Protein Levels of Several Arabidopsis Auxin Response Factors Are Regulated by Multiple Factors and ABA Promotes ARF6 Protein Ubiquitination

Abstract

The auxin response factor (<i>ARF</i>) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. <i>ARF</i>s are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of <i>ARF</i> protein levels. We expressed five different <i>ARF</i>s 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 <i>HA-ARF5</i>, <i>HA-ARF6</i>, and <i>HA-ARF10</i>, but not that of <i>HA-ARF19</i>, while 37 °C treatment increased the levels of the four <i>HA-ARF</i>s, suggesting that the <i>ARF</i> protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of <i>HA-ARF6</i> level by ABA and 4 °C treatments, suggesting that these treatments decrease <i>HA-ARF6</i> level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased <i>HA-ARF6</i> ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on <i>ARF</i>s, which could serve to integrate multiple hormonal and environmental signals into the <i>ARF</i>-regulated gene expression.