Identification and characterization of TMV-induced volatile signals in Nicotiana benthamiana: evidence for JA/ET defense pathway priming in congeneric neighbors via airborne (E)-2-octenal.

Plants release a mixture of volatile compounds when subjects to environmental stress, allowing them to transmit information to neighboring plants. Here, we find that Nicotiana benthamiana plants infected with tobacco mosaic virus (TMV) induces defense responses in neighboring congeners. Analytical screening of volatiles from N. benthamiana at 7 days post inoculation (dpi) using an optimized SPME-GC-MS method showed that TMV triggers the release of several volatiles, such as (E)-2-octenal, 6-methyl-5-hepten-2-one, and geranylacetone. Exposure to (E)-2-octenal enhances the resistance of N. benthamiana plants to TMV and triggers the immune system with upregulation of pathogenesis-related genes, such as NbPR1a, NbPR1b, NbPR2, and NbNPR1, which are related to TMV resistance. Furthermore, (E)-2-octenal upregulates jasmonic acid (JA) that levels up to 400-fold in recipient N. benthamiana plants and significantly affects the expression pattern of key genes in the JA/ET signaling pathway, such as NbMYC2, NbERF1, and NbPDF1.2, while the salicylic acid (SA) level is not significantly affected. Our results show for the first time that the volatile (E)-2-octenal primes the JA/ET pathway and then activates immune responses, ultimately leading to enhanced TMV resistance in adjacent N. benthamiana plants. These findings provide new insights into the role of airborne compounds in virus-induced interplant interactions.

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.

CAR modulates plasma membrane nano-organization and immune signaling downstream of RALF1-FERONIA signaling pathway.

In Arabidopsis, the receptor-like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive responses. However, the molecular interplay of these diverse processes is largely unknown. Here, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM. During this process, the RALF1-FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to the PM. This acts as a rapid feedforward loop that stabilizes the PM liquid-ordered phase. FER interacts with and phosphorylates CARs, thereby reducing their lipid-binding ability and breaking the feedback regulation at later time points. The formation of the flg22-induced FLS2-BAK1 immune complex, which depends on the integrity of FER-containing nanodomains, is impaired in fer and pentuple car14569 mutant. Together, we propose that the FER-CAR module controls the formation of PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedback.

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.

FERONIA phosphorylates E3 ubiquitin ligase ATL6 to modulate the stability of 14-3-3 proteins in response to the carbon/nitrogen ratio.

The ratio between carbon (C) and nitrogen (N) utilization must be precisely coordinated to enable plant growth. Although numerous physiological studies have examined carbon/nitrogen (C/N) ratios, the mechanisms of sensing the C/N balance and C/N signaling remain elusive. Here, we report that a mutation of FERONIA (FER), a receptor kinase that plays versatile roles in plant cell growth and stress responses, caused hypersensitivity to a high C/N ratio in Arabidopsis. In contrast, FER-overexpressing plants displayed more resistant phenotypes. FER can interact with and phosphorylate ATL6, an E3 ubiquitin ligase that has been shown to regulate plant C/N responses. FER-mediated ATL6 phosphorylation enhanced the interaction between ATL6 and its previously identified target 14-3-3 proteins, thus decreasing 14-3-3 protein levels, leading to an increased insensitivity to high C/N ratios. Further analyses showed that the rapid alkalinization factor peptide (RALF1), which is a ligand of FER, also influenced the stability of 14-3-3 proteins via a FER-ATL6-mediated pathway. These findings reveal a novel regulatory mechanism that links the RALF1/FER-ATL6 pathway to whole-plant C/N responses and growth.

OsMSR3, a Small Heat Shock Protein, Confers Enhanced Tolerance to Copper Stress in Arabidopsis thaliana.

Copper is a mineral element essential for the normal growth and development of plants; however, excessive levels can severely affect plant growth and development. Oryza sativa L. multiple stress-responsive gene 3 (OsMSR3) is a small, low-molecular-weight heat shock protein (HSP) gene. A previous study has shown that OsMSR3 expression improves the tolerance of Arabidopsis to cadmium stress. However, the role of OsMSR3 in the Cu stress response of plants remains unclear, and, thus, this study aimed to elucidate this phenomenon in Arabidopsis thaliana, to further understand the role of small HSPs (sHSPs) in heavy metal resistance in plants. Under Cu stress, transgenic A. thaliana expressing OsMSR3 showed higher tolerance to Cu, longer roots, higher survival rates, biomass, and relative water content, and accumulated more Cu, abscisic acid (ABA), hydrogen peroxide, chlorophyll, carotenoid, superoxide dismutase, and peroxidase than wild-type plants did. Moreover, OsMSR3 expression in A. thaliana increased the expression of antioxidant-related and ABA-responsive genes. Collectively, our findings suggest that OsMSR3 played an important role in regulating Cu tolerance in plants and improved their tolerance to Cu stress through enhanced activation of antioxidative defense mechanisms and positive regulation of ABA-responsive gene expression.

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.

Deciphering the physiological and molecular mechanisms for copper tolerance in autotetraploid Arabidopsis.

KEY MESSAGE: Autotetraploid Arabidopsis line esd and 4COL exhibit enhanced tolerance to Cu stress by enhancing activation of antioxidative defenses, altering expression of genes related to Cu transport, chelation, and ABA-responsive. Autopolyploidy is ubiquitous among angiosperms and often results in better adaptation to stress conditions. Although copper (Cu) is an essential trace element, excess amounts can inhibit plant growth and even result in death. Here, we report that autotetraploid Arabidopsis thaliana esd and 4COL exhibit higher tolerance to Cu stress. Under such conditions, tetraploid plants had lower Cu contents and significantly more biomass compared with diploid plants. When exposed to excess Cu for 24 h, levels of superoxide anions, hydrogen peroxide, and malondialdehyde were lower in tetraploids than in diploids. Moreover, activities of the antioxidant enzymes superoxide dismutase and peroxidase were stimulated and glutathione content was maintained at a relative higher level in the tetraploids. The expression of genes related to Cu transport and chelation was altered in autotetraploid Arabidopsis under Cu stress, and several key genes involved in the response to abscisic acid (ABA) were significantly up-regulated. Our results indicate that tetraploid Arabidopsis esd and 4COL acquire improved tolerance to Cu stress through enhanced activation of antioxidative defense mechanisms, altered expression of genes related to Cu transport and chelation, and positive regulation of expression for ABA-responsive genes.

NtERF283 positively regulates water deficit tolerance in tobacco (Nicotianatabacum L.) by enhancing antioxidant capacity.

Ethylene responsive factor (ERF) is a plant-specific transcription factor that plays a pivotal regulatory role in various stress responses. Although the genome of tobacco harbors 375 ER F genes, the functional roles of the majority of these genes remain unknown. Expression pattern analysis revealed that NtERF283 was induced by water deficit and salt stresses and mainly expressed in the roots and leaves. Subcellular localization and transcriptional activity assays confirmed that NtERF283 was localized in the nucleus and exhibited transcriptional activity. In comparison to the wild-type (WT), the NtERF283-overexpressing transgenic plants (OE) exhibited enhanced water deficit tolerance, whereas the knockout mutant erf283 displayed contrasting phenotypes. Transcriptional analysis demonstrated that several oxidative stress response genes were significantly altered in OE plants under water deficit conditions. 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining showed that erf283 accumulated a higher level of reactive oxygen species (ROS) compared to the WT under water deficit conditions. Conversely, OE plants displayed the least amount of ROS accumulation. Furthermore, the activities of POD and SOD were higher in OE plants and lower in erf283, suggesting that NtERF283 enhanced the capacity to effectively eliminate ROS, consequently enhancing water deficit tolerance in tobacco. These findings strongly indicate the significance of NtERF283 in promoting tobacco water deficit tolerance through the activation of the antioxidant system.

Temporal transcriptome analysis reveals several key pathways involve in cadmium stress response in Nicotiana tabacum L.

Tobacco has a strong cadmium (Cd) enrichment capacity, meaning that it can absorb large quantities from the environment, but too much Cd will cause damage to the plant. It is not yet clear how the plant can dynamically respond to Cd stress. Here, we performed a temporal transcriptome analysis of tobacco roots under Cd treatment from 0 to 48 h. The number of differentially expressed genes (DEGs) was found to change significantly at 3 h of Cd treatment, which we used to define the early and middle stages of the Cd stress response. The gene ontology (GO) term analysis indicates that genes related to photosynthesis and fatty acid synthesis were enriched during the early phases of the stress response, and in the middle phase biological process related to metal ion transport, DNA damage repair, and metabolism were enriched. It was also found that plants use precursor mRNA (pre-mRNA) processes to first resist Cd stress, and with the increasing of Cd treatment time, the overlapped genes number of DEGs and DAS increased, suggesting the transcriptional levels and post-transcriptional level might influence each other. This study allowed us to better understand how plants dynamically respond to cadmium stress at the transcriptional and post-transcriptional levels and provided a reference for the screening of Cd-tolerant genes in the future.

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.

Comprehensive analysis of the carboxylesterase gene reveals that NtCXE22 regulates axillary bud growth through strigolactone metabolism in tobacco.

Carboxylesterases (CXE) are a class of hydrolytic enzymes with α/ß-folding domains that play a vital role in plant growth, development, stress response, and activation of herbicide-active substances. In this study, 49 Nicotiana tabacum L. CXE genes (NtCXEs) were identified using a sequence homology search. The basic characteristics, phylogenetic evolution, gene structure, subcellular location, promoter cis-elements, and gene expression patterns of the CXE family were systematically analyzed. RNA-seq data and quantitative real-time PCR showed that the expression level of CXEs was associated with various stressors and hormones; gene expression levels were significantly different among the eight tissues examined and at different developmental periods. As a new class of hormones, strigolactones (SLs) are released from the roots of plants and can control the germination of axillary buds.NtCXE7, NtCXE9, NtCXE22, and NtCXE24 were homologous to Arabidopsis SLs hydrolase AtCXE15, and changes in their expression levels were induced by topping and by GR24 (a synthetic analogue of strigolactone). Further examination revealed that NtCXE22-mutant (ntcxe22) plants generated by CRISPR-Cas9 technology had shorter bud outgrowth with lower SLs content. Validation of NtCXE22 was also performed in NtCCD8-OE plants (with fewer axillary buds) and in ntccd8 mutant plants (with more axillary buds). The results suggest that NtCXE22 may act as an efficient SLs hydrolase and affects axillary bud development, thereby providing a feasible method for manipulating endogenous SLs in crops and ornamental plants.

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.

The RALF1-FERONIA complex interacts with and activates TOR signaling in response to low nutrients.

Target of rapamycin (TOR) kinase is an evolutionarily conserved major regulator of nutrient metabolism and organismal growth in eukaryotes. In plants, nutrients are remobilized and reallocated between shoots and roots under low-nutrient conditions, and nitrogen and nitrogen-related nutrients (e.g., amino acids) are key upstream signals leading to TOR activation in shoots under low-nutrient conditions. However, how these forms of nitrogen can be sensed to activate TOR in plants is still poorly understood. Here we report that the Arabidopsis receptor kinase FERONIA (FER) interacts with the TOR pathway to regulate nutrient (nitrogen and amino acid) signaling under low-nutrient conditions and exerts similar metabolic effects in response to nitrogen deficiency. We found that FER and its partner, RPM1-induced protein kinase (RIPK), interact with the TOR/RAPTOR complex to positively modulate TOR signaling activity. During this process, the receptor complex FER/RIPK phosphorylates the TOR complex component RAPTOR1B. The RALF1 peptide, a ligand of the FER/RIPK receptor complex, increases TOR activation in the young leaf by enhancing FER-TOR interactions, leading to promotion of true leaf growth in Arabidopsis under low-nutrient conditions. Furthermore, we showed that specific amino acids (e.g., Gln, Asp, and Gly) promote true leaf growth under nitrogen-deficient conditions via the FER-TOR axis. Collectively, our study reveals a mechanism by which the RALF1-FER pathway activates TOR in the plant adaptive response to low nutrients and suggests that plants prioritize nutritional stress response over RALF1-mediated inhibition of cell growth under low-nutrient conditions.

A novel rice calmodulin-like gene, OsMSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis.

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²âº signals are perceived by different Ca²âº sensors, and calmodulin (CaM) is one of the best-characterized Ca²âº sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei'ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²âº in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.

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.

OsDSSR1, a novel small peptide, enhances drought tolerance in transgenic rice.

Small signaling peptides play important roles in plant development and responses to abiotic and biotic stresses. We have identified a novel small peptide gene in rice, OsDSSR1, which is expressed mainly in the root, stem, node, leaf, and panicle. OsDSSR1 expression is also induced by drought, salinity, ABA, and H2O2 treatment. OsDSSR1 is localized in the nucleus and cytoplasm. Transgenic plants overexpressing OsDSSR1 exhibited enhanced drought stress tolerance and decreased ABA sensitivity as compared to the wild type. Overexpression of OsDSSR1 promoted the accumulation of compatible osmolytes, such as free proline and soluble sugars. OsDSSR1-overexpressing plants displayed enhanced OsSodCc2 and OscAPX expression and superoxide dismutase and ascorbate peroxidase activities under drought stress. RNA-sequencing data revealed that the expression of 72 abiotic stress-responsive genes was significantly altered in homozygous transgenic plants. These stress-responsive candidate genes will aid in expanding our understanding of the mechanisms by which small peptides mediate tolerance in crop species.

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.