Identification and Characterisation of the CircRNAs Involved in the Regulation of Leaf Colour in Quercus mongolica.

Circular RNAs (circRNAs) are important regulatory molecules involved in various biological processes. However, the potential function of circRNAs in the turning red process of Quercus mongolica leaves is unclear. This study used RNA-seq data to identify 6228 circRNAs in leaf samples from four different developmental stages and showed that 88 circRNAs were differentially expressed. A correlation analysis was performed between anthocyanins and the circRNAs. A total of 16 circRNAs that may be involved in regulating the colour of Mongolian oak leaves were identified. CircRNAs may affect the colour of Q. mongolica leaves by regulating auxin, cytokinin, gibberellin, ethylene, and abscisic acid. This study revealed the potential role of circRNAs in the colour change of Q. mongolica leaves.

Association Analysis of Transcriptome and Targeted Metabolites Identifies Key Genes Involved in Iris germanica Anthocyanin Biosynthesis.

The anthocyanin biosynthetic pathway is the main pathway regulating floral coloration in Iris germanica, a well-known ornamental plant. We investigated the transcriptome profiles and targeted metabolites to elucidate the relationship between genes and metabolites in anthocyanin biosynthesis in the bitone flower cultivar 'Clarence', which has a deep blue outer perianth and nearly white inner perianth. In this study, delphinidin-, pelargonidin-, and cyanidin-based anthocyanins were detected in the flowers. The content of delphinidin-based anthocyanins increased with the development of the flower. At full bloom (stage 3), delphinidin-based anthocyanins accounted for most of the total anthocyanin metabolites, whereas the content of pelargonidin- and cyanidin-based anthocyanins was relatively low. Based on functional annotations, a number of novel genes in the anthocyanin pathway were identified, which included early biosynthetic genes IgCHS, IgCHI, and IgF3H and late biosynthetic genes Ig F3'5'H, IgANS, and IgDFR. The expression of key structural genes encoding enzymes, such as IgF3H, Ig F3'5'H, IgANS, and IgDFR, was significantly upregulated in the outer perianth compared to the inner perianth. In addition, most structural genes exhibited their highest expression at the half-color stage rather than at the full-bloom stage, which indicates that these genes function ahead of anthocyanins synthesis. Moreover, transcription factors (TFs) of plant R2R3-myeloblastosis (R2R3-MYB) related to the regulation of anthocyanin biosynthesis were identified. Among 56 R2R3-MYB genes, 2 members belonged to subgroup 4, with them regulating the expression of late biosynthetic genes in the anthocyanin biosynthetic pathway, and 4 members belonged to subgroup 7, with them regulating the expression of early biosynthetic genes in the anthocyanin biosynthetic pathway. Quantitative real-time PCR (qRT-PCR) analysis was used to validate the data of RNA sequencing (RNA-Seq). The relative expression profiles of most candidate genes were consistent with the FPKM of RNA-seq. This study identified the key structural genes encoding enzymes and TFs that affect anthocyanin biosynthesis, which provides a basis and reference for the regulation of plant anthocyanin biosynthesis in I. germanica.

Geographical provenance variation of growth and wood properties of 18-year-old Schima superba.

We analyzed the variation patterns of growth and wood properties of 24 different provenances of 18-year-old Schima superba in Jian'ou, Fujian Province. A total of 11 growth and wood indices were measured, including tree height, diameter at breast height, wood basic density and anatomical structure. We analyzed the geographical variation patterns of growth and wood properties, and the provenance areas were divided. Further, the excellent timber provenances were selected according to different uses. The results showed that the variation of growth traits, which was 17.6%-27.3% with mean value of 22.4%, was larger than that of wood properties (7.0%-21.0%, mean 12.7%). Growth properties and some wood properties (fiber length, fiber lumen diameter and fiber cell wall thickness) had significant differences among provenances. Growth traits were not correlated with fiber traits, and they could be selected independently without emphasis on other traits. There was significant correlation between the longitudinal and radial growth indicators of wood properties, but they were not correlated with the wood basic density, which could be selected independently. In addition, the growth and wood properties were significantly influenced by temperature and precipitation, which showed a latitudinal variation pattern. According to Q-type clustering analysis, 24 provenances could be divided into four categories, of which southern provenances from distribution area of S. superba had vigorous growth and supper wood properties. They had smaller microfibril angle, higher maturity, longer fiber length, and thicker fiber cell wall. Finally, five excellent provenances were selected according to pulpwood and building use.

Integrated physiological and metabolomic analyses reveal changes during the natural senescence of Quercus mongolica leaves.

Quercus mongolica is a common landscape, afforestation, and construction timber species in northern China with high ecological, economic, and ornamental value. Leaf senescence is a complex process that has important implications for plant growth and development. To explore changes of metabolites during the ageing of Quercus mongolica leaves, we investigated physiological responses and metabolite composition in ageing leaves harvested from 15-20-year-old Quercus mongolica. Leaf samples of Q. mongolica were collected when they were still green (at maturity) (stage 1), during early senescence (stage 2), and during late senescence (stage 3). These leaves were then subjected to physiological index and metabolome sequencing analyses. The physiological analysis showed that the leaves of Q. mongolica changed from green to yellow during senescence, which induced significant accumulation of soluble sugar and significant reductions in the concentration of soluble protein and chlorophyll. Peroxidase and catalase were the main antioxidant enzymes mitigating leaf senescence. Metabolomic analysis identified 797 metabolites during leaf senescence. Compared to stage 1, 70 differential metabolites were screened in stage 2 and 72 were screened in stage 3. Differential metabolites in the two senescent stages were principally enriched in amino acid metabolism, lipid metabolism and secondary metabolite biosynthesis. The contents of N-oleoylethanolamine and N, N-dimethylglycine were significantly increased only in stage 2, while the contents of trifolin, astragalin, valine, isoleucine, leucine, and citric acid were significantly increased only in stage 3. Histidine, homoserine, tryptophan, tyrosine, phenylalanine, proline, norleucine, N-glycyl-L-leucine, linoleic acid, linolenic acid, gallic acid, 3-indoleacrylic acid, 3-amino-2-naphthoic acid, 3-hydroxy-3-methylpentane-1,5-dioic acid, 2,3,4-trihydroxybenzoic acid, trifolin, astragalin, DL-2-aminoadipic acid, pinoresinol dimethyl ether, dimethylmatairesinol, and lysophosphatidylcholine increased during both stage 2 and stage 3. Increasing contents of these metabolites may constitute the main mechanism by which Q. mongolica leaves adapt to senescence.

Drought-responsive WRKY transcription factor genes IgWRKY50 and IgWRKY32 from Iris germanica enhance drought resistance in transgenic Arabidopsis.

Drought greatly affects the growth and development of garden plants and affects their ornamental value. WRKY transcription factors make up one of the largest transcription factor families in plants and they play an important role in the plant response to drought stress. However, the function of the WRKY gene in response to drought stress in Iris germanica, which is commonly used in landscaping, has not been studied. In this study, we isolated two WRKY transcription factor genes from Iris germanica, IgWRKY50 and IgWRKY32, which belong to Group II and Group III of the WRKY family, respectively. IgWRKY50 and IgWRKY32 could be induced by PEG-6000, high temperature and ABA in Iris germanica. IgWRKY50 and IgWRKY32 could quickly respond to drought and they peaked at 3 h after PEG-6000 treatment (19.93- and 23.32-fold). The fusion proteins IgWRKY50-GFP and IgWRKY32-GFP were located in the nucleus of mesophyll protoplasts of Arabidopsis. The overexpression of the IgWRKY50 and IgWRKY32 genes improved the osmotic tolerance of transgenic Arabidopsis, mainly exhibited by the transgenic plants having a higher germination rate and a longer total root length on 1/2 MS medium containing mannitol. Under PEG-6000 stress, the transgenic plants had higher stomatal closure than the wild type (WT). Under natural drought stress, the water loss rate of the isolated leaves of transgenic Arabidopsis was lower than that of WT, the contents of proline (Pro) and soluble protein (SP) and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the transgenic plants were higher, but the content of malondialdehyde (MDA) was lower. Furthermore, the expression of several stress-related genes (RD29A, DREB2A, PP2CA, and ABA2) was significantly increased in IgWRKY50- and IgWRKY32- overexpressing transgenic Arabidposis plants after drought treatment. These results suggest that IgWRKY50 and IgWRKY32, as two positive regulators, enhance the drought resistance of transgenic Arabidopsis by mediating the ABA signal transduction pathway. IgWRKY50 and IgWRKY32 can be used as candidate genes for molecular breeding of drought resistance in Iris.

Nitrogen and sulfur fertilizers promote the absorption of lead and cadmium with Salix integra Thunb. by increasing the bioavailability of heavy metals and regulating rhizosphere microbes.

Fertilization is an effective agronomic strategy to improve the efficiency of phytoextraction by Salix integra Thunb. However, the specific effects of the simultaneous application of nitrogen (N) and sulfur (S) fertilizers in the rhizosphere remain unclear. We investigated the bioavailability of lead (Pb) and Cadmium (Cd) along with the microbial metabolic functions and community structure in the rhizosphere soil of S. integra after the application of N (0, 100, and 200 kg·ha-1·year-1) and S (0, 100, and 200 kg·ha-1·year-1) fertilizers for 180 days. The simultaneous application of N and S fertilizers significantly enhanced the absorption of Pb and Cd by S. integra, whereas this effect was not observed for the single application of N or S fertilizer. The contents of acid-soluble Pb and Cd in the rhizosphere soil significantly increased after either single or combined fertilize applications. The microbial metabolic activity was enhanced by the N and S fertilizers, whereas the microbial diversity markedly decreased. The metabolic patterns were mainly affected by the concentration of N fertilizer. The dominant fungi and bacteria were similar under each treatment, although the relative abundances of the dominant and special species differed. Compared to the N200S100 and N200S200 treatments, the N100S100 and N100S200 treatments resulted in fewer pathogenic fungi and more rhizosphere growth-promoting bacteria, which promoted phytoremediation by S. integra. Redundancy analysis indicated that the pH and nitrate content were the key factors affecting the structure of the microbial community. Collectively, the results suggest interactive effects between N and S fertilizers on the rhizosphere soil, providing a potential strategy for plant-microbial remediation by S. integra.

Inoculation With Indigenous Rhizosphere Microbes Enhances Aboveground Accumulation of Lead in Salix integra Thunb. by Improving Transport Coefficients.

The application of plant-microbial remediation of heavy metals is restricted by the difficulty of exogenous microbes to form large populations and maintain their long-term remediation efficiency. We therefore investigated the effects of inoculation with indigenous heavy-metal-tolerant rhizosphere microbes on phytoremediation of lead (Pb) by Salix integra. We measured plant physiological indexes and soil Pb bioavailability and conducted widespread targeted metabolome analysis of strains to better understand the mechanisms of enhance Pb accumulation. Growth of Salix integra was improved by both single and co-inoculation treatments with Bacillus sp. and Aspergillus niger, increasing by 14% in co-inoculated plants. Transfer coefficients for Pb, indicating mobility from soil via roots into branches or leaves, were higher following microbial inoculation, showing a more than 100% increase in the co-inoculation treatment over untreated plants. However, Pb accumulation was only enhanced by single inoculation treatments with either Bacillus sp. or Aspergillus niger, being 10% greater in plants inoculated with Bacillus sp. compared with uninoculated controls. Inoculation mainly promoted accumulation of Pb in aboveground plant parts. Superoxide dismutase and catalase enzyme activities as well as the proline content of inoculated plants were enhanced by most treatments. However, soil urease and catalase activities were lower in inoculated plants than controls. Proportions of acid-soluble Pb were 0.34 and 0.41% higher in rhizosphere and bulk soil, respectively, of plants inoculated with Bacillus sp. than in that of uninoculated plants. We identified 410 metabolites from the microbial inoculations, of which more than 50% contributed to heavy metal bioavailability; organic acids, amino acids, and carbohydrates formed the three major metabolite categories. These results suggest that both indigenous Bacillus sp. and Aspergillus niger could be used to assist phytoremediation by enhancing antioxidant defenses of Salix integra and altering Pb bioavailability. We speculate that microbial strains colonized the soil and plants at the same time, with variations in their metabolite profiles reflecting different living conditions. We also need to consider interactions between inocula and the whole microbial community when applying microbial inoculation to promote phytoremediation.

Evaluation of drought resistance and transcriptome analysis for the identification of drought-responsive genes in Iris germanica.

Iris germanica, a species with very high ornamental value, exhibits the strongest drought resistance among the species in the genus Iris, but the molecular mechanism underlying its drought resistance has not been evaluated. To investigate the gene expression profile changes exhibited by high-drought-resistant I. germanica under drought stress, 10 cultivars with excellent characteristics were included in pot experiments under drought stress conditions, and the changes in the chlorophyll (Chl) content, plasma membrane relative permeability (RP), and superoxide dismutase (SOD), malondialdehyde (MDA), free proline (Pro), and soluble protein (SP) levels in leaves were compared among these cultivars. Based on their drought-resistance performance, the 10 cultivars were ordered as follows: 'Little Dream' > 'Music Box' > 'X'Brassie' > 'Blood Stone' > 'Cherry Garden' > 'Memory of Harvest' > 'Immortality' > 'White and Gold' > 'Tantara' > 'Clarence'. Using the high-drought-resistant cultivar 'Little Dream' as the experimental material, cDNA libraries from leaves and rhizomes treated for 0, 6, 12, 24, and 48 h with 20% polyethylene glycol (PEG)-6000 to simulate a drought environment were sequenced using the Illumina sequencing platform. We obtained 1, 976, 033 transcripts and 743, 982 unigenes (mean length of 716 bp) through a hierarchical clustering analysis of the resulting transcriptome data. The unigenes were compared against the Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KEGG, and gene ontology (GO) databases for functional annotation, and the gene expression levels in leaves and rhizomes were compared between the 20% PEG-6000 stress treated (6, 12, 24, and 48 h) and control (0 h) groups using DESeq2. 7849 and 24,127 differentially expressed genes (DEGs) were obtained from leaves and rhizomes, respectively. GO and KEGG enrichment analyses of the DEGs revealed significantly enriched KEGG pathways, including ribosome, photosynthesis, hormone signal transduction, starch and sucrose metabolism, synthesis of secondary metabolites, and related genes, such as heat shock proteins (HSPs), transcription factors (TFs), and active oxygen scavengers. In conclusion, we conducted the first transcriptome sequencing analysis of the I. germanica cultivar 'Little Dream' under drought stress and generated a large amount of genetic information. This study lays the foundation for further exploration of the molecular mechanisms underlying the responses of I. germanica to drought stress and provides valuable genetic resources for the breeding of drought-resistant plants.

Pollen morphology of different species of Iris barbata and its systematic significance with scanning electron microscopy methods.

Forty-eight cultivars of Iris barbata were used as research materials, and observations of their pollen morphologies were made using scanning electron microscopy (SEM). The pollen of I. barbata consisted of a single grain, which was subspheroidal or subprolate and boat-shaped in equatorial view and oval or nearly round in polar view. The pollen was symmetrical or radially symmetrical on both sides, and there were five types of germination furrows: monocolpate, dicolpate, monocolpate-colpoidal, 2-syncolpate, and parasyncolpate. The exine ornamentation was mostly crass-reticulate and occasionally verrucate or pilate. Variation in the equatorial axis length of I. barbata pollen was the lowest, and variation in pollen morphology was stable. Q-type cluster analysis was conducted using seven indexes: polar axis length (P), equatorial axis length (E), P/E, mesh diameter (D), net ridge width (W), D/W, and germination furrow width (WG). The 48 cultivars were divided into three groups. Three dwarf cultivars were clustered in one group, and the degree of evolution of this group was higher than that of the other two groups. This paper systematically describes the characteristics of I. barbata for the first time, and thus provides important palynological insights into the classification and cross-breeding of I. barbata.

Indigenous Bacteria Have High Potential for Promoting Salix integra Thunb. Remediation of Lead-Contaminated Soil by Adjusting Soil Properties.

Salix integra Thunb., a fast-growing woody plant species, has been used for phytoremediation in recent years. However, little knowledge is available regarding indigenous soil microbial communities associated with the S. integra phytoextraction process. In this study, we used an Illumina MiSeq platform to explore the indigenous microbial composition after planting S. integra at different lead (Pb) contamination levels: no Pb, low Pb treatment (Pb 500 mg kg-1), and high Pb treatment (Pb 1500 mg kg-1). At the same time, the soil properties and their relationship with the bacterial communities were analyzed. The results showed that Pb concentration was highest in the root reaching at 3159.92 ± 138.98 mg kg-1 under the high Pb treatment. Planting S. integra decreased the total Pb concentration by 84.61 and 29.24 mg kg-1, and increased the acid-soluble Pb proportion by 1.0 and 0.75% in the rhizosphere and bulk soil under the low Pb treatment compared with unplanted soil, respectively. However, it occurred only in the rhizosphere soil under the high Pb treatment. The bacterial community structure and microbial metabolism were related to Pb contamination levels and planting of S. integra, while the bacterial diversity was only affected by Pb contamination levels. The dominant microbial species were similar, but their relative abundance shifted in different treatments. Most of the specific bacterial assemblages whose relative abundances were promoted by root activity and/or Pb contamination were suitable for use in plant-microbial combination remediation, especially many genera coming from Proteobacteria. Redundancy analysis (RDA) showed available nitrogen and pH having a significant effect on the bacteria relating to phytoremediation. The results indicated that indigenous bacteria have great potential in the application of combined S. integra-microbe remediation of lead-contaminated soil by adjusting soil properties.

[Physiological response and salt-tolerance of Gleditsia microphylla under NaCl stress].

In order to exploit the salt-tolerance ability and mechanism of Gleditsia microphylla, the plant growth, cell membrane permeability, the activities of cell protective enzymes, and the distri- butions of Na+ and K+ in different tissues were investigated under various NaCl stress (0.053%, 0.15%, 0.3%, 0.45% and 0.6%) with potted two-year seedlings. The results were as follows: With the increase of NaCl concentration, the seedling growth decreased while the salt injured index in- creased, and the salt-tolerance thresholds of seedling was 0.42% NaCl. With the NaCl concentration increasing, the membrane permeability, superoxide anion radical generating rate and MDA content increased grandly, while the activities of SOD, POD and CAT demonstrated an increase-decrease curve which reached the peak at 0.3% or 0.45%. Under the high salt stress condition, the supero- xide anion could be consumed timely by increasing the activities of SOD, POD and CAT enzymes, which was useful to avoid cell injure. Under salt stress condition, the Na+ content in different tissues increased gradually, following the order of root > leaf > stem, and the K+ content and K+/Na+ in different tissues decreased, following the order of leaf > root > stem. The K+-Na+ selective transpor- tation coefficients (S(K+) · Na+) of stem and leaf tissues under the soil NaCl stress condition were both increased, following the order of leaf > stem. In conclusion, the findings suggested that the salt- adaptation mechanisms of G. microphylla were root salt-rejection by Na+ accumulation and restriction in root tissue and leaf salt-tolerance by a remarkably increased ability of K+ selective absorption and accumulation in leaf tissue.

[Effects of drought stress on the biomass distribution and photosynthetic characteristics of cluster mulberry].

One-year-old Morus alba seedlings were cultivated in pots containing soil with different water contents to study the biomass, chlorophyll fluorescence parameters, net photosynthesis rate, stomatal conductance, transpiration rate and water use efficiency of the seedlings in response to diverse water stress. The results showed that the aboveground biomass and total biomass decreased with increasing water stress. However, more photosynthetic products were transferred to the roots in relation to the aboveground parts to make the root-shoot ratio increase. The fluorescence parameters, such as F(o), F(v) and F(v)/F(m), and stomatal conductance, transpiration rate and water use efficiency decreased with increasing water stress.