Rapid Profiling and Characterization of the Multicomponents from the Root and Rhizome of <i>Salvia miltiorrhiza</i> by Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry in Combination with Computational Peak Annotation Workflows

Herbal components characterization represents a challenging task because of the co-existing of multiple classes of naturally occurring compounds with wide spans of polarity, molecular mass, and the ubiquitous isomerism. The root and rhizome of Salvia miltiorrhiza have been utilized as a reputable traditional Chinese medicine Salviae Miltiorrhizae Radix et Rhizoma (Dan-Shen) in the treatment of cardiovascular disease. Herein, a dimension-enhanced ultra-high performance liquid chromatography/ion mobility/quadrupole time-of-flight mass spectrometry approach in combination with intelligent peak annotation workflows was established aimed to rapidly characterize the multicomponents from S. miltiorrhiza. Due to the sufficient optimization, satisfactory chromatography separation was enabled on an HSS T3 column within 33 min using 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phase, while the data-independent HDMS^E in both the negative and positive electrospray ionization modes was utilized for the high-coverage MS² data acquisition. Streamlined automatic peak annotation by searching an in-house library (recording 198 known compounds) followed by the subsequent confirming steps (e.g., comparison with the reference compounds, fragmentation pathways analysis, and retention behavior comparison, etc.), allowed us to identify or tentatively characterize a total of 86 components (including 50 terpenoids, 21 phenolic acids, and 15 others) from S. miltiorrhiza. Importantly, three-dimensional structure information, such as the retention time, MS¹ and MS² data, and collision cross section (CCS), was provided, which can facilitate the more reliable characterization of herbal components.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

<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.     

Efficient Evergreen Plant Regeneration of <i>Cinnamomum japonicum</i> Sieb. through <i>in vitro</i> Organogenesis

Cinnamomum japonicum Sieb. is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value. In this study, we successfully developed a large-scale micropropagation protocol for C. japonicum for the first time. Sterilized shoots were excised and used as explants for shoot induction on several basal media, supplemented with different concentrations of plant growth regulators (PGRs), such as Thidiazuron (TDZ), N⁶ -Benzyladenine (6-benzylaminopurine) (BA), α-naphthaleneacetic acid (NAA) and Gibberellic acid (GA₃). After comparison, the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^–1 BA, 0.05 mg L^–1 NAA and 0.2 mg L^–1 GA₃, which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d, respectively. Then, elongated adventitious shoots were transferred to induce roots. 86.7% of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^–1 NAA and 0.1 mg L^–1 BA. The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d. Our study shows that C. japonicum can be successfully regenerated through de novo organogenesis, which lays a foundation for future transformation research on this tree.     

Phenotypic Variation among and within Three Peppers Species (<i>Capsicum</i>) from Mexico

The phenotypic variation and its distribution among species, morphotypes, and among and within populations was estimated in 71 populations pertaining to 15 morphotypes of three domesticated species of Capsicum from Mexico. Collections were made in the states of Sinaloa, Nayarit, Oaxaca, Tabasco, and Yucatán in two agroecosystems: Backyard-garden and monoculture. Fifteen phenotypic characteristics were analyzed through one-way variance analysis and multivariate analyses of principal components analysis (PCA) and hierarchical clustering using Ward’s method. The study was performed in a uniform greenhouse experiment. A high variation was found among and within populations in all the measured characteristics. Of the total variation, 13.0% was distributed among species, 27.9% among morphotypes, 8.1% among populations, and 51.0% within populations. Because plants grew in a uniform environment, these results indicate that the differences observed among and within species, morphotypes, and populations have a genetic basis. Univariate and multivariate analyses clearly differentiated morphotypes, suggesting that the category of morphotypes must be used to nominate the infraspecific variation in the domesticated Capsicum. The principal components analysis identified a total of 15 principal components that contributed to explain the total variation. The first two components explained 59.64% of the total variation and seven components explained more than 90% of the total variation. Among the measured characteristics, number of seed per fruit, weight of the fruit, width of the fruit, length of the fruit, stem diameter, days to flowering, and height of the plant contributed to component 1 variation, whereas width of the leaf, length of the leaf, and number of locules, number of fruits and number of seeds per plant contributed to component 2. The hierarchical clustering separated the populations and the morphotypes in two large different groups. One group consisted of populations collected in monoculture conditions and the other group corresponded to population collected from backyard-garden conditions. The monoculture populations were characterized mainly by their longer, wider, and higher weight fruits, plants were of less height, had smaller stem diameters, and lower number of fruits than the populations collected from the backyard-garden conditions. The backyard-garden populations of the Capsicum annuum and Capsicum frutescens species, considered wild or semi-domesticated, constituted a non-differentiated phenotypic group that does not allow dividing them in different species.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Genome-Wide Identification and Expression Profiling of the Shaker K⁺ Channel and HAK/KUP/KT Transporter Gene Families in Grape (<i>Vitis vinifera</i> L.)

Potassium (K⁺) is an essential macronutrient for plants to maintain normal growth and development. Shaker-like K⁺ channels and HAK/KUP/KT transporters are critical components in the K⁺ acquisition and translocation. In this study, we identified 9 Shaker-like K⁺ channel (VvK) and 18 HAK/KUP/KT transporter (VvKUP) genes in grape, which were renamed according to their distributions in the genome and relative linear orders among the distinct chromosomes. Similar structure organizations were found within each group according to the exon/intron structure and protein motif analysis. Chromosomal distribution analysis showed that 9 VvK genes and 18 VvKUP genes were unevenly distributed on 7 or 10 putative grape chromosomes. Three pairs of tandem duplicated genes and one pair of segmental duplicated genes were observed in the expansion of the grape VvKUP genes. Gene expression omnibus (GEO) data analysis showed that VvK and VvKUP genes were expressed differentially in distinct tissues. Various cis-acting regulatory elements pertinent to phytohormone responses and abiotic stresses, including K⁺ deficiency response and drought stress, were detected in the promoter region of VvK and VvKUP genes. This study provides valuable information for further functional studies of VvK and VvKUP genes, and lays a foundation to explore K⁺ uptake and utilization in fruit trees.     

Effects of Different Geographical Aspects and Ontogenetic Variability on Total Hypericin Content of <i>Hypericum triquetrifolium</i> Turra. and <i>Hypericum scabrum</i> L.

The present study was conducted to determine the total hypericin contents of Hypericum triquetrifolium Turra. and Hypericum scabrum L. species which are naturally distributed in the flora of Siirt province, Turkey. Hypericin contents of Hypericum species grown in different geographical aspects (North, South, East, and West), and it was measured at different harvest times (full blooming and post blooming period). In the current study, it has been determined that total hypericin content varies considerably according to aspects, plant developmental stages (ontogenetic variance), and species. According to species x aspect interaction, the highest total hypericin content was recorded from the west aspect (3.13 mg/g) in Hypericum triquetrifolium, while, the lowest hypericin content was also obtained from the west aspect (1.22 mg/g) in Hypericum scabrum. When the highest total hypericin content was analyzed according to aspect x species x harvest time interaction, the highest total hypericin content was produced from Hypericum triquetrifolium at the harvest of west aspect with 5.28 mg/g, while the minimum amount of hypericin was obtained from the same aspect in Hypericum scabrum with 0.50 mg/g. In species x harvest time interaction, the highest total hypericin content was obtained from the full bloom (3.10 mg/g) harvest in Hypericum triquetrifolium, while the lowest hypericin was obtained from the full bloom (1.26 mg/g) harvest in Hypericum scabrum. The data suggest that the average total hypericin content was 2.26 mg/g in Hypericum triquetrifolium and 1.28 mg/g in Hypericum scabrum.     

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to <i>Sclerotinia sclerotiorum</i> in <i>Brassica napus</i>

Invertase (INV), a key enzyme in sucrose metabolism, irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose, thus playing important roles in plant growth, development, and biotic and abiotic stress responses. In this study, we identified 27 members of the BnaINV family in Brassica napus. We constructed a phylogenetic tree of the family and predicted the gene structures, conserved motifs, cis-acting elements in promoters, physicochemical properties of encoded proteins, and chromosomal distribution of the BnaINVs. We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B. napus cultivar Zhongshuang 11 using qRT-PCR. In addition, we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum. We used WGCNA (weighted coexpression network analysis) to uncover BnaINVregulatory networks. Finally, we explored the expression patterns of several BnaINV genes in cultivars with long (Zhongshuang 4) and short (Ningyou 12) siliques. Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens. Our findings could facilitate the breeding of high-yielding B. napus cultivars with strong disease resistance.     

Participation of Auxin Transport in the Early Response of the <i>Arabidopsis</i> Root System to Inoculation with <i>Azospirillum brasilense</i>

The potential of Plant Growth Promoting Rhizobacteria (PGPR) has been demonstrated in the case of plant inoculation with bacteria of the genus Azospirillum which improves yield. A. brasilense produces a wide variety of molecules, including the natural auxin indole-3-acetic acid (IAA), as well as other phytoregulators. However, several studies have suggested that auxin induces changes in plant development during their interaction with the bacteria. The effects of A. brasilense Sp245 on the development of Arabidopsis thaliana root were investigated to help explain the molecular basis of the interaction. The results obtained showed a decrease in primary root length from the first day and remained so throughout the exposure, accompanied by a stimulation of initiation and maturation of lateral root primordia and an increase of lateral roots. An enhanced auxin response was evident in the vascular tissue and lateral root meristems of inoculated plants. However, after five days of bacterization, the response disappeared in the primary root meristems. The role of polar auxin transport (PAT) in auxins relocation involved the PGP1, AXR4-1, and BEN2 proteins, which apparently mediated A. brasilense-induced root branching of Arabidopsis seedlings.     

Characterization and Expression of Ammonium Transporter in Peach (<i>Prunus persica</i>) and Regulation Analysis in Response to External Ammonium Supply

As the preferred nitrogen (N) source, ammonium (NH₄⁺ ) contributes to plant growth and development and fruit quality. In plants, NH₄⁺ uptake is facilitated by a family of NH₄⁺ transporters (AMT). However, the molecular mechanisms and functional characteristics of the AMT genes in peach have not been mentioned yet. In this present study, excess NH₄⁺ stress severely hindered shoot growth and root elongation, accompanied with reduced mineral accumulation, decreased leaf chlorophyll concentration, and stunned photosynthetic performance. In addition, we identified 14 putative AMT genes in peach (PpeAMT). Expression analysis showed that PpeAMT genes were differently expressed in peach leaves, stems and roots, and were distinctly regulated by external NH₄⁺ supplies. Putative cis-elements involved in abiotic stress adaption, Ca²⁺ response, light and circadian rhythms regulation, and seed development were observed in the promoters of the PpeAMT family genes. Phosphorylation analysis of residues within the C-terminal of PpeAMT proteins revealed many conserved phosphorylation residues in both the AMT1 and AMT2 subfamily members, which could potentially play roles in controlling the NH₄⁺ transport activities. This study provides gene resources to study the biological function of AMT proteins in peach, and reveals molecular basis for NH₄⁺ uptake and N nutrition mechanisms of fruit trees.     

Chemical Constituents from Turnip and Their Effects on <i>α</i>-Glucosidase

Brassica rapa var. rapa (turnip) is an important crop in Qinghai-Tibet Plateau (QTP) with anti-hypoxic effect. Turnip is rich in glucosinolates, isothiocyanates and phenolic compounds with diverse biological activities, involving anti-oxidant, anti-tumor, anti-diabetic, anti-inflammatory, anti-microbial, hypolipidemic, cardioprotective, hepatoprotective, nephroprotective and analgesic properties. In this study, the ethyl acetate (EtOAc) and butanol parts of Brassica rapa were first revealed with inhibitory effects on α-glucosidase, whereas the water part was inactive. Subsequent bioassay-guided isolation on the EtOAc and butanol parts yielded 12 compounds, involving three indole derivatives, indole-3- acetonitrile (1) 4-methoxyindole-3-acetonitrile (2) and indole-3-aldehyde (3) two flavonoids, liquiritin (4) and licochalcone A (5) two phenylpropanoids, sinapic acid (6) and caffeic acid (7) two phenylethanol glycosides, 2-phenylethyl β- glucopyranoside (8) and salidroside (9) and three other compounds, syringic acid (10) adenosine (11) and (3β, 20E)-ergosta-5, 20 (22)-dien-3-ol (12) Licochalcone A (5) and caffeic acid (7) showed α-glucosidase inhibitory activity with IC₅₀ values of 62.4 ± 8.0 μM and 162.6 ± 3.2 μM, comparable to the positive control, acarbose (IC₅₀ = 142 ± 0.02 μM). Docking study suggested that licochalcone A (5) could well align in the active site of α-glucosidase (docking score = -52.88) by forming hydrogen bonds (Gln1372, Asp1420, Gln1372, Arg1510), hydrophobic effects (Tyr1251, Tyr1251, Trp1355, Phe1560, Ile1587, Trp1355, Phe1559, Phe1559) and π-π stacking interaction (Trp1355). This study provides valuable information for turnip as a new resource in searching anti-diabetic candidates.     

First Report of <i>Fusarium striatum</i> Causing Root Rot Disease of <i>Panax notoginseng</i> in Yunnan,China

Panax notoginseng is a traditional Chinese medicinal plant. Root rot of P. notoginseng is one of the most serious diseases affecting P. notoginseng growth and causes wilted leaves, fewer lateral roots and rotten roots. Root rot is a soil-borne disease, and mainly occurs from June to August in Yunnan Province when the temperatures are high and the air is humid. In this study, the endophytic fungal genus Fusarium isolate E-2018.1.22-#3.2 was obtained from a P. notoginseng embryo. Fusarium isolate E-2018.1.22-#3.2 was identified as Fusarium striatum based on morphological characteristics and molecular analysis. The fungus was found to have conidiophores and macroconidia, and its ITS, LSU and TEF-1α genes shared 100%, 99.2% and 99% identities with the homologous genes of Fusarium striatum, respectively. Isolate F. striatum E-2018.1.22-#3.2 can cause root rot symptoms, including black, soft roots, fewer lateral roots and leaf wilt, in 93% of the experimental P. notoginseng plants, and could be re-isolated, fulfilling Koch’s postulates. When the P. notoginseng plants were treated with the fungicide pyraclostrobin, isolate F. striatum E-2018.1.22-#3.2 was unable to cause root rot. We have therefore demonstrated that F. striatum E-2018.1.22-#3.2 is able to cause root rot disease in P. notoginseng. This is the first report of root rot disease caused by F. striatum on P. notoginseng in China.     

<i>VvAGAMOUS</i> Affect Development of Four Different Grape Species Ovary

Grape pistil has an important influence on fruit size and quality. However, there were few studies on grape ovary, and the development process of the ovary is still unclear. Therefore, in this paper, four different grape varieties with different lengths of small inflorescences, namely ‘Musct Hambourg’ grape (Vitis vinifera), ‘Concord’ grape (Vitis labrusca), ‘ShanPuTao’ grape (Vitis amurensis) and ‘GongNiang2Hao’ grape (Vitis amurensis × Vitis vinifera) were used as test materials. Four varieties ovary were significant differences by means of stereomicroscope, paraffin section. The expression of ovary determining gene VvAGAMOUS (VvAG) and its development related genes VvCRABS CLAW (VvCRC) andVvAGAMOUS-LIKE 11 (VvAGL11) with similar functions during the development of different grape varieties were preliminarily explored using fluorescence quantitative test. The relationship between VvAG and VvCRC, VvAG and VvAGL11 were analyzed using Y1H assay. Our results showed that there were obvious abdominal sutures on the surface of expect for ‘Musct Hambourg’ grape, and existing poly carpels. The ovary development of ‘ShanPuTao’ and ‘GongNiang2Hao’ grape was completed when the inflorescence length was less than 1 cm, while the ‘Concord’ and ‘Musct Hambourg’ grape were fully developed when the length of inflorescence was 3–4 and 4–5 cm, respectively. VvAG and VvCRC began to express in large quantities after the formation of stamen primordia, while VvAGL11 during the forming of ovule primordia. Therefore, VvAG and VvCRC mainly regulated the development of stamens and carpels and also promote the development of ovules, while VvAGL11 major regulated the development of ovules. The promoters of VvCRC and VvAGL11 were bound by VvAG. This study provides an important theoretical basis for further research on the molecular mechanism of grape ovary development.     

Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

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.