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Characterization of a small auxin-up RNA (SAUR)-like gene involved in Arabidopsis thaliana development.

Markakis MN, Boron AK, Van Loock B, Saini K, Cirera S, Verbelen JP, Vissenberg K - PLoS ONE (2013)

Bottom Line: From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated.Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane.SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Antwerp, Antwerpen, Belgium.

ABSTRACT
The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.

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qRT-PCR expression analysis of SAUR76 in ethylene and auxin-mutants in control conditions and after treatment with ACC or auxin.A) Effect of 3 hr IAA or B) ACC treatment on SAUR76 expression levels in wild type, ethylene and auxin mutants. * points to a statistical significance towards the untreated WT and ** towards the treated WT (n=3, mean ± SE).
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pone-0082596-g005: qRT-PCR expression analysis of SAUR76 in ethylene and auxin-mutants in control conditions and after treatment with ACC or auxin.A) Effect of 3 hr IAA or B) ACC treatment on SAUR76 expression levels in wild type, ethylene and auxin mutants. * points to a statistical significance towards the untreated WT and ** towards the treated WT (n=3, mean ± SE).

Mentions: To reveal whether the ACC-induced up regulation of SAUR76 occurred directly by ACC or by the ACC-induced increase in auxin, different mutants in ethylene and auxin signalling were treated with ACC and IAA, and the expression levels of SAUR76 were determined by qPCR (Figure 5). It is known that when ethylene binds to the ETR1 receptor, CTR1 may allow interaction of EIN2 with the kinase domain of ETR1. CTR1 is a negative regulator of ethylene by blocking the signalling downstream to EIN2, so the ctr1 mutant will have lost the blocking capacity resulting in a constitutive response to ethylene. Both etr1-3 and ein2-1 mutants are insensitive towards ethylene as they cannot sense or transduce the signal respectively (reviewed in 58). Under control conditions, SAUR76 was on average 10 times higher expressed in ctr1 than in the WT, whereas no significant difference in expression was seen between wild type and the ethylene-insensitive etr1-3, while expression of SAUR76 was approximately 40% less in ein2-1 plants. This agrees with the observed up regulation of SAUR76 in ACC-treated wild type seedlings, as the ctr1 plants mimic a wild type plant treated with ACC. Upon ACC addition, the increase in expression of SAUR76 was much higher in the WT than in the treated ctr1 plants, suggesting that efficient and working ethylene perception and signalling is required to increase the expression of SAUR76 (Figure 5A). As expected in the complete ethylene-insensitive etr1-3 background no ACC-effect on the expression levels was seen, while even a further reduction of the expression was apparent in ACC-treated ein2-1 plants. In the auxin-import mutant aux1-2 [59], SAUR76 expression is higher than in the WT under control conditions. SAUR76 expression shows only marginal increase in aux1-2 after ACC treatment, indicating that AUX1 plays a prominent role in the observed increase of SAUR76 and that maybe the ACC-induced increase of auxin [27–30] is the key-regulator of the observed increases in expression. Indeed, IAA treatments induced a significant up regulation of SAUR76 in all ethylene-related mutants and the WT (Figure 5B), providing further evidence that the increase of SAUR76 by ACC is mostly occurring through the up regulation of auxin signalling. As a consequence, it could be that different auxin levels are present in the ein2-1 and aux1-2 mutant, explaining the differences in SAUR76 expression under control and in ACC conditions. Furthermore, aux1-2 shows an up regulation of SAUR76 expression after IAA treatment. As auxin is not only transported into the cell by AUX1 transporters but also by diffusion through the membrane [60], the bulk addition of auxin in this case can lead to a higher auxin concentration in the cell even when AUX1 function is impaired, and hence an up regulation of SAUR76. Furthermore, the fact that SAUR76 expression levels in untreated aux1-2 plants is higher than in the wild type and that it is under control of auxin suggests that the endogenous auxin levels might be disturbed in aux1-2. This in turn suggests that there is a feedback mechanism between one/several of the auxin signaling components [61] and auxin biosynthesis itself. In addition, the increase in SAUR76 expression in overexpression plants treated with IAA or ACC is higher than in the WT treated with IAA or ACC, providing further evidence that there must be some kind of feedback-mechanism where the SAUR76 protein plays an active role. How exactly this feedback mechanism is regulated, remains to be uncovered. Nevertheless, the outcome of this analysis is that the ACC-induced pathway leading to an increase in expression of SAUR76 goes through changes in auxin concentration.


Characterization of a small auxin-up RNA (SAUR)-like gene involved in Arabidopsis thaliana development.

Markakis MN, Boron AK, Van Loock B, Saini K, Cirera S, Verbelen JP, Vissenberg K - PLoS ONE (2013)

qRT-PCR expression analysis of SAUR76 in ethylene and auxin-mutants in control conditions and after treatment with ACC or auxin.A) Effect of 3 hr IAA or B) ACC treatment on SAUR76 expression levels in wild type, ethylene and auxin mutants. * points to a statistical significance towards the untreated WT and ** towards the treated WT (n=3, mean ± SE).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842426&req=5

pone-0082596-g005: qRT-PCR expression analysis of SAUR76 in ethylene and auxin-mutants in control conditions and after treatment with ACC or auxin.A) Effect of 3 hr IAA or B) ACC treatment on SAUR76 expression levels in wild type, ethylene and auxin mutants. * points to a statistical significance towards the untreated WT and ** towards the treated WT (n=3, mean ± SE).
Mentions: To reveal whether the ACC-induced up regulation of SAUR76 occurred directly by ACC or by the ACC-induced increase in auxin, different mutants in ethylene and auxin signalling were treated with ACC and IAA, and the expression levels of SAUR76 were determined by qPCR (Figure 5). It is known that when ethylene binds to the ETR1 receptor, CTR1 may allow interaction of EIN2 with the kinase domain of ETR1. CTR1 is a negative regulator of ethylene by blocking the signalling downstream to EIN2, so the ctr1 mutant will have lost the blocking capacity resulting in a constitutive response to ethylene. Both etr1-3 and ein2-1 mutants are insensitive towards ethylene as they cannot sense or transduce the signal respectively (reviewed in 58). Under control conditions, SAUR76 was on average 10 times higher expressed in ctr1 than in the WT, whereas no significant difference in expression was seen between wild type and the ethylene-insensitive etr1-3, while expression of SAUR76 was approximately 40% less in ein2-1 plants. This agrees with the observed up regulation of SAUR76 in ACC-treated wild type seedlings, as the ctr1 plants mimic a wild type plant treated with ACC. Upon ACC addition, the increase in expression of SAUR76 was much higher in the WT than in the treated ctr1 plants, suggesting that efficient and working ethylene perception and signalling is required to increase the expression of SAUR76 (Figure 5A). As expected in the complete ethylene-insensitive etr1-3 background no ACC-effect on the expression levels was seen, while even a further reduction of the expression was apparent in ACC-treated ein2-1 plants. In the auxin-import mutant aux1-2 [59], SAUR76 expression is higher than in the WT under control conditions. SAUR76 expression shows only marginal increase in aux1-2 after ACC treatment, indicating that AUX1 plays a prominent role in the observed increase of SAUR76 and that maybe the ACC-induced increase of auxin [27–30] is the key-regulator of the observed increases in expression. Indeed, IAA treatments induced a significant up regulation of SAUR76 in all ethylene-related mutants and the WT (Figure 5B), providing further evidence that the increase of SAUR76 by ACC is mostly occurring through the up regulation of auxin signalling. As a consequence, it could be that different auxin levels are present in the ein2-1 and aux1-2 mutant, explaining the differences in SAUR76 expression under control and in ACC conditions. Furthermore, aux1-2 shows an up regulation of SAUR76 expression after IAA treatment. As auxin is not only transported into the cell by AUX1 transporters but also by diffusion through the membrane [60], the bulk addition of auxin in this case can lead to a higher auxin concentration in the cell even when AUX1 function is impaired, and hence an up regulation of SAUR76. Furthermore, the fact that SAUR76 expression levels in untreated aux1-2 plants is higher than in the wild type and that it is under control of auxin suggests that the endogenous auxin levels might be disturbed in aux1-2. This in turn suggests that there is a feedback mechanism between one/several of the auxin signaling components [61] and auxin biosynthesis itself. In addition, the increase in SAUR76 expression in overexpression plants treated with IAA or ACC is higher than in the WT treated with IAA or ACC, providing further evidence that there must be some kind of feedback-mechanism where the SAUR76 protein plays an active role. How exactly this feedback mechanism is regulated, remains to be uncovered. Nevertheless, the outcome of this analysis is that the ACC-induced pathway leading to an increase in expression of SAUR76 goes through changes in auxin concentration.

Bottom Line: From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated.Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane.SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Antwerp, Antwerpen, Belgium.

ABSTRACT
The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.

Show MeSH