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

Primer sequences used in the construction and analysis of B. napus transgenic lines. Table S1B. Blast of batch leaf Q-PCR primers to the B. rapa, B. oleracea, and B. napus genomes for five trichome regulatory genes and two control genes in B. napus. Table S1C. “Detectable” B. napus homologues of five trichome regulatory genes in first true leaves (from RNA sequencing). Table S1D. BlastP for five Arabidopsis trichome regulatory genes against the Brassica napus genome in NCBI. Table S2A. Differentially expressed leaf trichome ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy line K-5-8 relative to semi-glabrous cv. Westar. Table S2B. Leaf trichome genes with no significant expression differences (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy line K-5-8 relative to semi-glabrous cv. Westar. Table S3. Differentially expressed leaf flavonoid ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S4. Differentially expressed leaf phenylpropanoid and lignin ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S5. Differentially expressed leaf phenolic ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S6. Differentially expressed leaf shikimate ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S7. Differentially expressed leaf isoprenoid and terpene ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S8. Differentially expressed leaf glucosinolate-related and miscellaneous sulphur-related ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S9. Differentially expressed leaf alkaloid-related and miscellaneous N-metabolizing ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S10. Differentially expressed leaf cell wall structural carbohydrate ESTs ((p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S11. Differentially expressed leaf mucilage ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S12. Differentially expressed leaf wax ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S13. Differentially expressed leaf hormone ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S14. Differentially expressed leaf secondary metabolism ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S15. Differentially expressed leaf redox-related ESTs (p < 0.05)) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S16. Differentially expressed leaf protein modification ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S17. Differentially expressed leaf protein degradation ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. Table S18. Differentially expressed leaf transcription factor ESTs (p < 0.05) in hairy AtGL3+ B. napus or ultra-hairy K-5-8 relative to semi-glabrous cv. Westar. (XLSX 400 kb

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

The Establishment of thermotolerance in maize seedlings

Bibliography: p. 160-169

Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development1[C][W][OA]

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). Plant r-protein genes exist as families of multiple expressed members, yet only one r-protein from each family is incorporated into any given ribosome, suggesting that many r-protein genes may be functionally redundant or development/tissue/stress specific. Here, we characterized the localization and gene-silencing phenotypes of a large subunit r-protein family, RPL23a, containing two expressed genes (RPL23aA and RPL23aB). Live cell imaging of RPL23aA and RPL23aB in tobacco with a C-terminal fluorescent-protein tag demonstrated that both isoforms accumulated in the nucleolus; however, only RPL23aA was targeted to the nucleolus with an N-terminal fluorescent protein tag, suggesting divergence in targeting efficiency of localization signals. Independent knockdowns of endogenous RPL23aA and RPL23aB transcript levels using RNA interference determined that an RPL23aB knockdown did not alter plant growth or development. Conversely, a knockdown of RPL23aA produced a pleiotropic phenotype characterized by growth retardation, irregular leaf and root morphology, abnormal phyllotaxy and vasculature, and loss of apical dominance. Comparison to other mutants suggests that the phenotype results from reduced ribosome biogenesis, and we postulate a link between biogenesis, microRNA-target degradation, and maintenance of auxin homeostasis. An additional RNA interference construct that coordinately silenced both RPL23aA and RPL23aB demonstrated that this family is essential for viability

Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots.

Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species

Identification of Plasmodiophora brassicae effectors — A challenging goal

Clubroot is an economically important disease affecting Brassica plants worldwide. Plasmodiophora brassicae is the protist pathogen associated with the disease, and its soil-borne obligate parasitic nature has impeded studies related to its biology and the mechanisms involved in its infection of the plant host. The identification of effector proteins is key to understanding how the pathogen manipulates the plant’s immune response and the genes involved in resistance. After more than 140 years studying clubroot and P. brassicae, very little is known about the effectors playing key roles in the infection process and subsequent disease progression. Here we analyze the information available for identified effectors and suggest several features of effector genes that can be used in the search for others. Based on the information presented in this review, we propose a comprehensive bioinformatics pipeline for effector identification and provide a list of the bioinformatics tools available for such

The biochemical composition and transcriptome of cotyledons from Brassica napus lines expressing the AtGL3 transcription factor and exhibiting reduced flea beetle feeding

Abstract Background Previously, transgenic trichome-bearing (hairy leaf) Brassica napus lines expressing either the Arabidopsis thaliana GL3 gene (line AtGL3+) [1] or the AtGL3 gene in combination with an RNAi construct to down-regulate TTG1 (line K-5-8) [2] were developed. The leaves of these lines exhibited altered insect feeding (flea beetle) and oviposition (diamondback moth) behaviour compared to the non-transgenic semi-glabrous leaves of B. napus cv. Westar. Interestingly, the cotyledons of these lines remained glabrous, but also showed reduced feeding by flea beetles. Here we examine the composition and global transcriptome of the glabrous cotyledons from these transgenic lines to ascertain the mechanism(s) underlying this unexpected phenomenon. Results Approximately, 7500 genes were up-regulated in cotyledons of each hairy line, compared with < 30 that were down-regulated. The up-regulated genes included those involved in cell wall synthesis, secondary metabolite production, redox, stress and hormone-related responses that have the potential to impact host plant cues required to elicit defense responses toward insect pests. In particular, the expression of glucosinolate biosynthetic and degradation genes were substantially altered in the glabrous cotyledons of the two hairy leaf lines. The transcriptomic data was supported by glucosinolate and cell wall composition profiles of the cotyledons. Changes in gene expression were much more extreme in the AtGL3+ line compared with the K-5-8 line in terms of diversity and intensity. Conclusions The study provides a roadmap for the isolation and identification of insect resistance compounds and proteins in the glabrous cotyledons of these hairy leaf lines. It also confirms the impact of mis-expression of GL3 and TTG1 on types of metabolism other than those associated with trichomes. Finally, the large number of up-regulated genes encoding heat shock proteins, PR proteins, protease inhibitors, glucosinolate synthesis/breakdown factors, abiotic stress factors, redox proteins, transcription factors, and proteins required for auxin metabolism also suggest that these cotyledons are now primed for resistance to other forms of biotic and abiotic stress