The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling.During the transition from seed to seedling, the coordination of defense and development is critical for early survival (Finch-Savage and Leubner-Metzger, 2006; Holdsworth et al., 2008). After emerging from the seed coat, the embryo pushes through the soil to reach the surface; at this time, it is more vulnerable to biotic and abiotic stresses (Raven et al., 2005), and underlying actors of this transition are relatively unstudied (Warpeha and Montgomery, 2016). Phe-derived compounds, the phenylpropanoids, play an important role in the first line of defense by contributing to the reinforcement of the external cuticle layer and by conferring UV light protection properties to epicuticular waxes (Steyn et al., 2002; Pollard et al., 2008); in addition, phenylpropanoids influence wax production in response to UV light exposure (Rozema et al., 2002; Pollard et al., 2008; Warpeha et al., 2008). The activity of the phenylpropanoid pathway provides an additional line of defense, as phenolic compounds take part in a nonenzymatic mechanism to efficiently scavenge reactive oxygen species (ROS), whose levels increase as a result of metabolic reactions and when plants initiate a stress response (Sharma et al., 2012; Agati et al., 2013). Moreover, by influencing the cell’s ability to balance and modulate ROS production and scavenging, phenylpropanoids allow fluctuations in ROS levels that are required to elicit stress signaling pathways for specific defense strategies (Apel and Hirt, 2004; Mittler et al., 2011).AROGENATE DEHYDRATASE3 (ADT3)/PREPHENATE DEHYDRATASE1 belongs to the arogenate dehydratase protein family, whose members catalyze the last steps of the biosynthesis of Phe (Warpeha et al., 2006; Cho et al., 2007; Tzin and Galili, 2010; Bross et al., 2011). Activation of ADT3 leads to an increase in Phe content and in the production of phenylpropanoids (Warpeha et al., 2006). Accordingly, loss of ADT3 results in an enhanced sensitivity to UV irradiation in etiolated seedlings due to the reduced synthesis of photoprotective compounds and UV light-scattering epicuticular waxes (Warpeha et al., 2008). However, the physiological and molecular bases of this phenotype and the function of ADT3 in the seed-to-seedling transition remain to be elucidated.We sought to understand the role of ADT3 postgermination, in the seed-to-seedling transition. ADT3 is expressed early in seedling growth (Warpeha et al., 2006; Hruz et al., 2008). Localization studies in Arabidopsis (Arabidopsis thaliana) using protoplasts from cell suspension and light-grown rosette leaves have placed this enzyme within the chloroplast (Rippert et al., 2009), while we have reported ADT3 activity in the cytosolic fraction in young etiolated seedlings (Warpeha et al., 2006). These reports differ likely due to the different age and growth conditions of the studied plant material. Cytosolic forms of chorismate mutase, which act at the first committed step in the Phe and Tyr biosynthesis pathway, were found in Arabidopsis and other plants (d’Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996), suggesting the possibility of extraplastidic Phe biosynthesis.Here, we report in transgenic complementation experiments that ADT3 is expressed widely in the young shoot and largely accumulates in the cytosol. Based on the role of phenylpropanoids in plant defense, we hypothesize that the ADT3 regulation of Phe supply is required to coordinate defense and development at the seed-to-seedling transition. We found that, without ADT3, the cells of the epidermis cannot buffer and restrict ROS; moreover, adt3 cotyledons enter an aberrant developmental program that results in abnormal morphology and patterning as well as several alterations at the subcellular level. Proteomic analysis of adt3 seedlings provided insights into the molecular basis of adt3 phenotypes, as it uncovered a chronic inability to buffer an excess of ROS and maintain plastid integrity. It also revealed a failed attempt to control cell rheology through up-regulation of the biochemical pathway for cuticle biosynthesis and assembly, as indicated by the increase in the permeability of adt3 epidermal cells; we propose that this also could be the cause of the defecting epidermal patterning in adt3 cotyledons. In addition, we suggest an additional role of Phe in nutrient supply in etiolated seedlings.