Hairy Canola (Brasssica napus) re-visited: Down-regulating TTG1 in an AtGL3-enhanced hairy leaf background improves growth, leaf trichome coverage, and metabolite gene expression diversity.

BACKGROUND: Through evolution, some plants have developed natural resistance to insects by having hairs (trichomes) on leaves and other tissues. The hairy trait has been neglected in Brassica breeding programs, which mainly focus on disease resistance, yield, and overall crop productivity. In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 repeat protein), GL3 (a bHLH factor) and GL1 (a MYB transcription factor), activates trichome initiation and patterning. Introduction of a trichome regulatory gene AtGL3 from Arabidopsis into semi-glabrous Brassica napus resulted in hairy canola plants which showed tolerance to flea beetles and diamondback moths; however plant growth was negatively affected. In addition, the role of BnTTG1 transcription in the new germplasm was not understood. RESULTS: Here, we show that two ultra-hairy lines (K-5-8 and K-6-3) with BnTTG1 knock-down in the hairy AtGL3+ B. napus background showed stable enhancement of trichome coverage, density, and length and restored wild type growth similar to growth of the semi-glabrous Westar plant. In contrast, over-expression of BnTTG1 in the hairy AtGL3+ B. napus background gave consistently glabrous plants of very low fertility and poor stability, with only one glabrous plant (O-3-7) surviving to the T3 generation. Q-PCR trichome gene expression data in leaf samples combining several leaf stages for these lines suggested that BnGL2 controlled B. napus trichome length and out-growth and that strong BnTTG1 transcription together with strong GL3 expression inhibited this process. Weak expression of BnTRY in both glabrous and trichome-bearing leaves of B. napus in the latter Q-PCR experiment suggested that TRY may have functions other than as an inhibitor of trichome initiation in the Brassicas. A role for BnTTG1 in the lateral inhibition of trichome formation in neighbouring cells was also proposed for B. napus. RNA sequencing of first leaves identified a much larger array of genes with altered expression patterns in the K-5-8 line compared to the hairy AtGL3(+) B. napus background (relative to the Westar control plant). These genes particularly included transcription factors, protein degradation and modification genes, but also included pathways that coded for anthocyanins, flavonols, terpenes, glucosinolates, alkaloids, shikimates, cell wall biosynthesis, and hormones. A 2nd Q-PCR experiment was conducted on redox, cell wall carbohydrate, lignin, and trichome genes using young first leaves, including T4 O-3-7-5 plants that had partially reverted to yield two linked growth and trichome phenotypes. Most of the trichome genes tested showed to be consistant with leaf trichome phenotypes and with RNA sequencing data in three of the lines. Two redox genes showed highest overall expression in K-5-8 leaves and lowest in O-3-7-5 leaves, while one redox gene and three cell wall genes were consistently higher in the two less robust lines compared with the two robust lines. CONCLUSION: The data support the strong impact of BnTTG1 knockdown (in the presence of strong AtGL3 expression) at restoring growth, enhancing trichome coverage and length, and enhancing expression and diversity of growth, metabolic, and anti-oxidant genes important for stress tolerance and plant health in B. napus. Our data also suggests that the combination of strong (up-regulated) BnTTG1 expression in concert with strong AtGL3 expression is unstable and lethal to the plant.

Brassica villosa, a system for studying non-glandular trichomes and genes in the Brassicas.

Brassica villosa is a wild Brassica C genome species with very dense trichome coverage and strong resistance to many insect pests of Brassica oilseeds and vegetables. Transcriptome analysis of hairy B. villosa leaves indicated higher expression of several important trichome initiation genes compared with glabrous B. napus leaves and consistent with the Arabidopsis model of trichome development. However, transcripts of the TRY inhibitory gene in hairy B. villosa were surprisingly high relative to B. napus and relative transcript levels of SAD2, EGL3, and several XIX genes were low, suggesting potential ancillary or less important trichome-related roles for these genes in Brassica species compared with Arabidopsis. Several antioxidant, calcium, non-calcium metal and secondary metabolite genes also showed differential expression between these two species. These coincided with accumulation of two alkaloid-like compounds, high levels of calcium, and other metals in B. villosa trichomes that are correlated with the known tolerance of B. villosa to high salt and the calcium-rich natural habitat of this wild species. This first time report on the isolation of large amounts of pure B. villosa trichomes, on trichome content, and on relative gene expression differences in an exceptionally hairy Brassica species compared with a glabrous species opens doors for the scientific community to understand trichome gene function in the Brassicas and highlights the potential of B. villosa as a trichome research platform.

Comparison of five major trichome regulatory genes in Brassica villosa with orthologues within the Brassicaceae.

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.

Comprehensive sequence and expression profile analysis of PEX11 gene family in rice.

PEX11 gene family has been shown to be involved in peroxisome biogenesis but very little is known about this gene family in rice. Here we show that five putative PEX11 genes (OsPEX11-1-5) present in rice genome and each contain three conserved motifs. The PEX11 sequences from rice and other species can be classified into three major groups. Among the five rice PEX11 genes, OsPEX11-2 and -3 are most likely duplicated. Expression profile and RT-PCR analysis suggested that the members of PEX11 family in rice had differential expression patterns: OsPEX11-1 and OsPEX11-4 had higher expression levels in leaf tissues than in the other tissues, OsPEX11-2 was detected only in germinated seeds, OsPEX11-3 was expressed predominantly in endosperm and germinated seeds, and OsPEX11-5 was expressed in all the tissues investigated. We also observed that the rice PEX11 genes had differential expression patterns under different abiotic stresses. OsPEX11-1 and OsPEX11-4 were induced by abscisic acid (ABA), hydrogen peroxide (H2O2), salt and low nitrogen stress conditions. OsPEX11-3 was responsive to ABA and H2O2 treatments, and OsPEX11-5 was responsive to ABA, H2O2, and salt treatments. However, OsPEX11-2 had no response to any of the stresses. Our results suggest that the rice PEX11 genes have diversification not only in sequences but also in expression patterns under normal and various stress conditions.