INTRODUCTION
As one of the classical plant hormones, CK regulates several developmental programs in roots and shoots (Kieber & Schaller, 2018; Werner & Schmülling, 2009) and is of crucial importance to cope with a variety of biotic and abiotic stresses (Cortleven et al., 2019).
Recently, a novel type of abiotic stress caused by a prolongation of the light period has been described and was named photoperiod stress (previously circadian stress) (Nitschke et al., 2016; Nitschke, Cortleven, & Schmülling, 2017). During a typical photoperiod stress treatment, five-weeks-old short-day (SD) grown plants were exposed to a prolonged light period (PLP). In the experimental standard setup, a PLP of 32 h was used which caused a very strong stress response, but also a PLP of 12 h (i.e. 4 h of additional light) caused a stress response (Nitschke et al., 2016). Plants exposed to photoperiod stress responded by an increased expression of numerous stress marker genes (e.g.ZAT12 and BAP1 ) and by a decrease of genes involved in photosynthetic processes like CHLOROPHYLL A/B BINDING PROTEIN2(CAB2 ) about five hours after the beginning of the night following the PLP while control plants did not respond. The next day, stressed plants displayed a reduced photosynthetic efficiency and an increased percentage of water-soaked lesions that ultimately may enter programmed cell death compared to untreated and thus unaffected plants. It was found that a functional circadian clock is necessary to cope with a prolongation of the light period. Further, a particularly strong response to photoperiod stress was shown in plants with a reduced CK content or signaling suggesting that the hormone has a protective function (Nitschke et al., 2016).
Four different types of isoprenoid class CKs -N6 -isopentenyladenine (iP), tZ , dihydrozeatin (DHZ) and cis- zeatin (cZ) - have been identified in plants and are synthesized via two different pathways requiring either adenosine mono-/di-/triphosphate (AMP/ADP/ATP) or tRNA as a precursor. Different CK metabolites can be distinguished: the bioactive free bases and the non-active ribosides, ribotides, and O - andN -glucosides (Sakakibara, 2006). In Arabidopsis , iP andt Z are the biologically most relevant CKs and are initially synthesized by the addition of dimethylallyl diphosphate (DMAPP) to AMP/ADP/ATP. This reaction is catalyzed by ADENOSINE PHOSPHATE ISOPENTENYLTRANSFERASES (IPTs) (Kakimoto, 2001; Takei, Sakakibara, & Sugiyama, 2001). Two cytochrome P450 enzymes - CYP735A1 and CYP735A2 - convert the formed iP riboside mono-/di-/triphosphate (iPRMP/iPRDP/iPRTP) molecules into t Z nucleotides (Takei, Yamaya, & Sakakibara, 2004).CYP735A1 and CYP735A2are predominantly expressed in roots and both isoforms of the enzyme act redundantly (Kiba, Takei, Kojima, & Sakakibara, 2013). Bioactive iP andt Z are formed through dephosphoribosylation of iPRMP/t ZRMP by CK nucleoside 5’-monophosphate phosphoribohydrolase enzymes named LONELY GUY (LOGs) (Kurakawa et al., 2007; Kuroha et al., 2009; Tokunaga et al., 2012). CKs are synthesized in diverse root and shoot tissues (Miyawaki, Matsumoto-Kitano, & Kakimoto, 2004; Takei et al., 2004) and are transported through the vascular system. t Z-type CKs are mainly synthesized in the root and transported to the shoot via the xylem. ABCG14, an ATP-binding cassette transporter, is required for this translocation (Ko et al., 2014; Zhang et al., 2014). Root-derivedt Z-type CKs are essential for shoot development (Kiba et al., 2013) and t Z and t ZR have distinct functions in the shoot apical meristem (SAM) and the development of leaves (Osugi et al., 2017).
Bioactive CKs activate the CK signaling cascade (Kieber & Schaller, 2014; Werner & Schmülling, 2009) by binding to ARABIDOPSIS HISTIDINE KINASE (AHK) receptors, of which Arabidopsis possesses three (AHK2, AHK3 and CYTOKININ RESPONSE1 (CRE1)/AHK4 (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi, Sato, Kato, & Tabata, 2001; Yamada et al., 2001). Activated receptors autophosphorylate and then transfer the phosphoryl residue to AHPs (AHP1 - AHP5) (Hutchison et al., 2006). These activate type-B ARRs, which are transcription factors regulating CK-dependent gene expression (Mason, Li, Mathews, Kieber, & Schaller, 2004; Mason et al., 2005). In most cases type-B ARRs act as positive regulators of CK signaling, but one study suggested that gene regulation by type-B ARRs might be more complex (Mason et al., 2005).
The study of Nitschke et al. (2016) has shown that CK protects plants against photoperiod stress by mainly acting through the receptor AHK3 and the type-B response regulator ARR2. Further, a functional relevance of ARR10 and ARR12 as positive regulators of stress resistance was reported (Nitschke et al., 2016). However, the role of different CKs in photoperiod stress protection, the involvement of AHPs and the relationship between the different B-type ARRs has not been studied. Here, we provide evidence that plants increase their CK concentration in response to photoperiod stress and that root-derived t Z-type CKs protect against photoperiod stress requiring the action of AHP2, AHP3 and AHP5. The study of different type-B arr mutant combinations showed that ARR2, ARR10 and ARR12 together regulate the resistance to photoperiod stress.