Ca²⁺-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis inArabidopsis

Abstract

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca 2+ oscillations in Arabidopsis roots. These Ca 2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca 2+ -dependent protein kinases CPK21 and CPK23 as Ca 2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21 / 23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca 2+ -CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Plasma membrane‐associated calcium signaling modulates cadmium transport

“…Histochemical analysis of GUS activity of 7‐d‐old ProNRAMP6:GUS , ProCPK21:GUS , and ProCPK23:GUS transgenic plants showed that they have similar tissue‐specific expression patterns and all exhibited staining in root tips (Fig. S1), which was consistent with recently published research (Fu et al ., 2022). The results suggested that a physiological interaction between NRAMP6 and CPK21/23 is possible in Arabidopsis .…”

“…In addition, nramp1/6 double mutants enhanced the sensitive phenotype of nramp1 single mutant in response to Mn deficiency, which suggested that NRAMP6 and NRAMP1 cooperatively function under Mn deficient conditions, with NRAMP1 being the major player (Li et al ., 2019). Our recently published article has showed that CPK21/23 phosphorylate and activate Mn transporter NRAMP1 to enhance plant tolerance to Mn deficiency (Fu et al ., 2022). Although CPK21/23 act upstream of both NRAMP1 and NRAMP6, the results from heterologous yeast complementing assays and Mn concentration analysis of transgenic plants suggested that the roles of CPK21/23 on NRAMP1 and NRAMP6 are, to some extent, specific to Mn‐deficiency and Cd stress responses (Fig.…”

“…For the BiFC assay, the full‐length CDSs of CPK21/23/30/NRAMP6 and GUS were amplified and cloned into the nYFP or cYFP vector, other CPKs‐nYFP vectors were obtained from recently published research (Fu et al ., 2022).…”

“…The co‐immunoprecipitation assay in Arabidopsis was conducted according to a recently published method (Fu et al ., 2022). The 4‐d‐old transgenic lines were then transferred to ½MS medium supplemented with 80 μM CdCl 2 for 7 d, and the total proteins were extracted and resuspended.…”

Ca2+ signaling in plant manganese uptake: CPK21/23 kinases phosphorylate and activate manganese transporter NRAMP1

“…However, whether Ca 2+ signaling plays an important role in plant response and tolerance to Mn deficiency remains unclear. This brief article highlights the recent results of Fu et al (2022), who revealed a Ca 2+ -CPK21/23-NRAMP1 axis as an important mechanism for plant tolerance and adaptation to low Mn.…”