Galactinol synthase (GolS) is a key enzyme in raffinose family oligosaccharide (RFO) biosynthesis. The finding that GolS accumulates in plants exposed to abiotic stresses indicates RFOs function in environmental adaptation. However, the evolutionary relationships and biological functions of GolS family in rapeseed (Brassica napus) and tobacco (Nicotiana tabacum) remain unclear. In this study, we identified 20 BnGolS and 9 NtGolS genes. Subcellular localization predictions showed that most of the proteins are localized to the cytoplasm. Phylogenetic analysis identified a lost event of an ancient GolS copy in the Solanaceae and an ancient duplication event leading to evolution of GolS4/7 in the Brassicaceae. The three-dimensional structures of two GolS proteins were conserved, with an important DxD motif for binding to UDP-galactose (uridine diphosphate-galactose) and inositol. Expression profile analysis indicated that BnGolS and NtGolS genes were expressed in most tissues and highly expressed in one or two specific tissues. Hormone treatments strongly induced the expression of most BnGolS genes and homologous genes in the same subfamilies exhibited divergent-induced expression. Our study provides a comprehensive evolutionary analysis of GolS genes among the Brassicaceae and Solanaceae as well as an insight into the biological function of GolS genes in hormone response in plants.

Leaf senescence is a vital aspect of plant physiology and stress responses and is induced by endogenous factors and environmental cues.. The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factor family influences growth, development, and stress responses in Arabidopsis (Arabidopsis thaliana) and other species. However, the roles of NACs in tobacco (Nicotiana tabacum) leaf senescence are still unclear. Here, we report that NtNAC56 regulates leaf senescence in tobacco. Transgenic plants overexpressing NtNAC56 (NtNAC56-OE) showed induction of senescence-related genes and exhibited early senescence and lower chlorophyll content compared to wild-type (WT) plants and the Ntnac56-19 mutant. In addition, root development and seed germination were inhibited in the NtNAC56-OE lines. Transmission electron microscopy observations accompanied by physiological and biochemical assays revealed that NtNAC56 overexpression triggers chloroplast degradation and reactive oxygen species accumulation in tobacco leaves. Transcriptome analysis demonstrated that NtNAC56 activates leaf senescence-related genes and jasmonic acid (JA) biosynthesis pathway genes. In addition, the JA content of NtNAC56-OE plants was higher than in WT plants, and JA treatment induced NtNAC56 expression. We performed DNA affinity purification sequencing to identify direct targets of NtNAC56, among which we focused on LIPOXYGENASE 5 (NtLOX5), a key gene in JA biosynthesis. A dual-luciferase reporter assay and a yeast one-hybrid assay confirmed that NtNAC56 directly binds to the TTTCTT motif in the NtLOX5 promoter. Our results reveal a mechanism whereby NtNAC56 regulates JA-induced leaf senescence in tobacco and provide a strategy for genetically manipulating leaf senescence and plant growth.

Glandular trichomes are universal epidermal structures that produce abundant specialized metabolites. However, knowledge of the initiation of glandular heads in glandular trichomes is limited. Herein, we found an intrinsic link of morphogenesis between glandular trichomes and non-glandular trichomes. Two novel homeodomain leucine zipper II members in tobacco (Nicotiana tabacum), NtHD9 and NtHD12, played important roles in long glandular trichome formation: NtHD9 was responsible for glandular head formation, while NtHD12 simultaneously controlled the formation of stalks and glandular heads. DAP-seq analysis suggested that NtHD9 can bind to the KKGCATTWAWTR motif of the cytochromes P450 94C1 (NtCYP94C1) promoter, which is involved in jasmonoyl-isoleucine oxidation. RNA-seq analysis of non-transformed tobacco and nthd9 plants revealed that NtHD9 modulates the expression of jasmonate (JA) signaling- and six trichome development-related genes. Notably, MeJA treatment restored the morphogenesis of long glandular trichomes in nthd9 and nthd12 plants, and the size of glandular heads increased with increasing MeJA concentration. However, the phenotype of long glandular trichome absence in double mutants of NtHD9 and NtHD12 could not be restored by MeJA. Our data demonstrate that NtHD9 and NtHD12 have distinct major functions yet overlapping roles in long glandular trichome formation via JA signaling.