Acquisition (Jakoby et al., 2004). This displays the

Acquisition of trace elements, such
as iron (Fe), is indispensable for metabolic pathways, crop yield and high-quality
nutritious food. Biofortification offers the chance to reduce Fe deficiency
anemia in humans (Haas et al., 2016) but relies on elaborate knowledge of plant nutrient sensing, uptake and
signaling. Under Fe deficient conditions, Arabidopsis
thaliana, like other non-graminaceous angiosperms, induces Fe uptake and
homeostasis genes, and follows a reduction-based Fe acquisition strategy (Strategy
I) (Marschner et al.,
1986). Due to high amounts of insoluble Fe in the soil (Guerinot and Yi, 1994), plants first acidify the rhizosphere via proton
extrusion, chelate ferric Fe (Fe3+) and subsequently reduce it to ferrous
Fe (Fe2+), which is mediated by FERRIC REDUCTION OXIDASE 2 (FRO2) (Brumbarova et al.,
2015). IRON REGULATED TRANSPORTER 1 (IRT1), which displays a robust Fe
deficiency marker gene (Ivanov et al., 2012), then takes up Fe2+ into root epidermal
cells (Eide et al., 1996;
Vert et al., 2002). The expression of components involved in Strategy I Fe uptake is controlled
by a complex regulatory network comprising the central regulator FER-LIKE IRON
DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), a subgroup IIIa basic helix-loop-helix
(bHLH) protein (Heim et al., 2003;
Colangelo and Guerinot, 2004; Jakoby et al., 2004). FIT robustly controls the transcription of 34 Fe responsive genes (Mai et al., 2016). The fit-3 loss-of-function
mutant allele develops a strong Fe deficiency leaf chlorosis and has a lethal
phenotype (Jakoby et al., 2004). This displays the
pivotal role of FIT in the Fe deficiency response.

An interplay between external and
internal signaling pathways affects the activity of FIT on a molecular level. FIT
protein activity is controlled by Fe availability (Jakoby et al., 2004) and is modulated by different signaling molecules as
well as hormones (Brumbarova et al.,
2015; Le et al., 2016). FIT activity is further controlled by different protein-protein
interactions (Brumbarova et al.,
2015; Le et al., 2016; Gratz et al., 2018 (submitted)). BHLH proteins are known to form homo- and hetero-dimers (Heim et al., 2003). The hetero-dimerization of FIT and members of subgroup Ib bHLH
proteins, bHLH038/039/100/101 is needed for downstream activation of FIT target
gene expression (Heim et al., 2003;
Wang et al., 2007; Yuan et al., 2008; Wang et al., 2013). Interestingly, FIT
expression requires the presence of FIT protein, which displays the existence
of a FIT auto-regulatory loop (Jakoby et al., 2004;
Wang et al., 2007). Hence, the dynamic
addition of FIT interactors enables the cell to quickly react to changes in Fe
availability by fine-tuning FIT protein activity.

In a FIT overexpression situation, the FIT transcript is present under both Fe sufficient and deficient
conditions. However, induction of FIT target genes, FRO2 and IRT1, only occurs
upon Fe deficiency. Thus, it was suggested that FIT is divided into active and inactive
pools (Lingam et al., 2011;
Meiser et al., 2011; Sivitz et al., 2011). Further, it was shown that FIT undergoes proteasomal degradation (Lingam et al., 2011;
Meiser et al., 2011; Sivitz et al., 2011). Probably, mostly active FIT forms are degraded, but in order to
maintain Fe deficiency responses, however, inactive FIT is constantly
activated. This might be beneficial for the cell to remain responsive to quick changes
in nutrient abundance (Lingam et al., 2011;
Meiser et al., 2011; Sivitz et al., 2011). This raises the question what the molecular difference between active
and inactive FIT forms is. One potential mechanism is the involvement of post-translational
modifications. This would be in agreement with the finding that the
experimentally observed molecular weight of FIT is higher than predicted (Sivitz et al., 2011).

 

Recently, we identified CBL-INTERACTING
PROTEIN KINASE CIPK11 as a novel
FIT interaction partner. We demonstrated that phosphorylation of the C-terminal
part of FIT (FIT-C) at position Ser272 by CIPK11 has a positive impact on its
localization, mobility and dimerization capacity. Overall, Ser272
phosphorylation renders the FIT protein active, which displays a valuable
molecular tool for the fine-tuning of the Fe uptake from the soil (Gratz et al., 2018
(submitted)).

The activity of many
transcription factors, such as the
bHLH protein INDUCER OF CBF EXPRESSION 1
(ICE1), involved in cold-stress responses (Chinnusamy et al.,
2003), is regulated by phosphorylation. Cold-induced phosphorylation of
Ser278, mediated by SnRK2.6 / OPEN STOMATA 1 (OST1), increases ICE1 stability
and hence promotes downstream target gene expression (Ding et al., 2015). Likewise, MITOGEN-ACTIVATED PROTEIN KINASEs (MAPKs)
MAPK3/MAPK6 facilitates additional ICE1 phosphorylation. However, this has
diametrical effects on its stability and leads to proteasomal degradation of
ICE1 (Li et al., 2017; Zhao
et al., 2017). Hence, activating phosphorylation events can contribute to the
activity of a protein, but an antagonistic mechanism is likewise needed to
ensure responsiveness to quickly changing conditions by a balanced protein
activity.

Plant tyrosine phosphorylation is
mainly associated with proteins having kinase or transferase activity and is
overrepresented on nuclear proteins (Sugiyama et al., 2008). Only a few transcription factors were identified to
be tyrosine-phosphorylated until now, such as Coptis japonica WRKY-type transcription factor CjWRKY1, involved in
alkaloid biosynthesis (Kato et al., 2007;
Yamada and Sato, 2016). A CjWRKY1 tyrosine phospho-mimicking mutant displays enhanced
cytosolic localization and reduced transactivation activity. Additionally,
protein turn-over was shown to be connected to the tyrosine-phosphorylation
status (Yamada and Sato, 2016).

This prompted us to question
whether FIT activity is similarly regulated. Hence, it is of interest to
investigate, whether FIT possesses additional phosphorylation target sites and whether
one site reflects a negative regulatory unit. We further asked if a tyrosine phosphorylation-site
can be identified that might contribute to FIT stability. It is also conceivable
that different phosphorylation sites exhibit different significances and hence
display a certain hierarchy.