Background: MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are two major classes of small RNAs. They play important regulatory roles in plants and animals by regulating transcription, stability and/or translation of target genes in a sequence-complementary dependent manner. Over 4,000 miRNAs and several classes of siRNAs have been identified in plants, but in tobacco only computational prediction has been performed and no tobacco-specific miRNA has been experimentally identified. Wounding is believed to induce defensive response in tobacco, but the mechanism responsible for this response is yet to be uncovered. Results: To get insight into the role of small RNAs in damage-induced responses, we sequenced and analysed small RNA populations in roots and leaves from wounding or topping treated tobacco plants. In addition to confirmation of expression of 27 known miRNA families, we identified 59 novel tobacco-specific miRNA members of 38 families and a large number of loci generating phased 21- or 24-nt small RNAs (including ta-siRNAs). A number of miRNAs and phased small RNAs were found to be responsive to wounding or topping treatment. Targets of small RNAs were further surveyed by degradome sequencing. Conclusions: The expression changes of miRNAs and phased small RNAs responsive to wounding or topping and identification of defense related targets for these small RNAs suggest that the inducible defense response in tobacco might be controlled by pathways involving small RNAs.

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

Maca (Lepidium meyenii Walp.) is a traditional Andean crop with great potential for various sanitarian and medical functions, which is attracting increased research attention. The majority of previous Maca studies were focused on biochemistry and pharmacodynamics, while the genetic basis of its unique characteristics lagged due to a lack of genome information. The authors perform gas chromatography-mass spectrometry (GC/MS) analysis in the hypocotyls of three Maca ecotypes and identify 79 compounds. Among them, 62 compounds have distinct profiles among Maca ecotypes. To reveal the underlying regulatory mechanism of the chemical composition differences, de novo transcriptome sequencing is performed and the transcription profiles of three Maca ecotypes are comparatively analyzed. Functional analysis indicates several key pathways, including “starch and sucrose metabolism,” “phenylpropanoid biosynthesis,” “phenylalanine metabolism” and “plant-pathogen interaction,” are involved in regulating the chemical compositions of Maca. Combining metabolomics and transcriptomics analysis indicates transcription factors such as MYB and WRKY and mediators such as protein kinase and bifunctional inhibitors might be critical regulators of chemical composition in Maca. The transcriptome reference genome and differentially expressed genes (DEGs) obtained in this study might serve as an initial step to illustrate the genetic differences in nutrient component, secondary metabolites content, medicinal function and stress resistance in Maca.