Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development
Article first published online: 15 OCT 2012
© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd
The Plant Journal
Volume 72, Issue 5, pages 707–720, December 2012
How to Cite
Lin, L., Zhong, S.-H., Cui, X.-F., Li, J. and He, Z.-H. (2012), Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development. The Plant Journal, 72: 707–720. doi: 10.1111/j.1365-313X.2012.05109.x
- Issue published online: 22 NOV 2012
- Article first published online: 15 OCT 2012
- Accepted manuscript online: 17 JUL 2012 12:02PM EST
- Received 23 April 2012; revised 11 July 2012; accepted 13 July 2012; published online 15 October 2012.
Figure S1. Map-based cloning of theSCM/SUB gene.
(a) The mutant locus was mapped to a 112-kb region between two markers cer443489 and cer445705 on chromosome 1 using a F2 population from the cross of sub-2 with Ler. Numbersin brackets represent recombination events.
(b) Upper, diagram of the genomic locus of SCM/SUBincluding exons (blue boxes), introns (lines) and untranslationalregions (grey boxes). Lower, diagram of structural domains of theSCM/SUB protein. The G-to-A mutation in sub-2 that creates apremature stop codon TGA (*) at amino acid 337 before transmembranedomain and the T-DNA insertion position in scm-2 were indicated.
(c) Genetic complementation of sub-2 at 22°C and30°C. Note that the incomplete leaf in sub-2 was rescued in a transgenic line (arrows).
(d) scm-2 with leaf phenotypes similar to sub-2 atboth 22°C and 30°C. Scale bars = 1cm.
Figure S2. Temperature-sensitive phenotypes ofthe sub mutants in Ler background.
(a) Morphological phenotypes of Ler, sub-2 andsub-3 plants at the rosette stage. The leaves of thesub mutants were only slightly smaller than those ofLer grown at 22°C; whereas at 30°C, the submutants produced narrow and twisted leaves similar to sub-2 in Col-0 background.
(b) Comparison of Ler and sub plants during theflowering stage at 22°C and 30°C. Note that the submutant plants are shorter than Ler at both temperatures andextremely dwarf at 30°C. Inset shows a magnified sub plant for details.
(c) Statistical analysis of plant heights of Ler andsub mutants grown at 22°C and 30°C. Values aremeans ± SD, Student’s t-test, ** P≤0.001(n=12). Scale bars = 1cm.
Figure S3. Time course of early leaf growth.
The leaf width (a), leaf length (b), leaf area (c) and leaf index (d) were measured on the first-pair leaves of Col-0 and sub-2 seedlings grown at 30°C from 3 to 8 DAG. Valuesare means ± SD (n≥12).
Figure S4. CYCB1;1-GUS activity in the Col-0and sub-2 leaves at 22°C.
The accumulation of CYCB1;1-GUS is decreased and diminished earlier in the first leaf from 6- to 8-day-old sub-2seedlings (b) grown at 22°C than that in Col-0(a). Scale bars = 200μm.
Figure S5. Venation pattern in NPA-treatedleaves of sub-2.
(a, b) Cleared first leaves of the 5μM NPA-treated 12-day-oldCol-0 and sub-2 seedlings grown at 22°C.
(c, d) Cleared first leaves of the 5μM NPA-treated 12-day-oldCol-0 and sub-2 seedlings grown at 30°C. Arrowheadindicates a gap of vascular strands along leaf margin ofsub-2.
Figure S6. DR5-GUS expression in Col-0and sub-2 seedling with or without exogenous IAA treatment.
(a, b) DR5-GUS expression in 4- and 5-day-old firstleaves at 30°C. Note that DR5-GUS expression wasslightly reduced in sub-2 compared with Col-0. Arrowindicates DR5-GUS expression in ectopic hydathodes insub-2.
(c) Induction of the DR5-GUS expression by 5μM IAA for6 hours in the leaf primordia of 4-day-old Col-0 and sub-2seedlings grown at 30°C, with non-IAA treatment as control(Ctrl).
(d) Induction of the DR5-GUS expression by 5μM IAA for6 hours in the roots of 4-day-old Col-0 and sub-2 seedlingsgrown at 30°C, with non-IAA treatment as control.
Figure S7. Analysis of leaf developmental stage at the time of temperature shift
SEM and light-microscope images show the leaves that wereharvested from 2- to 12-day-old Col-0 seedlings continuously grownat 30°C (a) and 22°C (b) in temperature-shift experiments.Leaf initiation has happened at 2 DAG. The leaf developmentalstages during primary morphogenesis (PM) were estimated based onleaf length and morphological characteristics such as trichomeinitiation, the appearance of midrib and petiole, and theelongation of petiole as described by Carland and McHale (1996).The transition from PM to secondary morphogenesis was determined bythe disappearance of CYCB1;1-GUS accumulation in leaves.
Figure S8. Comparison of wild-type andSUB-OE seedlings.
(a) The SUB-OE seedlings (OE-78 and OE-44) are smallerthan wild-type Col-0.
(b) The roots of the SUB-OE seedlings are shorter thatthose of the wild type.
(c) Statistical analysis of root length of Col-0 and SUB-OE seedlings in (b). Values are means ± SD,Student’s t-test, ** P≤0.001 (n =12). Scale bars = 1mm in (a), 1cm in (b).
Table S1. Primers used in this study.
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