Glabrous Brassica napus cv Westar and very hairy AtGL3+ B. napus were transformed using Agrobacterium tumefaciens and either a full length trichome regulatory gene BnTTG1 (isoform 1 coding region called O-TTG1) or an RNAi cassette with 260 bp of a conserved region between isoform I and II (called K-TTG1), each driven by the CaMV 35S promoter. Agronomic and trichome phenotypes were observed in the resulting lines. Transformed lines developed in the glabrous Westar background showed no changes in growth or trichome density and transformation efficiency was similar to that of an empty vector control construct. Over-expression of BnTTG1 in the AtGL3+ B. napus background resulted in low transformant survival and poor seed viability, with the only surviving line O-3-7 being taller than non-transformed lines and with a completely glabrous phenotype. The two knock-down lines with the lowest BnTTG1 expression showed a dramatic increase in trichome density, with longer trichomes and expanded trichome density (up to the 12th leaf in the K-5-8 line) compared to the AtGL3+ hairy background line, which showed increased trichome density only on the first three leaves. Moreover, K-5-8 plants were healthy, with both vegetative and reproductive growth similar to that of Westar non-transgenic control plants under both greenhouse and field conditions. The relative expression of five B. napus primary trichome regulatory genes and AtGL3 was measured in three different tissues of B. napus Westar, and the AtGL3+, K-5-8 and O-3-7 transgenic lines. Over-expression of AtGL3 resulted in changes in the expression of BnGL3, BnGL2 and BnTRY. Manipulation of BnTTG1 levels also resulted in changes in expression of these three genes in addition to AtGL3. AtGL3+ plants and O-3-7 also showed increased red pigment accumulation in several above ground vegetative tissues including cotyledons, hypocotyl and leaves, whereas the K-5-8 line (knock down of TTG1) had less anthocyanin in the same tissues. The level of anthocyanin accumulation corresponded to the relative expression of the three primary anthocyanin regulatory genes BnDFR, BnANS and BnGST. In a laboratory bioassay, diamondback moth (DBM) adults (Plutella xylostella) laid more eggs on hairy leaves of K-5-8 than glabrous Westar. However, more feeding damage from young DBM larvae was observed on Westar leaves than K-5-8 in both choice and no-choice feeding assays. In a field test comparing Phyllotreta flea beetle feeding, the hairy K-5-8 leaves showed between a 30-50% reduction in feeding over four ratings on 14 to 28 day old seedlings. Curiously, the glabrous cotyledons of the two hairy lines (AtGL3+ and K-5-8) proved to be more resistant than wild type B. napus Westar cotyledons from Helix XTra® insecticide-treated or non-treated seed. These data support the introduction of AtGL3 and the knockdown of BnTTG1 to induce a dense trichome phenotype, into otherwise glabrous B. napus, resulting in an increased host plant resistance to crucifer insects, without agronomic penalties.

Studies on epidermal cell fate determination have been important for gaining insight into the genetic and molecular mechanisms leading to the differentiation and patterning of cells. In Arabidopsis, the organization and development of many epidermal characters including trichomes, root hairs and the seed coat have been found to be controlled by a single combinatorial transcription factor complex consisting of a WD-repeat containing protein, Transparent Testa Glabra 1 (TTG1), and various MYB and bHLH proteins. The work here consists of identification of Glabra2 (GL2) and Transparent Testa Glabra2 (TTG2) as direct transcriptional targets of the TTG1 combinatorial complex, further characterization of GL2 function, and identification of transcriptional targets of GL2 and TTG2. Both GL2 and TTG2 are important in the regulation of trichomes, root hairs and seed coat development. vii GL2 has been identified as an important regulator of epidermal cell fate for over fifteen years yet there is little known about its function and only three transcriptional targets are identified, all involved in root hair patterning. Through the examination of its function a nuclear localization signal was verified and shown that GL2 homodimerizes. Through analysis of available expression databases and differential sequence analysis using SOLiD sequencing technology, several direct targets of GL2 and many more possible transcriptional targets of both GL2 and TTG2 were identified in trichomes. Some of these targets are members of the TTG1 complex, and they are all specialized in the maturation of trichomes, suggesting that GL2 switches the focus of the complex by activating the TTG1 complex members involved in maturation of the trichome through a feedback mechanism. Examination of gl2 mutants shows that they do not produce trichome accessory cells which usually surround the trichome. An additional target of GL2 is At5g65300 which when overexpressed results in the elongation and proliferation of trichome accessory cells into a tall pillar of cells. This suggests that GL2 is involved in the regulation of accessory cell development through At5g65300. The work presented here represents important advances of our knowledge of epidermal cell fate through characterization of the major downstream regulators of epidermal development. / text