Cytokinin-facilitated proteolysis of ARABIDOPSIS RESPONSE REGULATOR 2 attenuates signaling output in two-component circuitry
Version of Record online: 12 DEC 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 69, Issue 6, pages 934–945, March 2012
How to Cite
Kim, K., Ryu, H., Cho, Y.-H., Scacchi, E., Sabatini, S. and Hwang, I. (2012), Cytokinin-facilitated proteolysis of ARABIDOPSIS RESPONSE REGULATOR 2 attenuates signaling output in two-component circuitry. The Plant Journal, 69: 934–945. doi: 10.1111/j.1365-313X.2011.04843.x
- Issue online: 16 MAR 2012
- Version of Record online: 12 DEC 2011
- Accepted manuscript online: 3 NOV 2011 12:40PM EST
- Received 8 September 2011; revised 31 October 2011; accepted 1 November 2011; published online 12 December 2011.
Figure S1. Rapid degradation of ARR1 is independent on cytokinin in protoplasts and transgenic Arabidopsis. (a) ARR1-HA was transiently expressed in mesophyll protoplasts of ahk2-1/ahk3-2 mutants, in which cytokinin perception was abolished, and (b) ARR1-HA was detected in seedlings of transgenic Arabidopsis. Protoplasts and young seedlings were treated with 100 mM of cycloheximide for 30 min and then incubated with 100 and 1 mM of t-zeatins, respectively. Total proteins were isolated and subjected to SDS-PAGE followed by immunoblot analysis with anti-HA antibody conjugated to HRP. ND, nonspecifically detected protein by anti-HA antibody and used as loading control; NT, non-transfected control; Col, Col-0 wild type Arabidopsis. In case of transgenic lines expressing ARR1-HA, two independent immunoblots were presented together.
Figure S2. Cytokinin-mediated degradation of ARR2 is not altered by leupeptin, a cysteine/serine protease inhibitor. ARR2-HA was transiently expressed in mesophyll protoplasts of Col-0. Transfected protoplasts were treated with 100 mM cycloheximide for 30 min and then incubated with 100 nM t-zeatins and 10 mM leupeptin for the designated time. Total proteins were subject to SDS-PAGE followed by immunoblot with anti-HA antibody conjugated to horse radish peroxidase. RBC, Coomassie-stained Rubisco large subunit, as a loading control. Relative amount of ARR2-HA proteins are designated on the bottom.
Figure S3. ARR2 is likely an ubiquitinated protein. HA-tagged ARR10 and various versions of ARR2 proteins transiently expressed in protoplasts were pulled down with anti-UBQ antibody using protein A/G conjugated agarose beads and then immunodetected by anti-HA antibody. ARR2-HA and ARR10-HA proteins are indicated with arrows. NT, non-transfected control; P, preimmune washing with mouse IgG; U, anti-UBQ antibody.
Figure S4. Transgenic lines expressing ARR2K90G-HA exhibited relatively high sensitivity to cytokinin during primary root elongation. ARR2K90G-HA overexpressing lines are more sensitive to cytokinin in primary root elongation than ARR2-HA overexpressing lines. Vertically-grown 5-day-old Arabidopsis seedlings were transferred to 6-Benzylaminopurine (BAP)-containing medium and grown for another 9 days. (a) Root elongation in various concentrations of BAP was measured as relative ratio to 0 nM of BAP. Primary root lengths were measured from at least 30 individual seedlings. Error bars denote the SD. (b) Representative relative rooth lengths to 0 nM of BAP. Line K11 expressing ARR2K90G-HA showed the greater inhibition in primary root growth than ARR2-HA OX1 lines at 10 and 50 nM of BAP. (c) Primary root elongation patterns of transgenic Arabidopsis expressing ARR2-HA and ARR2K90G-HA, respectively. Col-0, Col-0 wild-type control; arr2-4, arr2-4 mutant; OX1, 2, and 8, Pro35S:ARR2-HA line; K2, K11, and K44, Pro35S:ARR2K90G-HA lines.
Figure S5. ARR2K90G–HA rapidly induces callus formation in response to exogenous cytokinins. Various Arabidopsis lines were germinated and grown in darkness for 5 days in half strength of MS media. Then, 10 pieces of explants cut from hypocotyls were stacked on callus induction media containing fixed concentration of 2,4-d (0.05 μM) and varied amount of t-zeatins. (a) The protein accumulation levels in transgenic lines. RBL, Rubisco large subunit as loading control. Relative amount of ARR2-HA are designated. (b) After 2 weeks, callus formation was visually examined. Transgenic Arabidopsis expressing ARR2K90G induced calli in lower concentration of cytokinin than lines expressing ARR2, arr2-4, and Col-0. (c) After 1 month, green calli were developed even in the absence of exogenous cytokinin treatment. (d) The relative chlorophyll contents in the presence and absence of t-zeatin (10 mM). Different letters designate the significantly different lines (P < 0.05 for t-tests).
Figure S6. ARR2K90G-HA overexpressing plants exhibit greater outgrowth of floral tissues than ARR2 overexpressing plants. (a) The floral morphology of Arabidopsis expressing various versions of ARR2. (b) The pistil and anther of Arabidopsis expressing various versions of ARR2. (c) Expression patterns of ProARR2:ARR2-GUS in transgenic Arabidopsis. (i) Matured flowers, (ii) Young flowers, (iii) Seedling Leaf, (iv) Root tips, (v) Whole roots, (vi) Young silique, (vii) Matured silique. Seeds were not stained.
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