Efficient plant regeneration through direct shoot organogenesis and two-step rooting in Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver (E. ulmoides Oliver), a multipurpose woody plant, holds great economic significance due to its expansive medicinal, food and industrial applications. The rapid advancement of E. ulmoides in various fields has resulted in the inadequacy of existing breeding methods to meet its growth and annual production demands. Consequently, there is an urgent need for innovative propagation strategies. This study introduces an optimized micropropagation protocol for E. ulmoides, facilitating direct shoot organogenesis from nodal segments with axillary buds. We systematically examined the impact of basal medium composition, plant growth regulators, photosynthetic photon flux density, and sucrose concentration on bud sprouting. Employing cuttings with axillary buds as propagation material, we achieved a shortened cultivation period of merely 4 weeks for bud elongation and proliferation, marking a substantial enhancement in propagation efficiency. Notably, the Driver Kuniyuki Walnut medium, supplemented with 20.0 g L-1 sucrose and 2.0 mg L-1 trans-zeatin, induced shoots sprouting with a 100% success rate and an average length of 5.18 cm per nodal segment, equating to a great bud propagation rate of approximately 500%. Furthermore, a light source with an intensity of 80 µmol m-2 s-1 was shown the most economical choice. To address the primary challenge of inducing roots in regenerated plants, we employed a refined two-step rooting technique. This method yielded the optimal rooting frequency of 93.02%, producing an average of 5.90 adventitious roots per plantlet, each with an average length of 2.77 cm. The micropropagation program developed in this work will be the cornerstone for the preservation of the germplasm of E. ulmoides and its long-term use in medicinal and industrial applications.

In vitro regeneration, photomorphogenesis and light signaling gene expression in Hydrangea quercifolia cv. 'Harmony' under different LED environments.

MAIN CONCLUSION: Plant growth regulators, sucrose concentration, and light quality significantly impact in vitro regeneration of 'Harmony'. Blue light promotes photomorphogenesis by enhancing light energy utilization, adjusting transcription of light signal genes, and altering hormone levels. Hydrangea quercifolia cv. 'Harmony', celebrated for lush green foliage and clusters of white flowers, has been extensively researched for its regenerative properties. Regeneration in stem segments, leaves, and petioles is facilitated by exogenous auxin and cytokinins (CTKs), with the concentration of sucrose (SC) being a key determinant for shoot regeneration from leaves. The study also highlights the significant impact of light conditions on photomorphogenesis. With an increase in the proportion of red (R) light, there is an inhibitory effect, leading to a reduction in leaf area, a decrease in the quantum yield of PSII (ΦPSII), and an increase in non-photochemical quenching (ΦNPQ) and non-regulated energy dissipation in PSII (ΦNO). Conversely, blue (B) light enhances growth, characterized by an increase in leaf area, elevated ΦPSII, and stable ΦNPQ and ΦNO levels. Additionally, B light induces the upregulation of HqCRYs, HqHY5-like, HqXTH27-like, and HqPHYs genes, along with an increase in endogenous CTKs levels, which positively influence photomorphogenesis independent of HqHY5-like regulation. This light condition also suppresses the synthesis of endogenous gibberellins (GA) and brassinosteroids (BR), further facilitating photomorphogenesis. In essence, B light is fundamental in expediting photomorphogenesis in 'Harmony', demonstrating the vital role in plant growth and development.

Plant morphology, secondary metabolites and chlorophyll fluorescence of Artemisia argyi under different LED environments.

Different light spectra from light-emitting diodes (LEDs) trigger species-specific adaptive responses in plants. We exposed Artemisia argyi (A. argyi) to four LED spectra: white (the control group), monochromatic red light (R), monochromatic blue light (B), or a mixture of R and B light of photon flux density ratio is 3 (RB), with equivalent photoperiod (14 h) and light intensity (160 µmol s-1 m-2). R light accelerated photomorphogenesis but decreased biomass, while B light significantly increased leaf area and short-term exposure (7 days) to B light increased total phenols and flavonoids. HPLC identified chlorogenic acid, 3,5-dicaffeoylquinic acid, gallic acid, jaceosidin, eupatilin, and taxol compounds, with RB and R light significantly accumulating chlorogenic acid, 3,5-dicaffeoylquinic acid, and gallic acid, and B light promoting jaceosidin, eupatilin, and taxol. OJIP measurements showed that B light had the least effect on the effective quantum yield ΦPSII, with higher rETR(II), Fv/Fm, qL and PIabs, followed by RB light. R light led to faster photomorphology but lower biomass than RB and B lights and produced the most inadaptability, as shown by reduced ΦPSII and enlarged ΦNPQ and ΦNO. Overall, short-term B light promoted secondary metabolite production while maintaining effective quantum yield and less energy dissipation.

Correction: Wang, Y.T., et al. Selenium Nanoparticle Synthesized by Proteus mirabilis YC801: An Efficacious Pathway for Selenite Biotransformation and Detoxification. Int. J. Mol. Sci. 2018, 19, 3809.

The authors wish to make the following corrections to this paper [...].

Karrikin Improves Osmotic and Salt Stress Tolerance via the Regulation of the Redox Homeostasis in the Oil Plant Sapium sebiferum.

Karrikins are reported to stimulate seed germination, regulate seedling growth, and increase the seedling vigor in abiotic stress conditions in plants. Nevertheless, how karrikins alleviate abiotic stress remains largely elusive. In this study, we found that karrikin (KAR1) could significantly alleviate both drought and salt stress in the important oil plant Sapium sebiferum. KAR1 supplementation in growth medium at a nanomolar (nM) concentration was enough to recover seed germination under salt and osmotic stress conditions. One nanomolar of KAR1 improved seedling biomass, increased the taproot length, and increased the number of lateral roots under abiotic stresses, suggesting that KAR1 is a potent alleviator of abiotic stresses in plants. Under abiotic stresses, KAR1-treated seedlings had a higher activity of the key antioxidative enzymes, such as superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase, in comparison with the control, which leads to a lower level of hydrogen peroxide, malondialdehyde, and electrolyte leakage. Moreover, the metabolome analysis showed that KAR1 treatment significantly increased the level of organic acids and amino acids, which played important roles in redox homeostasis under stresses, suggesting that karrikins might alleviate abiotic stresses via the regulation of redox homeostasis. Under abiotic stresses, applications of karrikins did not increase the endogenous abscisic acid level but altered the expression of several ABA signaling genes, such as SNF1-RELATED PROTEIN KINASE2.3, SNF1-RELATED PROTEIN KINASE2.6, ABI3, and ABI5, suggesting potential interactions between karrikins and ABA signaling in the stress responses. Conclusively, we not only provided the physiological and molecular evidence to clarify the mechanism of karrikins in the regulation of stress adaptation in S. sebiferum but also showed the potential value of karrikins in agricultural practices, which will lay a foundation for further studies about the role of karrikins in abiotic stress alleviation in plants.

Correction: Wang, Y.T., et al. Selenite Reduction and the Biogenesis of Selenium Nanoparticles by Alcaligenes faecalis Se03 Isolated from the Gut of Monochamus alternatus (Coleoptera: Cerambycidae). Int. J. Mol. Sci. 2018, 19, 2799.

The authors wish to make the following corrections to this paper [...].

Genome-wide identification, characterization and expression pattern analysis of APYRASE family members in response to abiotic and biotic stresses in wheat.

APYRASEs, which directly regulate intra- and extra-cellular ATP homeostasis, play a pivotal role in the regulation of various stress adaptations in mammals, bacteria and plants. In the present study, we identified and characterized wheat APYRASE family members at the genomic level in wheat. The results identified a total of nine APY homologs with conserved ACR domains. The sequence alignments, phylogenetic relations and conserved motifs of wheat APYs were bioinformatically analyzed. Although they share highly conserved secondary and tertiary structures, the wheat APYs could be mainly categorized into three groups, according to phylogenetic and structural analysis. Additionally, these APYs exhibited similar expression patterns in the root and shoot, among which TaAPY3-1, TaAPY3-3 and TaAPY3-4 had the highest expression levels. The time-course expression patterns of the eight APYs in response to biotic and abiotic stress in the wheat seedlings were also investigated. TaAPY3-2, TaAPY3-3, TaAPY3-4 and TaAPY6 exhibited strong sensitivity to all kinds of stresses in the leaves. Some APYs showed specific expression responses, such as TaAPY6 to heavy metal stress, and TaAPY7 to heat and salt stress. These results suggest that the stress-inducible APYs could have potential roles in the regulation of environmental stress adaptations. Moreover, the catalytic activity of TaAPY3-1 was further analyzed in the in vitro system. The results showed that TaAPY3-1 protein exhibited high catalytic activity in the degradation of ATP and ADP, but with low activity in degradation of TTP and GTP. It also has an extensive range of temperature adaptability, but preferred relatively acidic pH conditions. In this study, the genome-wide identification and characterization of APYs in wheat were suggested to be useful for further genetic modifications in the generation of high-stress-tolerant wheat cultivars.

Overexpression of the Stress-Inducible SsMAX2 Promotes Drought and Salt Resistance via the Regulation of Redox Homeostasis in Arabidopsis.

Recent studies have demonstrated that strigolactones (SLs) also participate in the regulation of stress adaptation; however, the regulatory mechanism remains elusive. In this study, the homolog of More Axillary Branches 2, which encodes a key component in SL signaling, in the perennial oil plant Sapium sebiferum was identified and functionally characterized in Arabidopsis. The results showed that the expression of SsMAX2 in S. sebiferum seedlings was stress-responsive, and SsMAX2 overexpression (OE) in Arabidopsis significantly promoted resistance to drought, osmotic, and salt stresses. Moreover, SsMAX2 OE lines exhibited decreased chlorophyll degradation, increased soluble sugar and proline accumulation, and lower water loss ratio in response to the stresses. Importantly, anthocyanin biosynthesis and the activities of several antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX), were enhanced in the SsMAX2 OE lines, which further led to a significant reduction in hydrogen peroxide levels. Additionally, the SsMAX2 OE lines exhibited higher expression level of several abscisic acid (ABA) biosynthesis genes, suggesting potential interactions between SL and ABA in the regulation of stress adaptation. Overall, we provide physiological and biochemical evidence demonstrating the pivotal role of SsMAX2 in the regulation of osmotic, drought, and salt stress resistance and show that MAX2 can be a genetic target to improve stress tolerance.

Selenium Nanoparticle Synthesized by Proteus mirabilis YC801: An Efficacious Pathway for Selenite Biotransformation and Detoxification.

Selenite is extremely biotoxic, and as a result of this, exploitation of microorganisms able to reduce selenite to non-toxic elemental selenium (Se°) has attracted great interest. In this study, a bacterial strain exhibiting extreme tolerance to selenite (up to 100 mM) was isolated from the gut of adult Monochamus alternatus and identified as Proteus mirabilis YC801. This strain demonstrated efficient transformation of selenite into red selenium nanoparticles (SeNPs) by reducing nearly 100% of 1.0 and 5.0 mM selenite within 42 and 48 h, respectively. Electron microscopy and energy dispersive X-ray analysis demonstrated that the SeNPs were spherical and primarily localized extracellularly, with an average hydrodynamic diameter of 178.3 ± 11.5 nm. In vitro selenite reduction activity assays and real-time PCR indicated that thioredoxin reductase and similar proteins present in the cytoplasm were likely to be involved in selenite reduction, and that NADPH or NADH served as electron donors. Finally, Fourier-transform infrared spectral analysis confirmed the presence of protein and lipid residues on the surfaces of SeNPs. This is the first report on the capability of P. mirabilis to reduce selenite to SeNPs. P. mirabilis YC801 might provide an eco-friendly approach to bioremediate selenium-contaminated soil/water, as well as a bacterial catalyst for the biogenesis of SeNPs.

Selenite Reduction and the Biogenesis of Selenium Nanoparticles by Alcaligenesfaecalis Se03 Isolated from the Gut of Monochamus alternatus (Coleoptera: Cerambycidae).

In this study, a bacterial strain exhibiting high selenite (Na2SeO3) tolerance and reduction capacity was isolated from the gut of Monochamus alternatus larvae and identified as Alcaligenes faecalis Se03. The isolate exhibited extreme tolerance to selenite (up to 120 mM) when grown aerobically. In the liquid culture medium, it was capable of reducing nearly 100% of 1.0 and 5.0 mM Na2SeO3 within 24 and 42 h, respectively, leading to the formation of selenium nanoparticles (SeNPs). Electron microscopy and energy dispersive X-ray analysis demonstrated that A. faecalis Se03 produced spherical electron-dense SeNPs with an average hydrodynamic diameter of 273.8 ± 16.9 nm, localized mainly in the extracellular space. In vitro selenite reduction activity and real-time PCR indicated that proteins such as sulfite reductase and thioredoxin reductase present in the cytoplasm were likely to be involved in selenite reduction and the SeNPs synthesis process in the presence of NADPH or NADH as electron donors. Finally, using Fourier-transform infrared spectrometry, protein and lipid residues were detected on the surface of the biogenic SeNPs. Based on these observations, A. faecalis Se03 has the potential to be an eco-friendly candidate for the bioremediation of selenium-contaminated soil/water and a bacterial catalyst for the biogenesis of SeNPs.

Flower Development and Sex Determination between Male and Female Flowers in Vernicia fordii.

Vernicia fordii is a monoecious and diclinous species with male and female flowers on the same inflorescence. Low female to male flower ratio is one of the main reasons for low yield in this species. However, little is known of its floral development and sex determination. Here, according to the results of scanning electron microscopy and histological analysis, the floral development of V. fordii was divided into 12 stages and the first morphological divergence between the male and female flowers was found to occur at stage 7. The male flowers are always unisexual, but the female flowers present bisexual characteristics, with sterile stamen (staminode) restricted to pre-meiosis of mother sporogenous cells and cell death occurring at later development stages. To further elucidate the molecular mechanism underling sex determination at the divergence stage for male and female flowers, comparative transcriptome analysis was performed. In total, 56,065 unigenes were generated and 608 genes were differentially expressed between male and female flowers, among which 310 and 298 DEGs (differentially expressed genes) showed high expression levels in males and females, respectively. The transcriptome data showed that the sexual dimorphism of female flowers was affected by jasmonic acid, transcription factors, and some genes related to the floral meristem activity. Ten candidate genes showed consistent expression in the qRT-PCR validation and DEGs data. In this study, we provide developmental characterization and transcriptomic information for better understanding of the development of unisexual flowers and the regulatory networks underlying the mechanism of sex determination in V. fordii, which would be helpful in the molecular breeding of V. fordii to improve the yield output.

Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum.

Chinese tallow (Sapium sebiferum L.) is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB) were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt). TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae).

Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure-function relationships between enzymatic properties, toxicity and structural characterization of SEBIN.

Flower bud transcriptome analysis of Sapium sebiferum (Linn.) Roxb. and primary investigation of drought induced flowering: pathway construction and G-quadruplex prediction based on transcriptome.

Sapium sebiferum (Linn.) Roxb. (Chinese Tallow Tree) is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA) and triacylglycerol (TAG) biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.

Generating DNA sequences encoding tandem peptide repeats suitable for expression and immunological application.

Tandem repeats of single short peptide sequences are useful for many purposes. Here we describe a method called ligation-PCR to construct DNA sequences encoding numerous tandem peptide repeats that can stably produce such repeats in both prokaryotic and eukaryotic cells. The method employs double-strand target monomers consisting of a short peptide coding sequences. These sequences contain 3-bp cohesive overhangs to ensure correct repeat orientation and reading frame during ligation. The ligation products are PCR amplified and directly cloned into a new TA-cloning vector, pZeroT. Constructs containing tandem 10-amino-acid myc-tag peptide coding sequence repeats that ranged from approximately 0.45-1.2 kb, representing 15-40 copies of the corresponding peptide, were successfully obtained by this method. When one of the constructs was subcloned into prokaryotic vector pET-28 c (+) and eukaryotic vector rGHpcDNA3.0, and introduced into E. Coli and COS-7 cells, respectively, proteins containing tandem myc-tag peptide repeats were expressed with expected molecular weights. Purified proteins from E. Coli could successfully stimulate a peptide specific immune response. This method provides a means to manipulate peptides at the nucleic acid level, and can serve as the basis for biological peptide synthesis, epitope-specific antibody production, and epitope-based DNA vaccine construction.