Ligand-binding properties and subcellular localization of maize cytokinin receptors

The ligand-binding properties of the maize (Zea mays L.) cytokinin receptors ZmHK1, ZmHK2, and ZmHK3a have been characterized using cytokinin binding assays with living cells or membrane fractions. According to affinity measurements, ZmHK1 preferred N(6)-(Δ(2)-isopentenyl)adenine (iP) and had nearly equal affinities to trans-zeatin (tZ) and cis-zeatin (cZ). ZmHK2 preferred tZ and iP to cZ, while ZmHK3a preferred iP. Only ZmHK2 had a high affinity to dihydrozeatin (DZ). Analysis of subcellular fractions from leaves and roots of maize seedlings revealed specific binding of tZ in the microsome fraction but not in chloroplasts or mitochondria. In competitive binding assays with microsomes, tZ and iP were potent competitors of [(3)H]tZ while cZ demonstrated significantly lower affinity; adenine was almost ineffective. The binding specificities of microsomes from leaf and root cells for cytokinins were consistent with the expression pattern of the ZmHKs and our results on individual receptor properties. Aqueous two-phase partitioning and sucrose density-gradient centrifugation followed by immunological detection with monoclonal antibody showed that ZmHK1 was associated with the endoplasmic reticulum (ER). This was corroborated by observations of the subcellular localization of ZmHK1 fusions with green fluorescent protein in maize protoplasts. All these data strongly suggest that at least a part of cytokinin perception occurs in the ER.     

Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.     

Antisenescent Activity of Natural Cytokinins

The antisenescent activity of naturally occurring cytokinins (bases and ribosides) has been evaluated by measuring chlorophyll retention in detached wheat (Triticum vulgare) leaf segments. 6-(3-Methyl-2-butenylamino)-2-methylthiopurine (ms2ip) was the most active cytokinin followed by 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine (tZ). 6-(4-Hydroxy-3-methyl-cis-2-butenylamino)-9-β-D-ribofuranosylpurine (cZR), 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-2-methylthio-9β-D-ribofuranosylpurine (MstZR), and 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthio-9-β-D-ribofuroanosylpurine (mscZR) were essentially inactive. 9-Ribosyl substitution did not affect the activity of tZ, (±)-6-(4-hydroxy-3-methylbutylamino)purine (DHZ), or 6-(3-methyl-2-butenylamino)purine (2ip), but lowered the activity of 6-(o-hydroxybenzylamino)purine (OHBA) and 6-(4-hydroxy-3-methyl-cis-2-butenylamino)purine (cZ). 2-Methylthio substitution increased the activity of 2ip and DHZ, decreased the activity of tZ, and had no effect on the activity of cZ. The activities of the simultaneously substituted 2-methylthio-9-ribosyl compounds are lower than those of their corresponding unsubstituted or 2-methylthio substituted bases with the exception of DHZ. Structure-activity relationships for chlorophyll retention did not parallel many of the relationships found for callus tissue growth stimulation.     

Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay

The cytokinin receptor AHK3 of Arabidopsis thaliana plays a predominant role in shoot development. A study of the hormone-binding characteristics of AHK3 compared with the mainly root-confined receptor CRE1/AHK4 has been accomplished using a live-cell binding assay on transgenic bacteria expressing individual receptor proteins. Both receptors bound trans-zeatin (tZ) with high affinity. Scatchard analysis showed a linear function corresponding to an apparent K(D) of 1-2 nM for the AHK3 receptor-hormone complex, which is close to the K(D) (2-4 nM) for the CRE1/AHK4 receptor-hormone complex. The specific binding of tZ to both receptors was pH dependent, AHK3 being more sensitive to pH changes than CRE1/AHK4. Hormone binding was reversible, at least for the bulk of (3)H-zeatin, and influenced by monovalent cations, while divalent cations (Ca(2+), Mg(2+), Mn(2+)) at physiological concentrations had no significant effect. AHK3 differed significantly from CRE1/AHK4 in relative affinity to some cytokinins. AHK3 had an approximately 10-fold lower affinity to isopentenyladenine (iP) and its riboside, but a higher affinity to dihydrozeatin than CRE1/AHK4. For AHK3, cytokinin ribosides (tZR, iPR) and cis-zeatin had true binding activity, although lower than that of tZ. The phenylurea-derived cytokinin thidiazuron was a strong competitor and bound to the same site as did adenine-derived cytokinins. The inhibitor of cytokinin action butan-1-ol had little effect on cytokinin-receptor complex formation. The revealed properties of AHK3 suggest its specific function in root-to-shoot communication.     

Functional identification of OsHk6 as a homotypic cytokinin receptor in rice with preferential affinity for iP

Cytokinins are involved in key developmental processes in rice (Oryza sativa), including the regulation of cell proliferation and grain yield. However, the in vivo action of histidine kinases (OsHks), putative cytokinin receptors, in rice cytokinin signaling remains elusive. This study examined the function and characteristics of OsHk3, 4 and 6 in rice. OsHk6 was highly sensitive to isopentenyladenine (iP) and was capable of restoring cytokinin-dependent ARR6 reporter expression in the ahk2 ahk3 Arabidopsis mutant upon treatment with 1 nM iP. OsHk4 recognized trans-zeatin (tZ) and iP, while OsHk3 scarcely induced cytokinin signaling activity. OsHk4 and OsHk6 mediated the canonical two-component signaling cascade of Arabidopsis to induce phosphorylation of ARR2. OsHk4 and OsHk6 were highly expressed in spikelets, suggesting that tZ and iP might play key roles in grain development. OsHk6 formed a self-interacting homomer in rice protoplasts, although the trans-phosphorylation activity between subunits was much lower than the intra-molecular trans-phosphorylation activity. This indicates that the action mechanism of OsHks is evolutionarily diverged from bacterial histidine kinases. Ectopic expression of OsHk6 in rice calli promoted green pigmentation and subsequent shoot induction, further supporting an OsHk6 in planta function as a cytokinin receptor. From the results of this study, OsHks are homomeric cytokinin receptors with distinctive cytokinin preferences in rice.     

Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes

Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N?-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N?-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes.     

Characterization of cytokinin and adenine transport in Arabidopsis cell cultures

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H(+)-mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.     

Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydroxylases that catalyze the biosynthesis of trans-Zeatin

Cytokinins (CKs), a group of phytohormones, are adenine derivatives that carry either an isoprene-derived or an aromatic side chain at the N(6) terminus. trans-Zeatin (tZ), an isoprenoid CK, is assumed to play a central physiological role because of its general occurrence and high activity in bioassays. Although hydroxylation of isopentenyladenine-type CKs is a key step of tZ biosynthesis, the catalyzing enzyme has not been characterized yet. Here we demonstrate that CYP735A1 and CYP735A2 are cytochrome P450 monooxygenases (P450s) that catalyze the biosynthesis of tZ. We identified the genes from Arabidopsis using an adenosine phosphate-isopentenyltransferase (AtIPT4)/P450 co-expression system in yeast. Co-expression of AtIPT4 and CYP735A enabled yeast to excrete tZ and the nucleosides to the culture medium. In vitro, both CYP735As preferentially utilized isopentenyladenine nucleotides rather than the nucleoside and free base forms and produced tZ nucleotides but not the cis-isomer. The expression of CYP735A1 and CYP735A2 was differentially regulated in terms of organ specificity and response to CK. Root-specific induction of CYP735A2 expression by CK suggests that the trans-hydroxylation is involved in the regulation of CK metabolism and signaling in roots.     

Divergent expression of cytokinin biosynthesis,signaling and catabolism genes underlying differences in feeding sites induced by cyst and root‐knot nematodes

Cyst and root‐knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root‐knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.     

Cytokinin biosynthesis and transport for systemic nitrogen signaling

The plasticity of growth and development in response to environmental changes is one of the essential aspects of plant behavior. Cytokinins play an important role as signaling molecules in the long-distance communication between organs in systemic growth regulation in response to nitrogen. The spatial distribution of the expression sites of cytokinin biosynthesis genes leads to structural differences in the molecular species transported through the xylem and phloem, giving root-borne trans-hydroxylated cytokinins, namely trans-zeatin (tZ) type, a specialized efficacy in regulating shoot growth. Furthermore, root-to-shoot translocation via the xylem, tZ, and its precursor, the tZ riboside, controls different sets of shoot growth traits to fine-tune shoot growth in response to nitrogen availability. In addition to nitrogen, photosynthetically generated sugars positively regulate de novo cytokinin biosynthesis in the roots, and contribute to plant growth under elevated CO₂ conditions. In shoot-to-root signaling, cytokinins also play a role in the regulation of nutrient acquisition and root system growth in cooperation with other types of signaling molecules, such as C-TERMINALLY ENCODED PEPTIDE DOWNSTREAMs. As cytokinin is a key regulator for the maintenance of shoot apical meristem, deepening our understanding of the regulatory mechanisms of cytokinin biosynthesis and transport in response to nitrogen is important not only for basic comprehension of plant growth, but also to ensure the stability of agricultural production.     

Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to cis-zeatin

Genes for cytokinin-responsive His-protein kinases (ZmHK1, ZmHK2, and ZmHK3a) were isolated from maize (Zea mays). Heterologous expression of each of the ZmHKs in Escherichia coli having the DeltarcsC and cpslacZ genetic background conferred cytokinin-inducibility of lacZ expression on the bacteria. In the recombinant E. coli system, ZmHK1 and ZmHK3a were more sensitive to free-base cytokinins than to the corresponding nucleosides; isopentenyladenine was most effective for ZmHK1, while ZmHK2 tended to be most sensitive to trans-zeatin and the riboside. In contrast to a known cytokinin receptor of Arabidopsis (AHK4/CRE1/WOL), all ZmHKs responded to cis-zeatin (cZ), which generally is believed to be inactive or only weakly active. In cultured maize cells, expression of ZmRR1, a cytokinin-inducible response regulator, was induced by cZ as well as by trans-zeatin. These results strongly suggest that maize cytokinin receptors differ in ligand preference, and that cZ is an active cytokinin at least in maize.     

Multi-omics analysis reveals the ontogenesis of basal chichi in Ginkgo biloba L.

Chichi is a unique biological phenomenon observed in Ginkgo biloba L.. In this study, multi-omics analysis was used to compare basal chichi (C) with roots (R) and stems (S) to explore the regulatory mechanisms of basal chichi ontogenesis. The results showed that compared with roots and stems, the tZ, SA and ABA contents in basal chichi were the highest, and the ratio of IAA/tZ was the lowest. Nucleotides and their derivatives in basal chichi were upregulated, and phenylpropane metabolites were downregulated. Some differentially expressed genes (DEGs) strongly correlated to plant hormones were screened. We speculate that auxin and cytokinin are involved in the morphogenesis of basal chichi and that cytokinin plays a major role. The ontogenesis of basal chichi is closely related to environmental stress, and it may be a coping strategy of G. biloba in the face of environmental stress.     

Cytokinins in cut carnation flowers. VII. The effect of zeatin and dihydrozeatin derivatives on flower longevity

Dihydrozeatin, at 4×10^–5 M, delayed the senescence of carnation flowers while tZ, at the same concentration, accelerated it. cis-Zeatin was ineffective. The DHZ derivatives as well as the Z derivatives gave responses very similar to those observed for the parent free bases. While additional experimentation with radiolabelled derivatives is clearly called for, the similarity between the responses observed for the respective derivatives and the free bases, suggests that in the carnation flower there is a great deal of metabolic interconversion.Abbreviations DHZ dihydrozeatin - DHZR ribosyldihydrozeatin - DHZOG glucosyl-O-dihydrozeatin - DHZ9G glucosyl-9-dihydrozeatin - DHZROG glucosyl-O-ribosyldihydrozeatin - cZ cis-zeatin - tZ trans-zeatin - ZR ribosylzeatin - Z9G glucosyl-9-zeatin - ZOG glucosyl-O-zeatin - ZROG glucosyl-O-ribosylzeatin     

Cytokinins in cut carnation flowers. IX. Transport and metabolism of iso-pentenyladenine and the effect of its derivatives on flower longevity

Of the four 2iP derivatives applied to cut carnations 2iP, IPA and IPAMP delayed the rate of flower senescence but did not delay longevity. 2iP9G was the least effective. [³H]2iP was rapidly transported to all flower components. The receptacular tissue apparently plays a major role in the distribution of the applied cytokinin to the rest of the flower parts. Over and above possible interconversion between 2iP, IPA and IPAMP the applied [³H]2iP was also converted to 2iP9G, Ade, Ado, tZ and DHZ. The presence of Ade indicates the involvement of cytokinin oxidase-type enzymes while the presence of tZ and DHZ indicates a degree of hydroxylation. These latter steps may contribute to the efficiency of 2iP in the system.     

Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins

Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N(6)-benzyladenosine (BAR), N(6)-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5'-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N(6)-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.     

The phenylquinazoline compound S-4893 is a non-competitive cytokinin antagonist that targets Arabidopsis cytokinin receptor CRE1 and promotes root growth in Arabidopsis and rice

We identified two phenylquinazoline compounds in a large-scale screening for cytokinin antagonists in yeast expressing the Arabidopsis cytokinin receptor cytokinin response 1/histidine kinase 4 (CRE1). After chemical modifications, we obtained compound S-4893, which non-competitively inhibited binding of the natural ligand 2-isopentenyladenine to CRE1. S-4893 antagonized cytokinin-induced activation of the Arabidopsis response regulator 5 promoter in Arabidopsis. Importantly, S-4893 had no detectable intrinsic cytokinin agonist activity in Arabidopsis or in the transformed yeast system. Cytokinin bioassay further demonstrated that S-4893 antagonized cytokinin-induced stimulation of callus formation and inhibition of root elongation. S-4893 also promoted seminal, crown and lateral root growth in rice, suggesting that S-4893 could potentially promote root growth in a variety of agronomically important plants. We believe S-4893 will be a useful tool in functional studies of cytokinin action in a wide range of plants and a lead compound for the development of useful root growth promoters in agriculture.