EFHD2 suppresses intestinal inflammation by blocking intestinal epithelial cell TNFR1 internalization and cell death.

TNF acts as one pathogenic driver for inducing intestinal epithelial cell (IEC) death and substantial intestinal inflammation. How the IEC death is regulated to physiologically prevent intestinal inflammation needs further investigation. Here, we report that EF-hand domain-containing protein D2 (EFHD2), highly expressed in normal intestine tissues but decreased in intestinal biopsy samples of ulcerative colitis patients, protects intestinal epithelium from TNF-induced IEC apoptosis. EFHD2 inhibits TNF-induced apoptosis in primary IECs and intestinal organoids (enteroids). Mice deficient of Efhd2 in IECs exhibit excessive IEC death and exacerbated experimental colitis. Mechanistically, EFHD2 interacts with Cofilin and suppresses Cofilin phosphorylation, thus blocking TNF receptor I (TNFR1) internalization to inhibit IEC apoptosis and consequently protecting intestine from inflammation. Our findings deepen the understanding of EFHD2 as the key regulator of membrane receptor trafficking, providing insight into death receptor signals and autoinflammatory diseases.

Direct Z-Scheme Heterostructure of Vertically Oriented SnS2 Nanosheet on BiVO4 Nanoflower for Self-Powered Photodetectors and Water Splitting.

The construction of nanostructured Z-scheme heterostructure is a powerful strategy for realizing high-performance photoelectrochemical (PEC) devices such as self-powered photodetectors and water splitting. Considering the band structure and internal electric field direction, BiVO4 is a promising candidate to construct SnS2 -based heterostructure. Herein, the direct Z-scheme heterostructure of vertically oriented SnS2 nanosheet on BiVO4 nanoflower is rationally fabricated for efficient self-powered PEC photodetectors. The Z-scheme heterostructure is identified by ultraviolet photoelectron spectroscopy, photoluminescence spectroscopy, PEC measurement, and water splitting. The SnS2 /BiVO4 heterostructure shows a superior photodetection performance such as excellent photoresponsivity (10.43 mA W-1 ), fast response time (6 ms), and long-term stability. Additionally, by virtue of efficient Z-scheme charge transfer and unique light-trapping nanostructure, the SnS2 /BiVO4 heterostructure also displays a remarkable photocatalytic hydrogen production rate of 54.3 µmol cm-2 h-1 in Na2 SO3 electrolyte. Furthermore, the synergistic effect between photo-activation and bias voltage further improves the PEC hydrogen production rate of 360 µmol cm-2 h-1 at 0.8 V, which is an order of magnitude above the BiVO4 . The results provide useful inspiration for designing direct Z-scheme heterostructures with special nanostructured morphology to signally promote the performance of PEC devices.

Two genes encoded by mulberry crinkle leaf virus (MCLV): The V4 gene enhances viral replication, and the V5 gene is needed for MCLV infection in Nicotiana benthamiana.

Mulberry crinkle leaf virus (MCLV) is a member of the genus Mulcrilevirus, family Geminiviridae. The expression and functions of the V4 and V5 genes encoded by the MCLV genome remain unknown. Here, we confirmed the expression of V4 and V5 by analyzing the V4 and V5 mRNAs and the promoter activity of individual ORFs upstream sequences. The functions of V4 and V5 were investigated by constructing Agrobacterium-mediated infectious clones of wild-type MCLV variant П (MCLV vII), MCLVwt and MCLV vП mutants, such as MCLVmV4 (start codon of V4 ORF mutated), MCLVdV4 (5'-end partial deletion of V4 ORF sequence) and MCLVmV5 (V5 ORF start codon mutated). Although MCLVwt, MCLVmV4, and MCLVdV4 could infect natural host mulberry and experimental tomato plants systematically, the replication of the MCLVmV4 and MCLVdV4 genomes was obviously reduced compared to MCLVwt in both mulberry and tomato plants. MCLV vП expressing V5 could infect Nicotiana benthamiana plants systematically, but MCLVmV5 could not, implying that V5 is needed for MCLV vП to infect N. benthamiana plants. Taken together, V4 is involved in replication of the MCLV genome in host plants, and V5 potentially might extend the host range. Our findings lay a foundation for in-depth insight into the functions of MCLV-encoded proteins and provide a novel perspective for the subsequent study of MCLV-host plant interactions.

Functional Characterization of a (E)-ß-Ocimene Synthase Gene Contributing to the Defense against Spodoptera litura.

Soybean is a worldwide crop that offers valuable proteins, fatty acids, and phytonutrients to humans but is always damaged by insect pests or pathogens. Plants have captured sophisticated defense mechanisms in resisting the attack of insects and pathogens. How to protect soybean in an environment- or human-friendly way or how to develop plant-based pest control is a hotpot. Herbivore-induced plant volatiles that are released by multiple plant species have been assessed in multi-systems against various insects, of which (E)-ß-ocimene has been reported to show anti-insect function in a variety of plants, including soybean. However, the responsible gene in soybean is unknown, and its mechanism of synthesis and anti-insect properties lacks comprehensive assessment. In this study, (E)-ß-ocimene was confirmed to be induced by Spodoptera litura treatment. A plastidic localized monoterpene synthase gene, designated as GmOCS, was identified to be responsible for the biosynthesis of (E)-ß-ocimene through genome-wide gene family screening and in vitro and in vivo assays. Results from transgenic soybean and tobacco confirmed that (E)-ß-ocimene catalyzed by GmOCS had pivotal roles in repelling a S. litura attack. This study advances the understanding of (E)-ß-ocimene synthesis and its function in crops, as well as provides a good candidate for further anti-insect soybean improvement.

Occurrence and Pathogenicity of Hop Stunt Viroid Infecting Mulberry (Morus alba) Plants in China.

To investigate the presence of hop stunt viroid (HSVd) in mulberry (Morus alba) plants in China, HSVd was detected by reverse transcription (RT)-PCR using dsRNAs extracted from symptomatic or asymptomatic mulberry leaf samples collected from a mulberry field located in Zhenjiang, China, as a template and the primer pairs for HSVd detection. The primer pairs were designed based on the conserved sequence of 25 HSVd variants deposited in the GenBank database. Four out of a total of 53 samples were HSVd-positive, confirming that HSVd is present in mulberry plants in China. The consensus full-length nucleotide (nt) sequence of two HSVd variants determined by sequencing the HSVd variants in these four HSVd-positive samples consisted of 296 nt and shared the highest nt identity of 96.8% with that from plum in Turkey but relatively low identity with those from mulberry in Iran (87.3 to 90.8%). Phylogenetic analysis showed that these HSVd variants clustered together with those of the HSVd-hop group. Analysis of the infectivity and pathogenicity to hosts by the constructed Agrobacterium-mediated dimeric head-to-tail HSVd cDNA infectious clones demonstrated that one of the HSVd variants identified in this study infects the natural host, mulberry plants, and also infects experimental plants, cucumber, and tomato. It probably induces stunting symptoms in HSVd-infected tomatoes but does not induce symptoms on mulberry leaves or in cucumbers. Although HSVd infecting mulberry has been found in Iran, Italy, and Lebanon, this is the first study to report this viroid in naturally infected mulberry plants in China.

Whole genome sequence of mulberry crinivirus, a new member of the genus Crinivirus.

The whole genome sequence of mulberry crinivirus (MuCV), a novel member of the genus Crinivirus (family Closteroviridae) identified in mulberry (Morus alba L), was determined. The virus possesses a bipartite genome. RNA1 contains 8571 nucleotides (nt) with four open reading frames (ORFs). ORF1a encodes a putative polyprotein with papain-like protease, methyltransferase, and RNA helicase domains. ORF1b putatively encodes an RNA-dependent RNA polymerase (RdRp), which is probably expressed via a + 1 ribosomal frameshift. RNA2 consists of 8082 nt, containing eight ORFs that are similar in size and position to orthologous genes of other criniviruses. Phylogenetic analysis based on RdRp amino acid sequences of criniviruses placed MuCV in group 1.

Bi2 Te3 /Bi2 Se3 /Bi2 S3 Cascade Heterostructure for Fast-Response and High-Photoresponsivity Photodetector and High-Efficiency Water Splitting with a Small Bias Voltage.

Large-scale multi-heterostructure and optimal band alignment are significantly challenging but vital for photoelectrochemical (PEC)-type photodetector and water splitting. Herein, the centimeter-scale bismuth chalcogenides-based cascade heterostructure is successfully synthesized by a sequential vapor phase deposition method. The multi-staggered band alignment of Bi2 Te3 /Bi2 Se3 /Bi2 S3 is optimized and verified by X-ray photoelectron spectroscopy. The PEC photodetectors based on these cascade heterostructures demonstrate the highest photoresponsivity (103 mA W-1 at -0.1 V and 3.5 mAW-1 at 0 V under 475 nm light excitation) among the previous reports based on two-dimensional materials and related heterostructures. Furthermore, the photodetectors display a fast response (≈8 ms), a high detectivity (8.96 × 109 Jones), a high external quantum efficiency (26.17%), and a high incident photon-to-current efficiency (27.04%) at 475 nm. Due to the rapid charge transport and efficient light absorption, the Bi2 Te3 /Bi2 Se3 /Bi2 S3 cascade heterostructure demonstrates a highly efficient hydrogen production rate (≈0.416 mmol cm-2  h-1 and ≈14.320 µmol cm-2  h-1 with or without sacrificial agent, respectively), which is far superior to those of pure bismuth chalcogenides and its type-II heterostructures. The large-scale cascade heterostructure offers an innovative method to improve the performance of optoelectronic devices in the future.

CYP24A1 Involvement in Inflammatory Factor Regulation Occurs via the Wnt Signaling Pathway.

OBJECTIVE: While the upregulation of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene expression has been reported in colon cancer, its role in tumorigenesis remains largely unknown. In this study, we aimed to investigate the involvement of CYP24A1 in Wnt pathway regulation via the nuclear factor kappa B (NF-κB) pathway. METHODS: The human colon cancer cell lines HCT-116 and Caco-2 were subjected to stimulation with interleukin-6 (IL-6) as well as tumor necrosis factor alpha (TNF-α), with subsequent treatment using the NF-κB pathway-specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC). Furthermore, CYP24A1 expression was subjected to knockdown via the use of small interfering RNA (siRNA). Subsequently, NF-κB pathway activation was determined by an electrophoretic mobility shift assay, and the transcriptional activity of ß-catenin was determined by a dual-luciferase reporter assay. A mouse ulcerative colitis (UC)-associated carcinogenesis model was established, wherein TNF-α and the NF-κB pathway were blocked by anti-TNF-α monoclonal antibody and NF-κB antisense oligonucleotides, respectively. Then the tumor size and protein level of CYP24A1 were determined. RESULTS: IL-6 and TNF-α upregulated CYP24A1 expression and activated the NF-κB pathway in colon cancer cells. PDTC significantly inhibited this increase in CYP24A1 expression. Additionally, knockdown of CYP24A1 expression by siRNA could partially antagonize Wnt pathway activation. Upregulated CYP24A1 expression was observed in the colonic epithelial cells of UC-associated carcinoma mouse models. Anti-TNF-α monoclonal antibody and NF-κB antisense oligonucleotides decreased the tumor size and suppressed CYP24A1 expression. CONCLUSION: Taken together, this study suggests that inflammatory factors may increase CYP24A1 expression via NF-κB pathway activation, which in turn stimulates Wnt signaling.

The influence of different proton pump inhibitors and potassium-competitive acid blockers on indomethacin-induced small intestinal injury.

BACKGROUND AND AIM: The influence of gastric acid inhibitors (GAIs) on nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy is controversial. Herein, the influences of different GAIs on NSAID-induced intestinal injury and the underlying mechanisms are clarified. METHODS: Indomethacin (IND; 10 mg/kg/day) was administered to mice to induce small intestinal injury. Disease activity was examined macroscopically and histologically. The permeability of small intestine was evaluated by measuring plasma lipopolysaccharide levels. 16S rDNA sequencing was performed to determine the composition of intestinal flora. RESULTS: Among the four GAIs, ilaprazole (IPZ) significantly attenuated IND-induced small intestinal injury and maintained the integrity of the mucosal barrier. Omeprazole (OPZ) and vonoprazan (VPZ) ameliorated ulceration without significant differences, while rabeprazole (RPZ) failed to protect against the injury. To explore the potential mechanism, we investigated changes in the gut microbiota mediated by GAIs. After 5-day administration, GAIs significantly altered the composition of the gut microbiota. The IND group had a significant decrease in alpha diversity compared with the control group, and this decrease was reversed by OPZ and IPZ treatment, respectively. After IPZ treatment, the community membership was more assembled in the control group than the IND group. Further, we found that Lactobacillus was significantly increased in the groups of OPZ, IPZ, and VPZ, while Bacteroides was significantly increased in the RPZ group. CONCLUSION: Our results indicated that GAIs have different influences on the mucosal barrier, possibly by altering the composition of intestinal microbiota, and the impacts mediated by various GAIs in the IND-induced intestinal damage model seem different.

Vertically oriented SnS2 on MoS2 nanosheets for high-photoresponsivity and fast-response self-powered photoelectrochemical photodetectors.

Van der Waals heterostructures have great potential for the emerging self-powered photoelectrochemical photodetectors due to their outstanding photoelectric conversion capability and efficient interfacial carrier transportation. By considering the band alignment, structural design, and growth optimization, the heterostructures of vertically oriented SnS2 with different densities on MoS2 nanosheets are designed and fabricated using a two-step epitaxial growth method. Compared with SnS2, MoS2, and low density-vertical SnS2/MoS2 heterostructure, the high density-vertical SnS2/MoS2 heterostructure exhibits largely enhanced self-powered photodetection performances, such as a giant photocurrent density (∼932.8 µA cm-2), an excellent photoresponsivity (4.66 mA W-1), and an ultrafast response/recovery time (3.6/6.4 ms) in the ultraviolet-visible range. This impressive enhancement of high density-vertical SnS2/MoS2 photodetectors is mainly ascribed to the essentially improved charge transfer and carrier transport of type-II band alignment heterostructures and the efficient light absorption from the unique light-trapping structure. In addition, the photoelectrocatalytic water splitting performance of the high density-vertical SnS2/MoS2 heterostructure also benefits from the type-II band alignment and the light-trapping structure. This work provides valuable inspiration for the design of two-dimensional optoelectronic and photoelectrochemical devices with improved performance by the morphology and heterostructure design.

Sb2S3/Sb2Se3 heterojunction for high-performance photodetection and hydrogen production.

Photoelectrochemical (PEC)-type devices provide promising ways for harvesting solar energy and converting it to electric and chemical energy with a low-cost and simple manufacturing process. However, the high light absorption, fast carrier separation, and low carrier recombination are still great challenges in reaching high performance for PEC devices. As emergent two-dimensional (2D) materials, Sb2Se3 and Sb2S3 exhibit desirable photoelectric properties due to the narrow bandgap, large optical absorption, and high carrier mobility. Herein, Sb2S3/Sb2Se3 heterojunction is synthesized by a two-step physical vapor deposition method. The type-II Sb2S3/Sb2Se3 heterojunction displays excellentphotoelectric properties such as a high photocurrent density (Iph âˆ¼ 162 µA cm-2), a high photoresponsivity (Rph âˆ¼ 3700 µA W-1), and a fast time response speed (rising time ∼ 2 ms and falling time ∼ 4.5 ms) even in harsh environment (H2SO4 electrolyte). Especially, the Sb2S3/Sb2Se3 shows an excellent self-powered photoresponse (Iph âˆ¼ 40 µA cm-2, Rph âˆ¼ 850 µA W-1). This increment is attributed to the improvement in light absorption, charge separation, and charge transfer efficiency. Taking these advantages, the Sb2S3/Sb2Se3 heterojunction also exhibits higher PEC water splitting synergically, which is approximately 3 times larger than that of Sb2Se3 and Sb2S3. These results pave the way for high-performance PEC devices by integrating 2D narrow bandgap semiconductors.

Enhanced UV-Vis photodetector performance by optimizing interfacial charge transportation in the heterostructure by SnS and SnSe2.

Optimizing interfacial charge transfer in type-II heterostructures, is one promising solution to improve efficiency of the solar energy conversion in photodetectors and solar cells. Herein, the SnS/SnSe2/ITO and SnSe2/SnS/ITO heterostructures are prepared by two-step physical vapor epitaxial growth. X-ray photoelectron spectroscopy confirms the SnS/SnSe2 heterostructure belongs to type-II band-alignment. The SnS/SnSe2 based photodetector shows higher photoresponsivity, which is approximately 2, 9, and 14 times larger than that of SnSe2/SnS, SnSe2, and SnS, respectively. The improvement of SnS/SnSe2 in photoelectric response mainly comes from high light harvesting and efficient charge transportation than individual SnSe2 and SnS, which is verified by UV-Vis absorption spectra. Electrochemical impedance spectroscopy, open circuit potentials, and Mott-Schottky characterization results further confirm that the better photodetection performance of SnS/SnSe2/ITO than that of SnSe2/SnS/ITO heterostructure is from the appropriate energy level cascade facilitating electron transport. These results provide an effective way to further improve the performance of heterostructure-based optoelectronic devices by an appropriate interface design.

Characterization of a strong constitutive promoter from paper mulberry vein banding virus.

Paper mulberry vein banding virus (PMVBV), a member of the genus Badnavirus in the family Caulimoviridae, infects paper mulberry (Broussonetia papyrifera), a dicotyledonous plant. Putative promoter regions in the PMVBV genome were tested using recombinant plant expression vectors, revealing that the promoter activity of three genome fragments was about 1.5-fold higher than that of the 35S promoter of cauliflower mosaic virus in Nicotiana benthamiana. In transformed transgenic Arabidopsis thaliana plants, these promoter constructs showed constitutive expression. Based on the activity and gene expression patterns of these three promoter constructs, a fragment of 384 bp (named PmVP) was deduced to contain the full-length promoter of the PMVBV genome. The results suggest that the PMVBV-derived promoter can be used for the constitutive expression of transgenes in dicotyledonous plants.

The Identification of Tautoneura mori as the Vector of Mulberry Crinkle Leaf Virus and the Infectivity of Infectious Clones in Mulberry.

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus identified from mulberry. The pathogenicity and natural vector transmission of MCLV remain unknown. Here, infectious clones consisting of the complete tandem dimeric genome of MCLV in a binary vector were constructed and agroinoculated into young mulberry plants. The results showed that the infectious clones of MCLV were systemically infectious in mulberry, but the infected mulberry plants did not show any virus infection-like symptoms. The natural transmission vectors of MCLV were also identified from possible vector insects occurring on the MCLV-infected mulberry plants. The vector ability of Tautoneura mori was identified through an inoculation assay. Three of 21 (14.3%) plants inoculated with T. mori collected from MCLV-infected mulberry plants grown naturally were found to be MCLV-positive 50 days postinoculation. These MCLV-positive mulberry plants did not show any virus infection-like symptoms. Collectively, these results suggest that MCLV is infectious to mulberry plants but, by itself, does not induce infection symptoms. The leafhopper T. mori was experimentally determined to be a transmission vector of MCLV for the first time.

Bifidobacterium infantis Maintains Genome Stability in Ulcerative Colitis via Regulating Anaphase-Promoting Complex Subunit 7.

Probiotics represents a promising intestinal microbiota-targeted therapeutic method for the treatment of ulcerative colitis (UC). Several lines of evidence implicate that Bifidobacterium infantis serves as a probiotic strain with proven efficacy in maintaining the remission of UC. However, the exact mechanisms underlying the beneficial effects of B. infantis on UC progression have yet to be elucidated. Herein, we provide evidence that B. infantis acts as a key predisposing factor for the maintenance of host genome stability. First, we showed that the fecal microbiota transplantation (FMT) of UC-derived feces contributes to more severely DNA damage in dextran sodium sulfate (DSS)-induced mice likely due to mucosa-associated microbiota alterations, as reflected by the rapid appearance of DNA double strand breaks (DSBs), a typical marker of genome instability. Genomic DNA damage analysis of colon tissues derived from healthy controls, patients with UC or dysplasia, and colitis associated cancer (CAC) patients, revealed an enhanced level of DSBs with aggravation in the degree of the intestinal mucosal lesions. To evaluate whether B. infantis modulates the host genome stability, we employed the DSS-induced colitis model and a TNFα-induced intestinal epithelial cell model. Following the administration of C57BL/6 mice with B. infantis via oral gavage, we found that the development of DSS-induced colitis in mice was significantly alleviated, in contrast to the colitis model group. Notably, B. infantis administration decreased DSB levels in both DSS-induced colitis and TNF-treated colonial cell model. Accordingly, our bioinformatic and functional studies demonstrated that B. infantis altered signal pathways involved in ubiquitin-mediated proteolysis, transcriptional misregulation in cancer, and the bacterial invasion of epithelial cells. Mechanistically, B. infantis upregulated anaphase-promoting complex subunit 7 (APC7), which was significantly suppressed in colitis condition, to activate the DNA repair pathway and alter the genome stability, while downregulation of APC7 abolished the efficiency of B. infantis treatment to induce a decrease in the level of DSBs in TNFα-induced colonial cells. Collectively, our results support that B. infantis orchestrates a molecular network involving in APC7 and genome stability, to control UC development at the clinical, biological, and mechanistic levels. Supplying B. infantis and targeting its associated pathway will yield valuable insight into the clinical management of UC patients.

Identification of key genes and biological processes contributing to colitis associated dysplasia in ulcerative colitis.

BACKGROUND: Ulcerative colitis-associated colorectal cancer (UC-CRC) is a life-threatening complication of ulcerative colitis (UC). The mechanisms underlying UC-CRC remain to be elucidated. The purpose of this study was to explore the key genes and biological processes contributing to colitis-associated dysplasia (CAD) or carcinogenesis in UC via database mining, thus offering opportunities for early prediction and intervention of UC-CRC. METHODS: Microarray datasets (GSE47908 and GSE87466) were downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) between groups of GSE47908 were identified using the "limma" R package. Weighted gene co-expression network analysis (WGCNA) based on DEGs between the CAD and control groups was conducted subsequently. Functional enrichment analysis was performed, and hub genes of selected modules were identified using the "clusterProfiler" R package. Single-gene gene set enrichment analysis (GSEA) was conducted to predict significant biological processes and pathways associated with the specified gene. RESULTS: Six functional modules were identified based on 4929 DEGs. Green and blue modules were selected because of their consistent correlation with UC and CAD, and the highest correlation coefficient with the progress of UC-associated carcinogenesis. Functional enrichment analysis revealed that genes of these two modules were significantly enriched in biological processes, including mitochondrial dysfunction, cell-cell junction, and immune responses. However, GSEA based on differential expression analysis between sporadic colorectal cancer (CRC) and normal controls from The Cancer Genome Atlas (TCGA) indicated that mitochondrial dysfunction may not be the major carcinogenic mechanism underlying sporadic CRC. Thirteen hub genes (SLC25A3, ACO2, AIFM1, ATP5A1, DLD, TFE3, UQCRC1, ADIPOR2, SLC35D1, TOR1AIP1, PRR5L, ATOX1, and DTX3) were identified. Their expression trends were validated in UC patients of GSE87466, and their potential carcinogenic effects in UC were supported by their known functions and other relevant studies reported in the literature. Single-gene GSEA indicated that biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to angiogenesis and immune response were positively correlated with the upregulation of TFE3, whereas those related to mitochondrial function and energy metabolism were negatively correlated with the upregulation of TFE3. CONCLUSIONS: Using WGCNA, this study found two gene modules that were significantly correlated with CAD, of which 13 hub genes were identified as the potential key genes. The critical biological processes in which the genes of these two modules were significantly enriched include mitochondrial dysfunction, cell-cell junction, and immune responses. TFE3, a transcription factor related to mitochondrial function and cancers, may play a central role in UC-associated carcinogenesis.

MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmentation of seed coat in soybean.

Soybean has undergone extensive selection pressures for seed nutrient composition and seed color during domestication, but the major genetic loci controlling seed coat color have not been completely understood, and the transcriptional regulation relationship among the loci remains elusive. Here, two major regulators, GmMYBA2 and GmMYBR, were functionally characterized as an anthocyanin activator and repressor, respectively. Ectopic expression of GmMYBA2 in soybean hairy roots conferred the enhanced accumulation of delphinidin and cyanidin types of anthocyanins in W1t and w1T backgrounds, respectively, through activating anthocyanin biosynthetic genes in the reported loci. The seed coat pigmentation of GmMYBA2-overexpressing transgenic plants in the W1 background mimicked the imperfect black phenotype (W1/w1, i, R, t), suggesting that GmMYBA2 was responsible for the R locus. Molecular and biochemical analysis showed that GmMYBA2 interacted with GmTT8a to directly activate anthocyanin biosynthetic genes. GmMYBA2 and GmMYBR might form a feedback loop to fine-tune seed coat coloration, which was confirmed in transgenic soybeans. Both GmTT8a and GmMYBR that were activated by GmMYBA2 in turn enhanced and obstructed the formation of the GmMYBA2-GmTT8a module, respectively. The results revealed the sophisticated regulatory network underlying the soybean seed coat pigmentation loci and shed light on the understanding of the seed coat coloration and other seed inclusions.

MYB repressors and MBW activation complex collaborate to fine-tune flower coloration in Freesia hybrida.

Floral anthocyanin has multiple ecological and economic values, its biosynthesis largely depends on the conserved MYB-bHLH-WD40 (MBW) activation complex and MYB repressors hierarchically with the MBW complex. In contrast to eudicots, the MBW regulatory network model has not been addressed in monocots because of the lack of a suitable system, as grass plants exhibit monotonous floral pigmentation patterns. Presently, the MBW regulatory network was investigated in a non-grass monocot plant, Freesia hybrida. FhMYB27 and FhMYBx with different functional manners were confirmed to be anthocyanin related R2R3 and R3 MYB repressors, respectively. Particularly, FhMYBx could obstruct the formation of positive MBW complex by titrating bHLH proteins, whereas FhMYB27 mainly defected the activator complex into suppressor via its repression domains in C-terminus. Furthermore, the hierarchical and feedback regulatory loop was verified, indicating the synergistic and sophisticated regulatory network underlying Freesia anthocyanin biosynthesis was quite similar to that reported in eudicot plants.

The spatio-temporal biosynthesis of floral flavonols is controlled by differential phylogenetic MYB regulators in Freesia hybrida.

Floral flavonols play specific pivotal roles in pollinator attraction, pollen germination and fertility, in addition to other functions in vegetative organs. For many plants, the process of flavonol biosynthesis in late flower development stages and in mature flower tissues is poorly understood, in contrast to early flower development stages. It is thought that this process may be regulated independently of subgroup 7 R2R3 MYB (SG7 MYB) transcription factors. In this study, two FLS genes were shown to be expressed synchronously with the flower development-specific and tissue-specific biosynthesis of flavonols in Freesia hybrida. FhFLS1 contributed to flavonol biosynthesis in early flower buds, toruses and calyxes, and was regulated by four well-known SG7 MYB proteins, designated as FhMYBFs, with at least partial regulatory redundancy. FhFLS2 accounted for flavonols in late developed flowers and in the petals, stamens and pistils, and was targeted directly by non SG7 MYB protein FhMYB21L2. In parallel, AtMYB21 and AtMYB24 also activated AtFLS1, a gene highly expressed in Arabidopsis anthers and pollen, indicating the conserved regulatory roles of MYB21 against FLS genes in these two evolutionarily divergent angiosperm plants. Our results reveal a novel regulatory and synthetic mechanism underlying flavonol biosynthesis in floral organs and tissues which may be exploited to investigate supplementary roles of flavonols in flowers.