Cytokinins: metabolism and function in plant adaptation to environmental stresses

In plants, the cytokinin (CK) phytohormones regulate numerous biological processes, including responses to environmental stresses, via a complex network of CK signaling. By an unknown mechanism, stress signals are perceived and transmitted through the His-Asp phosphorelay, an important component of the CK signal transduction pathway, triggering CK-responsive genes. Because of the intensive crosstalk between CKs and abscisic acid (ABA), modulation of CK levels and their signal transduction affects both ABA-dependent and ABA-independent pathways, enabling plant adaptation to adverse conditions. This review presents our current understanding of the functions of CKs and CK signaling in the regulation of plant adaptation to stress. Biotechnological strategies based on the modulation of CK levels have been examined with the aim of stabilizing agriculture yields.     

Identification and Expression Analysis of Cytokinin Metabolic Genes in Soybean under Normal and Drought Conditions in Relation to Cytokinin Levels

Cytokinins (CKs) mediate cellular responses to drought stress and targeted control of CK metabolism can be used to develop drought-tolerant plants. Aiming to manipulate CK levels to improve drought tolerance of soybean cultivars through genetic engineering of CK metabolic genes, we surveyed the soybean genome and identified 14 CK biosynthetic (isopentenyltransferase, GmIPT) and 17 CK degradative (CK dehydrogenase, GmCKX) genes. Comparative analyses of GmIPTs and GmCKXs with Arabidopsis counterparts revealed their similar architecture. The average numbers of abiotic stress-inducible cis-elements per promoter were 0.4 and 1.2 for GmIPT and GmCKX genes, respectively, suggesting that upregulation of GmCKXs, thereby reduction of CK levels, maybe the major events under abiotic stresses. Indeed, the expression of 12 GmCKX genes was upregulated by dehydration in R2 roots. Overall, the expressions of soybean CK metabolic genes in various tissues at various stages were highly responsive to drought. CK contents in various organs at the reproductive (R2) stage were also determined under well-watered and drought stress conditions. Although tRNA-type GmIPT genes were highly expressed in soybean, cis-zeatin and its derivatives were found at low concentrations. Moreover, reduction of total CK content in R2 leaves under drought was attributable to the decrease in dihydrozeatin levels, suggesting a role of this molecule in regulating soybean's responses to drought stress. Our systematic analysis of the GmIPT and GmCKX families has provided an insight into CK metabolism in soybean under drought stress and a solid foundation for in-depth characterization and future development of improved drought-tolerant soybean cultivars by manipulation of CK levels via biotechnological approach.     

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.     

Elucidating cytokinin response mechanisms using mutants

Cytokinins (CKs) have powerful effects on many elements of plant development, but little is known about the cellular and molecular mechanisms of CK action. This review describes recent progress in identification and characterization of mutants of flowering plants that may permit elucidation of CK response mechanisms. Several Nicotiana plumbaginifolia mutants that are resistant to high levels of exogenous CK have been isolated. Characterization of these mutants has led to information about relationships between CKs and other hormones, and CKs and nutrient metabolism. Two Arabidopsis thaliana mutants that are specifically resistant to CKs in a root elongation assay, cyr1 and stp1, have been described, and may represent lesions in the CK signal transduction pathway. A mutant that produces elevated levels of CKs, amp1, has provided surprising information about the role of CKs in cotyledon formation. A set of tagged mutations that result in CK independent growth in culture has been identified, and the affected gene, CKI1, cloned. The possibility that this gene encodes a CK receptor is discussed. Continued molecular/genetic analysis of CK responses is predicted to result in rapid progress in the next few years in understanding how CKs act.     

A member of the maize isopentenyl transferase gene family, <Emphasis Type="Italic">Zea mays isopentenyl transferase 2</Emphasis> (<Emphasis Type="Italic">ZmIPT2</Emphasis>), encodes a cytokinin biosynthetic enzyme expressed during kernel development

Cytokinins (CKs) are plant hormones that regulate a large number of processes associated with plant growth and development such as induction of stomata opening, delayed senescence, suppression of auxin-induced apical dominance, signaling of nitrogen availability, differentiation of plastids and control of sink strength. In maize, CKs are thought to play an important role in establishing seed size and increasing seed set under normal and unfavorable environmental conditions therefore influencing yield. In recent years, the discovery of isopentenyl transferase (IPT) genes in plants has shed light on the CK biosynthesis pathway in plants. In an effort to increase our understanding of the role played by CKs in maize development and sink-strength, we identified several putative IPT genes using a bioinformatics approach. We focused our attention on one gene in particular, ZmIPT2, because of its strong expression in developing kernels. The expression of the gene and its product overlays the change in CK levels in developing kernels suggesting a major role in CK biosynthesis for kernel development. We demonstrate that at 8–10 days after pollination (DAP) the endosperm and especially the basal transfer cell layer (BETL) is a major site of ZmIPT2 expression, and that this expression persists in the BETL and the developing embryo into later kernel development stages. We show that ectopic expression of ZmIPT2 in calli and in planta created phenotypes consistent with CK overproduction. We also show that ZmIPT2 preferentially uses ADP and ATP over AMP as the substrates for dimethylallyl diphosphate (DMAPP) IPT activity. The expression pattern of ZmIPT2 in the BETL, endosperm and embryo during kernel development will be discussed with an emphasis on the suggested role of CKs in determining sink-strength and grain production in crop plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea

Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot‐infected rye plants is also altered, although no pronounced changes occur in the expression of the host plant's CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT‐LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans‐zeatin, a reaction catalysed by a CK‐specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt‐log. Deletion of cpipt‐log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper‐sporulating phenotype, implying that CKs are environmental factors influencing fungal development.     

Genetic engineering of cytokinin metabolism: Prospective way to improve agricultural traits of crop plants

Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.