SummaryAuxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3-4 h. This halflife appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.

The success of the short-rotation coppice system in biomass willow (Salix spp.) relies on the activity of the shoot-producing meristems found on the coppice stool. However, the regulation of the activity of these meristems is poorly understood. In contrast, our knowledge of the mechanisms behind axillary meristem regulation in Arabidopsis (Arabidopsis thaliana) has grown rapidly in the past few years through the exploitation of integrated physiological, genetic, and molecular assays. Here, we demonstrate that these assays can be directly transferred to study the control of bud activation in biomass willow and to assess similarities with the known hormone regulatory system in Arabidopsis. Bud hormone response was found to be qualitatively remarkably similar in Salix spp. and Arabidopsis. These similarities led us to test whether Arabidopsis hormone mutants could be used to assess allelic variation in the cognate Salix spp. hormone genes. Allelic differences in Salix spp. strigolactone genes were observed using this approach. These results demonstrate that both knowledge and assays from Arabidopsis axillary meristem biology can be successfully applied to Salix spp. and can increase our understanding of a fundamental aspect of short-rotation coppice biomass production, allowing more targeted breeding.

Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short-rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time-consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone-related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot-branching traits. Single-nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%–99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.