AP2/ERF Transcription Factors Integrate Age and Wound Signals for Root Regeneration

By Bin-Bin Ye, Guan-Dong Shang | October 24, 2019

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Age and wounding are two major determinants for regeneration. In plants, the root regeneration is triggered by wound-induced auxin biosynthesis. As plants age, the root regenerative capacity gradually decreases. How wounding leads to the auxin burst and how age and wound signals collaboratively regulate root regenerative capacity are poorly understood. Here, we show that the increased levels of three closely-related miR156-targeted Arabidopsis (Arabidopsis thaliana) SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, SPL2, SPL10, and SPL11, suppress root regeneration with age by inhibiting wound-induced auxin biosynthesis. Mechanistically, we find that a subset of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors including ABSCISIC ACID REPRESSOR1 and ERF109 is rapidly induced by wounding and serves as a proxy for wound signal to induce auxin biosynthesis. In older plants, SPL2/10/11 directly bind to the promoters of AP2/ERFs and attenuates their induction, thereby dampening auxin accumulation at the wound. Our results thus identify AP2/ERFs as a hub for integration of age and wound signal for root regeneration.


Among the universal changes that occur with age in multicellular organisms is the decline in regenerative capacity. In mammals, for example, the heart quickly loses its capacity to regenerate within a brief period after birth (Porrello et al., 2011). Remyelination, the phenomenon by which new myelin sheaths are generated around axons in the adult central nervous system, declines with increasing age (Ruckh et al., 2012). The factors that are likely involved include accumulated DNA mutations, reduced number of stem cells, decreased cell proliferative capacity, and disturbed metabolic function (Wells and Watt, 2018). In plants, the regenerative capacity also progressively decreases with age. For example, the number of newly formed sprouts decreased significantly in Calluna vulgari plants that were more than 6 years old (Berdowski and Siepel, 1998). Similarly, the shoot regenerative capacity declines as Quercus euboica ages (Kartsonas and Papafotiou, 2007).

Plant hormones play pivotal roles in regeneration (Birnbaum and Sánchez Alvarado, 2008; Sena and Birnbaum, 2010; Perianez-Rodriguez et al., 2014; Pulianmackal et al., 2014; Su and Zhang, 2014; Xu and Huang, 2014; Ikeuchi et al., 2016; Kareem et al., 2016; Sang et al., 2018). It is well known that cytokinin induces shoot regeneration whereas auxin promotes root regeneration (Skoog and Miller, 1957). There are different ways to regenerate roots. In the tissue culture experiments, high auxin/cytokinin in root induction medium triggers the reprogramming of callus, a group of dedifferentiated cells initiated from xylem-pole pericycle cells of root explants and pericycle-like cells of aerial organs, into roots (Atta et al., 2009; Sugimoto et al., 2010; Ikeuchi et al., 2016). By contrast, root tip regeneration follows the developmental stages of embryonic patterning and is guided by spatial information provided by complementary hormone domains (Efroni et al., 2016). The detached Arabidopsis (Arabidopsis thaliana) leaf or hypocotyl can also regenerate roots on hormone-free B5 medium (Liu et al., 2014). It is proposed that the regeneration-competent cells in the pericycle around the wound undergo four sequential steps to initiate adventitious roots (Xu, 2018).

The molecular mechanism by which shoot regenerative capacity is affected by age during tissue culture has been well documented. Our previous work reveals an important role of microRNA156 (miR156), the master regulator of juvenility, in shoot regeneration (Poethig, 2009; Yu et al., 2015c). miR156 targets a group of transcription factors named SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL; Rhoades et al., 2002; Schwab et al., 2005; Wu and Poethig, 2006; Gandikota et al., 2007). Juvenile plants exhibit a high cytokinin response and regenerative capacity. As a plant ages, miR156 levels decline, alleviating the repression of its SPL targets (Wang et al., 2009; Yamaguchi et al., 2009; Xu et al., 2018). SPL directly inhibits the transcriptional activity of B-type ARABIDOPSIS RESPONSE REGULATORs and thereby impairs shoot regenerative capacity (Zhang et al., 2015). miR156 is also involved in de novo root regeneration in Arabidopsis (Xu et al., 2016; Ye et al., 2019). The hypocotyls from the plants with low miR156 activity produce fewer adventitious roots than those from wild type. Notably, this phenomenon seems to be evolutionarily conserved. The same positive relationship between miR156 level and rooting capacity has been found in Malus xiaojinensis and Medicago sativa (Aung et al., 2017; Xu et al., 2017). However, the molecular mechanism by which miR156/SPL regulates root regenerative capacity is poorly understood.

Here, we find that miR156-targeted SPLs have divergent roles in plant regeneration. Whereas the SPL9 subfamily suppressed shoot regeneration, the SPL10 subfamily specifically suppressed root regeneration by inhibiting auxin burst in response to wounding. By the combination of RNA-sequencing (RNA-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq), we further identified a subset of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors as the proxy for wound signal to induce auxin biosynthesis, and these transcription factors act as a hub for integration of age and wound signal.