Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay

Strains of Escherichia coli that express two different cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, were used to study the relative sensitivity of these receptors to various cytokinins. Both receptors were most sensitive to the bases of the isoprenoid-type cytokinins trans-zeatin and isopentenyladenine but differed significantly in the recognition of other cytokinin compounds. In particular, CRE1/AHK4 recognized at 1 microm concentration only trans-zeatin while AHK3 recognized cis-zeatin and dihydrozeatin as well, although with a lower sensitivity. Similarly, CRE1/AHK4 was not activated by cytokinin ribosides and ribotides, but AHK3 was. Comparisons using the ARR5::GUS fusion gene as a cytokinin reporter in Arabidopsis showed similar relative degrees of responses in planta, except that cytokinins with aromatic side chains showed much higher activities than in the bacterial assay. These results indicate that the diverse cytokinin compounds might have specific functions in the numerous cytokinin-regulated processes, which may depend in turn on different receptors and their associated signalling pathways. The importance of precise control of local concentrations of defined cytokinin metabolites to regulate the respective downstream event is corroborated.     

Exogenous brassinosteroids activate cytokinin signalling pathway gene expression in transgenic Arabidopsis thaliana

Transgenic Arabidopsis thaliana plants carrying the GUS reporter gene fused to the promoter of the gene of primary response to cytokinins (CKs), ARR5, were used to estimate the influence of several brassinosteroids (BRs): brassinolide (BL), epibrassinolide (EBL), homobrassinolide (HBL), and 6-o-carboxymethyloxohomocastasterone (CHC) on the expression of CK signalling genes. BRs tested differed in their ability to activate the ARR5 gene promoter in 4-day-old seedlings and 3-week-old plants. BL caused the most prominent effect, yet it was considerably less than that of 6-benzylaminopurine (BA). An increase in GUS activity was observed in both dark and light conditions; however, the rate of elevation was higher in dark conditions. The activation of the P _ARR5 :GUS fusion was accompanied by a moderate induction of the P _AHK :GUS constructs, in which the reporter GUS gene was fused to the promoter of one of the CK receptor histidine kinases. The effects of BL on the AHK gene promoters were organ specific and correlated with the ability of a particular AHK gene to respond to BA treatment. BL activated the AHK3 promoter in 4-day-old seedlings and in shoots and roots of 3-week-old plants without any effect in detached leaves. The AHK2 gene promoter was activated by BA and BL only in seedlings, whereas the AHK4 gene promoter was activated only in roots. BL treatment caused the coordinate elevation of the CK levels in leaves to the same degree as the activation of the P _AHK :GUS construct, suggesting that the accumulation of CKs was the reason for the activation by BRs of the CK signalling genes. The data obtained provide the evidence for the involvement of BRs in the regulation of the genes of the CK signalling pathway through an increase in the CK levels. However, the exact molecular mechanisms underlying BR-induced elevation of the CK content are unclear and warrant identification in the future.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.     

Cytokinin receptor antagonists derived from 6-benzylaminopurine

Recently we reported 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) as the first molecule to antagonize cytokinin activity at the receptor level. Here we report the synthesis and in vitro biological testing of eleven BAP derivatives substituted in the benzyl ring and in the C2, N7 and N9 positions of the purine moiety. The ability of the compounds to interact with Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 was tested in bacterial receptor and in live-cell binding assays, and in an Arabidopsis ARR5:GUS (Arabidopsis response regulator 5) reporter gene assay. Cytokinin activity of the compounds was determined in classical cytokinin biotests (tobacco callus, wheat leaf senescence and Amaranthus bioassays). 6-(2,5-Dihydroxybenzylamino)purine (LGR-991) was identified as a cytokinin receptor antagonist. At the molecular level LGR-991 blocks the cytokinin receptor CRE1/AHK4 with the same potency as PI-55. Moreover, LGR-991 acts as a competitive inhibitor of AHK3, and importantly shows reduced agonistic effects in comparison to PI-55 in the ARR5:GUS reporter gene assay and in cytokinin bioassays. LGR-991 causes more rapid germination of Arabidopsis seeds and increases hypocotyl length of dark-grown seedlings, which are characteristics of plants with a reduced cytokinin status. LGR-991 exhibits a structural motive that might lead to preparation of cytokinin antagonists with a broader specificity and reduced agonistic properties.     

Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1

Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.     

The altered responses of a new mutant long life span 1 to cytokinin in Arabidopsis thaliana

Cytokinins are a class of essential plant hormones regulating plant growth and development.Although the two-component phosphorelay pathway of cytokinin has been well characterized,the intact cytokinin responses regulation picture still needs to be fully depicted.Here we report a new mutant,long life span 1(lls1),which displays dwarf stature,curled leaves,numerous axillary branches and nearly 5-month life span.Exogenous cytokinin could not recover the phenotypes of the mutant.Moreover,mutation in lls1 suppressed the cytokinin-responsive phenotypes,including root and hypocotyl growth inhibition,anthocyanin accumulation,metaxylem promotion in primary root development.The induction of cytokinin-responsive genes,ARR5,AHP5,and CKX3,was also suppressed in lls1.According to quantitative RT-PCR(qRT-PCR) and microarray results,the basal expression of positive factors AHP5,ARR1,and ARR10 were down-regulated,while the negative factors ARR4 and ARR5 were up-regulated.Our results suggested that LLS1 gene might be involved in the regulation of cytokinin signaling.It was mapped to chromosome 4 where no other cytokinin relevant gene has been reported.     

Characterization of the ARR15 and ARR16 response regulators with special reference to the cytokinin signaling pathway mediated by the AHK4 histidine kinase in roots of Arabidopsis thaliana

The cytokinin receptor AHK4 histidine kinase, identified in Arabidopsis thaliana, presumably acts in concert with downstream components, such as histidine-containing phosphotransfer (HPt) factors (AHPs) and response regulators (ARRs). In this respect, we characterized a loss-of-function mutant of the AHK4 gene, named cre1-1, which showed a reduced cell number within the vascular tissues in roots. Among the 10 type-A ARR members, the expression of ARR15 and ARR16 in roots was specifically and markedly reduced in cre1-1, suggesting a link between these response regulators and the AHK4-mediated signal transduction in roots. The results for transgenic plants expressing promoter::GUS or promoter::LUC fusion genes showed that both the ARR15 and the ARR16 gene products are accumulated upon cytokinin treatment in roots. The results of GFP-fusion experiments with onion epidermal cells further showed that ARR15 was found in the nucleus, and ARR16 mainly in the cytoplasm. Together, it was suggested that ARR15 and ARR16 are distinctly implicated in the presumed AHK4-mediated signaling pathway in roots.     

Proteasome-dependent proteolysis has a critical role in fine-tuning the feedback inhibition of cytokinin signaling

The ubiquitin/26S proteasome-dependent proteolysis of response regulators is a critical element of many plant hormone signaling pathways. We have recently shown that cytokinin signaling requires the AXR1 component of the related to ubiquitin (RUB) protein modification pathway to promote the proteasome-dependent degradation of the cytokinin response inhibitor ARR5. Here, we show that ARR5 also accumulates in the 26S proteasome mutant rpn12a-1, and leads to a marked resistance to cytokinins. Collectively, these results suggest that proteasome-dependent proteolysis of feedback inhibitors such as ARR5 is essential for the maintenance of optimal responsivity and plasticity in cytokinin signaling.     

Cytokinin-dependent expression of the ARR5::GUS construct during transgenic arabidopsis growth

Growth and glucuronidase (GUS) activity were followed in the cotyledons and rosette leaves of Arabidopsis thaliana (L.) Heynh (ecotype Wassilewskija) plants transformed with the GUS gene under the control of the cytokinin-dependent promoter of the ARR5 gene. The presence of active cytokinins in plant tissues was assessed from GUS activity. Plants were grown for three weeks on the nitrate-or ammonium-containing nutrient medium. In plants grown on ammonium nutrition, cotyledon and leaf growth was substantially suppressed as compared with plants feeding with nitrates. In correspondence with this growth inhibition, GUS activity was markedly lower in plant leaves grown on the ammonium-containing medium. This indicated a reduction in these leaves of active cytokinin forms capable of activation of the promoter for the ARR5 gene. On both nitrogen sources, GUS activity increased during leaf growth and dropped sharply after growth ceasing. This indicated that leaf growth depended on the cytokinin content in them. High GUS activity was detected in petioles and leaf conductive system, indicating leaf providing with cytokinins along the conductive vessels. A sharp drop in the GUS activity after leaf growth stoppage coincided in time with GUS activation in the leaf positioned above this leaf. This indicated possible cytokinin redistribution in the plant; its content could be a limiting factor for leaf growth. A higher growth rate in plants on nitrate nitrogen nutrition and corresponding high GUS activity in them are discussed in terms of cytokinin signaling role in leaf growth regulation mediated by nitrate.     

AXR1 promotes the Arabidopsis cytokinin response by facilitating ARR5 proteolysis

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multi‐step phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): type B ARRs (response activators) and type A ARRs (negative‐feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the RUB (related to ubiquitin) modification pathway, lead to decreased cytokinin sensitivity. Here we show that the cytokinin resistance of axr1 seedlings is suppressed by loss of function of the type A ARR family member ARR5. Based on the established role of the RUB pathway in ubiquitin‐dependent proteolysis, these data suggest that AXR1 promotes the cytokinin response by facilitating type A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity.     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.