Do Arabidopsis <i>Squamosa promoter binding Protein‐Like</i> genes act together in plant acclimation to copper or zinc deficiency?

Abstract

The genome of <i>Arabidopsis thaliana</i> encodes 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 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 <i>spl1 spl12</i> double mutant and the <i>spl1 spl7 spl12</i> triple mutant with both the wild type and the <i>spl7</i> 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 <i>Chlamydomonas reinhardtii</i>, 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 <i>spl7</i>, <i>spl1 spl12</i> and <i>spl1 spl7 spl12</i> mutants. We observed that CuZnSOD activity is strongly downregulated in Zn-deficient <i>A. thaliana</i>, in association with an about 94% reduction in the abundance of the <i>CSD2</i> transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of <i>SPL7</i> and not associated with an upregulation of <i>MIR398b</i> primary transcript levels. Our data suggest that there is no conservation in <i>A. thaliana</i> of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 of 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 the regulation of either Cu deficiency responses or growth and development of land plants.