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 (<i>A</i>) and Brachypodium (<i>B</i>).

<p>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 (<i>A</i>) and Brachypodium (<i>B</i>).</p

Effect of L-kynerunine (L-kyn) treatment on root elongation of different genotypes.

<p>(<i>A</i>) Representative images of 4-day-old seedlings, transferred onto media with indicated L-kyn concentration at 2 dag. (<i>B</i>) Quantification of root length after 2 days of indicated L-kyn treatment. (<i>C</i>) 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 3^rd cortex layer; (<i>D</i>) Quantification of mature cortex cell length after 2 days of indicated L-kyn treatment. (<i>E–F</i>) 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 (<i>A</i>) or 100 µm (<i>C</i>); 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.</p

Isolation of the <i>Bdtar2l^hypo</i> mutant and characterization of macroscopic phenotypes.

<p>(<i>A</i>) 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 <i>Bdtar2l^hypo</i> mutation. (<i>B</i>) Seminal root length quantification of the different genotypes at 4 days after germination (dag). (<i>C</i>) Schematic presentation of the <i>BdTAR2L</i> gene and the location of the T-DNA insertion in the <i>Bdtar2l^hypo</i> mutant. (<i>D–E</i>) Relative expression level of <i>BdTAR2L</i> in different genotypes at 4 dag as determined by qPCR and normalized with respect to the housekeeping gene, <i>BdUBC18</i>. (<i>F</i>) Root elongation in wild type, control and homozygous <i>Bdtar2l^hypo</i> mutants, assayed at 4 dag. (<i>G–H</i>) Quantification of seedling phenotypes at 4 dag. (<i>I</i>) Leaf number at 18 dag. (<i>J–L</i>) Different size parameters of the 5^th leaf of plants, assayed at 18 dag. (<i>M</i>) 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.</p

Auxin homeostasis in <i>Bdtar2l^hypo</i> roots and its relation to the ethylene pathway.

<p>(<i>A</i>) Free auxin (IAA) content in wild type and <i>Bdtar2l^hypo</i> root segments at 4 dag. The root tip comprised the terminal 8 mm of the roots, the elongated parts all above this. (<i>B</i>) Expression levels of the homologs of various genes encoding rate limiting enzymes in alternative auxin biosynthesis pathways in wild type and <i>Bdtar2l^hypo</i> roots at 4 dag. (<i>C</i>) Expression levels of <i>YUCCA</i> homologs in wild type and <i>Bdtar2l^hypo</i> roots at 4 dag. (<i>D</i>) Expression levels of <i>BdTAR1L</i> and <i>BdTAR2L</i> in wild type at 3 dag and after 3 h of ACC treatment. (<i>E</i>) Expression levels of <i>YUCCA</i> 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, <i>BdUBC18</i>; 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.</p

Phenotypes of the <i>Bdtar2l^qnull</i> mutant in comparison to its wild type background, Bd21-3.

<p>(<i>A</i>) Relative expression level of <i>BdTAR2L</i> in different genotypes at 4 dag as determined by qPCR and normalized with respect to the housekeeping gene, <i>BdUBC18</i>. (<i>B–C</i>) Root elongation in wild type and <i>Bdtar2l^qnull</i> mutants, assayed at 4 dag. (<i>D</i>) Representative Nomarski optics images of mature root portions. Arrowheads point out top and bottom of individual cells in a cortex layer; (<i>E</i>) Quantification of mature cortex cell length at 4 dag. (<i>F</i>) Representative microscopy images of root hairs at 4 dag. (<i>G</i>) Representative light microscopy images of transverse sections across the mature root. (<i>H–I</i>) Quantification of total transverse area and stele area in sections from mature roots. (<i>J</i>) Relative seminal root length in <i>Bdtar2l^qnull</i> mutants during root growth progression. (<i>K</i>) Progressive breakdown of root meristem as indicated by shrinkage of the meristematic zone. (<i>L</i>) Seedling shoot length at 4 dag. (<i>M</i>) Adult shoots. Size bars are 1 cm (<i>B, M</i>) or 100 µm (<i>D,F,G</i>); 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.</p

Additional file 1 of Profiling of 1-aminocyclopropane-1-carboxylic acid and selected phytohormones in Arabidopsis using liquid chromatography-tandem mass spectrometry

Supplementary Material

Model of auxin transport-mediated communication in stems.

<p>Schematic figure illustrating models of auxin transport in an Arabidopsis stem segment bearing two buds. Pink arrows indicate auxin transport; purple shading indicates concentration of auxin exported from the left-hand bud. Blue shading indicates the position of the high-conductance polar auxin transport stream (PATS), dark green shading indicates low-conductance polar transport, and light green shading indicates low-conductance non-polar auxin transport. The green areas together constitute connective auxin transport (CAT). Auxin is exchanged between PATS and CAT (red crossed arrows). <b>A)</b> Auxin transport scheme with only high-conductance polar auxin transport around the vascular bundles. Auxin exported from the bud on the left is rapidly taken up into the PATS on the left. The mechanism of communication between buds (cyan arrow) is unclear. <b>B)</b> Auxin transport scheme with widespread auxin transport in the stem. There is widespread exchange of auxin between PATS and CAT tissues, particularly in the younger part of the stem (top). Auxin exported from the bud on the left can move across the stem, providing a mechanism for bud-bud communication. In older parts of the stem (bottom), the decline in PIN4/PIN7 expression reduces auxin exchange between PATS and CAT tissues.</p

PIN3, PIN4, and PIN7 influence shoot branching.

<p><b>A)</b> Rosette branching in Col-0, <i>pin4-3 pin7-1</i>, and <i>pin3-3 pin4-3 pin7-1</i> after decapitation. <i>n</i> = 21–22, bars indicate s.e.m. For each time point, bars with different letters are significantly different from each other (ANOVA, Tukey HSD, <i>p</i> < 0.05). <b>B)</b> Bud-bud communication in Col-0, <i>pin4-3 pin7-1</i>, and <i>pin3-3 pin4-3 pin7-1</i>. Explants were decapitated and left for 10 d. The mean relative growth index (longest branch/total branch length) was calculated for each genotype. <i>n</i> = 21–24; bars indicate s.e.m. Relative growth index is significantly reduced in <i>pin4-3 pin7-1</i> and <i>pin3-3 pin4-3 pin7-1</i> relative to Col-0 (ANOVA, <i>p</i> < 0.05). <b>C)</b> The angle formed between secondary cauline branches and the primary stem at the point of emergence in Col-0 and <i>pin3-3 pin4-3 pin7-1</i>. <i>n</i> = 45–46 cauline branches from at least 10 plants per genotype; bars indicate s.e.m. The angle is significantly different between the two genotypes (<i>t</i> test, <i>n</i> = 45–46, <i>p</i> < 0.005). <b>D)</b> Phenotype of the <i>pin3-3 pin4-3 pin7-1</i> shoot system compared to Col-0 (6 wk old). <b>E–H)</b> Leaf phenotypes in 4-wk-old rosettes of Col-0 (E), <i>pin3-3 pin4-3</i> (F), <i>pin4-3 pin7-1</i> (G), and <i>pin3-3 pin4-3 pin7-1</i> (H).</p

Auxin efflux dynamics from stem segments are nonlinear.

<p><b>A)</b> Endogenous auxin eluted from 2 cm stem segments from the basal inflorescence internode of 6-wk-old plants was quantified by GC-MS at different points post excision. The cumulative auxin collected (pg) is shown relative to time. The red line indicates the approximate free auxin content of the stem segments at t = 0. <b>B)</b> Distribution of radio-labelled IAA (measured as CPM) in 2 mm intervals of 24 mm long stem segments after a 10 min pulse of 5 μM radio-labelled IAA was supplied to the apical end of the segment. Stems were dissected and analyzed by scintillation 30 min (blue line), 60 min (red line) or 90 min (green line) after the application of the pulse; <i>n</i> = 8 per time point, bars indicate standard error of the mean (s.e.m.). <b>C)</b> Distribution of radio-labelled IAA (measured as CPM) in 2 mm intervals of 24 mm long stem segments after a 10 min pulse of 5 μM radio-labelled IAA was supplied to the apical end of the segment. Stems were dissected and analyzed by scintillation 120 min (purple line), 150 min (light blue line) or 180 min (orange line) after the application of the pulse; <i>n</i> = 8 per time point, bars indicate s.e.m. <b>D)</b> Auxin transport assay to measure cross-stem auxin movement. Two schemes were trialed (b–c and d–e), in both of which the apical end of 18 mm stem segments were dissected so that radio-labelled auxin could be supplied to only half of the stem (see illustration). Longitudinal incisions were made across the diameter of the stem (top image, red dotted line), to a depth of 5 mm, followed by a second transverse incision to remove half the stem. Control segments (a) were left intact. In the d–e scheme, the basal end of the segment was similarly treated at the start of the experiment to leave either half the stem directly beneath the site of auxin application (d) or diametrically opposite the site of application (e), while in the b–c scheme the basal end was left intact during the assay. The apical end of the stem segments was then immersed in 2μM ^14CIAA for 6 hours. At the end of the assay, the basal 5 mm of stem was dissected from the stems. In the b–c scheme, the basal 5 mm was longitudinally bisected, to separate the tissue directly under the site of auxin application (b) from the tissue diametrically opposite (c). The amount of radio-label transported into the basal 5 mm in each of a, b, c, d, and e was then measured by scintillation. The graph shows the auxin transported in each of these dissections (measured as CPM), <i>n</i> = 16, bars indicate s.e.m.</p

Anatomy of Arabidopsis inflorescence stems.

<p><b>A)</b> Light micrograph of transverse section through the basal internode of a 6-wk-old Arabidopsis inflorescence stem. Vascular bundles are clearly visible as dark green triangles. Longitudinal sections used in this study were made across the center of the stem, between two vascular bundles, as indicated by the white line. <b>B, C)</b> Close up of cellular anatomy in a transverse section of an Arabidopsis vascular bundle and surrounding tissue. (C) is shaded to indicate tissue types. <b>D)</b> Light micrograph of longitudinal section through the basal internode of a 6-wk-old Arabidopsis inflorescence stem (along the type of transect indicated in A). Vascular bundles are visible as continuous white lines (indicated by white arrow heads). Inset shows the whole 2 cm segment. White box indicates the region shown in E, F. <b>E,F)</b> Close up of cellular anatomy in longitudinal section of an Arabidopsis vascular bundle and surrounding tissue. (F) is shaded to indicate tissue types. Unshaded/P = pith, purple/XP = xylem parenchyma, yellow/X = xylem, green/C = cambium, red/Ph = phloem, orange/E = epidermis, blue/IFF = interfascicular fibers.</p