DISCUSSION
The CK concentration is increased in response to photoperiod
stress
Here we reported on the functional relevance of root-derived CK in the
response to photoperiod stress. Wild-type plants grown under short-day
conditions and experiencing a PLP responded by increasing the CK
concentration in their leaves (Fig.1, Table S1). As plants with a
reduced CK concentration or signaling are particularly sensitive to
photoperiod stress (Nitschke et al., 2016), this response may be part of
a defense mechanism enabling wild-type plants to react appropriately to
photoperiod stress and to cope with its consequences. Altered CK
concentrations are often part of the response to abiotic stress and they
may either increase or decrease. A decreased CK concentration was found
after exposure to several abiotic stresses like heat, salt or drought
stress (Bano, Hansen, Dörffling, & Hahn, 1994; Caers, Rüdelsheim, van
Onckelen, & Horemans, 1985; Itai, Ben‐Zioni, & Ordin, 1973; Nishiyama
et al., 2011). Plants with a lower CK status were more stress resistant
indicating a functional relevance of the reduced CK level ( Nishiyama et
al., 2011). In contrast, under high light stress CK has a protective
function. It represses excessive starch grain and plastoglobuli
formation and is required for a functional D1 repair cycle (Cortleven et
al., 2014). In the response to biotic stress such as Pseudomonasinfection, CK is required for an effective defense regulating the
oxidative burst through ARR2 (Arnaud et al., 2017; Choi et al., 2010).
CK is known also from other instances to regulate the response to
oxidative stress (Pavlů et al., 2018; Cortleven et al., 2019) which is a
hallmark of the response to photoperiod stress (Nitschke et al., 2016).
We propose that one function of the enhanced CK formation could be to
properly respond to oxidative stress caused by PLP treatment (Nitschke
et al., 2016).
Root-derived tZ-type CKs act as
protectants against photoperiod
stress
Root-derived t Z-type CKs were shown to be the most relevant to
protect plants from the negative consequences of photoperiod stress.
This result supports the role of CK in root-to-shoot communication and
extends its function to a specific response to stress beyond its better
known roles in regulating development and growth. The results show also
that tZ-type CKs are particularly relevant for stress protection. The
major transport form, t ZR, as well as to a minor extent its
bioactive derivative t Z, are transported from the root to the
shoot via the xylem flow requiring the transporter ABCG14 (Ko et al.,
2014; Zhang et al., 2014). abcg14 mutants are thus deficient intZ in the shoot (Ko et al., 2014; Zhang et al., 2014) and
consistently these mutants showed a very strong response to photoperiod
stress. In cypDM mutants, the lower levels of t Z-type CKs
in the shoot are compensated by an increased level of iP-type CK (Kiba
et al., 2013). The inability of these higher levels of iP-type CKs to
compensate the sensitive photoperiod stress response of cypDMmutants corroborates the functional relevance of t Z-type CKs.
Consistent with a major role of t Z-type CKs is also the
functional relevance of AHK3 in photoperiod stress signaling (Nitschke
et al., 2016). AHK3 displays an about tenfold higher sensitivity tot Z than to iP while AHK2 and AHK4/CRE1 have similar affinities to
both iP and t Z (Lomin et al., 2015; Romanov, Lomin, &
Schmülling, 2006; Stolz et al., 2011). It has been proposed that the
affinity profile of AHK3 is particularly set to respond to root-derived
CK (Romanov et al., 2006).
Further support for a role of root-derived CK in photoperiod stress
protection came from supplementation experiments. Watering ofcypDM plants with either t ZR or t Z demonstrated
that both metabolites can protect plants against photoperiod stress
although t Z was more effective (Fig. 3). Both t Z andt ZR supplementation rescued the decrease in type-A ARRtranscript abundance in these plants demonstrating that after
application through roots they reached the shoot in a biologically
effective concentration. Different roles for root-derived t Z andt ZR have been reported by Osugi et al. (2017). It might be that
the ability of certain tissues to convert inactive t ZR to activet Z, as discussed in Romanov et al. (2018), might have an impact
on the plant´s response to photoperiod stress.
The functional relevance of root-derived CK in the response to
photoperiod stress raises the question how information about a stress
perceived and acting primarily in the shoot is relayed to the root. One
possibility is that the light signal is perceived and interpreted in the
root directly (Sun, Yoda, & Suzuki, 2005; Sun, Yoda, Suzuki, & Suzuki,
2003). Another possibility is that an instructive chemical signal is
formed in the shoot and transported to the root to induce synthesis oft Z CK. This signal could be iP-type CK as these are mainly formed
in the shoot and known to be transported to the root through the phloem
(Hirose et al., 2008; Kudo, Kiba, & Sakakibara, 2010). iP-type CKs
could then positively regulate t Z-type CK formation as they not
only serve as a precursor for t Z-formation but also induce the
expression of CYP735A2 (Takei et al., 2004). Another candidate
for a chemical signal is jasmonic acid which is increased in response to
photoperiod stress in sensitive genotypes (Nitschke et al., 2016) and
which has recently been shown to be a shoot-to-root signal (Schulze et
al., 2019).
ARR2, ARR10 and ARR12 regulate the response to photoperiod
stress in a complex
manner
AHP2, AHP3 and AHP5 act redundantly in the response to photoperiod
stress (Fig.4). The functional redundancy of these AHPs has been shown
before in the context of seed, primary root and hypocotyl development
(Hutchison et al., 2006). Our results integrate AHPs into the
CK-dependent photoperiod stress signaling pathway that so far involved
AHK3 and ARR2 as the main signaling components (Nitschke et al., 2016).
Downstream of the AHPs act several transcription factors to realize the
transcriptional output of the photoperiod stress response. ARR2 has a
predominant role in mediating CK activity in leaves (Hwang & Sheen,
2001) but its redundant function with ARR10 and ARR12 has not yet been
described. The latter two ARRs are better known for their role in
regulating most CK-related vegetative developmental processes together
with ARR1 (Argyros et al., 2008; Ishida, Yamashino, Yokoyama, & Mizuno,
2008). Analysis of single and double mutants showed that loss of eitherARR10 or ARR12 rescued the stress phenotype of arr2plants while the loss of both factors enhanced the stress response ofarr2 (Fig. 5). This hints to a complex regulatory mechanism
between these three transcription factors during photoperiod stress
signaling. A complex relationship among these type-B ARRs has also been
described for their role in regulating root elongation. arr12 andarr10,12 root elongation was less affected by CK treatment than
that of arr2,12 and arr2,10,12 (Mason et al., 2005). For
type-B ARR dependent gene regulation, a model has been proposed in which
simultaneous binding of multiple/different type-B ARRs and unknown
factors to certain promoter regions is crucial (Ramireddy, Brenner,
Pfeifer, Heyl, & Schmülling, 2013). However, experimental evidence for
a direct interaction between members of the type-B ARR family is rare.
An interaction of ARR2 and ARR14 has been described using a two-hybrid
system in yeast (Dortay, Mehnert, Bürkle, Schmülling, & Heyl, 2006).
Recently, it was found that the C-termini of ARR1 and ARR12 interact to
regulate auxin synthesis (Yan et al., 2017). It could also be that
interactions between type-B ARRs are context-dependent as it is known
for the phosphorylation-dependent homodimerization of bacterial RRs
(Mack, Gao, & Stock, 2009). Similarly, ARR18 can homodimerize when both
ARR18 proteins are either both phosphorylated or both not phosphorylated
(Veerabagu et al., 2012).
The different phenotypic and in part molecular responses to photoperiod
stress of arr mutants could be explained by a model, in which
ARR2, ARR10 and ARR12 interact with a yet unknown interaction partner
(X) that is essential for photoperiod stress resistance (Fig. 6). It is
predicted that the affinity of ARR2 to X would be higher than the
affinities of ARR10 and ARR12 to X. In addition, we propose a direct or
indirect interaction of ARR10 and ARR12. In photoperiod stress-treated
wild-type plants, ARR2 would interact with X resulting in photoperiod
stress resistance while ARR10 and ARR12 together would have independent
auxiliary functions. In arr2 plants, X would not have an
interaction partner and thus would be unable to function in stress
protection because ARR10 and ARR12 would not be available as interaction
partners. Consequently, stress resistance would be lowered. Resistance
of arr2,10 and arr2,12 plants would be caused by the loss
of the ARR10-ARR12 association and the resulting interaction of X with
ARR10 or ARR12. Ultimately, the enhanced stress phenotype ofarr2,10,12 plants would be caused by the complete loss of
interaction partners for X.
Beside the interaction amongst ARRs, interactions between several type-B
ARRs and other proteins exist. For example, ARR1, ARR2 and ARR14
interact with the DELLA proteins RGA1 and GAI to regulate root
development and photomorphogenesis (Marín-de la Rosa et al., 2015; Yan
et al., 2017). During the regulation of auxin synthesis, EIN3 interacts
with the C-terminus of ARR1 and thereby increases ARR1 activity (Yan et
al., 2017). As part of the crosstalk between CK and abscisic acid, ARR1,
ARR11 and ARR12 directly interact with SUCROSE NON‐FERMENTING‐1
(SNF1)‐RELATED PROTEIN KINASE2 (SnRK2) kinases and thereby inhibit their
function prior to drought stress (Huang et al., 2018). Future
experiments might resolve whether and how type-B ARRs interact with each
other or with other proteins during photoperiod stress.