Chilling stress increases the amount of polyphenols, especially lignin, which protects tobacco (Nicotiana tabacum L. cv. k326) from chilling stress. To clarify the molecular biosynthesis mechanism of the key representative compounds, specifically lignin, RNA sequencing and ultra-high pressure liquid chromatography coupled to quadrupole-time of flight mass spectrometry technologies were used to construct transcriptomic and metabolomic libraries from the leaves of tobacco plants subjected to normal (25°C) and chilling (4°C) temperature treatments. Transcriptomic libraries from the different samples were sequenced, generating more than 40 million raw reads. Among nine samples, metabolomic analysis identified a total of 97 encoding enzymes that function in the key steps of pathways related to polyphenol biosynthesis, where 42 metabolites were also located. An integrated analysis of metabolic and transcriptomic data revealed that most of the intermediate metabolites and enzymes related to lignin biosynthesis were synthesised in the leaves under chilling stress, which suggests that the biosynthesis of lignin plays an important role in the response of tobacco leaves to cold temperatures. In addition, the cold insensitivity of chalcone synthase genes might be considered to be an important rate-limiting factor in the process of precursor substance flow to flavonoid biosynthesis under chilling stress. Furthermore, the upregulated expression of phenylalanine ammonia lyase (PAL), hydroxycinnamoyl transferase (HCT) and cinnamyl-alcohol dehydrogenase (CAD) under chilling stress is the key to an increase in lignin synthesis. This study provides a hypothetical basis for the screening of new active metabolites and the metabolic engineering of polyphenols in tobacco.

Background: Drought stress is the most harmful one among other abiotic stresses with negative impacts on crop growth and development. Drought-hardening is a feasible and widely used method in tobacco seedlings cultivation. It has gained extensive interests due to its role in improving drought tolerance. This research aimed to investigate the role of drought-hardening and to unravel the multiple mechanisms underlying tobacco drought tolerance and adaptation. Results: This study was designed in which various drought-hardening treatments (CK (no drought-hardening), T1 (drought-hardening for 24 h), T2 (drought-hardening for 48 h), and T3 (drought-hardening for 72 h)) were applied to two tobacco varieties namely HongHuaDaJinYuan (H) and Yun Yan-100 (Y). The findings presented a complete framework of drought-hardening effect at physiological, biochemical, and gene expression levels of the two tobacco varieties under drought stress. The results showed that T2 and T3 significantly reduced the growth of the two varieties under drought stress. Similarly, among the various drought-hardening treatments, T3 improved both the enzymatic (POD, CAT, APX) and non-enzymatic (AsA) defense systems along with the elevated levels of proline and soluble sugar to mitigate the negative effects of oxidative damage and bringing osmoregulation in tobacco plants. Finally, the various drought-hardening treatments (T1, T2, and T3) showed differential regulation of genes expressed in the two varieties, while, particularly T3 drought-hardening treatment-induced drought tolerance via the expression of various stress-responsive genes by triggering the biosynthesis pathways of proline (P5CS1), polyamines (ADC2), ABA-dependent (SnRK2, AREB1), and independent pathways (DREB2B), and antioxidant defense-related genes (CAT, APX1, GR2) in response to drought stress. Conclusions: Drought-hardening made significant contributions to drought tolerance and adaptation in two tobacco variety seedlings by reducing its growth and, on the other hand, by activating various defense mechanisms at biochemical and molecular levels. The findings of the study pointed out that drought-hardening is a fruitful strategy for conferring drought tolerance and adaptations in tobacco. It will be served as a useful method in the future to understand the drought tolerance and adaptation mechanisms of other plant species. Graphical Drought-hardening improved drought tolerance and adaptation of the two tobacco varieties. T1 indicates drought-hardening for 24 h, T2 indicates drought-hardening for 48 h, T3 indicates drought-hardening for 72 h

Drought stress is one of the main factors limiting crop production, which provokes a number of changes in plants at physiological, anatomical, biochemical and molecular level. To unravel the various mechanisms underpinning tobacco (Nicotiana tabacum L.) drought stress tolerance, we conducted a comprehensive physiological, anatomical, biochemical and transcriptome analyses of three tobacco cultivars (i.e., HongHuaDaJinYuan (H), NC55 (N) and Yun Yan-100 (Y)) seedlings that had been exposed to drought stress. As a result, H maintained higher growth in term of less reduction in plant fresh weight, dry weight and chlorophyll content as compared with N and Y. Anatomical studies unveiled that drought stress had little effect on H by maintaining proper leaf anatomy while there were significant changes in the leaf anatomy of N and Y. Similarly, H among the three varieties was the least affected variety under drought stress, with more proline content accumulation and a powerful antioxidant defense system, which mitigates the negative impacts of reactive oxygen species. The transcriptomic analysis showed that the differential genes expression between HongHuaDaJinYuan, NC55 and Yun Yan-100 were enriched in the functions of plant hormone signal transduction, starch and sucrose metabolism, and arginine and proline metabolism. Compared to N and Y, the differentially expressed genes of H displayed enhanced expression in the corresponding pathways under drought stress. Together, our findings offer insights that H was more tolerant than the other two varieties, as evidenced at physiological, biochemical, anatomical and molecular level. These findings can help us to enhance our understanding of the molecular mechanisms through the networks of various metabolic pathways mediating drought stress adaptation in tobacco.

Annual removal of tobacco residues and insufficient input of organic materials have exacerbated total organic carbon (TOC) depletion and soil degradation in a tobacco field in the Huanghuai area. Straw residue and biochar application may be effective ways to increase TOC accumulation and improve soil fertility. In this field experiment, wheat straw (WS) and wheat-straw-derived biochar (BC) with mineral fertilizer were compared with mineral fertilizer alone (CK), and we assessed their effects on soil organic carbon fractions, enzyme activities, and nutrients in Shandong Province, China, during 2016 and 2017. At 0–20 cm depth, the WS treatment had a greater overall effect on the measured soil properties. Compared with the control, the WS treatment significantly increased the concentrations of microbial biomass carbon (MBC), hot-water-extractable carbon (HWC), and permanganate-oxidizable carbon concentrations (POXC; by 252.41%, 107.02%, and 65.53%, respectively); the activities of sucrase, urease, and phosphatase (by 112.52%, 7.81%, and 34.33%, respectively); and the contents of alkaline hydrolysable nitrogen, available phosphorus, and available potassium (by 92.22%, 100.78%, and 10.57%, respectively). Compared with the control, the BC treatment significantly increased TOC content, MBC content, light fraction organic carbon (LFOC), and potassium (TK) concentration (by 74.93%, 86.24%, 153.73%, and 21.92%, respectively). Most soil enzyme activity and nutrient parameters were significantly correlated with MBC. Thus, straw application improved soil fertility by increasing the concentrations of high labile organic carbon fractions (HWC, MBC, and POXC), stimulating soil enzyme activities, and releasing more soil available nutrients, and BC addition contributed to the accumulation of TOC, MBC, LFOC, and TK.