Arabidopsis Response Regulators Research Articles

Identification of Cytokinin-Responsive Genes Using Microarray Meta-Analysis and RNA-Seq in Arabidopsis

Cytokinins are N(6)-substituted adenine derivatives that play diverse roles in plant growth and development. We sought to define a robust set of genes regulated by cytokinin as well as to query the response of genes not represented on microarrays. To this end, we performed a meta-analysis of microarray data from a variety of cytokinin-treated samples and used RNA-seq to examine cytokinin-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Microarray meta-analysis using 13 microarray experiments combined with empirically defined filtering criteria identified a set of 226 genes differentially regulated by cytokinin, a subset of which has previously been validated by other methods. RNA-seq validated about 73% of the up-regulated genes identified by this meta-analysis. In silico promoter analysis indicated an overrepresentation of type-B Arabidopsis response regulator binding elements, consistent with the role of type-B Arabidopsis response regulators as primary mediators of cytokinin-responsive gene expression. RNA-seq analysis identified 73 cytokinin-regulated genes that were not represented on the ATH1 microarray. Representative genes were verified using quantitative reverse transcription-polymerase chain reaction and NanoString analysis. Analysis of the genes identified reveals a substantial effect of cytokinin on genes encoding proteins involved in secondary metabolism, particularly those acting in flavonoid and phenylpropanoid biosynthesis, as well as in the regulation of redox state of the cell, particularly a set of glutaredoxin genes. Novel splicing events were found in members of some gene families that are known to play a role in cytokinin signaling or metabolism. The genes identified in this analysis represent a robust set of cytokinin-responsive genes that are useful in the analysis of cytokinin function in plants.

Functional Characterization of Type-B Response Regulators in the Arabidopsis Cytokinin Response

Cytokinins play critical roles in plant growth and development, with the transcriptional response to cytokinin being mediated by the type-B response regulators. In Arabidopsis (Arabidopsis thaliana), type-B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]) form three subfamilies based on phylogenic analysis, with subfamily 1 having seven members and subfamilies 2 and 3 each having two members. Cytokinin responses are predominantly mediated by subfamily 1 members, with cytokinin-mediated effects on root growth and root meristem size correlating with type-B ARR expression levels. To determine which type-B ARRs can functionally substitute for the subfamily 1 members ARR1 or ARR12, we expressed different type-B ARRs from the ARR1 promoter and assayed their ability to rescue arr1 arr12 double mutant phenotypes. ARR1, as well as a subset of other subfamily 1 type-B ARRs, restore the cytokinin sensitivity to arr1 arr12. Expression of ARR10 from the ARR1 promoter results in cytokinin hypersensitivity and enhances shoot regeneration from callus tissue, correlating with enhanced stability of the ARR10 protein compared with the ARR1 protein. Examination of transfer DNA insertion mutants in subfamilies 2 and 3 revealed little effect on several well-characterized cytokinin responses. However, a member of subfamily 2, ARR21, restores cytokinin sensitivity to arr1 arr12 roots when expressed from the ARR1 promoter, indicating functional conservation of this divergent family member. Our results indicate that the type-B ARRs have diverged in function, such that some, but not all, can complement the arr1 arr12 mutant. In addition, our results indicate that type-B ARR expression profiles in the plant, along with posttranscriptional regulation, play significant roles in modulating their contribution to cytokinin signaling.

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): typeB ARRs (response activators) and typeA 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 typeA 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 typeA 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.

Phloem development in nematode-induced feeding sites: the implications of auxin and cytokinin

Sedentary plant parasitic nematodes such as root-knot nematodes and cyst nematodes induce giant cells or syncytia, respectively, in their host plant's roots. These highly specialized structures serve as feeding sites from which exclusively the nematodes withdraw nutrients. While giant cells are symplastically isolated and obtain assimilates by transporter-mediated processes syncytia are massively connected to the phloem by plasmodesmata. To support the feeding sites and the nematode during their development, phloem is induced around syncytia and giant cells. In the case of syncytia the unloading phloem consists of sieve elements and companion cells and in the case of root knots it consists exclusively of sieve elements. We applied immunohistochemistry to identify the cells within the developing phloem that responded to auxin and cytokinin. Both feeding sites themselves did not respond to either hormone. We were able to show that in root knots an auxin response precedes the differentiation of these auxin responsive cells into phloem elements. This process appears to be independent of B-type Arabidopsis response regulators. Using additional markers for tissue identity we provide evidence that around giant cells protophloem is formed and proliferates dramatically. In contrast, the phloem around syncytia responded to both hormones. The presence of companion cells as well as hormone-responsive sieve elements suggests that metaphloem development occurs. The implication of auxin and cytokinin in the further development of the metaphloem is discussed.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

Arabidopsis Response Regulator1 and Arabidopsis Histidine Phosphotransfer Protein2 (AHP2), AHP3, and AHP5 Function in Cold Signaling

The Arabidopsis (Arabidopsis thaliana) two-component signaling system, which is composed of sensor histidine kinases, histidine phosphotransfer proteins, and response regulators, mediates the cytokinin response and various other plant responses. We have previously shown that ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and cold-inducible type A ARABIDOPSIS RESPONSE REGULATORS (ARRs) play roles in cold signaling. However, the roles of type B ARRs and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHPs) have not been investigated in cold signaling. Here, we show that ARR1 and AHP2, AHP3, and AHP5 play positive roles in the cold-inducible expression of type A ARRs. arr1 mutants showed greatly reduced cold-responsive expression of type A ARRs compared with the wild type, whereas ARR1-overexpressing Arabidopsis exhibited the hypersensitive cold response of type A ARRs as well as enhanced freezing tolerance with cytokinin, suggesting that ARR1 functions as a positive factor of cold signaling. Transgenic Arabidopsis expressing ARR1ΔDDK:GR lacking the amino-terminal receiver domain showed wild-type expression levels of type A ARRs in response to cold, indicating that the signal receiver domain of ARR1 might be important for cold-responsive expression of type A ARRs. ahp2 ahp3 ahp5 triple mutations greatly reduced type A ARR expression in response to cold, whereas the single or double ahp mutants displayed wild-type levels of ARR expression, suggesting that AHP2, AHP3, and AHP5 are redundantly involved in cold signaling. Taken together, these results suggest that ARR1 mediates cold signal via AHP2, AHP3, or AHP5 from AHK2 and AHK3 to express type A ARRs. We further identified a cold transcriptome affected by ahk2 ahk3 mutations by microarray analysis, revealing a new cold-responsive gene network regulated downstream of AHK2 and AHK3.

Transcriptional changes of antioxidant responses, hormone signalling and developmental processes evoked by the Brassica napus SHOOTMERISTEMLESS during in vitro embryogenesis

Previous work showed that alterations in Brassica napus (Bn) SHOOTMERISTEMLESS (BnSTM) expression levels influence microspore-derived embryogenesis in B. napus. While over-expression of BnSTM increased microspore-derived embryo (MDE) yield and quality, down-regulation of BnSTM repressed embryo formation [16]. Transcriptional analyses were conducted to investigate the molecular mechanisms underpinning these responses. The induction of BnSTM resulted in a heavy transcriptional activation of genes involved in antioxidant responses, hormone signalling and developmental processes. Several antioxidant enzymes, including catalases, superoxide dismutases, and components of the Halliwell-Asada cycle were induced in embryos ectopically expressing BnSTM and contributed to the removal of reactive oxygen species (ROS). These changes were accompanied by elevated levels of ascorbate and glutathione, which have been shown to promote embryonic growth and development. Induction or repression of BnSTM altered the early cytokinin response, whereas late responses, modulated by Type-A Arabidopsis response regulators (ARRs), were induced in MDEs over-expressing BnSTM. Major differences between transgenic MDEs were also observed in the expression pattern of several auxin transporters and key developmental factors required for normal embryogenesis. While some of these factors, BABYBOOM1 (BBM1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), play a key role during early embryogeny, others, CYP78A5, LEAFY COTYLEDON1 and 2 (LEC1 and LEC2), as well as WOX2 and 9, are required for proper embryo development. Collectively these results demonstrate the involvement of BnSTM in novel developmental processes which can be utilized to enhance invitro embryogenesis.

In vitro shoot organogenesis and hormone response are affected by the altered levels of Brassica napus meristem genes

Arabidopsis shoot meristem activity is regulated by a molecular network involving the participation of several components, including SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1), and ZWILLE (ZLL). In an effort to identify the role of these genes during in vitro shoot formation Brassica and Arabidopsis plants were transformed with the Brassica napus (Bn) STM, CLV1, ZLL1 and ZLL2 identified in previous work [1]. In both systems shoot organogenesis was promoted by the over-expression of BnSTM, BnZLL1, and BnZLL2, and repressed by the over-expression of BnCLV1. This distinct regulation, analogous to that occurring during in vivo meristem formation where STM and ZLL encourage stem cell formation while CLV1 accelerates transition to differentiation, suggests similar regulatory mechanisms governing shoot formation in vivo and in vitro. While the BnZLL1 and BnZLL2 induction of shoot organogenesis correlated only to changes in auxin signaling, BnSTM and BnCLV1 evoked major transcriptional alterations in cytokinin response. Besides increasing the transcript levels of two cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE4 (AHK4) and CYTOKININ INDEPENDENT KINASE (CKI1), ectopic expression of BnSTM induced Type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) and repressed Type-A ARRs. Opposite transcriptional patterns occurred in explants over-expressing BnCLV1, characterized by a decreased ability to produce shoots. The role played by Type-A and Type-B ARRs during shoot organogenesis was further examined using a genetic approach which revealed the requirement of ARR12 for the BnSTM positive regulation of shoot organogenesis. Collectively these results expand our knowledge on the function of meristem genes, and provide new tools for enhancing in vitro propagation systems.

Cytokinin‐facilitated proteolysis of ARABIDOPSIS RESPONSE REGULATOR 2 attenuates signaling output in two‐component circuitry

Cytokinins propagate signals via multiple phosphorelays in a mechanism similar to bacterial two-component systems. In Arabidopsis, signal outputs are determined by the activation state of transcription factors termed type-B Arabidopsis response regulators (ARRs); however, their regulatory mechanisms are largely unknown. In this study, we demonstrate that the proteolysis of ARR2, a type-B ARR, modulates cytokinin signaling outputs. ARR2-hemagglutinin (HA) is rapidly degraded by cytokinin treatment, but other type-B ARRs, such as ARR1-HA, ARR10-HA, ARR12-HA and ARR18-HA, are not. ARR2 degradation is mediated by the 26S proteasome pathway, and requires cytokinin-induced phosphorylation of Asp80 residue in the receiver domain. Through mutational analysis of amino acid residues in the receiver domain, we found that substitution of Lys90 with Gly inhibits ARR2 degradation. ARR2(K90G) -HA in transgenic Arabidopsis conferred enhanced cytokinin sensitivity in various developmental processes, including primary root elongation, callus induction, leaf senescence and hypocotyl growth. ARR2(K90G) -HA increased the expression of type-A ARRs, primary cytokinin-responsive genes and indicators of signaling output in two-component circuits. Expression of ARR2(K90G) -HA from the native ARR2 promoter in the arr2-4 knock-out mutant also increased cytokinin sensitivity. In conclusion, ARR2 proteolysis is involved in the maintenance of the primary signaling output for normal developmental processes mediated by cytokinin in Arabidopsis.

Cytokinin Interplay with Ethylene, Auxin, and Glucose Signaling Controls Arabidopsis Seedling Root Directional Growth

Optimal root architecture is established by multiple intrinsic (e.g. hormones) and extrinsic (e.g. gravity and touch) signals and is established, in part, by directed root growth. We show that asymmetrical exposure of cytokinin (CK) at the root tip in Arabidopsis (Arabidopsis thaliana) promotes cell elongation that is potentiated by glucose in a hexokinase-influenced, G protein-independent manner. This mode of CK signaling requires the CK receptor, ARABIDOPSIS HISTIDINE KINASE4 and, at a minimum, its cognate type B ARABIDOPSIS RESPONSE REGULATORS ARR1, ARR10, and ARR11 for full responsiveness, while type A response regulators act redundantly to attenuate this CK response. Ethylene signaling through the ethylene receptor ETHYLENE RESISTANT1 and its downstream signaling element ETHYLENE INSENSITIVE2 are required for CK-induced root cell elongation. Negative and positive feedback loops are reinforced by CK regulation of the expression of the genes encoding these elements in both the CK and ethylene signaling pathways. Auxin transport facilitated by PIN-FORMED2 as well as auxin signaling through control of the steady-state level of transcriptional repressors INDOLE-3-ACETIC ACID7 (IAA7), IAA14, and IAA17 via TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN are involved in CK-induced root cell elongation. This action lies downstream of ethylene and CK induction. Intrinsic signaling in this response operates independently of the extrinsic signal touch, although actin filament organization, which is important in the touch response, may be important for this response, since latrunculin B can induce similar growth. This root growth response may have adaptive significance, since CK responsiveness is inversely related to root coiling and waving, two root behaviors known to be important for fitness.

Type‐A response regulators are required for proper root apical meristem function through post‐transcriptional regulation of PIN auxin efflux carriers

The phytohormones cytokinin and auxin regulate a diverse array of plant processes, often acting together to modulate growth and development. Although much has been learned with regard to how each of these hormones act individually, we are just beginning to understand how these signals interact to achieve an integrated response. Previous studies indicated that exogenous cytokinin has an effect on the transcription of several PIN efflux carriers. Here we show that disruption of type-A Arabidopsis response regulators (ARRs), which are negative regulators of cytokinin signalling, alters the levels of PIN proteins and results in increased sensitivity to N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport. Disruption of eight of the 10 type-A ARR genes affects root development by altering the size of the apical meristem. Furthermore, we show that the effect of cytokinin on PIN abundance occurs primarily at the post-transcriptional level. Alterations of PIN levels in the type-A ARR mutants result in changes in the distribution of auxin in root tips as measured by a DR5::GFP reporter, and an altered pattern of cell division and differentiation in the stem cell niche in the root apical meristem. Together, these data indicate that cytokinin, acting through the type-A ARRs, alters the level of several PIN efflux carriers, and thus regulates the distribution of auxin within the root tip.

The AP2/ERF Transcription Factor WIND1 Controls Cell Dedifferentiation in Arabidopsis

Many multicellular organisms have remarkable capability to regenerate new organs after wounding. As a first step of organ regeneration, adult somatic cells often dedifferentiate to reacquire cell proliferation potential, but mechanisms underlying this process remain unknown in plants. Here we show that an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), is involved in the control of cell dedifferentiation in Arabidopsis. WIND1 is rapidly induced at the wound site, and it promotes cell dedifferentiation and subsequent cell proliferation to form a mass of pluripotent cells termed callus. We further demonstrate that ectopic overexpression of WIND1 is sufficient to establish and maintain the dedifferentiated status of somatic cells without exogenous auxin and cytokinin, two plant hormones that are normally required for cell dedifferentiation. In vivo imaging of a synthetic cytokinin reporter reveals that wounding upregulates the B-type ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin response and that WIND1 acts via the ARR-dependent signaling pathway to promote cell dedifferentiation. This study provides novel molecular insights into how plants control cell dedifferentiation in response to wounding.

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.

CLE Peptides can Negatively Regulate Protoxylem Vessel Formation via Cytokinin Signaling

Cell–cell communication is critical for tissue and organ development. In plants, secretory CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides function as intercellular signaling molecules in various aspects of tissue development including vascular development. However, little is known about intracellular signaling pathways functioning in vascular development downstream of the CLE ligands. We show that CLE peptides including CLE10, which is preferentially expressed in the root vascular system, inhibit protoxylem vessel formation in Arabidopsis roots. GeneChip analysis displayed that CLE10 peptides repressed specifically the expression of two type-A Arabidopsis Response Regulators (ARRs), ARR5 and ARR6, whose products act as negative regulators of cytokinin signaling. The arr5 arr6 roots exhibited defective protoxylem vessel formation. These results indicate that CLE10 inhibits protoxylem vessel formation by suppressing the expression of type-A ARR genes including ARR5 and ARR6. This was supported by the finding that CLE10 did not suppress protoxylem vessel formation in a background of arr10 arr12, a double mutant of type-B ARR genes. Thus, our results revealed cross-talk between CLE signaling and cytokinin signaling in protoxylem vessel formation in roots. Taken together with the indication that cytokinin signaling functions downstream of the CLV3/WUS signaling pathway in the shoot apical meristem, the cross-talk between CLE and cytokinin signaling pathways may be a common feature in plant development.

Hormonal control of the shoot stem-cell niche

The classic phytohormones cytokinin and auxin play essential roles in the maintenance of stem-cell systems embedded in shoot and root meristems, and exhibit complex functional interactions. Here we show that the activity of both hormones directly converges on the promoters of two A-type ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, ARR7 and ARR15, which are negative regulators of cytokinin signalling and have important meristematic functions. Whereas ARR7 and ARR15 expression in the shoot apical meristem (SAM) is induced by cytokinin, auxin has a negative effect, which is, at least in part, mediated by the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP) transcription factor. Our results provide a mechanistic framework for hormonal control of the apical stem-cell niche and demonstrate how root and shoot stem-cell systems differ in their response to phytohormones.

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.

Characterization of Arabidopsis Response Regulator Genes with Regard to Bisphenol A Signaling

Arabidopsis response regulators (ARRs) are involved in the His-to-Asp phosphotransfer signal transduction mechanism induced by cytokinins, a class of phytohormones in Arabidopsis thaliana. Previous studies have reported ARR mRNA accumulation in response to cytokinins. ARRs consist of 2 families (types A and B). The expression of type-A ARR genes can be induced by cytokinins, while that of type-B genes is constitutive. Recently, we have shown that plant growth induced by the endocrine disruptor bisphenol A (4,4� -isopropylidenediphenol) was similar to that induced by cytokinins. The present study is the first to show that ARR (type-A family; ARR15 and ARR16) mRNA accumulation is influenced by bisphenol A, indicating that bisphenol A is involved in inducing intracellular signal transduction.

Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration

In Arabidopsis, the network responsible for the maintenance of the shoot apical meristem (SAM) is built on a negative feedback loop involving the peptide ligand CLAVATA3 (CLV3) and the homeodomain transcription factor WUSCHEL (WUS). The local WUS/CLV3 regulatory module is linked to the organism-wide cytokinin signalling system by direct transcriptional control of A-type ARABIDOPSIS RESPONSE REGULATOR genes ( ARRs) by WUS. Here we investigate two A-type ARR genes , ARR7 and ARR15, which are negative regulators of cytokinin signalling. We show that the expression of ARR7, WUS and CLV3 is dependent on cytokinin signalling. While ARR7 expression strongly responds to variations in cytokinin activity, WUS and CLV3 appeared to be much more buffered against this type of variation. As earlier studies had shown that pertubation of A-type ARR activity only causes mild effects on SAM function, we employed tissue regeneration assays as a sensitised background. Root explants pre-treated on auxin and cytokinin-rich callus-inducing medium showed severely suppressed shoot regeneration when ARR7 and ARR15 were overexpressed, whereas loss of function of these genes had a strongly promoting effect. This phenotype was even aggravated in the arr3,4,5,6,7,8,9 septuple mutant. Futhermore, loss-of A-type ARR function in arr7 and arr3,4,5,6,7,8,9 mutants strongly stimulated callus development, indicating that cell proliferation is repressed by A-type ARRs. To elucidate the mechanisms underlying the enhanced capacity of the arr3,4,5,6,7,8,9 septuple mutant to develop shoot tissue in culture, we used whole-genome expression profiling. Among the transcripts with increased abundance in arr3,4,5,6,7,8,9 inflorescence apices a strong enrichment for functions in pollen development was apparent, while the reduced transcripts showed a more heterogeneous distribution of functional categories, ranging from development to pathogen defence.

Ectopic expression of ARR1ΔDDK in tobacco: alteration of cell fate in root tip region and shoot organogenesis in cultured segments

A specific deleted version of ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) lacking the signal receiver domain (1–152 amino acids)-coding sequence, referred to as ARR1ΔDDK, was amplified using Arabidopsis thaliana cDNA prepared from adult leaves and transferred into the genome of Nicotiana tabacum cv. Samsun under the transcriptional control of a β-estradiol-inducible expression system. The ectopic expression of ARR1ΔDDK affected the morphology of transgenic seedlings and their segments in vitro. In the presence of an inducer, β-estradiol, ectopic expression of ARR1ΔDDK induced only the formation of soft, pseudo-bulbous tissue in the root tip region of intact seedlings, which appeared similar to callus generated on a hypocotyl segment in the presence of 2,4-d and 6-benzyladenine (BA), both at 1 μM. Those callus tissues on the root tip region could not generate shoots unless 1 μM BA was supplied. In segment culture, ectopic expression of ARR1ΔDDK induced callus-like tissue around the cut-end of cotyledon and hypocotyl segments with occasional shoot formation, suggesting that the expression of ARR1ΔDDK could substitute for the effects of cytokinin on these segments. Additionally, treatment with only β-estradiol induced NtWUS, a WUS ortholog in tobacco, which was detected during the process of callus tissue formation in the root tip region and also in cotyledon or hypocotyl segments. These findings suggest that the NtWUS might be associated in the transdifferentiation process caused by the functional regulation of ARR1ΔDDK in transgenic tobacco seedlings.

Genome-wide comparative analysis of type-A Arabidopsis response regulator genes by overexpression studies reveals their diverse roles and regulatory mechanisms in cytokinin signaling

Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cytokinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from the receptors, through histidine phosphotransfer proteins, to the downstream response regulators (ARRs). Of these ARRs, type-A ARR genes, whose transcription can be rapidly induced by cytokinin, act as negative regulators of cytokinin signaling. However, because of functional redundancy, the function of type-A ARR genes in plant growth and development is not well understood by analyzing loss-of-function mutants. In this study, we performed a comparative functional study on all ten type-A ARR genes by analyzing transgenic plants overexpressing these ARR genes fused to a MYC epitope tag. Overexpression of ARR genes results in a variety of cytokinin-associated phenotypes. Notably, overexpression of different ARR transgenes causes diverse phenotypes, even between phylogenetically closely-related gene pairs, such as within the ARR3-ARR4 and ARR5-ARR6 pairs. We found that the accumulation of a subset of ARR proteins (ARR3, ARR5, ARR7, ARR16 and ARR17; possibly ARR8 and ARR15) is increased by MG132, a specific proteasomal inhibitor, indicating that stability of these proteins is regulated by proteasomal degradation. Moreover, similar to that of previously characterized ARR5, ARR6 and ARR7, stability of ARR16 and ARR17, possibly including ARR8 and ARR15, is regulated by cytokinin. These results suggest that type-A ARR proteins are regulated by a combinatorial mechanism involving both the cytokinin and proteasome pathways, thereby executing distinctive functions in plant growth and development.