Abstract
The genome of Arabidopsis thaliana 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 spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with both 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 CSD2 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 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.
Highlights
Micronutrient metals act as cofactors in a multitude of proteins and are essential for plant growth, development and reproduction
We confirmed by RT‐PCR that SPL1 and SPL12 were expressed in 21‐day old wild type (WT) seedlings under both Cu‐ and Zn‐deficient growth conditions (Fig. S1), which we generated by removing trace metal contaminations arising from agar and glassware and by omitting Cu and Zn, respectively, from the synthetic growth media (Schulten and Krämer 2018)
The role of Squamosa promoter binding Protein‐Like 7 (SPL7) in the Cu deficiency response of Arabidopsis thaliana is well established, but according to genome‐wide transcriptomics only 13% of about 1,500 Cu deficiency‐responsive genes were regulated in an SPL7‐dependent manner (Yamasaki et al 2009; Bernal et al 2012; Garcia‐Molina et al 2014)
Summary
Micronutrient metals act as cofactors in a multitude of proteins and are essential for plant growth, development and reproduction. In Arabidopsis, the closest homolog of CRR1 is Squamosa promoter binding Protein‐Like 7 (SPL7) and is, the major known transcription factor mediating Cu deficiency responses (Yamasaki et al 2009; Bernal et al 2012). Both CRR1 and SPL7 activate the transcription of genes acting to increase cellular Cu uptake under Cu‐deficient growth conditions. This constitutes a regulatory module in diverse developmental processes such as developmental phase transition, the specification of floral meristem identity, shoot branching and lateral root development (Wu and Poethig 2006; Schwarz et al 2008; Wu et al 2009; Yamaguchi et al 2009; Yu et al 2015; Xu et al 2016; He et al 2018)
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