María Bernal

Corrigendum: Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk

SUMMARY: IRON‐REGULATED TRANSPORTER1 (IRT1) is the root high‐affinity ferrous iron (Fe) uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous Fe supplementation. Here we provide evidence supporting a second role of IRT1 in root‐to‐shoot partitioning of Fe. We show that irt1 mutants overaccumulate Fe in roots, most prominently in the cortex of the differentiation zone in irt1‐2, compared to the wild type. Shoots of irt1‐2 are severely Fe‐deficient according to Fe content and marker transcripts, as expected. We generated irt1‐2 lines producing IRT1 mutant variants carrying single amino‐acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. Root short‐term ⁵⁵Fe uptake rates were uninformative concerning IRT1‐mediated transport. Overall irt1‐like concentrations of the secondary substrate Mn suggested that the transgenic Arabidopsis lines also remain incapable of IRT1‐mediated root Fe uptake. Yet, IRT1_S206A partially complements rosette dwarfing and leaf chlorosis of irt1‐2, as well as root‐to‐shoot Fe partitioning and gene expression defects of irt1‐2, all of which are fully complemented by wild‐type IRT1. Taken together, these results suggest a regulatory function for IRT1 in root‐to‐shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1‐dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve Fe nutrition and the nutritional quality of agricultural crops.

Numerous central biological processes depend on the participation of the essential elements iron (Fe) or copper (Cu), including photosynthesis, respiration, cell wall remodeling and oxidative stress protection. Yet, both Fe and Cu metal cations can become toxic when accumulated in excess. Because of the potent ligand-binding and redox chemistries of these metals, there is a need for the tight and combined homeostatic control of their uptake and distribution. Several known examples pinpoint an inter-dependence of Fe and Cu homeostasis in eukaryotes, mostly in green algae, yeast and mammals, but this is less well understood in multicellular plants to date. In Arabidopsis, Cu deficiency causes secondary Fe deficiency, and this is associated with reduced in vitro ferroxidase activity and decreased root-to-shoot Fe translocation. Here we summarize the current knowledge of the cross-talk between Cu and Fe homeostasis and present a partial characterization of LACCASE12 (LAC12) that encodes a member of the multicopper oxidase (MCO) protein family in Arabidopsis. LAC12 transcript levels increase under Fe deficiency. The phenotypic characterization of two mutants carrying T-DNA insertions suggests a role of LAC12 in root-to-shoot Fe partitioning and in maintaining growth on Fe-deficient substrates. A molecular understanding of the complex interactions between Fe and Cu will be important for combating Fe deficiency in crops and for advancing biofortification approaches.

SUMMARY: Zinc is an essential nutrient at low concentrations, but toxic at slightly higher ones. This could be used by plants to fight pathogens colonization. Elemental distribution in Arabidopsis thaliana leaves inoculated with the necrotrophic fungus Plectosphaerella cucumerina BMM ( PcBMM ) was determined and compared to mock-inoculated ones. Infection assays were carried out in wild type and long-distance zinc trafficking double mutant hma2hma4 , defective in root-to-shoot zinc partitioning. Expression levels of genes involved in zinc homeostasis or in defence phytohormone-mediated pathways were determined. Zinc and manganese levels increased at the infection site. Zinc accumulation was absent in hma2hma4. HMA2 and HMA4 transcription levels were upregulated upon PcBMM inoculation. Consistent with a role of these genes in plant immunity, hma2hma4 mutants were more susceptible to PcBMM infection, phenotype rescued upon zinc supplementation. Transcript levels of genes involved in the salicylic acid, ethylene and jasmonate pathways were constitutively upregulated in hma2hma4 plants. These data are consistent with a role of zinc in plant immunity not only of hyperaccumulator plants, but also of plants containing ordinary levels of zinc. This new layer of immunity seems to run in parallel to the already characterized defence pathways, and its removal has a direct effect on pathogen resistance.

The genome ofArabidopsis thalianaencodes approximately 260 copper (Cu)‐dependent proteins, which includes enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu‐deficient growth conditions, Squamosa promoter binding Protein‐Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems, and it mediates a metabolic reorganization to economize on Cu. The transcription factorSPL7 groups among comparably large proteins in theSPLfamily, which additionally comprises a second group of smallSPLproteins targeted by miRNA156 with roles in plant development.SPL7 shares extended regions of sequence homology withSPL1 andSPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of largeSPLfamily proteins. We compared thespl1 spl12double mutant and thespl1 spl7 spl12triple mutant with both the wild type and thespl7single mutant under normal and Cu‐deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement ofSPL7, but provided no indication for important roles ofSPL1 orSPL12 in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green algaChlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn‐deficient wild type plants. Known Zn deficiency responses were unaltered inspl7,spl1 spl12andspl1 spl7 spl12mutants. We observed that CuZnSODactivity is strongly downregulated in Zn‐deficientA. thaliana, in association with an about 94% reduction in the abundance of theCSD2transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent ofSPL7and not associated with an upregulation ofMIR398bprimary transcript levels. Our data suggest that there is no conservation inA. thalianaof the crosstalk between Zn and Cu homeostasis mediated by the singleSPLfamily proteinCRR1 of Chlamydomonas. In the future, resolving how the specificity ofSPLprotein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of differentSPLfamily proteins in the regulation of either Cu deficiency responses or growth and development of land plants.

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)-dependent proteins, which include enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu-deficient growth conditions, Squamosa promoter binding Protein-Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems and mediates a metabolic reorganization to economize on Cu. The transcription factor SPL7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by miRNA156 with roles in plant development. SPL7 shares extended regions of sequence homology with SPL1 and SPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with the wild type and the spl7 single mutant under normal and Cu-deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL7, but provided no indication for important roles of SPL1 or SPL12, in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn-deficient wild-type plants. Known Zn deficiency responses were unaltered in spl7, spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZnSOD activity is strongly downregulated in Zn-deficient A. thaliana, in association with an about 94% reduction in the abundance of the CDS2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL7 and not associated with an upregulation of MIR398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 in Chlamydomonas. In the future, resolving how the specificity of SPL protein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of different SPL family proteins in either Cu deficiency response regulation or growth and development of land plants.