Abstract
Coding sequences for major trichome regulatory genes, including the positive regulators GLABRA 1(GL1), GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) and the negative regulator TRIPTYCHON (TRY), were cloned from wild Brassica villosa, which is characterized by dense trichome coverage over most of the plant. Transcript (FPKM) levels from RNA sequencing indicated much higher expression of the GL2 and TTG1 regulatory genes in B. villosa leaves compared with expression levels of GL1 and EGL3 genes in either B. villosa or the reference genome species, glabrous B. oleracea; however, cotyledon TTG1 expression was high in both species. RNA sequencing and Q-PCR also revealed an unusual expression pattern for the negative regulators TRY and CPC, which were much more highly expressed in trichome-rich B. villosa leaves than in glabrous B. oleracea leaves and in glabrous cotyledons from both species. The B. villosa TRY expression pattern also contrasted with TRY expression patterns in two diploid Brassica species, and with the Arabidopsis model for expression of negative regulators of trichome development. Further unique sequence polymorphisms, protein characteristics, and gene evolution studies highlighted specific amino acids in GL1 and GL2 coding sequences that distinguished glabrous species from hairy species and several variants that were specific for each B. villosa gene. Positive selection was observed for GL1 between hairy and non-hairy plants, and as expected the origin of the four expressed positive trichome regulatory genes in B. villosa was predicted to be from B. oleracea. In particular the unpredicted expression patterns for TRY and CPC in B. villosa suggest additional characterization is needed to determine the function of the expanded families of trichome regulatory genes in more complex polyploid species within the Brassicaceae.
Highlights
Trichomes are protruding structures which protect plant surfaces against dehydration and insect pests, and provide a storage organ to handle metal toxicity [1,2]
Trichome Regulatory Gene Amplification in B. villosa Amplification of the coding regions for B. villosa orthologues to the GL1, GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), TESTA GLABRA 1 (TTG1) and TRY regulatory genes using primers based on available B. napus and B. rapa sequences gave single PCR bands
Three independent clones each were sequenced for the BvGL1, BvGL2, and BvTTG1 genes from B. villosa, and this was expanded to 10 clones for BvEGL3, and thirty clones for BvTRY
Summary
Trichomes are protruding structures which protect plant surfaces against dehydration and insect pests, and provide a storage organ to handle metal toxicity [1,2]. A number of major positive regulatory genes for trichome initiation have been studied in detail, including GLABRA 1(GL1), GLABRA 2 (GL2), GLABRA 3 (GL3), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) [3]. GL1, TTG1 and GL3/EGL3 are positive patterning proteins which form an activator MYB-bHLH-WD40 (MBW) triprotein complex that induces the expression of an immediate downstream target gene, GLABRA 2 (GL2). Mutations in any of these four abovementioned genes reduce trichome initiation and the density of trichome patterning in A. thaliana [11,12,13,14] A yeast two hybrid assay demonstrated a direct physical interaction between GL3 and GL1, TTG1, and itself; GL1 and GL3 co-overexpression confirmed their interaction, but GL1 and TTG1 do not physically interact [15]. Egl mutant plants have no obvious trichome defects, but gl3egl double mutants show a complete glabrous phenotype [14] due to some functional overlap between the two genes [16]
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