Cytokinin at the Crossroads of Abiotic Stress Signalling Pathways

Cytokinin is a multifaceted plant hormone that plays major roles not only in diverse plant growth and development processes, but also stress responses. We summarize knowledge of the roles of its metabolism, transport, and signalling in responses to changes in levels of both macronutrients (nitrogen, phosphorus, potassium, sulphur) and micronutrients (boron, iron, silicon, selenium). We comment on cytokinin’s effects on plants’ xenobiotic resistance, and its interactions with light, temperature, drought, and salinity signals. Further, we have compiled a list of abiotic stress-related genes and demonstrate that their expression patterns overlap with those of cytokinin metabolism and signalling genes.


Introduction
As sessile organisms, plants have evolved elaborate mechanisms that enable them to sense and respond to changes in environmental conditions, and are thus crucial for their adaptation and survival. These mechanisms involve abiotic stimuli triggering wide arrays of local and long-distance signals initiating developmental processes and stress responses that are regulated and coordinated by common integrative pathways. One of the key transmitted signals is cytokinin: a multifaceted plant hormone that plays major roles in diverse plant growth and development processes. Cytokinin signalling cascades are evolutionarily related to the two-component systems in unicellular organisms that participate in transduction of signals that are triggered by various environmental stimuli, for example, changes in temperature, nutrient levels, chemoattractants, or osmotic conditions [1][2][3]. In contrast to ethylene, another phytohormone that is involved in a two-component signalling pathway, cytokinin was not traditionally considered part of the primary stress response machinery. However, more recently, cytokinin crosstalk with ethylene and other so-called "stress hormones" (jasmonate, salicylic acid and abscisic acid) has been recognized (e.g., [4]), and current evidence indicates that it could be a primary perceptor in temperature or nutrient sensing. In the following text, we present an overview of cytokinin crosstalk with abiotic stimuli, as outlined in Figure 1. The presented evidence includes findings from compilation of a list of abiotic stress-related genes and analyses showing that their expression patterns overlap with those of cytokinin metabolism and signalling Figure 1. Crosstalk between abiotic stress signals and cytokinin. Summary of interactive points between cytokinin metabolism and signalling pathways (as currently modelled in Arabidopsis [5]) and abiotic stress response pathways. See corresponding sections of the text for details and references.

Nutrient Stress
Plants require a number of elements for their growth and development. Besides carbon, hydrogen, and oxygen, which are primarily obtained from carbon dioxide and water, plants actively take up at least 20 elements. These include both macronutrients (nitrogen, phosphorus, sulphur, potassium), and micronutrients (including boron, iron, silicon and selenium). As discussed in the following text, cytokinin plays a pivotal role in plants' uptake of these nutrients, and their responses to toxic metal(loid)s, including cadmium, aluminium, and arsenite.

Nitrogen
Nitrogen is one of the most strongly growth-limiting nutrients for plants. Thus, their internal nitrogen status and both the availability and distribution of nitrogen in their growth media are sensed by a complex network of signalling pathways that generate and regulate integrated responses to local and long-distance signals, including several phytohormones [6][7][8][9]. A well-known connection between Figure 2. Similarity of expression patterns of genes related to nutrient stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33].

Figure 2.
Similarity of expression patterns of genes related to nutrient stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33].  Figure 3. Similarity of expression patterns of genes related to xenobiotic stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33]. . Similarity of expression patterns of genes related to temperature/drought stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33]. . Similarity of expression patterns of genes related to xenobiotic stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33].  Figure 3. Similarity of expression patterns of genes related to xenobiotic stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33]. . Similarity of expression patterns of genes related to temperature/drought stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33]. . Similarity of expression patterns of genes related to temperature/drought stress and genes involved in cytokinin metabolism or signalling. Asterisks indicate profile similarities >85%. The heatmap was generated using R software and available data from Araport [33].

Phosphorus
Like nitrogen sensing, a complex signalling system is required to maintain inorganic phosphate (Pi) homeostasis, and plants' responses to Pi-limiting conditions involve multiple phytohormones [34,35]. As in nitrate sensing, one of the strongly affected cytokinin genes is the biosynthetic gene IPT3. Pi shortage causes the downregulation of IPT3 [36] and cytokinin signalling components, including the cytokinin receptor AHK4 [37]. Conversely, the resupply of Pi after a shortage causes upregulation of IPT3, CRF5, and CRF6 [38]. Moreover, reductions in root cytokinin levels upregulate the expression of Pi transporters [39][40][41][42] and the exogenous supply of cytokinin can suppress Pi uptake and Pi starvation responses in Arabidopsis and rice [37,[43][44][45][46][47][48], presumably by mobilizing Pi from internal sources (preferentially stores in shoot tissues) [46]. This may temporarily reduce Pi starvation signalling and contribute to the relief of Pi deficiency symptoms, including the reported moderation of shoot growth inhibition of Pi-starved plants in the presence of cytokinin [49]. It has been proposed that the level of cell-cycle activity governs the magnitude of Pi demand in Pi-starved plants. This would fit well with cytokinin's opposite effects on cell cycling in shoot and root meristems, where it, respectively, stimulates and represses cell division [50]. Further, auxin-cytokinin crosstalk via the auxin responsive factor OsARF16 regulates Pi signalling, and transport of Pi from roots to shoots [47].

Potassium
Potassium is the most abundant inorganic cation in plants, and it is one of the primary macronutrients that are generally added (together with nitrogen and phosphorus) to soil in fertilizers. Analysis of Arabidopsis plants has shown that potassium deprivation reduces cytokinin contents, and cytokinin signalling regulates root growth inhibition and potassium uptake [51]. The cited authors also found that cytokinin-deficient plants have enhanced the tolerance of potassium deficiency, which they attributed to the stimulation of ROS accumulation, root hair growth, and expression of HAK5, which encodes a potassium uptake transporter. This transporter connects multiple phytohormonal networks, as it is also regulated by ethylene [52] and participates in the modulation of the auxin transporter PIN1's localization [53]. We found no significant similarity between expression patterns of HAK5 and any cytokinin metabolism/signalling genes. However, expression patterns of genes encoding seven potassium-deficiency-related genes (four potassium transporters, two antiporters, and a potassium channel) showed ≥85% similarity to those of candidate cytokinin genes (Table 1, Figure 2).

Sulphur
The availability of sulphur in soil is directly associated with crop yields and quality, and sulphur deficiency induces a number of adaptive responses [54]. A link between sulphur deficiency and responses in cytokinin status is indicated by IPT3 downregulation in roots of Arabidopsis plants grown on sulphur-deficient media [36], and observed changes in cytokinin contents triggered by sulphur deficiency in poplar [55]. In addition, exogenous application of cytokinin upregulates expression of sulphur-responsive genes in leaves [36]. By contrast, cytokinin downregulates the root expression of sulphate transporters (SULTR1;1 and SULTR1;2) that are involved in sulphate acquisition from the soil [39,56], and the arr1,10,12 triple mutant displays sulphur-deficiency-like gene expression patterns [57]. Thus, complex interplay between cytokinin and sulphur signalling, which is possibly mediated by independent regulatory circuits, is likely involved. The sulphur-deficiency marker gene GGCT2;1 encodes a key enzyme of glutathione degradation and it is a highly cytokinin-responsive gene [58], suggesting that cytokinin may participate in glutathione homeostasis and cytokinin-mediated glutathione decomposition may play a physiologically important role in nutrient mobilization.

Boron
Boron is an essential micronutrient for the growth of higher plants but there is a very narrow range between deficient and toxic concentrations [59]. Symptoms of severe boron deficiency include root growth inhibition, perturbances in root morphology, and reductions in vegetative and reproductive growth. Early detectable changes in boron-deficient plants include disturbances of hormonal metabolism and several lines of evidence suggest that ethylene and auxin are involved in the regulation of boron stress responses [60]. Boron deprivation induces the downregulation of cytokinin signalling genes [61,62], and our meta-analysis showed that BOR4, encoding a boron transporter, has a similar expression pattern to ARR1 and the cytokinin metabolism gene LOG7. Moreover, in oilseed rape, the shoot boron concentration reportedly correlates closely with cytokinin content, and boron enhances both cytokinin synthesis and the conversion of weakly active cytokinins to highly active forms [63]. Conversely, recent analysis indicates that boron deficiency inhibits root meristem growth via a molecular mechanism involving the cytokinin-mediated repression of cyclin CYCD3 [64].

Iron
Cytokinin suppresses expression of several genes that respond to iron deficiency in Arabidopsis [65]. This cytokinin-induced repression is mediated via AHK3 and AHK4 receptors, and it targets genes encoding components of the iron-uptake machinery (FRO2, IRT1) and the iron-deficiency induced transcription factor FIT1. The repression does not reflect the plant's iron nutritional status, and analysis of a fit1 loss-of-function mutant indicates that it acts via a distinct, FIT1-independent signalling pathway [65]. This could be mediated by the ARF16 transcription factor, which is required for iron deficiency responses in rice [66] and participates in the auxin-cytokinin control of phosphate homeostasis [47]. Only five of 38 selected genes that are related to iron-deficiency had similar expression profiles to cytokinin regulatory genes. Moreover, the expression pattern of NRAMP4 (encoding a transporter of iron and several other metals) is similar to that of CKX (encoding a cytokinin degradation enzyme) and ARR1, but NRAMP4 expression is not reportedly upregulated by exogenous application of cytokinin [65]. Thus, this coregulation is unlikely to reflect iron status signalling.

Silicon
Silica minerals are major soil components, and high silicon uptake, boosted by root silicon transporters, promotes plants' tolerance to many biotic and abiotic stresses. Mineralized (insoluble) silica provides structural support for many plants, but it can also enhance various defence mechanisms of plants and influence their stress responses by modulating their hormonal balance [67,68]. The beneficial effects of silicon are partially mediated by cytokinin [69]. Inter alia, silicic acid induces the cytokinin synthesis gene IPT7 and silicon accumulation delays dark-induced leaf senescence through the activation of cytokinin pathways in sorghum and Arabidopsis [70].

Selenium
At low concentrations, selenium promotes plant growth and stress resistance [71,72], but elevated levels can be toxic. Selenate and selenite, the two major forms of selenium that are found in the environment, are readily absorbed by plants via sulphate and phosphate transporters, respectively [73]. In this respect, cytokinin-regulated sulphate and Pi pathways might form a point of cross-talk between selenium and cytokinin signalling. For instance, the cytokinin-responsive sulphate transporter SULTR1;2 is a determinant of selenium tolerance in Arabidopsis [74]. Cytokinin signalling is promoted in the root tip of selenite-exposed Arabidopsis plants, and high cytokinin levels reportedly improve the performance of selenite-exposed roots, whereas reductions in cytokinin status or sensitivity enhance selenite sensitivity [75][76][77]. Recently, a selenium-tolerant Arabidopsis mutant with a loss-of-function mutation in a terpenoid synthase gene (TPS22) has been described. Observed effects of the mutation include reductions in cytokinin levels and the expression of cytokinin receptors AHK3 and AHK4, while the application of exogenous cytokinin upregulated selenocysteine methyltransferase (as well as high-affinity phosphate transporters) and decreased selenium tolerance of the mutant [42].

Xenobiotics
Strict control of processes that are involved in plants' absorption, translocation, and storage of essential metals is crucial for the maintenance of their concentrations within physiological ranges and the avoidance of toxicity. Nevertheless, despite the transport systems' selectivity, they may also take up toxic, non-essential metals and metalloids, such as arsenic, cadmium, chromium, lead, and mercury. Responses to these toxic xenobiotics, including cadmium [78] and aluminium [79], involve increases in cytokinin biosynthesis and signalling that inhibit root growth. Accordingly, application of substances that reduce active cytokinin contents or signalling can mitigate the adverse effects of cadmium [80]. Similarly, the cytokinin signalling component CRF6 is induced by organic xenobiotics, including the herbicide atrazine [81,82] and atrazine inhibition is weaker in the crf6 insertional mutant line than in wild-type plants [83]. Moreover, cytokinin-deficient plants grown in cadmium-contaminated soil reportedly accumulate more cadmium [39] and display enhanced arsenate tolerance [41], which is likely due to higher levels of thiol compounds [41]. Cytokinin also induces the upregulation of glutathione-S-transferase GSTU26 [84,85] and may thus play a role in glutathione conjugation.

Cytokinin Roles in Drought and Salinity Tolerance
Drought and salinity stress are the most frequent abiotic stresses and both impair crop production on a global scale [86]. Analysis of natural variants of Arabidopsis has shown that even mild drought can adversely affect plants if they are not evolutionarily adapted to it [87]. Plants react to water-limiting conditions by reducing their cytokinin levels, mainly through the modulation of cytokinin metabolism-as shown (inter alia) in Arabidopsis, creeping bentgrass, soybean, tobacco, and sunflower [88][89][90][91][92][93]-and/or the regulation of cytokinin receptors' expression [94,95]. However, other mechanisms, including activation of the negative regulators of cytokinin signalling AHP6 and ARR5 also probably participate in this process [94,96,97]. Appropriate modulation of cytokinin metabolism and signalling has been known to improve drought and salt tolerance for many years [92,95,98,99], and at least five mechanisms may contribute to cytokinin-mediated enhancement of tolerance of water deficiency. These are: protection of the photosynthetic machinery, enhancement of antioxidant systems, improvement in water balance regulation, modulation of plant growth and differentiation, and modulation of activities of stress-related phytohormones.

Cytokinin Modulates Photosynthesis under Water-Limiting Conditions and Salt Stress
Changes in cytokinin status (mainly increases in cytokinin levels) reportedly enhance photosynthesis and related processes under water-deficiency or salt stress in many plant species [99][100][101][102][103][104], by increasing the expression of genes that are involved in photosynthesis, chlorophyll levels, photochemical efficiency, photochemical quenching, electron transport rates, and/or CO 2 assimilation. Accordingly, in transgenic barley plants ectopically expressing the cytokinin-degradation enzyme AtCKX1, reductions in CO 2 assimilation rates, accompanied by lower stomatal conductance, have been recorded [105]. Conversely, increases in CO 2 assimilation have been observed in barley lines overexpressing CKX under a different promoter resulting in localization to different compartments. However, the cited authors only presented results from plants with elevated concentrations of tZ-type cytokinins [106]. It has been previously demonstrated that CKX overexpression stimulates cytokinin biosynthesis [107], so the observed positive effect on CO 2 assimilation was likely due to increases in cytokinin content.

Cytokinin Enhances Capacities of Antioxidant Systems
Ectopic expression of ipt reportedly increases the capacities of plants' antioxidant systems, including levels of antioxidants during severe drought stress [100]. This could protect their cells from excessive stress-induced ROS accumulation, thereby preserving chloroplast integrity [100,108,109] and reducing electrolyte leakage and/or rises in malondialdehyde levels [57,89,110]. On the other hand, ectopic expression of CKX in barley has been found to activate genes putatively involved in flavonoid biosynthesis [105] and flavonoids also participate in drought tolerance [111]. These effects of cytokinin in drought stress tolerance could involve indirect priming of antioxidant systems in response to manipulation of cytokinin homeostasis. In accordance with this hypothesis, significant enhancement of cytokinin biosynthesis can induce hypersensitivity-like responses and ROS-mediated cell death [112].

Cytokinin Influences Water Balance Regulation
Clearly, water management is crucial for drought tolerance, and plants with low levels of cytokinin or weak cytokinin signalling generally have higher water contents during drought stress than counterparts with higher cytokinin contents or stronger signalling [92,95,105]. This could be due to better root systems, since cytokinin is a known negative regulator of root growth and lateral root formation [106,110]. The improved water uptake in these plants is clearly complemented with reductions in transpiration rates and stomatal apertures, which could protect them from severe water losses during stress periods [57,105,110]. Ectopic expression of ipt also reduces water losses in plants that are exposed to drought, even when they have higher transpiration rates and stomatal conductance, but the mechanisms that are involved are elusive [90,102].

Cytokinin Effects on Growth
As cytokinins play key roles in root and shoot development they also participate in expression of growth and architectural traits that are required for tolerance of water-limiting conditions [113]. Cytokinins are well known to reduce root to shoot hypocotyl ratios [39,114,115], and one of the approaches for enhancing plants' drought tolerance is to decrease cytokinin levels in order to modify root morphology and enhance root biomass [116]. Root-specific overexpression of CKX can also enhance root growth, nutrient uptake, and drought tolerance [106], as well as improving recovery after drought stress [116] without adverse effects on shoot growth. Similarly, one of the dehydration-responsive element binding factors in Malus (MdDREB6.2) activates the expression of MdCKX, mainly in roots, and overexpression of this factor can enhance drought tolerance [110]. Several studies indicate that not only quantitative features but also qualitative traits of root tissues could be important factors in cytokinin-regulated responses to water-limiting conditions, including the differentiation of vascular tissue [117] and lignification [116].

Abscisic Acid
Rapid accumulation of the phytohormone abscisic acid plays a crucial role in regulating plants' defensive responses to drought stress, including stomatal closure, growth modulation, and synthesis of protective metabolites. It has been known for more than a decade that cytokinin and abscisic acid have antagonistic functions in diverse physiological processes, including stress tolerance, germination, and hypocotyl greening [95,118,119]. Cytokinin signalling has been shown to be dramatically inhibited by abscisic acid application [120,121] and cytokinin facilitates degradation of the abscisic acid signalling component transcription factor ABI5 [118]. It has been reported that under drought stress plants with decreased levels of cytokinin or attenuated cytokinin signalling have decreased levels of abscisic acid, but higher sensitivity to this stress-related hormone and greater drought tolerance [57,92,122]. However, elucidation of the molecular mechanism involved in this interaction has begun only recently. Experiments with a series of cytokinin and abscisic acid signalling mutants have demonstrated that cytokinin and abscisic acid interact directly through their signalling components, as plants constitutively expressing HA-Flag-ARR5 and arr5 loss-of-function mutants respectively showed increased and attenuated sensitivity to abscisic acid treatment [97]. ARR5 stability is promoted by phosphorylation catalysed by SnRK2 protein kinases that are key components of the abscisic acid signalling pathway. In contrast, type-B ARR1, 11, and 12 interact with these SnRK2s and repress their kinase activity, and the abscisic acid hypersensitivity of the triple mutant arr1,11,12 can be completely rescued by mutation of SnRK2s [97]. Interestingly, the same authors found that expression of ARR1∆DDK (a constitutively activated form of ARR1), but not constitutive expression of ARR1-Myc, was associated with slight insensitivity to abscisic acid, suggesting that the modulation of ARR1's phosphorylation status by cytokinin signalling may also be important. Since cytokinin is essential for normal growth of plants [10] the SnRK2-ARR regulatory module is clearly a recently discovered signalling hub that balances growth and defence in response to environmental cues.

Jasmonates
Jasmonic acid is known to play a role in drought tolerance [123,124]. Inter alia, drought-induced xylem differentiation is negatively and positively regulated by cytokinin and jasmonic acid, respectively, and jasmonic acid attenuates cytokinin signalling by repressing the cytokinin receptor AHK4 and stimulating expression of AHP6, a negative regulator of cytokinin signalling [117]. In addition, cytokinin may influence jasmonate metabolism. Ectopic expression of AtCKX1 in barley plants has been found to induce expression of lipoxygenases, which participate in the release of volatile compounds, including jasmonates [105], but an increase in jasmonic acid has also been observed in tobacco plants with highly increased levels of cytokinin [112].

Temperature and Cytokinin
Temperature is one of the most important abiotic factors influencing plants' growth, development, productivity, and yields. Plants can only grow within taxa-specific temperature ranges, thus suboptimal temperatures cause stress, and temperature limits their geographical distributions. The mechanisms that are involved in temperature perception and signalling in plants are far from completely understood, but key aspects of associated morphological changes are clearly mediated by phytohormones.

Low Temperature Stress: Cold and Freezing
Most reported responses of cytokinin metabolism or signalling systems to low temperatures in plants are repressive [94,[125][126][127][128], but there are some documented exceptions, notably cold-mediated upregulation of AHK3 [95]. However, the responses are complex, and roles of cytokinin and cytokinin signalling pathways in cold tolerance are unclear. Cold-induced attenuation of cytokinin signalling seems to impair plants' tolerance of low temperature because the exogenous application of cytokinin significantly promotes cold tolerance in Arabidopsis [129][130][131]. Accordingly, recent hormonal analysis of Zoysia grass has shown that a genotype from relatively high latitude retained higher cytokinin levels during low-temperature treatment and exhibited higher freezing tolerance than a genotype from a lower latitude [128]. However, this seems to conflict with a reported negative role of AHK cytokinin receptors in cold tolerance [129]. The mechanisms whereby cytokinin could both promote cold tolerance and activate negative regulators of cold stress responses is unclear, but it seems to be at least partially independent of the cold-induced CBF/DREBs regulatory system [129].
Cold has also been shown to transiently activate expression of type-A ARRs in a cytokinin-and ethylene-dependent manner [129,130]. Mutation of ARR5, ARR6, and ARR7 leads to higher freezing tolerance [129], but the overexpression of ARR7 reportedly has both negative [129] and positive effects [130]. Results of overexpression studies indicate that other type-A ARRs [130] and ARR22, a cold-inducible type-C ARR [131], may also play positive roles in freezing tolerance. As shown by these conflicting results, the molecular mechanisms involved are unclear and further research is required. Besides ARRs, cytokinin response factors (CRFs) that act downstream of the primary cytokinin signalling pathway participate in responses to low temperature. More specifically, CRFs are induced by cold in Arabidopsis and tomato [130,132], and detailed analysis of Arabidopsis overexpressors and mutants has shown that CRF4 mediates freezing tolerance in non-acclimated plants [132], while CRF2 and CRF3 regulate lateral root development in response to cold stress [133]. Our meta-analysis revealed two novel candidates for interactive points in cytokinin-cold stress crosstalk: the MAP kinase MKK2, and the component of the ubiquitin-proteasome pathway, HOS1. The gene encoding HOS1 has a similar expression pattern to four and five genes that are involved in cytokinin metabolism and signalling, respectively (Figure 4). Cytokinin and its signalling evidently play important roles in cold stress responses, but various aspects of the molecular mechanism of their action regarding (for example) the duration of the period of cytokinin modulation prior to the stress require clarification

High Temperature and Heat Stress
Observed responses of Arabidopsis to heat stress treatments include a rapid but transient increase in active cytokinin contents [134,135]. A rapid proteomic heat-shock response that could be mimicked to some extent by cytokinin treatment at standard temperature has also been reported [136], indicating that cytokinin may play a role in temperature perception. Moreover, the accumulation of cytokinin has been observed in Pinus radiata under prolonged heat stress and in recovered plants [137,138]. Plants with increased levels of cytokinins show a higher accumulation of heat-shock proteins [107,139,140] and enhanced activity of the antioxidant system [88,112]. Accordingly, transgenic lines with inactivated components of cytokinin signalling pathways or reductions in pools of active cytokinin have displayed increased tolerance to high temperatures [141,142]. Further, analyses of temperature-induced hypocotyl growth in cytokinin-deficient transgenic plants and cytokinin receptor ahk double mutants have shown that impairment of the cytokinin pathway strongly inhibits growth at high temperatures [136]. This indicates that cytokinin could serve as a signal for thermomorphogenesis. It is also likely that a higher temperature sensitizes cytokinin signalling, which could explain why a transient increase in the active cytokinin pool is followed by its significant depletion [134,135], and downregulation in cytokinin metabolism genes and the expression of ARR-type A orthologs in strawberry (Fragaria vesca) [143,144]. It has also been proposed that heat stress-induced cytokinin depletion can promote stomatal closure, as this process is inhibited in plants with increased cytokinin levels [135].

Light Signalling and the Circadian Clock Interact with Cytokinin
As described in a recent review [145], soon after its discovery it was found that cytokinin promotes chlorophyll synthesis and chloroplast development. There is increasing evidence of direct interactions between cytokinin and light via the light photoreceptor phyB [146,147]. Moreover, cytokinin-mediated development in Arabidopsis is modulated by the expression of the sensor histidine kinase CKI1 (Cytokinin Independent-1), which is regulated by phyA (and thus light) via the phyA interacting factor (PIF3) and Circadian Clock Associated 1 (CCA1) [148]. Further, levels of the cytokinin pool in tobacco leaves vary diurnally, with the main peak occurring around midday [149], and a key component of the circadian clock in plants, Late Elongated Hypocotyl (LHY), modulates cytokinin levels in Populus trees [150]. Reductions in cytokinin status or sensitivity enhance circadian stress in Arabidopsis and cytokinin-deficient plants display a highly similar expression of clock output genes to that of clock mutants [151]. Light conditions may also influence contents of specific cytokinins, as recently demonstrated in detached leaf experiments [152]. Moreover, light-cytokinin interactions are not limited to cytokinin metabolism and components of the two-component cytokinin responsive pathway. They also influence a bZIP transcription factor, Elongated Hypocotyl 5 (HY5), which participates in photomorphogenesis [94,153]. Cytokinin is also apparently involved in photoprotection mechanisms as plants with deficiencies in cytokinin receptors or cytokinin signalling are more susceptible to light stress than wild-type counterparts [154,155]. To identify new candidate participants in light-cytokinin interactions, we subjected Arabidopsis transcriptomic expression profiles to association analysis. Data were collected from the publicly available database Thalemine (available online: http://apps.araport.org/thalemine/), then normalized, and degrees of similarity between expression patterns were visualized in a heatmap ( Figure 5). In total, we tested similarities in expression profiles of 70 candidate cytokinin genes and 31 genes that were putatively involved in light signalling. The analysis revealed that 10 of the latter had similar expression patterns (>70%) to candidate cytokinin genes. The results confirmed the previously described relation between ARR and COP1, but also highlighted several novel putative interactions, including a connection between the UV-B receptor UVR8 and the AHP2 component of cytokinin signalling. This is consistent with a recent finding that cytokinin regulates UV-B-induced damage in tomato seedlings [156].
receptors or cytokinin signalling are more susceptible to light stress than wild-type counterparts [154,155]. To identify new candidate participants in light-cytokinin interactions, we subjected Arabidopsis transcriptomic expression profiles to association analysis. Data were collected from the publicly available database Thalemine (available online: http://apps.araport.org/thalemine/), then normalized, and degrees of similarity between expression patterns were visualized in a heatmap ( Figure 5). In total, we tested similarities in expression profiles of 70 candidate cytokinin genes and 31 genes that were putatively involved in light signalling. The analysis revealed that 10 of the latter had similar expression patterns (>70%) to candidate cytokinin genes. The results confirmed the previously described relation between ARR and COP1, but also highlighted several novel putative interactions, including a connection between the UV-B receptor UVR8 and the AHP2 component of cytokinin signalling. This is consistent with a recent finding that cytokinin regulates UV-B-induced damage in tomato seedlings [156].

Figure 5.
Heatmap showing degrees of similarity between expression profiles of cytokinin (signalling and metabolism) genes and genes involved in light perception. The heatmap was generated using R software and data available from Araport [33]. Asterisks indicate >70% similarity. Heatmap showing degrees of similarity between expression profiles of cytokinin (signalling and metabolism) genes and genes involved in light perception. The heatmap was generated using R software and data available from Araport [33]. Asterisks indicate >70% similarity. Table 1. List of all abiotic stress-related genes, with references, and their putative interactions with cytokinin according to the expression profile analysis outlined in Figures 2-4. Genes in bold indicate profile similarities >85% to cytokinin-related genes, and numbers indicate the number of detected co-expressed cytokinin signalling/metabolism genes.  [182,193,194] F17A17.6 AT3G07720 AT3g07720/F17A17_6 [182,193,194]

Summary
Generally, it can be concluded that cytokinin metabolism and signalling play important roles in abiotic stress tolerance and the manipulation of these processes in crops could be beneficial for sustainable agriculture. However, recent studies have mainly focused on global transcriptomic, proteomic and metabolomic changes in various plant species with modulated cytokinin levels [105,122,250]. Thus, further detailed analysis is required to confirm the importance of identified candidate genes/proteins and validate their roles in stress tolerance. Moreover, current models have substantial gaps. There is mounting evidence of intensive crosstalk in phytohormonal signalling, including redox and proteasome-ubiquitin pathways [251]. Thus, any disruption in a single phytohormone signalling pathway will probably affect the whole hormonome, but current limits in the sensitivity and spatiotemporal scope of analyses constrain our ability to detect all of the changes. Ongoing advances in hormonome analyses will undoubtedly improve our understanding [252], but another limitation is that most presented findings are based solely on transcriptomic analyses, in some cases supplemented with results of knocking out or overexpressing specific genes. Furthermore, posttranslational modifications play important roles in regulatory networks [253], and thus abiotic responses [254]. Thus, they must also be considered. Similarly, to fully understand phytohormonal interactions in abiotic stress responses, it will be crucial to integrate protein-protein interactions and the associated signalling hubs and networks [255].

Conflicts of Interest:
The authors declare no conflict of interest.