Effect of Low Red-to-Far-Red Light on Stem Elongation and Pith Cell Development in Dicots

Abstract

In dense canopies, light becomes a limiting factor for plant growth. Many plants respond to neighbor cues by growing taller to improve light capture, a phenomenon known as the shade avoidance syndrome (SAS). The major neighbor detection is via enrichment of far-red (FR) light that leads to a low red:far-red light ratio (R:FR), suppressing phytochrome activity. In tomato, low R:FR induces elongation of the internodes, but study into the role of different cell types in this response has remained limited. We characterized changes in cellular anatomy of the tomato internode in response to low R:FR and its accompanying changes in gene expression. We observed changes to the pith traits, including increases in pith layer number, pith cell diameter, and longitudinal cell length. We profiled the transcriptome in the entire internodes and in the hand-dissected pith in the central cylinder of the internode in response to low R:FR treatment and identified transcription factors (TFs) of interest that were upregulated in the central cylinder, mostly GATA, TCP, and bZIPs. We then characterized FR responses in eight dicotyledonous species. Significant pith elongation was observed in species that exhibited a strong internode elongation response. The FR-responsive expression of homologs of target GATA, TCP, and bZIP TFs in the central cylinder was conserved within the Solanaceae family. Overall, we discovered central cylinder gene expression patterns in SAS that are distinct from those of the entire internode, suggesting that some responses are unique and likely specific to vascular cell types such as pith. These patterns were conserved with close relatives of tomato but not in other dicot families we sampled, indicating that different molecular mechanisms drive FR responses in different dicots.

Keywords: central cylinder; elongation; pith; shade avoidance syndrome; stem; transcription factors.

FIGURE 1

Tomato internode cellular morphology in response to low R:FR. (a) Comparison of Solanum lycopersicum cultivars under different light conditions. Two 24‐day‐old M82 plants after 10 days of treatment of either white light (WL) or white light supplemented with far‐red (WL + FR). (b) Stem length analysis in M82 and Moneymaker (MM) of 3‐week‐old plants following 7‐day treatments under WL and WL + FR. Notable differences between WL and FR + WL conditions are indicated with asterisks (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). This analysis was conducted with 18 biological replicates and repeated three times. MM data are from Li et al. (2024). (c) Cellular anatomy of M82 first internode. The cross section details the cell types of interest including epidermis (E), collenchyma (C), parenchyma (Pa), and interfascicular cambium (IC), Vasculature (V, combined xylem and phloem) and pith (Pi). (d,e) FR‐responsive changes in cellular anatomy traits in the tomato first internode. The heatmaps show (d) logFC and (e) ‐log(p‐value) for each trait comparing WL + FR to WL. The layer number of each cell type is indicated from the outermost layer moving inward. Measurements of interfascicular cambium thickness were done in areas without vascular bundles. Cell length was captured from longitudinal sections, whereas the rest of the measurements were taken from cross sections. A logFC (logarithm of fold change) of 0 is depicted in white to signify no change, with upregulation shown in red and downregulation in blue. A‐log Pval of ‐log0.05 is set to white, indicating no statistical significance.

FIGURE 2

Transcriptomic responses of tomato first internode to low R:FR. (a) The experimental design of the transcriptomics experiment, including the timing of harvest and the specific tissues harvested. White bar represents the WL treatment, red bars represent periods when the supplemental FR light is added, while black bars indicate night phases, and alternating light/dark bars depict the light/dark cycle. (b) DEG analysis of FR‐responsive genes for each timepoint for whole internode and central cylinder samples. Upregulated genes are marked in red and downregulated genes in blue. (c) DEG analysis of central cylinder‐enriched and cylinder‐depleted genes, as identified by DE analysis for central cylinder versus whole internode for specific timepoints and light treatments. Central cylinder‐enriched genes marked in red and central cylinder‐depleted genes in blue. (d) Overlap of FR‐responsive upregulated DEGs between the central cylinder and whole internode at 30 and 48 h of light treatment. (e–g) The expression patterns of the three genes encoding for TFs of interest. (e) Solyc01g09760, (f) Solyc07g053459, and (g) Solyc08g080150. The significant differences are determined using two‐way ANOVA, and error bars represent standard errors. (h–k) GUS staining of 2‐week‐old proSolyc01g090760::nlsGFPGUS seedling with 6‐h FR treatment. Cross sections show (i) the first internode, (j) petiole of the first true leaf, and (k) the hypocotyl. (l–n) GUS staining of 2‐week‐old proSolyc07g053450::nlsGFPGUS seedling with 6‐h FR treatment. Cross sections show (m) the first internode and (n) petiole of the first true leaf.

FIGURE 3

FR‐responsive internode elongation is accompanied with pith cell changes across multiple dicots. Representative images of 7‐day WL and WL + FR‐treated plants (a,d,g,j,m,p), measurements of the first internode or inflorescence length (b,e,h,k,n,w), and pith cell length measurements (c,f,i,l,n,r). The data are given by species as follows: (a–c) bell pepper ( Capsicum annuum ), (d–f) eggplant ( Solanum melongena ), (g–i) soybean ( Glycine max ), (j–l) pea ( Pisum sativum ), (m–o) black mustard ( Brassica nigra ), and (p–r) Arabidopsis (Col‐0). Asterisks in the figure denote significant differences between WL and WL + FR conditions, with: * represents p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. The sample size for each phenotyping comparison was approximately 12, and each experiment was repeated twice. The sample size for each cellular phenotyping comparison was approximately 50.

FIGURE 4

FR‐responsiveness of target TF expression is conserved in Solanaceae. (a) Phylogeny and expression patterns of Solyc07g053450 homologs: tomato (Solyc07g053450), bell pepper (CA00g71840:1‐921), eggplant (SMEL4.1_07g019740.1.01), Arabidopsis (AT2G42380), four black mustard homologs (BniB06g001480.2N.1, BniB08g027470.2N.1, BniB08g072820.2N.1, BniB06g055220.2N.1), soybean (Gm06:49537899..49539195), and two pea homologs (XM_051027770.1, XM_051045459.1). (b) Phylogeny and expression patterns of Solyc08g080150 homologs: tomato (Solyc08g080150), bell pepper (TCP19 XM_016712397.2), eggplant (chr8:85852001‐85853900), two Arabidopsis homologs (AT2G45680, AT5G51910), black mustard (BniB08g029800.2N.1), two soybean homologs (Glyma.07G080300.1, Glyma07g08710.2), and two pea homologs (XM_051030490.1, XM_051030491.1). (c) Phylogeny and expression patterns of Solyc01g090760 homologs: tomato (Solyc01g090760), two bell pepper homologs (chr3:267174801‐267176100, chr8:35902201‐35904000), eggplant (chr8:35902201‐35904000), Arabidopsis (AT2G45050), black mustard (BniB07g039570.2N.1), soybean (GlysoPI483463.06G078800.1), and three pea homologs (XM_051027377, XM_051030552.1, XM_051038772.1). Each section utilizes Mega X software for phylogenetic analysis, adopting the Tamura‐Nei model with a bootstrap analysis (1000 replicates) for enhanced accuracy. The gene expression data from qRT‐PCR is represented as heatmaps of log fold changes in gene expression under WL + FR versus WL conditions in the internode or pith at 6 and 24 h after FR treatment, with red indicating upregulation, blue for downregulation, and white for no change (logFC = 0).