Research progress on the types, functions, biosynthesis, and metabolic regulation of ginkgo terpenoids.

Ginkgo biloba L. is a relict plant endemic to China that is commonly considered a "living fossil". It contains unique medicinal compounds that play important roles in its response to various stresses and help maintain human health. Ginkgo terpenoids are known to be important active ingredients but have received less attention than flavonoids. Hence, this review focuses on recent progress in research on the pharmacological effects of ginkgo terpenoid and the bioactivities of different terpenoid monomers. Many key structural genes, enzyme-encoding genes, transcription factors, and noncoding RNAs involved in the ginkgo terpenoid pathway were identified. Finally, many external factors (ecological factors, hormones, etc.) that regulate the biosynthesis and metabolism of terpenoids were proposed. All these findings improve the understanding of the biosynthesis, accumulation, and medicinal functions of terpenoids. Finally, this review includes an in-depth discussion regarding the limitations of terpenoid-related studies and potential future research directions.

First-Principles Investigation into the Interaction of H2O with α-CsPbI3 and the Intrinsic Defects within It.

CsPbI3 possesses three photoactive black phases (α, ß, and γ) with perovskite structures and a non-photoactive yellow phase (δ) without a perovskite structure. Among these, α-CsPbI3 exhibits the best performance. However, it only exists at high temperatures and it tends to transform into the δ phase at room temperature, especially in humid environments. Therefore, the phase stability of CsPbI3, especially in humid environments, is the main obstacle to its further development. In this study, we studied the interaction of H2O with α-CsPbI3 and the intrinsic defects within it. It was found that the adsorption energy in the bulk is higher than that on the surface (-1.26 eV in the bulk in comparison with -0.60 eV on the surface); thus, H2O is expected to have a tendency to diffuse into the bulk once it adsorbs on the surface. Moreover, the intrinsic vacancy of VPb0 in the bulk phase can greatly promote H2O insertion due to the rearrangement of two I atoms in the two PbI6 octahedrons nearest to VPb0 and the resultant breaking of the Pb-I bond, which could promote the phase transition of α-CsPbI3 in a humid environment. Moreover, H2O adsorption onto VI+1 contributes to a further distortion in the vicinity of VI+1, which is expected to enhance the effect of VI+1 on the phase transition of α-CsPbI3. Clarifying the interaction of H2O with α-CsPbI3 and the intrinsic defects within it may provide guidance for further improvements in the stability of α-CsPbI3, especially in humid environments.

A review of changes at the phenotypic, physiological, biochemical, and molecular levels of plants due to high temperatures.

MAIN CONCLUSION: This review summarizes the physiological, biochemical, and molecular regulatory network changes in plants in response to high temperature. With the continuous rise in temperature, high temperature has become an important issue limiting global plant growth and development, affecting the phenotype and physiological and biochemical processes of plants and seriously restricting crop yield and tree growth speed. As sessile organisms, plants inevitably encounter high temperatures and improve their heat tolerance by activating molecular networks related to heat stress, such as signal transduction, synthesis of metabolites, and gene expression. Heat tolerance is a polygenic trait regulated by a variety of genes, transcription factors, proteins, and metabolites. Therefore, this review summarizes the changes in physiological, biochemical and molecular regulatory networks in plants under high-temperature conditions to lay a foundation for an in-depth understanding of the mechanisms involved in plant heat tolerance responses.

A review of the types, functions and regulatory mechanisms of plant spines.

Over a long period of evolution, plants have developed self-protection mechanisms, such as leaving seeds, dropping leaves, growing thorns, producing specific substances or emitting special odors to repel insects. Although studies on the taxonomic characteristics, functions and application of spines in spiny plants have been reported in China and abroad, a systematic overview of plant spines is currently lacking. This study therefore identifies the characteristics and types of plant spines based on domestic and international research on plant spines to provide clear criteria or bases for determining the types of plant spines. In addition, the functions, regulatory mechanisms, and factors influencing the formation of spines and the prospects for their development and application are described and summarized. This study will help to improve the understanding of the types, functions and regulatory mechanisms of plant spines and provide new ideas for the genetic improvement of plants from spiny to nonspiny varieties.

LC-MS and GC-MS Metabolomics Analyses Revealed That Different Exogenous Substances Improved the Quality of Blueberry Fruits under Soil Cadmium Toxicity.

Exogenous substances (ESs) can regulate plant growth and respond to environmental stress, but the effects of different ESs on blueberry fruit quality under soil cadmium (Cd) toxicity and related metabolic mechanisms are still unclear. In this study, four ES treatments [salicylic acid (SA), spermidine (Spd), 2,4-epibrassinolide (EBR), and melatonin (MT)] significantly increased blueberry fruit size, single-fruit weight, sweetness, and anthocyanin content under soil Cd toxicity and effectively reduced fruit Cd content to safe consumption levels by promoting mineral uptake (Ca, Mg, Mn, Cu and Zn). Furthermore, a total of 445, 360, 429, and 554 differentially abundant metabolites (DAMs) (LC-MS) and 63, 48, 79, and 73 DAMs (GC-MS) were identified from four comparison groups (SA/CK, Spd/CK, EBR/CK and MT/CK), respectively. The analyses revealed that ESs improved blueberry fruit quality and tolerance to Cd toxicity mainly by regulating the changes in metabolites related to ABC transporters, the TCA cycle, flavonoid biosynthesis, and phenylpropanoid biosynthesis.

Overview and Recent Progress on the Biosynthesis and Regulation of Flavonoids in Ginkgo biloba L.

Flavonoids and their derivatives play important roles in plants, such as exerting protective activity against biotic and abiotic stresses, functioning in visual signaling to attract pollinators, and regulating phytohormone activity. They are also important secondary metabolites that are beneficial to humans. Ginkgo biloba L. is a well-known relict plant considered to be a "living fossil". Flavonoids present in ginkgo leaves have antioxidant and anti-aging capacities and show good therapeutic effects on a variety of neurological diseases. To date, studies on flavonoids have mainly focused on their extraction, pharmacological effects, and component analysis and on the expression levels of the key genes involved. However, a systematic review summarizing the biosynthesis and regulatory mechanisms of ginkgo flavonoids is still lacking. Thus, this review was conducted to comprehensively introduce the biological characteristics, value, and utilization status of ginkgo; summarize the effects, biosynthetic pathways, and transcriptional regulation of flavonoids; and finally, discuss the factors (ecological factors, hormones, etc.) that regulate the biosynthesis of flavonoids in ginkgo. This review will provide a reference basis for future research on the biosynthesis and efficient utilization of flavonoids in ginkgo.

Fruit Quality and Metabolomic Analyses of Fresh Food Accessions Provide Insights into the Key Carbohydrate Metabolism in Blueberry.

Blueberry is a nutrient-rich berry, and its taste and flavor directly determine the consumer preference. Until now, few studies have focused on the comparison of fresh food quality and the key metabolites in superior fresh-eating blueberry cultivars. Herein, fruit quality indicators of 10 highbush blueberry cultivars were evaluated using 'Bluerain' as the control. Appearance quality analysis of fruits showed that 'Brigitta' had a larger fruit size and 'Anna' was the smallest. 'Anna' fruits, followed by 'O'Neal', had the highest ratio of soluble solids to acidity because of their lowest titratable acidity content. Despite the high soluble sugar content, the antioxidants in 'Anna' fruits such as total flavonoids, anthocyanins and vitamin C were lowest among all cultivars, while 'Duke' seemed to have opposite patterns. Furthermore, a total of 553 and 557 metabolites were identified by non-targeted metabolomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative ion mode, respectively. Particularly, the numbers of differentially accumulated metabolites (DAMs) were the most between the 'O'Neal' vs. 'Bluerain' group. The DAMs involved in the metabolic pathways, sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, galactose metabolism, starch and sucrose metabolism, may be mainly related to the synthesis of flavor and carbohydrate substances. Moreover, the expression patterns of genes involved in sugar metabolism were verified by quantitative real-time PCR (qRT-PCR) analysis in different cultivars. Therefore, the systematical comparison of the quality characteristics, metabolites and expression profiles of related genes in highbush blueberries with good flavor could provide some basis for further research on fresh fruit breeding of blueberries.

Screening and Evaluation of Excellent Blackberry Cultivars and Strains Based on Nutritional Quality, Antioxidant Properties, and Genetic Diversity.

To screen and evaluate excellent blackberry cultivars and strains, 17 indexes of plant growth and fruit horticultural and nutritional characteristics were measured, 20 simple sequence repeat (SSR) markers were analyzed, the fingerprints of 23 blackberry cultivars and strains were constructed, and the processing characteristics of 10 excellent cultivars and strains were evaluated. The results showed that 'Chester' and 'Shuofeng' had the highest plant yield (6.5 kg per plant), of which the 'Chester' fruit also had the highest hardness (2.78 kg/cm2). 'Kiowa' had the highest single fruit weight (10.43 g). '10-5n-2' had the highest total anthocyanin content (225.4 mg/100 g FW) and total polyphenol content (3.24 mg/g FW), but a low plant yield. These results suggest that 'Shuofeng' and 'Chester' are the top two blackberry cultivars planted in Nanjing, with the best growth and comprehensive quality. Moreover, a total of 119 alleles were detected with an average number of 6 alleles per locus. The polymorphism information content (PIC) was 0.374~0.844, with an average of 0.739, indicating a high genetic diversity among the 23 blackberry cultivars and strains. This study provides insight into the plant growth, fruit characteristics and genetic diversity of the 23 blackberry cultivars and strains, and is thus conducive to the protection and utilization of blackberry cultivars and strains.

Quality analysis and metabolomic profiling of the effects of exogenous abscisic acid on rabbiteye blueberry.

Blueberry is a characteristic berry fruit shrub of the genus Vaccinium in the Rhododendron family. The fruit is rich in anthocyanins and has a variety of nutritional and health functions. This study aimed to systematically study the effect of exogenous abscisic acid (ABA) application on ripening and metabolites in blueberry fruits. Blueberry fruit ripening was divided into six stages for further analysis. In this study, nontarget metabolomics was performed to demonstrate the effect on metabolite levels. The results showed that 1000 mg/L ABA significantly promoted fruit ripening and increased anthocyanin content. Moreover, exogenous ABA treatment can affect endogenous ABA levels and improve its antioxidant capacity. Important metabolites of the flavonoid pathway were detected, and the results showed that anthocyanin synthesis increased, and some other bioactive metabolite levels decreased. After comprehensive assessments, we believe that 1000 mg/L exogenous ABA application will have positive impacts on blueberry fruit quality and economic benefits.

Intrinsic defects on α, γ and δ-CsPbI3 (001) surfaces and implications for the α/γ to δ phase transition.

Compared with traditional organic-inorganic hybrid perovskites, CsPbI3 is considered to be a better solar photovoltaic absorption material. However, under environmental conditions, it will undergo the phase transition from the α phase to the γ phase and finally to the non-perovskite phase (δ), especially in a humid environment. Considering the important role of surface intrinsic defects in the phase transition process, we investigated the intrinsic defects on α, γ and δ-CsPbI3 (001) surfaces by first-principles calculations based on density functional theory (DFT). The formation energy of most defects on the surface is similar to that in the bulk in all three phases except for VPb and VI. The formation energy of VPb and VI on the α-CsPbI3 (001) surface is significantly increased, and the formation energy of VPb on the γ-CsPbI3 (001) surface is also increased, due to the relaxation and distortion of the surface Cs and the Pb-I octahedron. The formation energy of interstitial defects on the α-CsPbI3 (001) surface is the lowest due to the remaining large dodecahedral void, even though the Pb-I octahedron distortion has largely enhanced the stability of the α-CsPbI3 (001) surface. The formation energy of VCs is the lowest in all three phases, indicating that Cs ions in CsPbI3 are indeed flexible in CsPbI3. The results are expected to provide a theoretical basis and guidance for the stability improvement of all-inorganic halide perovskites especially in a humid environment.

Root Niches of Blueberry Imprint Increasing Bacterial-Fungal Interkingdom Interactions along the Soil-Rhizosphere-Root Continuum.

Plant root-associated microbiomes play critical roles in promoting plant health, productivity, and tolerance to biotic/abiotic stresses. Blueberry (Vaccinium spp.) is adapted to acidic soils, while the interactions of the root-associated microbiomes in this specific habitat under various root microenvironments remain elusive. Here, we investigated the diversity and community composition of bacterial and fungal communities in various blueberry root niches (bulk soil, rhizosphere soil, and root endosphere). The results showed that blueberry root niches significantly affected root-associated microbiome diversity and community composition compared to those of the three host cultivars. Deterministic processes gradually increased along the soil-rhizosphere-root continuum in both bacterial and fungal communities. The co-occurrence network topological features showed that both bacterial and fungal community complexity and intensive interactions decreased along the soil-rhizosphere-root continuum. Different compartment niches clearly influenced bacterial-fungal interkingdom interactions, which were significantly higher in the rhizosphere, and positive interactions gradually dominated the co-occurrence networks from the bulk soil to the endosphere. The functional predictions showed that rhizosphere bacterial and fungal communities may have higher cellulolysis and saprotrophy capacities, respectively. Collectively, the root niches not only affected microbial diversity and community composition but also enhanced the positive interkingdom interactions between bacterial and fungal communities along the soil-rhizosphere-root continuum. This provides an essential basis for manipulating synthetic microbial communities for sustainable agriculture. IMPORTANCE The blueberry root-associated microbiome plays an essential role in its adaptation to acidic soils and in limiting the uptake of soil nutrients by its poor root system. Studies on the interactions of the root-associated microbiome in the various root niches may deepen our understanding of the beneficial effects in this particular habitat. Our study extended the research on the diversity and composition of microbial communities in different blueberry root compartment niches. Root niches dominated the root-associated microbiome compared to that of the host cultivar, and deterministic processes increased from the bulk soil to the endosphere. In addition, bacterial-fungal interkingdom interactions were significantly higher in the rhizosphere, and those positive interactions progressively dominated the co-occurrence network along the soil-rhizosphere-root continuum. Collectively, root niches dominantly affected the root-associated microbiome and the positive interkingdom interactions increased, potentially providing benefits for the blueberry.

Breeding and Growth Performance of 'Ningzhi 4', a New Blackberry Cultivar with High Yield Potential and Good Quality in China.

The thornless blackberry cultivar 'Ningzhi 4' was developed by the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-Sen). The new blackberry cultivar was selected from the 'Kiowa' (female parent) and 'Hull Thornless' (male parent) F1 hybrid. 'Ningzhi 4' had excellent plant characteristics, including thornlessness, semi-erect to erect canes, vigorous growth and good disease resistance. 'Ningzhi 4' had large fruit and high yield. In addition, the parents of the superior hybrid plant were further identified by SSR markers, which provided the basis for the fingerprint of the new blackberry cultivar 'Ningzhi 4'. This is a commercial cultivar to be grown for fruit production for either shipping or local sales. It also has value as a home-garden plant. This unique type of blackberry fruit was a traditional summer fruit. This new cultivar has thornless semi-erect to erect canes and produces high-quality berries with large size, good firmness, excellent flavor, and potential for shipping and postharvest storage. The new blackberry cultivar 'Ningzhi 4' is adapted to all areas of southern China and is expected to replace or complement 'Kiowa', 'Hull Thornless', 'Chester Thornless' and 'Triple Crown'. A local cultivar patent has been approved by the Jiangsu Variety Approval Committee as 'Rubus spp. Ningzhi 4' in 2020 (S-SV-RS-014-2020). In the future, 'Ningzhi 4' could be promoted as an advantageous thornless blackberry cultivar in the main production regions of China.

GbFLSa overexpression negatively regulates proanthocyanin biosynthesis.

Flavonoids are important secondary metabolites with extensive pharmacological functions. Ginkgo biloba L. (ginkgo) has attracted extensive attention because of its high flavonoid medicinal value. However, little is understood about ginkgo flavonol biosynthesis. Herein, we cloned the full-length gingko GbFLSa gene (1314 bp), which encodes a 363 amino acid protein that has a typical 2-oxoglutarate (2OG)-Fe(II) oxygenase region. Recombinant GbFLSa protein with a molecular mass of 41 kDa was expressed in Escherichia coli BL21(DE3). The protein was localized to the cytoplasm. Moreover, proanthocyanins, including catechin, epicatechin, epigallocatechin and gallocatechin, were significantly less abundant in transgenic poplar than in nontransgenic (CK) plants. In addition, dihydroflavonol 4-reductase, anthocyanidin synthase and leucoanthocyanidin reductase expression levels were significantly lower than those of their CK counterparts. GbFLSa thus encodes a functional protein that might negatively regulate proanthocyanin biosynthesis. This study helps elucidate the role of GbFLSa in plant metabolism and the potential molecular mechanism of flavonoid biosynthesis.

Iron-Cobalt-Cerium Multimetallic Oxides Derived from Prussian Blue Precursors: Enhanced Oxygen Evolution Electrocatalysis.

Exploring non-precious metal-based electrocatalysts is still challenging in 21st century. In this work, a series of hexagonal bipyramidal Ce-based PBA materials as precursors with different Fe/Co metal ratios, namely as CeFex Co1-x -PBA, are successfully constructed via co-precipitation method and converted into corresponding metal oxides (denoted as Fex Co1-x CeOy ) via thermal treatment. Then, they as electrocatalysts realize highly efficient oxygen evolution reaction (OER). Especially, as-synthesized Fe0.7 Co0.3 CeOy electrocatalyst shows very low overpotentials of 320 mV at the current density of 10 mA cm-2 and the Tafel slop of 98.4 mV dec-1 in 1 M KOH with remarkable durability for 24 h, which was due to the synergistic effect of multi-metal FeCoCe centers. Furthermore, a two-electrode cell of Fe0.7 Co0.3 CeOy /NF||Pt/C/NF realizes outstanding overall water splitting with a voltage of only 1.71 V at 10 mA cm-2 and remarkable long-term durability, that is even superior to benchmark IrO2 /NF||Pt/C/NF counterpart.

Analysis of flavonoid-related metabolites in different tissues and fruit developmental stages of blackberry based on metabolome analysis.

Blackberry is an economically important shrub species of Rubus in the Rosaceae family. It is rich in phenolic compounds, which have many health effects and pharmaceutical value. The utilization of metabolites from various blackberry tissues is still in the primary stage of development, so investigating the metabolites in various tissues is of practical significance. In this study, nontargeted LC - MS metabolomics was used to identify and measure metabolites in the roots, stems, leaves and fruits (green, red, and black fruits) of blackberry "Chester". We found that 1,427 and 874 metabolites were annotated in the positive and negative ion modes (POS; NEG), respectively. Differentially abundant metabolites (DAMs) between the leaf and root groups were the most abundant (POS: 249; NEG: 141), and the DAMs between the green and red fruit groups were the least abundant (POS: 21; NEG: 14). Moreover, the DAMs in different fruit development stages were far less than those in different tissues. There were significant differences in flavonoid biosynthesis-related pathways among the comparison groups. Trend analysis showed that the profile 10 had the largest number of metabolites. This study provides a scientific basis for the classification and efficient utilization of resources in various tissues of blackberry plants and the directional development of blackberry products.

Integrative physiological, metabolomic and transcriptomic analysis reveals nitrogen preference and carbon and nitrogen metabolism in blackberry plants.

Nitrogen (N) is an indispensable element for plant growth and development. To understand the regulation of underlying carbon (C) and N metabolism in blackberry plants, we performed integrated analyses of the physiology, metabolome and transcriptome. Blackberry plants were subjected to no N, nitrate (NO3⁻)-N, ammonium (NH4+)-N and urea treatments. Our results showed that the NH4⁺-N treatment yielded higher values for the biomass, chlorophyll, antioxidants, N contents and antioxidant enzyme activities, as well as lower levels of free radicals and the C/N ratio compared with other treatments. Transcriptome analysis showed that different N forms significantly affected photosynthesis, flavonoid biosynthesis and the TCA cycle. Metabolome analysis indicated that the levels of lipids, carbohydrates, flavonoids and amino acids were markedly changed under different N treatments. Integrated transcriptomic and metabolomic data revealed that amino acids, including proline, arginine, L-isoleucine, L-aspartate, threonine, and L-glutamate, played important roles in maintaining normal plant growth by regulating N metabolism and amino acid metabolism. Overall, blackberry plants preferentially take up NH4⁺-N. Under the NH4⁺-N treatment, N assimilation was stronger, flavonoid biosynthesis was decreased, and the promoting influence of NH4⁺-N on N metabolism was better than that of NO3⁻-N. However, the NO3⁻-N treatment enhanced the C/N ratio, accelerated the process of C metabolism and increased the synthesis of flavonoids, thereby accelerating the flow of N metabolism to C metabolism. These results provide deeper insight into coordinating C and N metabolism and improving N use efficiency in blackberry plants.

Transcriptomic and Metabolomic Profiling Reveals the Variations in Carbohydrate Metabolism between Two Blueberry Cultivars.

Blueberry is a high-quality fruit tree with significant nutritional and economic value, but the intricate mechanism of sugar accumulation in its fruit remains unclear. In this study, the ripe fruits of blueberry cultivars 'Anna' and 'Misty' were utilized as experimental materials, and physiological and multi-omics methodologies were applied to analyze the regulatory mechanisms of the difference in sugar content between them. The results demonstrated that the 'Anna' fruit was smaller and had less hardness than the 'Misty' fruit, as well as higher sugar content, antioxidant capability, and lower active substance content. A total of 7067 differentially expressed genes (DEGs) (3674 up-regulated and 3393 down-regulated) and 140 differentially abundant metabolites (DAMs) (82 up-regulated and 58 down-regulated) were identified between the fruits of the two cultivars. According to KEGG analysis, DEGs were primarily abundant in phenylpropanoid synthesis and hormone signal transduction pathways, whereas DAMs were primarily enriched in ascorbate and aldarate metabolism, phenylpropanoid biosynthesis, and the pentose phosphate pathway. A combined multi-omics study showed that 116 DEGs and 3 DAMs in starch and sucrose metabolism (48 DEGs and 1 DAM), glycolysis and gluconeogenesis (54 DEGs and 1 DAM), and the pentose phosphate pathway (14 DEGs and 1 DAM) were significantly enriched. These findings suggest that blueberries predominantly increase sugar accumulation by activating carbon metabolism network pathways. Moreover, we identified critical transcription factors linked to the sugar response. This study presents new understandings regarding the molecular mechanisms underlying blueberry sugar accumulation and will be helpful in improving blueberry fruit quality through breeding.

Integrated Physiological and Metabolomic Analyses Reveal the Differences in the Fruit Quality of the Blueberry Cultivated in Three Soilless Substrates.

With improving living standards, traditional blueberry planting modes cannot meet commercial demands, and blueberry cultivation with soilless substrate has become a popular solution in the blueberry industry. In this study, different soilless substrate treatments were found to markedly influence fruit appearance and intrinsic quality. The fruit in the 50:50 peat/pine bark (v/v) (FPB) treatment group had the maximum single fruit weight, largest vertical diameter, and brightest color, as well as the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) value, solid-acid ratio and anthocyanin content. The fruit in the 50:50 pine bark/rice husk (v/v) (FBR) treatment group had the highest total phenol and flavonoid levels, largest drip loss value, and lowest total pectin content and firmness value. Metabolomic analysis showed that flavonoid, carbohydrate, and carbohydrate conjugate, and amino acid, peptide, and analog levels were significantly different between groups. Fruit in the FPB group had the highest sucrose, D-fructose 1,6-bisphosphate, salidroside, tectorigenin, naringenin chalcone, trifolirhizin, and galangin contents. The increase in the relative expression of phenylalanine (Phe) promoted the synthesis of fruit polyphenols in the FBR group. Our results provide new insights into the effects of different substrates on the quality of blueberries and a reference for the soilless substrate cultivation of blueberries.

Comprehensive resistance evaluation of 15 blueberry cultivars under high soil pH stress based on growth phenotype and physiological traits.

High soil pH is one of the main abiotic factors that negatively affects blueberry growth and cultivation. However, no comprehensive evaluation of the high soil pH tolerance of different blueberry cultivars has been conducted. Herein, 16 phenotypic and physiological indices of 15 blueberry cultivars were measured through pot experiments, and the high-pH soil tolerance coefficient (HSTC) was calculated based on these indices to comprehensively evaluate the high-soil-pH tolerance of plants. The results demonstrated that high soil pH stress inhibited blueberry 77.growth, and MDA, soluble sugar (SS), and soluble protein (SP) levels increased in leaves. Moreover, in all cultivars, CAT activity in the antioxidant system was enhanced, whereas SOD activity was reduced, and the relative expression levels of the antioxidant enzyme genes SOD and CAT showed similar changes. In addition, the leaf chlorophyll relative content (SPAD), net photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (Gs) decreased, while changes in the intercellular CO2 concentration (Ci) were noted in different cultivars. Finally, according to the comprehensive evaluation value D obtained from the combination of principal component analysis (PCA) and membership function (MF), the 15 blueberry cultivars can be divided into 4 categories: high soil pH-tolerant type ['Briteblue' (highest D value 0.815)], intermediate tolerance type ('Zhaixuan 9', 'Zhaixuan 7', 'Emerald', 'Primadonna', 'Powderblue' and 'Chandler'), low high soil pH-tolerant type ('Brightwell', 'Gardenblue', 'Plolific' and 'Sharpblue') and high soil pH-sensitive type ['Legacy', 'Bluegold', 'Baldwin' and 'Anna' (lowest D value 0.166)]. Stepwise linear regression analysis revealed that plant height, SS, E, leaf length, Ci, SOD, and SPAD could be used to predict and evaluate the high soil pH tolerance of blueberry cultivars.

Overexpression of RuFLS2 Enhances Flavonol-Related Substance Contents and Gene Expression Levels.

As an emerging third-generation fruit, blackberry has high nutritional value and is rich in polyphenols, flavonoids and anthocyanins. Flavonoid biosynthesis and metabolism is a popular research topic, but no related details have been reported for blackberry. Based on previous transcriptome data from this research group, two blackberry flavonol synthase genes were identified in this study, and the encoded proteins were subjected to bioinformatics analysis. RuFLS1 and RuFLS2 are both hydrophobic acidic proteins belonging to the 2OG-Fe(II) dioxygenase superfamily. RuFLS2 was expressed at 27.93-fold higher levels than RuFLS1 in red-purple fruit by RNA-seq analysis. Therefore, RuFLS2-overexpressing tobacco was selected for functional exploration. The identification of metabolites from transgenic tobacco showed significantly increased contents of flavonoids, such as apigenin 7-glucoside, kaempferol 3-O-rutinoside, astragalin, and quercitrin. The high expression of RuFLS2 also upregulated the expression levels of NtF3H and NtFLS in transgenic tobacco. The results indicate that RuFLS2 is an important functional gene regulating flavonoid biosynthesis and provides an important reference for revealing the molecular mechanism of flavonoid accumulation in blackberry fruit.