Identification of CEP peptides encoded by the tobacco (Nicotiana tabacum) genome and characterization of their roles in osmotic and salt stress responses.

Members of the CEP (C-terminally Encoded Peptide) gene family have been shown to be involved in various developmental processes and stress responses in plants. In order to understand the roles of CEP peptides in stress response, a comprehensive bioinformatics approach was employed to identify NtCEP genes in tobacco (Nicotiana tabacum L.) and to analyze their potential roles in stress responses. Totally 21 NtCEP proteins were identified and categorized into two subgroups based on their CEP domains. Expression changes of the NtCEP genes in response to various abiotic stresses were analyzed via qRT-PCR and the results showed that a number of NtCEPs were significant up-regulated under drought, salinity, or temperature stress conditions. Furthermore, application of synthesized peptides derived from NtCEP5, NtCEP13, NtCEP14, and NtCEP17 enhanced plant tolerance to different salt stress treatments. NtCEP5, NtCEP9 and NtCEP14, and NtCEP17 peptides were able to promote osmotic tolerance of tobacco plants. The results from this study suggest that NtCEP peptides may serve as important signaling molecules in tobacco's response to abiotic stresses.

Characterization of NAC transcription factor NtNAC028 as a regulator of leaf senescence and stress responses.

NAC proteins constitute one of the largest transcription factor families and are involved in regulation of plant development and stress responses. Our previous transcriptome analyses of tobacco revealed a significant increase in the expression of NtNAC028 during leaf yellowing. In this study, we found that NtNAC028 was rapidly upregulated in response to high salinity, dehydration, and abscisic acid (ABA) stresses, suggesting a vital role of this gene in abiotic stress response. NtNAC028 loss-of-function tobacco plants generated via CRISPR-Cas9 showed delayed leaf senescence and increased tolerance to drought and salt stresses. Meanwhile NtNAC028 overexpression led to precocious leaf senescence and hypersensitivity to abiotic stresses in Arabidopsis, indicating that NtNAC028 functions as a positive regulator of natural leaf senescence and a negative regulator of stress tolerance. Furthermore, NtNAC028-overexpressing Arabidopsis plants showed lower antioxidant enzyme activities, higher reactive oxygen species (ROS), and H2O2 accumulation under high salinity, resulted in more severe oxidative damage after salt stress treatments. On the other hand, NtNAC028 mutation in tobacco resulted in upregulated expression of ROS-scavenging and abiotic stress-related genes, higher antioxidant enzyme activities, and enhanced tolerance against abiotic stresses, suggesting that NtNAC028 might act as a vital regulator for plant stress response likely by mediating ROS scavenging ability. Collectively, our results indicated that the NtNAC028 plays a key regulatory role in leaf senescence and response to multiple abiotic stresses.

Bioinformatics and Expression Analysis of IDA-Like Genes Reveal Their Potential Functions in Flower Abscission and Stress Response in Tobacco (Nicotiana tabacum L.).

The inflorescence deficient in abscission-like (IDL) genes have been shown to play critical roles in floral organ abscission, lateral root formation and various stress responses in Arabidopsis. The IDL gene family has been characterized in a number of plant species, while limited information is available about IDL genes of tobacco. In the current study, 15 NtIDL members were identified in the tobacco genome, and were classified into six groups together with IDL members from other species. Evolution analysis suggested that the NtIDL members form group VI might have originated from duplication events. Notably, NtIDL06 shared high similarities with AtIDA in the EPIP sequence, and its encoding gene was highly expressed in the abscission zone of flowers at late developmental stages, implying that NtIDL06 might regulate tobacco flower abscission. In addition, the results from cis-elements analysis of promoters and expression after stress treatments suggested that NtIDL members might be involved in various stress responses of tobacco. The results from this study provide information for further functional analysis related to flower abscission and stress responses of NtIDL genes.

Study on metabolic variation in whole grains of four proso millet varieties reveals metabolites important for antioxidant properties and quality traits.

Proso millet (Panicum miliaceum L.) is a minor cereal crop that has been considered as health-promoting food. Little information is available however, about the metabolic basis of nutritional values of proso millet. In this study, using a UHPLC-QqQ-MS/MS-based metabolomics approach, we compared the metabolomes of whole grains from four proso millet varieties with different bran color, namely White, Black, Gray and Red. In total, 672 metabolites were identified, among which 121, 116 and 148 metabolites showed differential accumulation in the three comparison groups (White vs. Black/Gray/Red). The results demonstrated the main pathways that were differentially activated included: tryptophan metabolism, flavonoid, isoflavonoid, flavone, and flavonol biosynthesis. Considerable difference between varieties was observed in accumulation of phenolic acids and flavonoids, which might lead to difference in antioxidant activities. The results of this study provide useful information for further investigation of proso millet food chemistry and for sufficient utilization of this special crop.

Characterization and Mapping of a Novel Premature Leaf Senescence Mutant in Common Tobacco (Nicotiana tabacum L.).

As the last stage of plant development, leaf senescence has a great impact on plant's life cycle. Genetic manipulation of leaf senescence has been used as an efficient approach in improving the yield and quality of crop plants. Here we describe an ethyl methane sulfonate (EMS) mutagenesis induced premature leaf senescence mutant yellow leaf 1 (yl1) in common tobacco (Nicotiana tabacum L.). The yl1 plants displayed early leaf yellowing. Physiological parameters and marker genes expression indicated that the yl1 phenotype was caused by premature leaf senescence. Genetic analyses indicated that the yl1 phenotype was controlled by a single recessive gene that was subsequently mapped to a specific interval of tobacco linkage group 11 using simple sequence repeat (SSR) markers. Exogenous plant hormone treatments of leaves showed that the yl1 mutant was more sensitive to ethylene and jasmonic acid than the wild type. No similar tobacco premature leaf senescence mutants have been reported. This study laid a foundation for finding the gene controlling the mutation phenotype and revealing the molecular regulation mechanism of tobacco leaf senescence in the next stage.

NAC Family Transcription Factors in Tobacco and Their Potential Role in Regulating Leaf Senescence.

The NAC family is one of the largest families of plant-specific transcription factors (TFs) and NAC proteins play important regulatory roles in a variety of developmental and stress response processes in plants. Members of the NAC family TFs have been shown to be important regulators of leaf senescence in a number of plant species. Here we report the identification of the NAC family in tobacco (Nicotiana tabacum) and characterization of the potential role of some of the tobacco NAC TFs in regulating leaf senescence. A total of 154 NAC genes (NtNACs) were identified and clustered together with the Arabidopsis NAC family into fifteen groups (a-o). Transcriptome data analysis followed by qRT-PCR validation showed that the majority of the senescence-up-regulated NtNACs fall into subgroups NAC-b and f. A number of known senescence regulators from Arabidopsis also belong to these two subgroups. Among these senescence-up-regulated NtNACs, NtNAC080, a close homolog of AtNAP, is a positive regulator of leaf senescence. Overexpression of NtNAC080 caused early senescence in Arabidopsis leaves and NtNAC080 mutation induced by Cas9/gRNA in tobacco led to delayed leaf senescence.

Intergrative metabolomic and transcriptomic analyses unveil nutrient remobilization events in leaf senescence of tobacco.

Leaf senescence in plants is a coordinated process that involves remobilization of nutrients from senescing leaves to sink tissues. The molecular events associated with nutrient remobilization are however not well understood. In this study the tobacco system with a source-sink relationship between different leaf positions was used in analyzing the spatiotemporal changes of 76 metabolites from leaves at 3 different stalk positions and 8 developmental stages. The metabolomic data was then compared with RNA-seq data from the same samples to analyze the activities of the metabolic pathways that are important for nutrient remobilization. Integrative analyses on metabolites accumulation and expression changes of enzyme-encoding genes in corresponding metabolic pathways indicated a significant up-regulation of the tricarboxylic acid cycle and related metabolism of sugars, amino acids and fatty acids, suggesting the importance of energy metabolism during leaf senescence. Other changes of the metabolism during tobacco leaf senescence include increased activities of the GS/GOGAT cycle which is responsible for nitrogen recycling, and increased accumulation of nicotine. The results also suggested that a number of compounds seemed to be transported from senescing leaves at lower positions to sink leaves at upper positions. Some of these metabolites could play a role in nutrient remobilization.

A MADS-box gene NtSVP regulates pedicel elongation by directly suppressing a KNAT1-like KNOX gene NtBPL in tobacco (Nicotiana tabacum L.).

Optimal inflorescence architecture is important for plant reproductive success by affecting the ultimate number of flowers that set fruits and for plant competitiveness when interacting with biotic or abiotic conditions. The pedicel is one of the key contributors to inflorescence architecture diversity. To date, knowledge about the molecular mechanisms of pedicel development is derived from Arabidopsis. Not much is known regarding other plants. Here, an SVP family MADS-box gene, NtSVP, in tobacco (Nicotiana tabacum) that is required for pedicel elongation was identified. It is shown that knockdown of NtSVP by RNA interference (RNAi) caused elongated pedicels, while overexpression resulted in compact inflorescences with much shortened pedicels. Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants. Disruption of NtBPL decreased pedicel lengths and shortened cortex cells. Consistent with the presence of a CArG-box at the NtBPL promoter, the direct binding of NtSVP to the NtBPL promoter was demonstrated by yeast one-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay, in which NtSVP may act as a repressor of NtBPL. Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin. These findings together suggest the function of a MADS-box transcription factor in plant pedicel development, probably via negative regulation of a BP-like class I KNOX gene. The present work thus postulates the conservation and divergence of the molecular regulatory pathways underlying the development of plant inflorescence architecture.