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.     

Molecular basis for cytokinin biosynthesis

Cytokinins (CKs) are a group of phytohormones that play a crucial role in the regulation of plant growth and development. Identification of the enzymes and the corresponding genes that are involved in CK metabolism allowed us to understand how plants synthesize CKs and adjust CK activity to optimal levels. A major accomplishment toward these goals was the identification of genes for the first enzyme in the CK biosynthetic pathway, adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis thaliana and Agrobacterium tumefaciens, detailed analyses of IPTs were conducted through not only enzymatic characterization but also molecular structural approaches. These studies revealed the molecular basis for the Agrobacterium-origin of IPT used for the efficient biosynthesis of trans-zeatin that promotes tumorigenesis in host plants. Another landmark in CK research was the identification of CYP735A as an enzyme that converts iP-nucleotide to tZ-nucleotide. Furthermore, the identification of a CK-activating enzyme, LOG, which catalyzes a novel activation pathway, is a remarkable recent achievement in CK research. Collectively, these advances have revealed the complexity of the entire metabolic scheme for CK biosynthesis.     

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis?trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

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.     

Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants

Cytokinins (CKs) are phytohormones that play an important role in plant growth and development. Although the first naturally produced CK, zeatin, was isolated almost four decades ago, no endogenous gene has been shown to produce active CKs in planta. In an activation tagging experiment we have identified a petunia line that showed CK-specific effects including enhanced shooting, reduced apical dominance and delayed senescence and flowering. This phenotype correlated with the enhanced expression of a gene we labelled Sho (Shooting). Sho, which encodes a protein with homology to isopentenyl transferases (IPTs), also causes CK-specific effects when expressed in other plant species. In contrast to the ipt gene from Agrobacterium, which primarily increases zeatin levels, Sho expression in petunia and tobacco especially enhances the levels of certain N6-(delta2-isopentenyl) adenosine (2iP) derivatives. Our data suggest that Sho encodes a plant enzyme whose activity is sufficient to produce active CKs in plants.     

Cytokinin conjugation: recent advances and patterns in plant evolution

Cytokinin (CK) conjugates are important in plant development because they regulate active CK concentrations, CK transport, storage, and irreversible inactivation. While numerous CK conjugates have been identified in higher plants, the biological functions of these compounds, their location within cells and tissues, and the enzymes and genes involved in their regulation are not clearly understood. In this paper, recent advances are reported which have occurred through the study of transgenic plants containing the ipt or rolC genes, the identification of new regulatory enzymes affecting CKs, and the characterization of new CK conjugates. In addition, a survey of the literature is presented which examines the pattern of CK conjugates found in different plant taxa. Based on current knowledge, it appears that green algae, mosses, and ferns contain relatively few CK conjugates of isopentenyl adenine (iP) and zeatin (Z). In contrast, higher land plants, such as gymnosperms and angiosperms, contain a more complex set of CKs, primarily conjugates of Z and dihydrozeatin (DHZ). This suggests that the pattern of CK conjugation has become more complex in parallel with the increasing complexity of higher plants.     

Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco

Cytokinins (CKs) are involved in the regulation of plant development including plastid differentiation and function. Partial location of CK biosynthetic pathways in plastids suggests the importance of CKs for chloroplast development. The impact of genetically modified CK metabolism on endogenous CK, indole-3-acetic acid, and abscisic acid contents in leaves and isolated intact chloroplasts of Nicotiana tabacum was determined by liquid chromatography/mass spectrometry and two-dimensional high-performance liquid chromatography, and alterations in chloroplast ultrastructure by electron microscopy. Ectopic expression of Sho, a gene encoding a Petunia hybrida isopentenyltransferase, was employed to raise CK levels. The increase in CK levels was lower in chloroplasts than in leaves. CK levels were reduced in leaves of tobacco harbouring a CK oxidase/dehydrogenase gene, AtCKX3. The total CK content also decreased in chloroplasts, but CK phosphate levels were higher than in the wild type. In a transformant overexpressing a maize beta-glucosidase gene, Zm-p60.1, naturally targeted to plastids, a decrease of CK-O-glucosides in chloroplasts was found. In leaves, the changes were not significant. CK-O-glucosides accumulated to very high levels in leaves, but not in chloroplasts, of plants overexpressing a ZOG1 gene, encoding trans-zeatin-O-glucosyltransferase from Phaseolus lunatus. Manipulation of the CK content affected levels of indole-3-acetic and abscisic acid. Chloroplasts of plants constitutively overexpressing Sho displayed ultrastructural alterations including the occasional occurrence of crystalloids and an increased number of plastoglobuli. The other transformants did not exhibit any major differences in chloroplast ultrastructure. The results suggest that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of CK metabolism.     

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.     

Effect of Blue Light on Endogenous Isopentenyladenine and Endoreduplication during Photomorphogenesis and De-Etiolation of Tomato (Solanum lycopersicum L.) Seedlings

Light is one of the most important factor influencing plant growth and development all through their life cycle. One of the well-known light-regulated processes is de-etiolation, i.e. the switch from skotomorphogenesis to photomorphogenesis. The hormones cytokinins (CKs) play an important role during the establishment of photomorphogenesis as exogenous CKs induced photomorphogenesis of dark-grown seedlings. Most of the studies are conducted on the plant model Arabidopsis, but no or few information are available for important crop species, such as tomato (Solanum lycopersicum L.). In our study, we analyzed for the first time the endogenous CKs content in tomato hypocotyls during skotomorphogenesis, photomorphogenesis and de-etiolation. For this purpose, two tomato genotypes were used: cv. Rutgers (wild-type; WT) and its corresponding mutant (7B-1) affected in its responses to blue light (BL). Using physiological and molecular approaches, we identified that the skotomorphogenesis is characterized by an endoreduplication-mediated cell expansion, which is inhibited upon BL exposure as seen by the accumulation of trancripts encoding CycD3, key regulators of the cell cycle. Our study showed for the first time that iP (isopentenyladenine) is the CK accumulated in the tomato hypocotyl upon BL exposure, suggesting its specific role in photomorphogenesis. This result was supported by physiological experiments and gene expression data. We propose a common model to explain the role and the relationship between CKs, namely iP, and endoreduplication during de-etiolation and photomorphogenesis.     

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.     

Regulatory Roles of Cytokinins and Cytokinin Signaling in Response to Potassium Deficiency in Arabidopsis

Potassium (K) is an important plant macronutrient that has various functions throughout the whole plant over its entire life span. Cytokinins (CKs) are known to regulate macronutrient homeostasis by controlling the expression of nitrate, phosphate and sulfate transporters. Although several studies have described how CKs signal deficiencies for some macronutrients, the roles of CKs in K signaling are poorly understood. CK content has been shown to decrease under K-starved conditions. Specifically, a CK-deficient mutant was more tolerant to low K than wild-type; however, a plant with an overaccumulation of CKs was more sensitive to low K. These results suggest that K deprivation alters CK metabolism, leading to a decrease in CK content. To investigate this phenomenon further, several Arabidopsis lines, including a CK-deficient mutant and CK receptor mutants, were analyzed in low K conditions using molecular, genetic and biochemical approaches. ROS accumulation and root hair growth in low K were also influenced by CKs. CK receptor mutants lost the responsiveness to K-deficient signaling, including ROS accumulation and root hair growth, but the CK-deficient mutant accumulated more ROS and exhibited up-regulated expression of HAK5, which is a high-affinity K uptake transporter gene that is rapidly induced by low K stress in ROS- and ethylene-dependent manner in response to low K. From these results, we conclude that a reduction in CK levels subsequently allows fast and effective stimulation of low K-induced ROS accumulation, root hair growth and HAK5 expression, leading to plant adaptation to low K conditions.     

Accumulation pattern of endogenous cytokinins and phenolics in different organs of 1‐year‐old cytokinin pre‐incubated plants: implications for conservation

A better understanding of phytohormone physiology can provide an essential basis to coherently achieve a conservation drive/strategy for valuable plant species. We evaluated the distribution pattern of cytokinins (CKs) and phenolic compounds in different organs of 1‐year‐old greenhouse‐grown Tulbaghia simmleri pre‐treated (during micropropagation) with three aromatic CKs (benzyladenine = BA, meta‐topolin = mT, meta‐topolin riboside = mTR). The test species is highly valuable due to its medicinal and ornamental uses. Based on UHPLC‐MS/MS quantification, mT and mTR pre‐treated plants had the highest total CK, mostly resulting from the isoprenoid CK‐type, which occurred at highest concentrations in the roots. Although occurring in much lower concentrations when compared to isoprenoid CKs, aromatic CKs were several‐fold more abundant in the root of mT pre‐treated plants than with other treatments. Possibly related to the enhanced aromatic CKs, free bases and ribonucleotides, plants pre‐treated with mT generally displayed better morphology than the other treatments. A total of 12 bioactive phenolic compounds, including four hydroxybenzoic acids, five hydroxycinnamic acids and three flavonoids at varying concentrations, were quantified in T. simmleri. The occurrence, distribution and levels of these phenolic compounds were strongly influenced by the CK pre‐treatments, thereby confirming the importance of CKs in phenolic biosynthesis pathways.     

The molecular basis of cytokinin action

Current understanding of cytokinin (CK) physiology at the cellular level results largely from the manipulation of endogenous CK levels by either application of exogenous CKs or the expression of CK biosynthetic transgenes, as well as the characterisation of single gene mutants. Cytokinins modulate changes in plant gene expression, which are in turn assumed to effect physiological and morphological changes with which CK action is associated. Presently, a major focus of investigation is elucidation of the biochemical events leading from the perception of CK to the manifestation of a response. Analysis of the expression patterns of CK-regulated genes and identification of their products provides one means of investigating CK action at the molecular level. Biochemical approaches have led to the identification of several soluble CK-binding proteins, although their functional roles in CK signalling largely remain uncertain. Conclusive identification of a bona fide CK receptor has yet to be achieved, although several potential candidates have been suggested. Pharmacological and molecular genetic strategies have implicated the involvement of signalling mechanisms likely to be involved in CK action. The apparent involvement of fluctuations in the concentration of intracellular Ca²⁺, changes in protein phosphorylation as well as DNA and/or protein methylation provide information concerning the types of proteins likely to be involved in the process. Dissection of CK signal transduction chains and elucidation of their interaction with other pathways that regulate plant growth and development is likely to be essential in understanding the mode of action of this poorly understood class of plant growth regulator. However, integration of this knowledge with an improved understanding of the mechanisms whereby overall hormone homeostasis is regulated at the metabolic level will be necessary for comprehensive appreciation of the influence of CKs on plant morphology and physiology.     

How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea?

Merwilla plumbea (Lindl.) Speta is a popular and highly sought after South African medicinal plant with diverse therapeutic values. Using Ultra performance liquid chromatography (UPLC), the effect of five cytokinins (CKs) [either isoprenoid = N ⁶-isopentenyladenine (iP) or aromatic = benzyladenine, meta-topolin (mT), meta-topolin riboside (mTR), and 6-(3-methoxybenzylamino)-9-tetrahydropyran-2-ylpurine] MemTTHP on growth and level of endogenous CKs during micropropagation and acclimatization stages was evaluated. Aromatic CK (mT/mTR) elicited the highest shoot proliferation (7–8 shoots per explant) during in vitro culture. Following acclimatization, iP-treated and the control plants were healthier with longer leaves, roots and higher fresh weight when compared to aromatic CKs. A total of 37 (22 isoprenoid and 15 aromatic) CK variants were quantified in both in vitro and acclimatized plants. Based on their metabolic function, these were grouped into five types including free bases, ribosides, ribotides, O- and 9-glucosides. In addition to enhancing our understanding of the hormone physiology in M. plumbea, the current findings are discussed in line with the effect of the exogenously applied CK on the observed differences in growth before and after the important stage of acclimatization. The observed dynamics in endogenous CK provide an avenue to manipulate in vitro growth and development of investigated species.     

Degradation of cytokinins by cytokinin oxidases in plants

The degradation metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This review attempts to collate the limited information available about this enzyme and introduce new facts, obtained in our laboratory, concerning the mechanism of degradation of cytokinins bearing unsaturated isoprene side chains. However, complete clarification of the effects of cytokinin oxidase on cytokinin regulation and its molecular and biochemical properties will be dependent upon the purification of the protein with cytokinin oxidase activity to homogeneity and progress in the development of requisite molecular probes.     

‘Real time’ genetic manipulation: a new tool for ecological field studies

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene‐related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose‐dependent effects. We applied the sensitive dexamethasone (DEX)‐inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin‐based DEX application method to facilitate ectopic gene expression and RNA interference‐mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field‐grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant–herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.