Sodium alginate enhances defense responses of Pinus sylvestris var. mongolica against Bursaphelenchus xylophilus by regulating chs genes and flavonoid metabolites.

Flavonoids are a major component of Artemisia argyi and play a crucial role in its pharmacological properties. However, the molecular mechanisms underlying flavonoid biosynthesis in A. argyi remain unclear. To address this, transcriptome and quantitative metabolome analyses were conducted across five developmental stages of A. argyi. In total, 85 flavonoid compounds were identified across these different stages. Differentially expressed candidate genes and metabolites involved in flavonoid biosynthesis were also identified. Differentially accumulated flavonoid metabolites (DFMs) were observed among the 10 comparison groups, with 29 DFMs identified from the five developmental stages of A. argyi leaves. The biosynthesis process identified 38 differentially expressed genes (DEGs) from seven gene families. Furthermore, 26 DEGs exhibited a significant correlation with the levels of seven active flavonoid metabolites, as revealed by weighted gene co-expression network analysis. These DEGs included eight HCT genes, six CHI genes, two CHS genes, three CCoAOMT genes, two F3'H genes, two C4H genes, two CYP98A genes, and one F3H gene. Based on preliminary analysis, HCT1 may be associated with accumulating hispidulin and jaceosidin. This study investigated the relationship between differential gene expression and flavonoid accumulation using an integrated transcriptomic and metabolomic approach, providing valuable insights into the mechanisms of flavonoid biosynthesis and quality formation in A. argyi.

珍稀濒危植物长白松(<i>Pinus sylvestris</i> var. <i>sylvestriformis</i>)天然种群生存压力

BackgroundRadix Ardisia (Jab Bik Lik Jib) is a common Miao medicine and is widely distributed in the Guizhou region of southern China. The botanical origin of Radix Ardisia includes the dry root and rhizome of Ardisia Crenata Sims (ACS) or Ardisia Crispa (Thunb.) A.DC. (AC), which are closely related species morphologically. However, the secondary metabolites in their roots are different from one another, especially the flavonoids, and these differences have not been thoroughly explored at the molecular level. This project preliminarily identified regulatory molecular mechanisms in the biosynthetic pathways of the flavonoids between ACS and AC using a multi-omics association analysis.MethodsIn this study, we determined the total levels of saponin, flavonoid, and phenolic in Radix Ardisia from different origins. Integrated transcriptome and metabolome analyses were used to identify the differentially expressed genes (DEGs) and differentially expressed metabolites (DEM). We also performed conjoint analyses on DEGs and DEMs to ascertain the degree pathways, and explore the regulation of flavonoid biosynthesis.ResultsThe total flavonoid and phenolic levels in ACS were significantly higher than in AC (P < 0.05). There were 17,685 DEGs between ACS vs. AC, 8,854 were upregulated and 8,831 were downregulated. Based on this, we continued to study the gene changes in the flavonoid biosynthesis pathway, and 100 DEGs involving flavonoid biosynthesis were differentially expressed in ACS and AC. We validated the accuracy of the RNA-seq data using qRT-PCR. Metabolomic analyses showed that 11 metabolites were involved in flavonoid biosynthesis including: Naringenin, Luteolin, Catechin, and Quercetin. A conjoint analysis of the genome-wide connection network revealed the differences in the types and levels of flavonoid compounds between ACS and AC. The correlation analysis showed that Naringenin, Luteolin, Catechin, and Quercetin were more likely to be key compounds in the flavonoid biosynthesis pathway also including 4CL, AOMT, CHS, CHI, DFR, F3’5’H, FLS, and LAR.ConclusionsThis study provides useful information for revealing the regulation of flavonoid biosynthesis and the regulatory relationship between metabolites and genes in the flavonoid biosynthesis pathway in Radix Ardisia from different origins.

Pinus sylvestris var. mongolica is widely planted in China as a windbreak and sand fixation tree. To improve the current situation of large-scale declines of forested areas planted as P. sylvestris var. mongolica monocultures, we investigated the biological and microbial effects of stand establishment using mixed tree species. The interactions during the mixed decomposition of the litter and leaves of different tree species are an important indicator in determining the relationships among species. In this experiment, a method of simulating the mixed decomposition of P. sylvestris var. mongolica and Morus alba litter under P. sylvestris var. mongolica forest was used to determine the total C, total N, and total P contents in the leaf litter, and the microbial structures were determined by using Illumina MiSeq high-throughput sequencing. It was found that with samples with different proportions of P. sylvestris var. mongolica and M. alba litters, the decomposition rate of P. sylvestris var. mongolica × M. alba litter was significantly higher than that of the pure P. sylvestris var. mongolica forest, and the microbial community and composition diversity of litter in a pure P. sylvestris var. mongolica forest could be significantly improved. The possibility of using M. alba as a mixed tree species to address the declines of pure P. sylvestris var. mongolica forest was verified to provide guidance for pure P. sylvestris var. mongolica forests by introducing tree species with coordinated interspecific relationships and creating a mixed forest.

BackgroundGrape (Vitis vinifera L.) is one of the most important fruit products globally and has a high nutritional value with potent antioxidant and anti-cancer activities. In current years, phenylalanine application has been particularly noticed for enhancing the nutritional quality of horticultural crops. With the aim of quality improvement, the effects of foliar application of phenylalanine at 5 concentrations (0, 100, 500, 1000, and 2000 μM) on Vitis vinifera cv. ‘Qzl Ouzum’ berry compositions were studied. The studied parameters included antioxidant activity, phenolics, flavonoids, anthocyanin, catalase and phenylalanine ammonia-lyase enzyme activity, and phenolic compounds content based on HPLC analyses (myricetin, quercetin, kaempferol, syringetin, catechin, gallic acid, caffeic acid, p-coumaric acid, resveratrol).ResultsPhenylalanine at 1000 μM increased total phenols, flavonoids, anthocyanins, and PAL enzyme activity. In addition, HPLC analysis revealed a significant accumulation of individual phenolic compounds by phenylalanine treatment. The highest values were recorded in the treatments with 100 and 500 μM phenylalanine for most of the phenolic components. Real-time quantitative RT-PCR results showed that the expression of PAL and CHS genes was induced by phenylalanine. The highest PAL and CHS gene expression was observed at 500 μM and then at 1000 μM phenylalanine treatment.ConclusionsWith the use of phenylalanine, the activity of the PAL and CHS enzyme and PAL and CHS gene expression were significantly increased and led to a greater accumulation of phenolic compounds and antioxidant activiety. This study suggests that the use of phenylalanine as a main precursor for the synthesis of phenolic compounds, can improve the phenolic composition of grapes and could be a practical approach to advance fruit quality in the production of grape cv. ‘Qzl Ouzum’.

Daxing'an Mountains is one of regions in China with the most significant climate change. Larix gmelinii and Pinus sylvestris var. mongolica are the most important species in this area. The study of their radial growth response to climate change would provide a scientific basis for predicting the dynamics of boreal forests under climate change. A total of 451 tree-ring cores of L. gmelinii and P. sylvestris var. mongolica were collected from six sites in the Daxing'an region, and 12 standard chronologies were established. We compared the tree growth trend since 1900, and analyzed their response to the climate factor in each site using Pearson correlation analysis. Effects of temperature and precipitation on the annual radial growth of L. gmelinii and P. sylvestris var. mongolica were investigated by linear mixed models. The time stability of the relationship between two species growth-climate was compared by moving correlation. The results showed that the radial growth of L. gmelinii was negatively correlated with mean temperature in March and positively correlated with precipitation in the previous winter and July of current year. The radial growth of P. sylvestris var. mongolica was positively correlated with temperature in August and precipitation in the growing season (from May to September) of current year. Snow in winter played an important role in promoting the radial growth of L. gmelinii, whereas precipitation in summer limited the radial growth of P. sylvestris var. mongolica. The responses of L. gmelinii and P. sylvestris var. mongolica to climate change were significantly different, which affected tree growth, species composition, and spatial distribution in the boreal forests.

With six Pinus sylvestris var. mongolica plantations (Huinan, Xifeng, Fujia, Zhanggutai, Naiman and Wulanaodu) along an aridity gradient in the Horqin sandy land, we examined the changes in non-structural carbohydrates (NSCs) and nitrogen (N) contents of current and one-year-old needles and twigs, to explore the carbon supply and demand status as well as the nutrient accumulation strategies of P. sylvestris var. mongolica under drought. The results showed that the contents of NSCs and soluble sugars in needles and twigs of P. sylvestris var. mongolica plantations significantly decreased with increasing aridity. From the most humid site (Huinan) to the most aridity site (Wulanaodu), the soluble sugar contents in current and one-year old needles of P. sylvestris var. mongolica decreased from 12.8% and 12.5% to 9.0% and 9.5%, respectively. The soluble sugar contents in current-year old twigs decreased from 15.6% to 9.2%. With increasing aridity, the starch contents in needles and twigs remained relatively stable, soluble sugars/starch ratio in current and one-year old needles decreased, the N contents in current and one-year old twigs significantly increased. The P. sylvestris var. mongolica plantations in the Horqin sandy land consumed soluble sugar storage under drought, resulting in a risk of mortality from 'carbon starvation'. P. sylvestris var. mongolica tended to maintain stable starch storage and accumulate N in twigs to cope with long-term drought stress.

Walnut somatic embryos (Juglans nigra x Juglans regia) were transformed with a vector containing a neomycin phosphotransferase II, a beta-glucuronidase and an antisense chalcone synthase (chs) gene. This antisense construct included a 400 bp cDNA fragment of a walnut chs gene under the control of the duplicated CaMV-35S promoter. Molecular, biochemical and biological characterizations were performed both on transformed embryos propagated by secondary somatic embryogenesis and on microshoots developed by in vitro culture of embryonic epicotyls from somatic embryos. Thirteen transformed lines with the vector containing the antisense chs gene, one line with only the gus and nptII genes and one untransformed line were maintained in tissue culture. Six of the antisense lines were shown to be flavonoid-deficient. They exhibited a strongly reduced expression of chs genes, very low chalcone synthase activity and no detectable amounts of quercitrin, myricitrin, flavane-3-ols and proanthocyanidins in stems. Rooting tests showed that decreased flavonoid content in stems of antisense chs transformed lines was associated with enhanced adventitious root formation. Free auxin and conjugated auxin contents were determined during the latter phase of the micropropagation, and no variations were detected between control and antisense chs transformed lines. The in vitro plants developed a large basal callus and apical necrosis upon auxinic induction and the transformed lines highly deficient in flavonoids were more sensitive to exogenous application of indolebutyric acid (IBA).

To understand the contents of various phosphorus forms, phosphorus solubilizing bacte-rial community structure and the relationship between them in soils after restoration from the seriously burning, we collected soil samples from artificial restoration (Pinus sylvestris var. mongolica plantation, Larix gmelinii plantation), artificial accelerated natural restoration (secondary forest) and natural restoration (natural secondary forest) stands in Greater Khingan Mountain area. Using methods of Sui et al. modified from Hedley phosphorus fractionation, we measured the contents of different phosphorus forms in rhizosphere soil and bulk soil (0-10, 10-20 cm). Abundances of phosphorus solubilizing bacteria were quantified by high-throughput sequencing method. The results showed that the contents of H2O-Pi, NaHCO3-Pi and NaHCO3-Poin 0-10 cm bulk soil and NaHCO3-Po in rhizosphere soil followed the order of L. gmelinii plantation : P. sylvestris var. mongolica plantation : natural secondary forest : secondary forest. The contents of H2O-Pi, NaHCO3-Pi, NaHCO3-Po in 10-20 cm bulk soil and H2O-Pi, NaHCO3-Pi in rhizosphere soil followed the order of L. gmelinii plantation : P. sylvestris var. mongolica plantation : secondary forest : natural secondary forest. The ratios of contents of H2O-Pi, NaHCO3-Pi and NaHCO3-Po in rhizosphere to those in bulk soil (R/S) were higher than 1 in all forest stands. The moderately labile NaOH-P included NaOH-Pi and NaOH-Po. The content of NaOH-P was in order of L. gmelinii plantation : natural secondary forest : secondary forest : P. sylvestris var. mongolica plantation in 0-10 cm layer of bulk and rhizosphere soil, and ranked as L. gmelinii plantation : P. sylvestris var. mongolica plantation : secondary forest : natural secondary forest in 10-20 cm layer of bulk soil. There was rhizosphere effect of NaOH-P in the soil. The stable HCl-P included HCl-Pi and HCl-Po. The content of HCl-P followed the order of L. gmelinii plantation : natural secondary forest : P. sylvestris var. mongolica plantation : secondary forest in 0-10 cm layer of bulk soil,and ranked as L. gmelinii plantation : P. sylvestris var. mongolica plantation : natural secondary forest : secondary forest in the 10-20 cm layer. The content of residual-P in the soil was not sensitive to restoration methods. Bradyrhizobium, Streptomyces, Burkholderia and Bacillus were the main phosphorus solubilizing bacteria across all forest stands. The abundances of phosphorus solubilizing bacteria in soil of L. gmelinii plantation and P. sylvestris var. mongolica plantation were significantly higher than that of secondary forest and natural secondary forest. Results of redundancy analysis showed that the correlation between phosphorus solubilizing bacteria and various phosphorus forms was different. Our results showed that artificial afforestation was more conducive in improving the availability of phosphorus in soil and the abundance of phosphorus solubilizing bacteria.

BackgroundDrought stress severely impedes plant growth, and only a limited number of species exhibit long-term resistance to such conditions. Pinus sylvestris var. mongolica, a dominant tree species in arid and semi-arid regions of China, exhibits strong drought resistance and plays a crucial role in the local ecosystem. However, the molecular mechanisms underlying this resistance remain poorly understood.ResultsHere, we conducted transcriptome sequence and physiological indicators analysis of needle samples during drought treatment and rehydration stages. De-novo assembly yielded approximately 114,152 unigenes with an N50 length of 1,363 bp. We identified 6,506 differentially expressed genes (DEGs), with the majority being concentrated in the heavy drought stage (4,529 DEGs). Functional annotation revealed enrichment of drought-related GO terms such as response to water (GO:0009415: enriched 108 genes) and response to water deprivation (GO:0009414: enriched 106 genes), as well as KEGG categories including MAPK signaling pathway (K04733: enriched 35 genes) and monoterpenoid biosynthesis (K21374: enriched 27 genes). Multiple transcription factor families and functional protein families were differentially expressed during drought treatment. Co-expression network analysis identified a potential drought regulatory network between cytochrome P450 genes (Unigene4122_c1_g1) and a core regulatory transcription factor Unigene9098_c3_g1 (PsNAC1) with highly significant expression differences. We validated PsNAC1 overexpression in Arabidopsis and demonstrated enhanced drought resistance.ConclusionsThese findings provide insight into the molecular basis of drought resistance in P. sylvestris var. mongolica and lay the foundation for further exploration of its regulatory network.

Combined metabolomic and transcriptomic analysis reveals key candidate genes involved in the regulation of flavonoid accumulation in Anoectochilus roxburghii

Flower color is one of the most important ornamental traits of chrysanthemums. Previous studies have shown that high temperatures can cause the petals of some chrysanthemum varieties to fade; however, the molecular mechanisms behind this phenomenon remain poorly understood. This study examines the mechanisms of color change in purple chrysanthemums under high-temperature stress using combined metabolomic and transcriptomic analyses. Four chrysanthemum varieties—two heat-stable (‘Zi Feng Che’ and ‘Chrystal Regal’) and two heat-sensitive (‘Zi Hong Tuo Gui’ and ‘Zi Lian’)—were analyzed. High-temperature conditions (35 °C) significantly downregulated key anthocyanins in heat-sensitive varieties, particularly cyanidin-3-O-(3″,6″-O-dimalonyl)glucoside and pelargonidin-3-O-(3″,6″-O-dimalonyl)glucoside. Transcriptome analysis revealed differential gene expression involved in anthocyanin biosynthesis and degradation, with significant enrichment in the MAPK signaling, phenylpropanoid biosynthesis, flavonoid biosynthesis, and anthocyanin biosynthesis pathways. The study highlighted the differential expression of CHS, DFR, ANS, GT1, 3AT, and UGT75C1 genes in anthocyanin synthesis between heat-sensitive and heat-tolerant varieties. Compared to heat-stable varieties, the petals of heat-sensitive varieties exhibited greater differential expression of heat-responsive transcription factors, including HSFs, ERFs, MYBs, and WRKYs. Genes that show a significant negative correlation with the downregulated anthocyanins, including Cse_sc012959.1_g030.1 (βG), Cse_sc001798.1_g020.1 (MYB), Cse_sc006944.1_g010.1 (MYB), and Cse_sc000572.1_g090.1 (HSF), might regulate anthocyanin accumulation in chrysanthemums in response to high-temperature stress. These results provide guidance for the cultivation management and variety selection of chrysanthemums under high-temperature conditions. Additionally, they lay the foundation for elucidating the molecular mechanisms of flower color stability under heat stress and for breeding new heat-tolerant varieties.

Pinus densiflora var. zhangwuensis grows fast, and its drought and salinity resistance are better than Pinus sylvestris var. mongolica. We compared cold hardiness and mechanisms of cold hardiness between the two species, to provide a theoretical basis for promoting and applying P. densiflora var. zhangwuensis in cold regions. A cold stress experiment was carried out on 3-year-old plantlets of P. densiflora var. zhangwuensis and P. sylvestris var. mongolica after hardening at five temperature regimes, 5, −10, −20, −40, and −60 °C, respectively. Some indices of needle samples for both species were measured, such as relative conductivity (REL), maximum photochemical efficiency (Fv/Fm), malondialdehyde (MDA), catalase (CAT), proline (Pro), soluble sugar (SS), and stomata density. REL and MDA values of both species after hardening had the same trend of increasing, but the trend was opposite in Fv/Fm value with increasing cold stress. Compared with P. sylvestris var. mongolica, the P. densiflora var. zhangwuensis had smaller increases in REL and MDA, and a smaller decline in Fv/Fm during cold stress. Compared to the control, REL growth of P. densiflora var. zhangwuensis and P. sylvestris var. mongolica at −60 °C were 0.41 and 0.60, and MDA growth was 29.94 mol g−1 FW and 47.80 mol g−1 FW, and Fv/Fm declines were 0.08 and 0.27. Half-lethal temperatures (LT50) calculated by logistic equation for P. densiflora var. zhangwuensis and P. sylvestris var. mongolica were −58.23 and −50.34 °C, respectively. These data suggest that cold resistance of P. densiflora var. zhangwuensis is stronger than that of P. sylvestris var. mongolica. Cold-resistance mechanisms of the two species differed. In response to cold stress, P. sylvestris var. mongolica had strong osmotic adjustment ability because of higher Pro and SS content, while P. densiflora var. zhangwuensis had strong antioxidant ability due to stronger CAT activity. Stomata density and diameter of P. densiflora var. zhangwuensis were smaller, as were single leaf area and number of leaves per plant, both characteristics promoting survival in a cold environment. Greater shoot height and total biomass of seedlings of P. densiflora var. zhangwuensis might be another reason for its stronger cold tolerance.

With global climate warming and the alteration of precipitation regime, the Three-North Shelter Forest in China has been degraded in recent years, which is a primary challenge for shelterbelt construction. Our understan-ding of the physiological mechanisms behind forest decline remains limited. In this study, we employed dendrochronological methods to measure the basal area increment (BAI) and tree-ring width of both healthy and degraded Pinus sylvestris var. mongolica and Populus L. We developed corresponding standardized chronologies (STD) to analyze their responses to climatic factors and ecological resilience during drought periods, and to elucidate the mechanisms of degradation in P. sylvestris var. mongolica and Populus L. under climate change, as well as their differences in drought adaptability. The results showed that radial growth in healthy P. sylvestris var. mongolica had significantly increased since 1978, with higher mean BAI of healthy trees than the degraded trees. They both exhibited a significant positive correlation with the annual palmer drought severity index (PDSI). Healthy P. sylvestris var. mongolica was less sensitive to precipitation and more resistant and resilient during drought periods. Since 1983, there had been no significant trend in radial growth for either healthy or degraded Populus L., though the mean BAI of healthy Populus L. was significantly higher than that of declining trees. Healthy Populus L. trees exhi-bited weaker correlations with precipitation, PDSI, and vapor pressure deficit (VPD) compared to the degraded trees, yet they showed higher resistance and resilience during droughts. P. sylvestris var. mongolica showed signs of decline earlier than Populus L. and demonstrated greater sensitivity to the PDSI. Nevertheless, it exhibited higher resistance and resilience than Populus L. In conclusion, drought and precipitation emerge as the primary climatic factors contributing to the decline of P. sylvestris var. mongolica and Populus L., with degraded trees being more sensitive to climate change. P. sylvestris var. mongolica demonstrated stronger adaptability to cumulative drought than Populus L., making it more likely to survive under future scenarios of increased drought caused by climate warming. This study provided data support and scientific insights for species selection in the Three-North Shelter Forest Project.

Chitin is a component of the Rhodnius prolixus midgut