BRUTUS-LIKE (BTSL) E3 ligase-mediated fine-tuning of Fe regulation negatively affects Zn tolerance of Arabidopsis

Abstract The mineral micronutrients zinc (Zn) and iron (Fe) are essential for plant growth and human nutrition, but interactions between the homeostatic networks of these two elements are not fully understood. Here we show that loss of function of BTSL1 and BTSL2, which encode partially redundant E3 ubiquitin ligases that negatively regulate Fe uptake, confers tolerance to Zn excess in Arabidopsis thaliana. Double btsl1 btsl2 mutant seedlings grown on high Zn medium accumulated similar amounts of Zn in roots and shoots to the wild type, but suppressed the accumulation of excess Fe in roots. RNA-sequencing analysis showed that roots of mutant seedlings had relatively higher expression of genes involved in Fe uptake (IRT1, FRO2, and NAS) and in Zn storage (MTP3 and ZIF1). Surprisingly, mutant shoots did not show the transcriptional Fe deficiency response which is normally induced by Zn excess. Split-root experiments suggested that within roots the BTSL proteins act locally and downstream of systemic Fe deficiency signals. Together, our data show that constitutive low-level induction of the Fe deficiency response protects btsl1 btsl2 mutants from Zn toxicity. We propose that BTSL protein function is disadvantageous in situations of external Zn and Fe imbalances, and formulate a general model for Zn–Fe interactions in plants.


Gene name Primer name
Sequence (5' -3') Source   Figure S2 (continued from previous page) B) Total chlorophyll normalized to fresh weight (FW) in shoots from seedlings in (A).C) Shoot biomass, in mg fresh weight (FW), of seedlings in (A).D) Primary root length of 10-day-old seedlings.
E) The concentration of Mn in the roots and shoots of 14-d-old seedlings of wild type (Col-0) and btsl1x2 grown on standard (1 μM ZnSO 4 , 5 μM Fe(HBED)) or Zn excess (100 μM ZnSO 4 , 5 μM Fe(HBED)).Roots were desorbed with chelators to remove apoplastic cations prior to element analysis.F) Zn-to-Fe ratio of shoots and roots.For B-E data represent mean values (± SEM) from three independent experiments, each comprising ten plants per genotype and condition.Statistically significant differences are indicated by letter (p < 0.05) as determined by two-way ANOVA followed by Tukey HSD post-hoc test.

Figure S1 :
Figure S1: The btsl1 btsl2 double mutant displays Zn tolerance A) Representative photographs of 14-day-old seedlings of wild type (Col-0) and btsl1 btsl2 double mutant (btsl1x2), grown on standard medium (1 μM ZnSO4, 5 μM Fe(HBED)) or Zn excess (100 μM ZnSO4, 5 μM Fe(HBED)) B) Total chlorophyll normalized to fresh weight (FW) in shoots from seedlings in (A).C) Primary root length of 10-day-old seedlings.D) Shoot biomass, in mg fresh weight (FW), of seedlings in (A).For B -D, data represent mean values (± SEM) from three independent experiments, each comprising six plants per genotype and condition.Statistically significant differences are indicated by letters (p < 0.05) as determined by two-way ANOVA followed by Tukey HSD post-hoc test.

Figure S4 :
Figure S4: Fe deficiency-responsive genes are constitutively overexpressed in btsl1 btsl2 double mutant roots.A) Gene Ontology (GO) enrichment analysis of common and unique Zn-responsive DEGs in Col-0 and btsl1x2 roots.All significant (padj < 0.05) terms are shown on the vertical axis and grouped by similar function.The size of each point represents the number of DEGs associated with each term and the colour shows -log(p-value).C) upregulated and D) downregulated DEGs in four pairwise comparisons of interest (Col-0 1 μM Zn vs btsl1x2 1 μM Zn; Col-0 1 μM Zn vs Col-0 100 μM Zn; Col-0 1 μM Zn vs btsl1x2 100 μM Zn; btsl1x2 1 μM Zn vs btsl1x2 100 μM Zn) and the overlap between these gene sets.

Figure S5 :
Figure S5: Hierarchical clustering of root DEGs Major clusters are number I-VI and annotated for significantly (padj < 0.05) enriched GO terms and Fe/Zn homeostasis genes.Expression is represented as standard deviation from the mean.

Figure S6 :
Figure S6: Fe deficiency-responsive genes are not differentially expressed in btsl double mutant shoots under Zn excess Gene Ontology (GO) enrichment analysis of common and unique Zn-responsive DEGs in Col-0 and btsl1x2 shoots.All significant (padj < 0.05) terms are shown on the vertical axis and grouped by similar function.The size of each point represents the number of DEGs associated with each term and the color shows -log(p-value).