Article Figures & Data
Figures
- Fig. 1.
Effect of GL2-promoter fragments on GUS-reporter expression in roots and hypocotyls of Arabidopsis seedlings. The GL2-promoter fragments fused to the GUS-reporter gene are shown on the left. The ability of these constructs to drive GUS expression in a cell-position-dependent manner in the seedling root and/or hypocotyl was determined by histochemical staining with the X-Gluc substrate. +, Typical GUS expression pattern detected; −, abnormal/no GUS expression detected. The relative root/hypocotyl GUS-activity value was determined by comparing the GUS activity (calculated as millimoles of product per milligram of protein per minute) in root versus hypocotyl extracts from a common set of 3-d-old seedlings bearing the indicated transgene. Values are means ± sd. Xh, XhoI; H,HindIII; Hp, HpaI; RI, EcoRI; RV,EcoRV; X, XbaI; M, MscI; and ▵, deletion.
- Fig. 2.
Spatial expression pattern ofGL2::GUS-reporter-gene fusion construct during root development in Arabidopsis seedlings. Four-day-old seedlings were assayed for GUS activity by histochemical staining with the X-Gluc substrate. A, Wild-type root containing theGL2::GUS transgene. Bar = 50 μm. B, Wild-type root containing the GL2::GUStransgene. GUS-expressing cells are preferentially located in specific epidermal cell files. Bar = 20 μm. C, ttg-1mutant root containing the GL2::GUS transgene. Bar = 50 μm. D, ttg-1 mutant root containing theGL2::GUS transgene. GUS-expressing cells are preferentially located in specific epidermal cell files. Bar = 20 μm. E, 35S::R root containing theGL2::GUS transgene. Bar = 50 μm. F,35S::R root apex containing theGL2::GUS transgene. GUS-expressing cells are not clearly located in specific epidermal cell files. Bar = 20 μm. G, Wild-type root containing theGL2::GUS transgene; transverse plastic section taken from the late meristematic region. GUS expression is limited to epidermal cells located outside periclinal cortical cell walls (i.e. in contact with a single cortical cell). At this developmental stage, a single layer of lateral root cap cells surrounds the epidermis. Bar = 20 μm. H, ttg-1 mutant root containing theGL2::GUS transgene; transverse plastic section taken from the late meristematic region. No GUS expression is observed. At this developmental stage, a single layer of lateral root cap cells surrounds the epidermis. Bar = 20 μm. I,35S::R root containing theGL2::GUS transgene; transverse plastic section taken from the late meristematic region. GUS expression is observed throughout the epidermis, cortex, and lateral root cap. At this developmental stage, a single layer of lateral root cap cells surrounds the epidermis. Bar = 20 μm. J, ttg-1 mutant root containing the GL2::GUS transgene; thick transverse section from agarose-embedded root. GUS expression is observed in epidermal cells located outside periclinal cortical cell walls. Bar = 20 μm. K, Wild-type root apex containing theGL2::GUS transgene. Whole-mount root preparation showing GUS expression near the meristem initials but not within the lateral or columella root cap cells. The dense staining visible in the upper portion of this root is due to GUS-expressing epidermal cells above and below the plane of focus. Bar = 20 μm. L, 35S::R root apex containing theGL2::GUS transgene. Whole-mount root preparation showing GUS expression throughout region containing meristem initials and within the lateral root cap cells but not within the columella root cap cells. The dense staining visible in the upper root is due to GUS-expressing cells above and below the plane of focus. Bar = 20 μm. M, 35S::GUS root apex. Whole-mount root preparation showing preferential GUS expression throughout region containing meristem initials, the root cap, and the developing vascular tissue. Bar = 20 μm. TheseGL2::GUS seedlings all contain the full-length 4-kb GL2 promoter::GUS transgene. Note that root hairs are not visible in these images near the root apex; hairs form on epidermal cells at a later developmental age, just beyond the field of view shown in A, C, and E.
- Fig. 3.
The spatial-expression pattern of theGL2-GUS-reporter-gene fusion construct during hypocotyl development. Seedlings harboring the 4-kbGL2 promoter::GUS transgene were stained for GUS activity using X-Gluc. A, Wild-type hypocotyl from 3-d-old seedling. Bar = 100 μm. B, Wild-type hypocotyl epidermis from 3-d-old seedling. GUS-expressing cells are located within specific epidermal cell files. Bar = 50 μm. C, Wild-type 3-d-old seedling sectioned through the hypocotyl. A ring of GUS-expressing hypocotyl epidermal cells is visible. Bar = 100 μm. D, Wild-type hypocotyl from 3-d-old seedling; transverse agarose section. GUS expression is present in epidermal cells located outside a periclinal cortical cell wall. E, Wild-type hypocotyl from 5-d-old seedling. Note that cells in the GL2::GUS-expressing files are longer than cells in the nonexpressing files. Bar = 40 μm. F, Wild-type hypocotyl from 5-d-old seedling. Stomatal development (arrowhead) occurs in non-GL2::GUS-expressing cell files. Bar = 40 μm. G, Wild-type hypocotyl and cotyledons from 3-d-old seedling. GUS-expressing cells are visible at the margin of cotyledons. Bar = 100 μm. H, Wild-type hypocotyl/cotyledon junction region from 3-d-old seedling. Bar = 50 μm. I, Wild-type cotyledon; transverse section taken near cotyledon apex (3-d-old). Arrowhead indicates a GUS-staining epidermal cell. Bar = 50 μm. J, Wild-type hypocotyl from 7-d-old seedling. GUS expression is visible in the developing leaf primordia and trichomes. Bar = 200 μm. K,ttg-1 mutant hypocotyl from 3-d-old seedling. Bar = 100 μm. L, ttg-1 mutant hypocotyl from 3-d-old seedling. GUS-expressing cells appear to be located within specific epidermal cell files. Bar = 50 μm. M, ttg-1mutant hypocotyl from 3-d-old seedling; transverse agarose section. GUS expression is present in epidermal cells located outside a periclinal cortical cell wall (i.e. in contact with a single cortical cell). Bar = 50 μm. N, 35S::R hypocotyl from 3-d-old seedling; transverse agarose section. GUS expression is present in cells located throughout the epidermis. Bar = 50 μm. O,35S::R hypocotyl from 3-d-old seedling. Bar = 100 μm. P, 35S::R hypocotyl from 3-d-old seedling. GUS-expressing cells are located throughout the epidermis. Bar = 50 μm.
- Fig. 4.
Proposed pathway for the regulation of cell differentiation in the root and hypocotyl of Arabidopsis. The TTG is proposed to activate an R-like bHLH protein that positively controls the transcription of GL2. The GL2 homeodomain protein is proposed to control root and hypocotyl epidermal cell differentiation. See text for additional discussion. Arrows indicate positive action; blunted lines indicate negative regulation. RHD6, Root hair defective 6; and RHL, root hairless.
Tables
- Table I.
Root-hair formation and GL2-promoter activity in roots and hypocotyls of mutant and transgenic Arabidopsis seedlings
Genotype Hair Formation-a Ectopic Root-Hair Cells-b Proportion of Wild-Type GUS Activity-c Root Hypocotyl no. mm−1 % Landsberg (wild type) 61 ± 6 4 – – ttg-1 124 ± 12 47 0.29 ± 0.08 0.09 ± 0.06 ttg-w 94 ± 11 28 0.59 ± 0.15 0.25 ± 0.08 ttg-398 116 ± 19 44 0.36 ± 0.10 0.16 ± 0.09 35S::R 15 ± 6 6 0.78 ± 0.24 1.05 ± 0.18 gl2–1 132 ± 19 52 0.94 ± 0.20 1.21 ± 0.15 ttg-1 gl2–1 136 ± 20 56 n.d.-d n.d. gl2–1 35S::R 110 ± 25 41 n.d. n.d. -
↵F0-a Means ± sd.
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↵F0-b Percentage of the root-hair-bearing cells that are located over a periclinal cortical cell wall (ectopic position).
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↵F0-c GUS activity per milligram of protein in the mutant relative to the wild type within a common pool of F2 seedlings containing the full-length 4-kb GL2promoter::GUS fusion. Values are means ± sd.
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↵F0-d n.d., Not determined.
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- Table II.
Effect of the ttg mutation and 35S::R transgene on GUS activity in roots of GL2::GUS lines containing GL2-promoter deletions
GL2::GUSTransgene1-a GUS Expression1-b Proportion of Wild-Type GUS Activity1-c ttg-1 35S::R ttg-1 35S::R 4-kb GL2promoter +1-d + 0.29 ± 0.08 0.78 ± 0.24 ΔHd + + 0.19 ± 0.05 0.61 ± 0.20 ΔRI + + 0.21 ± 0.11 0.47 ± 0.23 ΔRV + + 0.24 ± 0.13 0.64 ± 0.13 ΔMHp −1-e − – – ΔMX + + 0.23 ± 0.11 0.94 ± 0.23 ΔMR − − – – -
↵F1-b GUS expression in roots, as assessed by histochemical staining.
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↵F1-c GUS activity per milligram of protein in the mutant relative to the wild type within a common pool of F2 seedlings containing the indicated GL2promoter::GUS fusion. Values are means ± sd.
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↵F1-d +, Typical qualitative pattern of GUS expression detected.
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↵F1-e −, No GUS expression detected.
- Table III.
Epidermal cell differentiation in hypocotyls of wild-type and mutant Arabidopsis seedlings
Genotype Relative Cell Length2-a Stomatal Development2-b Landsberg (wild type) 1.42 ± 0.12 0.95 ± 0.05 (146) ttg-1 1.29 ± 0.15 0.74 ± 0.05 (121) gl2–1 1.39 ± 0.11 0.79 ± 0.07 (119) -
↵F2-a Values represent the mean ratios (± sd) of the length of cells in files exhibitingGL2::GUS expression compared with the length of cells in non-GL2::GUS-expressing files in the hypocotyl epidermis.
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↵F2-b Values represent the mean proportions (± sd) of stomata located in non-GL2::GUS-expressing cell files. Total number of stomata analyzed are shown in parentheses.
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