Apical Dominance is controlled by Interaction between Cytokinin biosynthesis/degradation and Auxin in Stem. Mori, H. Nagoya University

3days after decapitation intact

Apical Dominance auxin cytokinin axillary bud dormant outgrowth decapitation

Northern Blot Analyses of Cell Cycle Related Genes cdc2 CycD3 ;1 CycB1 ;2 histoneH4 PCNA 2 4 8 6 10 12 14 16 18 20 22 24 Time after decapitation (hours) EtBr

1cm 2cm 4cm 6cm

Expresson of PCNA mRNA

auxin cytokinin axillary bud outgrowth dormant decapitation ・Auxin derived from terminal bud is transported basipetally. ・When auxin applied to the cut stump after decapitation, Auxin can repress axillary bud outgrowth. ・When auxin directly applied to axillary bud after decapitation, Auxin cannot repress axillary bud outgrowth.

Auxin is basipetally transported in stem. axillary bud IAA Scale leaf at 2nd node

The basipetal transport of auxin is essential for dormancy of axillary bud. Auxin acts at node or in stem, not in axillary bud.

The basipetal transport of IAA is essential for axillary buds dormancy intact decapitated IAA 3 hr 1cm IAA 0 hr 1 hr auxin transport rate: 1cm/hr

Subtracted cDNA clones 0hr-3hr Ps-IAA4/5, IAA7, Aux28, Aux22, GH3, F-box, cullin, auxin efflux carrier protein(PIN1 ), RD29B, LEA4, zeaxanthin epoxidase 3hr-0hr IAA-Ala hydrolase, GA2 oxidase, HMG-CoA synthase, squalene synthase, Isopentenyltransferase (IPT )

Current model of cytokinin metabolism in higher plants LOG LOG LOG LOG (revised)

1cm 2nd node

Expression of PsIPTs at the 2nd Node after Decapitation 0.5 4 3 2 1 5 6 9 15 18 24 12 PsIPT1 PsIPT2 rRNA time after decapitation (hr)

Is IPT expression repressed ? excise the segments 3hr after decapitation IPT is expressed incubate in MES buffer with or without IAA Is IPT expression repressed ? 1cm excise the segments from intact seedlings IPT is NOT expressed incubate in MES buffer with or without IAA Is IPT expressed?

PsIPTs Expression was Repressed by IAA 1 2 3 4 5 IAA Free 10-5 M IAA PsIPT1 PsIPT2 rRNA incubation time (hr)

Is IPT expression repressed? excise the segments after 3hr decapitation IPT is expressed incubate in MES buffer with or without IAA Is IPT expression repressed? 1cm excise the segments from intact seedlings IPT is NOT expressed incubate in MES buffer with or without IAA Is IPT expressed?

PsIPTs Expression was Induced in IAA Free Buffer 10-5 M IAA 1 6 5 4 3 2 incubation time (hr) 9 8 7 PsIPT1 PsIPT2 rRNA

10-5 M IAA IAA Free 10-5 M IAA excise the segments after 3hr decapitation IPT is expressed incubate in MES buffer with or without IAA Repress IPT expression? 1cm IAA Free 1 2 3 4 5 10-5 M IAA 1 2 3 4 5 IAA Free 10-5 M IAA 1 6 5 4 3 2 9 8 7 1 6 5 4 3 2 excise the segments from intact seedlings Not IPT expression incubate in MES buffer with or without IAA IPT express?

PsIPTs Expression was Repressed by IAA Transported Basipetally in the Stem PsPIN1 ＋ IAA Lanoline paste w or w/o IAA after decapitation 3hr Is IPT expression repressed? 1cm

pPsIPT2::GUS in Arabidopsis

pPsIPT2::GUS (2,4-D treated for 1 week)

pPsIPT2::GUS (2,4-D free for 3 days)

in Transformants harboring pPsIPT2::GUS GUS Transcripts Deceased by Auxin in Transformants harboring pPsIPT2::GUS IAA Free 10-5M IAA 1 2 3 4 5 incubation time (hr)

Cytokinin Levels in the Stems and Axillary Buds after Decapitation

Cytokinin Levels in the Excised Segments treated with/without IAA 100 200 300 iPMP iPA iP t-ZMP t-ZR t-zeatin 0h 3h 6h -IAA +IAA 100 200 300 0h 3h 6h (p mol/ gFW) (p mol/ gFW) iPMP iPA iP t-ZMP t-ZR t-zeatin excised the segments put on the agar plate ± 1%(w/v) IAA treatment

Conclusion One role of auxin in apical dominance is to repress the PsIPTs expression; local cytokinin biosynthesis in the nodal stem is negatively regulated by auxin through the control of IPT expression. Cytokinin, which is thought to be derived from the roots so far, is locally biosynthesized in the stem rather than in the roots after decapitation.

PsIPTs Expression was Repressed by Basipetally Transported IAA in the Stem PsPIN1 ＋ IAA 1% (w/v) IAA after decapitation 3hr Is IPT expression repressed? 1cm

Effect of IAA Applied to the Stump on PsPIN1 Expression actin 1 2 3 4 (cm) 1 2 4 (hours) 1 2 3 4 IAA

Can IAA from Axillary Buds induce PsPIN1? Biosynthesis decapitated intact IAA IAA 3hr 9hr 0hr

× IAA IAA Lower Stem Upper Stem PsPIN1 actin 2 4 6 8 10 12 PsPIN1 actin Time after decapitation ( hours )

Expression of PsIPTs at the 2nd Node after Decapitation 0.5 4 3 2 1 5 6 9 15 18 24 12 PsIPT1 PsIPT2 rRNA time after decapitation (hr)

upper stem node lower stem IAA 81.7 CK 2.9 44.0 9.1 19.3 135.5 20.8 105.5 60.5 3.6 35.4 175.2 175.3 47.6 79.5 1.9 50.5 6.3 27.7 76.6 33.6 158.0 34.6 94.8 39.9 50.7 55.2 23.8 72.2 7.8 1 3 12 18 24 36 48 upper stem node lower stem (p mol/gFW)

Cytokinin oxidase cDNAs were isolated from the stem 12 hr after decapitation. PsCKX1： was constantly expressed in stem before and after decapitation, was not induced by auxin. PsCKX2： was increased in stem after decapitation was induced by auxin.

Expression of PsCKX2 in Pea Stem 10 20 30 40 80 120 decapitation TIBA treatment Relative amounts of mRNA Time after treatment (hr)

PsCKX2 Expression in Excised Pea stem by Auxin 10 20 30 40 (x 103) Relative amounts of mRNA +NAA -NAA Time after treatment (hr)

PsCKX2 Expression in Pea stem - NAA 40 80 120 Relative amounts of mRNA 10 20 Time after treatment (hr)

PsCKX2 Expression in Excised Pea stem by Auxin 10 20 30 40 (x 103) Relative amounts of mRNA +NAA -NAA Time after treatment (hr)

upper stem node lower stem IAA 81.7 CK 2.9 44.0 9.1 19.3 135.5 20.8 105.5 60.5 3.6 35.4 175.2 175.3 47.6 79.5 1.9 50.5 6.3 27.7 76.6 33.6 158.0 34.6 94.8 39.9 50.7 55.2 23.8 72.2 7.8 1 3 12 18 24 36 48 upper stem node lower stem (p mol/gFW)

Expression of PsCKX2, PsPIN1, PsIPT2 in stem 120 PsCKX2 80 PsPIN1 Relative amounts of mRNA PsIPT2 40 g. 10 20 30 Time after decapitation (hr)

Model of Interaction Between Auxin and Cytokinin in Apical Dominance

Effect of Striolactone GR24 on outgrowth of axillary bud after decapitation control

Apical dominance Nagoya University ・Mina TANAKA ・Sae SATO-SHIMIZU ・Aiko ISHIWATA ・Yuka MADOKA RIKEN ・Mikiko KOJIMA ・Hitoshi SAKAKIBARA This work was done by my laboratory in Nagoya University and Dr.Sakakibara group in RIKEN. Thank you for your attention.