Functional Characterization of the Cystine-Rich-Receptor-like Kinases (CRKs) and Their Expression Response to Sclerotinia sclerotiorum and Abiotic Stresses in Brassica napus.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins that bind to the calcium ion to regulate stress-signaling and plant development-related pathways, as indicated by several pieces of evidence. However, the CRK gene family hasn't been inadequately examined in Brassica napus. In our study, 27 members of the CRK gene family were identified in Brassica napus, which are categorized into three phylogenetic groups and display synteny relationship to the Arabidopsis thaliana orthologs. All the CRK genes contain highly conserved N-terminal PKINASE domain; however, the distribution of motifs and gene structure were variable conserved. The functional divergence analysis between BnaCRK groups indicates a shift in evolutionary rate after duplication events, demonstrating that BnaCRKs might direct a specific function. RNA-Seq datasets and quantitative real-time PCR (qRT-PCR) exhibit the complex expression profile of the BnaCRKs in plant tissues under multiple stresses. Nevertheless, BnaA06CRK6-1 and BnaA08CRK8 from group B were perceived to play a predominant role in the Brassica napus stress signaling pathway in response to drought, salinity, and Sclerotinia sclerotiorum infection. Insights gained from this study improve our knowledge about the Brassica napus CRK gene family and provide a basis for enhancing the quality of rapeseed.

Plant Disease Resistance-Related Signaling Pathways: Recent Progress and Future Prospects.

Plant-pathogen interactions induce a signal transmission series that stimulates the plant's host defense system against pathogens and this, in turn, leads to disease resistance responses. Plant innate immunity mainly includes two lines of the defense system, called pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). There is extensive signal exchange and recognition in the process of triggering the plant immune signaling network. Plant messenger signaling molecules, such as calcium ions, reactive oxygen species, and nitric oxide, and plant hormone signaling molecules, such as salicylic acid, jasmonic acid, and ethylene, play key roles in inducing plant defense responses. In addition, heterotrimeric G proteins, the mitogen-activated protein kinase cascade, and non-coding RNAs (ncRNAs) play important roles in regulating disease resistance and the defense signal transduction network. This paper summarizes the status and progress in plant disease resistance and disease resistance signal transduction pathway research in recent years; discusses the complexities of, and interactions among, defense signal pathways; and forecasts future research prospects to provide new ideas for the prevention and control of plant diseases.

Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

Long-chain acyl-CoA synthetase 2 is involved in seed oil production in Brassica napus.

BACKGROUND: Triacylglycerols (TAGs) are the main composition of plant seed oil. Long-chain acyl-coenzyme A synthetases (LACSs) catalyze the synthesis of long-chain acyl-coenzyme A, which is one of the primary substrates for TAG synthesis. In Arabidopsis, the LACS gene family contains nine members, among which LACS1 and LACS9 have overlapping functions in TAG biosynthesis. However, functional characterization of LACS proteins in rapeseed have been rarely reported. RESULTS: An orthologue of the Arabidopsis LACS2 gene (BnLACS2) that is highly expressed in developing seeds was identified in rapeseed (Brassica napus). The BnLACS2-GFP fusion protein was mainly localized to the endoplasmic reticulum, where TAG biosynthesis occurs. Interestingly, overexpression of the BnLACS2 gene resulted in significantly higher oil contents in transgenic rapeseed plants compared to wild type, while BnLACS2-RNAi transgenic rapeseed plants had decreased oil contents. Furthermore, quantitative real-time PCR expression data revealed that the expression of several genes involved in glycolysis, as well as fatty acid (FA) and lipid biosynthesis, was also affected in transgenic plants. CONCLUSIONS: A long chain acyl-CoA synthetase, BnLACS2, located in the endoplasmic reticulum was identified in B. napus. Overexpression of BnLACS2 in yeast and rapeseed could increase oil content, while BnLACS2-RNAi transgenic rapeseed plants exhibited decreased oil content. Furthermore, BnLACS2 transcription increased the expression of genes involved in glycolysis, and FA and lipid synthesis in developing seeds. These results suggested that BnLACS2 is an important factor for seed oil production in B. napus.

Arabidopsis GDSL1 overexpression enhances rapeseed Sclerotinia sclerotiorum resistance and the functional identification of its homolog in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed (Brassica napus L.). To date, the genetic mechanisms of rapeseed' interactions with S. sclerotiorum are not fully understood, and molecular-based breeding is still the most effective control strategy for this disease. Here, Arabidopsis thaliana GDSL1 was characterized as an extracellular GDSL lipase gene functioning in Sclerotinia resistance. Loss of AtGDSL1 function resulted in enhanced susceptibility to S. sclerotiorum. Conversely, overexpression of AtGDSL1 in B. napus enhanced resistance, which was associated with increased reactive oxygen species (ROS) and salicylic acid (SA) levels, and reduced jasmonic acid levels. In addition, AtGDSL1 can cause an increase in lipid precursor phosphatidic acid levels, which may lead to the activation of downstream ROS/SA defence-related pathways. However, the rapeseed BnGDSL1 with highest sequence similarity to AtGDSL1 had no effect on SSR resistance. A candidate gene association study revealed that only one AtGDSL1 homolog from rapeseed, BnaC07g35650D (BnGLIP1), significantly contributed to resistance traits in a natural B. napus population, and the resistance function was also confirmed by a transient expression assay in tobacco leaves. Moreover, genomic analyses revealed that BnGLIP1 locus was embedded in a selected region associated with SSR resistance during the breeding process, and its elite allele type belonged to a minor allele in the population. Thus, BnGLIP1 is the functional equivalent of AtGDSL1 and has a broad application in rapeseed S. sclerotiorum-resistance breeding.

Genome-wide identification of the NPR1-like gene family in Brassica napus and functional characterization of BnaNPR1 in resistance to Sclerotinia sclerotiorum.

KEY MESSAGE: The BnaNPR1-like gene family was identified in B. napus, and it was revealed that repression of BnaNPR1 significantly reduces resistance toS. sclerotiorum, intensifies ROS accumulation, and changes the expression of genes associated with SA and JA/ET signaling in response to this pathogen. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and related NPR1-like genes play an important role in regulating plant defense. Oilseed rape (Brassica napus L.) is an important oilseed crop; however, little is known about the B. napus (Bna) NPR1-like gene family. Here, a total of 19 BnaNPR1-like genes were identified in the B. napus genome, and then named according to their respective best match in Arabidopsis thaliana (At), which led to the determination of B. napus homologs of every AtNPR1-like gene. Analysis of important protein domains and functional motifs indicated the conservation and variation among these homologs. Phylogenetic analysis of these BnaNPR1-like proteins and their Arabidopsis homologs revealed six distinct sub-clades, consequently indicating that their name classification totally conformed to their phylogenetic relationships. Further, B. napus transcriptomic data showed that the expression of three BnaNPR1s was significantly down-regulated in response to infection with Sclerotinia sclerotiorum, the most important pathogen of this crop, whereas BnaNPR2/3/4/5/6s did not show the expression differences in general. Further, we generated B. napus BnaNPR1-RNAi lines to interpret the effect of the down-regulated expression of BnaNPR1s on resistance to S. sclerotiorum. The results showed that BnaNPR1-RNAi significantly decreased this resistance. Further experiments revealed that BnaNPR1-RNAi intensified ROS production and changed defense responses in the interaction of plants with this pathogen. These results indicated that S. sclerotiorum might use BnaNPR1 to regulate specific physiological processes of B. napus, such as ROS production and SA defense response, for the infection.

Melatonin delays leaf senescence of Chinese flowering cabbage by suppressing ABFs-mediated abscisic acid biosynthesis and chlorophyll degradation.

Melatonin and abscisic acid (ABA) play contrasting roles in regulating leaf senescence in plants. The molecular mechanism underlying the interaction between melatonin and ABA involved in leaf senescence, however, remains poorly defined. Herein, we found that exogenous application of melatonin delayed the senescence of Chinese flowering cabbage, accompanied by reduced expression of chlorophyll catabolic and ABA biosynthetic genes, and a lower endogenous ABA level. Significantly, three nucleus-localized transcriptional activators BrABF1, BrABF4, and BrABI5 were identified, and their expressions were repressed by melatonin. In vitro and in vivo binding experiments revealed that BrABF1, BrABF4, and BrABI5 activated the transcription of a series of ABA biosynthetic and chlorophyll catabolic genes by physically binding to their promoters. Moreover, transient over-expression of BrABF1, BrABF4, and BrABI5 in tobacco leaves induced ABA accumulation and promoted chlorophyll degradation by upregulating tobacco ABA biosynthetic and chlorophyll catabolic genes, resulting in the accelerated leaf senescence. These effects were significantly attenuated by melatonin treatment. Our findings suggest that melatonin-mediated inhibition of leaf senescence involves suppression of ABFs-mediated ABA biosynthesis and chlorophyll degradation. Unraveling of the molecular regulatory mechanism of leaf senescence controlled by ABA and melatonin expands our understanding of the regulation of this phenomenon and offers potentially more effective molecular breeding strategies for extending the shelf-life of Chinese flowering cabbage.

Improving seed germination and oil contents by regulating the GDSL transcriptional level in Brassica napus.

KEY MESSAGE: Seed germination rate and oil content can be regulated at theGDSL transcriptional level by eitherAtGDSL1 orBnGDSL1 inB. napus. Gly-Asp-Ser-Leu (GDSL)-motif lipases represent an important subfamily of lipolytic enzymes, which play important roles in lipid metabolism, seed development, abiotic stress, and pathogen defense. In the present study, two closely related GDSL-motif lipases, Brassica napus GDSL1 and Arabidopsis thaliana GDSL1, were characterized as functioning in regulating germination rate and seed oil content in B. napus. AtGDSL1 and BnGDSL1 overexpression lines showed an increased seed germination rate and improved seedling establishment compared with wild type. Meanwhile, the constitutive overexpression of AtGDSL1 and BnGDSL1 promoted lipid catabolism and decreased the seed oil content. While RNAi-mediated suppression of BnGDSL1 (Bngdsl1) in B. napus improved the seed oil content and decreased seed germination rate. Moreover, the Bngdsl1 transgenic seeds showed changes in the fatty acid (FA) composition, featuring an increase in C18:1 and a decrease in C18:2 and C18:3. The transcriptional levels of six related core enzymes involved in FA mobilization were all elevated in the AtGDSL1 and BnGDSL1 overexpression lines, but strongly suppressed in the Bngdsl1 transgenic line. These results suggest that improving the seed germination and seed oil content in B. napus could be achieved by regulating the GDSL transcriptional level.

Activation of the Transcription of BrGA20ox3 by a BrTCP21 Transcription Factor Is Associated with Gibberellin-Delayed Leaf Senescence in Chinese Flowering Cabbage during Storage.

Several lines of evidence have implicated the involvement of the phytohormone gibberellin (GA) in modulating leaf senescence in plants. However, upstream transcription factors (TFs) that regulate GA biosynthesis in association with GA-mediated leaf senescence remain elusive. In the current study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP21 in GA-delayed leaf senescence in Chinese flowering cabbage. Exogenous GA3 treatment maintained a higher value of maximum PSII quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the repression of the expression of senescence-associated genes and chlorophyll catabolic genes, which led to the delay of leaf senescence. A class I member of TCP TFs BrTCP21, was further isolated and characterized. The transcript level of BrTCP21 was low in senescing leaves, and decreased following leaf senescence, while GA3 could keep a higher expression level of BrTCP21. BrTCP21 was further found to be a nuclear protein and exhibit trans-activation ability through transient-expression analysis in tobacco leaves. Intriguingly, the electrophoretic mobility shift assay (EMSA) and transient expression assay illustrated that BrTCP21 bound to the promoter region of a GA biosynthetic gene BrGA20ox3, and activated its transcription. Collectively, these observations reveal that BrTCP21 is associated with GA-delayed leaf senescence, at least partly through the activation of the GA biosynthetic pathway. These findings expand our knowledge on the transcriptional mechanism of GA-mediated leaf senescence.

BrTCP7 Transcription Factor Is Associated with MeJA-Promoted Leaf Senescence by Activating the Expression of BrOPR3 and BrRCCR.

The plant hormone jasmonic acid (JA) has been recognized as an important promoter of leaf senescence in plants. However, upstream transcription factors (TFs) that control JA biosynthesis during JA-promoted leaf senescence remain unknown. In this study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP7 in methyl jasmonate (MeJA)-promoted leaf senescence in Chinese flowering cabbage. Exogenous MeJA treatment reduced maximum quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the increased expression of senescence marker and chlorophyll catabolic genes, and accelerated leaf senescence. To further understand the transcriptional regulation of MeJA-promoted leaf senescence, a class I member of TCP TFs BrTCP7 was examined. BrTCP7 is a nuclear protein and possesses trans-activation ability through subcellular localization and transcriptional activity assays. A higher level of BrTCP7 transcript was detected in senescing leaves, and its expression was up-regulated by MeJA. The electrophoretic mobility shift assay and transient expression assay showed that BrTCP7 binds to the promoter regions of a JA biosynthetic gene BrOPR3 encoding OPDA reductase3 (OPR3) and a chlorophyll catabolic gene BrRCCR encoding red chlorophyll catabolite reductase (RCCR), activating their transcriptions. Taken together, these findings reveal that BrTCP7 is associated with MeJA-promoted leaf senescence at least partly by activating JA biosynthesis and chlorophyll catabolism, thus expanding our knowledge of the transcriptional mechanism of JA-mediated leaf senescence.

Down-regulation of BnDA1, whose gene locus is associated with the seeds weight, improves the seeds weight and organ size in Brassica napus.

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down-regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down-regulated in B. napus by over expressed of AtDA1R358K , which is a functional deficiency of DA1 with an arginine-to-lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.

BrWRKY65, a WRKY Transcription Factor, Is Involved in Regulating Three Leaf Senescence-Associated Genes in Chinese Flowering Cabbage.

Plant-specific WRKY transcription factors (TFs) have been implicated to function as regulators of leaf senescence, but their association with postharvest leaf senescence of economically important leafy vegetables, is poorly understood. In this work, the characterization of a Group IIe WRKY TF, BrWRKY65, from Chinese flowering cabbage (Brassica rapa var. parachinensis) is reported. The expression of BrWRKY65 was up-regulated following leaf chlorophyll degradation and yellowing during postharvest senescence. Subcellular localization and transcriptional activation assays showed that BrWRKY65 was localized in the nucleus and exhibited trans-activation ability. Further electrophoretic mobility shift assay (EMSA) and transient expression analysis clearly revealed that BrWRKY65 directly bound to the W-box motifs in the promoters of three senescence-associated genes (SAGs) such as BrNYC1 and BrSGR1 associated with chlorophyll degradation, and BrDIN1, and subsequently activated their expressions. These findings demonstrate that BrWRKY65 may be positively associated with postharvest leaf senescence, at least partially, by the direct activation of SAGs. Taken together, these findings provide new insights into the transcriptional regulatory mechanism of postharvest leaf senescence in Chinese flowering cabbage.

[Protective effect of succinic acid on cerebellar Purkinje cells of neonatal rats with convulsion].

OBJECTIVE: To investigate the protective effect of succinic acid (SA) on the cerebellar Purkinje cells (PCs) of neonatal rats with convulsion. METHODS: A total of 120 healthy neonatal Sprague-Dawley rats aged 7 days were randomly divided into a neonatal period group and a developmental period group. Each of the two groups were further divided into 6 sub-groups: normal control, convulsion model, low-dose phenobarbital (PB) (30 mg/kg), high-dose PB (120 mg/kg), low-dose SA (30 mg/kg), and high-dose SA (120 mg/kg). Intraperitoneal injection of pentylenetetrazole was performed to establish the convulsion model. The normal control group was treated with normal saline instead. The rats in the neonatal group were sacrificed at 30 minutes after the injection of PB, SA, or normal saline, and the cerebellum was obtained. Those in the developmental group were sacrificed 30 days after the injection of PB, SA, or normal saline, and the cerebellum was obtained. Whole cell patch clamp technique was used to record the action potential (AP) of PCs in the cerebellar slices of neonatal rats; the parallel fibers (PF) were stimulated at a low frequency to induce excitatory postsynaptic current (EPSC). The effect of SA on long-term depression (LTD) of PCs was observed. RESULTS: Compared with the normal control groups, the neonatal and developmental rats with convulsion had a significantly higher AP frequency of PCs (P<0.05), and the developmental rats with convulsion had a significantly decreased threshold stimulus (P<0.01) and a significantly greater inhibition of the amplitude of EPSC in PCs (P<0.05). Compared with the normal control groups, the neonatal and developmental rats with convulsion in the high-dose PB groups had a significantly decreased threshold stimulus (P<0.01), a significantly higher AP frequency of PCs (P<0.05), and a significantly greater inhibition of EPSC in PCs (P<0.05). Compared with the neonatal and developmental rats in the convulsion model groups, those in the high-dose SA groups had a significantly decreased AP frequency of PCs (P<0.05). The developmental rats in the low- and high-dose SA groups had a significantly higher AP threshold than those in the convulsion model group (P<0.05). CONCLUSIONS: The high excitability of PCs and the abnormal PF-PC synaptic plasticity caused by convulsion in neonatal rats may last to the developmental period, which can be aggravated by PB, while SA can reduce the excitability of PCs in neonatal rats with convulsion and repair the short- and long-term abnormalities of LTD of PCs caused by convulsion.

[Effect of Flos Daturae Alkaloids on TGF-beta1-induced Epithelial-Mesenchymal Transition of Human Pulmonary Adenocarcinoma A549 Cells].

OBJECTIVE: To study the effect of Flos Daturae alkaloids (FDA) on TGF-beta1-1uuuu;U epithelial-mesenchymal transition (EMT) of human pulmonary adenocarcinoma A549 cells. METHODS: A549 cells in vitro cultured were randomly divided into 5 groups, i.e., the blank control group, the TGF-beta1 group, the low dose FDA group, the medium dose FDA group, and the high dose FDA group. The morphologies of A549 cells were observed. Expressions of cytokeratin (CK)-19 and alpha-smooth muscle actin (alpha-SMA) were detected by Western blot and real-time PCR at 24, 48, and 72 h, respectively. RESULTS: A549 cells in the TGF-beta1, group turned from cobblestone to spindle shape gradually. Those in low, medium and high dose FDA groups showed similar shapes to those of the TGF-beta1 group. There was no statistical difference in the morphology of A549 cells among the 3 dose FDA groups (P > 0.05). Western blot showed that, when compared with the blank control group, the expression of CK-19 was down-regulated, but the expression of alpha-SMA was up-regulated in the TGF-beta1 group (P < 0.01). Compared with the TGF-beta1, group, the expression of CK-19 was up-regulated, but the expression of alpha-SMA was suppressed in low, medium and high dose FDA groups (P < 0.01). The CK-19 expression obviously increased, but the alpha-SMA expression was suppressed in high dose FDA group at 72 h (P < 0.01). Real-time PCR results showed, as compared with the TGF-beta1 group, the mRNA expression of CK-19 was increased, but the mRNA expression of alpha-SMA was reduced in low, medium and high dose FDA groups (P < 0.01). CONCLUSIONS: FDA had no effect on EMT morphological changes of TGF-beta1 induced A549 cells. FDA could reverse characteristic markers of A549 cells during EMT to some extent, such as expressions of CK-19 and alpha-SMA. The expression of CK-19 (as the epithelium marker) increased and the expression of alpha-SMA (as the mesenchymal marker) was reduced. Besides, they were most obviously seen in the high dose FDA group at 72 h in a dose- and time-dependent manner.

Research progress on the physiological response and molecular mechanism of cold response in plants.

Low temperature is a critical environmental stress factor that restricts crop growth and geographical distribution, significantly impacting crop quality and yield. When plants are exposed to low temperatures, a series of changes occur in their external morphology and internal physiological and biochemical metabolism. This article comprehensively reviews the alterations and regulatory mechanisms of physiological and biochemical indices, such as membrane system stability, redox system, fatty acid content, photosynthesis, and osmoregulatory substances, in response to low-temperature stress in plants. Furthermore, we summarize recent research on signal transduction and regulatory pathways, phytohormones, epigenetic modifications, and other molecular mechanisms mediating the response to low temperatures in higher plants. In addition, we outline cultivation practices to improve plant cold resistance and highlight the cold-related genes used in molecular breeding. Last, we discuss future research directions, potential application prospects of plant cold resistance breeding, and recent significant breakthroughs in the research and application of cold resistance mechanisms.

Dissection of quantitative trait nucleotides and candidate genes associated with agronomic and yield-related traits under drought stress in rapeseed varieties: integration of genome-wide association study and transcriptomic analysis.

Introduction: An important strategy to combat yield loss challenge is the development of varieties with increased tolerance to drought to maintain production. Improvement of crop yield under drought stress is critical to global food security. Methods: In this study, we performed multiomics analysis in a collection of 119 diverse rapeseed (Brassica napus L.) varieties to dissect the genetic control of agronomic traits in two watering regimes [well-watered (WW) and drought stress (DS)] for 3 years. In the DS treatment, irrigation continued till the 50% pod development stage, whereas in the WW condition, it was performed throughout the whole growing season. Results: The results of the genome-wide association study (GWAS) using 52,157 single-nucleotide polymorphisms (SNPs) revealed 1,281 SNPs associated with traits. Six stable SNPs showed sequence variation for flowering time between the two irrigation conditions across years. Three novel SNPs on chromosome C04 for plant weight were located within drought tolerance-related gene ABCG16, and their pleiotropically effects on seed weight per plant and seed yield were characterized. We identified the C02 peak as a novel signal for flowering time, harboring 52.77% of the associated SNPs. The 288-kbps LD decay distance analysis revealed 2,232 candidate genes (CGs) associated with traits. The CGs BIG1-D, CAND1, DRG3, PUP10, and PUP21 were involved in phytohormone signaling and pollen development with significant effects on seed number, seed weight, and grain yield in drought conditions. By integrating GWAS and RNA-seq, 215 promising CGs were associated with developmental process, reproductive processes, cell wall organization, and response to stress. GWAS and differentially expressed genes (DEGs) of leaf and seed in the yield contrasting accessions identified BIG1-D, CAND1, and DRG3 genes for yield variation. Discussion: The results of our study provide insights into the genetic control of drought tolerance and the improvement of marker-assisted selection (MAS) for breeding high-yield and drought-tolerant varieties.

Artificial cavernosa-like tissue based on multibubble Matrigel and a human corpus cavernous fibroblast scaffold.

Ex vivo tissue culture of the human corpus cavernosum (CC) can be used to explore the tissue structural changes and complex signaling networks. At present, artificial CC-like tissues based on acellular or three-dimensional (3D)-printed scaffolds are used to solve the scarcity of primary penis tissue samples. However, inconvenience and high costs limit the wide application of such methods. Here, we describe a simple, fast, and economical method of constructing artificial CC-like tissue. Human CC fibroblasts (FBs), endothelial cells (ECs), and smooth muscle cells (SMCs) were expanded in vitro and mixed with Matrigel in specific proportions. A large number of bubbles were formed in the mixture by vortexing combined with pipette blowing, creating a porous, spongy, and spatial structure. The CC FBs produced a variety of signaling factors, showed multidirectional differentiation potential, and grew in a 3D grid in Matrigel, which is necessary for CC-like tissue to maintain a porous structure as a cell scaffold. Within the CC-like tissue, ECs covered the surface of the lumen, and SMCs were located inside the trabeculae, similar to the structure of the primary CC. Various cell components remained stable for 3 days in vitro, but the EC content decreased on the 7th day. Wingless/integrated (WNT) signaling activation led to lumen atrophy and increased tissue fibrosis in CC-like tissue, inducing the same changes in characteristics as in the primary CC. This study describes a preparation method for human artificial CC-like tissue that may provide an improved experimental platform for exploring the function and structure of the CC and conducting drug screening for erectile dysfunction therapy.

Genetic breakthroughs in the Brassica napus-Sclerotinia sclerotiorum interactions.

Sclerotinia sclerotiorum (Lib.) de Bary is a highly destructive fungal pathogen that seriously damages the yield and quality of Brassica napus worldwide. The complex interaction between the B. napus and S. sclerotiorum system has presented significant challenges in researching rapeseed defense strategies. Here, we focus on the infection process of S. sclerotiorum, the defense mechanisms of rapeseed, and recent research progress in this system. The response of rapeseed to S. sclerotiorum is multifaceted; this review aims to provide a theoretical basis for rapeseed defense strategies.

Integration of genome-wide association studies, metabolomics, and transcriptomics reveals phenolic acid- and flavonoid-associated genes and their regulatory elements under drought stress in rapeseed flowers.

Introduction: Biochemical and metabolic processes help plants tolerate the adverse effects of drought. In plants accumulating bioactive compounds, understanding the genetic control of the biosynthesis of biochemical pathways helps the discovery of candidate gene (CG)-metabolite relationships. Methods: The metabolic profile of flowers in 119 rapeseed (Brassica napus) accessions was assessed over two irrigation treatments, one a well-watered (WW) condition and the other a drought stress (DS) regime. We integrated information gained from 52,157 single-nucleotide polymorphism (SNP) markers, metabolites, and transcriptomes to identify linked SNPs and CGs responsible for the genetic control of flower phenolic compounds and regulatory elements. Results: In a genome-wide association study (GWAS), of the SNPs tested, 29,310 SNPs were qualified to assess the population structure and linkage disequilibrium (LD), of which several SNPs for radical scavenging activity (RSA) and total flavanol content (TFLC) were common between the two irrigation conditions and pleiotropic SNPs were found for chlorogenic and coumaric acids content. The principal component analysis (PCA) and stepwise regression showed that chlorogenic acid and epicatechin in WW and myricetin in DS conditions were the most important components for RSA. The hierarchical cluster analysis (HCA) showed that vanillic acid, myricetin, gallic acid, and catechin were closely associated in both irrigation conditions. Analysis of GWAS showed that 60 CGs were identified, of which 18 were involved in stress-induced pathways, phenylpropanoid pathway, and flavonoid modifications. Of the CGs, PAL1, CHI, UGT89B1, FLS3, CCR1, and CYP75B137 contributed to flavonoid biosynthetic pathways. The results of RNA sequencing (RNA-seq) revealed that the transcript levels of PAL, CHI, and CYP75B137 known as early flavonoid biosynthesis-related genes and FLS3, CCR1, and UGT89B1 related to the later stages were increased during drought conditions. The transcription factors (TFs) NAC035 and ERF119 related to flavonoids and phenolic acids were upregulated under drought conditions. Discussion: These findings expand our knowledge on the response mechanisms to DS, particularly regarding the regulation of key phenolic biosynthetic genes in rapeseed. Our data also provided specific linked SNPs for marker-assisted selection (MAS) programs and CGs as resources toward realizing metabolomics-associated breeding of rapeseed.

Physiological and comparative transcriptome analyses reveal the mechanisms underlying waterlogging tolerance in a rapeseed anthocyanin-more mutant.

BACKGROUND: Rapeseed (Brassica napus) is the second largest oil crop worldwide. It is widely used in food, energy production and the chemical industry, as well as being an ornamental. Consequently, it has a large economic value and developmental potential. Waterlogging is an important abiotic stress that restricts plant growth and development. However, little is known about the molecular mechanisms underlying waterlogging tolerance in B. napus. RESULTS: In the present study, the physiological changes and transcriptomes of germination-stage rapeseed in response to waterlogging stress were investigated in the B. napus cultivar 'Zhongshuang 11' (ZS11) and its anthocyanin-more (am) mutant, which was identified in our previous study. The mutant showed stronger waterlogging tolerance compared with ZS11, and waterlogging stress significantly increased anthocyanin, soluble sugar and malondialdehyde contents and decreased chlorophyll contents in the mutant after 12 days of waterlogging. An RNA-seq analysis identified 1370 and 2336 differently expressed genes (DEGs) responding to waterlogging stress in ZS11 and am, respectively. An enrichment analysis revealed that the DEGs in ZS11 were predominately involved in carbohydrate metabolism, whereas those in the am mutant were particularly enriched in plant hormone signal transduction and response to endogenous stimulation. In total, 299 DEGs were identified as anthocyanin biosynthesis-related structural genes (24) and regulatory genes encoding transcription factors (275), which may explain the increased anthocyanin content in the am mutant. A total of 110 genes clustered in the plant hormone signal transduction pathway were also identified as DEGs, including 70 involved in auxin and ethylene signal transduction that were significantly changed in the mutant. Furthermore, the expression levels of 16 DEGs with putative roles in anthocyanin accumulation and biotic/abiotic stress responses were validated by quantitative real-time PCR as being consistent with the transcriptome profiles. CONCLUSION: This study provides new insights into the molecular mechanisms of increased anthocyanin contents in rapeseed in response to waterlogging stress, which should be useful for reducing the damage caused by waterlogging stress and for further breeding new rapeseed varieties with high waterlogging tolerance.