NtERF4 promotes the biosynthesis of chlorogenic acid and flavonoids by targeting PAL genes in Nicotiana tabacum.

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.

Bird Object Detection: Dataset Construction, Model Performance Evaluation, and Model Lightweighting.

The application of object detection technology has a positive auxiliary role in advancing the intelligence of bird recognition and enhancing the convenience of bird field surveys. However, challenges arise due to the absence of dedicated bird datasets and evaluation benchmarks. To address this, we have not only constructed the largest known bird object detection dataset, but also compared the performances of eight mainstream detection models on bird object detection tasks and proposed feasible approaches for model lightweighting in bird object detection. Our constructed bird detection dataset of GBDD1433-2023, includes 1433 globally common bird species and 148,000 manually annotated bird images. Based on this dataset, two-stage detection models like Faster R-CNN and Cascade R-CNN demonstrated superior performances, achieving a Mean Average Precision (mAP) of 73.7% compared to one-stage models. In addition, compared to one-stage object detection models, two-stage object detection models have a stronger robustness to variations in foreground image scaling and background interference in bird images. On bird counting tasks, the accuracy ranged between 60.8% to 77.2% for up to five birds in an image, but this decreased sharply beyond that count, suggesting limitations of object detection models in multi-bird counting tasks. Finally, we proposed an adaptive localization distillation method for one-stage lightweight object detection models that are suitable for offline deployment, which improved the performance of the relevant models. Overall, our work furnishes an enriched dataset and practice guidelines for selecting suitable bird detection models.

Genome-wide identification and analysis of the invertase gene family in tobacco (Nicotiana tabacum) reveals NtNINV10 participating the sugar metabolism.

Sucrose (Suc) is directly associated with plant growth and development as well as tolerance to various stresses. Invertase (INV) enzymes played important role in sucrose metabolism by irreversibly catalyzing Suc degradation. However, genome-wide identification and function of individual members of the INV gene family in Nicotiana tabacum have not been conducted. In this report, 36 non-redundant NtINV family members were identified in Nicotiana tabacum including 20 alkaline/neutral INV genes (NtNINV1-20), 4 vacuolar INV genes (NtVINV1-4), and 12 cell wall INV isoforms (NtCWINV1-12). A comprehensive analysis based on the biochemical characteristics, the exon-intron structures, the chromosomal location and the evolutionary analysis revealed the conservation and the divergence of NtINVs. For the evolution of the NtINV gene, fragment duplication and purification selection were major factors. Besides, our analysis revealed that NtINV could be regulated by miRNAs and cis-regulatory elements of transcription factors associated with multiple stress responses. In addition, 3D structure analysis has provided evidence for the differentiation between the NINV and VINV. The expression patterns in diverse tissues and under various stresses were investigated, and qRT-PCR experiments were conducted to confirm the expression patterns. Results revealed that changes in NtNINV10 expression level were induced by leaf development, drought and salinity stresses. Further examination revealed that the NtNINV10-GFP fusion protein was located in the cell membrane. Furthermore, inhibition of the expression of NtNINV10 gene decreased the glucose and fructose in tobacco leaves. Overall, we have identified possible NtINV genes functioned in leaf development and tolerance to environmental stresses in tobacco. These findings provide a better understanding of the NtINV gene family and establish the basis for future research.

The transcription factor NtERF13a enhances abiotic stress tolerance and phenylpropanoid compounds biosynthesis in tobacco.

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factors play multiple roles in modulating the biosynthesis of diverse specialized metabolites in response to various environmental stresses. ERF13 has been shown to participate in plant resistance to biotic stress as well as in repressing the synthesis of fatty acid. However, its full roles in regulating plant metabolism and stress resistance still remains to be further studied. In this study, we identified two NtERF genes from N. tabacum genome that belong to Ⅸa subgroup of ERF family. Over-expression and knock-out of NtERF13a showed that NtERF13a could enhance plant resistance to salt and drought stresses, as well as promoted the biosynthesis of chlorogenic acid (CGA), flavonoids, and lignin in tobacco. Transcriptome analysis between WT and NtERF13a-OE plants revealed 6 differentially expressed genes (DEGs) that encode enzymes catalyzing the key steps of phenylpropanoid pathway. Chromatin immunoprecipitation, Y1H, and Dual-Luc assays further clarified that NtERF13a could directly bind to the fragments containing GCC box or DRE element in the promoters of NtHCT, NtF3'H, and NtANS genes to induce the transcription of these genes. Knock-out of NtHCT, NtF3'H, or NtANS in the NtERF13a-OE background significantly repressed the increase of phenylpropanoid compound contents caused by over-expression of NtERF13a, indicating that the promotion of NtERF13a on the phenylpropanoid compound contents depends on the activity of NtHCT, NtF3'H, and NtANS. Our study demonstrated new roles of NtERF13a in promoting plant resistance to abiotic stresses, and provided a promising target for modulating the biosynthesis of phenylpropanoid compounds in tobacco.

Complete genome sequence of a new virus from Allium sativum L in China.

The complete genome of a new virus belonging to the family Betaflexiviridae was identified in garlic and sequenced by next-generation sequencing and reverse transcription PCR. The complete RNA genome (GenBank accession number OP021693) is 8191 nucleotides in length, excluding the 3' poly(A) tail, and contains five open reading frames (ORFs). These open reading frames encode the viral replicase, triple gene block, and coat protein, and the genome organization is typical of members of the subfamily Quinvirinae. The virus has been tentatively named "garlic yellow curl virus" (GYCV). Phylogenetic analysis suggested that it represents an independent evolutionary lineage in the subfamily, clustering with the currently unclassified garlic yellow mosaic associated virus (GYMaV) and peony betaflexivirus 1 (PeV1). Differences between the phylogenies inferred for the replicase and coat protein indicate that the new virus does not belong to any established genus of the family Betaflexiviridae. This is the first report of GYCV in China.

Transcription factor NtWRKY33a modulates the biosynthesis of polyphenols by targeting NtMYB4 and NtHCT genes in tobacco.

There are abundant polyphenols in tobacco leaves mainly including chlorogenic acid (CGA), rutin, and scopoletin, which not only influence plant growth, development, and environmental adaptation, but also have a great impact on the industrial utilization of tobacco leaves. Few transcription factors regulating the biosynthesis of polyphenols have been identified in tobacco so far. In this study, two NtWRKY33 genes were identified from N. tabacum genome. NtWRKY33a showed higher transcriptional activity than NtWRKY33b, and encoded a nuclear localized protein. Overexpression and knock-out of NtWRKY33a gene revealed that NtWRKY33a inhibited the accumulation of rutin, scopoletin, and total polyphenols, but meanwhile promoted the biosynthesis of CGA. Chromatin immunoprecipitation and Dual-Luc assays indicated that NtWRKY33a could directly bind to the promoters of NtMYB4 and NtHCT, and thus induced the transcription of these two genes. The contents of polyphenols in ntwrky33a, ntmy4, and ntwrky33a/ntmyb4 mutants further confirmed that the repression of NtWRKY33a on the biosynthesis of rutin, scopoletin, and total polyphenols depends on the activity of NtMYB4. Moreover, the promotion of NtHCT by NtWRKY33a modulates the distribution of metabolism flux into the synthesis of CGA. Ectopic expression of NtWRKY33a inhibit the expression of NtSAUR14, NtSAUR59, NtSAUR66, NtIAA4, NtIAA17, and NtIAA19 genes, indicating that NtWRKY33a might be involved in the regulation of plant auxin response. Our study revealed new functions of NtWRKY33a in regulating the synthesis of polyphenols, and provided a promising target for manipulating polyphenols contents in tobacco.

Genome-wide identification and expression profiling of sugar transporter genes in tobacco.

Sugars are both nutrients and important signal molecules in higher plants. Sugar transporters (STs) are involved in sugar loading and unloading and facilitate sugar transport across membranes. Tobacco (Nicotiana tabacum) is a model plant and one of the most significant plants economically. In our research, 92 N. tabacum ST (NtST) genes were identified and classified into eight distinct subfamilies in the tobacco genome based on phylogenetic analysis. Exon-intron analysis revealed that each subfamily manifested closely associated gene architectural features based on a comparable number or length of exons. Tandem repetition and purifying selection were the main factors of NtST gene evolution. A search for cis-regulatory elements in the promoter sequences of the NtST gene families suggested that they are probably regulated by light, plant hormones, and abiotic stress factors. We performed a comprehensive expression study in different tissues, viarious abiotic and phytohormone stresses. The results revealed different expression patterns and the functional diversification of NtST genes. The resulting data showed that NtSFP1 was highly expressed all measured five tobacco tissues, and also regulated by the MeJA, and temperature stress. In addition, the virus-induced NibenSFP1 silencing in tobacco and detected dramatically enhanced glucose content, indicating the NtSFP1 might regulate the glucose content and involved in MeJA signaling way to response the temperature stress. In general, our findings provide useful information on understanding the roles of STs in phytohormone signaling way and abiotic stresses in N. tabacum.

Identification of QTLs Associated With Agronomic Traits in Tobacco via a Biparental Population and an Eight-Way MAGIC Population.

Agronomic traits such as plant height (PH), leaf number (LN), leaf length (LL), and leaf width (LW), which are closely related to yield and quality, are important in tobacco (Nicotiana tabacum L.). To identify quantitative trait loci (QTLs) associated with agronomic traits in tobacco, 209 recombinant inbred lines (RILs) and 537 multiparent advanced generation intercross (MAGIC) lines were developed. The biparental RIL and MAGIC lines were genotyped using a 430 K single-nucleotide polymorphism (SNP) chip assay, and their agronomic traits were repeatedly evaluated under different conditions. A total of 43 QTLs associated with agronomic traits were identified through a combination of linkage mapping (LM) and association mapping (AM) methods. Among these 43 QTLs, three major QTLs, namely qPH13-3, qPH17-1, and qLW20-1, were repeatedly identified by the use of various genetically diverse populations across different environments. The candidate genes for these major QTLs were subsequently predicted. Validation and utilization of the major QTL qLW20-1 for the improvement of LW in tobacco were investigated. These results could be applied to molecular marker-assisted selection (MAS) for breeding important agronomic traits in tobacco.

Identifying loci controlling total starch content of leaf in Nicotiana tabacum through genome-wide association study.

Starch is an important primary metabolite in plants, which can provide bioenergy for fuel ethanol production. There are many studies focusing on starch metabolism in Arabidopsis, maize, and rice, but few reports have been made on the starch content of tobacco leaves. Hence, to identify the marker-trait associations and isolate the candidate genes related to starch content of tobacco leaf, the genome-wide association study (GWAS) was performed using a multiparent advanced generation intercross (MAGIC) population consisting of 276 accessions genotyped by a 430 K SNP array. In this study, we detected the leaf starch content of tobacco plants cultivated in two places (Zhucheng and Chenzhou), which showed a wide variation of starch content in the population. A total of 28 and 45 significant single-nucleotide polymorphism (SNP) loci associated with leaf starch content were identified by single-locus and multi-locus GWAS models, respectively, and the phenotypic variance explained by these loci varied from 1.80 to - 14.73%. Furthermore, among these quantitative trait loci (QTLs), one SNP, AX-106011713 located on chromosome 19, was detected repeatedly in multiple models and two environments, which was selected for linkage disequilibrium (LD) analysis to obtain the target candidate region. Through gene annotation, haplotype, and gene expression analysis, two candidate genes encoding E3 ubiquitin-protein ligase (Ntab0823160) and fructose-bisphosphate aldolase (Ntab0375050) were obtained. Results showed that the variety carrying the beneficial alleles of the two candidate genes had higher gene expression level and leaf starch content, suggesting the potential role of candidate genes in enhancing the level of tobacco leaf starch content. Furthermore, silencing of Ntab0823160 in tobacco leaves reduced the content of total starch to 39.41-69.75% of that in the wide type plants. Taken together, our results provide useful resources for further investigation of the starch metabolic pathway and are also beneficial for the creation of eco-friendly cultivars with increased accumulation of leaf starch content.

Molecular cloning and functional characterization of NtWRKY41a in the biosynthesis of phenylpropanoids in Nicotiana tabacum.

Phenylpropanoids are important secondary metabolites that have multifaceted effects on plant growth, development, and environmental adaptation. WRKY41 has been shown to repress anthocyanins synthesis in Arabidopsis, but its full roles in regulating plant phenylpropanoids metabolism still remains to be further studied. Here, we cloned two NtWRKY41 genes from N. tabacum genome, and NtWRKY41a showed higher expression levels than NtWRKY41b genes in all the tobacco tissues examined. Overexpression and knock-out of NtWRKY41a gene revealed that NtWRKY41a promoted the biosynthesis of Chlorogenic acid (CGA) and lignin, but repressed the accumulation of scopoletin and flavonoids in tobacco. Transcriptome analysis found 7 phenylpropanoids related differentially expressed genes (DEGs) between WT and NtWRKY41a-OE plants, among which the transcription of NtCCoAOMT and NtHST was significantly induced by posttranslational activation of NtWRKY41a, while those of NtF6'H1 and NtGT3 was significantly repressed by NtWRKY41a. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtWRKY41a could bind to the promoter regions of these four genes to regulate their transcription. Moreover, ectopic expression of NtWRKY41a also promoted the transcription of several NtLOX and NtHPL genes, which encode key enzymes involved in the oxylipin pathway. Our findings revealed new functions of NtWRKY41a in modulating the distribution of metabolism flux in phenylpropanoids pathway, and provided a promising target for manipulating phenylpropanoids contents in tobacco.

Hydrogen-rich water alleviates programmed cell death induced by GA in wheat aleurone layers by modulation of reactive oxygen species metabolism.

Hydrogen gas (H2) has been recently regarded as a novel gaseous signaling molecule that performs multiple functional roles in plant. Here, we demonstrate that hydrogen rich water (HRW)-an experimentally tractable reagent to assess the effects of the H2 significantly delays wheat aleurone layer programmed cell death (PCD) induced by gibberellic acid (GA). Endogenous H2 production exhibited lower level in aleurone layers under GA treatment, whereas the H2 production was apparently increased under abscisic acid (ABA) treatment. HRW not only increased H2 production but also delayed GA-induced PCD. We further observed that application of HRW substantially prevented the increases of hydrogen peroxide (H2O2) and superoxide anion radical (O2.-) triggered by GA. HRW also directly react with hydroxyl radical (·OH) to delay GA-induced PCD. Quantitative real-time PCR (qRT-PCR) and biochemical assays showed that HRW induced the transcripts and enzymatic activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) that metabolize reactive oxygen species (ROS); these increases coincided with the observed changes in O2.-, H2O2 and ·OH accumulation upon GA treatment. Our study therefore suggests that HRW-triggered alleviation of wheat aleurone layer PCD induced by GA results from a combination of H2-mediated decreases of ROS levels, including O2.-, H2O2, and ·OH.

Molecular cloning and functional characterization of NtWRKY11b in promoting the biosynthesis of flavonols in Nicotiana tabacum.

The biosynthesis of flavonols and anthocyanins is precisely regulated by different transcription factors in plants. WRKY11 promotes the biosynthesis of flavonoids in apple, but the molecular mechanism of WRKY11 regulating flavonols biosynthesis, and whether WRKY11 plays the same roles in other plants species remains to be further studied. Here, we cloned four NtWRKY11 genes from tobacco, which all contained the conserved WRKYGQK heptapeptide and a zinc-finger motif. The NtWRKY11b showed higher expression levels than the other NtWRKY11 genes in all the tobacco tissues examined, especially in tobacco leaves. Silencing of NtWRKY11b in tobacco leaves reduced the content of flavonols to 45.2 %-69.8 % of that in the WT plants, but overexpression of NtWRKY11b increased the flavonols content by 37.8 %-80.7 %. Transcriptome analysis revealed 8 flavonoids related differentially expressed genes (DEGs) between NtWRKY11b-OE and WT plants, among which the transcription of NtMYB12, NtFLS, NtGT5, and NtUFGT was significantly induced by posttranslational activation of NtWRKY11b with the presence of protein synthesis inhibitor, indicating a putative direct promotion of NtWRKY11b on the transcription of these flavonoids related genes. Chromatin immunoprecipitation assays further demonstrated that NtWRKY11b could bind to the promoter regions of NtMYB12, NtFLS, NtGT5, and NtUFGT to activate the transcription of these genes. Moreover, ectopic expression of NtWRKY11b also promoted the expression levels of NtCML38, NtCTL1, NtWRKY44, and NtCML37 genes, which have been shown to enhance plant resistance to various stresses. Our findings revealed the molecular mechanism of NtWRKY11b regulating flavonols biosynthesis, and provided a promising target for increasing flavonols content in tobacco.

Proteomic and Phosphoproteomic Analysis in Tobacco Mosaic Virus-Infected Tobacco (Nicotiana tabacum).

Tobacco mosaic virus (TMV) is a common source of biological stress that significantly affects plant growth and development. It is also useful as a model in studies designed to clarify the mechanisms involved in plant viral disease. Plant responses to abiotic stress were recently reported to be regulated by complex mechanisms at the post-translational modification (PTM) level. Protein phosphorylation is one of the most widespread and major PTMs in organisms. Using immobilized metal ion affinity chromatography (IMAC) enrichment, high-pH C18 chromatography fraction, and high-accuracy mass spectrometry (MS), a set of proteins and phosphopeptides in both TMV-infected tobacco and control tobacco were identified. A total of 4905 proteins and 3998 phosphopeptides with 3063 phosphorylation sites were identified. These 3998 phosphopeptides were assigned to 1311 phosphoproteins, as some proteins carried multiple phosphorylation sites. Among them, 530 proteins and 337 phosphopeptides corresponding to 277 phosphoproteins differed between the two groups. There were 43 upregulated phosphoproteins, including phosphoglycerate kinase, pyruvate phosphate dikinase, protein phosphatase 2C, and serine/threonine protein kinase. To the best of our knowledge, this is the first phosphoproteomic analysis of leaves from a tobacco cultivar, K326. The results of this study advance our understanding of tobacco development and TMV action at the protein phosphorylation level.

Genome-wide identification and analysis of the growth-regulating factor family in tobacco (Nicotiana tabacum).

Growth-regulating factors (GRFs) are plant-specific transcription factors that have important functions in regulating plant growth and development. GRF gene families have been described in several plant species, but a comprehensive analysis of the GRF gene family in tobacco has not yet been reported. In this study, we identified 25 NtabGRF genes in N. tabacum. The gene structures, motifs, and cis-acting regulatory elements of the NtabGRF genes were analyzed. Phylogenetic analysis divided the genes into six clusters. Additionally, highly conserved regions of microsynteny were identified in all of the sequenced tobacco species. Expression analysis showed that NtabGRF genes were highly expressed in actively growing tissues and responded to various hormone treatments. Our results provide foundational information about the GRF gene family in tobacco species, and open the door for future research on the functions of these genes.

Analysis of Nicotiana tabacum PIN genes identifies NtPIN4 as a key regulator of axillary bud growth.

The plant-specific PIN-FORMED (PIN) auxin efflux proteins have been well characterized in many plant species, where they are crucial in the regulation of auxin transport in various aspects of plant development. However, little is known about the exact roles of the PIN genes during plant development in Nicotiana species. This study investigated the PIN genes in tobacco (Nicotiana tabacum) and in two ancestral species (Nicotiana sylvestris and Nicotiana tomentosiformis). Genome-wide analysis of the N. tabacum genome identified 20 genes of the PIN family. An in-depth phylogenetic analysis of the PIN genes of N. tabacum, N. sylvestris and N. tomentosiformis was conducted. NtPIN4 expression was strongly induced by the application of exogenous indole-3-acetic acid (IAA), but was downregulated by the application of ABA, a strigolactone analogue, and cytokinin, as well as by decapitation treatments, suggesting that the NtPIN4 expression level is likely positively regulated by auxin. Expression analysis indicated that NtPIN4 was highly expressed in tobacco stems and shoots, which was further validated through analysis of the activity of the NtPIN4 promoter. We used CRISPR-Cas9 technology to generate mutants for NtPIN4 and observed that both T0 and T1 plants had a significantly increased axillary bud growth phenotype, as compared with the wild-type plants. Therefore, NtPIN4 offers an opportunity for studying auxin-dependent branching processes.

A Quantitative Real-Time PCR-Based Strategy for Molecular Evaluation of Nicotine Conversion in Burley Tobacco.

Nornicotine production in Nicotiana tabacum is undesirable because it is the precursor of the carcinogen N'-nitrosonornicotine. In some individual burley tobacco plants, a large proportion of the nicotine can be converted to nornicotine, and this process of nicotine conversion is mediated primarily by enzymatic N-demethylation of nicotine which is controlled mainly by CYP82E4. Here we report a novel strategy based on quantitative real-time polymerase chain reaction (qPCR) method, which analyzed the ratio of nicotine conversion through examining the transcript level of CYP82E4 in burley leaves and do not need ethylene induction before detected. The assay was linear in a range from 1 × 10¹ to 1 × 105 copies/mL of serially diluted standards, and also showed high specificity and reproducibility (93%-99%). To assess its applicability, 55 plants of burley cultivar Ky8959 at leaf maturing stage were analyzed, and the results were in accordance with those from gas chromatograph-mass spectrometry (GC-MS) method. Moreover, a linear correlation existed between conversion level and CYP82E4 transcript abundance. Taken together, the quantitative real-time PCR assay is standardized, rapid and reproducible for estimation of nicotine conversion level in vivo, which is expected to shed new light on monitoring of burley tobacco converter.

Analysis of the sucrose synthase gene family in tobacco: structure, phylogeny, and expression patterns.

MAIN CONCLUSION: Provide an evolutionary and an empirical molecular genetic foundation of the Sus gene family in tobacco and will be beneficial for further investigations of Sus gene functions Sucrose synthase (Sus) has been well characterized as the key enzyme participating in sucrose metabolism, and the gene family encoding different Sus isozymes has been cloned and characterized in several plant species. However, scant information about this gene family is available to date in tobacco. Here, we identified 14, 6, and 7 Sus genes in the genomes of Nicotiana tabacum, N. sylvestris and N. tomentosiformis, respectively. These tobacco Sus family members shared high levels of similarity in their nucleotide and amino acid sequences. Phylogenetic analysis revealed distinct evolutionary paths for the tobacco Sus genes. Sus1-4, Sus5, and Sus6-7 originated from three Sus precursors, respectively, which were generated by duplication before the split of monocots and eudicots. There were two additional duplications, before and after the differentiation of the Solanaceae, which separately gave rise to Sus3/4 and Sus1/2. Gene exon/intron structure analysis showed that the tobacco Sus genes contain varying numbers of conserved introns, resulting from intron loss under different selection pressures during the course of evolution. The expression patterns of the NtSus genes differed from each other in various tobacco tissues. Transcripts of Ntab0259170 and Ntab0259180 were detected in leaves at all tested developmental stages, suggesting that these two genes play a predominant role in sucrose metabolism during leaf development. Expression of Ntab0288750 and Ntab0234340 were conspicuously induced by low temperature and virus treatment, indicating that these two isozymes are important in meeting the increased glycolytic demand that occurs during abiotic stress. Our results provide an evolutionary and an empirical molecular genetic foundation of the Sus gene family in tobacco, and will be beneficial for further investigations of Sus gene functions in the processes of tobacco leaf development and tobacco resistance to environmental stresses.

Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern 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 ß-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 ß-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.