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![Page 1: A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis From: 2007 Jan SCIENCE Vol315 5.](https://reader036.fdocuments.us/reader036/viewer/2022062407/56649d355503460f94a0bc44/html5/thumbnails/1.jpg)
A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis
From: 2007 Jan SCIENCE Vol315 5
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Now We Known
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Sample Screen Lotus japonicus har1-1 hypernodulation.
Hyperinfected 1(HIT1) low nodulation, excessive formation of IT.
(hit1-1 har1-1), (hit1-1) Mesorhizobium loti carry reporter gene.
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hit1-1 har1-1 10dai
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J. Murray, 07
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hit1-1 har1-1 14dai
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hit1-1ª›®œ•Œ QuickTimeý ©M
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J. Murray, 07
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hit Lots IT, but less or no nodule organogenesis. ª›®œ•Œ QuickTimeý ©M
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Cortex Epidermis
J. Murray, 07
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Role of HIT1 in NP organogenesis ENOD40(cortical cell divison, mRNA) Nin(IT formation, initiation of primordia)
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Role of HIT1 in NP organogenesis
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inoculate
J. Murray, 07
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Role of HIT1 in NP organogenesis
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J. Murray, 07
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Role of HIT1 in NP organogenesis L. japonicus root hairless( Ljrhl1-1 )
- lots uncolonized NPs. - no root hairs and ITs. ( Ljrhl1-1 ) and (hit1-1) double mutant.
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Role of HIT1 in NP organogenesis
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J. Murray, 07
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Jens Stougaard’s group
- L. japonicus Spontaneous nodule formation (Lotus hitidine kinase - Lhk)
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har + AR hit har + AR hit har + Lhk1
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Expression of Lhk1 in various L. japonicus tissues as assayed by RT-
PCR.
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Phylogenetic analysis of LHK1 and LHK1-like proteins.
Analysis of the full-length Lhk1 cDNA revealeda 2979–base pair open reading frameencoding a predicted protein of 993 amino acids
The LHK1 protein had 64% identitywith the Arabidopsis cytokinin histidine kinasereceptor AHK4, and 49 and 45% identity withAHK2 and AHK3, respectively. Similar toLHK1, LHK2 was more closely related toAHK4 than to other Arabidopsis cytokininreceptors
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Lhk1 cDNA confers cytokinin responsiveness to yeast cells.
cells were transformed with either p415CYC carrying the Lhk1 cDNA, the hit1-2 cDNA or the vector alone
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hit1-1 roots are insensitive to exogenously applied cytokinin.
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We further investigated a role for the HIT1
locus in NP organogenesis by studying Early
Nodulin 40 (ENOD40) and Nin expression,
two markers for NP initiation
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50 nM benzyl adenine (BA) increased the steady-state level of Nin transcript
qPCR showing significant attenuation of Nin mRNA in hit1-1 versus wild-type roots upon exogenous application of 50nM BA. BA-stimulated accumulation of Nin transcripts in hit1 roots was significantly higher in comparison to water treatments
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Nodule organogenesis
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The LHK1 function as cytokinin receptors
hit1-1 roots are insensitive to exogenously applied cytokinin
50 nM benzyl adenine (BA) increased the steady-state level of Nin transcript
Nin mRNA in hit1-1 less then in wild-type
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The LHK1 function as cytokinin receptors, which may explain a leaky (formation of some nodules) symbiotic phenotype and lack of more general developmental abnormalities in mutants carrying hit1 alleles.
cytokinin biosynthesis and/or action are crucial downstream targets of NF perception recruitment of a cytokinin receptor could have been an essential event during the evolution of nitrogen-fixing nodule symbiosis
Nin expression, supporting IT formation, may be regulated by an Lhk1-independent mechanism in the root epidermis, possibly involving another cytokinin receptor.
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祥霖
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Arabidopsis ahk2 ahk3 ahk4 triple mutants display a reduced number of root vascular cell files→ impaired periclinal procambial cell divisions
snf2 mutant?
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Phenotypic characterization of the snf2 mutant
wt
snf2
5 week time 0 6 day 3 week
On hormone free medium
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Phenotypic characterization of the snf2 mutant
cytokinin
auxi
n
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ARR(type-A response regulator) gene was Cytokinin-induced cellular processes in plants
ARR5 promoter-GUS was expressed during nodulation
L266F receptor protein→ cytokinin- independent activity in E. coli
Lotus Lrr5?
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Expression of Lrr5, Lhk1, and Nin in intact wild-type and snf2 roots
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Expression of Lrr5 in wild-type and snf2 root explants
Medium with or without 0.5 mg/ml of BAP(cytokinin) for 10 days
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Expression of Nin in wild-type and snf2 rootexplants
Medium with or without 0.5 mg/ml of BAP(cytokinin) for 10 days
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Root length of wt and snf2 plants in the presence of increasing concentration of BAPBAP: 0 3 6 10 ng/ml
BAP: 30 50 100 500 ng/ml
Wt/snf2
• snf2 mutants growth were strongly affected by external cytokinin
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The phenotype of snf2 mutants suggests that cytokinin signaling acts downstream of Nod-factor signal transduction
signal Nod-factor snf2 nod formation
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Signal transduction: symRK mutants lacking Ca2+ spiking
ccamk (Ca2+- and calmodulin-dependent protein kinase)
develop spontaneous nodules in snf2 transgenic roots
symRKsnf2 nod formation
ccamk
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nfr1, nfr5, symrk, ccamk mutants→ unable to form nodules in response to rhizobia inoculation
nin, nsp2 mutants→ arrested before initiation of cell division induced by Nod-factor signaling, no spontaneous nodules were observed in snf2 transgenic rootsCytokinin signal perception acts upstream
of cell division initiation
symRKnod formation
ccamk
nin
nsp2snf2
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snf1-1 mutants(CCaMK protein impaired in autophosphorylation)→ 7±0.9
snf2 mutants→ 3±0.5 snf1-1+snf2→ 17±0.9
• Parallel signaling
• Deregulated signaling in snf1 results in a local increase in cytokinin levels transcriptionally up-regulating snf2
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har1-1(hypernodulating)→ no nodule snf2 mutant→ 3±0.5 snf2+har1-1 double mutants→ 14±1.4
•Only a few cells dedifferentiate •Only a few dedifferentiated cells sustain cell divisions during the snf2 nodule-initiation process
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Model for functional role of Lhk1 in nodulation
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Discuss Applications of auxin transport inhibitors resulted in empty nodule-like structures, which suggested that local inhibition of auxin transport sensitizes cells for division
Externally supplied cytokinin induces cortical cell division and activation genes, and expression of cytokinin biosynthesis gene in a nodulation-deficient Sinorhizobium meliloti resulted in nodule-like structures
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Conclude cytokinin signaling plays an important role in plant meristem formation and is directly involved in initiating root nodule organogenesis