SHORT-ROOT regulates vascular patterning,but not apical meristematic activity in the Arabidopsis root through cytokinin homeostasis

SHORT-ROOT (SHR) is a key regulator of radial patterning and stem-cell renewal in the Arabidopsis root. Although SHR is expressed in the stele, its function in the vascular tissue was not recognized until recently. In shr, the protoxylem is missing due to the loss of expression of microRNA165A (miR165A) and microRNA166B (miR165B). shr is also defective in lateral root formation, but the mechanism remains unclear. To dissect the SHR developmental pathway, we recently have identified its direct targets at the genome scale by chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). In further studies, we have shown that SHR regulates cytokinin homeostasis through cytokinin oxidase 3 and that this role of SHR is critical to vascular patterning in the root. In this communication we report that SHR also regulates miR165A and miR166B indirectly through its effect on cytokinin homeostasis. Although cytokinin is inhibitory to root growth, the root-apical-meristem defect in shr was not alleviated by reduction of endogenous cytokinin. These results together suggest that SHR regulates vascular patterning, but not root apical meristematic activity, through cytokinin homeostasis.     

Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death

The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B(1)-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.     

Plant growth promotion by Bacillus megaterium involves cytokinin signaling

Accumulating evidence indicates that plant growth promoting rhizobacteria (PGPR) influence plant growth and development by the production of phytohormones such as auxins, gibberellins, and cytokinins. Little is known on the genetic basis and signal transduction components that mediate the beneficial effects of PGPRs in plants. We recently reported the identification of a Bacillus megaterium strain that promoted growth of A. thaliana and P. vulgaris seedlings. In this addendum, the role of cytokinin signaling in mediating the plant responses to bacterial inoculation was investigated using A. thaliana mutants lacking one, two or three of the putative cytokinin receptors CRE1, AHK2 and AHK3, and RPN12 a gene involved in cytokinin signaling. We show that plant growth promotion by B. megaterium is reduced in AHK2-2 single and double mutant combinations and in RPN12. Furthermore, the triple cytokinin-receptor CRE1-12/AHK2-2/AHK3-3 knockout was insensitive to inoculation in terms of growth promotion and root developmental responses. Our results indicate that cytokinin receptors play a complimentary role in plant growth promotion by B. megaterium.Key words: Arabidopsis, plant growth stimulation, root development, rhizobacteria     

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.     

Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation

Degradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7–GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7‐expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis‐zeatin (cZ) and N‐glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ‐deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone.     

RcRR1, a Rosa canina Type-A Response Regulator Gene,Is Involved in Cytokinin-Modulated Rhizoid Organogenesis

In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina.     

A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation

Cytokinin is an adenine derivative plant hormone that generally regulates plant cell division and differentiation in conjunction with auxin. We report that a major cue for the negative regulation of sulfur acquisition is executed by cytokinin response 1 (CRE1)/wooden leg (WOL)/Arabidopsis histidine kinase 4 (AHK4) cytokinin receptor in Arabidopsis root. We constructed a green fluorescent protein (GFP) reporter system that generally displays the expression of the high-affinity sulfate transporter SULTR1;2 in Arabidopsis roots. GFP under the control of SULTR1;2 promoter showed typical sulfur responses that correlate with the changes in SULTR1;2 mRNA levels; accumulation of GFP was induced by sulfur limitation (-S), but was repressed in the presence of reduced sulfur compounds. Among the plant hormones tested, cytokinin significantly downregulated the expression of SULTR1;2. SULTR1;1 conducting sulfate uptake in sultr1;2 mutant was similarly downregulated by cytokinin. Downregulation of SULTR1;1 and SULTR1;2 by cytokinin correlated with the decrease in sulfate uptake activities in roots. The effect of cytokinin on sulfate uptake was moderated in the cre1-1 mutant, providing genetic evidence for involvement of CRE1/WOL/AHK4 in the negative regulation of high-affinity sulfate transporters. These data demonstrated the physiological importance of the cytokinin-dependent regulatory pathway in acquisition of sulfate in roots. Our results suggested that two different modes of regulation, represented as the -S induction and the cytokinin-dependent repression of sulfate transporters, independently control the uptake of sulfate in Arabidopsis roots.     

Melatonin Antagonizes Cytokinin Responses to Stimulate Root Growth in Arabidopsis

Melatonin functions as the key growth regulator in various plant species. The mechanisms of the interactions between melatonin and cytokinins remain largely unknown. In this study, the kinetic effects of melatonin over a range of concentrations were investigated. The results showed that melatonin functioned as a positive regulator of root growth ranged from 0.1 to 100 nM. In contrast, exogenous cytokinin at 0.5–1 nM and overexpression of cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE 8 (IPT8) inhibited primary root growth. Combined treatments with melatonin and cytokinin indicated that melatonin antagonized the inhibitory effect of cytokinin 6-benzylaminopurine (6-BA) on primary root elongation. Further analysis revealed that melatonin promotes primary root growth by modulating expression and distribution of auxin efflux transporters PIN2/3 and influx transporter AUX1. Moreover, the cytokinin signaling components AHK4, AHP2/3/5, and type-A ARR15 were down-regulated after melatonin treatment. The polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) impaired the promotive effect of melatonin on primary root growth, indicating that auxin transport is essential in melatonin-mediated root growth. Taken together, our data provided evidence to show that melatonin regulates primary root growth in coordination with cytokinin partially through auxin-dependent pathway.

     

AtSKIP functions as a mediator between cytokinin and light signaling pathway in Arabidopsis thaliana

Key message

AtSKIP participated in cytokinin-regulated leaf initiation. Putative phosphorylated AtSKIP (AtSKIP ^DD ) displayed the opposite function in the leaf development from AtSKIP transgenic seedlings.

Abstract

AtSKIP, as a multiple protein, is involved in many physiological processes, such as flowering, cell cycle regulator, photomorphogenesis and stress tolerance. However, the mechanism of AtSKIP in these processes is unclear. Here, we identify one gene, AtSKIP, which is associated with cytokinin-regulated leaf growth process in Arabidopsis. The expression of AtSKIP was regulated by cytokinin. Leaf development in AtSKIP overproduced seedlings was independent of light, but promoted by cytokinin, and phosphorylation of AtSKIP (AtSKIP^DD) partially interfered with AtSKIP function as a positive regulator in cytokinin signaling, indicative of true leaf formation, and the defects of AtSKIP^DD in the true leaf formation could be recovered to some extent by the addition of cytokinin. Moreover, different cytokinin-responsive gene Authentic Response Regulator 7 (ARR7) promoter-GUS activity further proved that expression of AtSKIP or AtSKIP^DD altered endogenous cytokinin signaling in plants. Together, these data indicate that AtSKIP participates in cytokinin-regulated promotion of leaf growth in photomorphogenesis, and that phosphorylation interferes with AtSKIP normal function.     

The MeJA-inducible copper amine oxidase AtAO1 is expressed in xylem tissue and guard cells

Copper amine oxidases oxidize the polyamine putrescine to 4-aminobutanal with the production of the plant signal molecule hydrogen peroxide (H₂O₂) and ammonia. The Arabidopsis (Arabidopsis thaliana) gene At4g14940 (AtAO1, previously referred to as ATAO1) encodes an apoplastic copper amine oxidase expressed in lateral root cap cells and developing xylem, especially in root protoxylem and metaxylem precursors. In our recent study, we demonstrated that AtAO1 expression is strongly induced in the root vascular tissues by the wound-signal hormone methyl jasmonate (MeJA). Furthermore, we also demonstrated that the H₂O₂ derived by the AtAO1-driven oxidation of putrescine, mediates the MeJA–induced early protoxylem differentiation in Arabidopsis roots. H₂O₂ may contribute to protoxylem differentiation by signaling developmental cell death and by acting as co-substrate in peroxidase-mediated cell wall stiffening and lignin polymerization. Here, by the means of AtAO1 promoter::green fluorescent protein-β-glucuronidase (AtAO1::GFP-GUS) fusion analysis, we show that a strong AtAO1 gene expression occurs also in guard cells of leaves and flowers. The high expression levels of AtAO1 in tissues or cell types regulating water supply and water loss may suggest a role of the encoded protein in water balance homeostasis, by modulating coordinated adjustments in anatomical and functional features of xylem tissue and guard cells during acclimation to adverse environmental conditions.     

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.     

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.     

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.