Ethylene, a key factor in the regulation of seed dormancy

1In soil, seeds are exposed to various environmental factors including temperature, moisture, oxygen and light, which regulate seed germination, and subsequent seedling growth. Phase I of the germination process is initiated by imbibition, which is required to activate the respiratory metabolism, and transcriptional and translational activities. In phase II called germination sensu stricto, water uptake ceases and reserve mobilization starts. Phase III is characterized by radicle protrusion. Germination requires specific temperatures, oxygen levels and light, the exact proportions being species specific. However, the seeds of species (or even within species) do not germinate, or do so with difficulty, even when incubated under apparently favorable conditions; these are considered as dormant and cannot germinate in the same conditions (i.e., water, air, temperature) under which non-dormant seeds do. Dormancy is a heritable trait, but its intensity at harvest and its maintenance after harvest is highly modulated by the environmental conditions throughout seed development and ripening on the plant, and during seed storage. Factors inhibiting germination can reside within the embryo itself (embryo dormancy) or can result from an inhibitory action of the covering structures (seed coat imposed dormancy;; ). Primary dormancy sets during seed development, but a secondary dormancy can develop in mature seeds with some degree of primary dormancy or in non-dormant seeds in response to unfavorable conditions for germination.

2The involvement of the hormonal balance between abscisic acid (ABA) and gibberellins (GAs) in the regulation of seed germination and dormancy in response to environmental signals is well documented and discussed in recent reviews. ABA is well known to play a crucial role in induction of dormancy in the developing seeds and in maintenance of dormancy during seed imbibition, while GAs are involved in dormancy release and/or germination. In addition to GAs and ABA, other hormones (ethylene, jasmonates, auxins) also play a role in the control of seed germination. Ethylene (C2H4) in particular regulates germination and dormancy of numerous species via a complex hormonal signaling network.

3The role of reactive oxygen species (ROS) in seed biology has progressively emerged and evolved this last decade. Originally considered as harmful compounds, causing deleterious reactions toward a wide range of biomolecules and thus to seeds, ROS are now widely acknowledged as signaling compounds regulating the germination process through an hormonal network.

4In this review, we describe how ethylene interacts with other plant hormones in regulation of germination and dormancy, concentrating on its interactions with ABA, GAs, and ROS.

5Ethylene production by seeds begins immediately after the onset of imbibition and increases with time of germination. There is, however, a peak in ethylene emission concomitant with the radicle protrusion through the seed coat. Seed ethylene production is species dependent, but is generally below levels detectable by gas chromatography during imbibition. Using a high sensitivity laser photo acoustic spectroscopy, have confirmed the occurrence of an ethylene peak at the end of the germination process in sunflower (Helianthus annuus) seeds. Interestingly, a close relationship between the ability to produce ethylene and seed vigor has been reported in various species including rape (Brassica napus; ), cotton (Gossypium spp.; ), peanut (Arachis hypogaea; ), cocklebur (Xanthium pennsylvanicum; ), snap bean (Phaseolus vulgaris; ), sunflower and pea (Pisum sativum; ), and 1-aminocyclopropane 1-carboxylic acid (ACC)-dependent C2H4 production was proposed as a marker of seed quality.

6The pathway of ethylene biosynthesis in seeds is the same as that described for other plant organs, in which S-adenosyl-methionine (S-AdoMet) and ACC are the main intermediates (;;; Figure 1). S-AdoMet synthesized from methionine by the S-AdoMet synthetase (or SAM synthetase), is converted to ACC, the direct precursor of ethylene, by ACC synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, ACS). The by-product 5′-methylthioadenosine (MTA) is recycled back to methionine through the Yang Cycle. S-AdoMet is also the precursor of the biosynthesis of polyamines, which can also play a role in seed germination. Ethylene production results from the oxidation of ACC by ACC oxidase (ACO), which also generates CO2 and hydrogen cyanide (HCN; Figure 1). Autocatalytic synthesis of ethylene via induction of ACC and ACO transcription is well known in fruit ripening, ethylene also regulates ACO expression in pea, beechnut (Fagus sylvatica; ), and turnip (Brassica rapa; ). In contrast, ethylene or ACC does not affect the abundance of ACO transcript in sugar beet (Beta vulgaris; ) and, expression of SoACS7 in Sisymbrium officinale and PsAC1 in pea.

7Increased ethylene production during germination is associated with an increase in ACO activity, as well as a progressive accumulation of ACS and ACO transcripts. Although ACS is considered to be a key enzyme in the regulation of ethylene production in most plant responses to abiotic and biotic stresses, it was demonstrated in seeds that ACO activity plays a fundamental role during germination. Both ACS and ACO are encoded by a multigene family, and the regulation of particular ACS and ACO genes differ among each other. In both Arabidopsis and cress (Lepidium sativum), ACO1 and ACO2 have been demonstrated to be the major ACOs involved in ethylene synthesis, and the correlation between the abundance of ACO transcripts and the ACO activity suggests its regulation at a transcriptional level during germination.

8Ethylene is involved in various developmental processes and responses to biotic and abiotic stresses in plants. The key components in its signaling pathway have been identified using a molecular dissection of ethylene responsiveness in Arabidopsis. Five membrane-localized ethylene receptors, ethylene resistant 1 (ETR1), ETR2, ethylene response sensor 1 (ERS1), ERS2, and ethylene insensitive 4 (EIN4) exist in Arabidopsis. Among them, ETR1 and ERS1 contain three transmembrane domains in the N-terminus and a histidine kinase domain in the C-terminus, when ETR2, EIN4, and ERS2 present four transmembrane regions and a serine-threonine kinase domain in the C-terminus. Binding of ethylene to its receptors results in inactivation of CTR1 (constitutive triple response 1) protein kinase, which in turn activates the kinase cascade controlling EIN2 and its transcription factors in the nucleus. These, such as EIN3, EILs, ethylene response element binding proteins (EREBPs)/ethylene responsive factors (ERFs) activate the transcription of ethylene response genes (;;;;,;; Figure 1). Recently, it was demonstrated that EIN2 is phosphorylated by CTR1 kinase in the absence of ethylene, and that EIN2 protein level is regulated through its degradation by the proteasome.

9Exogenous ethylene or ethephon, an ethylene releasing compound, improves germination in numerous species. It stimulates germination of non-dormant seeds incubated in non-optimal environmental conditions such as too high temperatures, osmotic stress, hypoxia, and salinity. It can also break primary and secondary dormancy (Table 1). It breaks the embryo dormancy in apple (Malus domestica;;; ) and beechnut, the dormancy of which is usually broken by chilling, and in sunflower, the dormancy of which is progressively alleviated during dry storage (after-ripening). It also promotes the germination of seeds exhibiting a seed coat imposed dormancy in various species such as cocklebur, subterranean clover (Trifolium subterraneum; ), Rumex crispus, and Arabidopsis. In particular, it can also overcome the secondary dormancy induced by high temperatures in lettuce (Lactuca sativa;; ), sunflower, Amaranthus caudatus and Amaranthus paniculatus. In Rhus coriaria, a post-fire pioneer, low ethylene concentrations (0.03–0.10 μL L-1) released by wet ash stimulates germination, although it does not improve that of many other species in which germination is smoke-induced. Ethylene also improves germination of seeds from parasitic plants such as Striga asiatica, Striga lutea and Striga hermonthica.

10The stimulatory effect of ethylene is dose dependent, the hormone being efficient when applied at concentration ranging from 0.1 to 200 μL L-1 depending on the species, the depth of dormancy and the environmental conditions. Breaking of dormancy either during chilling in apple or dry storage in sunflower, Amaranthus retroflexus and Stylosanthes humilis is associated with an increasing sensitivity to ethylene. At harvest, dormant sunflower seeds require 50 μL L-1 ethylene in order to germinate at 15°C, but only 10 and 3 μL L-1 after 8 and 15 weeks of dry-storage at 5°C, respectively. In contrast, the responsiveness to the hormone decreases progressively during seed incubation under environmental conditions that induce a secondary dormancy.

11Although ethylene stimulates the germination of numerous light sensitive seeds, it does not overcome the light requirement in Amaranthus retroflexus, celery (Apium graveolens; ), lettuce, and Spergula arvensis. Recently, demonstrate that loss of ETR1 reduces the inhibitory effect of far-red on the germination of Arabidopsis seeds through expression of genes involved in ABA and GAs metabolism. An epistasis analysis performed by the same authors also suggests that ETR1 may interact with phytochromes to control seed germination.

12An additive or synergistic effect of CO2 and ethylene has also been demonstrated in seeds of peanut, Spergula arvensis, cocklebur, and lettuce. In the case of cocklebur and sunflower seeds, it was suggested that the improving effect of CO2 results from an enhancement of C2H4 biosynthesis, since it is suppressed in the presence of inhibitors of ethylene synthesis.

13Numerous studies demonstrate that the ability to germinate correlates with ethylene production, suggesting that ethylene is involved in the regulation of seed germination and dormancy (reviewed in;; ). For example, induction of thermodormancy at high temperatures is associated with a reduced ethylene production in chickpea (Cicer arietinum; ), sunflower, and lettuce. This decrease in C2H4 production may result from an increase in ACC-malonyltransferase activity, thus from a decrease in ACC content as demonstrated in chickpea, an inhibition of ACO activity, or a reduced expression of ACS and ACO. In contrast, breaking of dormancy by various treatments (e.g., chilling, GAs, NO, HCN) leads to an increase in ethylene production. In Arabidopsis, the inductive effect of chilling is associated with a reduced expression of ACO, but in a transient induction of ACS. However, after-ripening of Sisymbrium officinale seeds inhibits expression of SoACS7 and SoACO2 which are involved in ethylene biosynthesis, during early seed incubation, but stimulates that of SoGA20ox2, SoGA3ox2, and SoGA2ox6 involved in GA metabolism.

14Data obtained using inhibitors of ethylene biosynthesis pathway or mutants altered in ethylene biosynthesis and signaling pathways demonstrated that endogenous ethylene plays a key role in the regulation of germination and dormancy. Incubation of seeds in the presence of aminoethoxyvinylglycine (AVG) and aminooxyacetic acid (AOA), inhibitors of ACS activity, CoCl2 and α-aminoisobutyric acid (α-AIB), inhibitors of ACO activity, or 2,5-norbornadiene (2,5 NBD) and silver thiosulfate (STS), inhibitors of ethylene action, allowed demonstration of the involvement of endogenous ethylene in germination and dormancy breakage. In contrast, seed incubation in the presence of ACC, the direct precursor of ethylene, improves seed germination in numerous species such as lettuce, sunflower, cocklebur, Amaranthus sp., chickpea, and sugar beet. This effect of ACC suggests that ACO is potentially active, and that dormancy might result from insufficient ACC level due to low ACS activity.

15It is important to notice that HCN, a co-product of ACC oxidation, can also break seed dormancy in apple, sunflower and Amaranthus retroflexus.

16Using Arabidopsis lines altered in ethylene biosynthesis and signaling allowed to characterize the regulation of dormancy by ethylene (Table 2). Seeds of ethylene insensitive etr1 (ethylene resistant) as well as ein2 (ethylene insensitive 2) mutants display enhanced primary dormancy relative to wild type, probably due to high ABA sensitivity, whereas ctr1 (constitutive triple responses) mutant have a slightly enhanced rate of germination. EIN2 plays a key role in the ethylene signaling pathway, and loss of its function results in a hypersensitivy to salt and osmotic stress during germination and early seedling development in Arabidopsis. ERFs genes might also play a pivotal role in ethylene responsiveness and regulation of germination. FsERF1 expression is minimal in dormant beechnut embryo, but increases during moist chilling which breaks dormancy. In sunflower, ERF1 expression is fivefold higher in non-dormant than in dormant embryos, and expression is markedly stimulated by HCN, which breaks dormancy. In tomato (Solanum lycopersicon), SlERF2 transcript abundance is higher in germinating seeds than in non-germinating ones, and its overexpression in transgenic lines results in premature seed germination.

17Transcriptional arrays have been used to draw a global view of gene expression in germinating and non-germinating seeds and core sets of genes were analyzed with respect to hormone responsive elements. The analysis of transcriptome data of dormant and after-ripened states in Arabidopsis performed by showed that ACS2 gene expression was up-regulated in the dormant state and AtERF5 was up-regulated in germinating state. In lettuce, have shown that ethylene responsive genes are regulated by thermo-inhibition; ACO and ACS gene expression is reduced while CTR1, EIN2, ETR1 expression is increased at high temperature. These results point out the gap that exists between hormone metabolism and signaling regulation at the level of gene expression. In wheat seeds, 78 probesets annotated as ethylene metabolism and signaling genes have been differentially expressed between dormant and after-ripened seeds. ACO is represented by four probesets that are up-regulated in after-ripened wheat seeds but no ACS corresponding probeset has been found. Ethylene signaling element as reversion to ethylene sensitivity 1, ERS1, EBF1 (EIN3 binding box protein1), prohibitin 3 or ERF have been shown to be up-regulated in after-ripened seeds at 12 or 24 h of imbibition. The number of genes related to ethylene involved in germination is underestimated since only direct known ethylene signaling components are targeted for analysis in omic studies. It has been shown that treatment with ACC of 7 days germinated seedlings triggers change in expression of 544 genes, among them 244 were common to seeds given an ABA treatment. These results have been used to compare genes regulated in Lepidium seed tissues during germination. Pectate Lyase 1, Argos-like, Expansin A2, B-1,3-glucanase and chitinase B which play an important role in endosperm weakening and/or radicle growth in germination of Lepidium seeds, are proposed as putative ethylene response down-stream genes. Cell wall loosening enzymes expressed in endosperm are also controlled by both ABA and GA.

18The antagonistic effects of ABA and ethylene in the regulation of seed germination and dormancy have been extensively studied. Ethylene overcomes the inhibitory action of ABA on germination of numerous species among which are Amaranthus caudatus, Chenopodium album, cotton, tobacco (Nicotiana tabacum), and Arabidopsis. In Arabidopsis and Lepidium sativum, ethylene also counteracts the inhibition by ABA of endosperm cap weakening and rupture. On the contrary, ABA increases the ethylene requirement in order to release dormancy in sunflower and Amaranthus caudatus. The negative interaction between ABA and ethylene is also supported by data obtained with various mutants affected in the signaling pathway of both hormones. Ethylene insensitive mutants (etr1, ein2, and ein6) are hypersensitive to ABA, whereas seeds of ein3, ein4, ein5, and ein7 germinate normally. Conversely, eto1, eto3, and ctr1 mutants (characterized by an increase in C2H4 production) exhibit a reduced sensitivity to ABA (Table 2;;; ). Loss of function of CRT1 enhances the tolerance to ABA of abi1-1 seeds. Genetic approaches, using double mutants obtained by crossing ethylene insensitive mutants (ctr1, ein1, ein3, and ein6) with aba2 mutant, demonstrate that ABA and C2H4 may also act in parallel, since they exibit phenotypes resulting from both ABA deficiency and altered ethylene sensitivity.

19Although ACC and exogenous ethylene do not affect ABA content in Lepidium sativum and sugar beet, Arabidopsis seeds from ethylene insensitive mutants, etr1 and ein2, have a higher ABA content than that of wild type seeds. For example, mutation in ETR1 results in an 8-fold higher ABA content in mature seeds than in wild type, probably due to a decrease in ABA conjugation. Loss of function of ACS7, one of type 3 ACS with a very short C-terminus and no phosphorylation site, results in reduced C2H4 emission and hyper-sensitivity to ABA, consequently conferring abiotic stress tolerance to Arabidopsis seeds. ABA accumulation is also associated with stimulation of ABA biosynthesis through an up-regulation of NCED and a down-regulation of CYP707A2 in seeds of the etr1 mutant, or an up-regulation of NCED3 associated with an up-regulation of ABA1 in ein2 seeds.

20Inhibition of germination by ABA is associated with an inhibition of in vivo ACO activity and is correlated with a reduction in ACO transcript accumulation, leading to a reduction of ethylene production. In Arabidopsis, accumulation of ACO1 and ACO2 transcripts during germination is inhibited by ABA, and the high level of ACO1 transcript in ABA-insensitive mutants suggest a tight regulation of ACO expression by ABA. In Lepidium sativum, ABA inhibits expression of both ACO1 and ACO2 in the endosperm cap. Up-regulation of ACO transcript has also been detected by microarray analysis in the aba2 mutant in Arabidopsis. In contrast, there is an ABA-mediated up-regulation of ACC accumulation and ACO expression in sugar beet seeds.

21Gibberellins improve the germination of dormant seeds in numerous species whose dormancy is broken by ethylene, ethephon, or ACC (c.f. Table 1). Both hormones promote the germination of primary dormant seeds of Arabidopsis, Amaranthus retroflexus, beechnut, apple, and Sisymbrium officinale. They also break secondary dormancy in Rumex crispus and cocklebur seeds, and thermodormancy in lettuce achenes. In Arabidopsis, C2H4 restores the germination of the GA-deficient mutant ga1-3, and GA3 stimulates that of the etr1 mutant, while no stimulatory effect is noted on the germination of the GA-deficient gib-1 mutant in tomato. All these data suggest that GAs and ethylene pathways interact.

22In beechnut, incubation of embryos in the presence of GA3 results in an accumulation of ACC and an increase in ACC oxidase activity and C2H4 production, concomitant with an increased expression of FsACO1. Similarly, the improving effect of GA4 on the germination of Arabidopsis ga1-3 mutant seeds is associated with an increase in AtACO. Decrease of the expression of FsACO1 in the presence of paclobutrazol, a GAs biosynthesis inhibitor, confirms that GAs activates the ethylene biosynthesis pathway. However, in Sisymbrium officinale, demonstrate that expression of SoACS7 and SoACO2 during germination is inhibited by paclobutrazol, but is not affected by application of either ethrel or GA4+7. In addition, the up-regulation of AtERS1 (ETHYLENE RESPONSE SENSOR encoding a member of ethylene receptor family) in Arabidopsis in the presence of GA4 and of an EIN-3 like in beechnut in the presence of GA3 suggest an effect of GAs on ethylene response.

23Numerous data also suggest that ethylene stimulates seed germination by affecting the GAs biosynthesis or signaling pathway. GA1, GA4, and GA7 strongly accumulate in dry mature seeds of the etr1-2 Arabidopsis mutant relative to wild type, and both GA4 and GA7 contents remain higher than in wild type during the two first days of imbibition. The changes in GA content during germination suggest that lack of ETR1, i.e., of ethylene signaling pathway, results (i) in alteration of GAs biosynthesis pathway, and (ii) in a requirement for higher levels of GAs than wild type, to promote germination. In beechnut, expression of FsGA20ox1, which is involved in the synthesis of active GAs, remains low in stratified seeds (i.e., non-dormant seeds) and seeds treated with GA3 or ethephon, but inhibition of ethylene biosynthesis by AOA (2-aminoxyacetic acid) results in an increase in this transcript indicating the involvement of ethylene in the regulation of GA biosynthesis. Studies of expression of genes involved in GA synthesis (SoGA3ox2 and SoGA20ox2) and degradation (SoGA2ox6) during imbibition of Sisymbrium officinale seeds in the presence of GA4+7, ethylene, and inhibitors of GA synthesis or ethylene synthesis and signaling, indicate that GA biosynthesis is strongly regulated by GA and ethylene.

24Gibberellin signaling pathways depend on DELLA proteins including GAI (GA INSENSITIVE), RGA (REPRESSOR OF ga1-3), RGL1 (RGA LIKE1), RGL2 and RGL3. GAs destabilizes the DELLA proteins, which act as growth repressors by targeting GAs for ubiquitination and degradation. In Arabidopsis,, ) reported that a part of ethylene action on hypocotyl growth and floral transition was mediated via its effects on the DELLA proteins. This may be true too in the control of germination since DELLA proteins seem to play a key role in the regulation of seed germination. Thus, the seed GA content and responsiveness may result from a regulation of DELLA accumulation by ethylene.

25It has been shown in various seed species, including Arabidopsis, that radicle protrusion is associated with and/or required a controlled accumulation of ROS. Regarding the role of plant hormones in seed germination and dormancy, several studies have investigated the possible relationship between metabolic and signaling pathways of these hormones, mainly ABA and GAs, and ROS homeostasis. Up-to-date, however, the relationship between ROS and ethylene has been scarcely studied within the context of seed germination, although this is well documented in other contexts such as plant pathogen interactions or cell death regulation.

26In sunflower embryos, whose dormancy is released by exogenous ethylene, it has been recently demonstrated that ethylene markedly enhanced ROS accumulation within dormant embryonic axes, probably through the activation of NADPH oxidase. Whether ethylene produced in response to ROS has a direct effect on cell wall properties and cell elongation or if it stimulates cell signaling pathways related to germination, is however not known. Contrasting results obtained by, ) demonstrates that ethylene decreases ROS content in Arabidopsis seeds germinating under salinity stress. In the case of the former species, ethylene has an antagonistic effect to ROS that are detrimental for germination, probably because their production increases to excessive levels in response to stress. This highlights the plasticity of seed responses to ROS but also the complexity of their interaction with ethylene.

27Several authors have also studied the effect of ROS on ethylene production during seed germination. Dormant sunflower embryos treated by methylviologen, a ROS generating compound, germinate rapidly at temperatures that would otherwise prevent their germination. However, this improving effect is not associated with an increase in ethylene production which peaks at the time radicle elongates, and which therefore must be considered as a post-germinative event. These authors propose that ethylene might participate in association with ROS to facilitate the initiation of cell elongation, the first visible symptom of germination. In dormant apple embryos, suggest that the improving effect of NO and HCN results from a transient ROS production leading to an ethylene production required in termination of the sensu stricto germination process before radicle elongation and propose that this might result from a non-enzymatic oxidation of ACC. have proposed that ROS produced in soybean (Glycine max) embryonic axes during imbibition induces ethylene production, which promotes cell elongation in the radicle. However, in that case, ethylene was measured after the onset of radicle protrusion, and this production was probably more related to the kinetics of seedling elongation than on a direct effect of H2O2. In contrast, in pea, which germination is not strongly regulated by ethylene, demonstrate that H2O2 treatment results in a reduction in PsACS2 transcript abundance consistent with a decrease in ACC content. These results suggest that ROS and ethylene probably do not interact directly, but rather through a complex hormonal network.

28All together these data suggest that the interaction between ROS and ethylene in seeds can operate in both directions, depending on the physiological context of germination, i.e., on the environmental conditions prevailing during imbibition, and is highly species related. One can predict that the use of seeds of the plant model Arabidopsis will help decipher the molecular bases of this interaction. In particular it will be interesting to determine whether ROS can trigger expression of the ethylene signaling pathway components and vice versa. For example, has demonstrated the occurrence of such a cross talk, since the treatment of dormant sunflower embryos by methylviologen induced the expression of ETR2 and of the transcription factor ERF1. The involvement of ethylene transcription factors in response to ROS appears to be worth investigating since studies with other plant systems have also implicated such a relationship.

29Seed germination is regulated by ethylene in a complex signaling network, which is also operational in numerous developmental processes, including vegetative growth, flowering timing, fruit ripening and organ senescence and abscission. As mentioned above, ethylene interacts with ABA and GAs, both hormones being essential regulators of germination and dormancy. Thus, the improving effect of ethylene may occur via the involvement of C2H4-GAs-ABA crosstalk but whether its action is direct or indirect needs clarification. Research on the effect of ABA and GAs on C2H4 biosynthesis and signaling pathways, especially in seeds, would then require further investigation, specifically in relation with ROS. Figure 2 summarizes the current data concerning ABA-GAs-C2H4 networks based on genetic analyses, microarray data, and physiological studies. ABA inhibits the C2H4 biosynthesis pathway via an inhibitory action on ACO activity and on the ACO transcript accumulation. On the contrary, C2H4 counteracts both ABA synthesis and signaling, ETR1 having a key role. In addition, C2H4 affects the synthesis of GAs via modification of expression of genes (GA3ox and GA20ox) involved in GAs synthesis. Ethylene also probably modifies the GAs signaling pathway via a regulation of DELLA proteins, as demonstrated in growth processes. To add to the complexity of the ABA-GAs-C2H4 network, there are antagonistic interactions between ABA and GAs, C2H4 and brassinosteroids, jasmonates and auxins. ROS also regulate seed germination through hormonal networks, in particular with ABA and GAs. It would be then important to discriminate the hierarchy of the different signaling pathways, and their role as sensor of environmental signals.

30Omics studies are now available in the field of seed germination but efforts to develop transcriptomic analysis of ethylene action are required to understand ethylene involvement in seed germination. Analysis of the effects of ethylene on specific cellular processes highlighted by dormancy and germination studies such as transcription regulation, cell cycle activity and endosperm weakening should help to understand the regulatory network of germination process in seeds. Moreover, although hormonal signaling network share common components, they may work in specific territories in seeds.

31The 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.