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To gain insight into the molecular mechanisms underpinning tobacco (Nicotiana tabacum) tolerance to drought stress, we integrated anatomical, physiological, and proteomic analyses of drought-tolerant (Yuyan6, [Y6]) and -sensitive (Yunyan87 [Y87]) varieties. In comparison to Y87, Y6 exhibited higher water retention capability, improved photosynthetic performance, delayed leaf-senescence, stable leaf ultrastructure, a stronger antioxidant defense, and lesser ROS accumulation when subjected to water stress. Using an iTRAQ-based proteomics approach, 405 and 1,560 differentially accumulated proteins (DAPs) were identified from Y6 and Y87 plants, respectively, of which 114 were found to be present in both cultivars. A subsequent functional characterization analysis revealed that these DAPs were significantly enriched in eight biological processes, six molecular functions, and six cellular components and displayed differential expression patterns in Y6 and Y87 plants, suggesting that the response to water stress between both varieties differed at the proteomic level. Furthermore, we constructed protein coexpression networks and identified hub proteins regulating tobacco defenses to water stress. Additionally, qPCR analysis indicated that the majority of genes encoding selected proteins showed consistency between mRNA levels and their corresponding protein expression levels. Our results provide new insights into the genetic regulatory mechanisms associated with drought response in tobacco plants.

Adverse environmental conditions, such as cold and drought, can inhibit plant growth, development, and productivity. The isolation and characterization of stress response genes from stress-tolerant plants can provide a better understanding of the underlying adaptive mechanisms. In this study, a novel cold-regulated gene, SikCOR413PM1, was isolated from Saussurea involucrata Kar. et Kir., which is a plant that survives at the high altitudes and in the low temperatures of alpine slopes in northwestern China. SikCOR413PM1 was induced in response to cold and drought in S. involucrata, and phylogenetic analysis revealed that the gene groups with a COR gene encoding a COR413PM protein family member. Subcellular localization of a SikCOR413PM1-green fluorescent fusion protein showed that SikCOR413PM1 was localized to the plasma membrane. A transgenic tobacco (Nicotiana tabacum) system was employed to investigate the possible role of SikCOR413PM1 in cold and drought tolerance. Analyses of growth, germination and survival rates, relative water content, malondialdehyde content, relative electrolyte leakage, and maximal photochemical efficiency of photosystem II showed that transgenic tobacco plants expressing SikCOR413PM1 were more tolerant to cold and drought stresses than WT plants. SikCOR413PM1 overexpression was also accompanied by constitutive activation of NtDREB1 and NtDREB3, two cold-responsive transcription factor genes, and NtERD10A and NtERD10B, two cold-induced genes. The expression levels of downstream transcription factor genes NtDREB3, NtERD10C, NtERD10D, and NtLEA5 were also induced in SikCOR413PM1-expressing transgenic plants under drought conditions. Our results suggest that the overexpression of SikCOR413PM1 induces changes in tobacco plants, and facilitates enhanced tolerance to cold and drought stresses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Agricultural production is greatly affected by environmental stresses, such as cold, drought and high-salinity. It is possible to produce tolerant genotypes by transferring genes encoding protective proteins or enzymes from other organisms. In this regard, the current study was aimed to clone a novel OeSRC1 gene identified during the transcriptome profiling of olives (Olea europaea L.) and to investigate the function of this gene in tobacco plants. Functional evaluation of OeSRC1 gene in putative transgenic tobacco plants were carried out under drought, cold and salt stress conditions by using molecular and biochemical tools. It was observed that the transgenic tobacco plants exhibited higher seed germination and survival rates, better root and shoot growth under cold, salt and drought stress treatments compared to wild type plants. Our results also demonstrated that, under stress conditions, transgenic plants accumulated more free proline while no significant changes were observed regarding electrolyte leakage. Ascorbate peroxidase activity of OeSRC1-overexpressing plants was higher than those of the WT plants under different stress conditions. The overall results demonstrate the explicit role of OeSRC1 gene in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional role of olive OeSRC1 gene looks good to enhance environmental stress tolerance in diverse plants.

Actin filament, also known as microfilament, is one of two major cytoskeletal elements in plants and plays important roles in various biological processes. Like in animal cells, actin filaments have been thought to participate in autophagy in plants. However, surprisingly, in this study we found that actin filaments are dispensable for the occurrence of autophagy in plants. Disruption of actin filaments by short term treatment with actin polymerization inhibitors, cytochalasin D and latrunculin B, or transient overexpression of Profilin 3 in Nicotiana benthamiana had no effect on basal autophagy as well as the upregulation of nocturnal autophagy and salt stress-induced autophagy. Furthermore, anti-microfilament drug treatment affected neither basal nor salt stress-induced autophagy in Arabidopsis . In addition, prolonged perturbation of actin filaments by silencing Actin7 or 24-h treatment with microfilament-disrupting agents in N. benthamiana caused endoplasmic reticulum (ER) disorganization and subsequent degradation via autophagy involving ATG2, 3, 5, 6 and 7. Our findings reveal that, unlike mammalian cells, actin filaments are unnecessary for bulk autophagy in plants. Abbreviations: ATG: autophagy-related; CD: cytochalasin D; Cvt pathway: cytoplasm to vacuole targeting pathway; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; LatB: latrunculin B; Nb: Nicotiana benthamiana ; PAS: phagophore assembly site; PRF3: Profilin 3; RER: rough ER; SER: smooth ER; TEM: transmission electron microscopy; TRV: Tobacco rattle virus ; VIGS: virus-induced gene silencing; wpi: weeks post-agroinfiltration.

Plasmodesmata (PD) are essential for plant development, but little is known about their regulation. Several studies have linked PD transport to chloroplast-centered signaling networks, but the physiological significance of this connection remains unclear. Here, we show that PD transport is strongly regulated by light and the circadian clock. Light promotes PD transport during the day, but light is not sufficient to increase rates of PD transport at night, suggesting a circadian gating mechanism. Silencing expression of the core circadian clock gene, LHY/CCA1 , allows light to strongly promote PD transport during subjective night, confirming that the canonical plant circadian clock controls the PD transport light response. We conclude that PD transport is dynamically regulated during the day/night cycle. Due to the many roles of PD in plant biology, this discovery has strong implications for plant development, physiology, and pathogenesis., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Key Message: This research revealed diverse PTG rates among intraspecific pollen-pistil interactions that showed variable dependency on the stigma and mature TT. Pollen-pistil interactions regulate pollen tube growth (PTG) rates and are determinants of fertilization and seed set. This research focuses on the diversity of intraspecific PTG rates and the spatial and temporal regulation of PTG among Nicotiana tabacum genotypes. Nonrandom mating within self-compatible species has been noted, but little is known on the mechanisms involved. To begin research on nonrandom mating, we took advantage of the model reproductive system of N. tabacum and used seventeen diverse N. tabacum genotypes in a complete pollination diallel to measure the diversity of intraspecific pollen-pistil interactions. The 289 intraspecific interactions showed surprisingly large differences in PTG rates. The interaction between specific males and females resulted in 18 specific combining abilities that were significantly different, indicating the importance of the specific genotype interaction in regulating intraspecific PTG. No single female or male genotype exerted overall control of PTG rates, as determined by a general combining ability analysis. Slow and fast pollen-pistil interactions showed spatial differences in growth rates along the style. Slower interactions had a slower initial PTG rate while fast interactions had faster consistent rates of growth indicating spatial regulation of PTG in the pistil. Removal of the stigma or the mature transmitting tissue (TT) showed the tissue-specific component of PTG regulation. Stigma removal resulted in slower or no change in PTG rate depending on the pollen and pistil genotypes. Removal of the TT, which necessitated removal of the stigma, showed no change, slower or unexpectedly, increased growth rates relative to growth rates without a stigma. These data show the diverse nature of pollen-pistil interactions in N. tabacum genotypes providing a system to further investigate the regulation of PTG.

Gut-expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, which encodes a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant-mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein and/or free amino acids in their honeydew. Larvae of Coccinella septempunctata (seven-spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi-mediated pest control., (© 2019 The Royal Entomological Society.)

SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptors) complex formation is necessary for intracellular membrane fusion and thus has a key role in processes such as secretion. However, little is known about the regulatory factors that bind to Qa-SNAREs, which are also known as syntaxins (SYPs) in plants. Here, we characterized Arabidopsis ( Arabidopsis thaliana ) Tomosyn protein (AtTMS) and demonstrated that it is a conserved regulator of SYPs in plants. AtTMS binds strongly via its R-SNARE motif-containing C terminus to the Qa domain of PM-resident, pollen-expressed SYP1s (SYP111, SYP124, SYP125, SYP131, and SYP132), which were narrowed down from 12 SYPs. AtTMS is highly expressed in pollen from the bicellular stage onwards, and overexpression of AtTMS under the control of the UBIQUITIN10 , MSP1 , or LAT52 promoter all resulted in defective pollen after the microspore stage in which secretion was inhibited, leading to the failure of intine deposition and cell plate formation during pollen mitosis I. In tobacco ( Nicotiana benthamiana ) leaf epidermal cells, overexpression of AtTMS inhibited the secretion of secreted GFP. The defects were rescued by mCherry-tagged SYP124, SYP125, SYP131, or SYP132. In vivo, SYP132 partially rescued the pMSP1 : AtTMS phenotype. In addition, AtTMS, lacking a transmembrane domain, was recruited to the plasma membrane by SYP124, SYP125, SYP131, and SYP132 and competed with Vesicle-Associated Membrane Protein721/722 for binding to, for example, SYP132. Together, our results demonstrated that AtTMS might serve as a negative regulator of secretion, whereby active secretion might be fine-tuned during pollen development., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory-elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory-elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory-elicited metabolic and hormone responses in CMNs-connected "receiver" plants after the elicitation of "donor" plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA-Ile) levels in N. attenuata roots but did not affect well-characterized JAs-regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS-elicited "receivers" with CMN connections with "donors" that had been W + OS-elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants., (© 2019 John Wiley & Sons Ltd.)

Rate of photosynthesis and related plant carbohydrate status are crucial factors affecting plant vigor. Sugars providing carbon and energy sources serve also as important signaling molecules governing plant growth and development through a complex regulatory network. These facts are often neglected when mixotrophic cultivation of plants in vitro is used, where artificial exogenous sugar supply hinders studies of metabolism as well as sugar-driven developmental processes. We compared the growth, selected gas-exchange parameters and sugar metabolism characteristics in four model plants, potato (Solanum tuberosum 'Lada'), tobacco (Nicotiana tabacum 'Samsun'), rapeseed (Brassica napus 'Asgard') and strawberry (Fragaria vesca), under both photomixotrophic (PM) and photoautotrophic (PA) conditions. To ensure PA conditions, we used our improved sun caps that serve as gas and light permeable covers for cultivation vessels. We found bigger biomass accumulation, larger leaf areas, higher stomatal conductance and higher instantaneous water use efficiency and lower root sugar contents in PA plants compared to PM ones. However, for other characteristics (root biomass, root/shoot ratio, pigment contents, leaf sugar and starch levels and transpiration rates), a strong species-dependent reactions to the exogenous sugar supply was noted, which does not allow to create a general view on the overall impact of PM nutrition under in vitro conditions., (© 2018 Scandinavian Plant Physiology Society.)

Background: The plant-specific homeodomain-leucine zipper class IV (HD-ZIP IV) gene family has been involved in the regulation of epidermal development., Results: Fifteen genes coding for HD-ZIP IV proteins were identified (NtHD-ZIP-IV-1 to NtHD-ZIP-IV-15) based on the genome of N. tabacum. Four major domains (HD, ZIP, SAD and START) were present in these proteins. Tissue expression pattern analysis indicated that NtHD-ZIP-IV-1, - 2, - 3, - 10, and - 12 may be associated with trichome development; NtHD-ZIP-IV-8 was expressed only in cotyledons; NtHD-ZIP-IV-9 only in the leaf and stem epidermis; NtHD-ZIP-IV-11 only in leaves; and NtHD-ZIP-IV-15 only in the root and stem epidermis. We found that jasmonates may induce the generation of glandular trichomes, and that NtHD-ZIP-IV-1, - 2, - 5, and - 7 were response to MeJA treatment. Dynamic expression under abiotic stress and after application of phytohormones indicated that most NtHD-ZIP IV genes were induced by heat, cold, salt and drought. Furthermore, most of these genes were induced by gibberellic acid, 6-benzylaminopurine, and salicylic acid, but were inhibited by abscisic acid. NtHD-ZIP IV genes were sensitive to heat, but insensitive to osmotic stress., Conclusion: NtHD-ZIP IV genes are implicated in a complex regulatory gene network controlling epidermal development and abiotic stress responses. The present study provides evidence to elucidate the gene functions of NtHD-ZIP IVs during epidermal development and stress response.

Drought is one of the major environmental stresses limiting crop growth and production. It is very important to exploit and utilize drought-tolerance genes to improve crop drought-resistance. In this study, we identified two homoeologs of a Nicotiana tabacum (Ntab) DELAY OF GERMINATION (DOG) 1 like gene, named as NtabDOG1L-T and NtabDOG1L-S, respectively. The NtabDOG1L genes were preferentially expressed in roots and their expression levels were induced by polyethylene glycol, high salt, cold, and abscisic acid treatments. Subcellular localization results indicated that NtabDOG1L-T was localized in the nucleus, cytoplasm and cell membrane. Overexpression of NtabDOG1L-T in tobacco resulted in roots growth enhancement in transgenic plants. Furthermore, overexpression of NtabDOG1L-T enhanced drought stress tolerance in transgenic tobacco. The transgenic tobacco lines exhibited lower leaf water loss and electrolyte leakage, lower content of malondialdehyde and reactive oxygen species (ROS), and higher antioxidant enzymes activities after drought treatment when compared with wild type (WT) plants. In addition, the expression levels of several genes encoding key antioxidant enzymes and drought-related proteins were higher in the transgenic plants than in the WT plants under drought stress. Taken together, our results showed that NtabDOG1L functions as a novel regulator that improves plant growth and drought tolerance in tobacco., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Research has begun to elucidate the signal transduction pathway(s) that control cellular responses to changes in mitochondrial status. Important tools in such studies are chemical inhibitors used to initiate mitochondrial dysfunction. This study compares the effect of different inhibitors and treatment conditions on the transcript amount of nuclear genes specifically responsive to mitochondrial dysfunction in leaf of Nicotiana tabacum L. cv. Petit Havana. The Complex III inhibitors antimycin A (AA) and myxothiazol (MYXO), and the Complex V inhibitor oligomycin (OLIGO), each increased the transcript amount of the mitochondrial dysfunction genes. Transcript responses to OLIGO were greater during treatment in the dark than in the light, and the dark treatment resulted in cell death. In the dark, transcript responses to AA and MYXO were similar to one another, despite MYXO leading to cell death. In the light, transcript responses to AA and MYXO diverged, despite cell viability remaining high with either inhibitor. This divergent response may be due to differential signaling from the chloroplast because only AA also inhibited cyclic electron transport, resulting in a strong acceptor-side limitation in photosystem I. In the light, chemical inhibition of chloroplast electron transport reduced transcript responses to AA, while having no effect on the response to MYXO, and increasing the response to OLIGO. Hence, when studying mitochondrial dysfunction signaling, different inhibitor and treatment combinations differentially affect linked processes (e.g. chloroplast function and cell fate) that then contribute to measured responses. Therefore, inhibitor and treatment conditions should be chosen to align with specific study goals., (© 2018 Scandinavian Plant Physiology Society.)

Main Conclusion: This study revealed novel insights into the function of MSP18 effector during root-knot nematode parasitism in rice roots. MSP18 may modulate host immunity and enhance plant susceptibility to Meloidogyne spp. Rice (Oryza sativa) production is seriously impacted by root-knot nematodes (RKN), including Meloidogyne graminicola, Meloidogyne incognita, and Meloidogyne javanica, in upland and irrigated culture systems. Successful plant infection by RKN is likely achieved by releasing into the host cells some effector proteins to suppress the activation of immune responses. Here, we conducted a series of functional analyses to assess the role of the Meloidogyne-secreted protein (MSP) 18 from M. incognita (Mi-MSP18) during rice infection by RKN. Developmental expression profiles of M. javanica and M. graminicola showed that the MSP18 gene is up-regulated throughout nematode parasitic stages in rice. Reproduction of M. javanica and M. graminicola is enhanced in rice plants overexpressing Mi-MSP18, indicating that the Mi-MSP18 protein facilitates RKN parasitism. Transient expression assays in onion cells suggested that Mi-MSP18 is localized to the cytoplasm of the host cells. In tobacco, Mi-MSP18 suppressed the cell death induced by the INF1 elicitin, suggesting that Mi-MSP18 can interfere with the plant defense pathways. The data obtained in this study highlight Mi-MSP18 as a novel RKN effector able to enhance plant susceptibility and modulate host immunity.

Electronic control of biological processes with bioelectronic devices holds promise for sophisticated regulation of physiology, for gaining fundamental understanding of biological systems, providing new therapeutic solutions, and digitally mediating adaptations of organisms to external factors. The organic electronic ion pump (OEIP) provides a unique means for electronically-controlled, flow-free delivery of ions, and biomolecules at cellular scale. Here, a miniaturized OEIP device based on glass capillary fibers (c-OEIP) is implanted in a biological organism. The capillary form factor at the sub-100 µm scale of the device enables it to be implanted in soft tissue, while its hyperbranched polyelectrolyte channel and addressing protocol allows efficient delivery of a large aromatic molecule. In the first example of an implantable bioelectronic device in plants, the c-OEIP readily penetrates the leaf of an intact tobacco plant with no significant wound response (evaluated up to 24 h) and effectively delivers the hormone abscisic acid (ABA) into the leaf apoplast. OEIP-mediated delivery of ABA, the phytohormone that regulates plant's tolerance to stress, induces closure of stomata, the microscopic pores in leaf's epidermis that play a vital role in photosynthesis and transpiration. Efficient and localized ABA delivery reveals previously unreported kinetics of ABA-induced signal propagation., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Natural vegetation is predicted to suffer from extreme heat events as a result of global warming. In this study, we focused on the immediate response to heat stress. Photosynthesis and volatile emissions were measured in the leaves of tobacco (Nicotiana tabacum cv. Wisconsin 38) after exposure to heat shock treatments between 46 °C and 55 °C. Exposure to 46 °C decreased photosynthetic carbon assimilation rates (A) by >3-fold. Complete inhibition of A was observed at 49 °C, together with a simultaneous decrease in the maximum quantum efficiency of PSII, measured as the Fv/Fm ratio. A large increase in volatile emissions was observed at 52 °C. Heat stress resulted in only minor effects on the emission of monoterpenes, but volatiles associated with membrane damage such as propanal and (E)-2-hexenal+(Z)-3-hexenol were greatly increased. Heat induced changes in the levels of methanol and 2-ethylfuran that are indicative of modification of cell walls. In addition, the oxidation of metabolites in the volatile profiles was strongly enhanced, suggesting the acceleration of oxidative processes at high temperatures that are beyond the thermal tolerance limit., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The primary pigments in flowers are anthocyanins, the biosynthesis of which is mainly regulated by R2R3-MYBs. Muscari armeniacum is an ornamental garden plant with deep cobalt blue flowers containing delphinidin-based anthocyanins. An anthocyanin-related R2R3-MYB MaAN2 has previously been identified in M. armeniacum flowers; here, we also characterized a novel R2R3-MYB MaMybA, to determine its function and highlight similarities and differences between MaMybA and MaAN2., Results: In this study, a novel anthocyanin-related R2R3-MYB gene was isolated from M. armeniacum flowers and functionally identified. A sequence alignment showed that MaMybA contained motifs typically conserved with MaAN2 and its orthologs. However, the shared identity of the entire amino acid sequence between MaMybA and MaAN2 was 43.5%. Phylogenetic analysis showed that they were both clustered into the AN2 subgroup of the R2R3-MYB family, but not in the same branch. We also identified a IIIf bHLH protein, MabHLH1, in M. armeniacum flowers. A bimolecular fluorescence complementation assay showed that MabHLH1 interacted with MaMybA or MaAN2 in vivo; a dual luciferase assay indicated that MaMybA alone or in interaction with MabHLH1 could regulate the expression of MaDFR and AtDFR, but MaAN2 required MabHLH1 to do so. When overexpressing MaMybA in Nicotiana tabacum 'NC89', the leaves, petals, anthers, and calyx of transgenic tobacco showed intense and magenta anthocyanin pigments, whereas those of OE-MaAN2 plants had lighter pigmentation. However, the ovary wall and seed skin of OE-MaMybA tobacco were barely pigmented, while those of OE-MaAN2 tobacco were reddish-purple. Moreover, overexpressing MaMybA in tobacco obviously improved anthocyanin pigmentation, compared to the OE-MaAN2 and control plants, by largely upregulating anthocyanin biosynthetic and endogenous bHLH genes. Notably, the increased transcription of NtF3'5'H in OE-MaMybA tobacco might lead to additional accumulation of delphinidin 3-rutinoside, which was barely detected in OE-MaAN2 and control plants. We concluded that the high concentration of anthocyanin and the newly produced Dp3R caused the darker color of OE-MaMybA compared to OE-MaAN2 tobacco., Conclusion: The newly identified R2R3-MYB transcription factor MaMybA functions in anthocyanin biosynthesis, but has some differences from MaAN2; MaMybA could also be useful in modifying flower color in ornamental plants.

CCCH-type zinc-finger proteins play essential roles in regulating plant development and stress responses. However, the molecular and functional properties of non-tandem CCCH-type zinc-finger (non-TZF) proteins have been rarely characterized in plants. Here, we report the biological and molecular characterization of a sweet potato non-TZF gene, IbC3H18. We show that IbC3H18 exhibits tissue- and abiotic stress-specific expression, and could be effectively induced by abiotic stresses, including NaCl, polyethylene glycol (PEG) 6000, H 2 O 2 and abscisic acid (ABA) in sweet potato. Accordingly, overexpression of IbC3H18 led to increased, whereas knock-down of IbC3H18 resulted in decreased tolerance of sweet potato to salt, drought and oxidation stresses. In addition, IbC3H18 functions as a nuclear transcriptional activator and regulates the expression of a range of abiotic stress-responsive genes involved in reactive oxygen species (ROS) scavenging, ABA signaling, photosynthesis and ion transport pathways. Moreover, our data demonstrate that IbC3H18 physically interacts with IbPR5, and that overexpression of IbPR5 enhances salt and drought tolerance in transgenic tobacco plants. Collectively, our data indicate that IbC3H18 functions in enhancing abiotic stress tolerance in sweet potato, which may serve as a candidate gene for use in improving abiotic stress resistance in crops., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na + /K + ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H 2 O 2 ) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H 2 O 2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H 2 O 2 scavenging, regulation of osmotic adjustment and ion homeostasis., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The highly conserved core circadian clock component TIMING OF CAB EXPRESSION1 ( TOC1 ) contextualizes environmental stress responses in plants, for example by gating abscisic acid signaling and suppressing thermoresponsive growth. Selective interaction of TOC1 with PHYTOCHROME B under far-red-enriched light suggests a connection between circadian gating of light responses and sensitivity to ABA, an important regulator of growth and stress responses, including under drought. However, the fitness consequences of TOC1 function, particularly in the root, are poorly understood. Here, we used the desert annual, Nicotiana attenuata , to investigate the function of TOC1 in shoots and roots for maintaining fitness under drought, in both field and glasshouse experiments. Despite marked decreases in leaf water loss, TOC1 -deficient lines failed to maintain fitness in response to drought stress as measured by total seed capsule production. Restoring TOC1 transcript levels in shoots via micrografting was sufficient to restore wild-type drought responses under field conditions. Microarrays identified a coexpression module in leaves strongly linking red and far-red light signaling to drought responses in a TOC1 -dependent manner, but experiments with phytochrome-deficient lines revealed that the effects of TOC1 deficiency under drought cannot be attributed to changes in red/far-red light perception alone. Taken together, these results elucidate the sophisticated, tissue-dependent role of the circadian clock in maintaining fitness in the face of long-term abiotic stresses such as drought., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Background: Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences., Results: In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5' deletion mutants of these promoters, it was determined that TaGAPCp2P (- 1312~ - 528) and TaGAPCp3P (- 2049~ - 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (- 1197~ - 635) and TaGAPCp3P (- 1456~ - 1144 and - 718~ - 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (- 1135 and - 985) and TaGAPCp3P (- 1414 and - 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB., Conclusion: The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.

Background: Tilletia horrida is a basidiomycete fungus that causes rice kernel smut, one of the most important rice diseases in hybrid rice growing areas worldwide. However, little is known about its mechanisms of pathogenicity. We previously reported the genome of T. horrida, and 597 genes that encoded secreted proteins were annotated. Among these were some important effector genes related to pathogenicity., Results: A secretome analysis suggested that five Tilletia fungi shared more gene families than were found in other smuts, and there was high conservation between them. Furthermore, we screened 597 secreted proteins from the T. horrida genome, some of which induced expression in host-pathogen interaction processes. Through transient expression, we demonstrated that two putative effectors could induce necrosis phenotypes in Nicotiana benthamiana. These two encoded genes were up-regulated during early infection, and the encoded proteins were confirmed to be secreted using a yeast secretion system. For the putative effector gene smut_5844, a signal peptide was required to induce non-host cell death, whereas ribonuclease catalytic active sites were required for smut_2965. Moreover, both putative effectors could induce an immune response in N. benthamiana leaves. Interestingly, one of the identified potential host interactors of smut_5844 was laccase-10 protein (OsLAC10), which has been predicted to be involved in plant lignification and iron metabolism., Conclusions: Overall, this study identified two secreted proteins in T. horrida that induce cell death or are involved in defense machinery in non-host plants. This research provides a useful foundation for understanding the interaction between rice and T. horrida.

Widespread application of silver nanoparticles (AgNPs), due to their antibacterial and antifungal properties, increases their release into the environment and potential detrimental impact on living organisms. Plants may serve as a potential pathway for AgNPs bioaccumulation and a route into the food chain, hence investigation of AgNP phytotoxic effects are of particular importance. Since proteins are directly involved in stress response, studies of their abundance changes can help elucidate the mechanism of the AgNP-mediated phytotoxicity. In this study, we investigated proteomic changes in tobacco (Nicotiana tabacum) exposed to AgNPs and ionic silver (AgNO 3 ). A high overlap of differently abundant proteins was found in root after exposure to both treatments, while in leaf, almost a half of the proteins exhibited different abundance level between treatments, indicating tissue-specific responses. Majority of the identified proteins were down-regulated in both tissues after exposure to either AgNPs or AgNO 3 ; in roots, the most affected proteins were those involved in response to abiotic and biotic stimuli and oxidative stress, while in leaf, both treatments had the most prominent effect on photosynthesis-related proteins. However, since AgNPs induced higher suppression of protein abundance than AgNO 3 , we conclude that AgNP effects can, at least partially, be attributed to nanoparticle form.

Main Conclusion: The absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.

Background: Abscisic acid (ABA) is an important phytohormone for plant growth, development and responding to stresses such as drought, salinity, and pathogen infection. Pyrabactin Resistance 1 (PYR1)/PYR1-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) (hereafter referred to as PYLs) has been identified as the ABA receptors. The PYL family members have been well studied in many plants. However, the members of PYL family have not been systematically identified at genome level in cultivated tobacco (Nicotiana tabacum) and its two ancestors. In this study, the phylogenic relationships, chromosomal distribution, gene structures, conserved motifs/regions, and expression profiles of NtPYLs were analyzed., Results: We identified 29, 11, 16 PYLs in the genomes of allotetraploid N. tabacum, and its two diploid ancestors N. tomentosiformis and N. sylvestris, respectively. The phylogenetic analysis revealed that NtPYLs can be divided into three subfamilies, and each NtPYL has one counterpart in N. sylvestris or N. tomentosiformis. Based on microarray analysis of NtPYL transcripts, four NtPYLs (from subfamily II, III), and five NtPYLs (from subfamily I) are highlighted as potential candidates for further functional characterization in N. tabacum seed development, response to ABA, and germination, and resistance to abiotic stresses, respectively. Interestingly, the expression profiles of members in the same NtPYL subfamily showed somehow similar patterns in tissues at different developmental stages and in leaves of seedlings under drought stress, suggesting particular NtPYLs might have multiple functions in both plant development and drought stress response., Conclusions: NtPYLs are highlighted for important functions in seed development, germination and response to ABA, and particular in drought tolerance. This work will not only shed light on the PYL family in tobacco, but also provides some valuable information for functional characterization of ABA receptors in N. tabacum.

Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit reproductive isolation in crosses with Nicotiana tabacum. In this study, we investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. We revealed that four N. suaveolens accessions comprised 64 chromosomes, while the other four accessions carried 32 chromosomes. Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12-17 days after pollination. A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, we confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.

Integration of environmental signals with endogenous biological processes is essential for organisms to thrive in their natural environment. Being entrained by periodic environmental changes, the circadian clock incorporates external information to coordinate physiological processes, phasing them to the optimal time of the day and year. Here, we present a pivotal role for the clock component GIGANTEA (GI) as a genome-wide regulator of transcriptional networks mediating growth and adaptive processes in plants. We provide mechanistic details on how GI integrates endogenous timing with light signaling pathways through the global modulation of PHYTOCHROME-INTERACTING FACTORs (PIFs). Gating of the activity of these transcriptional regulators by GI directly affects a wide array of output rhythms, including photoperiodic growth. Furthermore, we uncover a role for PIFs in mediating light input to the circadian oscillator and show how their regulation by GI is required to set the pace of the clock in response to light-dark cycles., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In flowering plants, intraspecific mate preference is frequently related to mating systems: the rejection of self pollen in self-incompatible (SI) plants that prevents inbreeding is one of the best described examples. However, in other mating systems, more nuanced patterns of pollen rejection occur. In the self-compatible (SC) Nicotiana attenuata, in which SI is not found and all crosses are compatible, certain pollen genotypes are consistently selected in mixed pollinations. However, the molecular mechanisms of this polyandrous mate selection remain unknown. Style-expressed NaS-like-RNases and pollen-expressed NaSLF-like genes, homologous to SI factors in Solanaceae, were identified and examined for a role in N. attenuata's mate selection. A comparison of two NaS-like-RNases and six NaSLF-like genes among 26 natural accessions revealed specific combinations of co-expression and direct protein-protein interactions. To evaluate their role in mate selection, we silenced the expression of specific NaS-like-RNases and NaSLF-like proteins and conducted diagnostic binary mixed pollinations and mixed pollinations with 14 different non-self pollen donors. Styles expressing particular combinations of NaS-like-RNases selected mates from plants with corresponding NaS-like-RNase expression patterns, while styles lacking NaS-like-RNase expression were non-selective in their fertilizations, which reflected the genotype ratios of pollen mixtures deposited on the stigmas. DNA methylation could account for some of the observed variation in stylar NaS-like-RNase patterns. We conclude that the S-RNase-SLF recognition mechanism plays a central role in polyandrous mate selection in this self-compatible species. These results suggest that after the SI-SC transition, natural variation of SI homologous genes was repurposed to mediate intraspecific mate selection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway., Results: In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H 2 O 2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT., Conclusions: Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.

Expansins play a pivotal role in plant adaptation to environmental stress via cell wall loosening. To evaluate the roles of expansin in response to different environmental stress conditions, the expansin gene PttEXPA8 from Populus tomentosa was transformed into tobacco. Analysis of physiological indices demonstrated the transgenic plants with improved resistance to heat, drought, salt, cold, and cadmium stress but to different extents. In mature plants, PttEXPA8 exerted the greatest effect on heat stress, with a response index value of 137.46%, followed by drought, cadmium, cold, and salt stress with response index values of 101.04%, 70.61%, 69.95%, and 54.68%, respectively. Over-expression of PttEXPA8 resulted in differential responses in physiological indices to the stresses. Soluble sugar content showed the highest response to the stresses, with an average response index value of 29.29%, whereas the absolute response index value for malondialdehyde content, relative electrolyte leakage, chlorophyll content, and superoxide dismutase activity ranged from 11.01% to 19.21%. The present results provide insight into the roles of expansin in stress resistance in Populus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study., Results: MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (F v /F m ) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into "photosynthesis" and "photosynthesis-antenna", such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into "spliceosome"., Conclusions: The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature.

It is well known that exogenous trehalose can improve resistances of plants to some abiotic and biotic stresses. Nonetheless, information respecting the molecular responses of tobacco leaves to Tre treatment is limited. Here we show that exogenous Tre can rapidly reduce stomatal aperture, up-regulate NADPH oxidase genes and increase O2•-andH2O2 on tobacco leaves at 2 h after treatment. We further demonstrated that imidazole and DPI, inhibitors of NADPH oxidase, can promote recovery of stomatal aperture of tobacco leaves upon trehalose treatment. Exogenous trehalose increased tobacco leaf resistance to tobacco mosaic disease significantly in a concentration-dependent way. To elucidate the molecular mechanisms in response to exogenous trehalose, the transcriptomic responses of tobacco leaves with 10 (low concentration) or 50 (high concentration) mM of trehalose treatment at 2 or 24h were investigated through RNA-seq approach. In total, 1288 differentially expressed genes (DEGs) were found with different conditions of trehalose treatments relative to control. Among them, 1075 (83.5%) were triggered by low concentration of trehalose (10mM), indicating that low concentration of Tre is a better elicitor. Functional annotations with KEGG pathway analysis revealed that the DEGs are involved in metabolic pathway, biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, protein processing in ER, flavonoid synthesis and circadian rhythm and so on. The protein-protein interaction networks generated from the core DEGs regulated by all conditions strikingly revealed that eight proteins, including ClpB1, HSP70, DnaJB1-like protein, universal stress protein (USP) A-like protein, two FTSH6 proteins, GolS1-like protein and chloroplastics HSP, play a core role in responses to exogenous trehalose in tobacco leaves. Our data suggest that trehalose triggers a signal transduction pathway which involves calcium and ROS-mediated signalings. These core components could lead to partial resistance or tolerance to abiotic and biotic stresses. Moreover, 19 DEGs were chosen for analysis of quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR for the 19 candidate genes coincided with the DEGs identified via the RNA-seq analysis, sustaining the reliability of our RNA-seq data., Competing Interests: The authors have declared that no competing interests exist.

Understanding the mechanism of abiotic-tolerance and producing germplasm of abiotic tolerance are important in plant research. Wild species often show more tolerance of environmental stress factors than their cultivated counterparts. Genes from wild species show potential abilities to improve abiotic resistance in cultivated species. Here, a tomato proline-, lysine-, and glutamic-rich type gene SpPKE1 was isolated from abiotic-resistant species ( Solanum pennellii LA0716) for over-expression in tomato and tobacco for salt tolerance. The protein encoded by SpPKE1 was predominantly localized in the cytoplasm in tobacco. SpPKE1 and SlPKE1 (from cultivated species S. lycopersicum cv. M82) shared 89.7% similarity in amino acid sequences and their transcripts abundance in flowers and fruits was reduced by the imposition of drought or oxidative stress and the exogenous supply of abscisic acid. The DNA of the PKE1 promoter was highly methylated in fruit and leaf, and the methylation of the coding sequence in leaf was significantly higher than that in fruit at different development stages. The over-expression of SpPKE1 under the control of a CaMV (Cauliflower Mosaic Virus) 35S promoter in transgenic tomato and tobacco plants enhanced their tolerance to salt stress. PKE1 was downregulated by abiotic stresses but enhanced the plant's salt stress tolerance. Therefore, this gene may be involved in post-transcriptional regulation and may be an important candidate for molecular breeding of salt-tolerant plants.

The basic helix-loop-helix (bHLH) family, one of the largest transcription factor groups in plants, regulates many critical developmental processes. However, their functions in plant defense have not been extensively studied in Nicotiana benthamiana, an important model plant species for phytopathology. Here, we identified N. benthamiana bHLH genes (NbbHLHs) using a whole-genome searching approach, and found that the NbbHLHs are highly enriched and some subfamilies are selectively expanded in N. benthamiana. The results showed that gene duplication may be responsible for bHLH family expansion in this plant. Furthermore, we analyzed their expression profiles upon infection with Phytophthora parasitica. Finally, 28 candidate NbbHLHs may play important roles in Phytophthora pathogen resistance using cis-element analysis and protein-interaction network prediction. Taken together, our results established a platform for future studies of the gene family and provide molecular insights into plant immune responses against P. parasitica., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Programmed cell death (PCD) is an essential process for development, and shows conserved cytological features in both plants and animals. Caspases are well-known critical components of the PCD machinery in animals. However, currently few typical counterparts have been identified in plants and only several caspase-like proteases are known to be involved in plant PCD, indicating the existence of great challenge for confirming new caspase-like proteases and elucidating the mechanisms regulating plant PCD. Here, we report a novel cysteine protease, NtTPE8, which was extracted from tobacco seeds and confirmed as a new caspase-like protease. Recombinant NtTPE8 exhibited legumain and caspase-like proteolytic activities, both of which could be inhibited by the pan-caspase inhibitor (Z-VAD-FMK). Notably, NtTPE8 possessed several caspase activities and the capacity to cleave the cathepsin H substrate FVR, indicating a unique character of NtTPE8. NtTPE8 was exclusively expressed in the integumentary tapetum and thus, is the first specific molecular marker reported to date for this cell type. Down-regulation of NtTPE8 caused seed abortion, via disturbing early embryogenesis, indicating its critical role in embryogenesis and seed development. In conclusion, we identified a novel caspase-like cysteine protease, NtTPE8, exclusively expressed in the integumentary tapetum that is involved in seed development., (© 2018 Institute of Botany, Chinese Academy of Sciences.)

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco ( Nicotiana tabacum ) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Background: Potassium is a nutrient element necessary for tobacco growth. Tobacco leaves with high potassium content are elastic and tough, rich in oil. And the same time, potassium can also improve the scent and aromatic value of flue-cured tobacco by regulating the synthesis of aromatic hydrocarbons in leaves.. It is an important quality indicator for flue-cured tobacco. However, the potassium concentration in tobacco leaves in most areas of China is generally lower than the global standard for high quality tobacco. Two tobacco genotypes were grafted to each other under different potassium levels to test whether potassium content and plant growth can be improved by grafting in tobacco., Results: The growth of tobacco in all treatments was inhibited under potassium starvation, and grafting significantly alleviated this potassium stress in 'Yunyan 87'. The trends in whole plant K + uptake and K + transfer efficiency to the leaves corresponded to the growth results of the different grafts. The nutrient depletion test results showed that the roots of 'Wufeng No.2' had higher K + absorption potential, K + affinity, and K + inward flow rate. K + enrichment circles appeared at the endoderm of the root section in the energy dispersive X-ray figure, indicating that the formation of Casparian strips may be partly responsible for the lower rate of lateral movement of K + in the roots of 'Yunyan 87'. Gene expression analysis suggested that energy redistribution at the whole plant level might constitute one strategy for coping with potassium starvation. The feedback regulation effects between scion 'Wufeng No.2' and rootstock 'Yunyan 87' indicated that the transmission of certain signaling substances had occurred during grafting., Conclusions: 'Wufeng No.2' tobacco rootstock grafting can increase the K + uptake and transport efficiency of 'Yunyan 87' and enhance plant growth under potassium stress. The physiological mechanism of the improved performance of grafted tobacco is related to higher K + uptake and utilization ability, improved xylem K + loading capacity, and up-regulated expression of genes related to energy supply systems.

Glutamyl-tRNA reductase1 (HEMA1) and ferrochelatase1 (FC1) are both expressed in response to salt stress in the biosynthetic pathway of tetrapyrroles. Peanut (Arachis hypogaea L.) HEMA1 and FC1 were isolated by RT-PCR. The amino acid sequence encoded by the two genes showed high similarity with that in other plant species. The AhFC1 fusion protein was verified to function in chloroplast using Arabidopsis mesophyll protoplast. Sense and wild-type (WT) tobaccos were used to further study the physiological effects of AhHEMA1 and AhFC1. Compared with WT, the Heme contents and germination rate were higher in AhFC1 overexpressing plants under salt stress. Meanwhile, overexpressing AhHEMA1 also led to higher ALA and chlorophyll contents and multiple physiological changes under salt stress, such as higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), lower contents of reactive oxygen species (ROS) and slighter membrane damage. In addition, the activities of CAT, POD and APX in the AhFC1 overexpressing plants were significantly higher than that in WT lines under salt stress, but the activity of SOD between the WT plants and the transgenic plants did not exhibit significant differences. These results suggested that, peanut can enhance resistance to salt stress by improving the biosynthesis of tetrapyrrole biosynthetic., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: The transmitting tissue of the style is the pathway for pollen tube growth to the ovules and has components that function in recognizing and discriminating appropriate pollen genotypes. In Nicotiana tabacum, the class III pistil extensin-like (PELPIII) arabinogalactan protein is essential for the inhibition of N. obtusifolia pollen tube growth. The transmitting tissue-specific (TTS) arabinogalactan protein amino acid sequence and expression pattern is similar to PELPIII, but it facilitates self-pollinated N. tabacum. The TTS and PELPIII arabinogalactan protein can be divided into the less conserved N-terminal (NTD) and the more conserved C-terminal (CTD) domains. This research tested whether the NTD is the key domain in determining PELPIII function in the inhibition of interspecific pollen tube growth. Three variant PELPIII gene constructs were produced where the PELPIII NTD was exchanged with the TTS NTD and a single amino acid change (cysteine to alanine) was introduced into the PELPIII NTD. The PELPIII variants of N. tabacum were tested for activity by measuring the inhibition N. obtusifolia pollen tube growth by using them to complement a 3'UTR RNAi transgenic line with reduced PELPIII mRNA., Results: The RNAi N. tabacum line had reduced PELPIII mRNA accumulation and reduced inhibition of N. obtusifolia pollen tube growth, but had no effect on self-pollen tube growth or pollen tube growth of 12 other Nicotiana species. The NTD of PELPIII with either the PELPIII or TTS CTDs complemented the loss PELPIII activity in the RNAi transgenic line as measured by inhibition of N. obtusifolia pollen tube growth. The TTS NTD with the PELPIII CTD and a variant PELPIII with a cysteine to alanine mutation in its NTD failed to complement the loss of PELPIII activity and did not inhibit N. obtusifolia pollen tube growth., Conclusion: The NTD is a key determinant in PELPIII's function in regulating interspecific pollen tube growth and is a first step toward understanding the mechanism of how PELPIII NTD regulates pollen tube growth.

Background: Heat shock proteins 90 (HSP90s) are a highly conserved protein family of cellular chaperones widely found in plants; they play a fundamental role in response to biotic and abiotic stresses. The genome-wide analysis of HSP90 gene family has been completed for some species; however, it has been rarely reported for the tobacco HSP90 genes., Results: In this study, we systematically conducted genome-wide identification and expression analysis of the tobacco HSP90 gene family, including gene structures, evolutionary relationships, chromosomal locations, conserved domains, and expression patterns. Twenty-one NtHSP90s were identified and classified into eleven categories (NtHSP90-1 to NtHSP90-11) based on phylogenetic analysis. The conserved structures and motifs of NtHSP90 proteins in the same subfamily were highly consistent. Most NtHSP90 proteins contained the ATPase domain, which was closely related to conserved motif 2. Motif 5 was a low complexity sequence and had the function of signal peptide. At least 6 pairs of NtHSP90 genes underwent gene duplication, which arose from segment duplication and tandem duplication events. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of HSP90s. Dynamic expression analysis indicated that some NtHSP90 genes may play fundamental roles in regulation of abiotic stress response. The expression of NtHSP90-4, NtHSP90-5, and NtHSP90-9 were up-regulated, while NtHSP90-6, and NtHSP90-7 were not induced by ABA, drought, salt, cold and heat stresses. Among the five treatments, NtHSP90s were most strongly induced by heat stress, and weakly activated by ABA treatment. There was a similar response pattern of NtHSP90s under osmotic stress, or extreme temperature stress., Conclusions: This is the first genome-wide analysis of Hsp90 in N. tabacum. These results indicate that each NtHSP90 member fulfilled distinct functions in response to various abiotic stresses.

APETALA2/ethylene-responsive factor superfamily (AP2/ERF) is a transcription factor involved in abiotic stresses, for instance, cold, drought, and low oxygen. In this study, a novel ethylene-responsive transcription factor named AdRAP2.3 was isolated from Actinidia deliciosa 'Jinkui'. AdRAP2.3 transcription levels in other reproductive organs except for the pistil were higher than those in the vegetative organs (root, stem, and leaf) in kiwi fruit. Plant hormones (Salicylic acid (SA), Methyl-jasmonate acid (MeJA), 1-Aminocyclopropanecarboxylic Acid (ACC), Abscisic acid (ABA)), abiotic stresses (waterlogging, heat, 4 °C and NaCl) and biotic stress ( Pseudomonas Syringae pv. Actinidiae , Psa ) could induce the expression of AdRAP2.3 gene in kiwi fruit. Overexpression of the AdRAP2.3 gene conferred waterlogging stress tolerance in transgenic tobacco plants. When completely submerged, the survival rate, fresh weight, and dry weight of transgenic tobacco lines were significantly higher than those of wile type (WT). Upon the roots being submerged, transgenic tobacco lines grew aerial roots earlier. Overexpression of AdRAP2.3 in transgenic tobacco improved the pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzyme activities, and improved the expression levels of waterlogging mark genes NtPDC , NtADH , NtHB1 , NtHB2 , NtPCO1 , and NtPCO2 in roots under waterlogging treatment. Overall, these results demonstrated that AdRAP2.3 might play an important role in resistance to waterlogging through regulation of PDC and ADH genes in kiwi fruit.

Dehydroascorbate reductase (DHAR) plays an important role in stress responses, but the transcriptional regulation of DHAR in response to abiotic stress is still poorly understood. In this study, we isolated a novel R2R3-type MYB transcription factor from Pyrus betulaefolia by yeast one-hybrid screening, designated as PbrMYB5. PbrMYB5 was localized in the nucleus and could bind specifically to the promoter of PbrDHAR2. PbrMYB5 was greatly induced by cold and salt but slightly by dehydration. Overexpression of PbrMYB5 in tobacco conferred enhanced tolerance to chilling stresses, whereas down-regulation of PbrMYB5 in P. betulaefolia by virus-induced gene silencing resulted in elevated chilling sensitivity. Transgenic tobacco exhibited higher expression levels of NtDHAR2 and accumulated larger amount of ascorbic acid (AsA) than the wild-type plants. Virus-induced gene silencing of PbrMYB5 in P. betulaefolia down-regulated PbrDHAR2 abundance and decreased AsA level, accompanied by an increased sensitivity to the chilling stress. Taken together, these results demonstrated that PbrMYB5 was an activator of AsA biosynthesis and may play a positive role in chilling tolerance, at least in part, due to the modulation of AsA synthesis by regulating the PbrDHAR2 expression., (© 2018 John Wiley & Sons Ltd.)

Cultivars of purple tea (Camellia sinensis) that accumulate anthocyanins in place of catechins are currently attracting global interest in their use as functional health beverages. RNA-seq of normal (LJ43) and purple Zijuan (ZJ) cultivars identified the transcription factor CsMYB75 and phi (F) class glutathione transferase CsGSTF1 as being associated with anthocyanin hyperaccumulation. Both genes mapped as a quantitative trait locus (QTL) to the purple bud leaf color (BLC) trait in F 1 populations, with CsMYB75 promoting the expression of CsGSTF1 in transgenic tobacco (Nicotiana tabacum). Although CsMYB75 elevates the biosynthesis of both catechins and anthocyanins, only anthocyanins accumulate in purple tea, indicating selective downstream regulation. As glutathione transferases in other plants are known to act as transporters (ligandins) of flavonoids, directing them for vacuolar deposition, the role of CsGSTF1 in selective anthocyanin accumulation was investigated. In tea, anthocyanins accumulate in multiple vesicles, with the expression of CsGSTF1 correlated with BLC, but not with catechin content, in diverse germplasm. Complementation of the Arabidopsis tt19-8 mutant, which is unable to express the orthologous ligandin AtGSTF12, restored anthocyanin accumulation, but did not rescue the transparent testa phenotype, confirming that CsGSTF1 did not function in catechin accumulation. Consistent with a ligandin function, transient expression of CsGSTF1 in Nicotiana occurred in the nucleus, cytoplasm and membrane. Furthermore, RNA-Seq of the complemented mutants exposed to 2% sucrose as a stress treatment showed unexpected roles for anthocyanin accumulation in affecting the expression of genes involved in redox responses, phosphate homeostasis and the biogenesis of photosynthetic components, as compared with non-complemented plants., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Cadmium (Cd) toxicity induces oxidative burst and leads to programmed cell death (PCD) in plant cells. The role of salicylic acid-induced protein kinase (SIPK) signaling pathway in Cd-induced oxidative stress was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Barley 21). The cells were pretreated with 40 μM PD98059 (inhibitor of MAPKK) and then exposed to 50 μM Cd for 24 h. The percentages of cell viability, apoptosis, necrosis, and the content of reactive oxygen species (ROS) were monitored by flow cytometry. Expression of PCD related gene (Hsr203J) and the contents of certain signaling molecules were measured as well. The results showed that Cd increased the expression of SIPK, Hsr203J, and CAT genes, the activities of catalase and caspase-3-like enzymes. Addition of PD98059 inhibitor reduced the expression of Hsr203J and CAT genes, decreased CAT activity, but increased ROS and SA contents, and caspase-3-like activity and apoptosis rate. The highest apoptosis level was accompanied by the highest level of Hsr203J gene expression. From the results it can be suggested that upon treatment of tobacco cells with Cd, internal SA content increased and induced the SIPK signaling pathway, thereby inhibited the antioxidant system and led to PCD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

β-Aminobutyric acid (BABA) pre-treatment has been shown to alter both biotic and abiotic stress responses. The present study extends this observation to acclimative UV-B-response, which has not been explored in this context so far. A single soil application of 300 ppm BABA modified the non-enzymatic antioxidant capacities and the leaf hydrogen peroxide levels in tobacco (Nicotiana tabacum L.) leaves in response to a 9-day treatment with 5.4 kJ m-2 d-1 biologically effective supplementary UV-B radiation in a model experiment that was performed in a growth chamber. BABA decreased leaf hydrogen peroxide levels both as a single factor and in combination with UV-B, but neither BABA nor UV-B affected leaf photochemistry significantly. The total antioxidant capacities were increased by either BABA or UV-B, and this response was additive in BABA pre-treated leaves. These results together with the observed changes in hydroxyl radical neutralising ability and non-enzymatic hydrogen peroxide antioxidant capacities show that BABA pre-treatment (i) has a long-term effect on leaf antioxidants even in the absence of other factors and (ii) modifies acclimative readjustment of prooxidant-antioxidant balance in response to UV-B. BABA-inducible antioxidants do not include phenolic compounds as a UV-B-induced increase in the adaxial leaf flavonoid index and total leaf extract UV absorption were unaffected by BABA.

Recent strong restrictions on the use of pesticides has prompted the search for safer alternatives, being antimicrobial peptides promising candidates. Herein, with the aim of identifying new agents, 15 peptides reported as plant defense elicitors, promiscuous, multifunctional or antimicrobial were selected and tested against six plant pathogenic bacteria of economic importance. Within this set, KSL-W (KKVVFWVKFK-NH 2 ) displayed high antibacterial activity against all the tested pathogens, low hemolysis and low phytotoxicity in tobacco leaves. This peptide was taken as a lead and 49 analogues were designed and synthesized, including N-terminal deletion sequences, peptides incorporating a d-amino acid and lipopeptides. The screening of these sequences revealed that a nine amino acid length was the minimum for activity. The presence of a d-amino acid significantly decreased the hemolysis and endowed KSL-W with the capacity to induce the expression of defense-related genes in tomato plants. The incorporation of an acyl chain led to sequences with high activity against Xanthomonas strains, low hemolysis and phytotoxicity. Therefore, this study demonstrates that KSL-W constitutes an excellent candidate as new agent to control plant diseases and can be considered as a lead to develop derivatives with multifunctional properties, including antimicrobial and plant defense elicitation., (Copyright © 2018 Elsevier Inc. All rights reserved.)