The shoot regeneration capacity of excised Arabidopsis cotyledons is established during the initial hours after injury and is modulated by a complex genetic network of light signalling

Abstract

Excised plant tissues (explants) can regenerate new shoot apical meristems in vitro, but regeneration rates can be inexplicably variable. Light affects rates of shoot regeneration, but the underlying mechanisms are poorly understood. Here, excised Arabidopsis cotyledons were dark-light shifted to define the timing of explant light sensitivity. Mutants and pharmacological agents were employed to uncover underlying physiological and genetic mechanisms. Unexpectedly, explants were most light sensitive during the initial hours post-excision with respect to shoot regeneration. Only~100 mmol m -2 s -1 of fluorescent light was sufficient to induce reactive oxygen species (ROS) accumulation in new explants. By 48 h post-excision, induction of ROS, or quenching of ROS by xanthophylls, increased or decreased shoot regeneration, respectively. Phytochrome A-mediated signalling suppressed light inhibition of regeneration. Early exposure to blue/UV-A wavelengths inhibited regeneration, involving photoreceptor CRY1. Downstream transcription factor HY5 mediated explant photoprotection, perhaps by promoting anthocyanin accumulation, a pigment also induced by cytokinin. Surprisingly, early light inhibition of shoot regeneration was dependent on polar auxin transport. Early exposure to ethylene stimulated dark-treated explants to regenerate, but inhibited light-treated explants. We propose that variability in longterm shoot regeneration may arise within the initial hours post-excision, from inadvertent, variable exposure of explants to light, modulated by hormones.

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

“…The incubation in dark conditions increased the organogenetic capacity of T. belorussicum hypocotyls and especially of the cotyledons. A similar effect of light was reported in Arabidopsis cotyledons (Nemeth et al 2013).…”

Plant regeneration: cellular origins and molecular mechanisms

“…Light is required for shoot regeneration in some plant species (Reuveni and Evenor, 2007) and can also trigger organ regeneration (Saitou et al, 1992;Sorin et al, 2006;Gutierrez et al, 2009). On the other hand, exposure to light can have an inhibitory effect on root or shoot regeneration in some contexts (Bellini et al, 2014;Nameth et al, 2013). A series of tissue culture experiments using Arabidopsis cotyledons showed that light exposure during the first hours after tissue excision is mostly deleterious to shoot regeneration, and keeping explants in darkness for as little as 2-6 h is sufficient to improve regeneration (Nameth et al, 2013).…”

“…At least two photoreceptors are implicated in the light response of shoot regeneration: the blue/UV-A light receptor CRYPTOCHROME1 (CRY1), which mediates the strong inhibition of shoot regeneration; and the far-red light receptor PHYTOCHROME A (PHYA), which protects explants against initial light inhibition (Nameth et al, 2013). A key regulator acting downstream of light signaling is the transcription factor ELONGATED HYPOCOTYL5 (HY5), which appears to protect explants against light exposure by inducing anthocyanin accumulation.…”

Natural Variation in Plant Pluripotency and Regeneration

“…It is likely that part of the regenerative variation among explants from the same individual or subspecies can be attributed to fluctuations in environmental conditions, such as light, temperature, osmotic stress, and medium composition. For instance, it was found that variable light exposure of cotyledon explants during the first 5 days after excision affects shoot regeneration in Arabidopsis , and this response was different in 4 accessions [ 157 ]. More specifically, high fluorescent light intensities and early dark–light shifts impeded callus formation and regeneration, which involved the UV-A photoreceptor CRY1 and photo-oxidative damage caused by ROS accumulation.…”