The epidermis coordinates auxin-induced stem growth in response to shade

Abstract

Growth of a complex multicellular organism requires coordinated changes in diverse cell types. These cellular changes generate organs of the correct size, shape, and functionality. In plants, the growth hormone auxin induces stem elongation in response to shade; however, which cell types of the stem perceive the auxin signal and contribute to organ growth is poorly understood. Here, we blocked the transcriptional response to auxin within specific tissues to show that auxin signaling is required in many cell types for correct hypocotyl growth in shade, with a key role for the epidermis. Combining genetic manipulations in Arabidopsis thaliana with transcriptional profiling of the hypocotyl epidermis from Brassica rapa, we show that auxin acts in the epidermis in part by inducing activity of the locally acting, growth-promoting brassinosteroid pathway. Our findings clarify cell-specific auxin function in the hypocotyl and highlight the complexity of cell type interactions within a growing organ.

Keywords: auxin; brassinosteroid; epidermis; shade avoidance; stem growth.

Figure 1.

axr3-1 mutants of Arabidopsis have defects in shade-induced hypocotyl growth. (A) Hypocotyl length of 9-d-old seedlings grown in white (W) light or shade. Data show mean ± SEM. (**) P < 0.005; (ns) not significant, Tukey's HSD. The control genotype is plants hemizygous for UBQ10pro::GAL4::VP16. (B) Representative images of wild-type (Col-0) and axr3-1 mutants grown as in A. (C) Image of a GUS-stained 9-d-old white light-grown whole seedling carrying an IAA17pro::GUS::mCit transcriptional reporter. The red arrowhead marks the hypocotyl. (D) Schematic representation of cell types in a young Arabidopsis hypocotyl cross-section. (E) Fluorescence image showing GUS::mCit protein localization in a cross-section through the mid–upper portion of the hypocotyl from a 9-d-old IAA17pro::GUS::mCit plant. Note the GUS::mCit fluorescence in the epidermis and cortex. Bars: B,C, 5 mm; E, 50 µm.

Figure 2.

axr3-1 expression in different cell types alters hypocotyl growth in shade. (A–F) Representative fluorescence images of hypocotyl cross-sections of 9-d-old seedlings expressing axr3-1::mCit protein (green) under the control of different cell-specific promoters and counterstained with propidium iodide (red). CER6pro was used to drive axr3-1 expression in the epidermis (A), CAB3pro in green tissue (including strong cortical and weak epidermal expression) (B), SCRpro in the endodermis (C), SHRpro throughout the stele (D), PXYpro in procambium cells (E), and SUC2pro in the phloem companion cells (F). Arrows mark examples of nuclear-localized axr3-1::mCit. Not all nuclei of a given cell layer are in the plane of focus. In our hands, PXYpro had an expression pattern similar to that of SHRpro, including expression in the meristematic pericycle and procambium and perhaps other unidentified cells of the vasculature. SUC2pro also had occasional weak expression in the epidermis. For each promoter, the cell-specific pattern of axr3-1::mCit expression was unaffected by shade, and multiple independent lines had similar phenotypes that correlated with expression level (Supplemental Figs. S2, S3). In addition to hypocotyl expression, these promoters drove axr3-1::mCit in equivalent cell types of the cotyledons and, in some cases, various zones of the root. Bars, 50 µm. (G) Hypocotyl length in white (W) light and shade of 9-d-old seedlings expressing axr3-1::mCit under the control of a cell-specific promoter. Control is hemizygous for UAS::axr3-1::mCit. Data show mean ± SEM. Means are compared with the control in the same light condition using Tukey's HSD. (**) P < 0.005; (ns) not significant.

Figure 3.

Blocking auxin responses in the epidermis feeds back onto auxin production and gene expression. (A) Levels of free IAA in 5-d-old white light-grown Arabidopsis seedlings. Data show mean ± SEM. (**) P < 0.005; (ns) not significant, Student's t-test. (B) Box plots showing gene expression values (left) and fold change in shade (right) of 5-d-old white (W) light-grown seedlings treated for 4 h of white light or shade measured by RNA sequencing (RNA-seq). Seedlings were either hemizygous for the UAS::axr3-1::mCit transgene only (control) or expressed axr3-1::mCit under the CER6pro (hemizygous for both UAS::axr3-1::mCit and CER6pro::GAL4::VP16 transgenes). Of 104 previously identified shade-induced, SAV3-dependent genes (Li et al. 2012), 12 were significantly up-regulated by shade in our control. P < 0.005, one-sided Fisher's exact test. Expression values for these 12 genes are shown. (**) P < 0.005; (ns) not significant, Student's t-test. (C) Same as in B, except showing SAV3-independent shade-induced genes (20 of 40 such genes were significantly induced by shade in our control). P < 0.005, one-sided Fisher's exact test. (D) Same as in B, except showing BR genes. Of 1005 genes previously identified as BR-activated (Sun et al. 2010), 46 were significantly induced by shade in our control and are shown here. P < 0.005, one-sided Fisher's exact test. Of these, three were also classified as SAV3-dependent genes, and two were classified as SAV3-independent genes.

Figure 4.

Auxin signaling is required in the epidermis for hypocotyl growth. (A) Hypocotyl length of 7-d-old white light-grown wild-type (Col-0) and axr3-1 mutant Arabidopsis seedlings grown on picloram (PIC). Data points show mean ± SEM. One-way ANOVA, Col-0, P < 0.005; axr3-1, not significant. (B) Representative images of Col-0 and axr3-1 seedlings grown as in A. Picloram concentrations were (from left to right) 0, 0.05, 0.1, 0.5, 1, 5, 10, and 50 µM. axr3-1 seedlings were agravitropic but are arranged vertically here for comparison with the Col-0 strain. Bars, 5 mm. (C) Hypocotyl length of 7-d-old white light-grown seedlings grown in the presence of 5 µM picloram or without picloram (DMSO vehicle). (D) Hypocotyl length of white light-grown seedlings grown for 5 d at 22°C followed by 4 d at either 22°C or 28°C. In C and D, the control genotype was plants hemizygous for the UAS::axr3-1::mCit transgene. Data show mean ± SEM. Tukey's HSD was calculated within a group. (**) P < 0.005; (ns) not significant.

Figure 5.

Shade increases the expression of auxin and BR target genes in the hypocotyl epidermis of Brassica plants. (A) Overlap of genes with epidermal peel-enriched expression (in either white [W] light or shade) with shade-induced and shade-repressed genes in epidermal peel samples. Tissue was collected from 4-d-old white light-grown Brassica seedlings treated with 9 h of white light or shade. Gene expression was measured by RNA-seq. (B) Overlap of genes expressed at higher levels in the whole hypocotyl tissue relative to epidermal peels (referred to here as “inner tissue-enriched genes”) with shade-induced and shade-repressed genes in whole hypocotyl tissue samples. In A and B, all overlapping regions are significant. P < 0.005, one-sided Fisher's exact tests. (C) Of 174 putative orthologs of SAV3-dependent shade-induced genes (Li et al. 2012), 77 were significantly induced by shade in both epidermal peel and whole hypocotyl tissue. Box plot shows expression values for these genes. (D) Of 1702 putative orthologs of BR-activated genes (Sun et al. 2010), 475 were significantly induced by shade in both epidermal peels and whole hypocotyl tissue. The box plot shows the expression values for these genes. Twenty-nine of these genes are also represented in C. (E–G) Fold enrichment of gene expression in epidermal peels compared with whole hypocotyl tissue for white light and shade treatments for putative orthologs of Aux/IAA (E), ARF (F), and TIR1/AFB (G) family members. Only genes with fragments per kilobase per million mapped fragments (FPKM) > 0 in all sample types are shown. Purple and green diamonds indicate expression values in epidermal peels that are significantly different from whole hypocotyl tissue (false discovery rate, q < 0.05). Purple indicates epidermis-enriched, and green indicates inner tissue-enriched. Lines connect genes that have significantly enriched expression in either the epidermis or inner tissues in both light conditions.

Figure 6.

The Brassica epidermis is enriched for expression of auxin and BR target genes with high sensitivity to 9 h of shade treatment. (A) Fold enrichment of gene expression in Brassica epidermal peels compared with whole hypocotyl tissue in white (W) light-treated and shade-treated seedlings for genes associated with the GO term “auxin-activated signaling pathway.” Purple and green diamonds indicate expression values in epidermal peels that are significantly different from whole hypocotyl tissue (false discovery rate, q < 0.05). Purple indicates epidermis-enriched, and green indicates inner tissue-enriched. Lines connect genes that have significantly enriched expression in either the epidermis or inner tissues in both light conditions. Note that most genes do not change the tissue type of enrichment. Only those genes with FPKM > 0 in all sample types are shown. (B) Fold change in shade of gene expression in whole hypocotyl tissue (Y-axis) plotted against fold change in epidermal peel samples (X-axis). “Auxin-activated signaling pathway” genes that are significantly enriched in either the epidermis or inner tissues (in either white light or shade) are shown. The boxed region represents a cluster of 29 epidermis-enriched genes highly induced by shade. (C) Box plot of genes shown in B, showing fold change in shade. Note that the epidermis-enriched genes are more sensitive to shade. (**) P < 0.005; (*) P < 0.05; (ns) not significant, Student's t-test. (D–F) Same as in A–C, except showing genes associated with the GO term “response to BR stimulus.” (G,H) Same as in B and C except showing SAUR family genes. Genes with FPKM > 0 in all samples but that are not enriched in any particular tissue type are also shown. In H, comparisons are Tukey's HSD. (I) Hypocotyl cross-section of a 6-d-old Arabidopsis seedling showing GFP localization (green) in a SAUR19pro::GFP::GUS plant. Chlorophyll autofluorescence (blue) shows the position of the cortical cells. Note the strong GFP localization to the epidermis. Bar, 50 µm.

Figure 7.

Auxin functions to regulate hypocotyl growth in part through the BR pathway, upstream of BZR1. (A) Hypocotyl length of 9-d-old Arabidopsis seedlings treated with white (W) light or shade in the presence of BL or without BL (DMSO vehicle). Data show mean ± SEM. Statistical comparisons are shown for each mean compared with the control genotype grown in the same light condition. (**) P < 0.005; (ns) not significant, Tukey's HSD. The CER6pro>>axr3-1::mCit line shown is number 4. Control plants were CER6pro::GAL4::VP16 hemizygotes. (B) Representative images of plants from A. For each genotype and pharmacological treatment, white light-grown is at the left, and shade-treated is at the right. Bars, 5 mm. (C,D) Representative fluorescence images of nuclear BZR1::YPET protein in hypocotyl epidermal cells of 5-d-old white light-grown plants treated for 24 h with DMSO, 1 µM BL, or 5 µM picloram (PIC) (C), or, in a separate experiment, 6-d-old white light-grown plants treated with 24 h of white light or shade (D). Images were taken near the center of the hypocotyl and with the same exposure in a given panel. Bars, 10 µm. The graph shows the corrected total nuclear fluorescence for plants treated as in D. Nuclear fluorescence was measured from 54 and 59 nuclei from 12 white light-treated and 12 shade-treated plants, respectively. Data show mean ± SEM. (**) P < 0.005, Student's t-test. (E) A model for auxin-induced hypocotyl growth in shade. Shade stimulates auxin production in the cotyledons, which is transported to the hypocotyl (Tao et al. 2008). As auxin travels down the hypocotyl via the vasculature, it is transported laterally and is perceived by the different cell layers (Keuskamp et al. 2010). Auxin perception by nonepidermal cell types has minor contributions to hypocotyl elongation but may have greater roles to play in other auxin-dependent phenotypes not assessed here. In the epidermis (red), auxin induces auxin and BR target genes and cellular growth through both BR-dependent (upstream of BZR1) and BR-independent mechanisms. How the epidermis signals back to instruct the inner cell layers on how to grow is unknown but may involve a chemical or mechanical signal. In addition, auxin signaling in the epidermis negatively feeds back onto auxin production. We were not able to resolve whether this feedback occurs in the cotyledons, from the hypocotyl, or from another organ.