Plant hormones cytokinins regulate various aspects of plant growth and development. For their positive effects on branching, delaying of senescence, nutrient remobilisation, flower and seed set control they became interesting substances in search for potential agrochemicals. From the 1970' of the last century exogenous application of cytokinins have been tested in field conditions to improve yield traits of world-wide important crops such as wheat, rice, maize, barley, and soybean. Despite the extensive testing summarized in this work, so far cytokinins haven't found their stable place among commercialized plant growth regulators, mainly due to the complexity of their effects. Here we bring an overview of the outcomes obtained in pot and field experiments using cytokinin exogenous treatments, summarize the ways of application and point to the affected traits in various field crops, vegetables, cotton and fruit trees. Further, we present here outcomes of field trials performed with a derivative of N(6)-benzyladenine, 2-chloro-6-(3-methoxybenzyl)aminopurine, in spring barley and winter wheat. The effect on yield forming traits such as number of tillers, grains per ear, number of ears and the final yield was evaluated and compared after spraying of the both crops in different phenological stages.

Plant cells are connected by plasmodesmata (PD), nanoscopic channels in cell walls that allow diverse cytosolic molecules to move between neighboring cells. PD transport is tightly coordinated with physiology and development, although the range of signaling pathways that influence PD transport has not been comprehensively defined. Several plant hormones, including salicylic acid (SA) and auxin, are known to regulate PD transport, but the effects of other hormones have not been established. In this study, we provide evidence that cytokinins promote PD transport in leaves. Using a green fluorescent protein (GFP) movement assay in the epidermis of Nicotiana benthamiana, we have shown that PD transport significantly increases when leaves are supplied with exogenous cytokinins at physiologically relevant concentrations or when a positive regulator of cytokinin responses, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 5 (AHP5), is overexpressed. We then demonstrated that silencing cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE 3 (AHK3) or AHK4 or overexpressing a negative regulator of cytokinin signaling, AAHP6, significantly decreases PD transport. These results are supported by transcriptomic analysis of mutants with increased PD transport (ise1-4), which show signs of enhanced cytokinin signaling. We concluded that cytokinins contribute to dynamic changes in PD transport in plants, which will have implications in several aspects of plant biology, including meristem patterning and development, regulation of the sink-to-source transition, and phytohormone crosstalk.

Cytokinins (CKs), primarily trans-zeatin (tZ) and isopentenyladenine (iP) types, play critical roles in plant growth, development, and various stress responses. Long-distance transport of tZ-type CKs meidated by Arabidopsis ATP-binding cassette transporter subfamily G14 (AtABCG14) has been well studied; however, less is known about the biochemical properties of AtABCG14 and its transporter activity toward iP-type CKs. Here we reveal the biochemical properties of AtABCG14 and provide evidence that it is also required for long-distance transport of iP-type CKs. AtABCG14 formed homodimers in human (Homo sapiens) HEK293T, tobacco (Nicotiana tabacum), and Arabidopsis cells. Transporter activity assays of AtABCG14 in Arabidopsis, tobacco, and yeast (Saccharomyces cerevisiae) showed that AtABCG14 may directly transport multiple CKs, including iP- and tZ-type species. AtABCG14 expression was induced by iP in a tZ-type CK-deficient double mutant (cypDM) of CYP735A1 and CYP735A2. The atabcg14 cypDM triple mutant exhibited stronger CK-deficiency phenotypes than cypDM. Hormone profiling, reciprocal grafting, and 2H6-iP isotope tracer experiments showed that root-to-shoot and shoot-to-root long-distance transport of iP-type CKs were suppressed in atabcg14 cypDM and atabcg14. These results suggest that AtABCG14 participates in three steps of the circular long-distance transport of iP-type CKs: xylem loading in the root for shootward transport, phloem unloading in the shoot for shoot distribution, and phloem unloading in the root for root distribution. We found that AtABCG14 displays transporter activity toward multiple CK species and revealed its versatile roles in circular long-distance transport of iP-type CKs. These findings provide new insights into the transport mechanisms of CKs and other plant hormones.

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.

Cytokinins (CKs) are a class of compounds that have long been thought to be exclusively plant growth regulators. Interestingly, some species of phytopathogenic bacteria and fungi have been shown to, and gall-inducing insects have been hypothesized to, produce CKs and use them to manipulate their host plants. We used high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-MS/MS) to examine concentrations of a wide range of CKs in 17 species of phytophagous insects, including gall- and non-gall-inducing species from all six orders of Insecta that contain species known to induce galls: Thysanoptera, Hemiptera, Lepidoptera, Coleoptera, Diptera, and Hymenoptera. We found CKs in all six orders of insects, and they were not associated exclusively with gall-inducing species. We detected 24 different CK analytes, varying in their chemical structure and biological activity. Isoprenoid precursor nucleotide and riboside forms of trans-zeatin (tZ) and isopentenyladenine (iP) were most abundant and widespread across the surveyed insect species. Notably, the observed concentrations of CKs often markedly exceeded those reported in plants suggesting that insects are synthesizing CKs rather than obtaining them from the host plant via tissue consumption, compound sequestration, and bioaccumulation. These findings support insect-derived CKs as means for gall-inducing insects to manipulate their host plant to facilitate cell proliferation, and for both gall- and non-gall-inducing insects to modify nutrient flux and plant defenses during herbivory. Furthermore, wide distribution of CKs across phytophagous insects, including non-gall-inducing species, suggests that insect-borne CKs could be involved in manipulation of source-sink mechanisms of nutrient allocation to sustain the feeding site and altering plant defensive responses, rather than solely gall induction. Given the absence of any evidence for genes in the de novo CK biosynthesis pathway in insects, we postulate that the tRNA-ipt pathway is responsible for CK production. However, the unusually high concentrations of CKs in insects, and the tendency toward dominance of their CK profiles by tZ and iP suggest that the tRNA-ipt pathway functions differently and substantially more efficiently in insects than in plants.

Cytokinins (CKs) are thought to play important roles in fruit development, especially cell division. However, the mechanisms and regulation of CK activity have not been well investigated. This study analysed CK concentrations and expression of genes involved in CK metabolism in developing tomato (Solanum lycopersicum) ovaries. The concentrations of CK ribosides and isopentenyladenine and the transcript levels of the CK biosynthetic genes SlIPT3, SlIPT4, SlLOG6, and SlLOG8 were high at anthesis and decreased immediately afterward. In contrast, trans-zeatin concentration and the transcript levels of the CK biosynthetic genes SlIPT1, SlIPT2, SlCYP735A1, SlCYP735A2, and SlLOG2 increased after anthesis. The expression of type-A response regulator genes was high in tomato ovaries from pre-anthesis to early post-anthesis stages. These results suggest that the CK signal transduction pathway is active in the cell division phase of fruit development. This study also investigated the effect of CK application on fruit set and development. Application of a synthetic CK, N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU), to unpollinated tomato ovaries induced parthenocarpic fruit development. The CPPU-induced parthenocarpic fruits were smaller than pollinated fruits, because of reduction of pericarp cell size rather than reduced cell number. Thus, CPPU-induced parthenocarpy was attributable to the promotion of cell division, not cell expansion. Overall, the results provide evidence that CKs are involved in cell division during development of tomato fruit.

The phenomenon of plant root tips sensing moisture gradient in soil and growing towards higher water potential is designated as root hydrotropism, which is critical for plants to survive when water is a limited factor. Molecular mechanisms regulating such a fundamental process, however, are largely unknown. Here we report our identification that cytokinins are key signaling molecules directing root growth orientation in a hydrostimulation (moisture gradient) condition. Lower water potential side of the root tip shows more cytokinin response relative to the higher water potential side. Consequently, two cytokinin downstream type-A response regulators, ARR16 and ARR17, were found to be up-regulated at the lower water potential side, causing increased cell division in the meristem zone, which allows the root to bend towards higher water potential side. Genetic analyses indicated that various cytokinin biosynthesis and signaling mutants, including the arr16 arr17 double mutant, are significantly less responsive to hydrostimulation. Consistently, treatments with chemical inhibitors interfering with either cytokinin biosynthesis or cell division completely abolished root hydrotropic response. Asymmetrically induced expression of ARR16 or ARR17 effectively led to root bending in both wild-type and miz1, a previously known hydrotropism-defective mutant. These data demonstrate that asymmetric cytokinin distribution is a primary determinant governing root hydrotropism.

STOP1 (sensitive to proton rhizotoxicity1) is a master transcription factor that governs the expression of a set of regulatory and structural genes involved in resistance to aluminum and low pH (i.e., proton) stresses in Arabidopsis. However, the mechanisms and regulatory networks underlying STOP1-mediated resistance to proton stresses are largely unclear. Here, we report that low-pH stresses severely inhibited root growth of the stop1 plants by suppressing root meristem activities. Interestingly, the stop1 plants were less sensitive to exogenous cytokinins at normal and low pHs than the wild type. Significantly, low concentrations of cytokinins promoted root growth of the stop1 mutant under low-pH stresses. Moreover, lateral and adventitious root formation was stimulated in stop1 and by low-pH stresses but suppressed by cytokinins. Further studies of the expression patterns of a cytokinin signaling reporter suggest that both the loss-of-function mutation of STOP1 and low-pH stresses suppressed cytokinin signaling outputs in the root. Furthermore, the expression of critical genes involved in cytokinin biosynthesis, biodegradation, and signaling is altered in the stop1 mutant in response to low-pH stresses. In conclusion, our results reveal a complex network of resistance to low-pH stresses, which involves coordinated actions of STOP1, cytokinins, and an additional low-pH-resistant mechanism for controlling root meristem activities and root growth upon proton stresses.

Cytokinins (CKs), a class of plant hormones, are central regulators of plant growth and development. Based on numerous physiological and genetic studies, the quantitative regulation of cytokinin levels is the major mechanism regulating cytokinin action in diverse developmental processes. Here, we identified a different mechanism with which the physiological function of CK is modulated through side-chain modification (trans-hydroxylation). The trans-hydroxylation that forms trans-zeatin (tZ)-type CK from N(6)-(Δ(2)-isopentenyl)adenine (iP)-type CK is catalyzed by the cytochrome P450 enzymes CYP735A1 and CYP735A2 in Arabidopsis. Deficiency in trans-hydroxylation activity results in dramatic retardation of shoot growth without affecting total CK quantity, while augmentation of the activity enhances shoot growth. Application of exogenous tZ but not iP recovers the wild-type phenotype in the mutants, indicating that trans-hydroxylation modifies the physiological function of CK. We propose that the control of cytokinin function by side-chain modification is crucial for shoot growth regulation in plants.

During primary growth, plant tissues increase their length, and as these tissues mature, they initiate secondary growth to increase thickness.1 It is not known what activates this transition to secondary growth. Cytokinins are key plant hormones regulating vascular development during both primary and secondary growth. During primary growth of Arabidopsis roots, cytokinins promote procambial cell proliferation2,3 and vascular patterning together with the hormone auxin.4-7 In the absence of cytokinins, secondary growth fails to initiate.8 Enhanced cytokinin levels, in turn, promote secondary growth.8,9 Despite the importance of cytokinins, little is known about the downstream signaling events in this process. Here, we show that cytokinins and a few downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors are rate-limiting components in activating and further promoting secondary growth in Arabidopsis roots. Cytokinins directly activate transcription of two homologous LBD genes, LBD3 and LBD4. Two other homologous LBDs, LBD1 and LBD11, are induced only after prolonged cytokinin treatment. Our genetic studies revealed a two-stage mechanism downstream of cytokinin signaling: while LBD3 and LBD4 regulate activation of secondary growth, LBD1, LBD3, LBD4, and LBD11 together promote further radial growth and maintenance of cambial stem cells. LBD overexpression promoted rapid cell growth followed by accelerated cell divisions, thus leading to enhanced secondary growth. Finally, we show that LBDs rapidly inhibit cytokinin signaling. Together, our data suggest that the cambium-promoting LBDs negatively feed back into cytokinin signaling to keep root secondary growth in balance.

Cytokinins play a relevant role in flower and fruit development and plant yield. Strawberry fruits have a high commercial value, although what is known as the "fruit" is not a "true" botanical fruit because it develops from a non-reproductive organ (receptacle) on which the true botanical fruits (achenes) are found. Given cytokinins' roles in botanical fruits, it is important to understand their participation in the development of a non-botanical or accessory "fruit". Therefore, in this work, the role of cytokinin in strawberry flowers and fruits was investigated by identifying and exploring the expression of homologous genes for different families that participate in the pathway, through publicly available genomic and expression data analyses. Next, trans-zeatin content in developing flowers and receptacles was determined. A high concentration was observed in flower buds and at anthesis and decreased as the fruit approached maturity. Moreover, the spatio-temporal expression pattern of selected CKX genes was evaluated and detected in receptacles at pre-anthesis stages. The results point to an important role and effect of cytokinins in flower and receptacle development, which is valuable both from a biological point of view and to improve yield and the quality of this fruit.

Cytokinins are phytohormones that are involved in many processes in plants, including growth, differentiation and leaf senescence. However, they also have various activities in animals. For example, kinetin and trans-zeatin can reduce levels of several aging markers in human fibroblasts. Kinetin can also protect mice against oxidative and glyoxidative stress, and prolong fruit flies' lifespan. Additionally, several cytokinins are currently used in cosmetics. To extend knowledge of the breadth of cytokinins' activities, we examined effects of natural cytokinin bases on the model nematode Caenorhabditis elegans. We found that kinetin, para-topolin and meta-topolin prolonged the lifespan of C. elegans. Kinetin also protected the organism against oxidative and heat stress. Furthermore, our results suggest that presence of reactive oxygen species, but not DAF-16 (the main effector of the insulin/insulin-like growth factor signaling pathway), is required for the beneficial effects of kinetin. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis showed that kinetin is unlikely to occur naturally in C. elegans, but the worm efficiently absorbs and metabolizes it into kinetin riboside and kinetin riboside-5'-monophosphate.

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

The regulation of photosynthetic acclimation to canopy density was investigated in tobacco canopies and in tobacco and Arabidopsis plants with part of their foliage experimentally shaded. Both species acclimated to canopy light gradients and partial shading by allocating photosynthetic capacity to leaves in high light and adjusting chloroplast organization to the local light conditions. An investigation was carried out to determine whether signalling mediated by photoreceptors, sugars, cytokinin, and nitrate is involved in and necessary for proper photosynthetic acclimation. No evidence was found for a role for sugars, or for nitrate. The distribution of cytokinins in tobacco stands of contrasting density could be explained in part by irradiance-dependent delivery of cytokinins through the transpiration stream. Functional studies using a comprehensive selection of Arabidopsis mutants and transgenics showed that normal wild-type responses to partial shading were retained when signalling mediated by photoreceptors or cytokinins was disrupted. This indicates that these pathways probably operate in a redundant manner. However, the reduction of the chlorophyll a/b ratio in response to local shade was completely absent in the Arabidopsis Ws-2 accession mutated in PHYTOCHROME D and in the triple phyAphyCphyD mutant. Moreover, cytokinin receptor mutants also showed a reduced response, suggesting a previously unrecognized function of phyD and cytokinins.

Cytokinins are one of the most important phytohormones and play essential roles in multiple life processes in planta. Root-derived cytokinins are transported to the shoots via long-distance transport. The mechanisms of long-distance transport of root-derived cytokinins remain to be demonstrated. In this study, we report that OsABCG18, a half-size ATP-binding cassette transporter from rice (Oryza sativa L.), is essential for the long-distance transport of root-derived cytokinins. OsABCG18 encodes a plasma membrane protein and is primarily expressed in the vascular tissues of the root, stem, and leaf midribs. Cytokinin profiling, as well as [14C]trans-zeatin tracer, and xylem sap assays, demonstrated that the shootward transport of root-derived cytokinins was significantly suppressed in the osabcg18 mutants. Transport assays in tobacco (Nicotiana benthamiana) indicated that OsABCG18 exhibited efflux transport activities for various substrates of cytokinins. While the mutation reduced root-derived cytokinins in the shoot and grain yield, overexpression of OsABCG18 significantly increased cytokinins in the shoot and improved grain yield. The findings for OsABCG18 as a transporter for long-distance transport of cytokinin provide new insights into the cytokinin transport mechanism and a novel strategy to increase cytokinins in the shoot and promote grain yield.

Following cell division, fruit growth is characterized by both expansion through increases in cell volume and biomass accumulation in cells. Fruit growth is limited by carbon starvation; however, the mechanism controlling fruit growth under restricted carbohydrate supply is poorly understood. In a previous study using red-fleshed kiwifruit, we showed that long-term carbon starvation had detrimental effects on carbohydrate, anthocyanin metabolism, and fruit growth. To elucidate the mechanisms underlying the reduction in fruit growth during kiwifruit development, we integrated phytohormone profiling with transcriptomic and developmental datasets for fruit under high or low carbohydrate supplies. Phytohormone profiling of the outer pericarp tissue of kiwifruit showed a 6-fold reduction in total cytokinin concentrations in carbon-starved fruit, whilst other hormones were less affected. Principal component analysis visualised that cytokinin composition was distinct between fruit at 16 weeks after mid bloom, based on their carbohydrate supply status. Cytokinin biosynthetic genes (IPT, CYP735A) were significantly downregulated under carbon starvation, in agreement with the metabolite data. Several genes that code for expansins, proteins involved in cell wall loosening, were also downregulated under carbon starvation. In contrast to other fleshy fruits, our results suggest that cytokinins not only promote cell division, but also drive fruit cell expansion and growth in kiwifruit.

Growth retardation and stress-induced premature plant senescence are accompanied by a severe yield reduction and raise a major agro-economic concern. To improve biomass and yield in agricultural crops under mild stress conditions, the survival must be changed to productivity mode. Our previous successful attempts to delay premature senescence and growth inhibition under abiotic stress conditions by autoregulation of cytokinins (CKs) levels constitute a generic technology toward the development of highly productive plants. Since this technology is based on the induction of CKs synthesis during the age-dependent senescence phase by a senescence-specific promoter (SARK), which is not necessarily regulated by abiotic stress conditions, we developed autoregulating transgenic plants expressing the IPT gene specifically under abiotic stress conditions. The Arabidopsis promoter of the stress-induced metallothionein gene (AtMT) was isolated, fused to the IPT gene and transformed into tobacco plants. The MT:IPT transgenic tobacco plants displayed comparable elevated biomass productivity and maintained growth under drought conditions. To decipher the role and the molecular mechanisms of CKs in reverting the survival transcriptional program to a sustainable plant growth program, we performed gene expression analysis of candidate stress-related genes and found unexpectedly clear downregulation in the CK-overproducing plants. We also investigated kinase activity after applying exogenous CKs to tobacco cell suspensions that were grown in salinity stress. In-gel kinase activity analysis demonstrated CK-dependent deactivation of several stress-related kinases including two of the MAPK components, SIPK and WIPK and the NtOSAK, a member of SnRK2 kinase family, a key component of the ABA signaling cascade. A comprehensive phosphoproteomics analysis of tobacco cells, treated with exogenous CKs under salinity-stress conditions indicated that >50% of the identified phosphoproteins involved in stress responses were dephosphorylated by CKs. We hypothesize that upregulation of CK levels under stress conditions desensitize stress signaling cues through deactivation of kinases that are normally activated under stress conditions. CK-dependent desensitization of environmental stimuli is suggested to attenuate various pathways of the avoidance syndrome including the characteristic growth arrest and the premature senescence while allowing normal growth and metabolic maintenance.

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited.