Plant responses to stress occur via abscisic acid (ABA) dependent or independent pathways. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) play a key role in plant stress signal transduction pathways. It is known that some SnRK2 members are positive regulators of ABA signal transduction through interaction with group A type 2C protein phosphatases (PP2Cs). Here, ten SnRK2s were isolated from wheat. Based on phylogenetic analysis using kinase domains or the C-terminus, the ten SnRK2s were divided into three subclasses. Expression pattern analysis revealed that all TaSnRK2s were involved in the responses to PEG, NaCl, and cold stress. TaSnRK2s in subclass III were strongly induced by ABA. Subclass II TaSnRK2s responded weakly to ABA, whereas TaSnRK2s in subclass I were not activated by ABA treatment. Motif scanning in the C-terminus indicated that motifs 4 and 5 in the C-terminus were unique to subclass III. We further demonstrate the physical and functional interaction between TaSnRK2s and a typical group A PP2C (TaABI1) using Y2H and BiFC assays. The results showed that TaABI1 interacted physically with subclass III TaSnRK2s, while having no interaction with subclass I and II TaSnRK2s. Together, these findings indicated that subclass III TaSnRK2s were involved in ABA regulated stress responses, whereas subclass I and II TaSnRK2s responded to various abiotic stressors in an ABA-independent manner.

Heat shock proteins 90 (HSP90s) are a highly conserved protein family of cellular chaperones widely found in plants; they play a fundamental role in response to biotic and abiotic stresses. The genome-wide analysis of HSP90 gene family has been completed for some species; however, it has been rarely reported for the tobacco HSP90 genes. In this study, we systematically conducted genome-wide identification and expression analysis of the tobacco HSP90 gene family, including gene structures, evolutionary relationships, chromosomal locations, conserved domains, and expression patterns. Twenty-one NtHSP90s were identified and classified into eleven categories (NtHSP90–1 to NtHSP90–11) based on phylogenetic analysis. The conserved structures and motifs of NtHSP90 proteins in the same subfamily were highly consistent. Most NtHSP90 proteins contained the ATPase domain, which was closely related to conserved motif 2. Motif 5 was a low complexity sequence and had the function of signal peptide. At least 6 pairs of NtHSP90 genes underwent gene duplication, which arose from segment duplication and tandem duplication events. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of HSP90s. Dynamic expression analysis indicated that some NtHSP90 genes may play fundamental roles in regulation of abiotic stress response. The expression of NtHSP90–4, NtHSP90–5, and NtHSP90–9 were up-regulated, while NtHSP90–6, and NtHSP90–7 were not induced by ABA, drought, salt, cold and heat stresses. Among the five treatments, NtHSP90s were most strongly induced by heat stress, and weakly activated by ABA treatment. There was a similar response pattern of NtHSP90s under osmotic stress, or extreme temperature stress. This is the first genome-wide analysis of Hsp90 in N. tabacum. These results indicate that each NtHSP90 member fulfilled distinct functions in response to various abiotic stresses.

Melatonin is a well-known signaling molecule that mediates a range of physiological activities and various stress reactions in plants. We comprehensively tested the effect of melatonin on the development of root hairs and glandular trichomes and found that melatonin pretreatment of tobacco seeds significantly increased the length of root hairs. Furthermore, melatonin-treated tobacco exhibited significantly higher density of trichomes and larger glandular heads on long-stalk glandular trichomes than untreated plants, which resulted in enhanced secretion in glandular trichomes. Exogenous melatonin enhanced the aphid resistance of plants by facilitating the accumulation of cembranoids in the glandular trichomes and alleviated cadmium toxicity by increasing the Cd-exudation capacity of long glandular trichomes. Metabolic analysis indicated that the contents of 108 metabolites significantly changed upon melatonin treatment, with the contents of those that are directly/indirectly involved in melatonin metabolism changing the most. Further, KEGG pathway analysis suggested that the metabolic pathways of amino acids, reducing sugar, secondary metabolites, indole alkaloid biosynthesis, purine, pyrimidine, and ABC transporters were greatly influenced by exogenous melatonin application. Moreover, metabolisms of melatonin-related antioxidants and pyrimidine nucleoside antibiotics were enhanced after melatonin treatment. Melatonin improved tobacco resistance to high salinity, drought, and extreme temperature stresses, as indicated by improved photosynthetic and antioxidant capacities in treated vs. untreated plants. This study lays a foundation for the comprehensive application of melatonin to increase the stress tolerance of plants.

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.