Considerations in engineering viral vectors for genome editing in plants.

Plant viruses have been engineered to express proteins and induce gene silencing for decades. Recently, plant viruses have also been used to deliver components into plant cells for genome editing, a technique called virus-induced genome editing (VIGE). Although more than a dozen plant viruses have been engineered into VIGE vectors and VIGE has been successfully accomplished in some plant species, application of VIGE to crops that are difficult to tissue culture and/or have low regeneration efficiency is still tough. This paper discusses factors to consider for an ideal VIGE vector, including insertion capacity for foreign DNA, vertical transmission ability, expression level of the target gene, stability of foreign DNA insertion, and biosafety. We also proposed a step-by-step schedule for excavating the suitable viral vector for VIGE.

Two novel transporters NtNRAMP6a and NtNRAMP6b are involved in cadmium transport in tobacco (Nicotiana tabacum L.).

Plant natural resistance-associated macrophage protein (NRAMP) plays important roles in metal transport and tolerance. Tobacco is a typical cadmium (Cd) accumulator, while research on NRAMP in tobacco has been limited. In the current study, two novel NRAMP genes (NtNRAMP6a and NtNRAMP6b) were identified from the allotetraploid plant Nicotiana tabacum L. Real time‒PCR and GUS (ß-glucuronidase) staining results showed that the two genes were expressed in roots, stems, leaves and flowers and induced by Cd stress. Subcellular localization revealed that they were located in the plasma membrane. Heterologously expressed NtNRAMP6a and NtNRAMP6b significantly increased the Cd sensitivity of the Δycf1 mutant, indicating that NtNRAMP6a and NtNRAMP6b had Cd transport functions in yeast. The difference in the manganese (Mn) transport activity of the two genes was demonstrated by point mutation, which was caused by the difference in the 18th amino acid. NRAMP6-N18K is a new key active site for manganese transport. After 50 µM Cd treatment for 7 days, the contents of Cd and Mn of the ntnramp6a/6b mutants was significantly lower than those of wild type in shoots, while the contents in roots were higher. Additionally, the mutant lines showed higher chorphyll contentration and lighter leaf damage. Knockout of NtNRAMP6a and NtNRAMP6b reduced Cd and Mn accumulation in tobacco shoots by influence root-to-shoot translocation. This provides new idea for cultivating tobacco varieties with low cadmium accumulation and high cadmium tolerance.

The OPEN STOMATA1-SPIRAL1 module regulates microtubule stability during abscisic acid-induced stomatal closure in Arabidopsis.

Drought stress triggers abscisic acid (ABA) signaling in guard cells and induces stomatal closure to prevent water loss in land plants. Stomatal movement is accompanied by reorganization of the cytoskeleton. Cortical microtubules disassemble in response to ABA, which is required for stomatal closure. However, how ABA signaling regulates microtubule disassembly is unclear, and the microtubule-associated proteins (MAPs) involved in this process remain to be identified. In this study, we show that OPEN STOMATA 1 (OST1), a central component in ABA signaling, mediates microtubule disassembly during ABA-induced stomatal closure in Arabidopsis thaliana. We identified the MAP SPIRAL1 (SPR1) as the substrate of OST1. OST1 interacts with and phosphorylates SPR1 at Ser6, which promotes the disassociation of SPR1 from microtubules and facilitates microtubule disassembly. Compared with the wild type, the spr1 mutant exhibited significantly greater water loss and reduced ABA responses, including stomatal closure and microtubule disassembly in guard cells. These phenotypes were restored by introducing the phosphorylated active form of SPR1. Our findings demonstrate that SPR1 positively regulates microtubule disassembly during ABA-induced stomatal closure, which depends on OST1-mediated phosphorylation. These findings reveal a specific connection between a core component of ABA signaling and MAPs.

Cloning, characterization and functional analysis of NtMYB306a gene reveals its role in wax alkane biosynthesis of tobacco trichomes and stress tolerance.

Trichomes are specialized hair-like organs found on epidermal cells of many terrestrial plants, which protect plant from excessive transpiration and numerous abiotic and biotic stresses. However, the genetic basis and underlying mechanisms are largely unknown in Nicotiana tabacum (common tobacco), an established model system for genetic engineering and plant breeding. In present study, we identified, cloned and characterized an unknown function transcription factor NtMYB306a from tobacco cultivar K326 trichomes. Results obtained from sequence phylogenetic tree analysis showed that NtMYB306a-encoded protein belonged to S1 subgroup of the plants' R2R3-MYB transcription factors (TFs). Observation of the green fluorescent signals from NtMYB306a-GFP fusion protein construct exhibited that NtMYB306a was localized in nucleus. In yeast transactivation assays, the transformed yeast containing pGBKT7-NtMYB306a construct was able to grow on SD/-Trp-Ade+X-α-gal selection media, signifying that NtMYB306a exhibits transcriptional activation activity. Results from qRT-PCR, in-situ hybridization and GUS staining of transgenic tobacco plants revealed that NtMYB306a is primarily expressed in tobacco trichomes, especially tall glandular trichomes (TGTs) and short glandular trichomes (SGTs). RNA sequencing (RNA-seq) and qRT-PCR analysis of the NtMYB306a-overexpressing transgenic tobacco line revealed that NtMYB306a activated the expression of a set of key target genes which were associated with wax alkane biosynthesis. Gas Chromatography-Mass Spectrometry (GC-MS) exhibited that the total alkane contents and the contents of n-C28, n-C29, n-C31, and ai-C31 alkanes in leaf exudates of NtMYB306a-OE lines (OE-3, OE-13, and OE-20) were significantly greater when compared to WT. Besides, the promoter region of NtMYB306a contained numerous stress-responsive cis-acting elements, and their differential expression towards salicylic acid and cold stress treatments reflected their roles in signal transduction and cold-stress tolerance. Together, these results suggest that NtMYB306a is necessarily a positive regulator of alkane metabolism in tobacco trichomes that does not affect the number and morphology of tobacco trichomes, and that it can be used as a candidate gene for improving stress resistance and the quality of tobacco.

Nitrogen metabolic rate and differential ammonia volatilization regulate resistance against opportunistic fungus Alternaria alternata in tobacco.

Nutritional correlations between plants and pathogens can crucially affect disease severity. As an essential macronutrient, the availability of nitrogen (N) and the types of N content play a fundamental part not only in energy metabolism and protein synthesis but also in pathogenesis. However, a direct connection has not yet been established between differences in the level of resistance and N metabolism. Pertinently, former studies hold ammonia (NH3) accountable for the development of diseases in tobacco (Nicotiana tabacum L.) and in some post-harvest fruits. With a purpose of pinpointing the function of NH3 volatilization on Alternaria alternata (Fries) Keissl pathogenesis and its correlation with both N metabolism and resistance differences to Alternaria alternata infection in tobacco, leaf tissue of two tobacco cultivars with susceptibility (Changbohuang; CBH), or resistance (Jingyehuang; JYH) were analyzed apropos of ammonia compensation point, apoplastic NH4 + concentration, pH value as well as activities of key enzymes and N status. At the leaf age of 40 to 60 d, the susceptible cultivar had a significantly higher foliar apoplastic ammonium (NH4 +) concentration, pH value and NH3 volatilization potential compared to the resistant one accompanied by a significant reduction in glutamine synthetase (GS), which in particular was a primary factor causing the NH3 volatilization. The NH4 + concentration in CBH was 1.44 times higher than that in JYH, and CBH had NH3 compensation points that were 7.09, 6.15 and 4.35-fold higher than those of JYH at 40, 50 and 60 d, respectively. Moreover, the glutamate dehydrogenase (GDH) activity had an upward tendency related to an increased NH4 + accumulation in both leaf tissues and apoplast but not with the NH3 compensation point. Collectively, our results strongly suggest that the accumulation of NH3 volatilization, rather than NH4 + and total N, was the primary factor inducing the Alternaria alternata infection in tobacco. Meanwhile, the susceptible cultivar was characterized by a higher N re-transfer ability of NH3 volatilization, in contrast to the disease-resistant cultivar, and had a stronger capability of N assimilation and reutilization. This study provides a deeper understanding of the pathogenicity mechanism induced by Alternaria alternata, which is useful for breeding Alternaria alternata-resistant varieties of tobacco, at the same time, our research is also conducive to control tobacco brown spot caused by Alternaria alternata in the field.

P25 and P37 proteins encoded by firespike leafroll-associated virus are viral suppressors of RNA silencing.

Firespike leafroll-associated virus (FLRaV) is a major pathogen associated with firespike (Odontonema tubaeforme) leafroll disease. Phylogenetic analysis showed that FLRaV possesses typical traits of subgroup II members of ampeloviruses, but encodes two additional proteins, P25 and P37. Here, we determined the microfilament localization of P25 protein. Posttranscriptional gene silencing (PTGS) assay showed that both FLRaV P25 and P37 were able to suppress the local and systemic PTGS and FLRaV P25 was capable of suppressing the green fluorescent protein (GFP) gene silencing triggered by both sense RNA-induced PTGS (S-PTGS) and inverted repeat RNA-induced PTGS (IR-PTGS). In contrast, FLRaV P37 was only able to inhibit the GFP silencing triggered by the S-PTGS but not the IR-PTGS. In the transcriptional gene silencing (TGS) assay, only FLRaV P25 was found to be able to reverse established TGS-mediated silencing of GFP in 16-TGS plants. We also found that FLRaV P25 could aggravate the disease symptom and viral titer of potato virus X in N. benthamiana. These results suggest that FLRaV P25 and P37 may have crucial roles in overcoming host RNA silencing, which provides key insights into our understanding of the molecular mechanisms underlying FLRaV infection.

Tandem application of endophytic fungus Serendipita indica and phosphorus synergistically recuperate arsenic induced stress in rice.

In the context of eco-sustainable acquisition of food security, arsenic (As) acts as a deterring factor, which easily infiltrates our food chain via plant uptake. Therefore, devising climate-smart strategies becomes exigent for minimizing the imposed risks. Pertinently, Serendipita indica (S. indica) is well reputed for its post-symbiotic stress alleviatory and phyto-promotive potential. Management of phosphorus (P) is acclaimed for mitigating arsenic toxicity in plants by inhibiting the uptake of As molecules due to the competitive cationic exchange in the rhizosphere. The current study was designed to investigate the tandem effects of S. indica and P in combating As toxicity employing two rice genotypes, i.e., Guodao-6 (GD-6; As-sensitive genotype) and Zhongzhe You-1 (ZZY-1; As-tolerant genotype). After successful fungal colonization, alone and combined arsenic (10 µ M L-1) and phosphorus (50 µ M L-1) treatments were applied. Results displayed that the recuperating effects of combined S. indica and P treatment were indeed much profound than their alone treatments; however, most of the beneficial influences were harnessed by ZZY-1 in comparison with GD-6. Distinct genotypic differences were observed for antioxidant enzyme activities, which were induced slightly higher in S. indica-colonized ZZY-1 plants, with or without additional P, as compared to GD-6. Ultrastructure images of root and shoot exhibited ravages of As in the form of chloroplasts-, nuclei-and cell wall-damage with enlarged vacuole area, mellowed mostly by the combined treatment of S. indica and P in both genotypes. Gene expression of PHTs family transporters was regulated at different levels in almost all treatments across genotypes. Conclusively, the results of this study validated the promising role of S. indica and additional P in mitigating As stress, albeit corroborated that the extent of relevant benefit exploitation is highly genotype-dependent. Verily, unlocking the potential of nature-friendly solutions will mend the anthropogenic damage already been done to our environment.

Integrated multi-omics analysis uncovers roles of mdm-miR164b-MdORE1 in strigolactone-mediated inhibition of adventitious root formation in apple.

Apple is one of the most important fruit crops in temperate regions and largely relies on cutting propagation. Adventitious root formation is crucial for the success of cutting propagation. Strigolactones have been reported to function in rooting of woody plants. In this study, we determined that strigolactones have inhibitory effects on adventitious root formation in apple. Transcriptome analysis identified 12 051 differentially expressed genes over the course of adventitious root initiation, with functions related to organogenesis, cell wall biogenesis or plant development. Further analysis indicated that strigolactones might inhibit adventitious root formation through repressing two core hub genes, MdLAC3 and MdORE1. Combining small RNA and degradome sequencing, as well as dual-luciferase sensor assays, we identified and validated three negatively correlated miRNA-mRNA pairs, including mdm-miR397-MdLAC3 and mdm-miR164a/b-MdORE1. Overexpression of mdm-miR164b and silencing MdORE1 exhibited enhanced adventitious root formation in tobacco and apple, respectively. Finally, we verified the role of mdm-miR164b-MdORE1 in strigolactone-mediated repression of rooting ability. Overall, the identified comprehensive regulatory network in apple not only provides insight into strigolactone-mediated adventitious root formation in other woody plants, but also points to a potential strategy for genetic improvement of rooting capacity in woody plants.

Triplex Immunostrip Assay for Rapid Diagnosis of Tobacco Mosaic Virus, Tobacco Vein Banding Mosaic Virus, and Potato Virus Y.

Mixed virus infection has increasingly become a problem in the production of Solanaceae crops in recent years; therefore, a fast and accurate detection method is needed. In this study, a novel triplex immunostrip assay was developed for the simultaneous detection of tobacco mosaic virus (TMV), tobacco vein banding mosaic virus (TVBMV), and potato virus Y (PVY). The limits of detection of this novel immunostrip reached 200 ppb (ng/ml), 1 ppm (µg/ml), and 2 ppm for TMV, PVY, and TVBMV particles, respectively. Importantly, no cross-reactivity was observed among TMV, TVBMV, and PVY or to a nontarget virus. When the assay was applied to suspected virus-infected tobacco, tomato, and potato samples collected from fields in Southwest China, samples of single or mixed virus infection were successfully identified. In conclusion, the triplex immunostrip assay provides a fast and easy to use on-site detection method for field epidemiological studies of TMV, TVBMV, and PVY, and for managing diseases that are caused by them.

Geminiviruses boost active DNA demethylation for counter-defense.

DNA methylation regulates gene expression under abiotic and biotic stresses. Recently, Gui et al. discovered that geminiviruses subverted DNA methylation-mediated defense through boosting the active DNA demethylation mediated by host DNA glycosylases to promote viral virulence. Their findings reveal a distinctive counter-defense strategy exploited by invading pathogens to achieve successful infection.

A novel signal amplification biosensor for detection of Cd2+ based on asymmetric PCR.

In this work, a novel signal amplification biosensor was utilized to detect Cd2+ based on asymmetric PCR. In the presence of Cd2+, it can bind with Cd2+-aptamer C1 which caused the complementary strand C2 to be released from double-stranded DNA C1-C2. Because the single-stranded C1 cannot be hydrolyzed by Exo III, it can be used as a template to take part in asymmetric PCR reaction. In the absence of Cd2+, the C1-C2 was digested by Exo III and no PCR template was left. During the experiment, an interesting phenomenon was found that the asymmetric PCR can obtain higher level of fluorescent signal than that of symmetric PCR. To the best of our knowledge, this is the first report of using asymmetric PCR to detect Cd2+. Through the asymmetric PCR amplification strategy, this biosensor had a low detection limit (19.93 nM) and a wide linear range (0-500 nM). Meanwhile, this biosensor showed a satisfactory selectivity and recovery rate.

Turnip mosaic virus manipulates DRM2 expression to regulate host CHH and CHG methylation for robust infection.

DNA methylation is an important epigenetic marker for the suppression of transposable elements (TEs) and the regulation of plant immunity. However, little is known how RNA viruses counter defense such antiviral machinery. In this study, the change of DNA methylation in turnip mosaic virus (TuMV)-infected cells was analyzed by whole genome bisulfite sequencing. Results showed that the total number of methylated sites of CHH and CHG increased in TuMV-infected cells, the majority of differentially methylated regions (DMRs) in the CHH and CHG contexts were associated with hypermethylation. Gene expression analysis showed that the expression of two methylases (DRM2 and CMT3) and three demethylases (ROS3, DML2, DML3) was significantly increased and decreased in TuMV-infected cells, respectively. Pathogenicity tests showed that the enhanced resistance to TuMV of the loss-of-function mutant of DRM2 is associated with unregulated expression of several defense-related genes. Finally, we found TuMV-encoded NIb, the viral RNA-dependent RNA polymerase, was able to induce the expression of DRM2. In conclusion, this study discovered that TuMV can modulate host DNA methylation by regulating the expression of DRM2 to promote virus infection.

Genome-wide characterization and expression analysis of TOPP-type protein phosphatases in soybean (Glycine max L.) reveal the role of GmTOPP13 in drought tolerance.

BACKGROUND: In response to various abiotic stressors such as drought, many plants engage different protein phosphatases linked to several physiological and developmental processes. However, comprehensive analysis of this gene family is lacking for soybean. OBJECTIVE: This study was performed to identify the TOPP-type protein phosphatase family in soybean and investigate the gene's role under drought stress. METHODS: Soybean genome sequences and transcriptome data were downloaded from the Phytozome v.12, and the microarray data were downloaded from NCBI GEO datasets GSE49537. Expression profiles of GmTOPP13 were obtained based on qRT-PCR results. GmTOPP13 gene was transformed into tobacco plants via Agrobacterium mediated method, and the drought tolerance was analyzed by water deficit assay. RESULTS: 15 GmTOPP genes were identified in the soybean genome database (GmTOPP1-15). GmTOPP genes were distributed on 9 of 20 chromosomes, with similar exon-intron structure and motifs arrangement. All GmTOPPs contained Metallophos and STPPase_N domains as well as the core catalytic sites. Cis-regulatory element analysis predicted that GmTOPPs were widely involved in plant development, stress and hormone response in soybean. Expression profiles showed that GmTOPPs expressed in different tissues and exhibited divergent expression patterns in leaf and root in response to drought stimulus. Moreover, GmTOPP13 gene was isolated and expression pattern analysis indicated that this gene was highly expressed in seed, root, leaf and other tissues detected, and intensively induced upon PEG6000 treatment. In addition, overexpression of GmTOPP13 gene enhanced the drought tolerance in tobacco plants. The transgenic tobacco plants showed regulation of stress-responsive genes including CAT, SOD, ERD10B and TIP during drought stress. CONCLUSIONS: This study provides valuable information for the study of GmTOPP gene family in soybean, and lays a foundation for further functional studies of GmTOPP13 gene under drought and other abiotic stresses.

A novel near-infrared optical and redox-active receptor for the multi-model detection of Hg2+ in water and living cells.

A key issue for constructing optical and redox-active receptors is how to conjugate a specific sensing kernel with a multi-signal-responsive system to carry out multi-feature analysis. Mercury is considered to be highly toxic to human health and ecological security. In this work, we present a novel near-infrared optical and redox-active receptor that can sense Hg2+ at ppb level in aqueous media via multi-model monitors with a low detection limit of 8.4 × 10-9 M (1.68 ppb). This receptor features a visible detection, 'off-on' fluorescence response, and efficient electrochemistry assessment, as well as pH-insensitivity to Hg2+ with high sensitivity. In view of its marked near-infrared emission and fluorescence enhancement, we successfully applied this receptor to visualize Hg2+ in live cells. Furthermore, a possible sensing model was established and rationalized with theoretical studies.

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector.

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.

High-Throughput Sequencing-Based Identification of Arabidopsis miRNAs Induced by Phytophthora capsici Infection.

MicroRNAs (miRNAs) are a group of small non-coding endogenous RNAs. In plants, miRNAs play vital functions in regulating growth, development, and stress response. However, the role of miRNAs in Arabidopsis-Phytophthora capsici (P. capsici) model pathosystem is poorly understood. Here, we used a high-throughput sequencing approach to identify pathogen-responsive miRNAs using 15 small RNA (sRNA) libraries prepared from Arabidopsis thaliana leaves collected at 0, 3, 6, 12, and 24 h post-inoculation with P. capsici. A total of 293 known miRNAs and 6 potential novel sRNAs (miRNAs or siRNAs) were identified, of which 33 miRNAs were differentially expressed at four different infection stages. To verify the reliability of the sRNA-seq results, we investigated the expression of five sRNAs upregulated throughout the four infection stages and their potential target genes using northern blot analysis and/or stem-loop quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the potential target genes of the differentially expressed miRNAs, both conserved and novel, were enriched in pathways such as starch and sugar metabolism, spliceosome, and plant-pathogen interaction, indicating that the splicing machinery and pathogenesis-related (PR) proteins play important roles in the response to P. capsici infection. Taken together, these results provide novel insights into the molecular mechanisms of pathogenesis by P. capsici. Additionally, these results will serve as a strong foundation for further in-depth analysis of miRNAs involved in the resistance to Phytophthora species in other crops.

New Pyridine-Bridged Ferrocene-Rhodamine Receptor for the Multifeature Detection of Hg2+ in Water and Living Cells.

A challenge in the design of optical and redox-active receptors is how to combine a specific recognition center with an efficient responsive system to facilely achieve multifeature detection in biological and environmental analyses. Herein, a novel ferrocene-rhodamine receptor conjugated with a pyridine bridge was designed and synthesized. This receptor can sensitively sense Hg2+ in aqueous media via chromogenic, fluorogenic, and electrochemical multisignal outputs with a low detection limit and fast response time. Moreover, it can be qualified as a fluorescent probe for effectively monitoring Hg2+ in living cells. A plausible recognition mode was proposed and rationalized with theoretical calculations.

Rapid microwave synthesis of dioxin-linked covalent organic framework for efficient micro-extraction of perfluorinated alkyl substances from water.

To synthesize covalent organic framework (COF) via irreversible reactions is more challenging than by reversible ones. In this work, microwave-assisted synthesis is used to facilitate the nucleophilic substitution of 2,3,5,6-tetrafluoro-4-pyridinecarbonitrile with 2,3,6,7,10,11-hexahydroxy triphenylene. The dioxin-linked COF, named TH-COF, was efficiently synthesized with extraordinarily large surface area of 1254 m2 g-1. With its high crystallinity, excellent thermal and chemical stabilities, TH-COF is used as the coating for the solid-phase micro-extraction (SPME) of perfluorinated alkyl substances (PFASs). The adsorptive mechanism was evaluated with adsorption isotherm and kinetic adsorption. Adsorption energies are calculated based on the density functional theory. Following SPME with TH-COF-coated fibers, PFASs were eluted using 1 mL of 0.6% trifluoroacetic acid/methanol and analyzed through the ultra-performance liquid chromatography equipped with triple quadrupole mass spectrometer (UPLC-MS/MS). When applied to spiked real water samples, this method demonstrates good linearity (0.01-1000 ng L-1) with R2 ≥ 0.9945. The TH-COF-SPME-UPLC-MS/MS technique provides low limits of detection (0.0020-0.0045 ng L-1), excellent precision (≤ 7.9%), and good fiber-to-fiber reproducibility (≤ 7.1%). The TH-COF-coated fibers can be reused at least 20 times without the loss of extraction performance. In addition, the relative recoveries from spiked real water samples are 89.5%-105%.

iTRAQ-based comparative proteomic analysis reveals high temperature accelerated leaf senescence of tobacco (Nicotiana tabacum L.) during flue-curing.

Tobacco (Nicotiana tabacum) is extensively cultivated all over the world for its economic value. During curing and storage, senescence occurs, which is associated with physiological and biochemical changes in postharvest plant organs. However, the molecular mechanisms involved in accelerated senescence due to high temperatures in tobacco leaves during curing need further elaboration. We studied molecular mechanisms of senescence in tobacco leaves exposed to high temperature during curing (Fresh, 38 °C and 42 °C), revealed by isobaric tags for relative and absolute quantification (iTRAQ) for the proteomic profiles of cultivar Bi'na1. In total, 8903 proteins were identified, and 2034 (1150 up-regulated and 1074 down-regulated) differentially abundant proteins (DAPs) were obtained from tobacco leaf samples. These DAPs were mainly involved in posttranslational modification, protein turnover, energy production and conversion. Sugar- and energy-related metabolic biological processes and pathways might be critical regulators of tobacco leaves exposed to high temperature during senescence. High-temperature stress accelerated tobacco leaf senescence mainly by down-regulating photosynthesis-related pathways and degrading cellular constituents to maintain cell viability and nutrient recycling. Our findings provide a valuable inventory of novel proteins involved in senescence physiology and elucidate the protein regulatory network in postharvest organs exposed to high temperatures during flue-curing.

Comparative Proteomic Analysis by iTRAQ Reveals that Plastid Pigment Metabolism Contributes to Leaf Color Changes in Tobacco (Nicotiana tabacum) during Curing.

Tobacco (Nicotiana tabacum), is a world's major non-food agricultural crop widely cultivated for its economic value. Among several color change associated biological processes, plastid pigment metabolism is of trivial importance in postharvest plant organs during curing and storage. However, the molecular mechanisms involved in carotenoid and chlorophyll metabolism, as well as color change in tobacco leaves during curing, need further elaboration. Here, proteomic analysis at different curing stages (0 h, 48 h, 72 h) was performed in tobacco cv. Bi'na1 with an aim to investigate the molecular mechanisms of pigment metabolism in tobacco leaves as revealed by the iTRAQ proteomic approach. Our results displayed significant differences in leaf color parameters and ultrastructural fingerprints that indicate an acceleration of chloroplast disintegration and promotion of pigment degradation in tobacco leaves due to curing. In total, 5931 proteins were identified, of which 923 (450 up-regulated, 452 down-regulated, and 21 common) differentially expressed proteins (DEPs) were obtained from tobacco leaves. To elucidate the molecular mechanisms of pigment metabolism and color change, 19 DEPs involved in carotenoid metabolism and 12 DEPs related to chlorophyll metabolism were screened. The results exhibited the complex regulation of DEPs in carotenoid metabolism, a negative regulation in chlorophyll biosynthesis, and a positive regulation in chlorophyll breakdown, which delayed the degradation of xanthophylls and accelerated the breakdown of chlorophylls, promoting the formation of yellow color during curing. Particularly, the up-regulation of the chlorophyllase-1-like isoform X2 was the key protein regulatory mechanism responsible for chlorophyll metabolism and color change. The expression pattern of 8 genes was consistent with the iTRAQ data. These results not only provide new insights into pigment metabolism and color change underlying the postharvest physiological regulatory networks in plants, but also a broader perspective, which prompts us to pay attention to further screen key proteins in tobacco leaves during curing.