Regulation of carbohydrate metabolism during anther development in a thermo-sensitive genic male-sterile wheat line.

Bainong sterility (BNS) is a thermo-sensitive genic male sterile wheat line, characterised by anther fertility transformation in response to low temperature (LT) stress during meiosis, the failure of vacuole decomposition and the absence of starch accumulation in sterile bicellular pollen. Our study demonstrates that the late microspore (LM) stage marks the transition from the anther growth to anther maturation phase, characterised by the changes in anther structure, carbohydrate metabolism and the main transport pathway of sucrose (Suc). Fructan is a main storage polysaccharide in wheat anther, and its synthesis and remobilisation are crucial for anther development. Moreover, the process of pollen amylogenesis and the fate of the large vacuole in pollen are closely intertwined with fructan synthesis and remobilisation. LT disrupts the normal physiological metabolism of BNS anthers during meiosis, particularly affecting carbohydrate metabolism, thus determining the fate of male gametophytes and pollen abortion. Disruption of fructan synthesis and remobilisation regulation serves as a decisive event that results in anther abortion. Sterile pollen exhibits common traits of pollen starvation and impaired starch accumulation due to the inhibition of apoplastic transport starting from the LM stage, which is regulated by cell wall invertase TaIVR1 and Suc transporter TaSUT1.

ARF2 positively regulates flavonols and proanthocyanidins biosynthesis in Arabidopsis thaliana.

MAIN CONCLUSION: Auxin response factor 2 acts as a positive regulator to fine-tune the spatial and temporal accumulation of flavonoid compounds, mainly flavonols and proanthocyanidins in Arabidopsis. Auxin response factor (ARF) proteins are reported to involve in auxin-mediated regulation of flavonoid biosynthesis. However, the detailed regulation mechanism of ARF remains still unknown. Here, we provide genetic and molecular evidence that one of the twenty-three ARF members-ARF2-positively regulates flavonoid biosynthesis at multi-level in tissue-specific manner in Arabidopsis thaliana. Loss-of-function mutation of ARF2 led to significant reduction in flavonoid content (e.g., flavonols and proanthocyanidins) in the seedlings and seeds of the Arabidopsis arf2 mutants. Over-expression of ARF2 increased flavonols and proanthocyanidins content in Arabidopsis. Additionally, the changes of flavonoid content correlate well with the transcript abundance of several regulatory genes (e.g., MYB11, MYB12, MYB111, TT2, and GL3), and key biosynthetic genes (e.g., CHS, F3'H, FLS, ANS, ANR, TT12, TT19, and TT15), in the arf2 mutant and ARF2 over-expression lines. Transient transactivation assays with site-directed mutagenesis confirmed that ARF2 directly regulates the expression of MYB12 and FLS genes in the flavonol pathway and ANR in the proanthocyanidin pathway, and indirectly regulates MYB11 and MYB111 genes in the flavonol pathway, and ANS, TT12, TT19 and TT15 genes in the proanthocyanidin pathway. Further genetic results indicated that ARF2 acts upstream of MYB12 to regulate flavonol accumulation, and of TT2 to regulate proanthocyanidins accumulation. In particular, yeast two-hybrid assays revealed that ARF2 physically interacts with TT2, a master regulator of proanthocyanidins biosynthesis. Combined together, these results indicated that ARF2 functions as a positive regulator for the fine-tuned spatial and temporal regulation of flavonoids (mainly flavonols and proanthocyanidins) accumulation in Arabidopsis.

Medicinal Values and Potential Risks Evaluation of Ginkgo biloba Leaf Extract (GBE) Drinks Made from the Leaves in Autumn as Dietary Supplements.

Ginkgo tea and ginkgo wine are two familiar Ginkgo biloba leaf extract (GBE) drinks in the form of dietary supplements (DS) used for healthcare in east Asia. Nevertheless, a comprehensive evaluation of their safety and efficacy is still lacking. In this study, GBE drinks were prepared from naturally newly senescent yellow leaves (YL) and green leaves (GL) in autumn. Their total flavonoids, antioxidant capacity and prescribed ingredients were investigated. In brief, the proportions of total flavonoids, total flavonol glycosides (TFs), total terpene trilactones (TTLs) and ginkgolic acids in the GBE drinks all did not meet the standards of worldwide pharmacopoeias. Specifically, the levels of TFs in the ginkgo tea prepared from YL were significantly higher than that prepared from GL. Further analyses revealed a substandard ratio of isorhamnetin/quercetin and an accumulation of leaf-age-related compounds, which were both unqualified. The proportions of specific TTLs varied between the ginkgo tea and ginkgo wine, although no significant differences were detected in terms of the total levels of TTLs. Noticeably, numerous biflavones and thousands of times over the limiting concentration of ginkgolic acids, including newly identified types, were only detected in ginkgo wine. Finally, the use of the GBE drinks as DSs was comprehensively evaluated according to the acceptable daily intake. This study showed the limited healthcare effects of GBE drinks despite their powerful antioxidant capacity.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings.

BACKGROUND: Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. RESULTS: In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a "Y- type" asymmetric structure and contained an axillary bud that was about 1-3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. CONCLUSIONS: Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

A Ricin B-Like Lectin Protein Physically Interacts with TaPFT and Is Involved in Resistance to Fusarium Head Blight in Wheat.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, has become one of the most serious diseases that damage wheat. The TaPFT (pore-forming toxin-like) and TaHRC (histidine-rich calcium-binding protein) genes at the quantitative trait locus Fhb1 were identified to confer resistance to FHB in the wheat cultivar Sumai 3. In this study, a wheat ricin B-like lectin gene (designated TaRBL) that interacted with TaPFT was isolated by a yeast two-hybrid screen of a wheat cDNA library. A yeast two-hybrid and bimolecular fluorescence complementation study further verified that TaRBL interacted with TaPFT but not with TaHRC. Gene expression studies showed that upon F. graminearum infection, TaRBL expression was upregulated in resistant cultivars but downregulated in susceptible cultivars. Furthermore, knockdown of TaRBL expression by barley stripe mosaic virus-induced gene silencing significantly reduced the resistance of wheat to FHB in both the resistant cultivar Sumai 3 and the susceptible cultivar Jimai 22. Thus, we conclude that TaRBL encodes a ricin B-like lectin protein that interacts with TaPFT and is involved in resistance to FHB in wheat.

GbMYBR1 from Ginkgo biloba represses phenylpropanoid biosynthesis and trichome development in Arabidopsis.

MAIN CONCLUSION: GbMYBR1, a new type of R2R3-MYB repressor from Ginkgo biloba, displayed pleiotropic effects on plant growth, phenylpropanoid accumulation, by regulating multiple related genes at different levels. Ginkgo biloba is a typical gymnosperm that has been thriving on earth for millions of years. MYB transcription factors (TFs) play important roles in diverse processes in plants. However, the role of MYBs remains largely unknown in Ginkgo. Here, an MYB TF gene from Ginkgo, designated as GbMYBR1, was found to act as a repressor in multiple processes. GbMYBR1 was mainly expressed in the leaves of Ginkgo. Over-expression of GbMYBR1 in Arabidopsis thaliana led to growth retardation, decreases in lignin content, reduced trichome density, and remarkable reduction in anthocyanin and flavonol contents in leaves. Proanthocyanidin content was decreased in the seeds of transgenic Arabidopsis, which led to light-brown seed color. Both qPCR and transcriptome sequencing analyses demonstrated that the transcript levels of multiple genes related to phenylpropanoid biosynthesis, trichome formation, and pathogen resistance were down-regulated in the transgenic Arabidopsis. In particular, we found that GbMYBR1 directly interacts with the bHLH cofactor GL3 as revealed by yeast two-hybrid assays. Our work indicated that GbMYBR1 has pleiotropic effects on plant growth, phenylpropanoid accumulation, and trichome development, mediated by interaction with GL3 or direct suppression of key pathway genes. Thus, GbMYBR1 represents a novel type of R2R3 MYB repressor.

In Situ Synthesis of CsPbX3/Polyacrylonitrile Nanofibers with Water-Stability and Color-Tunability for Anti-Counterfeiting and LEDs.

Inorganic CsPbX3 (X = Cl, Br, I) perovskite quantum dots (PQDs) have attracted widespread attention due to their excellent optical properties and extensive application prospects. However, their inherent structural instability significantly hinders their practical application despite their outstanding optical performance. To enhance stability, an in situ electrospinning strategy was used to synthesize CsPbX3/polyacrylonitrile composite nanofibers. By optimizing process parameters (e.g., halide ratio, electrospinning voltage, and heat treatment temperature), all-inorganic CsPbX3 PQDs have been successfully grown in a polyacrylonitrile (PAN) matrix. During the electrospinning process, the rapid solidification of electrospun fibers not only effectively constrained the formation of large-sized PQDs but also provided effective physical protection for PQDs, resulting in the improvement in the water stability of PQDs by minimizing external environmental interference. Even after storage in water for over 100 days, the PQDs maintained approximately 93.5% of their photoluminescence intensity. Through the adjustment of halogen elements, the as-obtained composite nanofibers exhibited color-tunable luminescence in the visible light region, and based on this, a series of multicolor anti-counterfeiting patterns were fabricated. Additionally, benefiting from the excellent water stability and optical performance, the CsPbBr3/PAN composite film was combined with red-emitting K2SiF6:Mn4+ (KSF) on a blue LED (460 nm), producing a stable and efficient WLED device with a color temperature of around 6000 K and CIE coordinates of (0.318, 0.322). These results provide a general approach to synthesizing PQDs/polymer nanocomposites with excellent water stability and multicolor emission, thereby promoting their practical applications in multifunctional optoelectronic devices and advanced anti-counterfeiting.

The APETALA2-MYBL2 module represses proanthocyanidin biosynthesis by affecting formation of the MBW complex in seeds of Arabidopsis thaliana.

Proanthocyanidins (PAs) are the second most abundant plant phenolic natural products. PA biosynthesis is regulated by the well-documented MYB/bHLH/WD40 (MBW) complex, but how this complex itself is regulated remains ill defined. Here, in situ hybridization and ß-glucuronidase staining show that APETALA2 (AP2), a well-defined regulator of flower and seed development, is strongly expressed in the seed coat endothelium, where PAs accumulate. AP2 negatively regulates PA content and expression levels of key PA pathway genes. AP2 activates MYBL2 transcription and interacts with MYBL2, a key suppressor of the PA pathway. AP2 exerts its function by directly binding to the AT-rich motifs near the promoter region of MYBL2. Molecular and biochemical analyses revealed that AP2 forms AP2-MYBL2-TT8/EGL3 complexes, disrupting the MBW complex and thereby repressing expression of ANR, TT12, TT19, and AHA10. Genetic analyses revealed that AP2 functions upstream of MYBL2, TT2, and TT8 in PA regulation. Our work reveals a new role of AP2 as a key regulator of PA biosynthesis in Arabidopsis. Overall, this study sheds new light on the comprehensive regulation network of PA biosynthesis as well as the dual regulatory roles of AP2 in seed development and accumulation of major secondary metabolites in Arabidopsis.

Multi-omics reveal key enzymes involved in the formation of phenylpropanoid glucosides in Artemisia annua.

Although mainly known for producing artemisinin, Artemisia annua is enriched in phenylpropanoid glucosides (PGs) with significant bioactivities. However, the biosynthesis of A. annua PGs is insufficiently investigated. Different A. annua ecotypes from distinct growing environments accumulate varying amounts of metabolites, including artemisinin and PGs such as scopolin. UDP-glucose:phenylpropanoid glucosyltransferases (UGTs) transfers glucose from UDP-glucose in PG biosynthesis. Here, we found that the low-artemisinin ecotype GS produces a higher amount of scopolin, compared to the high-artemisinin ecotype HN. By combining transcriptome and proteome analyses, we selected 28 candidate AaUGTs from 177 annotated AaUGTs. Using AlphaFold structural prediction and molecular docking, we determined the binding affinities of 16 AaUGTs. Seven of the AaUGTs enzymatically glycosylated phenylpropanoids. AaUGT25 converted scopoletin to scopolin and esculetin to esculin. The lack of accumulation of esculin in the leaf and the high catalytic efficiency of AaUGT25 on esculetin suggest that esculetin is methylated to scopoletin, the precursor of scopolin. We also discovered that AaOMT1, a previously uncharacterized O-methyltransferase, converts esculetin to scopoletin, suggesting an alternative route for producing scopoletin, which contributes to the high-level accumulation of scopolin in A. annua leaves. AaUGT1 and AaUGT25 responded to induction of stress-related phytohormones, implying the involvement of PGs in stress responses.

Characterization of a heat responsive UDP: Flavonoid glucosyltransferase gene in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) accumulates abundant flavonoid glycosides that are the major bioactive ingredients in tea. Biosynthesis of flavonoid glycosides are catalyzed by UDP-glucosyltransferases (UGTs) that are widely present in plants. Among one hundred and seventy-eight UGTs genes that we have previously identified in tea plant, few of them have been functionally characterized. In the present study, we further identified UGT73A17 gene that is responsible for the biosynthesis of a broad range of flavonoid glycosides. Sequence analysis revealed that the deduced UGT73A17 protein showed high identity with 7-O-glycosyltransferases at amino acid level and it was clustered into the clade containing several 7-O-glycosyltransferases from other plant species. Enzymatic assays revealed that the recombinant UGT73A17 protein (rUGT73A17) exhibited activity toward flavonols (kaempferol, quercetin, and myricetin), flavones (apigenin, luteolin, and tricetin), flavanone (naringenin), isoflavones (genistein) and epicatechin gallate, yielding 7-O-glucosides as the major in vitro products. In particular, rUGT73A17 displayed higher activity at high temperatures (eg. 50°C) than at low temperatures, which was consistent with its relatively high expression level at high temperatures. Two amino acid substitutions at I296L and V466A improved the enzymatic activity of rUGT73A17. Our study demonstrated that UGT73A17 is responsible for the biosynthesis of a broad range of flavonoid glucosides, which is also involved in heat response and quality of tea plant.

Characterization of UGT716A1 as a Multi-substrate UDP:Flavonoid Glucosyltransferase Gene in Ginkgo biloba.

Ginkgo biloba L., a "living fossil" and medicinal plant, is a well-known rich source of bioactive flavonoids. The molecular mechanism underlying the biosynthesis of flavonoid glucosides, the predominant flavonoids in G. biloba, remains unclear. To better understand flavonoid glucosylation in G. biloba, we generated a transcriptomic dataset of G. biloba leaf tissue by high-throughput RNA sequencing. We identified 25 putative UDP-glycosyltransferase (UGT) unigenes that are potentially involved in the flavonoid glycosylation. Among them, we successfully isolated and expressed eight UGT genes in Escherichia coli, and found that recombinant UGT716A1 protein was active toward broad range of flavonoid/phenylpropanoid substrates. In particular, we discovered the first recombinant UGT protein, UGT716A1 from G. biloba, possessing unique activity toward flavanol gallates that have been extensively documented to have significant bioactivity relating to human health. UGT716A1 expression level paralleled the flavonoid distribution pattern in G. biloba. Ectopic over-expression of UGT716A1 in Arabidopsis thaliana led to increased accumulation of several flavonol glucosides. Identification and comparison of the in vitro enzymatic activity of UGT716A1 homologs revealed a UGT from the primitive land species Physcomitrella patens also showed broader substrate spectrum than those from higher plants A. thaliana, Vitis vinifera, and Medicago truncatula. The characterization of UGT716A1 from G. biloba bridges a gap in the evolutionary history of UGTs in gymnosperms. We also discuss the implication of UGT716A1 for biosynthesis, evolution, and bioengineering of diverse glucosylated flavonoids.

Correlations among persistent viral infection, heart function and Chinese medicine syndromes in dilated cardiomyopathy patients.

OBJECTIVE: To investigate the correlations among persistent viral infection, heart function and Chinese medicine (CM) difined-syndromes in patients with dilated cardiomyopathy (DCM). METHODS: Fifty patients with DCM in the First Affiliated Hospital of Zhejiang Chinese Medical University from October 2009 to December 2011 were selected as the research subjects, and 30 healthy people were simultaneously selected as the normal control group to detect persistent viral infections after admission. The CM syndrome type and grade of heart function were then evaluated. The expression level of Coxsackie adenovirus receptor (CAR) was detected using the flow cytometry (FCM) technique, coxsackie virus RNA (CVB-RNA) using reverse transcription polymerase chain reaction (RTPCR), and the plasma brain natriuretic peptide (BNP) level with a Triage meter plus diagnosis instrument. Finally, the parameters such as left ventricular end diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) were measured by ultrasonic cardiogram. Person correlation analysis was used for measured data, Spearman correlation analysis for rating data, and the Chi-square test for numerical data. RESULTS: CVB-RNA was positive in 22 patients (44%) with DCM, while only 6 cases (20%) were CVB-RNA-positive in the normal control group, with a significant difference between the two groups (P<0.01). The expression level of CAR was significantly elevated in the DCM group compared with the normal control group (P<0.01). In CVB-RNA-positive patients (22 cases), the expression level of CAR was significantly higher than in CVB-RNA-negative patients (28 cases; P<0.01). In the DCM patients, there was a positive correlation between the CAR expression and the BNP level (r=0.34, P<0.05), while no significant difference was found between the CAR expression and the LVEF and LVEDd (r=-0.32, 0.30, P>0.05). There was no clear correlation between virus infection and the CM syndrome types in DCM patients (r=-0.22, P>0.05). According to the sequence of syndrome types: phlegm → qi deficiency → blood stasis → hydroretention with asthenic yang (from low to high), a positive correlation was existed between the BNP levels and CM syndrome types (r=0.139, P<0.05). CONCLUSION: The expression of CAR on the surface of white cells could be used to detect persistent viral infection. The expression level of CAR and heart function in DCM patients were highly correlated. The expression level of BNP may serve as an objective index for differentiating CM syndromes for patients with DCM.

Correlation between virus persistent infection and cardic function in patients with dilated cardiomyopathy.

In our study, 50 patients with dilated cardiomyopathy (DCM) were selected to investigate the correlation between virus persistent infection and cardic function. We found that 44% of patients with DCM were coxsackie virus B-RNA (CVB-RNA) positive, significantly different from that (20%) of the normal control group (P<0.05). The expression levels of coxsackie adenovirus receptor (CAR) in patients with DCM were significantly higher than those in the normal control group (P<0.01). In CVB-RNA-positive patients, expression levels of CAR were significantly higher than those in CVB-RNA-negative patients (P<0.01). There was a positive correlation between CAR expression and brain natriuretic peptide (BNP) level in patients with DCM, but no significant correlations between the CAR expression level and left ventricular ejection fraction (LVEF) or left ventricular end diastolic diameter (LVEDd). These results showed that expression levels of CAR on the surface of white cells can be used as an indicator for detecting persistent virus infection. We found that expression levels of CAR and heart function in patients with DCM were highly correlated.