tight regulation of tissue water status typically mediated by stomatal control (Tardieu & Simonneau, 1998; Hochberg et al., 2018). Consistent with this premise, analysis of gas exchange showed a consistent reduction of stomatal conductance under chronic water deficit, at the expense of photosynthesis (Fig. 1d,e). Moreover, when both lines were subjected to soil dehydration, shoot water potential decreased more rapidly in the naive line than in the trained line because naive plants were larger and thus consumed more water, but after 3 d of dry-down, water status was similar between lines due to feedback of stomatal regulation (Fig. 1f). To test for evidence of drought memory, we then performed a series of reciprocal transfer experiments. On generation #08, after a post-rooting period of 1 wk with all plants in well-watered conditions, we placed a batch of naive plants under water restriction and a batch of trained plants under well-watered conditions. We found no conspicuous effect of the clonal history on plant traits, neither in water deficit nor in well-watered conditions. There were no significant differences in shoot dry biomass, fresh biomass, branching, apparent area, or flower area between the naive and trained lines exposed to the same irrigation regimes during 3 wk (Figs 1g, S3a–d). Similar results were obtained from generations #09 and #10. We also measured the dynamics of leaf water content as water was withheld in the four conditions, corresponding to the first drought event for the well-watered plants. There was no significant difference between lines as soil desiccation progressed (Fig. 1h, three-way ANCOVA, Line × Time interaction). At incipient wilting, there was no difference in shoot water potential between the naive and the trained line (Fig. S3e). Overall, there were no strong beneficial or detrimental effects of the training regime experienced throughout 8–10 generations. Closer examination of the phenotypes revealed slight evidence of drought memory, but we did not find evidence it was adaptive. On generation #15, we compared the dynamics of response to the installation of chronic water deficit in naive and trained plants, through exposure to successive drought cycles. Biomass accumulated similarly between lines as the number of drought cycles progressed, yet the naive line performed slightly better (Fig. S4a). Total leaf area was also comparable between lines (Fig. S4b), but the trained line developed more leaves of smaller size compared to the naive line (Fig. 1i,j), suggesting plant morphology expressed a form of drought memory. Leaf water content in the trained line was slightly higher (by c. 1%), but no different behavior during soil desiccation was observed for this trait between the lines (Fig. 1k, three-way ANCOVA, Line × Time interaction). The same was true for quantum yield in darkness or light (Fig. S4e,f). Similar conclusions could be drawn from generation #17 (Fig. S5). Moreover, starch and sugar contents were not depleted in either line, even after 6 d of withholding water (Fig. 1l, generation #18). Reciprocal transfers at generations #20-24 showed virtually identical stomatal responses to light and leaf excision between the trained and the naive lines (Fig. 1m). No significant difference in net assimilation or intrinsic water use efficiency was observed either (Fig. S6). We noticed, however, that the trained line showed enhanced leaf thickness and a trend for higher mesophyll porosity (Figs 1n, S7a,b), also observed upon short-term drought hardening in tobacco (Khan et al., 2020). Compared to the naive line, leaf mass per area (LMA) was similar or higher in the trained line depending on the generation (Fig. S7c). These minor physiological and anatomical adjustments in the trained line did not translate into detectable changes in whole-plant morphology or water use efficiency when deficit irrigation was imposed in glasshouse conditions (Fig. S8). Instead, and not meeting our expectations, chronic exposure to drought across generations led insidiously to the extinction of the trained line. Line propagation started to become challenging, with more and more plants of the trained line showing impaired rooting, bud bursting, or subsequent growth: only 28% of cuttings collected from the trained line of generation #19 generated a viable plant for generation #20, most of them growing very slowly (Fig. S9). We then systematically scored the cutting success rate of subsequent generations, showing a gradual decline in the trained line up to generation #27, after which it was eventually extinct (Fig. 1o). We also established two new lines by switching their irrigation regimes. On the one hand, we generated a ‘late-trained line’ from cuttings taken on naive plants of generation #19 and then exposed to the same chronic drought as the trained line. This late-trained line could be easily propagated, and no decline was observed after 10 novel generations under chronic drought (Fig. 1o). Reciprocally, we built an ‘escaped line’ from cuttings of the trained line taken on generation #19 and then exposed to well-watered conditions as in the naive line. Strikingly, this line could not be propagated for more than 4 generations, suggesting the decline of the trained line was not reversible and even worsened upon return to conditions that are normally favorable (Fig. 1o). Transgenerational legacy of chronic exposure to sub-lethal drought therefore translated into a poor capacity for plantlet establishment at the cutting stage, which ultimately led to a gradual loss of the trained line. Since the escaped line died before the trained line, it was challenging to evaluate whether transgenerational drought memory was truly maladaptive. Chronic drought rather led petunia plants to a cul-de-sac, whereby firmly established memory of a predictable stress yielded plasticity with limited benefits under drought, but at other costs (e.g. impaired capacity for vegetative propagation) that were rapidly fatal upon return to well-watered conditions. Retrospectively, we could trace back the initiation of the decline of the trained line around generation #17, when we first noticed a small yet significant reduction in biomass, even before any stress application (Fig. S5a). This decline was unlikely driven by drought-induced starvation because carbohydrates and LMA maintained high levels in the trained line (Figs 1l, S7c), consistent with growth being more sensitive to water deficit than photosynthesis (McDowell, 2011; Muller et al., 2011). This is also in line with studies on adventitious rooting in petunia cuttings that emphasized the prominent role of sink establishment over source limitation (Ahkami et al., 2009, 2013). It is also unlikely that osmotic adjustment would make sugars unavailable for metabolism and thereby limit the establishment of plantlets since the latter were regenerated in well-watered conditions – releasing the constraint on osmotic potential build-up. We also excluded that the decline was due to increased susceptibility to pests and pathogens that could arise in plants under drought stress (Szczepaniec & Finke, 2019; Dixit et al., 2024), since there was no evidence of enhanced biotic attacks in the trained line. Alternatively, irremediable shifts at some key nodes of the hormonal network may underlie plant extinction across generations of drought. However, we do not speculate that it could have arisen from spontaneous mutation because a deleterious allele would not have been fixed through our propagation method that involved independent plants. The irreversible decline of the trained line may instead be subtended by drought-induced epigenetic drift, namely heritable changes that occur independently of sequence modifications. In populations of wild strawberry from different geographic regions, a large part of the variation in DNA methylation associated with climate of origin is inherited upon clonal propagation in a common garden (Sammarco et al., 2024). In this species, epimutations induced by heat stress can persist in at least three generations of successive stolons (López et al., 2024). Similarly, in the Lombardy poplar, natural variation in DNA methylation is largely transmitted through cutting propagation (Díez Rodríguez et al., 2022a). This natural variation overlaps with methylation patterns induced by different abiotic and biotic stresses within a single generation, suggesting functional and adaptive significance (Peña-Ponton et al., 2024). Nonetheless, the epigenetic legacy of geographical origin has a weak effect on phenotypic variation compared to that of current environmental conditions (Díez Rodríguez et al., 2022b; Vanden Broeck et al., 2024). Moreover, in Arabidopsis, repeated drought exposure across five sexual generations did not trigger consistent accumulation of epialleles and did not alter plant responses to drought (Ganguly et al., 2017). Thus, it remains to be proven whether drought-induced, stable epimutations actually accumulated at target loci across clonal generations and had any causal role in the gradual loss of petunia fitness. Overall, spanning 27 generations over 5 yr, our experiment demonstrates that chronic, sub-lethal drought may cause detrimental effects on plant fitness through gradual accumulation of deleterious legacy, without major benefit for the clonal progeny. Critically, this result is not in line with the positive effects that would be expected from an environmentally induced ‘soft inheritance’ (Richards, 2006; Youngson & Whitelaw, 2008). Nonetheless, it does not exclude that valuable training outcomes may be achieved using other genotypes or environmental scenarios, as transgenerational effects may be negative, null, or positive depending on these factors (Herman & Sultan, 2016; Groot et al., 2017; Rendina González et al., 2018; Deng et al., 2021). Perhaps the overpredictability of our controlled drought scenario prevented creating epigenetic noise that could have improved long-term fitness at the population level, following a ‘bet hedging’ strategy (Herman et al., 2014). Thus, the remaining gaps of our study – identification of molecular mechanisms and assessment of broader genotype × environment combinations – represent important opportunities for further investigation of how physiological and/or epigenetic memory may be leveraged to safeguard plants against recurrent drought. One single mother-plant was raised from a seed of Petunia hybrida cv ‘Carnival Red Star’ (kindly provided by Prof. Masayoshi Nakayama, Institute of Floricultural Science, NARO, Tsukuba, Japan). From this mother-plant (generation #00), single-node cuttings bearing one leaf were produced. Cutting bases were dipped into a β-indole to being into well-watered into a high and placed for 4 wk in a growth chamber at and with white light provided by cuttings were into growing of and with and with 10 and trace at were then for 1 wk in a glasshouse at and back in the growth chamber for a acclimation were then into two the naive and trained clonal lines (generation of these plants were for while part was either under well-watered conditions for the naive line or under chronic drought for the trained line (see in the Materials and Methods to the clonal generation from cuttings as To plantlet cuttings of both lines were in well-watered conditions during the propagation 1 this could have potential training it also to the morphological differences between lines at the of chronic drought at The same was to generation from generation and so independent plants of line were as for cuttings at Similarly, we generated an and a line from generation The escaped line was built from an batch of plants obtained from cuttings of the trained line at generation and then exposed to well-watered conditions as in the naive line. the late-trained line was built from an batch of plants obtained from cuttings of the naive line at generation and then exposed to the same chronic drought as the trained line. 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and from above (Fig. root at the of the the of the root was once the was (Fig. were using for and Total fresh biomass was from the fresh weight of all and of a plant together or depending on the were to the growing with and for fresh root biomass The were then for and their dry biomass dry mass was on leaf cutting success plantlets were observed 3 wk after before any drought was scored on the number of from the and bud was scored on the development of only the plantlets with at least 10 and 1 shoot were eventually leaves of the part of were for physiological leaf water content and water content a of was collected using a and for fresh weight Leaf were then water in a at for to being and for the of weight leaf were for at least and for dry weight to be as and as We noticed, however, that water could occur on some subjected to water and this was the were – and in these (Fig. We therefore only in this but source for gas exchange a with a 6 leaf by a light source was on leaves in the 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Fig. Chronic drought morphological of petunia across clonal generations. Fig. regime of the petunia clonal generations has weak legacy on petunia phenotypes at generations Fig. regime of the petunia clonal generations has only effects on petunia responses to chronic drought installation at generation Fig. regime of the petunia clonal generations has only effects on petunia responses to chronic drought installation at generation Fig. regime of the petunia clonal generations has no significant effects on petunia gas exchange at generations Fig. Chronic drought on numerous petunia generations to mesophyll porosity and leaf mass per Fig. Chronic drought on numerous petunia generations does not water use efficiency or morphological under deficit irrigation in glasshouse conditions. 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