Genome-wide analysis of long noncoding RNAs in response to salt stress in Nicotiana tabacum.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to play important roles in the response of plants to various abiotic stresses, including drought, heat and salt stress. However, the identification and characterization of genome-wide salt-responsive lncRNAs in tobacco (Nicotiana tabacum L.) have been limited. Therefore, this study aimed to identify tobacco lncRNAs in roots and leaves in response to different durations of salt stress treatment. RESULTS: A total of 5,831 lncRNAs were discovered, with 2,428 classified as differentially expressed lncRNAs (DElncRNAs) in response to salt stress. Among these, only 214 DElncRNAs were shared between the 2,147 DElncRNAs in roots and the 495 DElncRNAs in leaves. KEGG pathway enrichment analysis revealed that these DElncRNAs were primarily associated with pathways involved in starch and sucrose metabolism in roots and cysteine and methionine metabolism pathway in leaves. Furthermore, weighted gene co-expression network analysis (WGCNA) identified 15 co-expression modules, with four modules strongly linked to salt stress across different treatment durations (MEsalmon, MElightgreen, MEgreenyellow and MEdarkred). Additionally, an lncRNA-miRNA-mRNA network was constructed, incorporating several known salt-associated miRNAs such as miR156, miR169 and miR396. CONCLUSIONS: This study enhances our understanding of the role of lncRNAs in the response of tobacco to salt stress. It provides valuable information on co-expression networks of lncRNA and mRNAs, as well as networks of lncRNAs-miRNAs-mRNAs. These findings identify important candidate lncRNAs that warrant further investigation in the study of plant-environment interactions.

NCR343 is required to maintain the viability of differentiated bacteroids in nodule cells in Medicago truncatula.

Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

Reconstruction of the full-length transcriptome of cigar tobacco without a reference genome and characterization of anion channel/transporter transcripts.

BACKGROUND: Cigar wrapper leaves are the most important raw material of cigars. Studying the genomic information of cigar tobacco is conducive to improving cigar quality from the perspective of genetic breeding. However, no reference genome or full-length transcripts at the genome-wide scale have been reported for cigar tobacco. In particular, anion channels/transporters are of high interest for their potential application in regulating the chloride content of cigar tobacco growing on coastal lands, which usually results in relatively high Cl- accumulation, which is unfavorable. Here, the PacBio platform and NGS technology were combined to generate a full-length transcriptome of cigar tobacco used for cigar wrappers. RESULTS: High-quality RNA isolated from the roots, leaves and stems of cigar tobacco were subjected to both the PacBio platform and NGS. From PacBio, a total of 11,652,432 subreads (19-Gb) were generated, with an average read length of 1,608 bp. After corrections were performed in conjunction with the NGS reads, we ultimately identified 1,695,064 open reading frames including 21,486 full-length ORFs and 7,342 genes encoding transcription factors from 55 TF families, together with 2,230 genes encoding long non-coding RNAs. Members of gene families related to anion channels/transporters, including members of the SLAC and CLC families, were identified and characterized. CONCLUSIONS: The full-length transcriptome of cigar tobacco was obtained, annotated, and analyzed, providing a valuable genetic resource for future studies in cigar tobacco.

NtRLK5, a novel RLK-like protein kinase from Nitotiana tobacum, positively regulates drought tolerance in transgenic Arabidopsis.

Receptor-like protein kinase (RLKs) plays pivotal roles in plant growth and development as well as stress responses. However, little is known about the function of RLKs in Nitotiana tobacum. In the present study, we present data on NtRLK5, a novel RLK-like gene isolated from Hongda (Nitotiana tobacum L.). Expression profile analysis revealed that NtRLK5 was strongly induced by drought and salt stresses. Transient expression of NtRLK5-GFP fusion protein in protoplast showed that NtRLK5 was localized to plasma membrane. Overexpression of NtRLK5 conferred enhanced drought tolerance in transgenic Arabidopsis plants, which was attributed to not only the lower malondialdehyde (MDA) and H2O2 contents, but also the higher antioxidant enzymes activities. Moreover, the expression of several antioxidation- and stress-related genes was also significantly up-regulated in NtRLK5 transgenic plants under drought condition. Taken together, the results suggest that NtRLK5 functions as a positive regulator in drought tolerance.

Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

BACKGROUND: Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). RESULTS: In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed miRNAs (DEMs) and DEGs revealed 92 mRNA-miRNA interactions between CL and DL plants, and 32 mRNA-miRNA interactions between DL and WL plants. CONCLUSIONS: This study provides a global view of the transcriptional and the post-transcriptional responses of tobacco under drought stress and re-watering conditions. Our results establish an empirical foundation that should prove valuable for further investigations into the molecular mechanisms through which tobacco, and plants more generally, respond to drought stress at multiple molecular genetic levels.

A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion.

During legume-rhizobia symbiosis, differentiation of the symbiosome (engulfed intracellular rhizobia) is necessary for successful nitrogen fixation. To control symbiosome differentiation, host cell subcellular components, e.g., ER (endoplasmic reticulum), must adapt robustly to ensure large-scale host protein secretion to the new organelle. However, the key components controlling the adaption of ER in nodule cells remain elusive. We report that Medicago BID1, a nodule-specific signal peptide peptidase (SPP), is central to ER structural dynamics and host protein secretion. In bid1, symbiosome differentiation is blocked. BID1 localizes specifically to the ER membrane and expresses exclusively in nodule cells with symbiosomes. In the wild type ER forms proximal association structures with symbiosomes, but not in bid1. Consequently, in bid1 excessive ER stress responses are induced and ER-to-symbiosome protein secretion is impaired. In summary, a nodule-specific SPP is necessary for ER-symbiosome proximal association, host protein secretion, and symbiosome differentiation.

Metabolic engineering of a 1,8-cineole synthase enhances aphid repellence and increases trichome density in transgenic tobacco (Nicotiana tabacum L.).

BACKGROUND: The green peach aphid (Myzus persicae Sulzer) is a harmful agricultural pest that causes severe crop damage by directly feeding or indirectly vectoring viruses. 1,8-cineole synthase (CINS) is a multiproduct enzyme that synthesizes monoterpenes, with 1,8-cineole dominating the volatile organic compound profile. However, the relationship between aphid preference and CINS remains elusive. RESULTS: Here, we present evidence that SoCINS, a protein from garden sage (Salvia officinalis), enhanced aphid repellence and increased trichome density in transgenic tobacco. Our results demonstrated that overexpression of SoCINS (SoCINS-OE) led to the emission of 1,8-cineole at a level of up to 181.5 ng per g fresh leaf. Subcellular localization assay showed that SoCINS localized to chloroplasts. A Y-tube olfactometer assay and free-choice assays revealed that SoCINS-OE plants had a repellent effect on aphids, without incurring developmental or fecundity-related penalties. Intriguingly, the SoCINS-OE plants displayed an altered trichome morphology, showing increases in trichome density and in the relative proportion of glandular trichomes, as well as enlarged glandular cells. We also found that SoCINS-OE plants had significantly higher jasmonic acid (JA) levels than wild-type plants. Furthermore, application of 1,8-cineole elicited increased JA content and trichome density. CONCLUSION: Our results demonstrate that SoCINS-OE plants have a repellent effect on aphids, and suggest an apparent link between 1,8-cineole, JA and trichome density. This study presents a viable and sustainable approach for aphid management by engineering the expression of 1,8-cineole synthase gene in plants, and underscores the potential usefulness of monoterpene synthase for pest control. © 2023 Society of Chemical Industry.

Au nanocrystals grown on a better-defined one-dimensional tobacco mosaic virus coated protein template genetically modified by a hexahistidine tag.

In this paper, tobacco mosaic virus (TMV) coated protein (CP) was genetically modified by introducing a hexahistidine tag into it for a well-defined one-dimensional template, on which Au nanocrystals (NCs) were grown. The results showed that genetic modification could not only ameliorate the one-dimensional structure of the template, but also improve the growth density of Au NCs on the template. This indicated that genetic modification could be an effective method to modulate the structure of the TMVCP template-based nanocomposites allowing for a broader application of them.

Genome-wide identification and characterization of NPF family reveals NtNPF6.13 involving in salt stress in Nicotiana tabacum.

Proteins of the Nitrate Transporter 1/Peptide Transporter (NPF) family transport a diverse variety of substrates, such as nitrate, peptides, hormones and chloride. In this study, a systematic analysis of the tobacco (Nicotiana tabacum) NPF family was performed in the cultivated 'K326'. In total, 143 NtNPF genes were identified and phylogenetically classified into eight subfamilies, NPF1 to NPF8, based on the classification of NPF families in other plant species. The chromosomal locations and structures of the NtNPF genes were analyzed. The expression profiles of NtNPF genes under NaCl stress were analyzed to screen the possible NPF genes involving in chloride regulation in tobacco. Most NtNPF6 genes responded to salt stress in the roots and leaves. The expression of NtNPF6.13 was significantly down-regulated after salt stress for 12h. The chloride content was reduced in the roots of ntnpf6.13 mutant. These findings support the participation of NtNPF6.13 in chloride uptake. Several other NtNPF genes that play potential roles in chloride metabolism of tobacco require further study.

Overexpression of OsRLCK241 confers enhanced salt and drought tolerance in transgenic rice (Oryza sativa L.).

Receptor-like cytoplasmic kinases (RLCKs) have been demonstrated to be involved in the regulation of growth, development, and pathogen responses in plants. However, the identity of RLCKs involved in abiotic tolerance remains elusive. In this study, we present data on OsRLCK241, a receptor-like cytoplasmic kinase that is induced by salt and drought stresses. Subcellular localization revealed the presence of an OsRLCK241-GFP fusion protein at the plasma membrane. Under normal conditions, we did not observe any measurable discrepancies between the development and growth of WT and OsRLCK241 transgenic plants. In OsRLCK241 transgenic plants, the overexpression of OsRLCK241 conferred improved tolerance to salt and drought stresses. OsRLCK241 expression improved ROS detoxification by enhancing the activities of ROS scavengers as well as the accumulation of compatible osmolytes to alleviate the osmotic stress evoked by salt and drought stresses. Additionally, several stress-responsive genes showed higher expression levels in OsRLCK241 transgenic plants upon exposure to salt and drought conditions. Collectively, our observations suggest that OsRLCK241 improved salt and drought tolerance in rice is mainly due to improved ROS detoxification, increased accumulation of osmolytes, and altered expression of stress-responsive genes.

Integrating transcriptome and metabolome reveals molecular networks involved in genetic and environmental variation in tobacco.

Tobacco (Nicotiana tabacum) is one of the most widely cultivated commercial non-food crops with significant social and economic impacts. Here we profiled transcriptome and metabolome from 54 tobacco samples (2-3 replicates; n = 151 in total) collected from three varieties (i.e. genetic factor), three locations (i.e. environmental factor), and six developmental stages (i.e. developmental process). We identified 3,405 differentially expressed (DE) genes (DEGs) and 371 DE metabolites, respectively. We used quantitative real-time PCR to validate 20 DEGs, and confirmed 18/20 (90%) DEGs between three locations and 16/20 (80%) with the same trend across developmental stages. We then constructed nine co-expression gene modules and four co-expression metabolite modules , and defined seven de novo regulatory networks, including nicotine- and carotenoid-related regulatory networks. A novel two-way Pearson correlation approach was further proposed to integrate co-expression gene and metabolite modules to identify joint gene-metabolite relations. Finally, we further integrated DE and network results to prioritize genes by its functional importance and identified a top-ranked novel gene, LOC107773232, as a potential regulator involved in the carotenoid metabolism pathway. Thus, the results and systems-biology approaches provide a new avenue to understand the molecular mechanisms underlying complex genetic and environmental perturbations in tobacco.

[Effect of PIH1D1 on the degradation of its binding protein SNF5].

OBJECTIVE: To explore the effects of PIH1D1 on its binding protein SNF5, a core subunit of the SWI/SNF chromatin remodeling complex. METHOD: The degradation pathway of SNF5 was identified with protein synthesis inhibitor cycloheximide (CHX) and a potent proteasome inhibitor MG132, and then the PIH1D1 eukaryotic expression plasmid was transfected to explore its effect on the stability of SNF5. RESULTS: HEK293T cells were effectively treated with CHX (optimal concentration: 400 microg/ml) and MG132 (optimal concentration: 20 mmol/L). The degradation of SNF5 was mediated by the proteasome pathway. PIH1D1 regulated the protein level of SNF5 by attenuating its proteasome degradation. CONCLUSION: PIH1D1 may stabilize SNF5 by attenuating its proteasome degradation pathway.

Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum).

The chloride channel (CLC) protein family, which includes both chloride (Cl-) channels and chloride/proton (Cl-/H+) antiporters, is present in all domains of life, from prokaryotes to eukaryotes. However, there are no reported studies about this gene family in tobacco, an economically important global crop plant. In this study, we identified seventeen CLC genes in the genome of Nicotiana tabacum. A multiple sequence alignment showed that all of the predicted proteins shared a high sequence similarity and had a highly conserved GKxGPxxH motif. A gene structure analysis revealed that the NtCLC genes had highly divergent intron-exon patterns. A phylogenetic and conserved motif analysis revealed that the NtCLC family was divided into two clades, in a manner similar to other plants. We also evaluated the expression patterns of these NtCLC genes in different tissues and in plants treated with salt stress. The NtCLC genes had highly variable expression patterns, for example, the largely stem- and bud-specific expression patterns of NtCLC6 and NtCLC8, respectively. Salt stress treatment (300 mM NaCl) induced the expression of NtCLC2, NtCLC3, and NtCLC12, suggesting that these genes might play a role in tobacco responses to salt stress. Furthermore, the concentration of Cl- in the NtCLC2- and NtCLC13-silenced plants showed an obvious lower and higher level, respectively, than the control plants. Thus, we indicated that NtCLC2 or NtCLC13 might play an important role in chloride transport or metabolism in tobacco. Together, these findings establish an empirical foundation for the further functional characterization of the NtCLC genes in tobacco.

Human PIH1 associates with histone H4 to mediate the glucose-dependent enhancement of pre-rRNA synthesis.

Ribosome biogenesis is critical in the growth of eukaryotic cells, in which the synthesis of precursor ribosomal RNA is the first and rate-limiting step. Here, we show that human PIH1 domain-containing protein 1 (PIH1) interacts directly with histone H4 and recruits the Brg1-SWI/SNF complex via SNF5 to human rRNA genes. This process is likely involved in PIH1-dependent DNase I-hypersensitive chromatin remodeling at the core promoter of the rRNA genes. PIH1 mediates the occupancy of not only the Brg1 complex but also the Pol I complex at the core promoter and enhances transcription initiation of rRNA genes. Additionally, the interaction between PIH1 and H4K16 expels TIP5, a component of the silencing nucleolar remodeling complex (NoRC), from the core region, suggesting that PIH1 is involved in the derepression of NoRC-silenced rRNA genes. These data indicate that PIH1 is a positive regulator of human rRNA genes and is of great importance for the recovery of human cells from nutrient starvation and the transition to glucose-induced exponential growth in vivo.