Transcriptomic analysis of potato (Solanum tuberosum L.) tuber development reveals new insights into starch biosynthesis.

Potato tubers are rich sources of various nutrients and unique sources of starch. Many genes play major roles in different pathways, including carbohydrate metabolism during the potato tuber's life cycle. Despite substantial scientific evidence about the physiological and morphological development of potato tubers, the molecular genetic aspects of mechanisms underlying tuber formation have not yet been fully understood. In this study, for the first time, RNA-seq analysis was performed to shed light on the expression of genes involved in starch biosynthesis during potato tuber development. To this end, samples were collected at the hook-like stolon (Stage I), swollen tips stolon (Stage II), and tuber initiation (Stage III) stages of tuber formation. Overall, 23 GB of raw data were generated and assembled. There were more than 20000 differentially expressed genes (DEGs); the expression of 73 genes involved in starch metabolism was further studied. Moreover, qRT-PCR analysis revealed that the expression profile of the starch biosynthesis DEGs was consistent with that of the RNA-seq data, which further supported the role of the DEGs in starch biosynthesis. This study provides substantial resources on potato tuber development and several starch synthesis isoforms associated with starch biosynthesis.

Bunium Persicum Seeds Extract in Combination with Vincristine Mediates Apoptosis in MCF-7 Cells through Regulation of Involved Genes and Proteins Expression.

BACKGROUND: Bunium persicum seeds, a member of the Apiaceae family, have historically been consumed as part of the Iranian diet. OBJECTIVE: While many of this herb's biological properties have been fully investigated, there is currently no reliable information about its anticancer/cytotoxic properties. METHODS: Herein, we first determined the major bioactive compounds of B. persicum seed extract (BPSE) via GC-Mass analysis. We evaluated the cytotoxicity of the extract alone as well as in combination with vincristine (VCR), a commonly used chemotherapy drug, using MTT assays on two breast cancer cell lines, MCF-7 and MDA-MB-231, as well as a normal breast cancer cell line, MCF-10A. Moreover, these compounds were evaluated in vitro for their anticancer activity using ROS assays, Real-Time PCR, Western blots, flow cytometry, and cell cycle assays. RESULTS: As a result of our investigation, it was determined that the extract significantly reduced the viability of cancerous cells while remaining harmless to normal cells. The combination of BPSE and VCR also resulted in synergistic effects. BPSE and/or BPSE-VCR treatment increased the intracellular ROS of MCF-7 cells by over twofold. Moreover, the IC30 of BPSE (100 µg/ml) significantly increased the BAX/BCL-2 and P53 gene expression while reducing the expression of the MYC gene. Moreover, treated cells were arrested in the G2 phase of the cell cycle. The BPSE-VCR combination synergistically reduced the NF-κB and increased the Caspase-7 proteins' expression. The percent of apoptosis in the cells treated with the extract, VCR, and their combination was 27, 11, and 50, respectively. CONCLUSIONS: The present study demonstrated the anticancer activity of the BPSE and its potential for application in combination therapy with VCR.

Comparative transcriptome analysis to identify putative genes involved in carvacrol biosynthesis pathway in two species of Satureja, endemic medicinal herbs of Iran.

Satureja is rich in phenolic monoterpenoids, mainly carvacrol, that is of interest due to diverse biological activities including antifungal and antibacterial. However, limited information is available regarding the molecular mechanisms underlying carvacrol biosynthesis and its regulation for this wonderful medicinal herb. To identify the putative genes involved in carvacrol and other monoterpene biosynthesis pathway, we generated a reference transcriptome in two endemic Satureja species of Iran, containing different yields (Satureja khuzistanica and Satureja rechingeri). Cross-species differential expression analysis was conducted between two species of Satureja. 210 and 186 transcripts related to terpenoid backbone biosynthesis were identified for S. khuzistanica and S. rechingeri, respectively. 29 differentially expressed genes (DEGs) involved in terpenoid biosynthesis were identified, and these DEGs were significantly enriched in monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, carotenoid biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways. Expression patterns of S. khuzistanica and S. rechingeri transcripts involved in the terpenoid biosynthetic pathway were evaluated. In addition, we identified 19 differentially expressed transcription factors (such as MYC4, bHLH, and ARF18) that may control terpenoid biosynthesis. We confirmed the altered expression levels of DEGs that encode carvacrol biosynthetic enzymes using quantitative real-time PCR (qRT-PCR). This study is the first report on de novo assembly and transcriptome data analysis in Satureja which could be useful for an understanding of the main constituents of Satureja essential oil and future research in this genus.

Genome-wide transcriptional profiling provides clues to molecular mechanisms underlying cold tolerance in chickpea.

Chickpea is an important food legume cultivated in several countries. A sudden drop in autumn temperature, freezing winter temperature, and late spring cold events result in significant losses in chickpea production. The current study used RNA sequencing of two cold tolerant (Saral) and sensitive (ILC533) Kabuli chickpea genotypes to identify cold tolerance-associated genes/pathways. A total of 200.85 million raw reads were acquired from the leaf samples by Illumina sequencing, and around 86% of the clean reads (199 million) were mapped to the chickpea reference genome. The results indicated that 3710 (1980 up- and 1730 down-regulated) and 3473 (1972 up- and 1501 down-regulated) genes were expressed differentially under cold stress in the tolerant and sensitive genotypes, respectively. According to the GO enrichment analysis of uniquely down-regulated genes under cold stress in ILC533, photosynthetic membrane, photosystem II, chloroplast part, and photosystem processes were enriched, revealing that the photosynthesis is severely sensitive to cold stress in this sensitive genotype. Many remarkable transcription factors (CaDREB1E, CaMYB4, CaNAC47, CaTCP4, and CaWRKY33), signaling/regulatory genes (CaCDPK4, CaPP2C6, CaMKK2, and CaHSFA3), and protective genes (CaCOR47, CaLEA3, and CaGST) were identified among the cold-responsive genes of the tolerant genotype. These findings would help improve cold tolerance across chickpea genotypes by molecular breeding or genetic engineering.

Genome-wide identification and expression profiling of snakin/GASA genes under drought stress in barley (Hordeum vulgare L.).

Gibberellic Acid-Stimulated Arabidopsis (GASA) proteins are present in various plants and have a role in plant growth, stress responses, and hormone crosstalk. GASA coding sequences in barley were discovered in this study. We then investigated gene and protein structure, physicochemical characteristics, evolutionary and phylogenetic relationships, promoter region, post-translational modification, and in silico gene expression. Finally, real-time quantitative PCR (RT-qPCR) was used to examine the expression of GASA genes in root and shoot tissues under drought stress. We found 11 GASA genes spread across six of seven chromosomes in the barley genome. A conserved GASA domain and 12-cysteine residues at the C-terminus were included in the proteins. All GASA genes contained secretory signal peptides. The GASA genes in Hordeum vulgare (HvGASA) have been classified into three subfamilies based on evolutionary analysis. According to synteny analyses, segmental duplications are significant in forming the GASA gene family. According to the cis-elements analyses, GASA genes may be induced by a variety of phytohormones and stresses. Tissue-specific expression analysis indicated that GASA genes had varied expression patterns in different tissues. Contrary to common perception, the expression study of GASA genes under biotic and abiotic stresses revealed that GASA genes are more induced by abiotic stresses than biotic stresses. The qPCR confirmed the response of GASA genes to abiotic stresses and showed different expression patterns of these genes under drought stress. Overall, these results can improve our knowledge about the function of GASA genes and provide data for future researches. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03545-8.

Assessment of ACC and P450 Genes Expression in Wild Oat (Avena ludoviciana) in Different Tissues Under Herbicide Application.

Target-site resistance (TSR) and non-target-site resistance (NTSR) to herbicides in arable weeds are increasing rapidly all over the world and threatening universal food safety. Resistance to herbicides that inhibit ACCase activity has been identified in wild oat. In this study, expression of ACC1, ACC2, CYP71R4 and CYP81B1 genes under herbicide stress conditions were studied in two TSR (resistant in the residue Ile1781-Leu and Ile2041-Asn of ACCase) biotypes, two NTSR biotypes and one susceptible biotype of A. ludoviciana for the first time. Treated and untreated biotypes with ACCase-inhibitor clodinafop propargyl herbicide were sampled from the stem and leaf tissues at 24 h after treatment. Our results showed an increase in gene expression levels in different tissues of both types of resistance biotypes that occurred under herbicide treatment compared with non-herbicide treatment. In all samples, the expression levels of leaf tissue in all studied genes were higher than in stem tissue. The results of ACC gene expression showed that the expression level of ACC1 was significantly higher than that of ACC2. Also, expression levels of TSR biotypes were higher than NTSR biotypes for the ACC1 gene. For both CYP71R4 and CYP81B1 genes, the expression ratio increased significantly in TSR and NTSR biotypes in different tissues after herbicide treatment. In contrast, the expression levels of CYP genes in NTSR biotypes were higher than in TSR biotypes. Our results support the hypothesis that the reaction of plants to herbicide is carried out through a different regulation of genes, which can be the result of the interaction of resistance type in the target or non-target-site.

Transcriptome analysis of gall oak (Quercus infectoria): De novo assembly, functional annotation and metabolic pathways analysis.

Gall oak (Quercus infectoria) is a native tree of Iran, whose gall extract is used to treat many diseases. The presence of abundant secondary metabolites with various bioactivities in this plant has made it medically important. Despite its medicinal value, due to the lack of genomic information, the biosynthetic pathways of these compounds in this species are still unknown. The current research was aimed at observing, characterizing, and investigating the biosynthetic pathways of these compounds in Q.infectoria. De novo transcriptome assembly was conducted using the RNA sequencing technique. A total of 89,335 unigenes were generated, of which 6928 unigenes showed differential expression in leaves compared to root tissue. Gene ontology examination of DEGs revealed GO-term enrichment was related to cellular processes and enzyme activity. KEGG enrichment analysis for DEGs showed that most unigenes were related to metabolic pathways and biosynthesis of secondary metabolites. Moreover, 39 families of transcription factors were identified, of which the C2H2, bZIP, bHLH, and ERF TFs had the highest frequency. In the absence of a reference genome, the overall study of transcriptome will provide a reference for future functional and comparative studies. Moreover, the data obtained from sequencing and de novo assembly can be a valuable scientific resource for Q.infectoria.

Transcriptome Analysis of Persian Oak (Quercus brantii L.) Decline Using RNA-seq Technology.

Since the late 1980s, the oak decline has affected the Zagros oak forests in western Iran. Persian oak (Quercus brantii L.) the most important tree species of these forests has been damaged more than any other plant species. In the present study, the RNA sequencing technique was used for the first time to identify key genes and molecular mechanisms involved in Persian oak decline. The RNA was extracted from the leaves of healthy and declined oak trees, and sequenced using the Illumina HiSeq 2500 platform (2 × 150 bp paired-end reads). De novo transcriptome assembly of Persian oak revealed 56,743 unigenes and 6049 differentially expressed genes (DEGs) between declined and control samples. The results of gene ontology analysis showed that most of the DEGs involved in oak decline belong to the group of stress-responsive genes. In general, oak decline samples showed significant reductions in gene expression associated with "photosynthesis and storage of sugar" and "protein synthesis and related processes." Additionally, DEGs related to the starch degradation pathway were up-regulated, whereas DEGs associated with acetate-mevalonate (MVA), biosynthesis of lignin, and lignases pathways were down-regulated. The present study's findings can be an effective step in identifying the genes involved in oak decline and deciphering the relationship between this phenomenon and biotic and abiotic stresses.

CRISPR/Cas StNRL1 gene knockout increases resistance to late blight and susceptibility to early blight in potato.

With the development of genome editing technologies, editing susceptible genes is a promising method to modify plants for resistance to stress. NPH3/RPT2-LIKE1 protein (NRL1) interacts with effector Pi02860 of Phytophthora infestans and creates a protein complex, promoting the proteasome-mediated degradation of the guanine nucleotide exchange factor SWAP70. SWAP70, as a positive regulator, enhances cell death triggered by the perception of the P. infestans pathogen-associated molecular pattern (PAMP) INF1. Using a clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a construct was made to introduce four guide RNAs into the potato cultivar Agria. A total of 60 putative transgenic lines were regenerated, in which 10 transgenic lines with deletions were selected and analyzed. A mutant line with a four-allelic knockdown of StNRL1 gene was obtained, showing an ~90% reduction in StNRL1 expression level, resulting in enhanced resistance to P. infestans. Surprisingly, mutant lines were susceptible to Alternaria alternata, suggesting that StNRL1 may play a role as a resistance gene; hence, silencing StNRL1 enhances resistance to P. infestans.

Expression of apple MdMYB10 transcription factor in sugar beet with a screenable marker role and antimicrobial activity.

Selection of transgenic plants by using genes encoding screenable markers of plant origin with health benefit properties, such as anthocyanin is an important aim in plant genetic engineering. In this study, Malus domestica MYB10 (MdMYB10) gene, was used for Agrobacterium tumefaciens-mediated transformation of two SBS-02 and SBS-04 sugar beet lines. The impact of different light regimes on plant tissue culture from a combination of light, dark/light and dark was investigated. The results of this study showed that the MdMYB10 gene was successfully integrated into the selected purple transgenic lines, suggesting that the expression of MdMYB10 gene in sugar beet shoots can be used as a screenable markers for transformation, possibly replacing antibiotic resistant genes. Furthermore, the results of the antibacterial activity of transgenic plants extracts showed that the total extract obtained from transgenic lines significantly (P < 0.01) inhibited the growth and development of Enterococcus faecium and Enterococcus faecalis bacteria compared to the non-transgenic plants. The results of this study showed that the combination of betalain with vancomycin demonstrated a synergistic antimicrobial effect, also, suggesting that the expression of MdMYB10 may play a dual role by accumulating betalain and exhibiting a screenable markers function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03120-7.

Identification of key genes and molecular mechanisms associated with temperature stress in lentil.

Extreme temperature is one of the serious threats to crop production in present and future scenarios of global climate changes. Lentil (Lens culinaris) is an important crop, and there is a serious lack of genetic information regarding environmental and temperature stresses responses. This study is the first report of evaluation of key genes and molecular mechanisms related to temperature stresses in lentil using the RNA sequencing technique. De novo transcriptome assembly created 44,673 contigs and differential gene expression analysis revealed 7494 differentially expressed genes between the temperature stresses and control group. Basic annotation of generated transcriptome assembly in our study led to the identification of 2765 novel transcripts that have not been identified yet in lentil genome draft v1.2. In addition, several unigenes involved in mechanisms of temperature sensing, calcium and hormone signaling and DNA-binding transcription factor activity were identified. Also, common mechanisms in response to temperature stresses, including the proline biosynthesis, the photosynthetic light reactions balancing, chaperone activity and circadian rhythms, are determined by the hub genes through the protein-protein interaction networks analysis. Deciphering the mechanisms of extreme temperature tolerance would be a new way for developing crops with enhanced plasticity against climate change. In general, this study has identified set of mechanisms and various genes related to cold and heat stresses which will be useful in better understanding of the lentil's reaction to temperature stresses.

Transcriptome analysis of bread wheat leaves in response to salt stress.

Salinity is one of the main abiotic stresses limiting crop productivity. In the current study, the transcriptome of wheat leaves in an Iranian salt-tolerant cultivar (Arg) was investigated in response to salinity stress to identify salinity stress-responsive genes and mechanisms. More than 114 million reads were generated from leaf tissues by the Illumina HiSeq 2500 platform. An amount of 81.9% to 85.7% of reads could be mapped to the wheat reference genome for different samples. The data analysis led to the identification of 98819 genes, including 26700 novel transcripts. A total of 4290 differentially expressed genes (DEGs) were recognized, comprising 2346 up-regulated genes and 1944 down-regulated genes. Clustering of the DEGs utilizing Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that transcripts associated with phenylpropanoid biosynthesis, transporters, transcription factors, hormone signal transduction, glycosyltransferases, exosome, and MAPK signaling might be involved in salt tolerance. The expression patterns of nine DEGs were investigated by quantitative real-time PCR in Arg and Moghan3 as the salt-tolerant and susceptible cultivars, respectively. The obtained results were consistent with changes in transcript abundance found by RNA-sequencing in the tolerant cultivar. The results presented here could be utilized for salt tolerance enhancement in wheat through genetic engineering or molecular breeding.

Dissecting the molecular responses of lentil to individual and combined drought and heat stresses by comparative transcriptomic analysis.

Lentil cultivation could be challenged by combined heat and drought stress in semi-arid regions. We used RNA-seq approach to profile transcriptome changes of Lens culinaris exposed to individual and combined heat and drought stresses. It was determined that most of the differentially expressed genes observed in response to combined stress, could not be identified by analysis of transcriptome exposed to corresponding individual stresses. Interestingly, this study results revealed that the expression of ribosome generation and protein biosynthesis and starch degradation pathways related genes were uniquely up-regulated under the combined stress. Although multiple genes related to antioxidant activity were up-regulated in response to all stresses, variation in types and expression levels of these genes under the combined stress were higher than that of individual stresses. Using this comparative approach, for the first time, we reported up-regulation of several TF, CDPK, CYP, and antioxidant genes in response to combined stress in plants.

New Recombinant Antimicrobial Peptides Confer Resistance to Fungal Pathogens in Tobacco Plants.

Antimicrobial peptides have been long known to confer resistance to plant pathogens. In this study, new recombinant peptides constructed from a dermaseptin B1 (DrsB1) peptide fused to a chitin-binding domain (CBD) from Avr4 protein, were used for Agrobacterium tumefaciens-mediated transformation of tobacco plants. Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and western blotting analysis demonstrated the incorporation and expression of transgenes in tobacco genome and transgenic plants, respectively. In vitro experiments with recombinant peptides extracted from transgenic plants demonstrated a significant (P<0.01) inhibitory effect on the growth and development of plant pathogens. The DrsB1-CBD recombinant peptide had the highest antifungal activity against fungal pathogens. The expression of the recombinant peptides greatly protected transgenic plants from Alternaria alternata, Alternaria solani, Fusarium oxysporum, and Fusarium solani fungi, in comparison to Pythium sp. and Pythium aphanidermatum. Expression of new recombinant peptides resulted in a delay in the colonization of fungi and appearance of fungal disease symptoms from 6 days to more than 7 weeks. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, cling together, and crushed following the antimicrobial activity of the recombinant peptides. Greenhouse bioassay analysis showed that transgenic plants were more resistant to Fusarium and Pythium infections as compared with the control plants. Due to the high antimicrobial activity of the recombinant peptides against plant pathogens and novelty of recombinant peptides, this report shows the feasibility of this approach to generate disease resistance transgenic plants.

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco.

Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin-binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD-DrsB1 and pGSA/DrsB1-CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi-quantitative RT-PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD-DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1-CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

Transcriptome response of roots to salt stress in a salinity-tolerant bread wheat cultivar.

Salt stress is one of the major adverse environmental factors limiting crop productivity. Considering Iran as one of the bread wheat origins, we sequenced root transcriptome of an Iranian salt tolerant cultivar, Arg, under salt stress to extend our knowledge of the molecular basis of salinity tolerance in Triticum aestivum. RNA sequencing resulted in more than 113 million reads and about 104013 genes were obtained, among which 26171 novel transcripts were identified. A comparison of abundances showed that 5128 genes were differentially expressed due to salt stress. The differentially expressed genes (DEGs) were annotated with Gene Ontology terms, and the key pathways were identified using Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway mapping. The DEGs could be classified into 227 KEGG pathways among which transporters, phenylpropanoid biosynthesis, transcription factors, glycosyltransferases, glutathione metabolism and plant hormone signal transduction represented the most significant pathways. Furthermore, the expression pattern of nine genes involved in salt stress response was compared between the salt tolerant (Arg) and susceptible (Moghan3) cultivars. A panel of novel genes and transcripts is found in this research to be differentially expressed under salinity in Arg cultivar and a model is proposed for salt stress response in this salt tolerant cultivar of wheat employing the DEGs. The achieved results can be beneficial for better understanding and improvement of salt tolerance in wheat.

Correction to: Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity.

The original version of this article unfortunately contained a mistake in the unit "µg/l". The unit "µg/l" should be corrected to "µg/ml" throughout the paper.

Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity.

Expression of strong antimicrobial peptides in plants is of great interest to combat a wide range of plant pathogens. To bring the Dermaseptin B1 (DrsB1) peptide to the intimate contact of the plant pathogens cell wall surface, the DrsB1 encoding sequence was fused to the C-terminal part of the two copies of the chitin-binding domain (CBD) of the Avr4 effector protein and used for Agrobacterium rhizogenes-mediated transformation. The expression of the recombinant protein in the tobacco hairy roots (HRs) was confirmed by molecular analysis. Antimicrobial activity analysis of the recombinant protein purified from the transgenic HRs showed that the (CBD)2-DrsB1 recombinant protein had a significant (p < 0.01) antimicrobial effect on the growth of different fungal and bacterial pathogens. The results of this study indicated that the recombinant protein had a higher antifungal activity against chitin-producing Alternaria alternata than Pythium spp. Scanning electron microscopy images demonstrated that the recombinant protein led to fungal hypha deformation, fragmentation, and agglutination of growing hypha, possibly by dissociating fungal cell wall components. In vitro evidences suggest that the expression of the (CBD)2-DrsB1 recombinant protein in plants by generating transgenic lines is a promising approach to produce disease-resistant plants, resistance to chitin-producing pathogenic fungi.

Hydroponic grown tobacco plants respond to zinc oxide nanoparticles and bulk exposures by morphological, physiological and anatomical adjustments.

Zinc oxide nanoparticles (NPs) are the third highest in terms of global production among the various inorganic nanoparticles, and there are concerns because of their worldwide availability and accumulation in the environment. In contrast, zinc is an essential element in plant growth and metabolism, and ZnO NPs (nano-ZnO) may have unknown interactions with plants due to their small sizes as well as their particular chemical and physical characteristics. The present study examined the effect of nano-ZnO (25nm) and bulk or natural form (<1000nm, bulk-ZnO), compared with zinc in the ionic form (ZnSO4) on Nicotiana tabacum seedlings in a nutrient solution supplemented with either nano-ZnO, bulk-ZnO (0.2, 1, 5 and 25µM) or ZnSO4 (control) for 21 days. Results showed that nano-ZnO at most of the levels and 1µM bulk-ZnO positively affected growth (root and shoot length/dry weight), leaf surface area and its metabolites (auxin, phenolic compounds, flavonoids), leaf enzymatic activities (CAT, APX, SOD, POX, GPX, PPO and PAL) and anatomical properties (root, stem, cortex and central cylinder diameters), while bulk-ZnO caused decreases at other levels. The activities of enzymes were induced to a greater extent by intermediate nano-ZnO levels than by extreme concentrations, and were higher in nano-ZnO treated than in bulk treated tobacco. As the ZnO level increased, the vascular expansion and cell wall thickening of the collenchyma/parenchyma cells occurred, which was more pronounced when treated by NPs than by its counterpart. The Zn content of root and leaf increased in most of ZnO treatments, whereas the Fe content of leaves decreased. Our findings indicate that tobacco responded positively to 1µM bulk-ZnO and to nearly all nano-ZnO levels (with the best levels being at 0.2µM and 1µM) by morphological, physiological and anatomical adjustments.