Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem

Cytokinin phytohormones regulate a variety of developmental processes in the root such as meristem size, vascular pattern, and root architecture [1-3]. Long-distance transport of cytokinin is supported by the discovery of cytokinins in xylem and phloem sap [4] and by grafting experiments between wild-type and cytokinin biosynthesis mutants [5]. Acropetal transport of cytokinin (toward the shoot apex) has also been implicated in the control of shoot branching [6]. However, neither the mode of transport nor a developmental role has been shown for basipetal transport of cytokinin (toward the root apex). In this paper, we combine the use of a new technology that blocks symplastic connections in the phloem with a novel approach to visualize radiolabeled hormones in planta to examine the basipetal transport of cytokinin. We show that this occurs through symplastic connections in the phloem. The reduction of cytokinin levels in the phloem leads to a destabilization of the root vascular pattern in a manner similar to mutants affected in auxin transport or cytokinin signaling [7]. Together, our results demonstrate a role for long-distance basipetal transport of cytokinin in controlling polar auxin transport and maintaining the vascular pattern in the root meristem.     

Cytokinin receptors are required for normal development of auxin-transporting vascular tissues in the hypocotyl but not in adventitious roots

Plants alter the architecture of their root systems to adapt to the environment by modulating post-embryonic (lateral and adventitious) root formation and growth. To understand better the genetic basis of this regulation, we screened ethylmethane sulfonate-mutagenized lines of Arabidopsis thaliana for adventitious rooting mutants. One mutant showed retardation of the primary root growth, no production of lateral roots and enhanced formation of adventitious roots. Mapping and genetic complementation revealed that this mutant named wooden leg-3 (wol-3) was an allele of ARABIDOPSIS HISTIDINE KINASE 4 (AHK4), a locus known to encode a cytokinin receptor. Although the vascular system of the primary root and hypocotyl in the wol-3 mutant was aborted, that of the adventitious roots was normally developed. In the hypocotyl of the wol-3 mutant, auxin signals accumulated around the aborted vascular system. The application of auxin to primary roots induced lateral root formation in the wol-3 mutant. Transport of radiolabeled auxin from the top of the hypocotyl to the primary root was inhibited in wol-3. Although only a single amino acid alteration had occurred in AHK4, the root morphology in the wol-3 mutant was quite similar to that in the ahk2 ahk3 ahk4 triple mutant, which is a loss-of-function mutant of the three cytokinin receptors. This implies that the functional disturbance of AHK4 affects the function of the other receptors. Our results suggest that cytokinin receptors are necessary for the formation of auxin-transporting vascular tissues in the hypocotyl, but not in adventitious roots.     

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.     

Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3

Cytokinins control key processes during plant growth and development, and cytokinin receptors CYTOKININ RESPONSE 1/WOODEN LEG/ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/WOL/AHK4), AHK2, and AHK3 have been shown to play a crucial role in this control. The involvement of cytokinins in signaling the status of several nutrients, such as sugar, nitrogen, sulfur, and phosphate (Pi), has also been highlighted, although the full physiological relevance of this role remains unclear. To gain further insights into this aspect of cytokinin action, we characterized a mutant with reduced sensitivity to cytokinin repression of a Pi starvation-responsive reporter gene and show it corresponds to AHK3. As expected, ahk3 displayed reduced responsiveness to cytokinin in callus proliferation and plant growth assays. In addition, ahk3 showed reduced cytokinin repression of several Pi starvation-responsive genes and increased sucrose sensitivity. These effects of the ahk3 mutation were especially evident in combination with the cre1 mutation, indicating partial functional redundancy between these receptors. We examined the effect of these mutations on Pi-starvation responses and found that the double mutant is not significantly affected in long-distance systemic repression of these responses. Remarkably, we found that expression of many Pi-responsive genes is stimulated by sucrose in shoots and to a lesser extent in roots, and the sugar effect in shoots of Pi-starved plants was particularly enhanced in the cre1 ahk3 double mutant. Altogether, these results indicate the existence of multidirectional cross regulation between cytokinin, sugar, and Pi-starvation signaling, thus underlining the role of cytokinin signaling in nutrient sensing and the relative importance of Pi-starvation signaling in the control of plant metabolism and development.     

Over-expression of SOB5 suggests the involvement of a novel plant protein in cytokinin-mediated development

Cytokinins are a class of phytohormones that play a critical role in plant growth and development. sob5-D, an activation-tagging mutant, shows phenotypes typical of transgenic plants expressing the Agrobacterium tumefaciens isopentenyltransferase (ipt) gene that encodes the enzyme catalyzing the first step of cytokinin biosynthesis. The sob5-D mutant phenotypes are caused by over-expression of a novel gene, SOB5. Sequence analysis places SOB5 in a previously uncharacterized family of plant-specific proteins. A translational fusion between SOB5 and the green fluorescent protein reporter was localized in the cytoplasm as well as associated with the plasma membrane when transiently expressed in onion epidermal cells. Analysis of transgenic plants harboring an SOB5:SOB5-beta-glucuronidase (GUS) translational fusion under the control of the SOB5 promoter region showed GUS activity in vegetative tissues (hydathodes and trichomes of leaves, shoot meristems and roots) as well as in floral tissues (pistil tips, developing anthers and sepal vasculature). Cytokinin quantification analysis revealed that adult sob5-D plants accumulated higher levels of trans-zeatin riboside, trans-zeatin riboside monophosphate and isopentenyladenine 9-glucoside when compared to the wild-type. Consistent with this result, AtIPT3 and AtIPT7 were found to be up-regulated in a tissue-specific manner in sob5-D mutants. Physiological analysis of the sob5-D mutant demonstrated reduced responsiveness to exogenous cytokinin in both root-elongation and callus-formation assays. Taken together, our data suggest a role for the novel gene SOB5 in cytokinin-mediated plant development.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

Root development: cytokinin transport matters, too!

Unlike the plant hormone auxin, the mechanism and function of cytokinin transport is poorly characterised. Two new studies now demonstrate that cytokinins transported from shoot to roots via the phloem are critical for creating mutually exclusive auxin and cytokinin signalling domains that control root vascular patterning.     

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.     

Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism

We used loss-of-function mutants to study three Arabidopsis thaliana sensor histidine kinases, AHK2, AHK3, and CRE1/AHK4, known to be cytokinin receptors. Mutant seeds had more rapid germination, reduced requirement for light, and decreased far-red light sensitivity, unraveling cytokinin functions in seed germination control. Triple mutant seeds were more than twice as large as wild-type seeds. Genetic analysis indicated a cytokinin-dependent endospermal and/or maternal control of embryo size. Unchanged red light sensitivity of mutant hypocotyl elongation suggests that previously reported modulation of red light signaling by A-type response regulators may not depend on cytokinin. Combined loss of AHK2 and AHK3 led to the most prominent changes during vegetative development. Leaves of ahk2 ahk3 mutants formed fewer cells, had reduced chlorophyll content, and lacked the cytokinin-dependent inhibition of dark-induced chlorophyll loss, indicating a prominent role of AHK2 and, particularly, AHK3 in the control of leaf development. ahk2 ahk3 double mutants developed a strongly enhanced root system through faster growth of the primary root and, more importantly, increased branching. This result supports a negative regulatory role for cytokinin in root growth regulation. Increased cytokinin content of receptor mutants indicates a homeostatic control of steady state cytokinin levels through signaling. Together, the analyses reveal partially redundant functions of the cytokinin receptors and prominent roles for the AHK2/AHK3 receptor combination in quantitative control of organ growth in plants, with opposite regulatory functions in roots and shoots.     

SHY2 as a node in the regulation of root meristem development by auxin,brassinosteroids, and cytokinin

In multicellular organisms, the balance between cell division and differentiation determines organ size, and represents a central unknown in developmental biology. In Arabidopsis roots, this balance is mediated between cytokinin and auxin through a regulatory circuit converging on the IAA3/SHORT HYPOCOTYL 2 (SHY2) gene. Here, we show that crosstalk between brassinosteroids (BRs) and auxin occurs in the vascular transition zone to promote root meristem development. We found that BR increases root meristem size by up‐regulating expression of the PINFORMED 7 (PIN7) gene and down‐regulating expression of the SHY2 gene. In addition, BES1 could directly bind to the promoter regions of both PIN7 and SHY2, indicating that PIN7 and SHY2 mediate the BR‐induced growth of the root meristem by serving as direct targets of BES1. Moreover, the PIN7 overexpression and loss‐of‐function SHY2 mutant were sensitive to the effects of BR and could partially suppress the short‐root phenotypes associated with deficient BR signaling. Interestingly, BRs could inhibit the accumulation of SHY2 protein in response to cytokinin. Taken together, these findings suggest that a complex equilibrium model exists in which regulatory interactions among BRs, auxin, and cytokinin regulate optimal root growth.     

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.     

PvUPS1, an allantoin transporter in nodulated roots of French bean

Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system. In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen is used for synthesis of the ureides allantoin and allantoic acid, the major long-distance transport forms of organic nitrogen in these species. The purpose of this investigation was to identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots. A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid. In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats. In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin. In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature. These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.     

Isolation of Citrobacter sp. mutants defective in decolorization of brilliant green by transposon mutagenesis

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.     

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.     

The type-A response regulator,ARR15, acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana

The Arabidopsis thaliana AHK4 histidine kinase (also known as CRE1 or WOL) acts as a cytokinin signal transducer, presumably, in concert with downstream components, such as histidine-containing phosphotransfer factors (AHPs) and response regulators (ARRs), through the histidine-to-aspartate (His-->Asp) phosphorelay. Among 10 members of the type-A ARR family, the cytokinin-induced expression of ARR15 in roots is selectively impaired in the cre1-1 mutant, which carries a mutation in the AHK4 gene, suggesting a link between this type-A response regulator and the AHK4-mediated cytokinin signal transduction in roots. To address this issue further, we characterized a T-DNA insertion mutant of ARR15, and also constructed transgenic lines (referred to as ARR15-ox) that overexpress the ARR15 gene in a manner independent of cytokinin. While the T-DNA insertion mutant (arr15-1) showed no apparent phenotype, the cytokinin-independent overexpression of ARR15 in ARR15-ox plants resulted in a reduced sensitivity toward exogenously applied cytokinin, not only in elongation of roots in plants, but also in green callus formation (or shoot formation) in explants. Cytokinin-induced expressions of certain type-A ARRs were also down-regulated in ARR15-ox plants. These results support the view that ARR15 acts as a repressor that mediates a negative feedback loop in the cytokinin and AHK4-mediated His-->Asp phosphorelay.     

The shoot controls zeatin riboside export from pea roots. Evidence from the branching mutant rms4

The rms4 mutant of pea ( Pisum sativum L.) was used in grafting studies and cytokinin analyses of the root xylem sap to provide evidence that, at least for pea, the shoot can modify the import of cytokinins from the root. The rms4 mutation, which confers a phenotype with increased branching in the shoot, causes a very substantial decrease (down to 40-fold less) in the concentration of zeatin riboside (ZR) in the xylem sap of the roots. Results from grafts between wild-type (WT) and rms4 plants indicate that the concentration of cytokinins in the xylem sap of the roots is determined almost entirely by the genotype of the shoot. WT scions normalize the cytokinin concentration in the sap of rms4 mutant roots, whereas mutant scions cause WT roots to behave like those of self-grafted mutant plants. The mechanism whereby rms4 shoots of pea cause a down-regulation in the export of cytokinins from the roots is unknown at this time. However, our data provide evidence that the shoot transmits a signal to the roots and thereby controls processes involved in the regulation of cytokinin biosynthesis in the root.