A gradient of the HD-Zip regulator Woolly regulates multicellular trichome morphogenesis in tomato

Abstract

Homeodomain (HD) proteins regulate embryogenesis in animals such as the fruit fly (Drosophila melanogaster), often in a concentration-dependent manner. HD-leucine zipper (Zip) IV family genes are unique to plants and often function in the L1 epidermal cell layer. However, our understanding of the roles of HD-Zip IV family genes in plant morphogenesis is limited. In this study, we investigated the morphogenesis of tomato (Solanum lycopersicum) multicellular trichomes, a type of micro-organ in plants. We found that a gradient of the HD-Zip IV regulator Woolly (Wo) coordinates spatially polarized cell division and cell expansion in multicellular trichomes. Moreover, we identified a TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) transcription factor-encoding gene, SlBRANCHED2a (SlBRC2a), as a key downstream target of Wo that regulates the transition from cell division to cell expansion. High levels of Wo promote cell division in apical trichome cells, whereas in basal trichome cells, Wo mediates a negative feedback loop with SlBRC2a that forces basal cells to enter endoreduplication. The restricted high and low activities of Wo pattern the morphogenesis of tomato multicellular trichomes. These findings provide insights into the functions of HD-Zip IV genes during plant morphogenesis.

Figure 1.

Spatially polarized arrangement of cell division and endoreduplication in tomato trichomes. A) Model of the 7 types of tomato trichome. The maximum number of stalk cells is 8 to 9 for Type I/II trichomes, 3 to 4 for Type III trichomes, and 2 to 3 for Type IV/V trichomes, while all PTs have only 1 stalk cell. B) Scanning electron micrographs of trichomes on tomato leaves: Type I and Type VI trichomes are indicated by arrows. The more stalk cells of the trichome, the longer it is. bar = 200 μm. C to H) Division positions and statistics of dividing trichomes at different periods with 2, 3, and 4 cells, respectively (n = 5, biological replicates). It can be seen from the pictures that the division position of the trichomes in all periods occurred in the most apical cells. Bar = 20 μm. The blue dots represent the percentage of the dividing cells in individual trichomes, and the red lines show the trend of cell division from the bottom to the top of the trichomes. Note that only the top trichome cells divide. I) Fluorescence observation of the replication process in the nucleus of tomato trichomes at different developmental stages. H2B-GFP shows the similar size of the nucleus in the early stage of trichome development and dramatically enlarged nuclear size in the basal cells in more mature stage of trichome development. Bar = 50 μm. J) Quantification of nuclear volume of tomato trichomes at different developmental stages. Data are mean ± Sd (n > 3, biological replicates). Note the polyploidy state of basal cells of mature trichomes can reach up to 70C DNA content. Nuclear DNA content is normalized to that of the guard cell. Maximum intensity projection of confocal images of H2B-GFP was shown. “H2B FL” represent “H2B-GFP fluorescence.” Unpaired t-tests were used for statistical analysis (*P < 0.05, **P < 0.01, and ****P < 0.0001). The number indicates the position relative to basal cell.

Figure 2.

Wo protein gradient is associated with the pattern of division and expansion in tomato trichomes. A) High Wo levels are associated with actively dividing trichome cells. Note the Wo-GFP is seen in all developing trichomes but gradually disappears in mature trichomes (fully divided Type IV/V trichomes and differentiated PT trichomes are shown in this figure). Bar = 20 μm. Arrows indicate single cells of a trichome. B, C) Higher Wo levels in mtr1 mtr2 double mutants lead to a significant increase in the density of Type I/II/III trichomes with more stalk cells, probably generated by a higher cell division capacity. Data are mean ± Sd (n > 8, biological replicates). The diagram illustrates the regulatory relationship between Wo and MTRs. Positive regulations are represented by arrow-headed lines, and negative regulations are represented by bar-headed lines. Invalid regulatory relationships are indicated by dashed lines. Box plots display second and third quartiles and the median, bars indicate 1.5× the interquartile range, and the points represent each individual value. D) Expression patterns of MTR1 and MTR2 in developing tomato trichomes. E, F) Patterns of Wo protein gradient along trichome axis in developing trichomes, showing the highest Wo levels in apical cells E). Qualifications of Wo protein concentration in basal and apical cells of trichomes F). The color scale from dark blue to yellow reflects the intensity of protein concentration from weak to strong. Data are mean ± Sd (n = 5, biological replicates), and unpaired t-tests were used for statistical analysis (***P < 0.001). The number indicates the position relative to the basal cell. a.u., absorbance unit.

Figure 3.

Increased Wo concentration promotes cell division in tomato trichomes. A) RT-qPCR analysis of Wo expression in 2-wk-old WT, proWo:Wo^P635R-GFP, and proMX1:Wo^V-GFP seedlings. Total RNA was extracted from young leaves near the SAM. Data are mean ± Sd (n = 3, biological replicates). B) Quantification of fluorescence intensity of GFP-tagged Wo protein in the trichomes of 2-wk-old WT (proWo:Wo-GFP), proWo:Wo^P635R-GFP, and proMX1:Wo^V-GFP. Data are mean ± Sd (n > 14, biological replicates). Box plots display second and third quartiles and the median; bars indicate 1.5× the interquartile range; points represent each individual value. C) Confocal images showing the trichome cell division phenotype in 2-wk-old WT (proWo:Wo-GFP), proWo:Wo^P635R-GFP, and proMX1:Wo^V-GFP plants. Note the promoted trichome cell division induced by enhanced Wo protein stability. Bar = 50 μm. D) Mature trichomes from WT and proMX1:Wo^V-GFP lines. Note the dramatically increased cell division in proMX1:Wo^V-GFP trichomes in which cells do not enter endoreduplication. E) DAPI staining of proMX1:Wo^V-GFP and WT trichomes. Note the nucleus of the WT basal cells reached 24C DNA content, whereas almost all cells have 2C nucleus in the proMX1:Wo^V-GFP trichomes. F) Diagram showing the Wo protein gradient determines the polarized cell division and cell expansion during tomato trichome development. Wo protein maintains high concentration in the apical trichome cells, leading to active cell division in these cells. Reduced Wo concentration in the basal cells allows for endoreduplication in these cells. Additional unidentified factors may be involved in the transition from cell division to endoreduplication. Blue gradients represent the change of Wo concentration. Pink highlights the region of endoreduplication, and gray highlights the region of cell division. Unpaired t-tests were used for statistical analysis (**P < 0.01, ****P < 0.0001, and ns means no significance). a.u.: absorbance unit.

Figure 4.

SlBRC2a is a key regulator of the shift to cell expansion in basal cells. A) RT-qPCR analysis of SlBRC2a expression in the gain-of-function Wo mutant (Wo^P635R) and loss-of-function mutant (cr-wo). Total RNA was extracted from young leaves near SAM. Data are mean ± Sd (n = 3, biological replicates). B, C) Spatial expression pattern of SlBRC2a in trichomes. GUS staining of proSlBRC2a:GFP-GUSB) and confocal micrographs of proSlBRC2a:SlBRC2a-GFPC). Note the SlBRC2a gene is expressed mainly in basal cells of trichomes. D, E) Knockout of SlBRC2a gene (cr-slbrc2a) causes increased cell division in tomato trichomes. DIC images showing trichomes on the stem of cr-slbrc2a plants D) and quantification of cell numbers in Type I/II trichomes E). Data are mean ± Sd (n > 10, biological replicates). The black triangles indicate individual cells of trichome. F, G) SEM micrographs showing Type I/II trichomes in WT and cr-slbrc2a mutants. Note the increased cell division and forked trichomes in cr-slbrc2a mutants. The phenotypes were most pronounced in the long DT (Type I/II) with higher Wo protein concentration. Orange highlights a single mature trichome in the stem and leaf. H, I) Quantification of the nucleus size of Type I/II trichomes in different backgrounds. Note the significantly reduced cell size and nuclear size in the basal trichome in cr-slbrc2a mutants. The quantification indicates that the nucleus size of the first basal cells is 50C to 100C DNA content in WT but declines to 10C to 24C DNA content in cr-slbrc2a mutants I). Nuclear DNA content is normalized to that of the guard cells. Data are mean ± Sd (n > 10, biological replicates). “H2B FL” presented “H2B-GFP fluorescence.” Unpaired t-tests were used for statistical analysis (**P < 0.01, ***P < 0.001, and ****P < 0.0001). The number indicates the position relative to basal cell.

Figure 5.

SlBRC2a negatively affects Wo ability of promoting cell division. A) Venn diagram showing the overlap of differentially expressed transcription factors in cr-slbrc2a mutants and the loss-of-function mutant wo^W106R. Transcriptome analysis used stem tissue near the SAM region. B) Heat map of differentially expressed transcription factors from the transcriptome analyses. Note the almost opposite expression pattern of the transcription factors in cr-slbrc2a and wo^W106R mutants. The color scale from blue to magenta reflects downregulation to upregulation of gene expression level showed by the value of log₂ fold change. C) RT-qPCR analysis of MX1 expression in cr-slbrc2a and WT trichomes. Data are mean ± Sd (n = 3, biological replicates). Unpaired t-tests were used for statistical analysis (**P < 0.01). D to G) Confocal images showing Wo-GFP expression pattern in Type I/II trichomes of WT and cr-slbrc2a × proWo:Wo-GFP plants: the yellow arrows indicate dividing cells. E) and G) are outlined images shown in D) and F), respectively. Note the Wo protein gradient is disrupted when the SlBRC2a gene is knocked out. In cr-slbrc2a trichomes, bulging is often observed in the dividing basal cells where Wo protein levels are significantly higher. The color scale from dark blue to yellow reflects the intensity of protein concentration from weak to strong. TF, transcription factors.

Figure 6.

Interaction with SlBRC2a inhibits the transcriptional activity of Wo. A) SlBRC2a interaction with Wo shown by Y2H assay. Due to strong self-activation, SlBRC2a protein is truncated into 3 segments for Y2H. Both D1 and D2 segments show the interaction with Wo. DDO medium (SD/–Leu/–Trp) and QDO medium (SD/–Ade/–Leu/–His/–Trp). B) BiFC verification of SlBRC2a-Wo interaction. C) Co-IP validates the interaction between SlBRC2a and Wo. D) Y1H assay shows that Wo binds strongly to MX1 promoter, while SlBRC2a has a weak interaction. E) Diagram showing the constructs used in LUC activation assay. F) LUC assay showing that the addition of SlBRC2a significantly inhibits the activation of MX1 promoter by Wo. Data are mean ± Sd (n = 3, biological replicates). G, H)MX1 knockout leads to reduced trichome cell division in cr-slbrc2a mutants. SEM micrographs showing the trichome phenotype in different backgrounds G). Quantification of the cell number of Type I/II trichomes in cr-slbrc2a and cr-slbrc2a × cr-mx1 plants H). Data are mean ± Sd (n = 10, biological replicates). Unpaired t-tests were used for statistical analysis (***P < 0.001 and ****P < 0.0001).

Figure 7.

Trichome density and cell division are increased in MTR1/MTR2/SlBRC2a triple mutants. A) Phenotype of the single, double, and triple mutants of MTR1, MTR2, and SlBRC2a.B) SEM micrographs of the trichomes in mtr1 mtr2 and mtr1 mtr2 × cr-slbrc2a plants. The orange color was used for highlight a single mature trichome. C, D) Quantification of the cell number in Type IV/V C) and Type I/II D) trichomes. Data are mean ± Sd (n > 10, biological replicates). E) Quantification of the leaf trichome density. Data are mean ± Sd (n > 10, biological replicates). Unpaired t-tests were used for statistical analysis (**P < 0.01 and ****P < 0.0001). Box plots display second and third quartiles and the median; bars indicate 1.5× the interquartile range; points represent each individual value.

Figure 8.

SlBRC2a promotes cell expansion via suppressing CKX gene in basal cells. A) RT-qPCR showing CKX1 and CKX3 expression in WT and cr-slbrc2a trichomes. Data are mean ± Sd (n = 3, biological replicates). B) Liquid chromatography analysis of cytokinin concentration in WT and cr-slbrc2a trichomes. Data are mean ± Sd (n = 3, biological replicates). C)TCS:VENUS-NLS showing a gradient pattern of cytokinin along trichome axis. The number indicates the position relative to the basal cell. D) SEM micrographs of Type I/II trichomes in WT, proSlBRC2a:CKX1-GFP, and proWo:CKX1-GFP transgenic plants. The first basal cells of Type I/II trichomes are highlighted in yellow. Note the misexpressions of CKX1 expression leads to blocked expansion of basal trichome cells. Orange highlights the first cell at the position relative to the basal cell in the mature trichome. E) Quantitation of the first basal cells of Type I/II trichomes in proSlBRC2a:CKX1-GFP and proWo:CKX1-GFP transgenic plants. Data are mean ± Sd (n > 10, biological replicates). F, G) Nuclear size of the first basal cells of Type I/II trichomes in proSlBRC2a:CKX1-GFP and proWo:CKX1-GFP plants shown by DAPI staining. Nuclear DNA content is normalized to that in guard cells (2C). The red arrows indicate the nucleus. Data are mean ± Sd (n > 10). Unpaired t-tests were used for statistical analysis (**P < 0.01, ***P < 0.001, and ****P < 0.0001).

Figure 9.

Model of tomato trichome morphogenesis mediated by Wo-SlBRC2a pathway. Wo proteins form a gradient along trichome axis, with the highest Wo concentration in the apical dividing cells. High Wo protein levels promote the cell division of the apical cells by activating the expression of downstream genes including SlWox3b and MX1. In basal cells, Wo protein level declines, favoring the expression of SlBRC2a. SlBRC2a protein in turn binds to Wo proteins, inhibiting Wo transcriptional activity, and promoting endoreduplication and cell expansion in basal cells. In basal cells, SlBRC2a contributes to cytokinin accumulation by suppressing the expression of cytokinin-degrading enzyme CKX1 and CKX3. Cytokinin accumulation in basal cells contributes to increased endoreduplication and cell expansion in the basal part of tomato trichomes. In the apical cells, high level of Wo also activates downstream MTR genes, which in turn inhibits Wo protein level, forming a negative feedback brake to prevent excessive apical cell division. Blue gradients represent the change of Wo concentration. Pink highlights the region of endoreduplication, and gray highlights the region of cell division. Arrow-headed lines represent positive regulations, and bar-headed lines represent negative regulations. Dashed line with question mark represents hypothesized regulatory relationship. CK, cytokinin; CKX, cytokinin oxidase/dehydrogenase.