A suberized exodermis is required for tomato drought tolerance

Abstract

Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place.

Fig. 1. Suberin is deposited in the tomato exodermis and is regulated by ABA.

a, Graphical representation (left) of S. lycopersicum (cv. M82) root anatomy (the exodermis is highlighted in yellow) and representative cross-section (right) of a 7-day-old root stained with FY. Scale bar, 100 µm. b, Transmission electron microscopy cross-sections of 7-day-old roots obtained at 1 mm from the root–hypocotyl junction. Top: the epidermal (ep), exodermal (exo) and inner cortex (co) layers. Bottom: a close-up of the featured region (zone defined with blue dotted lines), showing the presence of suberin lamellae (SL). cw, cell wall; pm, plasma membrane. c, Fluorol yellow (FY) staining for suberin in wild-type 7-day-old plants treated with mock or 1 µM ABA for 48 h. Whole-mount staining of primary root (left) and mean fluorol yellow signal along the root (right), n = 6; error bars, s.d. Asterisks indicate significance with one-way analysis of variance (ANOVA) followed by a Tukey-Kramer post hoc test (***P < 0.005). NS, not significant. d, Developmental stages of suberin deposition of wild-type plants treated with mock or 1 µM ABA for 48 h. Zones were classified as non-suberized (white), patchy suberized (grey) and continuously suberized (yellow); letters indicate statistically different groups; apostrophes indicate different statistical comparisons; n = 6; error bars, s.d.

Fig. 2. The tomato suberin biosynthetic enzymes and transcriptional regulator are expressed in the mature exodermis.

a, Simplified diagram of the suberin biosynthesis pathway. Boxes indicate gene families involved in each step of the pathway (blue and yellow indicate biosynthetic enzymes and transcriptional regulators, respectively). Genes targeted in this study are outlined in red. TFs, transcription factors; VLCFAs, very-long-chain fatty acids. b, Annotated single-cell clusters from 3 cm of the tomato root tip displayed by an integrated UMAP. R.C., root cap. Q.C., quiescent centre; col, columella; procamb, procambium. c, UMAP of cortex/endodermis/exodermis-annotated cells that were extracted from the general projection and re-embedded. A small cluster of cells from the meristematic zone clusters were included to help anchor pseudotime estimations. CEEI, cortex–exodermis–endodermis initial. d, A pseudotime trajectory analysis for the cortex/endodermis/exodermis cell populations. e, Cell type or tissue-specific expression profiles for suberin biosynthetic pathway genes. Dot diameter represents the percentage of cells in which each gene is expressed (% Exp.) and colours indicate the average scaled expression of each gene in each developmental stage group with warmer colours indicating higher expression levels. f, Expression of SlMYB92 and SlASFT in the single-cell transcriptome data. The colour scale represents log₂-normalized corrected UMI counts.

Fig. 3. Loss-of-function mutant alleles of candidate genes disrupt exodermal suberization in tomato.

a, Graphical summary of the hairy root (HR; R. rhizogenes) mutant screen. b, Summary of mutant phenotypes of candidate genes in hairy roots. Top: representative cross-sections of mature portions of the roots stained with fluorol yellow. Bottom: overall quantification of the fluorol yellow signal across multiple cross-sections (wild-type n = 66; rest n = 6). Red line indicates statistically significant differences in fluorol yellow pixel intensity in the mutant versus wild type as determined with a one-way ANOVA followed by a Tukey–Kramer post hoc test (P_adj < 0.05); EV, empty vector. Box plot centres depict the median while the bottom and top box limits depict the 25th and 75th percentile, respectively. Whiskers represent minima and maxima. Dots depict individual samples. c, Fluorol yellow staining for suberin in 7-day-old wild-type (repeated from Fig. 1 for reference), slmyb92-1 and slasft-1 plants treated with mock or 1 µM ABA for 48 h. Whole-mount staining of primary roots across different sections (left) and mean intensity of fluorol yellow signal along the root (right) (n = 6). Letters indicate significant differences (one-way ANOVA followed by a Tukey–Kramer post hoc test (P_adj < 0.05). Error bars, s.d. d, Developmental stages of suberin deposition in the 7-day-old wild-type and mutant plants treated with mock or 1 µM ABA for 48 h. Zones were classified as non-suberized (white), patchy suberized (grey) and continuously suberized (yellow) (n = 6). Letters indicate statistically different groups; apostrophes indicate different statistical comparisons e, Representative transmission electron microscopy cross-sections of slasft-1 and slmyb92-1 mutants obtained at 1 mm from the root–hypocotyl junction. The slasft-1 mutant presents a deficit in suberin lamellar structure. cw, cell wall; dSL, defective suberin lamellae; exo, exodermis; pm, plasma membrane; SL, suberin lamellae.

Fig. 4. Impaired suberin deposition in slmyb92-1 and slasft-1 perturbs their whole-plant performances in response to water limitation.

a, Suberin composition in roots of mature 1-month-old wild type, slmyb92-1 and slasft-1 plants. Plants were exposed to 10 days of water-sufficient (WS) and water-limitation (WL) regimes (n = 4, Methods). Acid, fatty acids; alcohols, primary alcohols; ω-OH, ω-hydroxy fatty acids; DCA, dicarboxylic fatty acid; aromatics, ferulate and coumarate isomers. Error bars denote s.d. b–e, Dot plots of recorded values for stem water potential (stem Ψ) (b), relative water content (c), transpiration (d) and stomatal conductance (g_s) (e). Dotted line indicates zoom-in for better visual resolution of values. Black dots indicate mean values (n = 6). One-way ANOVAs for each treatment were performed followed by a Tukey–Kramer post-hoc test. ***P < 0.001 **P < 0.01 *P < 0.05 ‘.’ P < 0.1.

Extended Data Fig. 1. Suberin deposition and ultra-structure in tomato exodermal cells.

Transmission electron microscopy cross-sections of 7-day-old wild type (a-b), asft-1 (c-d) and myb92-1 (e-f) plants obtained at 1 mm from the root-hypocotyl junction. Overview images show the epidermal, exodermal and inner cortex layers. Close-up images (corresponding to the zones defined with blue dashed lines in the adjacent Overview images) show the presence or absence of suberin lamellae. Black arrows indicate the presence of suberin lamellae, white arrow indicates areas where suberin lamellae could not be detected. scale bars = 5 µm for overview and 200 nm for close-up. Close-ups of c & f are repeated in Fig. 3. co = cortex, exo = exodermis, ep = epidermis, SL = suberin lamellae, dSL = defective suberin lamellae, cw = cell wall, pm = plasma membrane.

Extended Data Fig. 2. Roots of tomato wild relative Solanum pennellii (LA0716) contain significantly more suberin than Solanum lycopersicum (cv. M82).

(a) Mean monomer abundance and (b) total suberin expressed as μg mg⁻¹ of total dry weight. n = 5, error bars: SD. Asterisks indicate significance with one-way ANOVA followed by a Tukey-Kramer post hoc test: P: ‘***’ <0.001 ‘**’ <0.01, n.s.: ‘not significant’. Acid: fatty acids; Alcohols: primary alcohols; ω-OH: ω-hydroxy fatty acids; DCA: dicarboxylic fatty acid; Aromatics: ferulate and coumarate derivatives.

Extended Data Fig. 3. ABA treatment increases suberin levels but not localization in Solanum lycopersicum, while levels remain unchanged in Solanum pennellii.

(a) Fluorol yellow staining of 7-day-old S. lycopersicum wild-type plants treated with mock or 1 µM ABA for 48 h. Cross-sections of roots at 1 cm from the hypocotyl junction. (b) Fluorol yellow staining for suberin in tomato wild relative S. pennellii (LA0716) 7-day-old plants treated with mock or 1 µM ABA for 48 h. Whole-mount staining of primary roots. (c) Developmental stages of suberin deposition of plants treated with mock or 1 µM ABA for 48 h. Zones were classified in non-suberized (white), patchy suberized (gray) and continuously suberized (yellow), n = 7, error bars: SD.

Extended Data Fig. 4. Single cell transcriptome atlas of the tomato root.

(a) Graphical depiction of a tomato root section with cell types profiled in the single cell population. (b) A pseudo-time trajectory analysis ran on the population. (c) Annotation of single cell clusters displayed by an integrated uniform manifold approximation and projection (UMAP). Circles indicate subpopulations clustered together. (d) Reproducibility of biological replicates as observed by UMAP and cluster identification. (e) Expression profiles for 39 genes expressed across the major root tissue types. Dot diameter represents the percentage of cells in which each gene is expressed (% Exp.); and colors indicate the average scaled expression of each gene in each developmental stage group with warmer colors indicating higher expression levels. Top row indicates whether the gene’s expression has been validated in S. lycopersicum in previously published work. R.C.: Root cap. Q.C.: Quiescent center. Col: Columella. Procamb: Procambium. Phlo: Phloem.

Extended Data Fig. 5. The SlASFT promoter drives exodermal-specific expression in roots.

Representative images depicting expression of the SlASFT promoter driving nuclear localized GFP (SlASFTp:NLS-2xGFP) in two independently-transformed hairy root lines. NLS-GFP and lignin autofluorescence (Green) and epidermal RFP autofluorescence (Magenta). (A and D) Transversal z-stack projections of mature regions of transgenic hairy roots. Red lines indicate the planes shown in the subsequent longitudinal sections. (B and E) Longitudinal section of a top plane of the z-stacks, showing the epidermal and exodermal cell layers. White arrows indicate some of the GFP-tagged nuclei in the exodermis. Yellow arrows indicate the lignin autofluorescence of the exodermal polar cap. (C and F) Longitudinal section of a bottom plane of the z-stacks, showing the epidermis, exodermis, cortex layers 1 and 2, and endodermis. White arrows indicate the GFP-tagged nuclei in the exodermis. Yellow arrows indicate the lignin autofluorescence of the exodermal polar cap and the endodermal Casparian Strip. EP: Epidermis; EXO: Exodermis; COR1&2: Cortex layer 1 and 2; ENDO: Endodermis, VASC: Vasculature.

Extended Data Fig. 6. R. rhizogenes-derived loss-of-function mutant alleles of candidate genes have impaired suberin deposition.

(a) Extended analysis of mutant phenotypes of candidate genes in hairy roots (HR). Mean fluorol yellow signal across multiple cross sections (wild type n = 66; rest n = 6). Red line indicates statistically significant difference (P < 0.05) in fluorol yellow pixel intensity in the mutant vs wild type as determined with a one-way ANOVA followed by a Tukey-Kramer post hoc test. In most cases, two independently generated HR lines were analyzed, as indicated in the plot. (b) Mean suberin abundance and (c) monomer composition of R. rhizogenes-generated mutants of suberin biosynthetic enzymes and transcriptional regulators. Acid: fatty acids; Alcohols: primary alcohols; ω-OH: ω-hydroxy fatty acids; DCA: dicarboxylic fatty acid; Aromatics: ferulate and coumarate derivatives. Error bars: SD. Letters indicate significant differences (one-way ANOVA followed by a Tukey-Kramer post hoc test, P < 0.05). (d) ABA treatment (1 µM for 48 h) does not restore suberin to wild type levels by fluorol yellow staining in slmyb41, slmyb92 and slmyb63 lines. Mean pixel intensities are not comparable between plots A and D as these were taken under different laser settings.

Extended Data Fig. 7. Impaired suberin deposition in the myb92-2 and asft-2 alleles and their impact on the response to ABA.

Fluorol Yellow (FY) staining for suberin in wild type (repeated from Fig. 1 for reference), myb92-2 and asft-2 plants treated with mock or 1 µM ABA.

Extended Data Fig. 8. Root length is not significantly affected by the ABA treatment.

Boxplot of total root length of 7-day-old wild-type plants treated with mock or 1 µM ABA for 48 h (n = 12). A one-way ANOVA analysis did not find any statistically significant differences.

Extended Data Fig. 9. Lignin polar cap in the exodermis is not affected in the myb41 myb63 and myb92 hairy root mutants.

a. Cross section of control hairy root stained with basic fuchsin. b. Cross section of myb92 hairy root mutant stained with basic fuchsin. c. Cross section of myb63 hairy root mutant stained with basic fuchsin. d. Cross section of myb41 hairy root mutant stained with basic fuchsin. Scale bars=50 µm.

Extended Data Fig. 10. Suberin deposition in mature roots of wild type, myb92-1, and asft-1.

Representative cross sections of mature portions of the roots stained with fluorol yellow (FY) of one-month-old plants exposed to 10 days of water sufficient (WS) and water limitation (WL) regimes (Methods).