The mutants mterf10-1 (SAIL_12A03), mterf10-2 (SALK_097699), mterf11-1 (FLAG_357F09), mterf11-2 (GABI_211D05), and mterf12-1 (GABI_407E04) were identified in the SIGnAL database (Alonso et al., 2003), the abi4-1 mutant was ordered from The European Arabidopsis Stock Centre (NASC; ID N8104). All mutants are in the Col-0 background except of mterf11-1 which is a WS line.

Arabidopsis thaliana plants were grown in long-day conditions (16 h light/8 h dark) on potting soil (Stender). Plants were illuminated with HQI Powerstar 400 W/D lamps and a fluence rate of approximately 100 μmol photons m⁻² s⁻¹. To accomplish salt and ABA stress experiments, seedlings were grown on plant agar (Sigma-Aldrich) containing half-strength MS medium and 1.5% (w/v) sucrose at 22°C under 100 μmol photons m⁻² s⁻¹ provided by white fluorescent lamps under continuous light or long-day conditions. For salt stress experiments, MS medium was supplemented with 125 mM or 175 mM NaCl as indicated. For ABA experiments, MS medium was supplemented with 1 μM ABA.

For DNA isolation, leaf tissue was homogenized in extraction buffer containing 200 mM Tris/HCl (pH 7.5), 25 mM NaCl, 25 mM EDTA, and 0.5% (w/v) SDS. After centrifugation, DNA was precipitated by adding isopropyl alcohol. After washing with 70% (v/v) ethanol, the DNA was dissolved in distilled water.

For RNA isolation, frozen tissue was ground in liquid nitrogen. Following the addition of TRIZOL (Invitrogen) and chloroform according to the manufacturer's instructions, RNA was precipitated from the aqueous phase with isopropyl alcohol, then washed with 70% (v/v) ethanol, and dissolved in RNase-free water. Concentration and purity of RNA samples were determined spectroscopically in a GeneQuant pro RNA/DNA Calculator (GE Healthcare Europe GmbH). Isolated RNA was stored at −80°C until further use.

cDNA synthesis and real-time PCR analysis were performed as outlined before (Voigt et al., 2010). All reactions were done in triplicate on three biological replicates. The target genes and the respective primers, are listed in Supplementary Table S1. The RCE1 gene was used as an internal reference in other studies (Voigt et al., 2010; Romani et al., 2015). RCE1 transcript levels are not changed upon diverse conditions, especially not under diverse stress conditions including lincomycin and norflurazon treatment which affect organellar gene expression.

To reduce MTERF12 mRNA levels by RNAi, a 145-bp fragment was amplified from genomic DNA with the primer pair AT4G09620-GST-attB1 and –attB2 (see Supplementary Table S1). The gel-purified PCR product was used for BP and LR Clonase reactions (GATEWAY Cloning; Invitrogen) which led to the final construct pB7GWIWG2/MTERF12 (for pB7GWIWG2, see Karimi et al., 2002). For overexpression and localization studies of mTERF10, mTERF11 and mTERF12, cDNAs encompassing the coding regions were amplified by PCR (see Supplementary Table S1 for primer information). Notably, in our Col-0 strain, MTERF11 has an additional triplet (CAT; coding for histidine) inserted after nucleotide 27 (relative to the start codon) compared with the coding sequence from The Arabidopsis Information Resource (TAIR; www.arabidopsis.org). MTERF10, MTERF11, and MTERF12 were cloned by GATEWAY technology (see above) into pB7FWG2 to generate fusions with enhanced GFP (eGFP), expression of which is controlled by the Cauliflower mosaic virus 35S promoter. For RAP-RFP fusions, the pENTR/RAP plasmid (Prof. Jörg Nickelsen, LMU Munich) was introduced into p2GWR7 by GATEWAY cloning. For overexpression of mTERF10, MTERF10 was introduced by classical cloning with the NcoI restriction enzyme into pCAMBIA1302. For RNAi experiments with MTERF12 and overexpression of mTERF10 and mTERF11, the plasmids pB7GWIWG2/MTERF12, pCAMBIA1302/MTERF10, and pB7FWG2/MTERF11 were independently transferred into Agrobacterium tumefaciens, and A. thaliana (ecotype Col-0 for MTERF10 overexpression and MTERF12 RNAi; ecotype WS for MTERF11 overexpression) plants were transformed by the floral-dip method (Clough and Bent, 1998). After seed set, transgenic plants were selected on the basis of their resistance to BASTA (pB7GWIWG2/MTERF12 and pB7FWG2/MTERF11) or hygromycin (pCAMBIA1302/MTERF10), respectively.

For fluorescence visualization, leaves of 3-week-old Col-0 plants grown on MS medium were cut into small pieces and incubated for 16 h at 24°C in the dark in a protoplasting solution (10 mM MES, 20 mM CaCl₂, 0.5 M mannitol (pH 5.8), 0.1 g ml⁻¹ macerozyme (Duchefa), 0.1 g ml⁻¹ cellulase (Duchefa). After isolation and transformation of protoplasts as described (Dovzhenko et al., 2003), preparations were examined with a Fluorescence Axio Imager microscope (Zeiss). Fluorescence was excited with the X-Cite Series 120 fluorescence lamp (EXFO) and images were collected at 500–550 nm (eGFP fluorescence), 570–640 nm (RFP fluorescence) and 670–750 nm (chlorophyll autofluorescence).

In vivo chlorophyll a fluorescence of whole plants was recorded using an imaging chlorophyll fluorometer (ImagingPAM, Walz GmbH, Effeltrich, Germany). Plants were dark adapted for 15 min and then exposed to a pulsed, blue measuring light (1 Hz, intensity 4) and a saturating light flash (intensity 5) to determine the maximum fluorescence F_m and the ratio (F_m-F₀)/F_m = F_v/F_m.

Protein sequences were retrieved from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) and The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org). Amino acid sequences were aligned using the ClustalW program (http://www.ebi.ac.uk/clustalw; Chenna et al., 2003). The unrooted tree was constructed with the Phylip server Mobyle at the Pasteur Institute (http://mobyle.pasteur.fr/cgi-bin/portal.py#welcome).

The localizations of almost all A. thaliana mTERF proteins have been investigated by fluorescence microscopy of mTERF-GFP fusions transiently expressed in isolated protoplasts, and in guard cells of transgenic plants (Babiychuk et al., 2011). These data indicated that mTERF10, -11, and -12 are targeted to chloroplasts. To confirm these results and if possible define the precise locations of the proteins within the chloroplast, the eGFP fluorescence of mTERF10-, mTERF11-, or mTERF12-eGFP fusions, transiently overexpressed in Col-0 protoplasts, was monitored. Localization of all three fusion proteins to chloroplasts was confirmed (Figure 1A). However, the fluorescence signals were not uniformly distributed, but appeared as small dots in the chloroplasts. The size and distribution of these dots were suggestive of nucleoids, which are associated with PGE (Majeran et al., 2012). Localization of the A. thaliana RNA-binding protein RAP to nucleoids was previously established using a transiently expressed RAP-eGFP fusion (Kleinknecht et al., 2014). Therefore, Col-0 protoplasts were co-transformed with a RAP-RFP fusion in combination with mTERF10-eGFP, mTERF11-eGFP, or mTERF12-eGFP. Indeed, for each mTERF-eGFP construct, signals were found in dots together with the RFP signal. Merging of both signals confirmed colocalization of the mTERF10, -11, and -12 fusions with RAP, and therefore localization of all three mTERFs to nucleoids (Figure 1B). It is noteworthy, that especially mTERF12—and a minor fraction of RAP—tend to be localized in the chloroplast stroma when both mTERF12-eGFP and RAP-RFP are expressed together in protoplasts.

To obtain insight into the physiological functions of mTERFs 10, 11, and 12, T-DNA insertion lines were identified in the SIGnAL database (Alonso et al., 2003). The insertions were confirmed by PCR (Figure 2A) and homozygous lines were selected. In the mutants mterf10-1 (SAIL_12A03) and mterf10-2 (SALK_097699) the T-DNAs are inserted in the 5′ UTR and the second exon, respectively (Figure 2B). The mterf11-1 (FLAG_357F09) and mterf11-2 (GABI_211D05) mutants both have their T-DNA insertion in the gene's single exon. For MTERF12, only one insertion line could be identified (mterf12-1, GABI_407E04), which contains a T-DNA in the promoter region (Figure 2B). To repress the MTERF12 gene by RNAi, Col-0 lines were generated that contained constructs with an inverted repeat of a fragment spanning the first exon and a part of exon 2 of MTERF12 (Figure 2A) which was under control of the constitutive Cauliflower mosaic virus 35S promoter.

Figure 2C shows the numbers and distributions of mTERF domains in the mTERF10, -11, and -12 proteins. In mTERF10 and mTERF11, six and five mTERF motifs are predicted by the Simple Modular Architecture Research Tool SMART (http://smart.embl-heidelberg.de/). One mTERF motif has been predicted for mTERF12 (our previous results, and see also Supporting Information of Babiychuk et al., 2011), but this domain is not annotated anymore with confidence by the SMART tool (http://smart.embl-heidelberg.de/smart/show_motifs.pl?ID=Q93ZZ2_ARATH). Thus, the classification of mTERF12 as an mTERF protein must be regarded as uncertain.

All mutants are in the Col-0 background except of mterf11-1 which is a WS line. Hence, in all following experiments, mterf11-1 was compared with WS, while Col-0 was used as the WT standard for the other lines. Real-time PCR analysis was employed to determine the extent of repression of MTERF transcripts in the different mutant lines (Figure 3A). In 3-week-old mterf10-1 and mterf10-2 plants, MTERF10 transcript levels were reduced to 29 and 4% of WT, respectively. To determine MTERF11 transcript levels, primer pair A was chosen to detect transcripts initiated 5′ of the T-DNA insertions (Figure 2A). Using this set-up, MTERF11 transcript levels were found to be unchanged (mterf11-1) and nearly 6-fold induced (mterf11-2) relative to their WT (Figure 3A). In the mterf11-1 allele (which is FLAG_357F09), the T-DNA of the pGKB5 vector integrated in the 5′LB–T-DNA–RB3′ direction. It is of note here that the pGKB5 vector used to generate the FLAGdb T-DNA insertion line collection contains the 35S promoter on the LB side (Samson et al., 2002). The 35S promoter drives the expression of PHOSPHINOTRICIN ACETYL TRANSFERASE (PAT) used to select transgenic plants, and the PAT transcripts are terminated by the G7 terminator. It was already shown with two independent FLAG lines as an example that the G7 terminator can be an inefficient terminator in the context of the pGKB5 vector, allowing transcription to continue through and beyond the terminator sequence (Ulker et al., 2008). However, real-time PCR carried out with a primer pair covering the region 3′ of the T-DNA insertion detected greatly reduced MTERF11 transcript levels in the mterf11 mutants: 0.09% of WT in mterf11-1 and 0.01% in mterf11-2 (Figure 3A). MTERF12 transcript levels were not affected in the mterf12-1 mutant (Figure 3A). Therefore, MTERF12 RNAi lines were tested for their ability to repress MTERF12 gene expression. Six independent lines were screened, but the most effectively repressed lines, mterf12i-1 and mterf12i-2, still retained 32% and 59% of WT (Col-0) amounts of MTERF12 transcripts, respectively (Figure 3A). Under normal growth conditions, all identified mutant lines were phenotypically indistinguishable from WT (Figure 3B). To look for subtle photosynthetic phenotypes, the maximum quantum yield of photosystem II (F_v/F_m) was measured in Col-0, WS and all mterf mutants (Figure 3B), but no deviations in this parameter were detected in the mutants.

To summarize, the expression of all mTERF motifs should be strongly reduced in the mterf10 mutants (particularly mterf10-2), while the mterf11 mutants produce truncated transcripts. Assuming the latter are translated, the protein products would lack the last two mTERF domains (mterf11-1) or mTERF domain 5 only (mterf11-2) (Figure 2C). In the mterf12i lines, transcripts including the single putative mTERF domain were—at best—reduced to one-third of Col-0 levels. At all events, none of the mterf mutant lines display any obvious phenotype under normal growth conditions.

Because the mterf10, -11, and -12 mutant lines lacked a clear phenotype under normal growth conditions (Figure 3), we asked whether this might be attributable to functional redundancy within the mTERF family. Several MTERF genes have undergone tandem duplications (on chromosome 1) and one block duplication (AT4G19650 and AT5G45113; Kleine, 2012). But neither MTERF12 nor MTERF10 or MTERF11 originated from a duplication event, so we can exclude the possibility of protein redundancy arising from gene duplication. To obtain an impression of the overall degree of sequence similarity within the mTERF protein family, we constructed a phylogenetic tree which included in addition to A. thaliana mTERFs, mTERF members from the green alga Chlamydomonas reinhardtii, Homo sapiens, Mus musculus, Drosophila melanogaster, and other organisms (Figure 4). The tree reveals four main clades. The mTERF members of C. reinhardtii, H. sapiens and M. musculus are all in the same clade. The majority of A. thaliana mTERFs form clade I which includes mTERF10 and -11, while mTERF12 along with five other A. thaliana mTERFs and one D. melanogaster mTERF constitute clade II. The mTERF10, -11, and -12 proteins are most closely related to mTERF1/SOLDAT10 (Meskauskiene et al., 2009), mTERF4/BSM/RUG (Babiychuk et al., 2011; Quesada et al., 2011) and mTERF15 (Hsu et al., 2014) proteins. Mutants for each of these three display phenotypes under normal growth conditions and have been shown to be involved in PGE or mitochondrial gene expression. Moreover, levels of sequence identity/similarity between mTERF10 and mTERF1 (38/68% over a stretch of 240 amino acids), mTERF11 and mTERF4 (26/41% over a stretch of 222 amino acids) and mTERF12 and mTERF15 (29/53% over a stretch of 77 amino acids), respectively, are noteworthy for the mTERF10/mTERF1 pair, but negligible for the other two.

To gain deeper insights into the functions of mTERF10, -11, and -12, their mRNA expression patterns were analyzed. Co-expression analysis of 26 MTERF genes and their corresponding gene ontology annotations have already been reported (Kleine, 2012). However, that study was designed to provide a global classification. Hence subsequent Genevestigator analyses only dealt with the numbers of conditions/treatments that altered MTERF gene expression. In the present study, we extracted MTERF transcript levels from the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) with “Abiotic Stress” as a data source (Winter et al., 2007). In these experiments, 18-day-old plants were subjected to different stresses, and samples were taken over a time course of 24 h from stress-treated and control plants. We calculated the relative changes in MTERF10, -11, and -12 transcript levels from plants exposed to drought, high salt, heat, or cold compared to control conditions (Figure 5A). Because mda1 (mterf5) and mterf9 mutants are known to exhibit altered stress responses (Robles et al., 2012, 2015), MTERF5 and -9 were included for reference. Under drought and heat stress, transcript levels of all investigated MTERF genes were only moderately changed (Figure 5A). With a 3-fold rise after 1 h of heat stress (MTERF10) and an approximately 0.3-fold change (MTERF5 and 9), those transcripts were the most responsive. Under salt and cold treatment, MTERF transcript levels tended to be reduced. Under both salt and cold stress, MTERF10 and MTERF11 levels were most responsive, and especially after 24 h of cold treatment MTERF5, 10, 11, and 12 transcript levels were reduced (Figure 5A). To confirm these data and to find other conditions under which the MTERFs of interest might be regulated, the Genevestigator Perturbations Tool (https://genevestigator.com/gv; Hruz et al., 2008) was employed on all deposited A. thaliana ATH1 arrays together with a 2-fold change filter and a p-value of < 0.05. An overview of all changes in MTERF10, MTERF11, and MTERF12 mRNA levels in response to perturbations (relative to untreated controls) can be found in Supplementary Figures S1–S3. In Figure 5B selected conditions are shown which are associated with changes in temperature and light, and with salt and drought stress conditions. Levels of MTERF10 mRNA were most susceptible to perturbation, being induced by light, raised after germination, and strongly reduced under drought conditions and various cold and high-light regimes, and on exposure to salt or ABA (Figure 5B).

To experimentally probe the involvement of mTERFs in stress responses, 3-week-old WT and mterf10, -11, and -12 mutant plants grown under standard conditions (22°C at 100 μmol photons m⁻² s⁻¹) were exposed for 7 days to moderate temperature stress (30°C, at a fluence rate of 100 μmol photons m⁻² s⁻¹) or moderate light stress (400 μmol photons m⁻² s⁻¹ at a temperature of 22°C). After 7 days of moderate temperature stress, the leaf petioles of WT and all mterf mutants were shortened, but otherwise all plants looked healthy (Figure 6A). After 3 days, F_v/F_m was slightly reduced in all mterf11 and mterf12 mutant lines, but was restored to normal levels after 7 days (Figures 6A,B). Also after 7 days of moderate light stress, the leaf petioles of WT and all mterf mutants were shortened—albeit to a lesser extent. Furthermore, the edges of older leaves in all lines began to show signs of necrosis (Supplementary Figure S4A). After 1 h of moderate light stress, F_v/F_m was slightly reduced in all lines (Supplementary Figure S4B). This reduction continued in the mterf11-1 mutant after 2 and 4 h, but all lines recovered to the initial F_v/F_m values after 96 h (Supplementary Figure S4B).

After 3 and 24 h of salt stress, MTERF5 and MTERF9 transcript levels were reduced to half of those in control conditions (Figure 5A), and indeed, mda1 (mterf5) and mterf9 seedlings are less sensitive to salt and osmotic stresses (Robles et al., 2012, 2015). Because MTERF10 and MTERF11 transcripts were reduced to an even larger extent than MTERF5 and MTERF9 RNAs following exposure to salt stress for 6 and 24 h (Figure 5A), we asked whether inactivation of MTERF10 or MTERF11 might enable the mutant plants to better tolerate salt stress. To this end, WT and mterf mutant lines were germinated on MS medium (control) and MS medium supplemented with 125 mM or 175 mM NaCl, and germination rates were scored after different time points (Figure 7A). All lines germinated to nearly 100% on the control MS medium. Germination rates of all lines with a Col-0 background grown for 48 h on MS medium supplemented with 125 mM NaCl or for 72 h on medium supplemented with 175 mM NaCl were very similar (Figure 7A). In the aforementioned conditions, germination rates of Col-0 seeds were approximately 60% (Figure 7A). The germination rates of mterf10-1 and -2, mterf11-2 and all mterf12 seeds were all lower (ranging from 26 to 47%) than those of Col-0 seeds. However, after 96 h on MS medium supplemented with 175 mM NaCl the germination rates of mterf12 seeds (76 to 87%) were comparable to that of Col-0 seeds (84%). Interestingly, mterf10-1, mterf10-2, and mterf11-2 still displayed enhanced sensitivity to salt inhibition, with germination rates of 54, 55, and 61%, respectively (Figure 7A). WS seeds were very susceptible to salt stress and failed to germinate under the conditions used to investigate the Col-0 descendant lines. For this reason, a milder salt stress treatment was applied to all lines with a WS background. Still, after 72 h growth on MS medium supplemented with 125 mM, the germination rate of WS was only 7% and raised to 48% after 96 h (Figure 7A). Although the mterf11-2 mutant was more sensitive to salt stress compared to Col-0, the germination rates of mterf11-1 seeds were comparable to their corresponding WT (WS; Figure 7A).

To ascertain whether the altered activity of MTERF10 was indeed responsible for the salt-stress phenotypes and whether overexpression of MTERF11 might lead to enhanced salt-stress tolerance, 35S:MTERF10:MGFP5 and 35S:MTERF11:EGFP constructs were introduced into Col-0 and WS, respectively, to generate oe-mTERF10 and oe-mTERF11 lines. Although MTERF10 mRNA levels were only approximately 2.3-fold higher in oe-mTERF10-1 and oe-mTERF10-2 lines than in Col-0 (Figure 8A), these lines—with germination rates of approximately 85 and 95%, respectively, after 48 h on MS medium with 125 mM NaCl and 72 h on MS medium with 175 mM NaCl—were nevertheless resistant to the deleterious effect of salt (Figure 7A). This confirms that the salt sensitivity of mterf10-1 and mterf10-2 mutants is indeed caused by knockdown of the MTERF10 gene. Moreover, we identified three oe-mTERF11 lines that displayed a high diversity of MTERF11 transcript overaccumulation which ranged from 12- to 117-fold (Figure 8B). Two of these lines were challenged with salt stress, and actually displayed much higher germination rates than WS and therefore, enhanced salt stress tolerance (Figure 7A).

To investigate this further, the performance of mterf mutants and mTERF overexpression lines was investigated during post-germination development. As shown in Figures 7B,C, the root lengths of mterf10-1 and -2 seedlings challenged with 125 mM NaCl were significantly shorter compared to Col-0, while the root lengths of oe-mTERF10 seedlings were longer (Figure 7C). Moreover, compared to WS, overexpression of MTERF11 results in longer root lengths, reflecting findings of the germination rates (Figure 7A). In addition, cotyledon greening—displayed as the ratio of the number of green cotyledons to the total number of cotyledons—of mterf10-1 and -2 seedlings was delayed by salt stress, while in contrast, overexpression of MTERF10 or MTERF11 enabled seedlings to display higher cotyledon greening rates than their corresponding wild types (Figure 7D).

ABA operates as a signal during developmental processes including seed germination, and moreover, in response to abiotic stresses including salt stress (Christmann et al., 2006). Furthermore, A. thaliana mutants in which the ABI4 (ABSCISIC ACID INSENSITIVE4) gene has been inactivated are more salt tolerant than WT (Quesada et al., 2000; Shkolnik-Inbar et al., 2013). To investigate whether reduced MTERF transcript levels in the mterf10, -11, and -12 mutant lines or overexpression of mTERF10 or mTERF11 alter ABA sensitivity, wild-type, mterf, oe-mTERF10, oe-mTERF11 and—as a control—abi4-1 mutant seedlings were grown on MS supplemented with 1 μM ABA, and germination rates were scored after 96 h (lines with a Col-0 background) and 120 h (lines with a WS background). With a 69% germination rate, the control line abi4-1 germinated better than Col-0 (49%; Figure 9A). All mterf12 lines displayed a slightly, but not significantly, higher germination rate than Col-0, but mterf10 and mterf11 lines were as sensitive as Col-0 to ABA stress. Importantly, especially oe-mTERF10 lines were also less susceptible to ABA stress (Figure 9A), like they were to salt stress (Figure 7A). After 120 h on 1 μM ABA, WS germinated to 32%, and both mterf11-1 and oe-mTERF11 lines displayed even lower germination rates (Figure 9A).

To follow this up, the phenotypes of seedlings grown on 1 μM ABA were examined after 7 days. Col-0 and WS seedlings displayed short roots and cotyledons had only started to emerge (Figure 9B). In contrast, abi4-1 seedlings had longer roots and fully expanded cotyledons. All mutant lines with reduced MTERF10, -11, or -12 transcript levels showed the same behavior as the wild types. However, the cotyledon phenotype of the oe-mTERF10 lines was comparable to that of the abi4-1 mutant (Figure 9B). This was also manifested in the higher relative cotyledon greening rate of oe-mTERF10 lines (25 and 30%) compared to Col-0 (Figure 9C).

It appears that challenging Arabidopsis seedlings with ABA or salt stress under continuous light reduces germination efficiencies or cotyledon greening of Col-0 to a greater extent (Reyes and Chua, 2007; Chen et al., 2008; Hwang et al., 2015) compared to long-day conditions (Figure 7; Hu et al., 2013). To tackle the oe-mTERF10 ABA phenotype, we grew Col-0, mterf mutant lines and oe-mTERF10 lines in continuous light and a temperature of 20°C on MS medium (control) and MS medium supplemented with 1 μM ABA, and the phenotypes were scored after 7 days (Figure 9D). Because lines with a WS background were already very sensitive to ABA in long-day conditions, these lines were omitted. Indeed, Col-0 and mterf mutant seedlings displayed even shorter roots compared to long-day conditions and the cotyledons that had started to emerge did not turn green to this time point (Figure 9D). In contrast, abi4-1 seedlings had longer roots and fully expanded green cotyledons. All mutant lines with reduced MTERF10, -11, or -12 transcript levels showed the same behavior as Col-0. However, also in continuous light the phenotype of oe-mTERF10 lines was comparable to that of the abi4-1 mutant, with longer roots and expanded green cotyledons (Figure 9D).

In sum, these results indicate that lower or higher MTERF11 levels result both in increased ABA sensitivity. But, strikingly, higher MTERF10 levels are associated with decreased sensitivity to ABA, which might in turn be linked to the higher salt tolerance of oe-mTERF10 lines.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.