N-Rich and Sulfur-Doped Nano Hollow Carbons with High Oxidase-like Activity Prepared Using a Green Template of CaCO3 for Bacteriostasis.

Nanozymes, enzyme-mimicking nanomaterials, have attracted increasing attention due to their low cost, high stability, and catalytic ability compared with natural enzymes. However, the catalytic efficiency of the nanozymes is still relatively low, and catalytic reaction mechanisms remain unclear. To address these issues, herein we prepared nitrogen-riched and sulfur-codoped nano hollow carbons (N/S-HCS) using a green and useful template of CaCO3. N/S-HCS exhibits enhanced oxidase-like activity and catalytic kinetic performance. It could directly oxidize the colorless 3,3',5,5'-tetramethylbenzidine (TMB) to the heavy blue colored ox-TMB without H2O2. The maximum reaction rate (Vmax) is 186.7 × 10-8 M·s-1, and Michaelis-Menten constant (Km) is 0.162 mM. DFT results show that N and S codoping could work synergistically to provide more active sites, resulting in the superior ability to adsorb oxygen and enhanced catalytic activity. Meantime, we develop a multispectral characterization strategy to unravel catalytic reaction mechanisms about N/S-HCS. It successfully induces the generation of superoxide (•O2-) and hydroxyl (•OH) during the colorimetric reaction which are the key intermediate products of the catalytic reaction. Furthermore, N/S-HCS increased the cellular reactive oxygen species level significantly and induced bacteriostasis to more than 95% of Escherichia coli.

IlAP2, an AP2/ERF Superfamily Gene, Mediates Cadmium Tolerance by Interacting with IlMT2a in Iris lactea var. chinensis.

Cadmium (Cd) stress has a major impact on ecosystems, so it is important to find suitable Cd-tolerant plants while elucidating the responsible molecular mechanism for phytoremediation to manage Cd soil contamination. Iris lactea var. chinensis is an ornamental perennial groundcover plant with strong tolerance to Cd. Previous studies found that IlAP2, an AP2/ERF superfamily gene, may be an interacting partner of the metallothionein gene IlMT2a, which plays a key role in Cd tolerance. To study the role of IlAP2 in regulating Cd tolerance in I. lactea, we analyzed its regulation function and mechanism based on a yeast two-hybrid assay, a bimolecular fluorescence complementation test, quantitative real-time PCR, transgenics and transcriptome sequencing. The results showed that IlAP2 interacts with IlMT2a and may cooperate with other transcription factors to regulate genes involved in signal transduction and plant hormones, leading to reduced Cd toxicity by hindering Cd transport. These findings provide insights into the mechanism of IlAP2-mediated stress responses to Cd and important gene resources for improving plant stress tolerance in phytoremediation.

Tobacco rattle virus-induced VcANS gene silencing in blueberry fruit.

Blueberry (Vaccinium corymbosum L.), a woody perennial bush in the genus Vaccinium, is an economically important and popular fruit crop worldwide. Development the superior cultivars, which including excellent fruit traits, not only means higher yielding and economic efficiency, but also produce fruit that to meet the preferences of different consumers. Excavating fruit quality-related genes, studying their functions, and using transgenic or molecular-assisted breeding are beneficial to the development of excellent blueberry varieties. Genetic transformation is an excellent way to study the function of genes in plants, however, it is a labor-intensive and time-consuming process to genetically transform many woody plants, including blueberry. Virus-induced gene silencing (VIGS) provides an efficient approach to knock-down the expression of target genes for functional analysis. In this study, tobacco rattle virus induced genes silencing (TRV-VIGS) was established in blueberry fruits using the VcANS gene as a reporter. The silenced sector of the skin of blueberry fruits injected with pTRV2 (plasmid Tobacco Rattle Virus, TRV-RNA2)::VcANS remained green or white at 25 days after agroinfiltration. In agroinfiltrated materials, the VcANS transcript levels were much lower in fruits with phenotypic changes (delayed color change) than in those infiltrated with the pTRV2 empty vector. Silencing of VcANS also affected the expression of other genes involved in the anthocyanin synthesis pathway. The experimental results support that VcANS can be used as an effective marker gene for VIGS system. In addition, the TRV-VIGS system has been successfully established in blueberry fruits, which provided an effective verification method for functional identification of unknown genes in blueberry fruits.

Genome-wide study of the GRAS gene family in Hibiscus hamabo Sieb. et Zucc and analysis of HhGRAS14-induced drought and salt stress tolerance in Arabidopsis.

GRAS proteins are widely distributed plant-specific transcription factors. In this study, we identified 59 GRAS proteins (HhGRASs) from the genomic and transcriptomic datasets of Hibiscus hamabo Sieb. et Zucc. These proteins were phylogenetically divided into nine subfamilies. RNA-seq analysis revealed that most HhGRASs were expressed in response to abiotic stresses. Results from quantitative real-time PCR analysis of nine selected HhGRASs suggested that HhGRAS14 was significantly upregulated under multiple abiotic stresses; therefore, this gene was selected for further study. Silencing HhGRAS14 in H. hamabo reduced the tolerance to drought and salt stress, while overexpression in Arabidopsis thaliana significantly increased the tolerance to drought and salt and reduced the sensitivity to abscisic acid (ABA). In summary, we analyzed the GRAS family of proteins in semi-mangrove plants for the first time and identified a gene that responds to drought and salt stress, which provided the basis for a comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in H. hamabo.

Genome-Wide Identification and Characterization of NAC Family in Hibiscus hamabo Sieb. et Zucc. under Various Abiotic Stresses.

NAC transcription factor is one of the largest plant gene families, participating in the regulation of plant biological and abiotic stresses. In this study, 182 NAC proteins (HhNACs) were identified based on genomic datasets of Hibiscus hamabo Sieb. et Zucc (H. hamabo). These proteins were divided into 19 subfamilies based on their phylogenetic relationship, motif pattern, and gene structure analysis. Expression analysis with RNA-seq revealed that most HhNACs were expressed in response to drought and salt stress. Research of quantitative real-time PCR analysis of nine selected HhNACs supported the transcriptome data's dependability and suggested that HhNAC54 was significantly upregulated under multiple abiotic stresses. Overexpression of HhNAC54 in Arabidopsis thaliana (A. thaliana) significantly increased its tolerance to salt. This study provides a basis for a comprehensive analysis of NAC transcription factor and insight into the abiotic stress response mechanism in H. hamabo.

Selection and validation of appropriate reference genes for RT-qPCR analysis of flowering stages and different genotypes of Iris germanica L.

Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in '00246' and 'Elizabeth', and IgTUB and IgUBC showed stable expression in '2010200'. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

Screening and Identification of Host Proteins Interacting with Iris lactea var. chinensis Metallothionein IlMT2a by Yeast Two-Hybrid Assay.

Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis) is a well-known cadmium (Cd)-tolerant plant and we have previously shown that the metallothionein gene, IlMT2a, of the plant may be playing a key role in conferring the Cd tolerance. In this study, we have identified several proteins interacting with the IlMT2a by screening yeast two-hybrid library constructed from cDNAs isolated from Cd-treated I. lacteal var. chinensis plants. Putative functions of these proteins include those involved in photosynthesis, ROS scavenge, nutrient transport, and transcriptional regulation, to name a few. In particular, another metallothionein, which we assigned the name of IlMT3, was identified as an interacting partner of the IlMT2a. Unlike IlMT2a, it did not provide any significant protection against Cd toxicity in transgenic Arabidopsis thaliana L. (A. thaliana). To our knowledge, this is the first time ever reporting the interaction of two metallothionein proteins in plants. Learning the biological significance of the interaction between IlMT2a and IlMT3 would be the focus of future study and would be able to provide valuable insights into the understanding plant metallothionein's diverse and complex roles in coordinating many important cellular physiologies including stress responses, gene regulations, and energy metabolisms.

Full-Length Transcriptome Sequencing and Comparative Transcriptome Analysis to Evaluate Drought and Salt Stress in Iris lactea var. chinensis.

Iris lactea var. chinensis (I. lactea var. chinensis) is a perennial herb halophyte with salt and drought tolerance. In this study, full-length transcripts of I. lactea var. chinensis were sequenced using the PacBio RSII sequencing platform. Moreover, the transcriptome was investigated under NaCl or polyethylene glycol (PEG) stress. Approximately 30.89 G subreads were generated and 31,195 unigenes were obtained by clustering the same isoforms by the PacBio RSII platform. A total of 15,466 differentially expressed genes (DEGs) were obtained under the two stresses using the Illumina platform. Among them, 9266 and 8390 DEGs were obtained under high concentrations of NaCl and PEG, respectively. In total, 3897 DEGs with the same expression pattern under the two stresses were obtained. The transcriptome expression profiles of I. lactea var. chinensis under NaCl or PEG stress obtained in this study may provide a resource for the same and different response mechanisms against different types of abiotic stress. Furthermore, the stress-related genes found in this study can provide data for future molecular breeding.

Phylogenetic and Transcription Analysis of Hibiscus hamabo Sieb. et Zucc. WRKY Transcription Factors.

WRKY transcription factors are known to play important roles in the regulation of various aspects of plant growth and development, including germination, stress resistance, and senescence. Nevertheless, there is little information about the WRKY genes in Hibiscus hamabo Sieb. et Zucc., an important semimangrove plant. In this study, HhWRKY genes in H. hamabo were identificated based on Illumina RNA-sequencing and isoform sequencing from salt-treated roots. Then phylogenetic analysis and conserved motif analysis of the WRKY family in H. hamabo and Arabidopsis thaliana were used to classify WRKY genes. Sixteen HhWRKY genes were selected from different groups to detect their expression patterns using real-time quantitative PCR in different organ (root, old leaf, tender leaf, receptacle, petal, or stamen) from 10-year-old H. hamabo plants grown in their natural environment and in seedlings with 8 to 10 true leaves challenged by phytohormone (salicylic acid, methyl jasmonate, or abscisic acid) and abiotic stress (drought, salt, or high temperature). As a result, the identified 78 HhWRKY genes were divided into two major groups and several subgroups based on their structural and phylogenetic features. Most transcripts of the selected 16 HhWRKY genes were more abundant in old than in tender leaves of H. hamabo. HhWRKY genes were regulated in reaction to abiotic stresses and phytohormone treatments and may participate in signaling networks to improve plant stress resistance. Some of HhWRKY genes behaved as would be predicted based on their homology with A. thaliana WRKY genes, but others showed divergent behavior. This systematic analysis lays the foundation for further identification of WRKY gene functions, with the aim of improving woody plants.

Iris lactea var. chinensis (Fisch.) cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

IlCDT1, a cysteine-rich protein, was isolated from Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis). Its transcription was up-regulated by the exogenous application of Cd. The truncated IlCDT1 (25-54) containing 14 Cys residues confers Cd tolerance to yeast as the intact IlCDT1, indicating that Cys residues are required for Cd tolerance presumably by chelating Cd. When the gene was constitutively expressed in A. thaliana, root length of transgenic lines was longer than that of wild-type under 100 µM or 200 µM Cd stress. However, Cd absorption in wild-type was more than in two trangenic lines under 100 µM Cd exposure. IlCDT1 may directly bind Cd, through chelating Cd and avoiding the Cd uptake into the cells. Together, IlCDT1 may be a promising gene for the Cd tolerance improvement. SUMMARY: Cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

De novo sequencing, assembly, and analysis of Iris lactea var. chinensis roots' transcriptome in response to salt stress.

As a halophyte, Iris lactea var. chinensis (I. lactea var. chinensis) is widely distributed and has good drought and heavy metal resistance. Moreover, it is an excellent ornamental plant. I. lactea var. chinensis has extensive application prospects owing to the global impacts of salinization. To better understand its molecular mechanism involved in salt resistance, the de novo sequencing, assembly, and analysis of I. lactea var. chinensis roots' transcriptome in response to salt-stress conditions was performed. On average, 74.17% of the clean reads were mapped to unigenes. A total of 121,093 unigenes were constructed and 56,398 (46.57%) were annotated. Among these, 13,522 differentially expressed genes (DEGs) were identified between salt-treated and control samples Compared to the transcriptional level of control, 7037 DEGs were up-regulated and 6539 down-regulated. In addition, 129 up-regulated and 1609 down-regulated genes were simultaneously detected in all three pairwise comparisons between control and salt-stressed libraries. At least 247 and 250 DEGs encoding transcription factors and transporter proteins were identified. Meanwhile, 130 DEGs regarding reactive oxygen species (ROS) scavenging system were also summarized. Based on real-time quantitative RT-PCR, we verified the changes in the expression patterns of 10 unigenes. Our study identified potential salt-responsive candidate genes and increased the understanding of halophyte responses to salinity stress.

De novo characterization of the Iris lactea var. chinensis transcriptome and an analysis of genes under cadmium or lead exposure.

Iris lactea var. chinensis (I. lactea var. chinensis) is tolerant to accumulations of cadmium (Cd) and lead (Pb). In this study, the transcriptome of I. lactea var. chinensis was investigated under Cd or Pb stresses. Using the gene ontology database, 31,974 unigenes were classified into biological process, cellular component and molecular function. In total, 13,132 unigenes were involved in enriched Encyclopedia of Genes and Genomes (KEGG) metabolic pathways, and the expression levels of 5904 unigenes were significantly changed after exposure to Cd or Pb stresses. Of these, 974 were co-up-regulated and 1281 were co-down-regulated under the two stresses. The transcriptome expression profiles of I. lactea var. chinensis under Cd or Pb stresses obtained in this study provided a resource for identifying common mechanisms in the detoxification of different heavy metals. Furthermore, the identified unigenes may be used for the genetic breeding of heavy-metal tolerant plants.

Transcriptome Profiling of Louisiana iris Root and Identification of Genes Involved in Lead-Stress Response.

Louisiana iris is tolerant to and accumulates the heavy metal lead (Pb). However, there is limited knowledge of the molecular mechanisms behind this feature. We describe the transcriptome of Louisiana iris using Illumina sequencing technology. The root transcriptome of Louisiana iris under control and Pb-stress conditions was sequenced. Overall, 525,498 transcripts representing 313,958 unigenes were assembled using the clean raw reads. Among them, 43,015 unigenes were annotated and their functions classified using the euKaryotic Orthologous Groups (KOG) database. They were divided into 25 molecular families. In the Gene Ontology (GO) database, 50,174 unigenes were categorized into three GO trees (molecular function, cellular component and biological process). After analysis of differentially expressed genes, some Pb-stress-related genes were selected, including biosynthesis genes of chelating compounds, metal transporters, transcription factors and antioxidant-related genes. This study not only lays a foundation for further studies on differential genes under Pb stress, but also facilitates the molecular breeding of Louisiana iris.

The heterologous expression of the Iris lactea var. chinensis type 2 metallothionein IlMT2b gene enhances copper tolerance in Arabidopsis thaliana.

Iris lactea var. chinensis (I. lactea var. chinensis) is a widely adapted perennial species with a high level of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in I. lactea var. chinensis, a full-length cDNA homologue of MT2, designated IlMT2b (GenBank accession No. AB907788), was cloned using the RACE-PCR method. The expression level of IlMT2b in the leaves and roots of I. lactea var. chinensis was induced in response to copper (Cu) treatment. Ectopic expression of IlMT2b in Arabidopsis thaliana increased the Cu concentration and reduced H2O2 production in the transgenic plants. After treatment with 50 and 100 µM Cu, the root length of two transgenic seedlings was respectively about 1.5- and 3-fold longer than that of the wild-type. Together, these results suggested that IlMT2b may represent a useful target gene for the phytoremediation of Cu-polluted soil.

Reference gene selection for quantitative real-time RT-PCR normalization in Iris. lactea var. chinensis roots under cadmium, lead, and salt stress conditions.

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1 α ), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (C t ) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

Overexpression of Iris. lactea var. chinensis metallothionein llMT2a enhances cadmium tolerance in Arabidopsis thaliana.

Metallothioneins (MTs) are cysteine-rich, low molecular weight, heavy metal-binding protein molecules. Here, a full-length cDNA homologue of MT2a (type 2 metallothionein) was isolated from the cadmium-tolerant species Iris. lactea var. chinensis (I. lactea var. chinensis). Expression of IlMT2a in I. lactea var. chinensis roots and leaves was up-regulated in response to cadmium stress. When the gene was constitutively expressed in Arabidopsis thaliana (A. thaliana), root length of transgenic lines was longer than that of wild-type under 50µM or 100µM cadmium stress. However, there was no difference of cadmium absorption between wild-type and trangenic lines. Histochemical staining by 3,3-diaminobenzidine (DAB) and nitroblue tetrazoliu (NBT) clearly demonstrated that transgenic lines accumulated remarkably less H2O2 and O2(-) than wild-type. Together, IlMT2a may be a promising gene for the cadmium tolerance improvement.