2. APLICACIÓN DE LA METODOGIA ABC DE CLASIFICACIÓN A LOS PRODUCTOS ALMACENADOS EN
2.1. Aplicación metodología ABC a los inventarios
Characterisation of the DR5::VENUS and DII-VENUS transgenic reporter lines has demonstrated that N23 has no effect on the expression of auxin-responsive genes or AUX/IAA degradation. Based on a knowledge of the auxin signalling cascade (Figure 4.5.), there are a number of possible scenarios in which N23 could potentially be altering this pathway and causing agravitropsim. Studies using the DR5::GUS reporter
a
b
DMSO IAA N23
Figure 4. 4. N23 does not induce DII:VENUS degradation.
(a) DII::VENUS fluorescence in the root tip 60 mins after treatment with
DMSO (mock), IAA (1 µM) and N23 (5 µM). (b) Quantification of
DII::VENUS fluorescence following treatment with DMSO, IAA or N23. Five-
day old seedlings were transferred to media containing IAA, N23 or mock
(DMSO) and imaged over 1 hour using a confocal microscope.
Fluorescence was quantified using Fiji software. As described in Brunoud
et al. (2012). Values are the average of 3 replicates (±SE).
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line have demonstrated that some ARFs are repressors (ARF1, -2, -3, -4, and -9) whereas others are activators (ARF5, -6, -7, and -8) (Tiwari et al., 2003). As N23 does not induces an increase in DR5::VENUS fluorescence it is possible that the chemical is either promoting the ARF repression or blocking ARF transcriptional activation. Tiwari et al. (2003) suggested that nuclear proteins could be increasing the stability of ARFs whilst bound to ARE in vivo. It is conceivable that N23 could also be decreasing ARF stability, which would result in decreased activation of auxin-responsive genes, such as DR5. Another potential mode of action for N23 is that it blocks the binding of AUX/IAA proteins with the repressor ARFs. This would prevent the inhibition of ARFs by the AUX/IAA proteins leading to constitutive deactivation of auxin-responsive gene expression (Figure 4.5.c.). An additional component of AUX/IAA-ARF binding is the protein TOPLESS (TPL) which has been shown to be required for IAA12/BDL repression of ARF5/MONOPTEROS (Szemenyei et al., 2008). TPL is known to bind both IAA12/BDL and ARF5/MONOPTEROS. If N23 was blocking ARF-AUX/IAA interaction it would also explain why it does not mimic IAA and lead to increased AUX/IAA degradation. As chemicals identified by previous chemical screens were predominantly found to be blocking protein-protein interactions (Armstrong et al., 2004; Kim et al., 2011), it was therefore decided to analyse the effect of N23 on ARF- AUX/IAA interactions.Yeast-2-hybrid assays provide a convenient heterologous system for monitoring ARF and AUX/IAA interactions (Tiwari et al., 2003). Previous studies of ARF7 and ARF19 have demonstrated that they are involved in positive transcriptional regulation of the auxin responsive genes through their interaction with the ARE, including DR5 (Tiwari et al., 2003; Wang et al., 2005b). They have also been demonstrated to have a role in regulating root gravitropism (Okushima et al., 2005). The AUX/IAA gene IAA14 also has an important role in controlling gravitropism, and has been demonstrated to interact with ARF7 and ARF19 in yeast 2-hybrid assays (Fukaki et al., 2002). These findings suggest that IAA14 may act to block ARF7 and ARF19 activity in planta to regulate gravitropism in response to auxin signalling (Fukaki et al., 2002). Given that
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N23 blocks gravitropism it was decided to analyse whether it perturbs the interaction between ARF19 and IAA14 in yeast-2-hybrid assays.Numerous other ARF-IAA interactions including ARF1 and IAA12 and -13 have been identified using the yeast-2-hybrid system (Ulmasov et al., 1997b), IAA14 with ARF7 and -19 (Fukaki et al., 2005) and more recently to test >1200 possible interactions among ARFs and Aux/IAA proteins (Vernoux et al., 2011). To test the interaction of ARF19 and IAA14 in the presence of N23 the Invitrogen ProQuest© yeast-2-hybrid system was used. This consists of the MaV203 yeast strain with the pDEST32 bait vector and the pDEST22 prey vector. In the case of this experiment the IAA14 was tested as a GAL4DB fusion and ARF19 as a GAL4AD fusion. This was because ARF19 self-activates expression of the reporter genes when it is expressed as a GAL4DB fusion protein due to its intrinsic transcriptional activation activity. Two ARF19 clones were used in these assays: a full length ARF19 (ARF19 FL) clone and one containing an N-terminally truncated clone, but which contained AUX/IAA interaction domains (ARF19 2-15). The reporter gene for the yeast-2-hybrid assays is HIS3, which allows the yeast strain to grow on plates lacking histidine. The competitive inhibitor of HIS3, 3AT is included in the assays as it provides some idea about the strength of the interactions based on the level of HIS3 expression.
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Yeast strains containing the IAA14 bait and both ARF19 prey constructs demonstrated growth on media lacking histidine with 50-100 mM 3AT, whereas the control strain (IAA14 bait and empty prey vector) only grew on those containing 10 mM 3AT (Figure 4.6.). This confirmed the previously report demonstrating an interaction between IAA14 and ARF19 in yeast-2-hybrid assays (Fukaki et al., 2005).Auxin responsive genes
ARF ARF AUX/IAA Auxin SCF TIR1 AUX/IAA degradation 26S- proteasome D C E A B
Figure 4. 5. Auxin signalling with possible sites of action of N23
highlighted in red.
The auxin responsive genes are up-regulated by the ARF which in turn
are inhibited by binding to the AUX/IAAs. This inhibition can be
overcome by the degradation of the AUX/IAA by the 26S-proteasome.
Degradation is mediated by auxin binding to the F-box component
(TIR1) of SCF
TIR1which forms a complex with the AUX/IAAs. Possible
modes of action of N23 are: (A) N23 increases the interaction of ARF
with the responsive genes, (B) N23 stabilises ARFs leading to increased
auxin responsive gene expression, (C) N23 blocks interaction of ARFs
with AUX/IAAs allowing ARFs to up-regulate DR5 expression without
affecting AUX/IAA degradation, (D) N23 up-regulates auxin responsive
genes (DR5), (E) N23 does not affect AUX/IAA degradation.
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When the assays were repeated in the presence of 50 µM N23 all of the strains continued to demonstrate growth at the same concentrations of 3AT (Figure 4.6). This indicates that N23 is not affecting the interaction between IAA14 and ARF19 in these assays. Whilst this assay implies N23 has no effect on IAA14-ARF19 interaction, the possibility that this chemical may be perturbing specific AUX/IAA-ARF interactions cannot be discounted. For example, IAA14 has also been demonstrated to interact with ARF7 which has been implicated in gravitropism (Fukaki et al., 2002; Fukaki et al., 2005). Taken together the results from the DR5::VENUS, DII-VENUS and ARF- IAA yeast screen rule out the likelihood of N23 acting primarily on components of the auxin signalling pathway.Figure 4. 6. Yeast-2-hybrid assay showing N23 does not block
IAA14-ARF19 interaction
Yeast two-hybrid study on the interaction between IAA14 and ARF19
FL and ARF19 2-15. Yeast colonies diluted in dH
2