Download The dehydratase ADT3 affects ROS homeostasis

Plant Physiology Preview. Published on August 18, 2016, as DOI:10.1104/pp.16.00464
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Running head: ADT3, ROS homeostasis and cotyledon development
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Corresponding Author: Katherine M. Warpeha, University of Illinois at Chicago,
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Biological Sciences, 900 S. Ashland Ave, Chicago, IL 60607, 312 996 7646,
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[email protected]
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Research Area: Cell Biology
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Secondary Research Area: Biochemistry and Metabolism or Signaling and
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Response
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The dehydratase ADT3 affects ROS homeostasis and cotyledon development
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Alessia Para1, DurreShahwar Muhammad2, Danielle A. Orozco-Nunnelly2, Ramis Memishi2,
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Sophie Alvarez3, Michael J. Naldrett3, Katherine M. Warpeha*2
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1. Weinberg College of Art and Science, Northwestern University, Evanston, IL.
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2. Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL.
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3. Proteomics and Mass Spectrometry Facility, Donald Danforth Plant Science Center, St.
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Louis, MO.
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Arabidopsis seedlings mutated in phenylalanine biosynthetic enzyme ADT3 cannot buffer reactive
oxygen species, produce a defective cuticle, and display impaired cotyledon development
Original project conceived by KMW; KMW supervised the experiments; KMW performed lab
experiments (except proteome) with technical assistance from DM, DO-N, RM, with SA and
MJN conducting the proteome on KMW samples, and AP handling data analysis of all
proteomic data; KMW designed the experiments with interpretation with input from the
authors especially AP, and AP and KMW analyzed the data; AP wrote the article with input
on proteome from SA and MJN, with editing and oversight from KMW.
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This work herein has been supported by National Science Foundation grant MCB-0848113
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(NSF: http://www.nsf.gov/div/index.jsp?div=MCB), awarded to Katherine M. Warpeha and
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Lon S. Kaufman.
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This work herein has been supported by National Science Foundation grant MCB-0848113
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(NSF: http://www.nsf.gov/div/index.jsp?div=MCB), awarded to Katherine M. Warpeha and
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Lon S. Kaufman.
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Corresponding author:
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Katherine Warpeha
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Biological Sciences, Molecular Cell and Developmental Biology Group
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University of Illinois at Chicago
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900 S. Ashland Ave
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Chicago, IL, 60612.
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ABSTRACT
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During the transition from seed to seedling, emerging embryos strategically balance the
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available resources between building up defenses against environmental threats, and
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initiating the developmental program that promotes the switch to autotrophy. We present
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evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE
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DEHYDRATASE 3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and
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cotyledon development in etiolated Arabidopsis thaliana seedlings. We show that ADT3 is
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expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and the
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epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of
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adt3 mutant revealed a general downregulation of plastidic proteins and ROS scavenging
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enzymes, corroborating the hypothesis that ADT3 supply of Phe is required to control of ROS
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concentration and distribution to protect cellular components. In addition, loss of ADT3
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disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement
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cells and stomata cell fate specification; we also observed severe alterations in mesophyll
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cells, which lack oil bodies and normal plastids. Interestingly, upregulation of the pathway
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leading to cuticle production is accompanied by an abnormal cuticle structure and/or
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deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and
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provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an
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additional role of Phe in supplying nutrients to the young seedling.
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INTRODUCTION
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During the transition from seed to seedling, the coordination of defense and
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development is critical for early survival (Finch-Savage and Leubner-Metzger, 2006;
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Holdsworth et al., 2008). After emerging from the seed coat, the embryo pushes through the
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soil to reach the surface, and at this time it is more vulnerable to biotic and abiotic stresses
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(Raven et al., 2005), where underlying actors of this transition are relatively unstudied
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(Warpeha
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phenylpropanoids, play an important role in the first line of defense by contributing to the
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reinforcement of the external cuticle layer and by conferring UV protection properties to
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epicuticular waxes (Steyn et al., 2002; Pollard et al., 2008); in addition, phenylpropanoids
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influence wax production in response to UV exposure (Rozema et al., 2002; Pollard et al.,
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2008; Warpeha et al., 2008). The activity of the phenylpropanoid pathway provides an
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additional line of defense as phenolic compounds take part in a non-enzymatic mechanism to
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efficiently scavenge reactive oxygen species (ROS), whose levels increase as a result of
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metabolic reactions, and when plants initiate a stress response (Sharma et al., 2012; Agati et
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al., 2013). Moreover, by influencing the cell’s ability to balance and modulate ROS production
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and scavenging, phenylpropanoids allow fluctuations in ROS levels that are required to elicit
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stress signaling pathways for specific defense strategies (Apel and Hirt, 2004; Mittler et al.,
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2011).
and
Montgomery
2016).
Phenylalanine
(Phe)-derived
compounds,
the
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AROGENATE DEHYDRATASE 3/PREPHENATE DEHYDRATASE 1 (ADT3/PD1)
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belongs to the arogenate dehydratase (ADT) protein family, whose members catalyze the last
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steps of the biosynthesis of Phe (Warpeha et al., 2006; Cho et al., 2007; Bross et al., 2011).
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Activation of ADT3 leads to an increase in Phe content and in the production of
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phenylpropanoids (Warpeha et al., 2006). Accordingly, loss of ADT3 results in an enhanced
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sensitivity to UV irradiation in etiolated seedlings due to reduced synthesis of photoprotective
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compounds and UV-scattering epicuticular waxes (Warpeha et al., 2008). However, the
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physiological and molecular basis of this phenotype and the function of ADT3 in the seed-to-
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seedling transition remain to be elucidated.
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We sought to understand the role of ADT3 post-germination, in the seed-to-seedling
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transition. ADT3 is expressed early in seedling growth (Warpeha et al., 2006; Hruz et al.,
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2008). Localization studies in Arabidopsis using protoplasts from cell suspension and light
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grown rosette leaves have placed this enzyme within the chloroplast (Rippert et al., 2009),
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while we have reported ADT3 activity in the cytosolic fraction in young etiolated seedlings
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(Warpeha et al., 2006). These reports differ likely due to the different age and growth
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conditions of the studied plant material. Cytosolic forms of chorismate mutase, which act at
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the first committed step in the Phe and Tyr biosynthesis pathway, were found in Arabidopsis
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and other plants (d'Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996),
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suggesting the possibility of extra-plastidic Phe biosynthesis.
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Herein, we report in transgenic complementation experiments that ADT3 is expressed
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widely in the young shoot and largely accumulates in the cytosol. Based on the role of
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phenylpropanoids in plant defense, we hypothesize that ADT3 regulation of Phe supply is
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required to coordinate defense and development at the seed-to-seedling transition. We found
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that, without ADT3, the cells of the epidermis cannot buffer and restrict ROS; moreover, adt3
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cotyledons enter an aberrant developmental program that results in abnormal morphology
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and patterning as well as several alterations at the subcellular level. Proteomic analysis of
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adt3 seedlings provided insights into the molecular basis of adt3 phenotypes as it uncovered
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a chronic inability to buffer an excess of ROS and maintain plastid integrity. It also revealed a
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failed attempt to control cell rheology through up-regulation of the biochemical pathway for
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cuticle biosynthesis and assembly, as indicated by the increase in the permeability of adt3
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epidermal cells; we propose that this could also be the cause of the defecting epidermal
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patterning in adt3 cotyledons. In addition, we suggest an additional role of Phe in nutrient
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supply in etiolated seedlings.
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RESULTS
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ADT3 is expressed in cotyledons of young etiolated seedlings, where its loss causes
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an imbalance in ROS homeostasis.
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To investigate the impact of loss of ADT3, we generated transgenic plants where
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native ADT3 fused to CFP was driven by its native promoter (ADT3::ADT3-CFP) in the adt3-1
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mutant background to examine ADT3 spatial expression pattern in 4-d-old dark-grown
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seedlings (Fig. 1). ADT3 is expressed in the shoot in developing mesophyll, and SAM (leaf
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primordial and dome) (Fig. 1A and S2) and epidermal cells, especially pavement cells (Fig.
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1B). In particular, ADT3 appeared to accumulate mainly in the cytosol (inset Fig,1A and 1B).
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An additional transgenic line harboring the ADT3::ADT3-GFP fusion in adt3-1 mutant
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background
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complementation of all adt3 defects reported herein (Fig. S1; see next section), and
recapitulated
the
ADT3::ADT3-CFP
expression
pattern,
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and
showed
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elsewhere (Warpeha et al., 2008). A similar expression pattern of ADT3::ADT3-GFP was
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observed in the WT background, where a more intense GFP signal is observed compared to
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the mutant background. ADT3 expression was also observed in the developing guard cells
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(Fig. S2).
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adt3 mutation has been previously shown to make etiolated seedlings vulnerable to
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UV irradiation and the defect was proposed to be due to a decrease in the production of
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phenylpropanoids upon exposure to both blue light and UV (Warpeha et al., 2006; Warpeha
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et al., 2008). To elucidate the physiological basis of UV sensitivity in relation to
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phenylpropanoids, we examined the accumulation of ROS in the epidermis of adt3-1
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cotyledons before and after UV treatment, as these compounds are produced in response to
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short-wavelength light (Wituszyńska and Karpiński, 2013; Consentino et al., 2015). 6-d-old
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dark-grown WT and adt3-1 seedlings were irradiated with a sublethal dose of UV-C radiation
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and the CellRoxTM Deep Red cell permeable fluorescent probe was used to detect ROS
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(Fig.2). This probe only fluoresces when in contact with ROS, and the signal can be captured
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when excited by the Cy5 LED (false-colored pink).
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Interestingly, probe fluorescence in the epidermis was detected in dark-grown
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unirradiated adt3-1 compared to unirradiated WT (control), indicating that basal ROS levels
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are elevated in adt3-1 (Fig. 2). Irradiation with a sublethal UV-C dose caused an increase in
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CellRoxTM basal signal from both WT and adt3-1 pavement cells (Fig. 2, bar graph). An
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additional ADT3 mutant (adt3-6) tested responded similarly to adt3-1 (Suppl. Fig. S3). Since
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ADT3 catalyzes the last step of the biosynthesis of Phe (Maeda and Dudareva, 2012), we
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assessed the requirement of Phe in counteracting ROS by transferring living, whole 6-d-old
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WT, adt3-1 and adt3-6 seedlings to Phe-supplemented medium for 3 h before subjecting
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them to a sublethal dose of UV-C radiation, immediately followed by incubation with
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CellRoxTM, where pre-incubation with Phe restored the seedling to control (WT unirradiated)
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levels of ROS in both adt3 alleles (Fig. 2; Suppl. Fig. S3). Another probe for ROS detection,
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SOSGreenTM (SOSG), indicated similar results for adt3-1 compared to WT, confirming that
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ROS production is significantly elevated as a consequence of loss of ADT3 (Suppl. Fig. S4),
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whereas adt3-6 was not significant.
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To test whether Phe metabolism is required to scavenge ROS, we UV-C irradiated 6-
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d-old, dark-grown seedlings of the fah1-7/tt4-1 double mutant (Li et al., 1993; Landry et al.,
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1995; Peer et al., 2001), which cannot metabolize Phe to produce flavonoids. Indeed, we
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observed that pretreatment with Phe was able to prevent UV-induced ROS production in the
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fah1-7/tt4-1 background (Suppl. Fig. S3). Accordingly, pre-treatment with Phe had the same
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effect as KI, a known ROS scavenger (Tsukagoshi et al., 2010), which was also able to
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prevent a ROS increase upon UV-C irradiation (Suppl. Fig. S5). Likewise, treatment with a
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non-metabolizable Phe analog, p-f-DL-Phenylalanine (Conway et al., 1963), caused a
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significant decrease in Cy5 fluorescence in UV-C-irradiated pavement cells (but not in the
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guard cells) in the adt3-1 epidermis (Suppl. Fig. S5). Taken together, these results indicate
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that ROS homeostasis is altered in dark-grown adt3 seedlings. In addition, we observed that
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Phe per se could prevent significant ROS accumulation as a result of UV-C irradiation.
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Loss of ADT3 disturbs epidermis development in the cotyledons of dark-grown
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seedlings.
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To further investigate the consequences of loss of ADT3, we examined the cellular
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makeup of the cotyledon’s epidermis. DAPI illumination of the epidermis of live 6-d-old dark-
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grown WT and adt3-1 seedlings enabled a clear distinction of cell lineages (pavement cells
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largely reflect, guard cell lineage largely absorbs DAPI), and revealed that adt3-1 mutants
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produced fewer but larger pavement cells than WT (Fig. 3, A); however, adt3-1 cotyledons
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are wider than the WT (Fig. 3, A and B). In addition, a reduced number of guard cells was
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observed for the adt3 mutant cotyledons (Fig. 3, A and B). Addition of Phe to the growth
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medium from sowing, rescued both the pavement cell and guard cell phenotypes in adt3-1
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mutants (Fig. 3, A and B). adt3-6 seedlings also presented similar phenotypes which could be
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rescued by Phe (Suppl. Fig. S6A). The defects persisted when exogenous L-tyrosine (L-Tyr),
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or L-tryptophan (L-Trp) (which derive from the shikimate pathway like Phe) were supplied
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instead of Phe (Suppl. Fig. S7).
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To address whether the guard cell defect was due to failure to specify the stomatal
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lineage, we supplied Phe to the mutants speechless and mute, which disrupt early and late
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steps of the stomata lineage progression, respectively (MacAlister et al., 2007; Pillitteri et al.,
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2007). Phe did not prevent the guard cell defects in these mutant, indicating that Phe is
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required upstream of these guard cell specification factors (Suppl. Fig. S7).
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In summary, we observed ADT3 expression in the cytoplasm of epidermal cells of the
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cotyledons and of the SAM. Loss of ADT3 severely affects the number and morphogenesis of
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pavement cells and disrupts guard cell development. The defects were rescued by
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supplementing Phe but not other amino acids, indicating that metabolism of ADT3-supplied
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Phe impacts cotyledon patterning in etiolated seedlings.
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adt3 cotyledons exhibit abnormal subcellular features.
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We also examined the ultrastructure of the epidermis and mesophyll cells in
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cotyledons of 6-d-old, dark-grown adt3-1 seedlings by transmission electron microscopy
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(TEM) (Fig. 4, A). A prominent feature we observed was the reduction in the number of
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globular oil bodies in adt3-1 mesophyll cells compared to WT (Fig. 4, A, inset c and f). In
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addition, adt3-1 cells were hyper-vacuolated (especially the epidermis), while most WT cells
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contained a large central vacuole (Fig. 4, A). Developing plastids were identified in WT, as
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they stain densely and contain prolamellar bodies with stromal strands extending from the
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thylakoid membrane (Fig. 4, A, inset b), whereas the adt3-1 plastids displayed atypical
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prolamellar bodies and loose stromal thylakoids (Fig. 4, A, inset e). The cytoplasm of adt3-1
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epidermal cells had a blackened appearance, indicating possible accumulation of osmiophilic
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material, and exhibited numerous round organelles, which are likely to be mitochondria, as
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suggested by the ribbed structure (Fig. 4, A, inset a and c).
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The distribution of oil bodies in WT and adt3-1 was further investigated using the
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lipophilic stain Nile Red (Greenspan et al., 1985). Optical sections of adt3 cotyledons showed
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a paucity of scattered oil bodies (Fig. 4, B), while in WT these organelles were more
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abundant, particularly at the tip (Fig. 4, B). Addition of Phe to the germination medium
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increased oil body production in adt3-1 (Fig. 4, B), rescuing the deficiency. This phenotype
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was also observed for the adt3-6 allele (Suppl. Fig. S6). Thus, ADT3 activity is required to
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preserve the subcellular structure of the cells of the cotyledons in etiolated seedlings.
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adt3 proteome reveals the molecular basis of the adt3 mutant phenotypes.
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In order to uncover the molecular phenotype underlying the adt3 phenotypes, we
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obtained a comparative protein expression profile of the aerial portions (upper hypocotyl and
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cotyledon) of adt3-1 and WT seedlings. We used the bioinformatics platform Virtual Plant for
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Gene Ontology (GO) terms analyses (Katari et al., 2010) and KEGG database for metabolic
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pathways (http://www.genome.jp/kegg/).
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The proteomic analysis revealed that adt3-1 mutation caused alteration of the
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expression of 1333 proteins, 608 of which were down-regulated, while 725 were up-regulated
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(Table S1). The maximum fold change was 1.69 for up-regulation and 0.646 for down-
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regulation, a dynamic range of expression in accordance with other proteomics studies
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(Table S1)(Alvarez et al., 2011; Alvarez et al., 2014). Given the high number of statistically
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significantly misregulated proteins in adt3-1 proteome, we investigated whether small
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changes in expression could be biologically significant by asking whether they occur in a
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specific tissue or at multiple steps within the same process, as it was conceptualized by
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metabolic control analysis (Kacser and Burns, 1995; Fell and Cornish-Bowden, 1997). First,
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we queried the Plant Ontology (PO) database in Virtual Plant to assess enrichment of
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proteins of different cell and organ types. For both adt3-1 up-regulated and down-regulated
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proteins, the most significant PO enrichment was observed for guard cell, shoot epidermal
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cell and epidermal cell (Table 1), indicating that the changes in protein expression were
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relevant to the cell types of the cotyledons where we observed ADT3 expression (Fig. 1, A).
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Next, plant-specific overrepresented terms for cellular compartment, molecular function and
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biological process were assessed by GO term analysis using a p value of 0.01 as the cutoff.
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The results of this analysis are summarized in Table 1, Table 2, and Tables S2-S11.
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GO term analysis of up-regulated proteins in adt3-1 proteome revealed enrichment
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for plasma membrane and vacuole factors involved in transport as well as in cell wall
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organization or biogenesis through the metabolism of the polysaccharides xylan and glucan
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(Table 1). Accordingly, the most represented molecular function for the cell wall category is
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“hydrolase activity”, since in these polymers the monosaccharide residues are joined by
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glycosidic linkages. The involvement of the cell wall in adt3 cell morphology is also
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underscored by the GO term “growth”, which showed one of the most significant enrichment
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for the up-regulated proteins in adt3-1 proteome (Table 1). The proteins belonging to this
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category are involved in cell expansion, like expansins (EXP3, 9, 11 and EXPL1), and DIM1
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(Takahashi et al., 1995). Interestingly, a protein that showed the highest increase in the adt3-
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1 mutant is AT14A, a protein that was shown to function like integrin in animals to regulate
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cell shape and cell-to-cell adhesion (Lu et al., 2012) (Table S1).
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For adt3-1 up-regulated proteins assigned to epidermal cells according to the PO
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database, we noticed an enrichment in proteins involved in carboxylic acid/oxoacid metabolic
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process (Table 1), encompassing enzymes for fatty acids conjugation (LACS4, LACS8,
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GPAT8 and GPAT4), and synthesis and elongation of very long chain fatty acids (VLCFAs)
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[ACC1/PAS3, CER2, CER6, CER10, ATT1 (CYP86A2), FDH (KCS10)]. We also found up-
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regulation of the 3-ketoacyl-CoA synthases (KCS) KCS8, 9, 16, 19 and CY86A4, as well as
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transporters of wax molecules and cuticle building blocks (ABCG11/DSO/CER5 and LTPG1).
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All of these functions were previously associated with cuticle formation (Kunst and Samuels,
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2009; Nawrath et al., 2013 and specific references therein).
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When considering down-regulated proteins, one of the most significant GO terms for
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cellular components is “plastid”, and the affected proteins are associated with the stroma,
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envelope and thylakoid membrane (Table 1). This is in agreement with the observations that
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the integrity of the plastids is compromised by loss of ADT3 (Fig. 4, A). Interestingly, we
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observed an overrepresentation of proteins associated with the stromule, an elusive plastidic
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structure that has been previously implicated in GPA1 signaling (Huang et al., 2006; Hanson
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and Sattarzadeh, 2011) (Table 1).
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One of the top GO terms that describes the overrepresented molecular functions for
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the group of down-regulated proteins in the epidermis of adt3-1 mutant is “oxidoreductase
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activity” (Table 1) and includes proteins of both the enzymatic and non-enzymatic scavenging
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system that controls ROS homeostasis like superoxide dismutases (MSD1, FSD2 and CSD2
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and CSD3), catalases (CAT2), enzymes of ascorbate-glutathione (AsA-GSH) cycle ascorbate
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peroxidase
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dehydroascorbate reductase (DHAR2), and glutathione reductase (ATGR2) (Sharma et al.,
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2012). GDP-Mannose 3′,5′-epimerase (GME), an important enzyme in the ascorbate
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biosynthetic pathway, is also down-regulated (Wheeler et al., 1998). In addition, of the 93
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down-regulated proteins that were assigned to gene families, 7 are members of the
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glutathione S-transferase family of proteins in adt3-1 (Table S1).
(SAPX,
APX1,
APX3),
monodehydroascorbate
reductase
(MDAR4),
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In addition to the GO terms listed above, “primary metabolic process” is highly
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significant for both up-regulated and down-regulated proteins in adt3-1 proteome (Table 1).
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Hence, we investigated the metabolic pathways that were affected by loss of ADT3 using the
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KEGG database. We observed down-regulation of key enzymes of the central carbohydrate
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and energy metabolism (Table 2), including enzymes of the glycolysis/gluconeogenesis
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pathway, the pentose phosphate pathway, and glyoxylate and dicarboxylate metabolism.
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However, some of the enzymes involved in oxidative phosphorylation are up-regulated,
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suggesting an intense mitochondrial activity. Also, the expression of proteins of starch and
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sucrose metabolism, pentose and glucuronate interconversions as well as in amino sugar
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and nucleotide sugar metabolism is increased in the adt3-1 mutant. The enrichment of
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enzymes from these pathways provides a link between cell wall metabolism and central plant
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metabolism (Seifert, 2004; Bar-Peled and O'Neill, 2011).
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In summary, the overrepresentation of terms for specific biological functions,
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localization or molecular components is consistent with the observed adt3 phenotypes,
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linking the physiological and morphological alterations to changes in the expression of key
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proteins or pathways. Moreover, in agreement with functional enrichment analysis, the
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pathways analysis suggests that alteration of cell morphology and subcellular structure might
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be linked to metabolic deficiencies as a result of loss of ADT3 activity.
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Cuticle formation and cell permeability are altered in adt3 cells.
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Many key proteins involved in the production of the cuticle were up-regulated in the
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adt3-1 mutant (Table 1 and Table S3). Higher magnification of the outermost cell wall
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revealed irregular deposition of electron-opaque material within the deeper layers (Fig. 5, A),
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indicating that cuticle biosynthesis and/or deposition is somehow defective. To further
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examine adt3-1 cuticular properties, we tested the permeability of adt3-1 cuticle under
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hyposmotic conditions using toludine Blue (TB) staining (Tanaka et al., 2004). Interestingly,
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diffuse TB staining was observed in adt3 seedlings after a 48 h incubation in water (Fig. 5, B)
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compared to the WT in the same conditions, a sign of incomplete or reduced cuticle in the
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mutant. TB staining of adt3-6 dark grown seedlings showed similar results (data not shown).
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Since it was found that mutations in genes of the wax biosynthetic pathway that affect the
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composition of the epicuticular waxes also perturb stomatal development and increase the
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number of guard cells (Gray et al., 2000; Guseman et al., 2010), we inspected the epidermis
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of white light grown adt3 seedlings and found adjacent stomata that were breaking the one-
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cell spacing rule (Fig. 5, C). Our results indicates that the cuticle of adt3 epidermis does not
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achieve a proper organization, and this causes a reduction in the isolating properties of the
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epidermis and is likely to impact cell patterning of adt3 cotyledons.
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Sucrose supply restores guard cell number while Asn and NH3Ac rescue pavement cell
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cell morphology in adt3 cotyledons.
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Proteomic analysis revealed that a significant number of proteins from primary
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metabolic pathways – in particular for carbon metabolism — are down-regulated in adt3-1
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seedlings (Table 1 and Table 2), so we investigated whether carbon input could alleviate the
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defects in adt3-1 cotyledons by transferring 4-d-old adt3-1 seedlings to medium containing
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1% sucrose. When we examined adt3-1 cotyledons 48 h later, we found that the pavement
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cell phenotype was not rescued by exogenous sucrose (Fig. 6, A); however, the number of
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guard cells was similar to the WT (Fig. 6, A). The rescue was not due to a change in osmotic
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potential, as it was not ameliorated by exogenous supply of 1% mannitol (Suppl. Fig. S8).
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330
Since ASPARAGINE SYNTHASE 1 (ASN1) is up-regulated in adt3-1 mutant (Table
331
S1), we next attempted to rescue adt3-1 phenotype by supplying exogenous asparagine
332
(Asn) from sowing. After 6 d of growth on Asn in darkness, we observed that the pavement
333
cell size and number as well as the cotyledon size were completely restored in the mutant
334
(Fig. 6, B). The same results were obtained when ammonium acetate (NH4Ac) was added to
335
the medium (Fig. 6, C). However, the guard cell lineage defect was not corrected by the
336
application of neither exogenous Asn nor NH4Ac (Fig. 6, B and C). To test whether the ability
337
of Asn and NH4Ac to rescue adt3-1 cell morphology phenotype could be linked to restored
338
fatty acid production (Fig. 4, B), we incubated Asn- and NH4Ac-supplied adt3-1 seedlings with
339
Nile Red (Fig. 6, C) and, similarly to the result we obtained by supplying Phe (Fig. 4, B),
340
addition of Asn and NH4Ac also resulted in an increase in Nile Red signal (Fig. 6, C),
341
indicating that these compounds could be used by adt3 to feed metabolic pathways that lead
342
to fatty acids production. These observations suggest that a nutrient imbalance could be part
343
of the defects in cell morphology and epidermal patterning of adt3 cotyledons (Fig. 3).
344
345
DISCUSSION
346
347
ADT3 accumulates in the cytosol and is required to maintain ROS homeostasis.
348
We previously reported that an arogenate dehydratase activity could be detected in the
349
cytosol of dark grown, young seedlings upon blue light irradiation. In this study, we used
350
transgenic lines expressing CFP tagged ATD3 to further investigate in vivo ADT3 localization
351
in 4-d-old dark-grown seedling. Here ADT3 expression was detected in the shoot, including
352
cotyledons and SAM (Fig.1; S Figure S2). We also examined the subcellular localization of
353
ADT3 and confirmed that the enzyme accumulates mainly in the cytosol at this stage of
354
seedling development (Fig 1). This observation adds to the growing body of evidence
355
supporting the existence of extra-plastidial pathways for Phe biosynthesis (Yoo et al., 2013).
356
Phe production in the cytosol has important implications since cytosolic Phe could be directly
357
channeled into secondary metabolism, as the enzymes of the pathways that utilize Phe as a
358
precursor are found outside the plastids. In particular, cytosolic synthesis of Phe would
359
ensure timely production of antioxidants and photoprotective molecules, which are required to
360
contain the damages caused by high-frequency radiations when etiolated seedlings are
361
exposed to solar light. Accordingly, we previously reported an overall reduction in UV and
362
Blue light-absorbing compounds in the cotyledon when Phe supply is compromised by loss of
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13
363
ADT3 (Warpeha et al., 2006). In addition to providing protection from photodamage, we show
364
herein that Phe metabolism could counteract the increase in ROS concentration as part of
365
the stress response to high frequency radiations (Fig. 2) (Mackerness et al., 2001; Kalbina
366
and Strid, 2006; Consentino et al., 2015).
367
However, ROS levels were significantly elevated in unirradiated adt3 cotyledon cells
368
compared to WT cells (Fig. 2; Suppl. Fig. S3-5), indicating that ADT3 activity is required to
369
maintain a low basal level of ROS in etiolated seedlings. Because of the high number of
370
mitochondria in the adt3 mutant cells and the decrease in oil bodies (Fig. 4), we suggest that
371
ROS over-production could be due to a combination of normal reoxygenation of the
372
germinating tissues when the embryo emerges from the seed coat (Sattler et al., 2004), and
373
the intense metabolic activity that is likely required for the catabolism of the oil bodies.
374
Increased sensitivity to light and oxidative stress has been reported previously for a
375
mutant of the ROS-scavenging enzyme ASCORBATE PEROXIDASE 1 (APX1) gene, which
376
exhibited a low cytosolic content of the antioxidant ascorbic acid (Davletova et al., 2005,
377
Miller et al., 2007). Similarly to apx1, the adt3 mutant phenotype could be a consequence of
378
high steady-state of cytosolic ROS levels, In Arabidopsis it has been shown that cytosolic
379
scavenging enzymes are induced upon light stress (Karpinski et al., 1997; Karpinski et al.,
380
1999; Pnueli et al., 2003; Davletova et al., 2005), even though ROS are thought to be
381
generated in chloroplasts and/or peroxisomes (Mittler, 2002). In addition, buffering ROS in
382
the cytosol was shown to be essential for protecting chloroplast from oxidative damage
383
(Davletova et al., 2005). Indeed, these chemical species can damage membranes of
384
organelles (Gill and Tuteja, 2010), in particular, plastids (Asada, 2000). Since we observed
385
localization of ADT3 in the cytosol of the cells in etiolated seedlings (Fig. 1), we concluded
386
that cytosolic Phe-derived antioxidants, and even cytosolic Phe per se, may be required to
387
protect the integrity of the plastids from the cytosolic side, a notion that is supported by the
388
abnormal structure of these organelles in adt3 plastids (Fig. 4), and to preserve their
389
functionality, as indicated by down-regulation of essential processes that take places in these
390
organelles such as nitrogen metabolism and response to oxidative stress (Table 1). Indeed,
391
when we irradiated dark-grown WT seedlings with high energy UV-B (300nm), we noticed
392
that the structure of the plastids was visibly affected by the treatment and inclusion of Phe to
393
the growth medium from sowing restored the characteristic pro-lamellar body organization
394
that was observed before irradiation (Suppl. Fig. S9). The effect is even more dramatic in
395
adt3-1 mutant, where plastids were hardly discernable upon UV-B irradiation, but were
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14
396
resistant to the treatment in presence of Phe (Suppl. Fig. S9). This is consistent with the
397
ability of the phenylpropanoids such as flavonols to bind to non-bilayer lipids such as
398
monogalactosyl diacyl glycerol (MGDG) (Erlejman et al., 2004). The outer membrane of
399
plastids is reported to be deficient in MGDG (Erlejman et al., 2004; Scheidt et al., 2004). Part
400
of the acute sensitivity of adt3 seedling to UV-C irradiation doses that are tolerated by WT
401
(Warpeha et al., 2008) could also be explained by the observation that other enzymes
402
involved in ROS removal are down-regulated in adt3 (Table 1 and Table S1). This could lead
403
to a chronic imbalance between the production of ROS upon high-energy UV treatment and
404
the ROS buffering capacity of adt3 seedlings. Failure to activate additional ROS-scavenging
405
pathways as part of the physiological response to increase in ROS concentration (Sharma et
406
al., 2012) would suggest that, without the contribution of ADT3 function, ROS would
407
accumulate above the threshold toxic levels in young etiolated seedlings, resulting in
408
irreversible cellular damage (Sharma et al., 2012).
409
Thus, Phe biosynthesis through ADT3 activity is required to protect the cotyledons of
410
etiolated seedlings from the deleterious effects of ROS by maintaining a low concentration of
411
these toxic compounds at the steady-state, and by buffering ROS increase as a consequence
412
of photostress. Control of ROS homeostasis is still poorly understood, and advancing the
413
knowledge of Phe and related metabolism will help elucidating the cellular signaling and
414
metabolic processes involved in this important mechanism (Geigenberger and Fernie, 2014).
415
416
A role for ADT3 supplied Phe in morphogenesis and cell proliferation through cuticle
417
assembly.
418
ADT3 was previously shown to participate in the production of epicuticular waxes
419
(Warpeha et al., 2008). In the present study, we have shown that adt3 epidermis bears signs
420
of alterations in the deposition process (Fig. 5, A). This is likely to cause a severe alteration in
421
the rheological properties of adt3 epidermal cells, particularly when the permeability of adt3
422
seedlings is challenged (Fig. 5, B). Increased levels of known factors involved in cuticle
423
production and transport indicates that the molecular machineries that act in this process are
424
in place in the adt3 epidermis (Table 1), so the defect might be due to a shortage of
425
substrates (like VLCFA), or impairment in cuticle assembly. Both hypotheses are plausible as
426
the VLCFA are produced by plastids (Yeats and Rose, 2013), whose structure and function
427
appear to be altered in adt3 (Fig. 4 and Table 1), or could derive from the degradation of
428
triacylglycerols stored in oil bodies, which are nearly absent in adt3 mutants (Fig. 4, A and B,
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15
429
Suppl. Fig. S6). The presence of osmiophilic compounds in the cytoplasm of adt3 epidermis
430
might suggest that a high amount of lipid substrates is available for the formation of cuticle
431
monomers (Fig. 4, B), but the observation that deposition of electron-opaque material occurs
432
within the deeper layers of the cell wall is a sign that the process is disrupted (Fig. 5, A). A
433
similar phenotype was previously seen in the bodyguard (bdg) mutant, a putative alpha-beta
434
hydrolase, which is thought to function in cutin biosynthesis, or as a cross-linking enzyme
435
(Kurdyukov et al., 2006). Similarly, Phe-derived phenolics components of cutin and cuticular
436
waxes (Nawrath et al., 2013) could participate in cuticle assembly as a cross-linker by
437
reacting with cutin monomers (Pollard et al., 2008). The involvement of phenylpropanoids in
438
cuticle formation has been previously reported for transparent testa mutants like ttg1, which
439
are regulators of the phenylpropanoid biosynthetic pathway (Koornneef, 1990; Xia et al.,
440
2010), strengthening the hypothesis that decreased Phe availability is linked to the adt3
441
defect in cuticle formation. TTG1 is also involved in epidermal development, as it participates
442
in trichomes and stomata patterning (Bean et al., 2002). Interestingly, mutations in other
443
genes involved in cuticle formation and up-regulated in adt3 mutant, like FIDDLEHEAD, also
444
affect the number of trichomes (Yephremov et al., 1999; Wellesen et al., 2001), while
445
different genes involved in the same process, like CER1 and CER6, affect stomatal number
446
(Bird and Gray, 2003). Thus, Phe derived compounds – and, hence, ADT3 function – could
447
play a role in cell differentiation by modulating cuticle assembly. Indeed, phenylpropanoids
448
appear to be more abundant in the cuticle of the guard cells, where the natural blue
449
fluorescence that they emit under UV light is higher than in the pavement cells because of
450
either the thickness or the richness of wax-bound phenolics in the epicuticular waxes of the
451
guard cells (Karabourniotis et al., 2001). Cuticle composition influences diffusion rates
452
(Kerstiens, 1996; Schreiber and Riederer, 1996), so tightening of the cuticular structure could
453
confine the effect of developmental cues within the stomatal cell lineage (Guseman et al.,
454
2010; Engineer et al., 2014), while a loose cuticle could facilitate exchanges of signaling
455
molecules to prevent pavement cells from acquiring stomatal identity at later stages of
456
stomatal development. Change of permeability due to a decrease in cell wall resistance could
457
then cause the arrest of stomatal development at the meristemoid stages in adt3, with a
458
consequent reduction in the number of stomata, or even at an earlier stage, when the initial
459
precursor stomatal lineage, the mother cells (MMC), first differentiate from the protoderm.
460
This would explain not only the reduced number of stomata in adt3 cotyledons, but also the
461
decreased number of pavement cells, as the MMCs divides asymmetrically to generate one
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
16
462
pavement cell and one meristemoid (Pillitteri and Torii, 2012). The defective cuticle could also
463
contribute to the enlargement observed for adt3 pavement cells, as they are incapable of
464
maintaining a correct water balance (Fig. 4, B). Interestingly, enlarged pavement cells are
465
also observed in stomata development mutants spch and mute (Tanaka et al., 2013),
466
suggesting that communication signals from the stomata cell lineage can influence the
467
morphology of proximal pavement cells. Hence, our work provides evidence that ADT3
468
function is required during early stages of seedling establishment to regulate the
469
development of the epidermal layer by controlling the size of pavement cells and stomatal cell
470
fate acquisition though the modulation of cuticle formation. Further analysis is required to
471
establish the chemical composition of the cuticle at this stage of cotyledon development.
472
473
Phe role in nutrient availability.
474
Our findings that loss of ADT3 compromises ROS homeostasis and alters cuticle
475
formation indicate that ADT3 function is required to coordinate defense and development in
476
the seed-to-seedling transition. Moreover, we speculate that Phe synthesis by ADT3 could be
477
implicated in energy production during early stages of plant growth. We have shown that
478
supply of Phe restores lipid synthesis in oil body-depleted adt3 cotyledons (Fig. 3, B,
479
Suppl.Fig. S6). As fatty acid biosynthesis takes place in chloroplasts (Yeats and Rose, 2013),
480
which are severely affected by loss of ADT3, Phe treatment of adt3 seedlings could support
481
this process by shielding the developing plastid membranes from the damage caused by
482
increased ROS levels at steady-state, therefore preserving their functionality. Alternatively,
483
de novo synthesis of fatty acids could be carried out using Phe as a substrate of the
484
tricarboxylic acid (TCA) cycle through Tyr conversion; however, resupplying adt3 mutant with
485
Tyr did not rescue adt3 phenotypes (Fig. S7). Although we cannot exclude that this may be
486
due to the inability of adt3 to uptake or metabolize Tyr in the form we supplied, it is tempting
487
to speculate that Phe could be metabolized by a pathway that does not encompass the Tyr
488
conversion step (Wang et al., 2013).
489
We could also rescue fatty acid biosynthesis by supplying the adt3-1 mutant with Asn
490
and NH4Ac (Fig. 5), which also led to the restoration of the pavement cell phenotype (Fig. 5).
491
Curiously, the pavement cell defect could not be ameliorated by addition of sucrose, which
492
was instead successful in rescuing the guard cell phenotype in adt3 cotyledons. Asn is known
493
to be an important nitrogen carrier for transport and storage and an increase in Asn through
494
up-regulation of ASN1 in dark-grown seedling was shown to be part of a metabolic strategy
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17
495
for the remobilization of metabolites in low energy states (Lam et al., 1998; Lam et al., 2003).
496
This seems to be the case for XANTHINE DEHYDROGENASE 1 (XHD1), whose activity
497
provides a source of nitrogen through purine catabolism (Brychkova et al., 2008). As both
498
proteins are significantly up-regulated in adt3 proteome (Table S1), we put forward that Phe
499
metabolism may provide an alternative source of nitrogen in etiolated seedlings. Indeed,
500
deamination by Phenylalanine Ammonia Lyase (PAL) is the first step of Phe metabolism and
501
the ammonia is thought to be recycled back into Phe biosynthesis (Singh et al., 1998; Corea
502
et al., 2012); however, under nitrogen shortage, glutamate from Phe metabolism could be
503
used as alternative nitrogen source (Lee et al., 2007) or as nitrogen donor for other
504
pathways, as observed for other organisms (Marusich et al., 1981; Sikora and Marzluf, 1982;
505
Vuralhan et al., 2003). Nitrogen requirement in adt3 seedlings could be needed to restore an
506
imbalance in carbon/nitrogen ratio that is caused by an increase in carbon availability as a
507
result of cell wall metabolism, as suggested by up-regulation of SUGAR TRANSPORTER
508
PROTEIN 1 (STP1) and the enrichment in enzymes involved in cell wall remodeling (Table 1
509
and Table S1)(Stadler et al., 2003; Baena-Gonzalez et al., 2007; Schofield et al., 2009).
510
511
ADT3 in signaling and development.
512
In Arabidopsis, α, β and γ subunits of G-proteins and their effectors are part of
513
complex signal transduction pathways that regulate defense mechanisms and developmental
514
processes (Huang et al., 2006; Pandey et al., 2006; Lee et al., 2013; Urano et al., 2013;
515
Urano and Jones, 2014). G-protein signaling is thought to participate in the developmental
516
program underlying the seed to seedling transition by influencing the structure of the
517
cotyledons. Mutations in GPA1, the sole Gα subunit found to date in Arabidopsis, result in low
518
stomata density, and in an increase in the size of epidermal cells in young seedlings (Ullah et
519
al., 2001; Zhang et al., 2008; Nilson and Assmann, 2010). These observations indicate a
520
defect in cell division and elongation as the possible cause of gpa1 morphological
521
phenotypes, which could also explain the short hypocotyl and open apical hook of gpa1
522
etiolated seedlings (Lease et al., 2001; Ullah et al., 2002; Ullah et al., 2003). Interestingly, the
523
assembly of a G-protein interactome has revealed that GPA1 interacts with proteins that are
524
involved in the modulation of cell wall composition or structure (Klopffleisch et al., 2011), like
525
those we reported misregulated in the adt3 proteome (Table I), providing a possible link
526
between G proteins and morphogenesis. ADT3 is part of a signaling module that includes
527
GPA1 and GCR1, whose activation by blue light leads to Phe synthesis (Warpeha et al.,
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
18
528
2006; 2008). gpa1 and gcr1 mutants are deficient in the production of Phe-derived
529
compounds, and share a high degree of phenotypic similarity with adt3, including reduced
530
number of stomata, broader cotyledons and poorly developed chloroplasts (Ullah et al., 2001;
531
Zhang et al., 2008; Nilson and Assmann, 2010), which can be rescued by supplying
532
exogenous Phe (Fig. 3; Suppl. Fig. S10). We therefore conclude that ADT3-derived Phe
533
could function as liaison between signaling and development at the early stages of seed
534
establishment.
535
536
CONCLUSIONS
537
In this study, We propose a simple model for ADT3 action, and the subsequent Phe produced
538
(Fig. 7, inset epidermis). We show that ADT3 localizes mainly in the cytosol of cotyledon cells
539
at the transition from seed to seedling, therefore placing Phe biosynthesis outside the plastid.
540
The existence of an extra-plastidic pathway for Phe biosynthesis was previously speculated
541
in Arabidopsis (d'Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996) and
542
evidence of a cytosolic route for Phe production was recently reported in petunia (Yoo et al.,
543
2013). Later in development, ADT3 appears to be localized in the chloroplast of green
544
tissues, suggesting that ADT3 localization appears to be age- and light-dependent; further
545
investigation will be required to unravel the molecular dynamics of ADT3 function.
546
Our work opens a window into an elusive phase of plant life, when the seedling has
547
emerged from the seed coat but is not yet capable to carry on photosynthesis. We
548
demonstrated that cytosolic Phe plays a critical role during the transition from heterotrophy to
549
autotrophy by protecting the cells from oxidative damage, providing substrates for defense
550
and, possibly, by fueling energy pathways. In laboratory conditions this transition is quite fast,
551
since germination is routinely achieved by planting seeds on synthetic medium in light
552
conditions; however, in natural environments variables such as light limitation due to dense
553
vegetation could extend the transition as well as the agricultural practice of burying seeds
554
deeper than natural dispersal, making accumulation of cytosolic Phe critical for survival.
555
Recently, a molecular switch that regulates allocation of resources to growth and to defense
556
has been described where the increase in uncharged tRNAPhe accumulation as a
557
consequence of the imbalance in Phe metabolism that accompanies pathogen challenge
558
switches off primary growth and development by repressing the expression of chloroplast
559
proteins and switch on the cellular program that lead to systemic acquired resistance (SAR)
560
(Pajerowska-Mukhtar et al., 2012). Through a similar mechanism, Phe availability could play
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
19
561
a critical role in etiolated seedlings by providing a physical, metabolic and defense support to
562
the emerging embryo.
563
564
MATERIALS AND METHODS
565
Chemicals. All chemicals, unless otherwise noted, were obtained from Sigma (St. Louis, MO).
566
Plant materials, seed stocks and accessions. Seeds of wild type (WT) Columbia (Col)
567
Arabidopsis thaliana, T-DNA insertion lines for AROGENATE DEHYDRATASE_3 (ADT3,
568
AT2G27820) adt3-1 (SALK_029949) and adt3-6 (SALK_071907) were obtained from the
569
Arabidopsis Biological Resource Center (Columbus, OH)(Alonso et al., 2003). Double mutant
570
(acc. CS8602) of CYTOCHROME P450 84A1/FERULIC ACID 5 HYDROXYLASE 1 (FAH1-7;
571
At4g36220) (Landry et al., 1995) and CHALCONE SYNTHASE/TRANSPARENT TESTA 4
572
(TT4-1; At5g13930) (Li et al., 1993) were an appreciated gift from Wendy Peer, University of
573
Maryland College Park. The mutant lines are homozygous null for the reported insertions.
574
The mutant speechless (SPCH; At5g53210) was an appreciated gift from Keiko Torii,
575
University of Washington, Seattle; mute (At3g06120) was an appreciated gift from Dominique
576
Bergmann, Stanford University.
577
Plant growth conditions for experiments. Seeds of Arabidopsis thaliana WT or T-DNA
578
insertion mutants were sown on 0.5X MS medium with no added sugars, hormones, vitamins
579
or other nutrients in complete darkness using a dim green safelight to prevent
580
photomorphogenesis (Mandoli and Briggs, 1982; Orozco-Nunnelly et al., 2014). For some
581
experiments, seeds were sown on top agarose supplemented with chemical (500μM NH3Ac,
582
500μM L-Phe, 500μM L-Tyr, 500μM L-Trp, L-Asparagine) then grown for 6 d. For other
583
experiments, 4-d post-sowing on 0.5X MS top agarose, disks bearing about 30 seedlings
584
were moved to filter paper soaked with 0.5X MS (control) or 1% sucrose or 1% mannitol (final
585
concentration in 0.5X MS), then analyzed after 48 h. Most replicates n=30 unless specified.
586
Contrast microscopy: deconvoluting. Living seedlings were mounted in sterile water to
587
be viewed on a Zeiss Observer.Z1 deconvoluting microscope utilizing the 20X objective or
588
63X (oil immersion) with DAPI excitation to view traditional fluorescence, or optical apotome
589
utilizing XCite 120 LED/Lumen Dynamics DAPI, FITC, CY5 LEDs, as managed by Zen pro
590
software (2012). Images were captured on a high resolution Axio503 mono camera. At least
591
30 seedlings were viewed per experimental condition for experiments unless described
592
otherwise (20-40 seedlings per experimental condition for analysis). A 150 μm square
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
20
593
superimposed on images was analyzed to quantitate cell-type numbers, and cell area was
594
quantified using Image J (http://imagej.nih.gov/ij/).
595
Cellular stress assays. 4 d after moving to 20oC, dark-grown seedlings were placed on
596
sterile filter paper for 3 h with either 0.5X MS (control), potassium iodide (KI), Phe, or the
597
analog para-DL-flouro-Phenylalanine (Conway et al., 1963). Seedlings received an empircally
598
determined sublethal (3 min) dose of 254nm (UV methods described in Warpeha et al.,
599
2008). Young cotyledons emit natural fluorescence after absorption of UV (DAPI)(Warpeha et
600
al., 2008) or blue/blue-green (FITC)(Tattini et al., 2004; Warpeha et al., 2008) irradiation, so
601
these LEDs were used to collect fluorescence information. The specific cell permeable dye
602
CellRox Deep RedTM (Life Technologies, Grand Island, NY) emitting in the red/far red region
603
was used as recommended by the manufacturer to detect ROS species dark-grown
604
seedlings (Cy5 channel, false-colored pink) (Ghura et al., 2016). In brief, dark-grown
605
seedlings of WT or adt3 (each replicate 30 seedlings) on d 6 were exposed to a sublethal
606
treatment of UV-C (254nm), then cut mid hypocotyl to be immediately immersed in 400 μL of
607
5 μM CellRoxTM in sterile DMSO in a glass well dish in darkness. Samples were orbitally
608
rotated (50 rpm) for 30 min. At that time CellRoxTM reagent was removed by p200 pipet tip to
609
avoid removing cut seedlings, then solution immediately replaced by sterile 1.0X PBS (pH
610
7.2) for 3 x 3 min washes under the same rotation. After washing, the PBS was removed and
611
seedlings were rinsed 1 time in sterile water for 3 min, then mounted in sterile water on glass
612
slides for immediate microscopy. Cotyledons were imaged at 63X (oil immersion), and for
613
some experiments 20X objectives with DAPI, FITC and Cy5 LEDs in 1 μm optical sections by
614
apotome (microscope described above). At least 20 seedlings were viewed per replicate, and
615
all exposure settings were based on the WT untreated cotyledons (that setting was used for
616
all seedling conditions and genotypes). For seedlings exposed to Phe, seedlings were moved
617
on d 6 to 500μM L-Phe for 3 h, then experienced the same conditions for CellRoxTM reagent
618
experiments. All growth conditions and seedling manipulations were identical for Singlet
619
Oxygen Sensor GreenTM (SOSG)(Thermo Fisher, Rockford, IL), however the preparations of
620
the reagent were as follows. SOSG is provided as a solid, and was dissolved as directed in
621
methanol to 5 mM. The working solution was utilized at 10 μM in 1X PBS (pH 7.2). Identically
622
to CellRox experiments, seedlings were immersed into the reagent immediately post-
623
irradiation, then washed in the same way, then mounted in sterile water for microscopy.
624
Microscopy was handled in the same way as for CellRox reagent, except that SOSG
625
responds to singlet oxygen by causing a large fluorescence that can be captured by FITC.
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
21
626
The LEDs utilized for these experiments were DAPI, FITC and Texas Red. As for CellRox,
627
the WT unirradiated was the replicate used to determine the best exposure upon which all
628
other samples used the identical exposure settings. From all fluorescence experiments
629
epidermal pavement cells were traced in ImageJ and the fluorescence level (artificial units)
630
was determined, then compared (Ghura et al., 2016). A minimum of 6 cells per cotyledon of
631
4-6 seedlings per replicate was assessed utilizing Prism (see Statistics).
632
Nile Red staining. 6-d-old dark-grown seedlings were cut into 1mM Nile Red stain
633
(saturated solution in acetone as directed by manufacturer (Sigma); working solution in 1X
634
PBS pH 7.2 made immediately before use, maintained in darkness throughout) and
635
incubated for 30 min under rotation, with all same experimental handling, timing as SOSG.
636
Seedlings then were mounted in sterile water as described for SOSG, then visualized by
637
optical sectioning (described above) at 20X magnification using FITC (false-colored gold).
638
n=30 each replicate.
639
Cloning. Standard molecular biology techniques and the Gateway system (Invitrogen)
640
were used for all cloning procedures as described (Orozco-Nunnelly et al., 2014). The 819 bp
641
ADT3 promoter fragment was cloned into pDONR P4-P1R (Invitrogen) and the ADT3 ORF
642
fragment was cloned into pENTR/D-TOPO (Invitrogen). pGreen (Hellens et al., 2000) was
643
used as the destination vector for the construct ADT3::ADT3-GFP or ADT3::ADT3-CFP. T3
644
homozygous transformed seedlings were used in experiments.
645
Visualization of transgene expression on spinning disk confocal. Transgenic and
646
untransformed 4-d-old dark-grown seedlings were mounted live and immediately viewed on
647
an Andor WD Spinning Disk confocal system using iQ3 software as described (Orozco-
648
Nunnelly et al., 2014). 1nm width LEDs used included 405nm (DAPI, Blue), 488nm (GFP,
649
Green), 445nm (CFP, magenta), or 561nm (Red). Images were captured on 30X or 60X as
650
described, with 1μm optical section thickness. Images were prepared with ImageJ software
651
(NIH, Bethesda, MD). n=20 each replicate.
652
TEM. Seedlings were harvested into 0.5X Karnovsky’s fixative for overnight storage at
653
4oC, buffer washed then fixed in 1% OSO4 for 2 h. After washes in 0.1M Cacodylate buffer,
654
the samples were embedded in 3% SeaPrep agar, dehydrated in a 10-100% ethanol series
655
and infiltrated with Spurr’s resin, which was let to polymerize at 70oC for 2 d. Thin sections
656
(between 70-80nm) of at least 5 seedlings for each sample were cut on a Leica UC7
657
ultramicrotome, collected on a 200 mesh formvar/carbon coated copper grid (Ted Pella, Inc.)
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
22
658
and stained with aq UA for 2 h and lead citrate for 5 min. The grids were viewed in a JEOL
659
JEM 1400 TEM and images taken at 120KV.
660
Toludine Blue staining. On d 4, dark-grown seedling islands of top agarose were
661
transplanted to 0.5XMS in phytatrays with disk annealed by 200μL top agarose drop to the
662
tray. The bottom of the phytatray was covered with sterile water only or 0.5XMS medium and
663
on d 6, the phytatray was filled with Toludine Blue solution to cover seedlings (Tanaka et al.,
664
2004). After 3 min seedlings were washed and immediately imaged on bright field Zeiss V.2
665
Stereo dissecting scope.
666
Statistics. Data shown in figures were entered into Prism v. 5.0 (GraphPad Software,
667
Inc., GraphPad.com), where mean and SE were calculated for relevant experiments.
668
Unpaired T test with Welch’s correction were used to assess significance.
669
Protein Extraction, Digestion, Labeling and Sub-fractionation. Protein was extracted
670
from each biological replicate of 6-d-old dark-grown WT and adt3 liquid nitrogen ground
671
samples, as previously described (Alvarez et al., 2011). The pellets were then dissolved in
672
150 µL of 3 M urea, 0.5 M triethylammonium bicarbonate (TEAB), pH 8.5, and assayed using
673
the CB-X™ protein assay kit (G-Biosciences, St Louis, MO). Each sample was reduced in a
674
final concentration of 5 mM tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) for 1 h at
675
37 °C and alkylated using 10 mM iodoacetamide for 30 min at 22 °C in the dark. Aliquots
676
containing 40 µg of protein were diluted to 1 M urea and digested with trypsin at a 1:20
677
enzyme:protein ratio for 16 h at 37 °C. Samples were desalted using 1 cc Sep-Pak® Vac
678
C18 solid-phase extraction (SPE) columns (Waters Ltd, Milford, MA) and vacuum centrifuged
679
(Centrivap™, Labconco, Kansas City, MO) to dryness. Stable isotope labeling was carried
680
out with TMT-6plex reagents (Thermo Fisher Scientific, Rockford, IL) according to the
681
manufacturer’s instructions and the six samples were combined, vacuum centrifuged and
682
desalted using SPE. Sub-fractionation was carried out on a 3100 OFFGEL™ fractionator
683
(Agilent Technologies, Santa Clara, CA) using pH 3-10 gradient strips and ampholytes
684
according to the manufacturer’s guidelines, but without the incorporation of glycerol. Focusing
685
was carried out for 50 kWh with a current limit of 50 µA and voltage limit of 8000 V. Fractions
686
were fully dried.
687
Protein identification and quantification by liquid chromatography–tandem mass
688
spectrometry (LC-MS/MS). LC-MS/MS was carried out on a LTQ-Orbitrap Velos Pro
689
(ThermoFisher Scientific, Waltham, MA) as previously described (Alvarez et al., 2013;
690
Alvarez et al., 2014) coupled with a U3000 RSLCnano HPLC (ThermoFisher Scientific,
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
23
691
Waltham, MA). The dried OFFGEL fractions were dissolved in 5% acetonitrile, 0.1% formic
692
acid and a portion loaded onto a C18 trap column (PepMap100, 300µm ID × 5mm, 5µm
693
particle size, 100Å; ThermoFisher Scientific, Waltham, MA) at a flow rate of 15 µL/min for 4
694
min equilibrated with 2% acetonitrile, 0.1% formic acid. Peptide separation was carried out on
695
a C18 column (Acclaim PepMap RSLC, 15cm × 75μm nanoViper™, C18, 2μm, 100 Å,
696
ThermoFisher Scientific) at a flow rate of 0.3μL/min and the following gradient: Time = 0-3
697
min, 2% B isocratic; 3-12 min, 2-15% B; 12-84 min, 15-38% B; 84-93 min, 38-50% B; 93-97
698
min 50-90% B. Mobile phase A, 0.1% formic acid; mobile phase B, 0.1% formic acid in 80:20
699
acetonitrile:water. The Orbitrap mass analyzer was operated as previously described
700
(Alvarez et al, 2014) except for the exclusion duration, which was set at 90 s, and the
701
minimum MS ion count for triggering MS/MS to 50,000 counts.
702
Database search and Data mining for proteome. Data processing was automated
703
using Mascot Daemon (Matrix Science, London, UK). Mascot Distiller, version 2.4.3.3 64 bit
704
(Matrix Science, London, UK) was used to create mgf files which Mascot Server, version 2.4
705
(Matrix Science, London, UK) searched against the TAIR10 Arabidopsis thaliana database
706
(35,386 entries) to identify and quantify the proteins with a false discovery rate of 1 %. The
707
search parameters used have been previously described (Alvarez et al., 2014) except for the
708
fixed modifications which were set as TMT (N terminal) and TMT (K). Only proteins with at
709
least two peptides were used for the analysis. Peptide and protein ratios were calculated
710
using the weighted method in Mascot where the intensity values of the set of peptides are
711
summed and the protein ratio(s) calculated from the summed values. Normalization of the
712
ratios was performed using the total labeling intensity of each species (summed intensities).
713
The ratios and standard deviations for all combinations of the replicates adt3/WT were
714
reported. An overall ratio for a protein hit was only reported if the minimum number of two
715
peptide matches was achieved. If the ratios for the peptide matches were not consistent with
716
a sample from a normal distribution, the ratios were not used further in the analysis. Only
717
protein ratios statistically different from 1 (p value < 0.05), detected from the replicates, were
718
averaged. Only proteins for which there were at least 3 protein ratios, of which at least one
719
was associated with each control for each of the three mutant replicates, were used for
720
further analyses. For these proteins, the standard deviation and coefficient of variation were
721
calculated.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
24
722
The proteome was analyzed using the bioinformatics platform Virtual Plant
723
(http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/)(Katari et al., 2010) and the KEGG database
724
(http://www.genome.jp/kegg/).
725
726
727
FIGURE LEGENDS
728
729
Figure 1. ADT3 localizes predominantly in the cotyledon of young, dark grown
730
seedlings.
731
Expression of ADT3::ADT3-CFP was analyzed in live 4-d-old adt3 seedlings by SD confocal
732
microscopy. A. (Top panels) In this view, CFP fluorescence (445 nm, false-colored magenta)
733
is detected in developing mesophyll cells of the cotyledon and in the SAM (yellow arrow)
734
(1μm thick optical slice). Autofluorescence from developing plastids (448nm, false-colored
735
green) is visible. Merged images of 445 and 488 indicate that ADT3 localizes mainly in the
736
cytosol. B. In the epidermal layer view, ADT3::ADT3-CFP accumulates in the pavement cells
737
(PC) and young guard cell (GC) lineage but not in mature GC. The signal captured by 488 is
738
due to accumulation of polyphenols (flavonoids) autofluorescence in the epidermis. Images
739
are representative, 60X. n=20. Scale bar = 50μm.
740
741
Figure 2. ROS levels are elevated in adt3 mutant seedlings compared to WT.
742
The cotyledon epidermis cells of live 6-d-old dark-grown seedlings incubated with
743
CellRoxTM Deep Red cell-permeable fluorescent dye are shown (merged DAPI, FITC and
744
Cy5). WT (top panels) and adt3-1 (middle panels) seedlings were either mock irradiated
745
(unirradiated control)(left panels) or irradiated with 254nm (+UV-C)(right panels), then
746
immediately immersed in CellRoxTM reagent, which emits fluorescence in Cy5 in presence of
747
ROS. adt3-1 were also treated with 500 μM Phe (+Phe) for 3h and then irradiated with
748
254nm (+UV-C) (bottom left panel). After washes and mounting in sterile water, seedlings
749
were imaged at 1μm optical slice thickness on a deconvoluting microscope in the epidermal
750
plane. CellRoxTM signal is low in untreated WT cells, and increases as a result of UV-C
751
irradiation, while signal is evident in unirradiated adt3-1 cells. Feeding Phe to adt3-1
752
seedlings before irradiation prevented ROS accumulation (bottom left panel and bar graph).
753
Scale bar= 10μm. Cy5 fluorescence from excited CellRoxTM (false-colored pink) was
754
quantified in PC (relative fluorescence, i.e. artificial units) (bottom right panel). In the bar
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
25
755
graph each value represents a minimum of 6 cells of 4 replicates, minimum 20 representative
756
seedlings viewed per replicate. Error bars are SEM. * = p<0.05.
757
758
Figure 3. Epidermal defects of adt3-1 seedlings.
759
A. Epidermis of WT (top) and adt3-1 live cotyledons (Cot; bottom) viewed on a deconvoluting
760
microscope (DAPI contrast). GC lineage (meristemoid and GC) cells appear dark gray, and
761
PCs appear light gray. Defects are prevented by inclusion of Phe in medium (right panels).
762
Scale bar = 25μm. B. Measurement of PC area and number, cotyledon width and GC number
763
in WT and adt3-1 without, and with added Phe. Cartoon indicates structure measured. Error
764
bars are SEM. ****<.0001. n=4 replicates (20-40 seedlings per experimental condition).
765
766
Figure 4. Ultrastructural phenotype of adt3-1 epidermal and mesophyll cells.
767
A. (Left) TEM micrographs of sections perpendicular to the adaxial surface of the cotyledons
768
of 6-day old, dark-grown WT (top) and adt3-1 (bottom) seedlings. (Right) Details of WT (a, b
769
and c) and adt3-1 (d, e and f) micrograph: cytoplasm of epidermal cells (a and c), plastids (b
770
and e) and cytoplasm of mesophyll cells (note the absence of oil bodies in f). n=5 seedlings
771
(~100 cells). Scale bar = 5 μm.
772
B. Adaxial side view of mesophyll cells in the cotyledons of 6-d-old dark grown WT (left),
773
adt3-1 (middle) and adt3-1 + Phe (right) seedling after staining with Nile Red (false-colored
774
gold). Merges of DAPI (blue), FITC (gold) and CY5 (pink) are shown. n=3 (30 seedlings).
775
Scale bar = 50 μm.
776
777
Figure 5. Defective cuticle formation, altered permeability and aberrant patterning of
778
GC in adt3-1 seedlings.
779
A. Ultrastructure of the cuticle of adt3-1 epidermis. TEM micrographs of WT (top) and adt3-1
780
(bottom) epidermis show deposition of electron-opaque material within adt3-1 cell wall (red
781
triangles).
782
B. Increased permeability of adt3-1 epidermis under hyposmotic treatment. TB penetration in
783
dark grown 6-d-old WT (top), and adt3-1 (bottom) seedlings after 48 h incubation in sterile
784
water. Scale bar = 50μm.
785
C. 16:8 light-grown, 6-d-old adt3-1 seedlings exhibit abnormal stomatal development; GC in
786
focal plane are circled. Scale bar = 25μm.
787
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
26
788
Figure 6. Effect of exogenous sucrose, Asn, and NH3Ac supply on adt3-1 epidermis.
789
A. GC number phenotype is rescued by sucrose, but not cotyledon width and PC defects of
790
adt3-1 seedlings. 4-d-old, dark grown seedlings were transferred to 1% sucrose and
791
epidermal phenotypes were quantified 48 h later (6-d-old). n=20-25. **** <.0001.
792
B. Asn supply rescues cotyledon width and PC number phenotypes but not the GC lineage in
793
adt3 seedlings. L-asparagine (Asn) was included in the top agarose at planting. Epidermal
794
phenotypes were measured on d 6. n=20-25. **** <.0001.
795
C. Asn and NH3Ac supply restores cotyledon morphology and oil bodies in adt3 cotyledons.
796
DAPI contrast images (top; Scale bar = 25μm), and Nile Red signal (bottom; Scale bar =
797
50μm) of 6-d-old, dark grown adt3 seedlings. Nile Red signal was false-colored gold. Merge
798
of DAPI (blue), FITC (gold) and CY5 (pink) is shown.
799
800
Figure 7. Model indicating multiple potential roles of Phe, mainly produced in the cytosol by
801
ADT3 in Arabidopsis cotyledons at the seed-to-seedling transition.
802
803
SUPPLEMENTAL FILES (SFigures and STables)
804
805
SFIGURES
806
SFigure S1. ADT3::ADT3-GFP restores adt3-1 cotyledon epidermis to WT appearance, and
807
confers survival to UV stress.
808
SFigure S2. Expression of ADT3-GFP fusions in adt3-1 and WT background and
809
ADT3::ADT3-GFP in the SAM.
810
SFigure S3. ROS detection in adt3-6 mutant by CellRoxTM.
811
SFigure S4. Singlet Oxygen Species (SOS) detection in adt3 mutant seedlings incubated
812
with Singlet Oxygen sensor GreenTM (SOSG).
813
SFigure S5. ROS levels detected by CellRoxTM from unirradiated and UV-C irradiated WT
814
and adt3-1 seedling cotyledons before and after pretreatment with Phe, the ROS scavenger
815
KI and a Phe analog.
816
SFigure S6. Epidermal defects in adt3-6 seedlings are rescued by Phe.
817
SFigure S7.
818
stomatal lineage mutants do not respond to Phe like adt3-1 mutants.
819
SFigure S8. Experiments indicate mannitol does not rescue GC lineage progression.
Phenotypic defects of adt3-1 seedlings not complemented by Tyr or Trp;
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
27
820
SFigure S9. Developing chloroplast ultrastructural phenotype rescue in adt3 mesophyll cells
821
by inclusion of Phe in planting medium,
822
SFigure S10. gpa1 and gcr1 mutants have cotyledon phenotypes that can be rescued by
823
Phe.
824
825
S TABLES
826
STable 1. Proteome data of 6-d dark-grown adt3-1 mutant compared to WT.
827
STable 2. Plant Ontology analysis for for upregulated proteins in adt3 proteome.
828
STable 3. GO term analysis for biological process for adt3 upregulated proteins in the
829
"epidermal cell" Plant Ontology category.
830
STable 4. GO term analysis for cell components for upregulated proteins in adt3 proteome.
831
STable 5. GO term analysis for biological process for adt3 upregulated proteins in the
832
“plasma membrane” category.
833
STable 6. GO term analysis for biological process for adt3 upregulated proteins in the
834
“vacuole” category.
835
STable 7. GO term analysis for biological process for adt3 upregulated proteins in the “cell
836
wall” category.
837
STable 8. Plant Ontology analysis for for downregulated proteins in adt3 proteome.
838
STable 9. GO term analysis for molecular function for adt3 downregulated proteins in the
839
“epidermal cell” Plant Ontology category.
840
STable 10. GO term analysis for cell components for downregulated proteins in adt3
841
proteome.
842
STable 11. GO term analysis for biological process for downregulated proteins in adt3
843
proteome.
844
845
ACKNOWLEDGEMENTS: We thank Jennifer Baek, Ashley Williams, Yang Chen, Huini Wu,
846
and Nadia Kukuruza for assistance with experiments; Dr. Jeremy Lynch and Dr. Teresa
847
Orenic for assistance with confocal, and deconvoluting microscopy, respectively; and, Jack
848
Gibbons for EM services featured in Supplementary data. We thank Stephen MacFarlane,
849
Sharmon Knecht, and especially Bobbie Schneider for TEM (Fred Hutchinson Cancer Center,
850
Seattle, WA). We thank John Cason for artwork services herein, except Fig. 7. We thank Dr.
851
Lon Kaufman, now at CUNY Hunter College, and Dr. Gary Gardner, University of Minnesota,
852
for helpful discussion.
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28
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854
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39
Table 1. Gene Ontology (GO) term analysis of UP-regulated and DOWN-regulated proteins
in adt3 proteome.
UP-regulated proteins in adt3 mutant
Plant Ontology (PO) Term
p-value
guard cell
1.28*10-78
shoot epidermal cell
2.08*10-77
epidermal cell
1.33*10-76
Transport
1.17*10-16
carboxylic acid/oxoacid metabolic process
6.54*10-10
fatty acid metabolic process
3.42*10-06
GO Term
plasma membrane
Transport
Vacuole
Transport
cell wall
cell wall organization or biogenesis
xylan catabolic process
glucan metabolic process
hydrolase activity
Growth
primary metabolic process
p-value
5.70*10-109
5.92*10-27
2.05*10-34
2.03*10-15
7.98*10-32
3.76*10-06
1.18*10-3
4.37*10-3
5.39*10-14
2.78*10-11
1.94*10-14
DOWN-regulated proteins in adt3 mutant
PO Term
shoot epidermal cell
guard cell
epidermal cell
p-value
1.19*10-86
4.38*10-86
3.35*10-84
oxidoreductase activity
GO Term
Plastid
plastid stroma
envelope
thylakoid
stromule
plastoglobule
nitrogen compound metabolic process
primary metabolic process
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1.92*10-14
p-value
5.61*10-128
6.38*10-127
2.77*10-50
6.28*10-46
1.18*10-19
3.15*10-10
1.8*10-17
7.25*10-25
40
Table 2. KEGG pathway analysis for up- and down-regulated proteins in adt3
mutant.
Number of genes
Total*
DOWN-
UP-
regulated
regulated
Carbohydrate metabolism
Glycolysis / Gluconeogenesis
29
19
10
Pyruvate metabolism
27
14
13
Starch and sucrose metabolism
27
6
21
Glyoxylate and dicarboxylate metabolism
26
23
3
19
4
15
Ascorbate and aldarate metabolism
15
7
8
Pentose and glucuronate interconversions
15
2
13
Citrate cycle (TCA)
14
9
5
Pentose phosphate pathway
11
10
1
Fructose and mannose metabolism
11
7
4
Inositol phosphate metabolism
7
2
5
Galactose metabolism
6
1
5
Carbon fixation in photosynthetic organisms
32
27
5
Oxidative phosphorylation (ATP synthase)
17
6
11
Photosynthesis
11
10
1
Nitrogen metabolism
9
8
1
Sulfur metabolism
6
6
0
Amino
sugar
and
nucleotide
sugar
metabolism
Energy metabolism
* number of input genes assigned by the KEGG database.
1157
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41
Para_Fig1
A 445
B
445
488
488
merge
merge
Figure 1. ADT3 localizes predominantly in the cotyledon of young, dark
grown seedlings.
Expression of ADT3::ADT3-CFP was analyzed in live 4-d-old adt3-1 seedlings
by SD confocal microscopy. A. (Top panels) In this view, CFP fluorescence (445
nm, false-colored magenta) is detected in developing mesophyll cells of the cotyledon and in the SAM (yellow arrow) (1μm thick optical slice). Autofluorescence
from developing plastids (448nm, false-colored green) is visible. Merged images
of 445 and 488 indicate that ADT3 localizes mainly in the cytosol. B. In the
epidermal layer view, ADT3::ADT3-CFP accumulates in the pavement cells (PC) and
young guard cell (GC) lineage but not in mature GC. The signal captured by
488 is due to polyphenols (flavonoids) autofluorescence in the epidermis.
Images are representative, 60X. n=20. Scale bar = 50μm.
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Para_Fig2
WT
WT + UV-C
adt3-1
adt3-1 + UV-C
adt3-1 + Phe + UV-C
fluorescence intensity from CellRox
Untreated
50
40
wt
adt3-1
UV-C
Phe+UV-C
*
*
30
20
10
0
Figure 2. ROS levels are elevated in adt3 mutant seedlings compared to WT.
The cotyledon epidermis cells of live 6-d-old dark-grown seedlings incubated with
TM Deep Red cell-permeable fluorescent dye are shown (merged DAPI, FITC and
CellRoxTM
Cy5). WT (top panels) and adt3-1 (middle panels) seedlings were either mock irradiated
(unirradiated control)(left panels) or irradiated with 254nm (+UV-C)(right panels), then
TM reagent, which emits fluorescence in Cy5 in presence
immediately immersed in CellRoxTM
of ROS. adt3-1 were also treated with 500μM Phe (+Phe) for 3h and then irradiated with
254nm (+UV-C) (bottom left panel). After washes and mounting in sterile water, seedlings
were imaged at 1μm optical slice thickness on a deconvoluting microscope in the epidermal
TM signal is low in untreated WT cells, and increases as a result UV-C irradiplane. CellRoxTM
ation, while signal is evident in unirradiated adt3-1 cells. Feeding Phe to adt3-1 seedlings
before irradiation prevented ROS accumulation (bottom left panel and bar graph). Scale bar
TM (false-colored pink) was quantified in
= 10μm. Cy5 fluorescence from excited CellRoxTM
PC (relative fluorescence, i.e. artificial units) (bottom right panel). In the bar graph each
value represents a minimum of 6 cells of 4 replicates, minimum 20 representative seedlings
viewed per replicate. Error
barsfrom
areonSEM.
= p<0.05.
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- Published by www.plantphysiol.org
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Para_Fig3
A
WT + Phe
WT
****
60
40
20
width of cotyledon day 6
0
****
400
350
300
250
stomata
200
+P
he
ad
t3
ad
t3
0
adt3-1 adt3-1+Phe
Ph
e
WT
W
T+
ad
t3
+P
he
U
nt
r
adt3-1 adt3-1+phe
ad
t3
w
tU
nt
r
WT
****
10
un
tr
200
20
ad
t3
ad
t3
300
GC (number)
ad
t3
****
Ph
e
ce
lls
W
T
ce
lls
400
W
T
30
500
U
nt
Cot width (µm) PC (number)
80
W
T
B
adt3-1 + Phe
cell #
PC area (µm2)
adt3-1
Figure 3. Epidermal defects of adt3-1 seedlings.
A. Epidermis of WT (top) and adt3-1 live cotyledons (Cot; bottom) viewed on a deconvoluting
microscope (DAPI contrast). GC lineage (meristemoid and GC) cells are dark gray, and
PCs appear light gray. Defects are prevented by inclusion of Phe in medium (right). Scale
bar= 25µm. B. Measurement of PC area and number, cotyledon width and GC number in
WT and adt3 without and with added Phe. Cartoon indicates structure measured. ****<.
0001. n=4 replicates (20-40 seedlings per experimental condition).
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Para_Fig4
A
a
WT
b
c
d
e
f
adt3-1
B
WT
adt3-1
adt3-1 + Phe
Figure 4. Ultrastructural phenotype of adt3-1 epidermal and mesophyll cells.
A. (Left) TEM micrographs of sections perpendicular to the adaxial surface of the
cotyledons of 6-day old, dark-grown WT (top) and adt3-1 (bottom) seedlings. (Right)
Details of WT (a, b and c) and adt3-1 (d, e and f) micrograph: cytoplasm of epidermal
cells (a and c), plastids (b and e) and cytoplasm of mesophyll cells (note the absence of
oil bodies in f). n=5 seedlings (~100 cells). Scale bar = 5µm.
B. Adaxial side view of mesophyll cells in the cotyledons of 6-d-old dark grown WT (left),
adt3-1 (middle) and adt3-1 + Phe (right) seedling after staining with Nile Red (false-colored
gold). Merges of DAPI (blue), FITC (gold) and CY5 (pink) are shown. n=3 (30 seedlings).
Scale bar= 50µm.
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Para_Fig5
A
WT
adt3-1
B
WT
adt3-1
C
WT
adt3-1
Figure 5. Defective cuticle formation, altered permeability and aberrant patterning of
GC in adt3-1 seedlings.
A. Ultrastructure of the cuticle of adt3-1 epidermis. TEM micrographs of WT (top) and
adt3-1 (bottom) epidermis show deposition of electron-opaque material within adt3-1 cell
wall (red triangles).
B. Increased permeability of adt3-1 epidermis under hyposmotic treatment. TB penetration
in dark grown 6-d-old WT (top), and adt3-1 (bottom) seedlings after 48 h incubation in
sterile water. Scale bars= 50µm.
C. 16:8 light-grown, 6-d-old adt3-1 seedlings exhibit abnormal stomatal development; GC in
focal plane are circled. Scale bar = 25µm.
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Para_Fig6
A
B
C
450
****
400
****
300
300
250
20
20
****
+A
sp
ad
t3
60
40
adt3-1 + Asn
0
0
30
15
****
****
ad
t3
+a
sp
ad
t3
un
tr
su
+
20
W
T
****
ad
t3
ad
t3
un
tr
25
W
T
adt3-1 + NH3Ac
10
10
5
0
0
WT adt3-1 adt3-1+Asn
+a
sp
ad
t3
un
tr
4d
ad
t+
su
c
ad
t3
W
T
WT adt3-1 adt3-1+Suc
W
T
GC (number)
un
tr
****
W
T
r
nt
U
T
****
ad
t3
+S
u
80
40
20
adt3-1 + NH3Ac
250
Data 10
W
60
adt3-1 + Asn
****
400
350
80
PC (number)
450
350
ad
t3
Cot width (mm)
Data 8
Figure 6. Effect of exogenous sucrose, Asn, and NH3Ac supply on adt3-1
epidermis.
A. GC number defect phenotype is rescued by sucrose (Suc), but not cotyledon width
and PC defects of adt3-1 seedlings. 4-d-old, dark grown seedlings were transferred to
1% sucrose and epidermal phenotypes were quantified 48 h later (6-d-old). n=20-25.
**** <.0001.
B. Asn supply rescues cotyledon width and PC number phenotypes but not the GC
lineage in adt3-1 seedlings. L-asparagine (Asn) was included in the top agarose at
planting. Epidermal phenotypes were measured on d 6. n=20-25. **** <.0001.
C. Asn and NH3Ac supply restores cotyledon morphology and oil bodies in adt3-1
cotyledon. DAPI contrast images (top; Scale bar = 25µm), and Nile Red signal (bottom;
Scale bar = 50µm) of 6-d-old, dark grown adt3-1 seedlings. Nile Red signal was falsecolored gold. Merge of DAPI (blue), FITC (gold) and CY5 (pink) is shown.
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Para_Fig7
ADT3
(cytosol)
Phe
(cytosol)
CELL
FATE
Figure 7. Model indicating multiple,
potential roles of Phe, mainly produced
in the cytosol by ADT3 in Arabidopsis
cotyledons at the seed-to-seedling transition.
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