Loss-of-function of MEDIATOR 12 or 13 subunits causes the swelling of root hairs in response to sucrose and abscisic acid in Arabidopsis

ABSTRACT Root hairs are epidermal cell extensions that increase the root surface for water and nutrient acquisition. Thus, both the initiation and elongation of root hairs are critical for soil exploration and plant adaptation to ever changing growth conditions. Here, we describe the critical roles of two subunits of the Mediator complex, MED12 and MED13, in root hair growth in response to sucrose and abscisic acid, which are tightly linked to abiotic stress resistance. When compared to the WT, med12 and med13 mutants showed increased sensitivity to sucrose and ABA treatments on root meristem and elongation zones that were accompanied with alterations in root hair length and morphology, leading to the isodiametric growth of these structures. The swollen root hair phenotype appeared to be specific, since med8 or med16 mutants did not develop rounded hairs when supplied with 4.8% sucrose. Under standard growth medium, MED12 and MED13 were mainly expressed in root vascular tissues and cotyledons, and their expression was repressed by sucrose or ABA. Interestingly, med12 and med13 mutants manifested exacerbated levels of nitric oxide under normal growth conditions, and upon sucrose supplementation in trichoblast cells, which coincided with root hair deformation. Our results indicate that MED12 and MED13 play non-redundant functions for maintenance of root hair integrity in response to sucrose and ABA and involve nitric oxide as a cellular messenger in Arabidopsis thaliana.


Introduction
In multicellular organisms, cell fate determination is controlled by environmental conditions and cell-specific cues that concertedly generate the distinct tissues. 1 In Arabidopsis roots, files of epidermal cells differentiate into root hairs that act as extensions influencing both soil physicochemical conditions through exudation of a wide variety of compounds and enhance absorptive potential. 2 Root hair length and density are key factors for plant growth and development owing its direct involvement in water and nutrient acquisition. [3][4][5] In the Arabidopsis root, there are two kinds of epidermal cell types, root-hair cells (also named H cells or trichoblasts), and non-hair cells (N cells or atrichoblasts), which are arranged depending upon external and internal factors, including nutrients such as phosphorus and nitrogen, water availability, phytohormones, and signaling through reactive nitrogen species (RNS) or reactive oxygen species (ROS), which operate through regulation of transcription factors. [3][4][5][6][7] The identification of Arabidopsis mutants defective on root hair initiation or elongation led to the cloning of the GLABRA (GL2) transcription factor that is expressed in atrichoblasts and blocks ROOT HAIR DEFECTIVE (RHD6) activity to generate the N cell identity. 8 RHD6 and its homologue RHD6-LIKE (RSL), two basic helix-loop-helix (bHLH) transcription factors, have been described as key regulators for root hair outgrowth. 9 Repression of GL2 allows RHD6 to activate downstream transcriptional components to start root hair formation. 9 The phytohormones auxin and ethylene and several microorganisms known to produce auxins or auxin-like compounds trigger root hair growth. 5,10,11 Consistently, reduced elongation of root hairs occurs in Arabidopsis mutants affected in auxin signaling or transport, including slr1/iaa14, aux1, arf7arf19, axr3, and tir1. 8,[12][13][14] The lack of root hairs in rhd6 mutants can be restored by auxin or ethylene, suggesting that these phytohormones act downstream of RHD6. 15 Abscisic acid (ABA) is a major phytohormone orchestrating the plant resistance to abiotic stress, including water deficit, salinity and drought. 16 ABA application reduces root hair length and activates the differentiation of trichoblasts, increasing root hair density, which was accompanied with an induction of nitric oxide (NO) within the hair protrusion. 6 This suggests that ABA and NO act as key modulators to regulate root hair development. A molecular mechanism by which ABA controls root hair formation came from the identification of DNA BINDING WITH ONE FINGER (DOF)-type transcriptional regulator, OBF BINDING PROTEIN (OBP4), whose overexpression inhibits cell elongation and control root hair development in Arabidopsis. 17 ABA application triggers the accumulation of OBP4 protein, which negatively regulates the expression of ROOT HAIR DEFECTIVE-LIKE (RSL2), a helix-loop-helix transcription factor involved in root hair elongation. 17 MEDIATOR (MED) is a multi-protein, transcriptional complex ubiquitous to eukaryotes including plants. In Arabidopsis, it is composed by 30 subunits, and organized in four modules; the core, the head, the tail, and a dissociable cyclin kinase module, called CDK8. 18 MED subunits are involved in multiple processes, including embryo and leaf development, fertility, root morphogenesis, mineral nutrition, and pathogen resistance. [19][20][21][22][23][24][25] and MED13 belong to the CDK8 module playing nonredundant roles in embryo patterning, vegetative and floral transitions, and flowering regulation. 19,20,25 Loss-offunction of either MED12 or MED13 produced a short root phenotype, with more lateral roots in response to auxin and sugar supplementation. 21 However, whether MED12 and MED13 may influence epidermal cell differentiation, and their relationship with plant hormones or second messengers remains unknown.
In this report, we analyzed the phenotypes of roots and root hairs in a suite of MED mutants including med8, med12, med13, and med16 exposed to either ABA or sucrose. Detailed cellular and structural analyses revealed that sucrose or ABA application to med12 and med13 mutants, reduced cell elongation and caused root hair swelling, which appeared to be specific, since med8 or med16 mutants did not manifest these alterations in response to sucrose. Mutant root hairs entered isodiametric growth which correlated with high nitric oxide levels as detected by confocal microscopy. Sucrose and ABA repressed MED12 and MED13 expression in both the shoot and root systems in a dose-dependent manner, positioning these proteins in the signal transduction cascade for epidermal cell elongation mediated by sucrose and ABA in Arabidopsis.

Plant material and growth conditions
Arabidopsis thaliana WT (Col-0), the transgenic Arabidopsis lines pMED12:GUS and pMED13:GUS were used to assess MED12 and MED13 expression, respectively, according to Gillmor et al. (2010). 19 The mutant lines, cct-2/crp-3/med12 19,25 , cct-3/crp-4/med13 19,25 , med8 (SALK_092406) 22 , and med16-2 (SALK_048091) 24 were employed for comparisons with the WT phenotype. To grow in vitro, seeds were disinfected with 95% (v/v) ethanol for 5 min and 20% (v/v) bleach for 7 min. After careful washing in distilled water, the seeds were stratified for 48 h at 4°C, and germinated and grown on agar plates containing 0.2× Murashige and Skoog (MS) basal salt mixture (PhytoTech Labs), lacking vitamins, and supplemented with 17.52 mM sucrose, referred as control condition. Plates were placed vertically at an angle of 65° to allow root growth along the agar surface and to enable proper shoot growth. Plates were placed in a plant growth chamber (Percival AR-95 L) with a photoperiod of 16 h of light/8 h darkness, light intensity of 300 μmol/m −2 /s −1 and temperature of 22°C.

Propidium iodide staining
Root tips and root hair morphology were analyzed via propidium iodide (PI) staining, which marks viable cells and tissue files in red color. Twelve-day-old Arabidopsis seedlings were incubated in 10 mg ml −1 of PI solution for 1 min, carefully washed and mounted in 1:1 glycerol/water proportion on microscope slides. The sample was recorded at wavelengths specific to PI fluorescence with a 568 nm excitation line and emission window of 585-610 nm, using a confocal microscope (Olympus FV1000 equipped with an objective lens Olympus PlanFlour N40× and a digital camera).

Detection of nitric oxide
The detection of nitric oxide (NO) in the primary root tip and root hairs was performed using the specific probe 4,5-diaminofluorescein diacetate (DAF-2 DA), which freely diffuses through root cells and is useful for NO detection. The seedlings were incubated in 10 µM DAF-2 DA for 30 min in darkness, washed 3 times with sterilized, distilled water and placed on slides. Fluorescence was detected using an Olympus FV1000 confocal microscope, and quantified with the Image J program and expressed in arbitrary units (AU).

Histochemical analysis
Detection of β-glucuronidase (GUS) activity in plant tissues was performed by incubating overnight Arabidopsis seedlings at 37°C in a GUS reaction solution, which contains the enzymatic substrate (0.5 mg ml −1 5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and 100 mM sodium phosphate, pH 7.0). Seedlings were cleared through 60 min incubation into 0.2 M HCl/20% methanol at 63°C, subsequently transferred into 1.75 M NaOH/60% ethanol solution by 30 min at room temperature, and finally, dehydrated by 40%, 20%, and 10% (v/v) ethanol dilutions for 20 min each at room temperature 26 . Seedlings were mounted with glycerol 50% on microscope slides and visualized via the Nomarsky optics in a LEICA DM500B microscope. The vasculature and root tips, as well as the leaves of at least 10 stained seedlings expressing pMED12: GUS and pMED13:GUS, were recorded and analyzed.

Data analysis
The data were statistically analyzed using the STATISTICA 12.0 program (Dell, StatSoft, Austin, Texas, USA). Significant differences among different traits and treatments of WT (Col-0) seedlings and mutants were determined by univariate and multivariate analyzes with Tukey's post hoc tests. Different letters were used to indicate means that differed significantly (p < 0.05) and were placed over the corresponding standard error bars.

MED12 and MED13 regulate cell elongation and differentiation in response to sucrose and ABA
ABA and sugars interact to control different plant developmental processes and abiotic stress-related responses. 27 To investigate whether MED12 and MED13 could play a role in cell elongation and differentiation mediated by ABA and sucrose within the Arabidopsis primary root, we applied 4.8% sucrose or 4 µM ABA to the growth medium of wild-type (WT; Columbia-0; Col-0), and med12 or med13 single mutants. As previously reported by Raya-González et al. (2017) 21 , under standard growth conditions, med12 and med13 mutants had shorter primary roots, primary root meristem, and cell elongation zone than WT plants (Figure 1a-c,j,k). Interestingly, these effects were accompanied with changes in root hair development. In WT seedlings, the sucrose or ABA treatments applied did not affect root hair morphology or structure, since they were comparable to control conditions (Figure 1l-m), but drastically influenced both root hair length and width in

Mutation of MED12 and MED13 led to the isodiametric growth of root hairs in response to sucrose and ABA
The swollen root hairs in med12 and med13 mutants prompted us to study the viability of these cells in the corresponding lines stained with propidium iodide (PI), which freely penetrates into the cytoplasm of cells upon damage. 23 Confocal microscopy analysis of the root hair forming zone showed that ABA or sucrose induced the nearly isodiametric growth of root hairs in med12 and med13 mutants without affecting cell viability (Figure 2ai). The formation of rounded root hairs of med12 and med13 mutants in response to sucrose treatments appeared to be specific, since mutation of MED8 or MED16 did not trigger this bulbous cell phenotype (Figure 3a-f). These data show not only the specificity of the root hair phenotype of med12 and med13 mutants but also the integrity of these cells under the applied treatments.

MED12 and MED13 modulate NO accumulation in Arabidopsis roots
Nitric oxide (NO) is a second messenger involved in distinct plant development processes. The accumulation of NO in primary root tips, lateral roots, adventitious roots, and root hairs is a hallmark of organogenesis. 6,28,29 To determine whether NO signaling could be regulated by MED12 and MED13 and analyze their relationship with sucrose responses, NO was visualized in primary roots by using the fluorescent probe 4, 5-diaminofluorescein diacetate (DAF-2 DA), which freely penetrates through the cell membrane and is hydrolyzed by esterases in the cytoplasm to produce 4,5-diaminofluorescein (DAF-2). DAF-2 specifically reacts with NO to produce a triazole compound, triazolofluorescein (DAF-2T). 30 Under our growth conditions, NO could be detected at the epidermis, close to the elongation zone in WT root tips, whereas med12 and med13 mutants showed up to four-fold higher NO accumulation, evidenced by a strong green fluorescence (Figure 4ac,g). Interestingly, 4.8% sucrose triggers NO production and accumulation in primary root tips in WT plants, whereas in med12 and med13 mutants, NO accumulation was comparable with the control condition (Figure 4a-g). NO was mainly accumulated at the elongation and differentiation zones, where root hair formation takes place (Figure 4a-g). This suggests that sucrose induces NO accumulation and that MED12 and MED13 orchestrate this process.

Sucrose induces NO accumulation in trichoblasts
The results described above suggest that NO could mediate root hair development in med12 and med13 mutants in response to sucrose. We next evaluated NO accumulation in root hairs in 12 d-old WT, med12 and med13 seedlings. Under standard growth conditions, NO levels were higher in trichoblast cells than in atrichoblast cells (Figure 5a). In contrast MED12 or MED13 mutation leads to much more detection of NO in trichoblast cells, which could be phenocopied by sucrose application in the WT (Figure 5a-d). Indeed, the round root hairs developed in med12 and med13 in response to sucrose had the strongest green fluorescence, indicating highest NO levels (Figures 5a-g, 6). These data show the correlation between NO and the root hair phenotype upon mutation of the Mediator complex subunits 12 and 13 in Arabidopsis.

Sucrose and ABA repress MED12 and MED13 expression in Arabidopsis
MED12 and MED13 could be involved in sucrose and ABA responses in epidermal cell differentiation. To determine this possible interaction, we analyzed the effect of sucrose on MED12 and MED13 expression, by using Arabidopsis transgenic plants, which express the pMED12:GUS and pMED13:GUS gene constructs. 31 For this purpose, pMED12:GUS and pMED13:GUS Arabidopsis seedlings were germinated and grown for 7d in MS 0.2× media supplemented with 0.6%, 1.2%, 2.4%, 4.8% and 9.6% sucrose and their expression patterns analyzed in three different regions of seedlings, including cotyledons, the root differentiation zone, and the primary root tip. Under 0.6% sucrose, MED12 and MED13 expression was preferentially located in vascular cells of cotyledons and roots (Figure 7a, b). Interestingly, sucrose clearly repressed MED12 and MED13 expression in a dose-dependent manner (Figure 7a, b). Next, we tested the effects of 0, 1, 2, 4 and 8 µM ABA on the expression of pMED12:GUS and pMED13:GUS transgenes. As the concentration of ABA increased, MED12 and MED13 expression decreased in the cotyledon and vasculature of seedlings (Figure 8a, b), indicating its repressing role on transcriptional expression of both MED12 and MED13 subunits. Together, these results suggest that sucrose and ABA signaling negatively regulates MED12 and MED13 expression in Arabidopsis seedlings.

Discussion
The Mediator complex is a multi-subunit co-activator of transcription in eukaryots, for which MED12 and MED13, two CDK subunits have been implicated in distinct plant development processes. Previously, we reported the functions of MED12 and MED13 in primary root growth of Arabidopsis thaliana seedlings and the growth response to sugars. 21 In this study, we went further to analyze the relationship of MED12 and MED13 with ABA and sucrose responses on root hair growth, an aspect of great importance for plants regarding the roles of these epidermal cells in nutrient and water uptake.
Root hairs are extensions of epidermal cells termed trichoblasts and the genetics of root hair initiation and elongation has been investigated for decades, leading to the identification of many genes implicated in these processes in the model plant Arabidopsis thaliana. Here, we found that Arabidopsis WT seedlings germinated and grown under high sucrose (4.8%) or ABA (4 µM) supplements were weakly affected. However, the primary roots of med12 and med13 mutants showed increased sensitivity to these compounds, because the root meristem and cell elongation zone of the primary root were drastically reduced, implying MED12 and MED13 in ABA and sucrose responses for primary root elongation. However, upon a microscopical inspection of the mature zone of primary roots of WT, med12 and med13 seedlings, we observed the nearly isodiametric growth of the root hairs that was absent in other Mediator mutants such as med8 Figure 6. Sucrose triggers nitric oxide accumulation in trichoblasts and atrichoblasts in WT, med12 and med13 seedlings. (a-f) Confocal images showing NO accumulation in trichoblasts (arrow heads) and atrichoblasts (asterisks) in WT (a, d), med12 (b, e) and med13 (c, f) roots through the specific probe DAF-2 DA in seedlings germinated and grown 12 d in Petri plates with 0.2× MS medium in control conditions (a-c) or in response to 4.8% sucrose (d-f). (g, h) Nitric oxide determinations represented as relative fluorescence in trichoblasts (g) and atrichoblasts (h). Scale bar in a = 100 µm. The standard error with different letters indicates statistical differences (P= ˂0.05). This experiment was repeated three times with comparable results. and med16. These data suggest that the formation of swollen root hairs is a MED12 and MED13 loss-of-function specific response to both sucrose and ABA.
Sucrose not only acts as a carbon source but also as a signaling input alone or in coordination with plant hormones, including ABA, to regulate gene expression. 32,33 In this context, we found that application of ABA to the plant growth media led trichoblasts to have much wider diameters that disturbs cell length and changes both the size and shape of the root hairs. Together, these data suggest that sucrose and ABA pathways could be interacting  to control root hair development via a MED12-MED13dependent mechanism in Arabidopsis. Root hair growth requires fine regulation at molecular level and involves polar cell wallloosening activity, which drives the elongation of the hair bulge. Interestingly, reduction of cellulosic and non-cellulosic polysaccharide synthesis leads to the isodiametric growth of trichoblasts. [34][35][36][37] Loss-of-function in KOJAK (KJK), SEVERAL RADIAL SWELLING (SRS), and PROCUSTE1 (PRC1) genes phenocopy the med12 and med13 phenotype in plants exposed to sucrose or ABA. [34][35][36] This suggests that MED12 and MED13 could be involved in cell wall organization perhaps acting directly on the expression of the above mentioned genes, and this possibility merits further investigation.
Root hair tip growth is a highly dynamic process requiring well-organized cytoskeleton through the coordination of actin filaments and microtubules to move organelles and vesicles. 38,39 Previous reports described that ROP members of the Rho family of GTPases are involved in the configuration of actin filaments and microtubules in leaf cell morphogenesis and in root tip growth. [40][41][42] Transgenic expression of the constitutively active CA1 form of ROP2 caused alterations on root hair growth and development through disruption of cell polarity. [40][41][42] Interestingly, in CA1 seedlings grown with 5% sucrose, about 30% of root hairs were bulbous, whereas at 1% sucrose no bulbous root hairs were evident. 43 A comparable phenotype in root hairs was found in med12 and med13 mutants in response to 4.8% sucrose. This suggests that MED12 and MED13 could act in actin filament and microtubule organization, which enables polar growth at the root hair tip.
Second messengers, such as nitric oxide (NO), play key roles in epidermal cell differentiation. 44 During root-hair-growth process, NO is produced and accumulated in high levels in trichoblasts, which is required for endocytosis, vesicle formation and trafficking. 7,45 We found that sucrose triggers a strong NO accumulation in WT, med12, and med13 roots, mainly in root hair cells, and these effects were exacerbated in the mutants. This shows a new role for MED12 and MED13 in coordinating NO levels in response to sucrose and ABA on root epidermal cells. Indeed, sucrose or ABA treatments drastically reduced cell size and root apical meristem length in med12 and med13 mutants, which was accompanied with defects in root hair structure and morphology and higher NO detection within these growth zones, but particularly in bulbous root hairs, in which NO strongly accumulated. A recent report by Lombardo and Lamattina (2018) demonstrated that ABA could trigger an increase of NO in root hairs 7 . Both ABA and NO deviated the orientation of microtubules from their longitudinal axis in control roots, to an oblique orientation upon ABA or NO treatments. In principle, these previous findings suggest that MED12/MED13 mutation and consequently, NO accumulation may affect the formation of root hairs through modifying cytoskeletal dynamics.
Previous studies on the regulation of MED gene expression showed that a large number of their subunits modulate transcription in response to ABA, which may be explained by the presence of two functional cis-acting elements, ACGT and SOSEM within their promoters 46 . However, whether ABA modulates MED12 and MED13 transcription remained unknown. Our analysis of gene expression revealed that sucrose and ABA negatively regulate MED12 and MED13 expression in Arabidopsis cotyledons and in the root vasculature. Being MEDIATOR of fundamental importance for transcription, it was of interest to find that either MED12 or MED13 are preferentially expressed in the cotyledons and root vasculature. This raised the question why the root hairs located at the epidermis manifest aberrant phenotypes if no expression is observed at this cell layer. Considering the strengths and possible limitations of using pMED12:GUS and pMED13:GUS analyses, we find plausible that pMED12 and pMED13 activity in epidermis and cortex may be below the limit of detection at the times assayed.
The repression of both subunits in cotyledons and vasculature was comparable and occurred in a dose-dependent manner in response to sucrose or ABA, which may affect the overall arrangement and functionality of the MED complex. Recently, sucrose application to the growth media negatively regulated the sucrose transporter SUC2 in the primary root tip and leaves. 47 SUC2 expression domain was found in sieve elements (SE) and phloem of the roots. 48 As shown in the current report, MED12 and MED13 expression pattern is mainly located in vascular tissues of roots in a comparable manner to SUC2, opening the possibility that MED12 and MED13 could regulate sucrose-transport and/or response to control root system configuration. The fact that other MED subunits, including med8 and med16 did not manifest root hair swelling upon sucrose or ABA treatments, indicate that some subunits play specific functions in response to different stimuli.