Mutational studies of the Aux/IAA proteins in Physcomitrella reveal novel insights into their function

Abstract

Summary • The plant hormone auxin regulates many aspects of plant growth and development. Auxin signaling involves hormone perception by the TRANSPORT INHIBITOR RESPONSE/AUXIN F-BOX (TIR1/AFB)–Aux/IAA co-receptor system, and the subsequent degradation of the Aux/IAA transcriptional repressors by the ubiquitin proteasome pathway. This leads to the activation of downstream gene expression and diverse physiological responses. Here, we investigate how the structural elements in the Aux/IAAs determine their function in Auxin perception and transcriptional repression. • We took advantage of the facile genetics of the moss Physcomitrella patens to determine the activity of wild-type and mutant PpIAA1a proteins in a Δaux/iaa null background. In this way, Aux/IAA function was characterized at the molecular and physiological levels without the interference of genetic redundancy. • We identified and characterized degron variants in Aux/IAAs that affect their stability and Auxin response. We also demonstrated that neither the Aux/IAA EAR motif nor Aux/IAA oligomerization is essential for the repressive function of Aux/IAA. • Our study demonstrates how key elements within the Aux/IAA proteins fine tune stability and repressor activity, as well as the long-term developmental outcome.

Minimal auxin sensing levels in vegetative moss stem cells revealed by a ratiometric reporter

“…The core mechanisms for auxin sensing are conserved in P. patens and M. polymorpha, indicating that they were already in place in the common ancestors of flowering plants and bryophytes (i.e. in the earliest land plants; Prigge et al, 2010;Lavy et al, 2012Eklund et al, 2015;Flores-Sandoval et al, 2015Kato et al, 2015Kato et al, , 2017Plavskin et al, 2016;Mutte et al, 2018;Tao & Estelle, 2018).…”

“…1b). In addition, point mutations in conserved residues of the DII domains in the PpAux/ IAAs result in stabilized protein versions conferring auxin resistance, just as in Arabidopsis, and a triple knockout of all moss Aux/IAA genes show dramatic phenotypes, indicating a constitutive auxin response which cannot be altered by changes in auxin concentrations (Prigge et al, 2010;Tao & Estelle, 2018). Transcriptome analysis of the triple mutant indicates that most, if not all, auxin-mediated changes on transcription are relayed through the Aux/IAAs, at least in filamentous protonema .…”

Regulation of auxin transcriptional responses

“…However, expression of nonoligomerizing IAA17 and IAA19 in protoplasts had an intermediate effect on repressive activity of these Aux/IAAs 53 ; expression of nonoligomerizing IAA1a in Physcomitrella had an intermediate effect on IAA1a repressive activity. 58 Further, expression of a stabilized nonoligomerizing IAA14 variant efficiently repressed auxin responses, 57 consistent with IAA14 oligomerization being unnecessary for ARF repression. In addition, Aux/IAA multimerization may be required for efficient recruitment of TPL, as structural studies have shown that binding affinity of the TPL/TPR co-repressor increases in the presence of oligomerized EAR-motif containing repressors.…”

“…Overexpression of a stabilized IAA16 with mutations disrupting either the basic or acidic face of the PB1 domain results in the loss of repressive activity of IAA16, suggesting that Aux/IAA multimerization is necessary for biological function. However, expression of nonoligomerizing IAA17 and IAA19 in protoplasts had an intermediate effect on repressive activity of these Aux/IAAs; expression of nonoligomerizing IAA1a in Physcomitrella had an intermediate effect on IAA1a repressive activity . Further, expression of a stabilized nonoligomerizing IAA14 variant efficiently repressed auxin responses, consistent with IAA14 oligomerization being unnecessary for ARF repression.…”

Back to basics: what is the function of an Aux/IAA in auxin response?

“…The authors had previously demonstrated that moss expressing only IAA1a have wild‐type auxin response and morphology (Lavy et al ., ). In the current study, Tao & Estelle perform quantitative structure/function analysis by substituting sequence variants at the IAA1a locus in the sensitized background of an iaa1b iaa2 double mutant. Their approach has made it possible to make definitive statements about a number of features of Aux/IAA function that have been the subject of debate since their initial discovery.…”