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. 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. 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.

Background: Aluminum (Al) toxicity severely limits Medicago sativa (alfalfa) production on acidic soils, resulting in major yield losses worldwide. The highly conserved miRNA156 (miR156) functions by downregulating at least 11 SQUAMOSA promoter-binding protein-like (SPL) transcription factors in alfalfa, including SPL13, but its role in Al stress remains unclear. This study aimed to investigate the miR156/SPL regulatory network’s function in alfalfa under Al stress. Methods: Gene expression analyses, histochemical staining, nutrient profiling, phenotypic assays, transcriptome profiling, and ChIP-seq were conducted on alfalfa plants with altered miR156 and SPL13 expression to assess their roles in the Al stress response. Results: Al stress induced SPL13 expression while repressing miR156 in the roots. Elevated miR156 intensified Al accumulation, lipid peroxidation, and plasma membrane damage, accompanied by reduced leaf nitrogen, magnesium, sulfur, and phosphorus content. Phenotypically, increased SPL13 enhanced the root length and Al tolerance, whereas SPL13 silencing reduced tolerance. Transcriptome profiling of SPL13-silenced plants identified differentially expressed genes involved in the Al response, including aluminum-activated malate transporters and various transcription factors (GRAS, Myb-related, bHLH041, NAC, WRKY53, bZIP, and MADS-box). ChIP-seq revealed that SPL13 directly regulates genes encoding a protein kinase, cytochrome P450, and fasciclin-like arabinogalactan proteins. Conclusions: The MsmiR156/MsSPL13 network plays a crucial regulatory role in alfalfa’s response to Al toxicity. These findings provide novel genetic targets and foundational knowledge to advance molecular breeding for enhanced Al tolerance in alfalfa.