PMC

EPFL4 and EPFL6 associate with ER in planta. Shown are co-IP assays of epitope-tagged ligand–receptor pairs expressed in N. benthamiana leaves. ER associates with EPFL4-FLAG and EPFL6-FLAG but not with a control, LURE2-FLAG. The faint, higher molecular bands in ligand inputs likely represent unprocessed/intermediate precursors. Molecular mass is expressed in kilodaltons. All experiments were repeated at least four times.

Endodermis-specific expression of EPFL4 or EPFL6 is sufficient to restore normal inflorescence architecture of epfl4 epfl6. (A–D) EPFL4 and EPFL6 promoters are predominantly active in the endodermis of inflorescence stems and pedicels. Shown are longitudinal sections of inflorescence tips expressing EPFL4pro:GUS (A) or EPFL6pro:GUS (B) and their stem sections at higher magnifications in longitudinal sections (C) or cross-sections (D). Arrowheads indicate endodermal GUS expression. Ep, epidermis; Co, cortex; En, endodermis; Ph, phloem. (Scale bars: 20 μm in A and B, 10 μm in C and D.) (E and F) GUS activities of EPFL4pro:GUS (E) and EPFL6pro:GUS (F) are severely reduced in shr mutant lacking endodermis. (G–J) Expression of EPFL4 and EPFL6 driven by the endodermis-specific SCR promoter fully rescues the inflorescence growth phenotype of epfl4 epfl6. Shown are top views of 4-wk-old inflorescence from WT (G), epfl4 epfl6/chal-2 (H), SCRpro:EPFL4 in epfl4 epfl6/chal-2 (I), and SCRpro:EPFL6 in epfl4 epfl6/chal-2 (J). (Insets) epfl4 epfl6/chal-2 exhibits characteristic flat inflorescence top with exposed SAM (H; arrowhead), while in rescued plants flower buds cover SAM (I). (Scale bar: 5 mm.) (K and L) Morphometric analysis showing heights of 5-wk-old plants (n = 7–8) and lengths of mature pedicels (n = 63) from 6-wk-old plants of each genotype. Bars indicate mean values; error bars indicate SD. *Significantly different from epfl4 epfl6/chal-2 (P < 0.0001, Student t test), but not from WT.

Phloem-specific expression of ER is sufficient to restore normal inflorescence architecture of er. (A and B) ERpro:GUS expression pattern in inflorescence (A) and stem cross- section (B). Ep, epidermis; Ph, phloem; Xy, xylem. (Scale bars: 1 mm in A; 50 μm in B.) (C) Five-wk-old plants of WT, er, and er-expressing AtSUC2pro:ER-FLAG, AtIRX3pro:ER-FLAG, and AtML1pro:ER-FLAG. (D) Top view of representative inflorescence from respective genotypes. (Scale bars: 1 mm.) (E and F) Morphometric analysis of plant height (E) and pedicel length (F) from each genotype. Five-wk-old plants (n = 7) were measured for plant height, and 6-wk-old mature pedicels (n = 20, from 5 plants; n = 14 for AtML1pro:ER-FLAG line 2) were measured. Bars indicate mean values; error bars indicate SD. *Significantly different from er (P < 0.0001, Student t test), but not from WT [Tukey's honestly significant difference (HSD) test]; ns, not significantly different from er. (G–J) Longitudinal sections of mature pedicels. Ep, epidermis; Co, cortex; En, endodermis; Va, vasculature. (Scale bar: 25 μm.) (K) Quantitative analysis of cortex cell length (n = 26–30). Bars indicate mean values; error bars indicate SD. *Significantly different from er (P < 0.0001, Student t test); ns, not significant from er. (L and M) Immunohistochemical analysis of inflorescence stem sections of er (L) and AtSUC2pro:ER-FLAG er (M) using anti-FLAG antibody. Strong orange-brown signals are detected in the phloem (Ph) tissues in AtSUC2pro:ER-FLAG (M, Inset; arrowheads).

EPFL4 and EPFL6 redundantly control inflorescence growth in a manner similar to ER. (A) Molecular phylogeny of EPFL family members. EPFL4, EPFL5, and EPFL6 cluster together. Shown is an unrooted neighbor-joining tree of C-terminal end amino acid sequence encompassing the predicted mature EPF (MEPF) domain. Branch lengths are scaled to the number of amino acid changes indicated on the scale bar. MEPF sequence alignment is shown in Fig. S8. (B) Promoter activities of EPFL4, EPFL5, and EPFL6 in inflorescence. (Scale bar: 1 mm.) (C) epfl4 epfl6/chal-2 confers compact inflorescence nearly identical to that of er. Shown are inflorescence tops of 5-wk-old Arabidopsis of WT, epfl4, epfl6, er-103 (intermediate allele), er-105 (null allele), and epfl4 epfl6/chal-2. (Scale bars: 1 cm.) (D and E) Morphometric analysis of plant height (D) and pedicel length (E) from each genotype. 5-wk-old plants were measured for plant height (n = 9), and 6-wk-old mature pedicels (n = 46, from 6 plants) were measured. Bars indicate mean values; error bars indicate SD. ns, not significantly different and grouped together (Tukey's HSD test). (F–I) Longitudinal sections of mature pedicels from WT (F), er-105 (G), er-103 (H), and epfl4 epfl6/chal-2 (I). Ep, epidermis; Co, cortex; En, endodermis; Va, vasculature. epfl4 epfl6 has large cortex cells similar to er (asterisks). (Scale bars: 25 μm.) (J) Quantitative analysis of cortex cell length (n = 30). Bars indicate mean values; error bars indicate SD. ns, not significantly different and grouped together (Tukey's HSD test). The images in each composite were obtained under the same magnification.