Figure 1.
Isolation of the Bdtar2lhypo mutant and characterization of macroscopic phenotypes.
(A) Four-day-old tissue culture grown seedlings of wild type (accession Bd21), an unrelated control transformant (control) (the unrelated transformant line was included in all our assays to control for any tissue culture regeneration effects) and the transgenic line segregating the Bdtar2lhypo mutation. (B) Seminal root length quantification of the different genotypes at 4 days after germination (dag). (C) Schematic presentation of the BdTAR2L gene and the location of the T-DNA insertion in the Bdtar2lhypo mutant. (D–E) Relative expression level of BdTAR2L in different genotypes at 4 dag as determined by qPCR and normalized with respect to the housekeeping gene, BdUBC18. (F) Root elongation in wild type, control and homozygous Bdtar2lhypo mutants, assayed at 4 dag. (G–H) Quantification of seedling phenotypes at 4 dag. (I) Leaf number at 18 dag. (J–L) Different size parameters of the 5th leaf of plants, assayed at 18 dag. (M) Representative image of adult plants at 18 dag. Size bars are 1 cm; differences as compared to wild type are not significant unless indicated otherwise; error bars indicate standard error; * = p<0.05; ** = p<0.01; *** = p<0.001.
Figure 2.
Cellular root phenotypes of Bdtar2lhypo mutants.
(A) Representative Nomarski optics images of mature root portions. s: stele; c: cortex layers; e: epidermis; arrowheads point out top and bottom of individual cells in the 3rd cortex layer; (B) Quantification of mature cortex cell length at 4 dag. (C) Confocal image of a 4-day-old Brachypodium wild type (Bd21) root meristem (top) and quantification of the progression of cell elongation per cell position (for the 60 cells above the stem cell niche) in the central metaxylem (cmx) (bottom). co: columella; DZ: division zone; TZ: transition zone; EZ: elongation zone; (D) Quantification of meristem size as number of metaxylem cells in the combined DZ and TZ. (E) Representative light microscopy images of transverse sections across the mature root. (F–H) Quantification of total area and stele area in root sections, as well as number of cells in the circumference of the innermost cortex layer. (I) Illustration of the segmentation process for quantification of root sections. Overlay of segmented cell shapes (green) on the section (top), labeling of cell layers after filtering (bottom). (J) Combined epidermal and cortical cell number in root sections. (K) Transverse cell area in root sections, for different cell layers from outside to inside. (L) Representative microscopy images of root hairs at 4 dag. (M) Quantification of root hair length in 4-day-old seedlings. Size bars are 100 µm; differences as compared to wild type or mock are not significant unless indicated otherwise; error bars indicate standard error; * = p<0.05; *** = p<0.001.
Figure 3.
Phenotypes of the Bdtar2lqnull mutant in comparison to its wild type background, Bd21-3.
(A) Relative expression level of BdTAR2L in different genotypes at 4 dag as determined by qPCR and normalized with respect to the housekeeping gene, BdUBC18. (B–C) Root elongation in wild type and Bdtar2lqnull mutants, assayed at 4 dag. (D) Representative Nomarski optics images of mature root portions. Arrowheads point out top and bottom of individual cells in a cortex layer; (E) Quantification of mature cortex cell length at 4 dag. (F) Representative microscopy images of root hairs at 4 dag. (G) Representative light microscopy images of transverse sections across the mature root. (H–I) Quantification of total transverse area and stele area in sections from mature roots. (J) Relative seminal root length in Bdtar2lqnull mutants during root growth progression. (K) Progressive breakdown of root meristem as indicated by shrinkage of the meristematic zone. (L) Seedling shoot length at 4 dag. (M) Adult shoots. Size bars are 1 cm (B, M) or 100 µm (D,F,G); differences as compared to wild type or mock are not significant unless indicated otherwise; error bars indicate standard error; * = p<0.05; *** = p<0.001.
Figure 4.
Effect of L-kynerunine (L-kyn) treatment on root elongation of different genotypes.
(A) Representative images of 4-day-old seedlings, transferred onto media with indicated L-kyn concentration at 2 dag. (B) Quantification of root length after 2 days of indicated L-kyn treatment. (C) Representative Nomarski optics images of mature root portions formed during indicated L-kyn treatment. Arrowheads point out top and bottom of individual cells in the 3rd cortex layer; (D) Quantification of mature cortex cell length after 2 days of indicated L-kyn treatment. (E–F) Relative root elongation of indicated mutants and their respective wild type backgrounds after 2 days of indicated 5-methyl-tryptophan treatment. Size bars are 1 cm (A) or 100 µm (C); differences as compared to wild type or mock are not significant unless indicated otherwise; error bars indicate standard error; * = p<0.05; ** = p<0.01; *** = p<0.001.
Figure 5.
Root branching phenotypes of Bdtar2lhypo mutants.
(A) Coleoptile node root formation in 25-day-old plants. (B) Quantification of coleoptile node root elongation. (C) Representative images of 8-day-old seminal roots, note emerged lateral roots. (D) Quantification of emerged lateral root number at 8 dag, normalized for seminal root length. (E) Representative images of 8-day-old seminal roots, 4 days after removal of the root tip. Note emerged lateral roots. (F) Quantification of emerged lateral root number after seminal root tip removal, normalized for seminal root length (per cm). Size bars are 1 cm; differences as compared to wild type are not significant unless indicated otherwise; error bars indicate standard error; ** = p<0.01; *** = p<0.001.
Figure 6.
Auxin homeostasis in Bdtar2lhypo roots and its relation to the ethylene pathway.
(A) Free auxin (IAA) content in wild type and Bdtar2lhypo root segments at 4 dag. The root tip comprised the terminal 8 mm of the roots, the elongated parts all above this. (B) Expression levels of the homologs of various genes encoding rate limiting enzymes in alternative auxin biosynthesis pathways in wild type and Bdtar2lhypo roots at 4 dag. (C) Expression levels of YUCCA homologs in wild type and Bdtar2lhypo roots at 4 dag. (D) Expression levels of BdTAR1L and BdTAR2L in wild type at 3 dag and after 3 h of ACC treatment. (E) Expression levels of YUCCA homologs in wild type at 3 dag and after 3 h of ACC treatment. All expression levels were determined by qPCR and normalized with respect to the housekeeping gene, BdUBC18; differences as compared to wild type or mock are not significant unless indicated otherwise; error bars indicate standard error; * = p<0.05; ** = p<0.01; *** = p<0.001.
Figure 7.
Manipulation of the ethylene pathway and its impact on root growth.
(A) Representative images of 4-day-old seedlings, transferred onto media with indicated ACC concentration at 2 dag. (B) Quantification of root length after 2 days of indicated ACC treatment. (C) Representative images of 4-day-old seedlings, transferred onto media with indicated AVG concentration at 2 dag. (D) Quantification of root length after 2 days of indicated AVG treatment. (E) Representative Nomarski optics images of mature root portions formed during indicated ACC or AVG treatment. Arrowheads point out top and bottom of individual cells in the 3rd cortex layer; (F–G) Quantification of mature cortex cell length after 2 days of indicated ACC or AVG treatment. (H) Schematic presentation of the BdEIN2L1 gene and the location of the T-DNA insertion in the Bdein2l1hypo mutant. (I) Relative expression level of BdEIN2L1 in the Bdein2l1hypo roots at 4 dag. (J) Representative seedlings of wild type and Bdein2l1hypo mutants at 4 dag. (K) Quantification of root length in wild type and Bdein2l1hypo mutants at 4 dag. (L) Expression levels of BdTAR1L and BdTAR2L in wild type and Bdein2l1hypo roots at 4 dag. (M) Expression levels of YUCCA homologs in wild type and Bdein2l1hypo roots at 4 dag. All expression levels were determined by qPCR and normalized with respect to the housekeeping gene, BdUBC18; size bars are 1 cm (A, C) or 100 µm (E); differences as compared to wild type or mock are not significant unless indicated otherwise; error bars indicate standard error; * = p<0.05; ** = p<0.01; *** = p<0.001.
Figure 8.
A schematic overview of the regulation of tryptophan-dependent auxin (indole-3-acetic acid) biosynthesis via indole-3-pyruvic acid (IPA) by ethylene action in Arabidopsis (A) and Brachypodium (B).