Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25

Abstract

Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

Keywords: Cell wall; Mediator; Papillae; Trichomes.

Figure 1. GLH1 Gene Identification

A. The glh1 phenotype. In comparison to wild type plants, glassy hair1 mutants possess trichomes with a low density of underdeveloped papillae on their cell wall surfaces. Scale bar=75μm. B. glh1 Mutation Mapping Interval. The glh1 mutation was mapped to an 82kb region on chromosome 1. The names of the markers and the number of recombinants in relation to the number of chromosomes tested are indicated. C. Mutation Location. MED25 was sequenced in the glh1 background and aligned with wild type Columbia sequences, revealing a point mutation at the beginning of the sixth intron that resulted in a Guanine to Adenine substitution. D. Mutation Effects on Transcript Splicing. PCR amplification of Arabidopsis MED25 cDNA using MED25 primers yielded a single band of 396bp for wild type plants and several bands ranging from approximately 300bp to 600bp for mutant plants. The PCR on wild type genomic DNA (g) using MED25 primers generated a single band of 634bp in length. Reactions on cDNA using EF1 primers was used as a control.

Figure 2. Complementation of the glh1 mutant

A–E. Scanning electron micrographs of whole trichomes at 500x magnification. Scale bars = 50μm. A′–E′ Scanning electron micrographs of trichome stalks at 1000x magnification. Scale bars = 30μm. A, A′. Wild type trichomes possess numerous fully formed papillae. B, B′. glh1 trichomes exhibit underdeveloped papillae at a dramatically reduced density compared to wild type. C, C′. SALK_129555c trichomes have underdeveloped papillae at a reduced density compared to wild type. D, D′. Trichomes of the glh1 mutants expressing MED25pro::MED25 construct display numerous fully developed papillae. E, E′. SALK_129555c x glh1 F1 trichomes possess underdeveloped papillae at a dramatically reduced density compared to wild type. F. Density of Papillae for glh1, Columbia, SALK_129555c, SALK_129555c x glh1 F1, and MED25pro::MED25 T2 trichomes. N=13 for glh1, N=10 for all other plant lines, **=P<0.01, ***=P<0.001.

Figure 3. Expression of the MED25pro::GUS in Seedlings

GUS staining in ten-day-old seedlings was evident primarily in A guard cells, B leaf vasculature, C roots, D developing trichomes, E trichomes of young leaves, and F cells surrounding mature trichomes. For A, D, E, F scale bars=100μm, for B and C scale bars=1 mm.

Figure 4. GL2pro::GUS and ETR2pro::GUS Expression in the glh1 Trichomes

Trichome and whole leaf images are shown for each parental and F3 line. Scale bar for trichome images=50μm. Scale bar for leaf images= 200μm.

Figure 5. Trichome Elemental Analysis

X-ray peaks for glh1 trichome surfaces (A), wild type inter-papillae regions (B), and wild type papillae (C) were measured to compare the elemental composition of wild type and mutant trichome cell wall surfaces. No significant differences in calcium were detected between X-ray peaks from the Columbia inter-papillae and Columbia papillae regions (D). Comparisons of X-ray peak heights (E) and ICP ppm values (F) between Columbia and glh1 samples revealed that mutant trichomes contain less calcium. N=5 trichomes for each plant line used in the X-ray dispersal spectra analysis. N=14 vials (3000 trichomes per vial) for each plant line used in the ICP analysis. *=P<0.05, **=P<0.01, ***=P<0.001.