1. Volume 25, Issue 23, Pages 3144-3150 (December 2015)
A Negative Feedback Loop Controlling bHLH Complexes Is Involved in Vascular Cell Division and Differentiation in the Root Apical Meristem 
Hirofumi Katayama, Kuninori Iwamoto, Yuka Kariya, Tomohiro Asakawa, Toshiyuki Kan, Hiroo Fukuda, Kyoko Ohashi-Ito 
Current Biology 
Volume 25, Issue 23, Pages (December 2015)
DOI: /j.cub
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2. Figure 1 LHW-T5L1 Induces Xylem Differentiation
Roots of plants harboring estrogen-inducible LHW-T5L1 treated with (B–F) or without (A) estrogen. Differential interference contrast images of the differentiation zone of roots (A–D) and propidium-iodide-stained images of the differentiation zone of root observed under a confocal microscopy (E and F) are shown. Scale bars, 50 μm.
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3. Figure 2 LHW-T5L1 Regulates ACL5 Expression
(A) qRT-PCR analysis of ACL5 in wild-type, lhw, and tmo5 t5l1 roots. Error bars indicate the SD. n = 3. ∗p < 0.01 (t test).
(B) GUS activity assay of the ACL5 promoter. Leaves injected with the pACL5-GUS reporter and estrogen-inducible effector genes were treated with estrogen and subjected to GUS activity measurements. Error bars indicate the SD. n = 3. ∗p < 0.01 (t test).
(C–H) Expression patterns of pACL5::YFP-nls in wild-type (C–E) and lhw (F–H) plants. Differentiation regions of roots (C and F), root tips (D and G), and cotyledons (E and H) are shown. Scale bars, 100 μm (C, D, F, and G) and 500 μm (E and H).
(I and J) Expression patterns of pACL5::YFP-nls in roots of plants harboring estrogen-inducible LHW-T5L1 with (I) or without (J) estrogen for 5 days. Scale bars, 100 μm.
(K–N) Images of vascular tissue in wild-type (K), acl5 (L), lhw (M), and lhw acl5 (N) roots. Scale bars, 50 μm.
(O–R) Transverse images of vascular bundles in wild-type (O), acl5 (P), lhw (Q), and lhw acl5 (R) roots. Asterisks indicate xylem vessels. Scale bars, 20 μm.
(S–V) Vein patterns in wild-type (S), acl5 (T), lhw (U), and lhw acl5 (V) cotyledons. Scale bars, 100 μm.
See also Figure S1.
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4. Figure 3 SACL3 Is a Target of LHW-T5L1 and Interacts with LHW
(A) Accumulation of SACL3 transcripts in plants harboring estrogen-inducible LHW-T5L1 treated with or without estrogen for 0, 4, 8, 12, and 24 hr. RNA gel blot analyses were repeated three times, and signal intensities were quantified and normalized relative to that at 0 hr in each analysis. Error bars indicate the SD. n = 3. ∗p < 0.05 (t test).
(B) Accumulation of SACL3 transcripts in wild-type, lhw, and tmo5 t5l1 roots. RNA gel blot analyses were repeated three times, and signal intensities were quantified and normalized relative to that of wild-type plants in each analysis. Error bars indicate the SD. n = 3. ∗p < 0.01 (t test).
(C–F) Expression patterns of pOL-SACL3::YFP-nls (C and D) and pOL-SACL2::YFP-nls (E and F) in roots.
(G and H) Subcellular localization of SACL3-GFP (G) and LHW-GFP (H) in epidermal cells of N. benthamiana.
(I and J) BiFC signal between SACL3 and LHW heterodimer (I) and SACL3 homodimers (J) in epidermal cells of N. benthamiana.
(K) Co-immunoprecipitation analysis. LHW-3×HA, SACL3-3×FLAG, T5L1-3×FLAG, and YFP were co-expressed in N. benthamiana, and total protein extracts were subjected to immunoprecipitation with an anti-HA antibody. The resulting immunocomplexes were detected by western blot analysis with anti-HA, anti-FLAG, or anti-GFP antibody.
(L–S) Phenotypes of wild-type (L, N, P, and R) and SACL3-overexpressing plants (M, O, Q, and S). Vascular patterns in cotyledons (L and M), roots (N–Q), and RAMs (R and S) are shown. Fractions of samples exhibiting a similar pattern are shown in (L)–(O). Note that a quarter of SACL3 overexpressing roots (7 out of 27) had no xylem vessels. The yellow asterisks indicate pericycle cells. The red asterisks indicate xylem vessels. Scale bars, 200 μm (L and M), 50 μm (N and O), and 20 μm (P–S).
See also Figure S2.
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5. Figure 4
The Negative Feedback Loop of ACL5/Thermospermine-SACL Suppresses LHW-T5L1 Function
(A) Seven-day-old wild-type and acl5 plants harboring estrogen-inducible LHW-T5L1 treated with or without 5 μM estrogen and 20 μM thermospermine (TMSP). Scale bars, 1 cm.
(B–G) Root sections of 7-day-old LHW-T5L1-overexpressing wild-type (B) and acl5 (C–G) plants. Arrows in (E) indicate the positions of cross sections shown in (C) and (F). (D) and (G) are magnified images of squares in (C) and (F), respectively. Red arrows indicate ectopic TEs. Scale bars, 50 μm (B, C, and F) and 1 mm (E).
(H) Ectopic TEs formed in the epidermal cells of LHW-T5L1-overexpressing acl5 root. Scale bar, 50 μm.
(I) Expression levels of LOG3 and LOG4 in wild-type, lhw, acl5, and lhw acl5 roots. Error bars indicate the SD. n = 3. The means, indicated by different letter superscripts in each column, were significantly (p < 0.01) different according to one-way ANOVA with the Tukey-Kramer post hoc test.
See also Figure S3.
Current Biology  , DOI: ( /j.cub )
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