Stunted plant 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 microM zeatin) or auxin (30 nM 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem.     

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.     

Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings.

Cytokinins have profound effects on seedling development in Arabidopsis thaliana. Benzyladenine (BA) inhibits root elongation in light- or dark-grown seedlings, and in dark-grown seedlings BA inhibits hypocotyl elongation and exaggerates the curvature of apical hooks. The latter are characteristic ethylene responses and, therefore, the possible involvement of ethylene in BA responses was examined in seedlings. It was found that the inhibitory effects of BA on root and hypocotyl elongation were partially blocked by the action of ethylene inhibitors or ethylene-resistant mutations (ein1-1 and ein2-1). Ethylene production was stimulated by submicromolar concentrations of BA and could account, in part, for the inhibition of root and hypocotyl elongation. It was demonstrated further that BA did not affect the sensitivity of seedlings to ethylene. Thus, the effect of cytokinin on root and hypocotyl elongation in Arabidopsis appears to be mediated largely by the production of ethylene. The coupling between cytokinin and ethylene responses is further supported by the discovery that the cytokinin-resistant mutant ckr1 is resistant to ethylene and is allelic to the ethylene-resistant mutant ein2.     

The Effects of Cytokinin and Light on Hypocotyl Elongation in Arabidopsis Seedlings Are Independent and Additive

Cytokinin has been reported to mimic some of the effects of light on de-etiolation responses in dark-grown Arabidopsis seedlings. The interaction between cytokinin and light was examined by analyzing cytokinin dose and light fluence effects on hypocotyl elongation in wild-type and mutant Arabidopsis seedlings with defects in light or hormone responses. It was found that (a) cytokinin and light-response systems have independent and additive effects on the inhibition of hypocotyl elongation and (b) either cytokinin or light can saturate the morphogenic responses. As a consequence, cytokinin has no effect on hypocotyl elongation under normal growth conditions because light levels saturate the hypocotyl inhibition response. To determine whether a functional light-response pathway is required for cytokinin responses, light-insensitive long hypocotyl (hy) mutants were tested for cytokinin responses. The hy mutants (hy1 to hy6) had normal cytokinin responses, except phyB-1 (hy3-1), in which hypocotyl elongation was insensitive to cytokinin. Cytokinin insensitivity in phyB-1 was attributed to an indirect effect of the mutation on cytokinin responses. The effects of cytokinin on the inhibition of hypocotyl elongation are largely mediated by ethylene, and blocking the ethylene-response pathway through the action of a cytokinin-resistant, ethylene-insensitive mutant (ckr1/ein2) had no effect on the light inhibition of hypocotyl elongation. These results do not support the idea that cytokinin mediates the action of light on hypocotyl elongation.     

Ethylene directs auxin to control root cell expansion

Root morphogenesis is controlled by the regulation of cell division and expansion. We isolated an allele of the eto1 ethylene overproducer as a suppressor of the auxin-resistant mutant ibr5, prompting an examination of crosstalk between the phytohormones auxin and ethylene in control of root epidermal cell elongation and root hair elongation. We examined the interaction of eto1 with mutants that have reduced auxin response or transport and found that ethylene overproduction partially restored auxin responsiveness to these mutants. In addition, we found that the effects of endogenous ethylene on root cell expansion in eto1 seedlings were partially impeded by dampening auxin signaling, and were fully suppressed by blocking auxin influx. These data provide insight into the interaction between these two key plant hormones, and suggest that endogenous ethylene directs auxin to control root cell expansion.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

The regulation of root growth in response to phosphorus deficiency mediated by phytohormones in a Tibetan wild barley accession

Low phosphorus (LP) causes a dramatic change of root system architecture in plants, which is possibly mediated by signaling pathways of hormones. In order to understand the regulatory mechanisms of the root development under LP, we examined the potential role of phytohormones in response to LP using three barley genotypes, differing in LP tolerance, namely 2 Tibetan wild barley genotypes XZ99 (LP tolerant) and XZ100 (LP sensitive), and a cultivated barley ZD9 (LP moderately tolerant). The results showed that LP stress caused a number of changes in root development, with XZ99 having less primary root growth inhibition, more lateral root and root hair formation than the other two genotypes. Meanwhile, LP stress also resulted in the dramatic changes in plant hormone contents, with changed extent and pattern differing among the three genotypes. The relative expression of genes responsible for indole acetic acid (IAA) and ethylene synthesis in roots also showed a significant difference among genotypes in both control and LP conditions. It can be concluded that the root system of Tibetan wild barley XZ99 adapts to phosphorus deficiency by changing the signal transduction pathway mediated by auxin, ethylene and cytokinins. However, further studies are needed to elucidate the behaviors of the key genes involved in the hormone-related response.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Role of cytokinin in the regulation of root gravitropism

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1381-8     

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.     

Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid

Etiolated pea seedlings ( Pisum sativum L. cv. Weibull's Marma) were used to investigate the effects of exogenous cytokinins on root growth. Benzylaminopurine (BAP) added to the growth solution inhibited the elongation and formation of lateral roots and stimulated swelling of the root tips. Similar effects were obtained with zeatin. The effects were obtained over a wide concentration range down to 0.01 μ M . Growth responses appeared only after treatment for several hours, and the duration of treatment had an important influence on the degree of the effects. BAP caused a moderate increase in ethylene production as measured in excised 10-mm-long root tips. Lowering ethylene production by treatment with cobalt ions counteracted both the inhibition and swelling caused by BAP. Treatment with silver ions also reversed the effect to some extent, indicating that ethylene is involved in the response of the roots to BAP. To further study the involvement of the increased ethylene production in the elongation and swelling response, the effects were compared with those obtained after application of 1-aminocyclopropane-1-carboxylic acid (ACC) in relation to the ethylene produced from this compound. This comparison showed that the increase in ethylene production caused by BAP was too low to explain the response of the roots. However, ACC treatment caused a considerable lowering of the content of indole-3-acetic acid (IAA) in the root tips, whereas BAP did not; instead, BAP increased the amount of IAA per root tip. It is concluded that cytokinins influence growth processes in roots via several mechanisms. A synergistic interaction between endogenous IAA, maintained at a high level by the cytokinin treatment, and the increased ethylene levels appears to explain most of the cytokinin effects during the first day of treatment.     

Characterization of a novel putative zinc finger gene MIF1: involvement in multiple hormonal regulation of Arabidopsis development

Phytohormones play crucial roles in regulating many aspects of plant development. Although much has been learned about the effects of individual hormones, cross-talk between and integration of different hormonal signals are still not well understood. We present a study of MINI ZINC FINGER 1 (MIF1), a putative zinc finger protein from Arabidopsis, and suggest that it may be involved in integrating signals from multiple hormones. MIF1 homologs are highly conserved among seed plants, each characterized by a very short sequence containing a central putative zinc finger domain. Constitutive overexpression of MIF1 caused dramatic developmental defects, including dwarfism, reduced apical dominance, extreme longevity, dark-green leaves, altered flower morphology, poor fertility, reduced hypocotyl length, spoon-like cotyledons, reduced root growth, and ectopic root hairs on hypocotyls and cotyledons. In addition, 35S::MIF1 seedlings underwent constitutive photomorphogenesis in the dark, with root growth similar to that in the light. Furthermore, 35S::MIF1 seedlings were demonstrated to be non-responsive to gibberellin (GA) for cell elongation, hypersensitive to the GA synthesis inhibitor paclobutrazol (PAC) and abscisic acid (ABA), and hyposensitive to auxin, brassinosteroid and cytokinin, but normally responsive to ethylene. The de-etiolation defect could not be rescued by the hormones tested. Consistent with these observations, genome-scale expression profiling revealed that 35S::MIF1 seedlings exhibited decreased expression of genes involved in GA, auxin and brassinosteroid signaling as well as cell elongation/expansion, and increased expression of ABA-responsive genes. We propose that MIF1, or the protein(s) with which MIF1 interacts, is involved in mediating the control of plant development by multiple hormones.     

The Effect of Ethylene on the Growth and Development of Wild-type and Mutant Arabidopsis thaliana (L.) Heynh

Some developmental responses of wild-type and mutant strainsof Arabidopsis thaliana to ethylene have been investigated (shootand root elongation, isodiametric cell expansion, plumular hookangle, ethylene biosynthesis, peroxidase activity). One suchmutant (eti 5) shows little or no response to ethylene at concentrationsup to 10000 µl 1^–1 in any test; the other mutantsshow reduced responses. The ability of germinated seedlingsto emerge through sand is directly proportional to their sensitivityto ethylene. It is suggested that this finding supports thehypothesis that increased ethylene production in response tomechanical impedance is involved, via its effects on stem thickeningand hook closure, in the emergence process. Arabidopsis thaliana (L.) Heynh., ethylene insensitive mutants, seedling emergence     

Hormonal Regulation of Cell Elongation in the Hypocotyl of Rootless Soybean: An Evaluation of the Role of DNA Synthesis

A method was developed where soybean seedlings were grown without roots to study the influence of hormones of root origin on shoot growth. Excision of the root resulted in inhibition of apical section growth and DNA synthesis and inhibited elongating section growth. A synthetic cytokinin restored DNA synthesis in the apical section, but did not influence growth in either the apical or elongating sections. Low concentrations of gibberellin with the cytokinin restored growth in the apical section. Gibberellin alone was sufficient to restore growth in the elongating section.An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, inhibited the increase in apical section DNA without inhibiting control or gibberellin-induced growth in the elongating section. Experiments with (14)C-thymidine resulted in no DNA labeling differences in the elongating section under conditions where gibberellin-induced elongation varied from 50% to 73% above controls. It was concluded that gibberellin-induced elongation in soybean hypocotyl occurred in the absence of DNA synthesis. Gibberellin does stimulate DNA synthesis in the apical tissue apart from its effect on cell elongation.Excised soybean hypocotyl elongated maximally at 10(-6)m auxin. At higher auxin concentrations, fresh weight and ethylene production increased, but elongation was reduced. Addition of GA to the higher auxin concentrations resulted in a 50% inhibition in auxin-induced ethylene production and resumption in maximal elongation. Added ethylene inhibited elongation 30% at 2 mul/l. Addition of up to 100 mul/l ethylene did not inhibit elongation with GA present in the incubation medium. Thus GA may counteract ehtylene inhibition of cell elongation in addition to inhibiting ethylene production in auxin-treated tissues.     

The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development

Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.     

ZEA3: A Negative Modulator of Cytokinin Responses in Plant Seedlings

In Nicotiana plumbaginifolia cytokinins affect seedling development by inhibiting root growth and hypocotyl elongation and by stimulating cotyledon expansion. The zea3.1 mutant was selected for its inability to grow in conditions of low nitrogen and for its ability to grow independently on inhibitory concentrations of zeatin (J.D. Faure, M. Jullien, M. Caboche [1994] Plant J 5: 481-491). The zea3.1 growth response to cytokinins is reflected by an increase in cotyledon expansion due to cell division and by a swelling of the hypocotyl due to cell enlargement. An analysis of the seedling's root length and fresh weight over a wide range of benzyladenine concentrations showed that zea3.1 plants exhibit a higher sensitivity and an amplified response to cytokinins. A similar response of zea3.1 to benzyladenine was also seen in the expression of msr1, a cytokinin-regulated gene. Regulation of msr1 expression by protein phosphorylation was unaffected by the zea3.1 mutation. No significant differences in cytokinin and auxin levels were found between zea3.1 and wild-type seedlings, suggesting that the mutant phenotype is not caused by an alteration of these hormone levels. The data presented suggest that ZEA3 negatively modulates cytokinin responses and may function as a broad regulator of seedling development.