Effects of Barium Stress in <i>Brassica juncea</i> and <i>Cakile maritima</i>: The Indicator Role of Some Antioxidant Enzymes and Secondary Metabolites

Soil contamination by toxic trace metal elements, like barium (Ba), may stimulate various undesirable changes in the metabolic activity of plants. The plant responses are fast and with, direct or indirect, generation of reactive oxygen species (ROS). To cope with the stress imposed by the ROS production, plants developed a dual cellular system composed of enzymatic and non-enzymatic players that convert ROS, and their by-products, into stable nontoxic molecules. To assess the Ba stress response of two Brassicaceae species (Brassica juncea, a glycophyte, and Cakile maritime, a halophyte), plants were exposure to different Ba concentrations (0, 100, 200, 300 and 500 μM). The plants response was evaluated through their morphology and development, the determination of plant leaves antioxidant enzymatic activities and by the production of plants secondary metabolites. Results indicated that the two Brassicaceae species have the ability to survive in an environment containing Ba (even at 500 μM). The biomass production of C. maritima was slightly affected whereas an increase in biomass B. juncea was noticed. The stress imposed by Ba activated the antioxidant defense system in the two species, noticed by the changes in the leaves activity of catalase (CAT), ascorbate peroxidase (APX) and guaicol peroxidase (GPX), and of the secondary metabolites, through the production of total phenols and flavonoids. The enzymatic response was not similar within the two plant species: CAT and APX seem to have a more important role against the oxidative stress in C. maritima while in B. juncea is GPX. Overall, total phenols and flavonoids production was more significant in the plants aerial part than in the roots, of the both species. Although the two Brassicaceae species response was different, in both plants catalytic and non-catalytic transformation of ROS occurs, and both were able to overcome the Ba toxicity and prevent the cell damage.     

Phytochemical Analysis and Antioxidant Activity of <i>Crocus speciosus</i> Leaves

The numerous studies indicate leaves of plants are a rich source of bioactive compounds that can be a valuable source of compounds used in the pharmaceutical and cosmetic industries. Aim of this study was to investigate the chemical composition and the antioxidant property of Crocus speciosus leaves. Primary phytochemical screening of C. speciosus leaves revealed the presence of some following compound categories such as phenolic compounds, aminoacids, saponins, proteins, tannins, triterpenoids, glycosides, polysaccharides. The total flavonoids and phenolic compounds content were determined spectrophotometrically and by HPLC-DAD and HPLC-MS. Antiradical activity was determined by ABTS radical-cation scavenging method, spectrophotometrically. The total amount of flavonoids in C. speciosus leaves was 1.07 ± 0.02 mg RE/g (p < 0.05), the total amount of phenolic compounds was 0.41 ± 0.01 mg GAE/g (p < 0.05). By HPLC-DAD-MS analysis the presence of the mangiferin, chlorogenic acid, isoorientin, kaempferol, hyperoside, and isoquercitin was established for the first time in Crocus leaves. The antiradical activity of C. speciosus leaves extracts was 150.08 ± 4.5 μmol/g (p < 0.05) and its was mainly attributed to phenolic compounds content. The high amounts of flavonoids and antiradical activity in C. speciosus leaves suggests promising phytochemical and pharmacological study of this Crocus species.     

Extraction,Antioxidant Activity and Identification of Flavonoids from Root Tubers of <i>Kosteletzkya virginica</i>

Kosteletzkya virginica (K. virginica) is used for revegetation of salt-affected coastal tidal flats and as a raw material of biodiesel. K. virginica root tuber, a biowaste with low economic value, is rich in bioactive compounds. This study aimed to extract and identify flavonoids from K. virginica root tubers. The optimal extraction conditions were 1/25 (w/v) solid/liquid ratio, 40% ethanol concentration at 40°C for 60 min. Under these conditions, 65.2 ± 3.7 mg/g total flavonoid content was extracted from the roots, which were collected from salinized soil in late autumn of the third year. Antioxidant activity was evaluated through 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical, and superoxide anion scavenging assays. The extracted flavonoids exhibited antioxidant activity in a dose-dependent manner. Five flavonoids, glucoliquiritin apioside, licoisoflavone B, 5-methoxy-7,8-diprenyl- flavone, 7,2′-dihydroxy-6,8-dimethyl-4′,5′-methylenedioxyflavan, and 5,7,4′-trihydroxy-3′-methoxy-6,8-di-Cmethylflavanone, were identified by ultra-performance liquid chromatography–tandem mass spectrometry. Our results suggest that the flavonoids of K. virginica root tubers might be potent antioxidants and can be effectively applied as an ingredient in food and natural medicine.     

Screening of <i>Bacillus subtilis</i> HAAS01 and Its Biocontrol Effect on <i>Fusarium</i> wilt in Sweet Potato

Fusarium wilt, a disease caused by Fusarium oxysporum f.sp batatas (Fob) is an important disease in sweet potato production. Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways. A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture. According to the biological characteristics, 16S rDNA sequence analysis, and physiological and biochemical analysis, the Bacillus subtilis HAAS01 was named. A pot experiment was conducted for the biological control experiment of strain HAAS01, and the endogenous hormone content, antioxidant enzyme activity, soluble protein content, and related gene expressions of sweet potato plants were detected. The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions. In summary, Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.     

Identification of Resistance to Pathogenesis Related Protein <i>GmPR1L</i> in Tobacco <i>Botrytis cinerea</i> Infection

Soybean (Glycine max (Linn.) Merr.) annual leguminous crop is cultivated all over the world. The occurrence of diseases has a great impact on the yield and quality of soybean. In this study, based on the RNA-seq of soybean variety M18, a complete CDS (Coding sequence) GmPR1L of the pathogenesis-related protein 1 family was obtained, which has the ability to resist fungal diseases. The overexpression vector and interference expression vector were transferred into tobacco NC89, and the resistance of transgenic tobacco (Nicotiana tabacum L.) to Botrytis cinerea infection was identified. The results show that: Compared with the control, the activities of related defense enzymes SOD (Superoxide dismutase), POD (Peroxidase), PAL (L-phenylalanine ammonia-lyase) and PPO (Polyphenol oxidase) in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees, and increased significantly at different infection time points. The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains. The results of resistance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line, and the disease spot rate of OEA2 was significantly lower than that of the control line. The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line. It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B. cinerea.     

Selection and Verification of Reference Genes for qRT-PCR Analysis in <i>Iris domestica</i> under Drought

Iris domestica is a plant of the Iridaceae family and is drought-tolerant, but its drought-resistance mechanism is not yet clear. Analysing the gene expression changes of I. domestica by qRT-PCR is an important mean to understand its drought resistance characteristics. Nevertheless, a lack of reference genes greatly hinders investigation and research on the adaptation of I. domestica to drought at the molecular and genetic levels. In this study, we assessed the expression stability of 11 candidate gene in I. domestica under drought stress conditions and different tissues using geNorm, NormFinder, BestKeeper and RefFinder tools. The results showed that EF1β was the most stable reference genes under drought stress and in different tissues. To validate further the stability of the identified reference genes, the expression patterns of VP gene in I. domestica was analysed. These results will be conducive to more accurate quantification of gene expression levels in I. domestica.     

<i>In vitro</i> Germination and Micropropagation of <i>Aconitum vilmorinianum</i>: An Important Medicinal Plant in China

Aconitum vilmorinianum, a well-known traditional Chinese herb, is recently being threatened by overexploitation and environment disturbance. This study was conducted to provide propagation methods through in vitro germination and explant cultivation. Germination was stimulated up to 66.00% on Murashige and Skoog (MS) medium containing 2.0 mg L⁻¹ 6-benzylaminopurine (BAP), 0.1 mg L⁻¹ 1-napthaleneacetic acid (NAA), and 30 g L⁻¹ sucrose. Three bacteria (Pantoea agglomerans, Erwinia persicina, and Pseudomonas tolaasii) would be responsible for consistent contamination during germination. The latter two were effectively eradicated after disinfected. The influence of explant types and hormone combinations on direct and indirect organogenesis was evaluated in the present work. The frequency of shoot induction from axillary bud explants was 100% on the MS fortified with 2.0 mg L⁻¹ BAP and 0.3 mg L⁻¹ NAA. Shoots multiplication was optimized on MS medium supplemented with 0.1 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA. High callus induction percentage (96.67%) was obtained from stem segments on MS medium with 2.0 mg L⁻¹ 2,4-D, then successfully regenerated into shoots on MS medium in the presence of 0.1 mg L⁻¹ TDZ and 0.2 mg L⁻¹ NAA. The present work could be useful for the utilization and conservation of this valuable species.     

Exogenous Spermidine Promotes Somatic Embryogenesis of <i>Cunninghamia lanceolata</i> by Altering the Endogenous Phytohormone Content

In order to study how exogenous hormones in C. lanceolata (gymnosperm) regulate somatic embryogenesis, we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic embryogenesis. By applying exogenous spermidine, we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone content. Based on these results, further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic embryogenesis. Our research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.     

Comparison of Morphological and Anatomical Characteristics of <i>Taxus chinensis</i> var. <i>mairei </i>Seedlings Root under Waterlogging Stress in Different Substrates

Four different ratios of river sand, ceramic pellets, vermiculite and perlite (1:1), and field soil were selected as the substrates in this experiment, and four gradient levels of root waterlogging, half waterlogging, full waterlogging and normal were set to investigate the effects of different gradients of waterlogging stress on the root morphology of Taxus chinensis var. mairei seedlings under different substrates. In this study, the root anatomical structure of Taxus chinensis var. mairei under different waterlogging stress was observed by the paraffin section method. The roots of T. chinensis var. mairei were diarch, with no pith and resin canals. There was a large number of tannins in the pericycle of the aerial adventitious roots of seedlings adapted to waterlogging. Also, the endodermis has obvious casparian strip thickening, and there were 4-5 layers of large parenchymatous cells in the close to the inner side of the pericycle in the vascular cylinder, which could increase the storage capacity, and transport capacity of the root. Under the treatment of root waterlogging stress, the development of plant roots in the mixed substrate of vermiculite and, perlite was the earliest. Under half waterlogging stress, T. chinensis var. mairei seedlings treated with various substrates all could better adapt to the environment of waterlogging stress. Under the stress of fully waterlogging, the roots of seedlings planted in river sand substrate developed secondary growth.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

Overexpression of <i>IbSINA5</i> Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Seven in absentia (SINA) family proteins play a central role in plant growth, development and resistance to abiotic stress. However, their biological function in plant response to cold stress is still largely unknown. In this work, a seven in absentia gene IbSINA5 was isolated from sweet potato. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato, with a predominant expression in fibrous roots, and was remarkably induced by cold, drought and salt stresses. Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus. Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants, which was associated with the up-regulated expression of IbCOR (cold-regulated) genes, increased proline production, and decreased malondialdehyde (MDA) and H₂O₂ accumulation in the leaves of transgenic plants. Furthermore, transient expression of IbCBF3, a C-repeat binding factor (CBF) gene, in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes. Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato, and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.     

Mycorrhizal Fungal Effects on Growth,Antioxidant Capacity,and Medicine Quality of <i>Paris polyphylla</i> var. <i>yunnanensis</i>

A field experiment was conducted to determine the effects of two commercial strains composed of mulple arbuscular mycorrhizal fungi (AMF) species on plant growth, antioxidant capacity, and medicine quality of Paris polyphylla var. yunnanensis in three subtropical soils from Wanzhou, Anshun and Baoshan in fields. The results showed that AMF inoculation enhanced the fungal colonization rate and activities of both succinate dehydrogenase and alkaline phosphatase, thereby, enhancing the mycorrhizal viability of P. polyphylla var. yunnanensis. The concentrations of photosynthetic pigments (chlorophyll a, b, a+b and carotenoids), soluble sugar, soluble protein and photosynthetic capacity were higher in AMF-inoculated plants than in non-AMF-treated plants in field. AMFtreated plants recorded higher activities of catalase, peroxidase and superoxide dismutase, and caused the reduction in malondialdehyde content, indicating lower oxidative damage, compared with non-AMF plants. Polyphyllin I, Polyphyllin II, Polyphyllin III, Polyphyllin IV and total polyphyllin contents were increased by AMF treatment. In conclusion, AMF improved the plant growth, antioxidant capacity and medicinal quality of P. polyphylla var. yunnanensis seedlings. Hereinto, AMF effects on the soil from Wanzhou was relatively greater than on other soils.     

Improvement of Selected Morphological,Physiological, and Biochemical Parameters of Banana (<i>Musa acuminata</i> L.) Using Potassium Silicate under Drought Stress Condition Grown <i>in vitro</i>

Drought stress has become more common in recent years as a result of climate change impacts on the production of banana crops and other fruit trees. The growth and productivity of Musa spp are severely impacted by the gradual degradation of water resources and the erratic distribution pattern of annual precipitation amount. The aim of the work includes increased drought tolerance in light of water scarcity in the world as a result of the bananas’ being gluttonous for water needs. This investigation was carried out from 2019 to 2020 to study the effect of potassium silicate on morphological growth and biochemical parameters of Musa acuminata L under drought stress by PEG. As a result, drought stress reduced the morphological characteristics such as shoots number, shoot length, roots number, and survival percentage and biochemical characteristics such as chlorophyll a, b, carotenoids, stomatal status, and RWC. While proline content increased in the leaf of M. acuminata L. Media complemented with K₂SiO₃ (2 to 6 mM) either individually or in combination with PEG led to an improvement in all morphological and biochemical characteristics. The activities of CAT, POD, and PPO enzymes increased signifi- cantly compared to control. Furthermore, the lowest PPO, CAT, and POD activity were achieved. Additionally, K₂SiO₃ treatments under drought stress successfully enhanced the leaf stomatal behavior. Our results suggest that K₂SiO₃ can help to maintain plant integrity in the tested cultivar under drought stress.     

Effects of Exogenous Manganese (Mn) on Mineral Elements,Polyamines and Antioxidants in Apple Rootstock <i>Malus robusta</i> Rehd.

Manganese (Mn) is one of the essential microelements in all organisms. However, high level of Mn is deleterious to plants. In this study, the effects of exogenous manganese application on mineral element, polyamine (PA) and antioxidant accumulation, as well as polyamine metabolic and antioxidant enzyme activities, were investigated in Malus robusta Rehd., a widely grown apple rootstock. High level of Mn treatments decreased endogenous Mg, Na, K and Ca contents, but increased Zn content, in a Mn-concentration-dependent manner. Polyamine metabolic assays revealed that, except the content of perchloric acid insoluble bound (PIS-bound) spermine, which increased significantly, the contents of putrescine (Put), spermidine (Spd) and spermine (Spm) all decreased progressively, accompanied with the decreased activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), and the increased activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.5.3.3). Further antioxidant capacity analyses demonstrated that contents of anthocyanin, non-protein thiols (NPT) and soluble sugar, and the activities of guaiacol peroxidase (POD, EC 1.11.1.7), catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1), also increased upon different concentrations of Mn treatments. Our results suggest that endogenous ion homeostasis is affected by high level of Mn application, and polyamine and antioxidant metabolism is involved in the responses of M. robusta Rehd. plants to high level of Mn stress.     

Identification and Evaluation of Insect and Disease Resistance in Transgenic <i>Cry1Ab13-1</i> and <i>NPR1</i> Maize

PCR detection, quantitative real-time PCR (q-RTPCR), outdoor insect resistance, and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations (T₂, T₃, and T₄) in transgenic maize germplasms (S3002 and 349) containing the bivalent genes (insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1) and their corresponding wild type. Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations; q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots, stems, and leaves of tested maize plants. In addition, S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type. Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit. These findings could be exploited for improving other cultivated maize varieties.