Abstract
The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.
Highlights
The auxin signaling cascade from auxin perception to activation of auxin-inducible genes has several core components including an SCF (SKP1–Cullin1–F-box) E3 complex, Aux/IAA and auxin response factor (ARF) (Auxin Response Factor) transcription factors [1]
A full-length HA-ARF protein band was detected in HA-ARF5, HA-ARF6, and HA-ARF19 transformants, while only a strong partial HA protein band was detected in ARF1 transformants and no band was detected in ARF10 transformants
The results indicated that HA-ARF6 transcript level did not change much when Arabidopsis seedlings were treated under 37 ◦C, ABA and NaCl conditions, but showed a slight increase at 4 ◦C compared to the control (Figure S2)
Summary
The auxin signaling cascade from auxin perception to activation of auxin-inducible genes has several core components including an SCF (SKP1–Cullin1–F-box) E3 complex, Aux/IAA and ARF (Auxin Response Factor) transcription factors [1]. The interactions of ARFs with the Aux/IAA proteins are an important aspect of auxin signaling and its regulation. Results from the yeast two-hybrid studies have shown that the activator ARFs (ARF5, 6, 7, 8, and 19) have more interactions with Aux/IAA proteins compared with the repressor-type ARFs [15,16]. ARF10 of Class C is found to be important for root cap formation [34], in vitro callus initiation and shoot regeneration [35,36] These results demonstrate that ARFs play important and diverse functions in plants. BIN2 has been reported to phosphorylate ARF7 and ARF19, and in contrast to reduce activity of ARF2, ARF7, and ARF19 phosphorylation results in the enhanced transactivation activity, which is attributed to reduced ARF7 and ARF19 interactions with the Aux/IAA repressors [41]. We further demonstrated that ABA treatment promotes the ubiquitination and degradation of ARF6
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