Cytokinins (CKs) are well-established as important phytohormonal regulators of plant growth and development. An increasing number of studies have also revealed the function of these hormones in plant responses to biotic and abiotic stresses. While the function of certain CK classes, including trans-zeatin and isopentenyladenine-type CKs, have been studied in detail, the role of cis-zeatin-type CKs (cZs) in plant development and in mediating environmental interactions is less well defined. Here we provide a comprehensive summary of the current knowledge about abundance, metabolism and activities of cZs in plants. We outline the history of their analysis and the metabolic routes comprising cZ biosynthesis and degradation. Further we provide an overview of changes in the pools of cZs during plant development and environmental interactions. We summarize studies that investigate the role of cZs in regulating plant development and defence responses to pathogen and herbivore attack and highlight their potential role as 'novel' stress-response markers. Since the functional roles of cZs remain largely based on correlative data and genetic manipulations of their biosynthesis, inactivation and degradation are few, we suggest experimental approaches using transgenic plants altered in cZ levels to further uncover their roles in plant growth and environmental interactions and their potential for crop improvement.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans‐zeatin O‐glucosyltransferase gene (35S::ZOG1), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT.As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves.During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.

Our phenotyping and hormonal study has been aimed to characterize the role of cytokinins (CK) in the drought and recovery responses of Arabidopsis thaliana. CK down-regulation was achieved by overexpression of the gene for CK deactivating enzyme cytokinin oxidase/dehydrogenase (CKX): constitutive (35S:CKX) or at the stress onset using a dexamethasone-inducible pOp/LhGR promoter (DEX:CKX). The 35S:CKX plants exhibited slow ontogenesis and higher expression levels of stress-associated genes, e.g. AtP5CS1, already at control conditions. CK down-regulation resulted during drought in higher stress tolerance (indicated by relatively low up-regulation of the expression of drought stress marker gene AtRD29B) accompanied with lower leaf water loss. Nevertheless, these plants exhibited slow and delayed recovery. CK levels were increased at the stress onset by stimulation of the expression of CK biosynthetic gene isopentenyl transferase (ipt) (DEX:IPT) or by application of exogenous CK meta-topolin. After water withdrawal, long-term CK elevation resulted in higher water loss in comparison with CKX transformants as well as with plants overexpressing ipt driven by senescence-inducible SAG12 promoter (SAG:IPT), which gradually enhanced CKs during the stress progression. In all cases, CK up-regulation resulted in fast and more vigorous recovery. All drought-stressed plants exhibited growth suppression associated with elevation of abscisic acid and decrease of auxins and active CKs (with the exception of SAG:IPT plants). Apart from the ipt overexpressers, also increase of jasmonic and salicylic acid was found.