On the Significance of Cytokinin Incorporation into RNA

The clarification of the following 2 questions was attempted: (a) are cytokinins precursors in the formation of sRNA, (b) is the observed incorporation of cytokinins into sRNA related to the action of the hormone? Although Escherichia coli contains cytokinins in its sRNA, no cytokinin auxotroph mutants of E. coli could be found and the statistical probability for the existence of such mutants is extremely low. This suggests that cytokinins are not precursors in the synthesis of sRNA. A radioactive cytokinin, 6-benzylamino-9-methyl-purine was synthesized and it was tested whether or not it is incorporated into sRNA of soybean callus tissue. Masking the 9-position of the purine inhibited the incorporation of this cytokinin into RNA while not affecting its biological activity. This is taken as an indication that the observed incorporation of cytokinins such as benzyladenine into sRNA is not related to the action of this hormone.     

Mutations at CRE1 impair cytokinin-induced repression of phosphate starvation responses in Arabidopsis

Plants display a number of responses to low phosphate availability, involving biochemical and developmental changes. Recently we have shown that many of these responses can be repressed in roots by exogenous addition of cytokinins. In order to understand the genetic basis to this effect of cytokinins, and its relation with the better known roles of cytokinins in the control of cell-cycle and differentiation, we have undertaken mutant screening and characterization using a transgenic line of Arabidopsis thaliana harbouring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS). One type of mutant identified displayed reduced sensitivity of AtIPS1::GUS to cytokinin repression. Several other Pi starvation response genes showed reduced cytokinin sensitivity in these lines. These mutants also showed reduced cytokinin repression of the anthocyanin accumulation induced by Pi starvation in the aerial part of the plants. Mapping and molecular characterization of these mutants showed that they were allelic of CRE1/WOL, a locus known to encode a cytokinin receptor. CRE1 is downregulated by Pi starvation and induced by cytokinins, both in the wild-type and in the cre1 mutants, in which cre1 mRNA levels are higher. These results reveal the existence of a positive feed-back loop, in addition to the already established negative feedback loop, in cytokinin signalling and indicate that the negative regulation of Pi starvation responses by cytokinins involves a two-component signalling circuitry, as it is the case of other types of cytokinin response.     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

植物体内的芳环细胞分裂素

芳环细胞分裂素包括6-苄基氨基嘌呤（BA）,6-（3-羟苄基氨基）-嘌呤（mT）,6-（2-羟苄基氨基）-嘌呤(oT)和它们的衍生物。它的代谢具有相互转换、羟基化作用、结合作用和氧化降解四个特征。本文从信号的感受,转导和应答阐明细胞分裂素在细胞和分子水平上的 功能。     

Cytokinins and plant immunity: old foes or new friends?

Cytokinins are plant growth promoting hormones involved in the specification of embryonic cells, maintenance of meristematic cells, shoot formation and development of vasculature. Cytokinins have also emerged as a major factor in plant-microbe interactions during nodule organogenesis and pathogenesis. Microbe-originated cytokinins confer abnormal hypersensitivity of cytokinins to plants, augmenting the sink activity of infected regions. However, recent findings have shed light on a distinct role of cytokinins in plant immune responses. Plant-borne cytokinins systemically induce resistance against pathogen infection. This resistance is orchestrated by endogenous cytokinin and salicylic acid signaling. Here, we discuss how plant- and pathogen-derived cytokinins inversely affect the plant defense response. In addition, we consider the molecular mechanisms underlying plant-derived cytokinin action in plant immunity.     

植物体内的芳环细胞分裂素

芳环细胞分裂素包括6—苄基氨基嘌呤(BA),6—(3—羟苄基氨基)—嘌呤(mT),6—(2—羟苄基氨基)—嘌吟(oT)和它们的衍生物。它的代谢具有相互转换、羟基化作用、结合作用和氧化降解四个特征。本文从信号的感受,转导和应答阐明细胞分裂素在细胞和分子水平上的功能。     

Cytokinins and tRNAs: a hypothesis on their competitive interaction via specific receptor proteins

Abstract. A hypothesis concerning the molecular mechanism of cytokinin action in plants is presented. Free cytokinins and some cytokinin-containing tRNAs are supposed to compete in the binding to a postulated receptor protein. As a result, the activity of some isoacceptor tRNAs would depend on cytokinin concentration in a cell. This explains a stimulatory effect of cytokinins on the translation of a definite set of cellular mRNAs. Various aspects of the hypothesis are briefly discussed.     

A Role for Cytokinins in De-Etiolation in Arabidopsis (det Mutants Have an Altered Response to Cytokinins)

When grown in the absence of light, Arabidopsis thaliana deetiolated (det) mutants develop many of the characteristics of light-grown plants, including the development of leaves and chloroplasts, the inhibition of hypocotyl growth elongation, and elevated expression levels of light-regulated genes. We show here that dark-grown wild-type seedlings exhibit similar phenotypic traits if any one of a variety of cytokinins are present in the growth medium. We further show that the striking phenotype of det mutants is unlikely to be caused by different levels of cytokinins in these mutants. The three major Arabidopsis cytokinins, zeatin, zeatin riboside, and isopentenyladenosine, accumulate to similar levels in wild-type seedlings grown in either the light or the dark. There is no consistently different pattern for the levels of these cytokinins in wild-type versus det1 or det2 mutants. However, det1 and det2 have an altered response to cytokinin in a detached leaf senescence assay and in tissue culture experiments. A model is proposed in which light and cytokinins act independently or sequentially through common signal transduction intermediates such as DET1 and DET2 to control the downstream light-regulated responses.     

Arabidopsis thaliana wild type, pho1, and pho2 mutant plants show different responses to exogenous cytokinins.

Despite the involvement of cytokinins in phosphate (Pi) signaling being highlighted, the physiological processes involved remain unclear. In this study, we have evaluated the effect of cytokinins on different physiological responses using wild type (wt) and two Arabidopsis mutants with altered shoot Pi content (pho1 and pho2). Physiological studies were related with those previously described as cytokinin-regulated: including hypocotyl elongation, root growth, anthocyanin accumulation, senescence and relative gene expression. Generally, pho1 mutants showed decreased sensitivity to cytokinin, whereas pho2 mutants showed increased sensitivity to the hormone. This observation applies to inhibition of hypocotyls and root growth and anthocyanin accumulation. However, this effect was not shown during senescence or in the expression of ARR6 (Arabidopsis response regulator, ARR). Interestingly, Pi content in shoot of pho1 mutants increased to wt levels after treatment with cytokinins. These results suggest that the interaction between phosphate signaling and cytokinin signaling may be bidirectional while the differential behavior in response to cytokinin is discussed further.     

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.     

Levels of Cytokinins in the Ovules of Cotton Mutants with Altered Fiber Development

Endogenous levels of cytokinin and abscisic acid (ABA) were determined in ovules of normal cotton (TM-1) and four fiber differentiation mutants (n2, Ligon lintless, H10, and Xu142) before and after flowering by enzyme-linked immunosorbent assays. The fluctuation patterns of ABA levels in ovules of normal cotton and mutants were similar. At the fiber elongation stage, ABA content was low, and from 1 day after flowering, the ABA content decreased steadily. On the other hand, the peaks of isopentenyladenine and isopentenyladenosine in ovules of TM-1 were observed 1 day before flowering. The level of cytokinins decreased after flowering in TM-1, whereas in the mutants it increased steadily. These results indicate that endogenous ABA is probably not the main inhibitor for fiber elongation and that endogenous cytokinins likely play a dual role in fiber development. Before flowering, cytokinins function as one of the stimuli for the initiation of fibers, but after flowering, cytokinins inhibit fiber growth. Received February 18, 1997; accepted June 11, 1997     

Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis

Cytokinins are plant hormones that may play essential and crucial roles in various aspects of plant growth and development. Although the functional significance of exogenous cytokinins as to the proliferation and differentiation of cells has been well documented, the biological roles of endogenous cytokinins have remained largely unknown. The recent discovery of the Arabidopsis Histidine Kinase 4 (AHK4)/CRE1/WOL cytokinin receptor in Arabidopsis thaliana strongly suggested that the cellular response to cytokinins involves a two-component signal transduction system. However, the lack of an apparent phenotype in the mutant, presumably because of genetic redundancy, prevented us from determining the in planta roles of the cytokinin receptor. To gain insight into the molecular functions of the three AHK genes AHK2, AHK3, and AHK4 in this study, we identified mutational alleles of the AHK2 and AHK3 genes, both of which encode sensor histidine kinases closely related to AHK4, and constructed a set of multiple ahk mutants. Application of exogenous cytokinins to the resultant strains revealed that both AHK2 and AHK3 function as positive regulators for cytokinin signaling similar to AHK4. The ahk2 ahk4 and ahk3 ahk4 double mutants and the ahk single mutants grew normally, whereas the ahk2 ahk3 double mutants exhibited a semidwarf phenotype as to shoots, such as a reduced leaf size and a reduced influorescence stem length. The growth and development of the ahk2 ahk3 ahk4 triple mutant were markedly inhibited in various tissues and organs, including the roots and leaves in the vegetative growth phase and the influorescence meristem in the reproductive phase. We showed that the inhibition of growth is associated with reduced meristematic activity of cells. Expression analysis involving AHK:beta-glucuronidase fusion genes suggested that the AHK genes are expressed ubiquitously in various tissues during postembryonic growth and development. Our results thus strongly suggest that the primary functions of AHK genes, and those of endogenous cytokinins, are triggering of the cell division and maintenance of the meristematic competence of cells to prevent subsequent differentiation until a sufficient number of cells has accumulated during organogenesis.     

The protective action of cytokinins on the photosynthetic machinery and productivity of plants under stress (review)

A putative way of the protective action of cytokinins on the photosynthetic processes in crops experiencing various stress factors is considered. Various cytokinins are characterized. Pathways of the multiple effects of cytokinin preparations mediating the protection of the photosynthetic machinery from stress are described. Cytokinins interact with receptor proteins, and then the signal is transduced to primary cellular targets (primary response genes). These genes, which possess receptor domains, induce synthesis of the corresponding mRNAs and photosynthesis-related proteins of chlorophyll-protein complexes, the electron-transport chain, and carbon metabolism, primarily, the key enzyme ribulose bisphosphate carboxylase/oxygenase. The protective action of cytokinins under stress conditions preserves the structure and function of the photosynthetic machinery. The application of cytokinins to improving crop yields is discussed.     

The Cytokinin-hypersensitive genes of Arabidopsis negatively regulate the cytokinin-signaling pathway for cell division and chloroplast development

We isolated Arabidopsis thaliana mutants that respond more sensitively than the wild type to cytokinins. The calli produced from the mutants exhibit typical cytokinin responses, including rapid proliferation and chloroplast development in response to lower levels of cytokinins than in the wild type. The mutations are recessive and belong to two complementation groups designated ckh1 and ckh2 for cytokinin-hypersensitive. CKH1 and CKH2 were mapped to the top of chromosome I and the middle of chromosome II, respectively. The cytokinin levels in these mutants were not increased. We speculate that the CKH1 and CKH2 gene products negatively regulate the signaling pathway leading from cytokinin perception to cell proliferation and chloroplast development.     

Dormancy regulation by morphactin in aerial tubers of Begonia evansiana

Summary The sprouting of aerial tubers of Begonia evansiana was promoted by treatment with morphactin. As with cytokinins, the promotion of sprouting occurred in both the immature and mature tubers. Unlike cytokinins, however, morphactin did not stimulate tuber enlargement. The sprout-inhibiting action of applied gibberellin (GA) was overcome by morphactin. The possible mechanism of the inhibitory action of GA is diseussed in relation to apical dominance.     

hoc: An Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity

A novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokinin-overproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth-regulator mutants. In darkness the hoc mutant is de-etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two- and sevenfold the cytokinin level of wild-type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the first cytokinin-overproducing Arabidopsis mutant capable of auto-regenerating shoots without exogenous growth regulators.     

Moss biology and phytohormones--cytokinins in Physcomitrella

Mosses present several advantages for the analysis of phytohormone physiology. Their enormous regeneration capacity, the possibility of controlling their whole life cycle under in vitro culture conditions, as well as the small number of cell types facilitate studies of hormone homeostasis. This review focuses on the metabolism and biosynthesis of cytokinins, mostly summarising data obtained using the moss Physcomitrella patens (Hedw.) B.S.G. which has served as a model system for cytokinin research for many years. A comparison of metabolic differences with respect to seed plants is presented, pointing out an important role of adenosine kinase for the formation of nucleotides during cytokinin interconversion in Physcomitrella. Results on cytokinin biosynthesis in Physcomitrella are summarised with respect to the OVE mutants, which can be considered unique in the plant kingdom due to their strong overproduction of cytokinins. The OVE phenotype is correlated with both increased activity in early stages of cytokinin biosynthesis as well as increased conversion of cytokinin riboside to the base. Cytokinin interconverting reactions can contribute to the increased levels of cytokinins in OVE mutants. Further studies on hormone physiology in moss will help to complete our understanding of hormonal homeostasis by elucidating the situation in an evolutionary early embryophyte.     

Cellular response of light/dark-grown green alga <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> Beijerinck (Chlorophyceae) to exogenous adenine- and phenylurea-type cytokinins

Treatment exposed to light Chlorella vulgaris Beijerinck (Chlorophyceae) with adenine- (BA, Kin, Z) and phenylurea-type (DPU) cytokinins effects positively on alga viability by 1.5- to twofold increase in cell number, chlorophylls, carotenoids, monosaccharides and glycolate content as well as NADH-dependent hydroxypyruvate reducing enzyme activity (NADH-HPR) extensively involved in carbon metabolism. Cytokinins enhance nitrogen assimilation by stimulation of NADH-dependent glutamate dehydrogenase (NADH-GDH) aminating activity finally leading to higher protein level and its secretion as well as polypeptide accumulation in the range of molecular weight 12–195 kDa. In the dark, cytokinins mimic the regulatory effect of light upon algal cell division, metabolite content and stimulate carbon recycling for Calvin cycle reactions by enhancing of light-dependent NADH-HPR activity. The delaying of protein degradation and stimulation of their secretion to environment, triggering polypeptide accumulation and twofold higher NADH-GDH activity catalysing amino acids biosynthesis are observed in the dark-grown microalgae in response to cytokinins. Chlorella vulgaris exhibits sensitivity on cytokinins in the following order of their stimulating properties: DPU > Z > Kin > BA in both light and dark conditions. Understanding of cytokinin role in lower plants under different light conditions could be a step toward the elucidation of the evolution of hormone regulation and their action at molecular level.     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.     

拟南芥细胞分裂素糖基转移酶UGT76C2的N端亮氨酸替换对酶活性的影响

UGT76C2是负责细胞分裂素N-糖基化修饰的糖基转移酶,该基因对于维持植物体内细胞分裂素动态平衡有重要作用。为了进一步研究UGT76C2酶蛋白结构与催化活性的关系,本文采用定点突变方法,将UGT76C2的N端第31位的保守亮氨酸替换为组氨酸。结果发现,突变型UGT76C2在离体实验中完全丧失了对细胞分裂素的糖基化修饰活性,该突变基因的过表达转基因植物出现与UGT76C2突变体类似的表型,转基因植物体内的两类主要细胞分裂素的N-糖苷含量显著降低。实验结果证明了UGT76C2 N端亮氨酸残基对于糖基化修饰活性的重要性。