the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX andZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type.
two rice authentic HPs (OsAHP1 and OsAHP2) were knocked down simultaneously via RNA interference (RNAi), and the transgenic OsAHPs-RNAi plants exhibited phenotypes expected for a deficiency in cytokinin signaling, including dwarfism with reduced internode lengths, enhanced lateral root growth, early leaf senescence, and reduced tiller numbers and fertility under natural conditions. The OsAHPs-RNAi seedlings were also hyposensitive to exogenous cytokinin.
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/AHK4receptor 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 to 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 suppressing35S: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 vascular tissue differentiation through CRE1/AHK4. Taken together, the distinct consequences of CKX7overexpression indicate that the cellular compartmentation of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters defining the activities of the hormone.
a novel subfamily of cytokinin receptors with members only from the early diverging land plants Marchantia polymorpha and Physcomitrella patens and then experimentally characterized two members of this subfamily. HTPs of charophyceae seemed to be more closely related to those of land plants than to other groups of green algae. Further down the signaling pathway, the type-B RRs were found across all plant clades, but many members lack either the canonical Asp residue or the DNA-binding domain. In contrast, the type-A RRs seemed to be limited to land plants. Finally, the analysis provided hints that one additional group of RRs, the type-C RRs, might be degenerated receptors and thus evolutionary of a different origin than bona fide response regulators.
feedback loop linking WOX5-mediated auxin production to IAA17-dependent repression of auxin responses. This WOX5–IAA17 feedback circuit further assures the maintenance of auxin response maximum in the root tip and thereby contributes to the maintenance of distal stem cell (DSC) populations. Our experimental studies and in silico computer simulations both demonstrate that the WOX5–IAA17 feedback circuit is essential for the maintenance of auxin gradient in the root tip and the auxin-mediated root DSC differentiation.
Myb-like domain in KAN1 binds the 6-bp motif GNATA(A/T) and that this motif alone is sufficient to squelch transcription of a linked reporter in vivo. In addition, we report that KAN1 acts as a transcriptional repressor. Among its targets are genes involved in auxin biosynthesis, auxin transport, and auxin response. Furthermore, we find that the adaxializing HD-ZIPIII transcription factor REVOLUTA has opposing effects on multiple components of the auxin pathway. We hypothesize that HD-ZIPIII and KANADI transcription factors pattern auxin accumulation and responsiveness in the embryo. Specifically, we propose the opposing actions of KANADI and HD-ZIPIII factors on cotyledon formation (KANADI represses and HD-ZIPIII promotes cotyledon formation) occur through their opposing actions on genes acting at multiple steps in the auxin pathway.
The efficiency of photoprotective mechanisms, such as non-enzymatic and enzymatic scavenging systems, was decreased in plants with a reduced cytokinin status which could be a cause for the increased photodamage and subsequent D1 degradation. Mutant analysis revealed that the protective function of cytokinin during light stress depends on the AHK2 and AHK3 receptors and the type-B response regulators ARR1 and ARR12. We conclude that proper cytokinin signaling and regulation of specific target genes is necessary to protect plants efficiently from light stress.
Lateral root formation in plants can be studied as the process of interaction between chemical signals and physical forces during development. Lateral root primordia grow through overlying cell layers that must accommodate this incursion. Here, we analyze responses of the endodermis, the immediate neighbor to an initiating lateral root. Endodermal cells overlying lateral root primordia lose volume, change shape, and relinquish their tight junction–like diffusion barrier to make way for the emerging lateral root primordium. Endodermal feedback is absolutely required for initiation and growth of lateral roots, and we provide evidence that this is mediated by controlled volume loss in the endodermis. We propose that turgidity and rigid cell walls, typical of plants, impose constraints that are specifically modified for a given developmental process.
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ARR22 is induced by cold and dehydration. Here, we show that inducible overexpression of ARR22 inArabidopsis enhanced dehydration, drought, and cold tolerance in a dexamethasone-dependent manner, whereas mutation of the putative phospho-accepting Asp to Asn in ARR22 (ARR22D74N) abolished these tolerance phenotypes. Overexpression of ARR22 decreased electrolyte leakage in dehydration-, drought-, or cold-stressed transgenic Arabidopsis plants compared with that of ARR22D74N or compared with wild-type plants. Transpiration rates and stomatal apertures were not affected by ARR22 overexpression. No significant difference in both dehydration and freezing tolerance was observed between wild-type and arr22 mutants with or without cytokinin preincubation, consistent with the lack of phenotypes of arr22 mutants in their vegetative development.
RETINOBLASTOMA-RELATED (RBR) protein regulates the differentiation of meristematic cells at the transition zone by allowing mRNA accumulation of AUXIN RESPONSE FACTOR19 (ARF19), a transcription factor involved in cell differentiation. We show that both RBR and the cytokinin-dependent transcription factor ARABIDOPSIS RESPONSE REGULATOR12 are required to activate the transcription of ARF19, which is involved in promoting cell differentiation and thus root growth.
cytokinin biosynthesis genes IPT3 and IPT5 and all three cytokinin receptor genes (AHK2, AHK3, and CRE1/AHK4) act redundantly during LR initiation. Mutation of AHK2 and AHK3 caused increased auxin sensitivity of LR formation, corroborating the functional relevance of auxin–cytokinin interaction during LR formation. In contrast, LR development of cytokinin receptor mutants in response to other hormones was mostly similar to that of the wild type, which is consistent with separate response pathways. A noticeable exception was an increased sensitivity of LR elongation to brassinolide in ahk2 ahk3 mutants indicating antagonistic action of cytokinin and brassinosteroid.
FBR12 genetically interacts with the cytokinin receptor CYTOKININ RESPONSE1 (CRE1) and downstream AHP genes, as double mutants show enhanced phenotypes. FBR12 forms a protein complex with CRE1 and AHP1, and cytokinin regulates formation of this protein complex. Intriguingly, ahp6 partially suppresses the fbr12 mutant phenotype, and the fbr12 mutation causes increased expression of AHP6, indicating that FBR12 negatively regulates AHP6. Consistent with this, ectopic expression of FBR12 in the CRE1-expressing domain partially rescues defective protoxylem development in fbr12, and overexpression of AHP6 causes an fbr12-like phenotype.
L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. lotiinfection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.
Detection of cycline B1 by GUS staining of a promoter-reporter construct revealed that low B led to a reduced zone of cell division. The expression of CRE1/WOL/AHK4, encoding an integral membrane protein with histidine kinase domain that mediates cytokinin signaling and root xylem differentiation, was inhibited under B deficiency resulting in arrested xylem development at the protoxylem stage. Because the transition from cell division to cell differentiation in apical root meristems is controlled by cytokinins, this result support the hypothesis that signaling mechanisms during cell differentiation and organogenesis are highly sensitive to B deficiency, and together with previous reports that link the micronutrient with auxin or ethylene control of root architecture, suggests that B could play a role in regulation of hormone mediated early plant development signaling.
ytokinin signaling increases the abundance of ARR1, a ubiquitously expressed RRB, by preventing its degradation by the 26S proteasome. We also show that the RRAs act to suppress ARR1 accumulation, thus providing an explanation for their inhibitory action in cytokinin signaling. Collectively, our results reveal an additional regulatory mechanism in the cytokinin response pathway that involves the cytokinin–dependent stability control of a major RRB response activator.
ERECTA (ER) pathway as a second receptor kinase signaling pathway that regulates WUS expression, and therefore shoot apical and floral meristem size, independently of the CLV pathway. We demonstrate that reduction in class III HD-ZIP and ER function together leads to a significant increase in WUS expression, resulting in extremely enlarged shoot meristems and a switch from spiral to whorled vegetative phyllotaxy. We further show that strong upregulation of WUS in the inflorescence meristem leads to ectopic expression of the AGAMOUS homeotic gene to a level that switches cell fate from floral meristem founder cell to carpel founder cell, suggesting an indirect role for ER in regulating floral meristem identity. This work illustrates the delicate balance between stem cell specification and differentiation in the meristem and shows that a shift in this balance leads to abnormal phyllotaxy and to altered reproductive cell fate.
operation of an auxin-GSL8 feedback circuit that regulates the level of plasmodesmal-localized callose in order to locally downregulate symplasmic permeability during hypocotyl tropic response. This system likely involves a plasmodesmal switch that would prevent the dissipation of a forming gradient by auxin diffusion through the symplasm. This regulatory system may represent a mechanism by which auxin could also regulate symplasmic delivery of a wide range of signaling agents.
5–(4–chlorophenyl)-4H-1,2,4–triazole-3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis inYUC-expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1-His suggested that yucasin strongly inhibited YUC1-His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over-expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss-of-function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l–kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin-treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development.
Root system architecture (RSA) is developmentally controlled by genetic pathways and their interaction with various environmental cues, in particular soil conditions.
the transcript levels of cytokinin genes in the root responds to changes in nutrient availability in distinct ways. IPT3,IPT5, CYP735A2, LOG5 and CKX4 are particularly responsive cytokinin metabolism genes, genes encoding different type-A response regulators and the transcriptions factor genes ARR10 and CRF6 are among the most responsive signalling genes. This finding supports the hypothesis that environmental cues operate through fine-tuned transcriptional regulation of cytokinin genes to modulate root development.
cytokinin specifically antagonizes ABA-mediated inhibition on cotyledon greening with minimal effects on seed germination in Arabidopsis (Arabidopsis thaliana). We found that the cytokinin-antagonized ABA effect is dependent on a functional cytokinin signaling pathway, mainly involved in the cytokinin receptor gene CRE1/AHK4, downstream AHP2, 3, 5 genes, and a type-B response regulator gene ARR12, which genetically acts upstream of ABI5 to regulate cotyledon greening. Cytokinin has no apparent effect on the transcription of ABI5. However, cytokinin efficiently promotes the proteasomal degradation of ABI5 protein in a cytokinin signaling-dependent manner. These results define a genetic pathway, through which cytokinin specifically induces the degradation of ABI5 protein, thereby antagonizing ABA-mediated inhibition on postgerminative growth.
bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1-GFP as well as development is hypersensitive to BFA. We found that in bex1 a member ofARF1 gene family, ARF1A1C was mutated. ARF1A1C localizes to TGN/EE and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes.
ZmCKX6 and ZmCKX9 could only be expressed successfully after the removal of putative sequence-specific vacuolar sorting signals (LLPT and LPTS, respectively), suggesting that these proteins are localized to the vacuole. Substrate specificity analyses revealed that the CKX isoforms can be grouped into two subfamilies: members of the first strongly prefer cytokinin free bases while members of the second degrade a broad range of substrates. The most active isoform was found to be ZmCKX1. One of the studied isoforms, ZmCKX6, seemed to encode a nonfunctional enzyme due to a mutation in a conserved HFG protein domain at the C-terminus. Site-directed mutagenesis experiments revealed that this domain is essential for CKX activity. The roles of the maize CKX enzymes in the development of maize seedlings during the two weeks immediately after radicle emergence were also investigated. It appears that ZmCKX1 is a key regulator of active cytokinin levels in developing maize roots. However, the expression of individual CKX isoforms in the shoots varied and none of them seemed to have strong effects on the cytokinin pool.
The trans-hydroxylation that forms trans-zeatin (tZ)-type CK from N6-(Δ2-isopentenyl)adenine (iP)-type CK is catalyzed by the cytochrome P450 enzymes CYP735A1 and CYP735A2 in Arabidopsis. Deficiency in trans-hydroxylation activity results in dramatic retardation of shoot growth without affecting total CK quantity, while augmentation of the activity enhances shoot growth. Application of exogenous tZ but not iP recovers the wild-type phenotype in the mutants, indicating that trans-hydroxylation modifies the physiological function of CK. We propose that the control of cytokinin function by side-chain modification is crucial for shoot growth regulation in plants.
The mutant showed multiple phenotypic changes including increased plant height, big seeds, and delayed flowering. The mutant also showed increased sensitivity to drought and salt stresses and treatments with kinetin and abscisic acid. OsPUP7 is expressed mainly in the vascular bundle, pistil, and stamens. The measurement of cytokinins (CKs) showed that CK content in the mutant spikelets accumulated higher than that in the wild type. Moreover, uptake experiment in the yeast fcy2 mutant suggested that OsPUP7 has the ability to transport caffeine, a CK derivative. Our results indicate that the PUP transport system also exists in rice, and OsPUP7 has an important role in the transport of CK, thus affecting developmental process and stress responses.