The involvement of J-protein AtDjC17 in root development in Arabidopsis

Abstract

In a screen for root hair morphogenesis mutants in Arabidopsis thaliana L. we identified a T-DNA insertion within a type III J-protein AtDjC17 caused altered root hair development and reduced hair length. Root hairs were observed to develop from trichoblast and atrichoblast cell files in both Atdjc17 and 35S::AtDJC17. Localization of gene expression in the root using transgenic plants expressing proAtDjC17::GUS revealed constitutive expression in stele cells. No AtDJC17 expression was observed in epidermal, endodermal, or cortical layers. To explore the contrast between gene expression in the stele and epidermal phenotype, hand cut transverse sections of Atdjc17 roots were examined showing that the endodermal and cortical cell layers displayed increased anticlinal cell divisions. Aberrant cortical cell division in Atdjc17 is proposed as causal in ectopic root hair formation via the positional cue requirement that exists between cortical and epidermal cell in hair cell fate determination. Results indicate a requirement for AtDJC17 in position-dependent cell fate determination and illustrate an intriguing requirement for molecular co-chaperone activity during root development.

Keywords: J-family; J-proteins; development; heat shock protein; root hair; root patterning.

FIGURE 1

Trichoblast and atrichoblast cells are arranged randomly in Atdjc17 and overexpressor mutants. (A–C) False colored stereomicroscope images of wild-type (WT; A), Atdjc17-1-1 (B) and 35S::AtDjC17 (C) illustrate the patterning of root hair cells (red) and non-root hair cells (green) in cell files. Stars represent ectopic root hairs in an otherwise atrichoblast cell file. Arrowheads represent the presence of non-root hair in an otherwise trichoblast cell file in Atdjc17-1-1. (D,E) Root hair and non-root hair cells in trichoblast and atrichoblast cell files were counted in WT, Atdjc17-1-1 and overexpressor 35S::AtDjC17.

FIGURE 2

Mutations and overexpression in ATDJC17 cause root hair alteration and ectopic root hair production. (A–C) Variation in number of root hairs is illustrated in Atdjc17-1-1 and 35S::AtDjC17 mutants as compared to WT plants. Green dots highlight the presence of root hair at the site. (G) Average number of root hairs determined from a total of 10 roots. An area approximately 2 mm from the root cap was chosen for the comparison covering 0.65 mm root length. (D–F) Stereomicroscope images of WT (D), Atdjc17-1-1 (E) and 35S::AtDjC17 (F) roots false colored to show the trichoblast (red) and atrichoblast (green) cell files. Arrows in Atdjc17-1-1 and 35S::AtDjC17 mutant highlights the presence of two trichoblast cell files adjacent to each other illustrating the presence of ectopic root hair. (H) Comparison of distance between adjacent root hairs in a single vertical trichoblast cell file in WT (D) Atdjc17-1-1 (E) and overexpressor 35S::AtDjC17 (F). *indicates significant difference (P ≤ 0.05).

FIGURE 3

GUS expression studies. (A,B) Stele-localized accumulation of proAtDjC17::GUS transcript in a 7-d old root. (A) Scale Bar 100 μm.(B) Scale Bar 50 μm. (C) Agarose embedded hand section of proAtDjC17::GUS expressing 7-d old roots displaying a clear stele accumulation of GUS, Scale Bar 50 μm.

FIGURE 4

Gene expression analysis for previously identified regulatory elements of root development. (A) RNA equal loading of WT and Atdjc17 mutant lines (Atdjc17-1-1, Atdjc17-1-2). (B) Relative fold changes determined on whole root sample for SCRAMBLE (SCM); GLABRA3 (GL3); Enhancer of GLABRA3 (ENGL3);WEREWOLF (WER); CAPLICE (CPC); TRANSPARENT TESTA 1 (TTG1);GLABRA2 (GL2); TRANSPARENT TESTA 2 (TTG2); SCARECROW (SCR); SHORTROOT (SHR); MAGPIE (MGP) and JAKDOW (JKD). Error bars indicate standard deviation. *indicates significant difference (P ≤ 0.05).

FIGURE 5

Examination of cell division in WT, Atdjc171-1 and 35S::AtDjC17 roots: agarose embedded hand sections stained with calcofluor-white stained and confocal microscopic images of propidium iodide stained WT, Atdjc17-1-1 and 35S::AtDjC17 mutant roots. (A) WT section. (B) Atdjc17-1-1 section showing additional cell division in the cortical and endodermal layers. Note that the divisional pattern seems anticlinal in nature. Arrow heads indicate ectopic divisions. (C) 35S::AtDjC17 section with no evidence of altered cortical and endodermal cell numbers. (D) 7-day old post-germination WT root tip and zone above the meristematic tip in WT root. (E) Atdjc17-1-1 root tip with associated zone above the meristematic tip.(F) 35S::AtDjC17 root tip and elongation zone. st, stele; en, endodermis; co, cortex; ep, epidermis.

FIGURE 6

Root expression analysis of the overexpressor of AtDjC17. Relative fold changes determined on whole root sample for 35S::AtDjC17 and 12 main regulators of root development. Error bars indicate standard deviation. *indicates significant difference (P ≤ 0.05).