Identification of Plasma hsa_circ_0001230 and hsa_circ_0023879 as Potential Novel Biomarkers for Focal Segmental Glomerulosclerosis and circRNA-miRNA-mRNA network analysis.

Introduction Focal segmental glomerulosclerosis (FSGS) is a common glomerulopathy with unclear mechanism. The demand for FSGS clinical diagnostic biomarkers has not yet been met. Circular RNA (circRNA) is a novel non-coding RNA with multiple functions, but its diagnostic value for FSGS remains unexplored. This study aimed to identify circRNAs that could aid in early clinical diagnosis and to investigate their mechanisms in podocyte injury. Methods The signature of plasma circRNAs for FSGS was identified by circRNA microarray. The existence of circRNAs was confirmed by qRT-PCR, RNase R assay, and DNA sequencing. Plasma levels of circRNAs were evaluated by qRT-PCR. The diagnostic value was appraised by receiver operating characteristic curve. The circRNA-miRNA-mRNA network was built with Cytoscape 7.3.2. Statistically significant differences were calculated by the Mann-Whitney U-test. Results A total of 493 circRNAs (165 upregulated, 328 downregulated) were differentially expressed in the plasma of FSGS patients (n = 3) and normal controls (n = 3). Eight candidate circRNAs were demonstrated to be circular and stable transcripts. Among them, hsa_circ_0001230 and hsa_circ_0023879 were significantly upregulated in FSGS patients (n = 29) compared to normal controls (n = 51). The areas under the curve value of hsa_circ_0001230 and hsa_circ_0023879 were 0.668 and 0.753, respectively, while that of two-circRNAs panel was 0.763. The RNA pull-down analysis revealed that hsa_circ_0001230 and hsa_circ_0023879 could sponge hsa-miR-106a. Additionally, hsa_circ_0001230 and hsa_circ_0023879 positively regulated hsa-miR-106a target genes phosphatase and tensin homolog (PTEN) and Bcl-2-like protein 11 (BCL2L11) in podocytes. Conclusion Hsa_circ_0001230 and hsa_circ_0023879 are novel blood biomarkers for FSGS. They may regulate podocyte apoptosis by competitively binding to hsa-miR-106a.

A novel peptide PDHK1-241aa encoded by circPDHK1 promotes ccRCC progression via interacting with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive. METHODS: The differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa. RESULTS: CircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway. CONCLUSIONS: Our data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.

Bioinformatics analysis and experimental validation reveal the anti-ferroptosis effect of FZD7 in acute kidney injury.

BACKGROUND: Ferroptosis plays an important role in acute kidney injury (AKI), but the specific regulatory mechanism of ferroptosis in AKI remains unclear. This study is expected to analyze ferroptosis-related genes (FRGs) in AKI and explore their underlying mechanisms. RESULTS: A total of 479 differentially expressed genes (DEGs), including 196 up-regulated genes and 283 down-regulated genes were identified in the AKI chip GSE30718. 341 FRGs were obtained from the Genecard, OMIM and NCBI database. Totally 11 ferroptosis-related DEGs in AKI were found, in which 7 genes (CD44, TIGAR, RB1, LCN2, JUN, ARNTL, ACSL4) were up-regulated and 4 genes (FZD7, EP300, FOXC1, DLST) were down-regulated. Three core genes (FZD7, JUN, EP300) were obtained by PPI and KEGG analysis, among which the function of FZD7 in AKI is unclear. The WGCNA analysis found that FZD7 belongs to a module that was negatively correlated with AKI. Further basic experiments confirmed that FZD7 is down-regulated in mouse model of ischemia-reperfusion-AKI and cellular model of hypoxia-reoxygenation(H/R). In addition, knockdown of FZD7 could further aggravate the down-regulation of cell viability induced by H/R and Erastin, while overexpression of FZD7 can rescue its down-regulation to some extent. Furthermore, we verified that knockdown of FZD7 decreased the expression of GPX4 and overexpression of FZD7 increased the expression of GPX4, suggesting that FZD7 may inhibit ferroptosis by regulating the expression of GPX4 and plays a vital role in the onset and development of AKI. CONCLUSIONS: This article revealed the anti-ferroptosis effect of FZD7 in acute kidney injury through bioinformatics analysis and experimental validation, suggesting that FZD7 is a promising target for AKI and provided more evidence about the vital role of ferroptosis in AKI.

CircNFATC3 promotes the proliferation of gastric cancer through binding to IGF2BP3 and restricting its ubiquitination to enhance CCND1 mRNA stability.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) is an RNA binding protein with multiple roles in regulation of gene expression at the post-transcriptional level and is implicated in tumorigenesis and progression of numerous cancers including gastric cancer (GC). Circular RNAs (circRNAs) are a diverse endogenous noncoding RNA population that have important regulatory roles in cancer. However, circRNAs that regulate the expression of IGF2BP3 in GC is largely unknown. METHODS: CircRNAs that bound to IGF2BP3 were screened in GC cells using RNA immunoprecipitation and sequencing (RIP-seq). The identification and localization of circular nuclear factor of activated T cells 3 (circNFATC3) were identified using Sanger sequencing, RNase R assays, qRT-PCR, nuclear-cytoplasmic fractionation and RNA-FISH assays. CircNFATC3 expression in human GC tissues and adjacent normal tissues were measured by qRT-PCR and ISH. The biological role of circNFATC3 in GC was confirmed by in vivo and in vitro experiments. Furthermore, RIP, RNA-FISH/IF, IP and rescue experiments were performed to uncover interactions between circNFATC3, IGF2BP3 and cyclin D1 (CCND1). RESULTS: We identified a GC-associated circRNA, circNFATC3, that interacted with IGF2BP3. CircNFATC3 was significantly overexpressed in GC tissues and was positively associated with tumor volume. Functionally, the proliferation of GC cells decreased significantly after circNFATC3 knockdown in vivo and in vitro. Mechanistically, circNFATC3 bound to IGF2BP3 in the cytoplasm, which enhanced the stability of IGF2BP3 by preventing ubiquitin E3 ligase TRIM25-mediated ubiquitination, thereby enhancing the regulatory axis of IGF2BP3-CCND1 and promoting CCND1 mRNA stability. CONCLUSIONS: Our findings demonstrate that circNFATC3 promotes GC proliferation by stabilizing IGF2BP3 protein to enhance CCND1 mRNA stability. Therefore, circNFATC3 is a potential novel target for the treatment of GC.

CircARID1A binds to IGF2BP3 in gastric cancer and promotes cancer proliferation by forming a circARID1A-IGF2BP3-SLC7A5 RNA-protein ternary complex.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumors in China. Circular RNAs (circRNAs) are novel non-coding RNAs with important regulatory roles in cancer progression. IGF2BP3 has been found to play oncogenic roles in various cancers including GC, while the exact mechanism of IGF2BP3 is largely unknown. METHODS: The expression of IGF2BP3 in GC was evaluated by Western Blot and bioinformatics analysis. CircRNA expression profiles were screened via IGF2BP3 RIP-seq in GC. Sanger sequencing, RNase R digestion, nucleo-plasmic separation and RNA-FISH assays were used to detect the existence and expression of circARID1A. RNA ISH assay was employed to test the expression of circARID1A in paraffin-embedded GC tissues. Moreover, the function of circARID1A on cellular proliferation was assessed by CCK-8, plate colony formation, EdU assays and GC xenograft mouse model in vivo. Furthermore, the location or binding of circARID1A, IGF2BP3 protein and SLC7A5 in GC was evaluated by RNA-FISH/IF or RNA pull-down assays. RESULTS: We identified a novel circRNA, circARID1A, that can bind to IGF2BP3 protein. CircARID1A was significantly upregulated in GC tissues compared with noncancerous tissues and positively correlated with tumor length, tumor volume, and TNM stage. CircARID1A knockdown inhibited the proliferation of GC cells in vitro and in vivo and circARID1A played an important role in the oncogenic function of IGF2BP3. Mechanistically, circARID1A served as a scaffold to facilitate the interaction between IGF2BP3 and SLC7A5 mRNA, finally increasing SLC7A5 mRNA stability. Additionally, circARID1A was able to directly bind SLC7A5 mRNA through complementary base-pairing and then formed the circARID1A-IGF2BP3-SLC7A5 RNA-protein ternary complex and promoted the proliferation of GC via regulating AKT/mTOR pathway. CONCLUSIONS: Altogether, our data suggest that circARID1A is involved in the function of IGF2BP3 and GC proliferation, and the circARID1A-IGF2BP3-SLC7A5 axis has the potential to serve as a novel therapeutic target for GC.

Circular RNA circ-TNPO3 inhibits clear cell renal cell carcinoma metastasis by binding to IGF2BP2 and destabilizing SERPINH1 mRNA.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a common malignant tumour of the urinary tract. The major causes of poor prognosis are the lack of early diagnosis and metastasis. Accumulating research reveals that circular RNAs (circRNAs) can play key roles in the development and the progression of cancer. However, the role of circRNAs in ccRCC is still uncertain. METHODS: The circRNAs microarray (n = 4) was performed to investigate the circRNAs with differential expression in ccRCC tissues. The candidate circRNA was selected based on the cut-off criteria, such as circRNA expression abundance, circRNA size and the design of divergent primers. The circ-transportin-3 (TNPO3) levels in ccRCC tissues were tested by quantitative real-time (qRT)-PCR (n = 110). The characteristics and subcellular localization of circ-TNPO3 were identified via RNase R assay, qRT-PCR and fluorescence in situ hybridization (FISH). Then, we explored the biological roles of circ-TNPO3 in ccRCC via the function experiments in vitro and in vivo. RNA pull-down, RNA immunoprecipitation, bioinformatic analysis, RNA-FISH assays and rescue assays were applied to validate the interactions between circ-TNPO3, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and serpin family H member 1 (SERPINH1) to uncover the underlying molecular mechanisms of circ-TNPO3. RESULTS: We detected the obvious downregulation of circ-TNPO3 in ccRCC compared to matched adjacent normal tissues (n = 110). The lower circ-TNPO3 expression was found in ccRCC patients with distant metastasis, higher World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) grade and more advanced tumour T stage. In vitro and in vivo, circ-TNPO3 significantly suppressed the proliferation and migration of ccRCC cells. Mechanistically, we elucidated that circ-TNPO3 directly bound to IGF2BP2 protein and then destabilized SERPINH1 mRNA. Moreover, IGF2BP2/SERPINH1 axis was responsible for circ-TNPO3's function of inhibiting ccRCC metastasis. Epithelial splicing regulatory protein 1 (ESRP1) was probably involved in the biogenesis of circ-TNPO3. CONCLUSIONS: Circ-TNPO3 can suppress ccRCC progression and metastasis via directly binding to IGF2BP2 protein and destabilizing SERPINH1 mRNA. Circ-TNPO3 may act as a potential target for ccRCC treatment.

Circular RNA circ-TNPO3 suppresses metastasis of GC by acting as a protein decoy for IGF2BP3 to regulate the expression of MYC and SNAIL.

Gastric cancer (GC) continues to be the most common gastrointestinal malignancy in China, and tumor metastases are a major reason for poor prognosis. Circular RNAs (circRNAs) are an intriguing type of noncoding RNAs with important regulatory roles. However, the roles of circRNAs in GC metastasis have not been fully elucidated. Here, we reported that circ-transportin 3 (TNPO3) was significantly downregulated in 103 pairs of GC tissues compared with matched noncancerous tissues. The level of circ-TNPO3 expression correlated with differentiation of GC, and plasma circ-TNPO3 could serve as a potential diagnostic biomarker. Functionally, circ-TNPO3 inhibited proliferation and migration of GC in vitro and in vivo. We further verified that circ-TNPO3 competitively interacted with insulin-like growth factor 2 binding protein 3 (IGF2BP3) protein; thus, the role of IGF2BP3 in stabilizing MYC mRNA was weakened, which inhibited the expression of MYC and its target SNAIL. Taken together, circ-TNPO3 acts as a protein decoy for IGF2BP3 to regulate the MYC-SNAIL axis, thereby suppressing the proliferation and metastasis of GC. Therefore, circ-TNPO3 has the potential to serve as a therapeutic target for GC.

MiR319a-targeted PtoTCP20 regulates secondary growth via interactions with PtoWOX4 and PtoWND6 in Populus tomentosa.

Secondary growth is a key characteristic of trees, which requires the coordination of multiple regulatory mechanisms including transcriptional regulators and microRNAs (miRNAs). However, the roles of microRNAs in the regulation of secondary growth need to be explored in depth. Here, the role of miR319a and its target, PtoTCP20, in the secondary growth of Populus tomentosa stem was investigated using genetic and molecular analyses. The expression level of miR319a gradually decreased from primary to secondary growth in P. tomentosa, while that of PtoTCP20 gradually increased. MiR319a overexpression in seedlings resulted in delayed secondary growth and decreased xylem production, while miR319a knockdown and PtoTCP20 overexpression promoted secondary growth and increased xylem production. Further analysis showed that PtoTCP20 interacted with PtoWOX4a and activated PtoWND6 transcription in vitro and in vivo. Our data show that PtoTCP20 controls vascular cambium proliferation by binding to PtoWOX4a, and promotes secondary xylem differentiation by activating PtoWND6 transcription, thereby regulating secondary growth in P. tomentosa. Our findings provide insight into the molecular mechanisms underlying secondary growth in trees.

miR319a/TCP module and DELLA protein regulate trichome initiation synergistically and improve insect defenses in Populus tomentosa.

Trichomes are specialized epidermal cells that contribute to plant resistance against herbivores. Their formation is controlled precisely by multiple genetic and environmental signals. Previous studies have shown that microRNA319 (miR319) and gibberellin (GA) signaling are involved in trichome development in Arabidopsis, but little is known about their interaction between these factors. Here we reported that the miR319a/TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) module participates in trichome initiation synergistically with GA signaling in Populus tomentosa. We demonstrated that overexpression of miR319a decreased transcription levels of its targeted TCPs and significantly elevated leaf trichome density in transgenic poplar, resulting in decreasing insect herbivory. Conversely, repressing miR319a by short tandem target mimics (STTM) elevated TCP expression levels and decreased trichome density in transgenic plants. The trichome phenotype of 35S:miR319a plants could be abolished by introducing a miR319a-resistant form of TCP19. Furthermore, the miR319a-targeted TCP19 interacted directly with REPRESSOR OF ga1-3 (RGA), a downstream repressor of GA signaling. TCP19 and RGA synergistically inhibited the GLABROUS1 (GL1)-induced expression of trichome marker gene GLABRA2 (GL2), thereby repressing leaf trichome initiation. Our results provide an insight into the molecular mechanism by which miR319/TCP19 module and GA signaling coordinated regulating trichome initiation in P. tomentosa.

R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynthesis but inhibits secondary cell wall formation in Populus tomentosa.

The secondary cell wall is an important carbon sink in higher plants and its biosynthesis requires coordination of metabolic fluxes in the phenylpropanoid pathway. In Arabidopsis (Arabidopsis thaliana), MYB75 and the KNOX transcription factor KNAT7 form functional complexes to regulate secondary cell wall formation in the inflorescence stem. However, the molecular mechanism by which these transcription factors control different branches of the phenylpropanoid pathway remains poorly understood in woody species. We isolated an R2R3-MYB transcription factor MYB6 from Populus tomentosa and determined that it was expressed predominately in young leaves. Overexpression of MYB6 in transgenic poplar upregulated flavonoid biosynthetic gene expression, resulting in significantly increased accumulation of anthocyanin and proanthocyanidins. MYB6-overexpression plants showed reduced secondary cell wall deposition, accompanied by repressed expression of secondary cell wall biosynthetic genes. We further showed that MYB6 interacted physically with KNAT7 and formed functional complexes that acted to repress secondary cell wall development in poplar and Arabidopsis. The results provide an insight into the transcriptional mechanisms involved in the regulation of the metabolic fluxes between the flavonoid and lignin biosynthetic pathways in poplar.

PtoMYB170 positively regulates lignin deposition during wood formation in poplar and confers drought tolerance in transgenic Arabidopsis.

Wood formation is a complex developmental process under multi-level transcriptional control executed by a large set of transcription factors. However, only limited members have been characterized to be key regulators of lignin biosynthesis in poplar. Here we report the conserved and unique functions of PtoMYB170, a transcription factor identified from Populus tomentosa (Chinese white poplar), in lignin deposition and drought tolerance in comparison with its duplicate paralog PtoMYB216. PtoMYB170 is preferentially expressed in young leaves and xylem tissues. Overexpression of PtoMYB170 in transgenic poplar plants resulted in stronger lignification and more thickened secondary wall in xylem compared with wild-type plants, whereas the CRISPR/Cas9-generated mutation of PtoMYB170 weakened lignin deposition, thereby leading to a more flexible and collapsed xylem phenotype. Transient expression experiments demonstrated that PtoMYB170 specifically activated the expression of lignin biosynthetic genes, consistent with the function of PtoMYB216. However, GUS staining assays revealed that PtoMYB170 was specifically expressed in guard cells of transgenic Arabidopsis while PtoMYB216 was not. Heterologous expression of PtoMYB170 in Arabidopsis enhanced stomatal closure in the dark and resulted in drought tolerance of the transgenic plants through reduced water loss, indicating a diversified role from PtoMYB216. These results revealed the PtoMYB170-dependent positive transcriptional regulation on lignin deposition in poplar and its coordinated function in enhancing drought tolerance by promoting dark-induced stomatal closure.

The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar.

Proanthocyanidins (PAs) are major defense phenolic compounds in the leaves of poplar (Populus spp.) in response to abiotic and biotic stresses. Transcriptional regulation of PA biosynthetic genes by the MYB-basic helix-loop-helix (bHLH)-WD40 complexes in poplar is not still fully understood. Here, an Arabidopsis TT2-like gene MYB115 was isolated from Populus tomentosa and characterized by various molecular, genetic and biochemical approaches. MYB115 restored PA productions in the seed coat of the Arabidopsis tt2 mutant. Overexpression of MYB115 in poplar activated expression of PA biosynthetic genes, resulting in a significant increase in PA concentrations. By contrast, the CRISPR/Cas9-generated myb115 mutant exhibited reduced PA content and decreased expression of PA biosynthetic genes. MYB115 directly activated the promoters of PA-specific structural genes. MYB115 interacted with poplar TT8. Coexpression of MYB115, TT8 and poplar TTG1 significantly enhanced the expression of ANR1 and LAR3. Additionally, transgenic plants overexpressing MYB115 had increased resistance to the fungal pathogen Dothiorella gregaria, whereas myb115 mutant exhibited greater sensitivity compared with wild-type plants. Our data provide insight into the regulatory mechanisms controlling PA biosynthesis by MYB115 in poplar, which could be effectively employed for metabolic engineering of PAs to improve resistance to fungal pathogens.

PtoMYB156 is involved in negative regulation of phenylpropanoid metabolism and secondary cell wall biosynthesis during wood formation in poplar.

Some R2R3 MYB transcription factors have been shown to be major regulators of phenylpropanoid biosynthetic pathway and impact secondary wall formation in plants. In this study, we describe the functional characterization of PtoMYB156, encoding a R2R3-MYB transcription factor, from Populus tomentosa. Expression pattern analysis showed that PtoMYB156 is widely expressed in all tissues examined, but predominantly in leaves and developing wood cells. PtoMYB156 localized to the nucleus and acted as a transcriptional repressor. Overexpression of PtoMYB156 in poplar repressed phenylpropanoid biosynthetic genes, leading to a reduction in the amounts of total phenolic and flavonoid compounds. Transgenic plants overexpressing PtoMYB156 also displayed a dramatic decrease in secondary wall thicknesses of xylem fibers and the content of cellulose, lignin and xylose compared with wild-type plants. Transcript accumulation of secondary wall biosynthetic genes was down-regulated by PtoMYB156 overexpression. Transcriptional activation assays revealed that PtoMYB156 was able to repress the promoter activities of poplar CESA17, C4H2 and GT43B. By contrast, knockout of PtoMYB156 by CRISPR/Cas9 in poplar resulted in ectopic deposition of lignin, xylan and cellulose during secondary cell wall formation. Taken together, these results show that PtoMYB156 may repress phenylpropanoid biosynthesis and negatively regulate secondary cell wall formation in poplar.

Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis.

KEY MESSAGE: An approach for restoring recombination activity of complementation split-Cre was developed to excise the transgene in hybrid progeny of GM crops. Growing concerns about the biosafety of genetically modified (GM) crops has currently become a limited factor affecting the public acceptance. Several approaches have been developed to generate selectable-marker-gene-free GM crops. However, no strategy was reported to be broadly applicable to hybrid crops. Previous studies have demonstrated that complementation split-Cre recombinase restored recombination activity in transgenic plants. In this study, we found that split-Cre mediated by split-intein Synechocystis sp. DnaE had high recombination efficiency when Cre recombinase was split at Asp232/Asp233 (866 bp). Furthermore, we constructed two plant expression vectors, pCA-NCre-In and pCA-Ic-CCre, containing NCre866-In and Ic-CCre866 fragments, respectively. After transformation, parent lines of transgenic Arabidopsis with one single copy were generated and used for hybridization. The results of GUS staining demonstrated that the recombination activity of split-Cre could be reassembled in these hybrid progeny of transgenic plants through hybridization and the foreign genes flanked by two loxP sites were efficiently excised. Our strategy may provide an effective approach for generating the next generation of GM hybrid crops without biosafety concerns.

PtoMYB92 is a Transcriptional Activator of the Lignin Biosynthetic Pathway During Secondary Cell Wall Formation in Populus tomentosa.

Wood is the most abundant biomass in perennial woody plants and is mainly made up of secondary cell wall. R2R3-MYB transcription factors are important regulators of secondary wall biosynthesis in plants. In this study, we describe the identification and characterization of a poplar MYB transcription factor PtoMYB92, a homolog of Arabidopsis MYB42 and MYB85, which is involved in the regulation of secondary cell wall biosynthesis. PtoMYB92 is specifically expressed in xylem tissue in poplar. Subcellular localization and transcriptional activation analysis suggest that PtoMYB92 is a nuclear-localized transcriptional activator. Overexpression of PtoMYB92 in poplar resulted in an increase in secondary cell wall thickness in stems and ectopic deposition of lignin in leaves. Quantitative real-time PCR showed that PtoMYB92 specifically activated the expression of lignin biosynthetic genes. Furthermore, transient expression assays using a ß-glucuronidase (GUS) reporter gene revealed that PtoMYB92 is an activator in the lignin biosynthetic pathway during secondary cell wall formation. Taken together, our results suggest that PtoMYB92 is involved in the regulation of secondary cell wall formation in poplar by controlling the biosynthesis of monolignols.

Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.

Split-Cre complementation restores combination activity on transgene excision in hair roots of transgenic tobacco.

The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. It was previously reported that inactive ''split-Cre'' fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro recombination reaction in which only the N-terminal domain (NCre) of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre) or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS) was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.