Comparative Transcriptomics Uncovers Upstream Factors Regulating BnFAD3 Expression and Affecting Linolenic Acid Biosynthesis in Yellow-Seeded Rapeseed (Brassica napus L.).

α-Linolenic acid (ALA) is an important nutrient component in rapeseed oil, and rapeseed breeders want to either restrain or enhance the function of fatty acid desaturases (FADs) in the ALA biosynthesis pathway. To determine the reason for the upregulation of rapeseed BnFAD genes in two high-ALA accessions, R8Q10 and YH25005, we compared their transcriptome profiles in the seed at 24 days after pollination (DAP) with those of two low-ALA lines, A28 and SW. The expression levels of twenty-eight important genes in the seed samples at 20, 27, and 34 DAP were also investigated using an RT-qPCR. The expression levels of genes involved in flavonoid and proanthocyanidin synthesis, including BnCHS, BnCHI, BnDFR, BnFLS1, BnLDOX, BnBAN, BnTT10, and BnTT12 and genes encoding the transcription factors BnTT1, BnTT2, BnTT8, and BnTT16 were lower in R8Q10 and YH25005 than in A28 and SW. The expression levels of genes encoding master transcription factors in embryo development, such as BnLEC1, BnABI3, BnFUS3, BnL1L, BnAREB3, and BnbZIP67, were elevated significantly in the two high-ALA accessions. Combined with previous results in the Arabidopsis and rapeseed literature, we speculated that the yellow-seededness genes could elevate the activity of BnLEC1, BnABI3, BnFUS3, and BnbZIP67, etc., by reducing the expression levels of several transparent testa homologs, resulting in BnFAD3 and BnFAD7 upregulation and the acceleration of ALA synthesis. Yellow-seededness is a favorable factor to promote ALA synthesis in the two high-ALA accessions with the yellow-seeded trait. These findings provide initial insights into the transcriptomic differences between high-/low-ALA germplasms and a theoretic basis for seed quality breeding.

E2F1-Induced lncRNA BAIAP2-AS1 Overexpression Contributes to the Malignant Progression of Hepatocellular Carcinoma via miR-361-3p/SOX4 Axis.

Currently, plenty of researches have revealed that long noncoding RNAs (lncRNAs) can act as crucial roles during the progression of various tumors, including hepatocellular carcinoma (HCC). Here, we measured the expression of lncRNA BAIAP2 antisense RNA 1(BAIAP2-AS1) as well as its contribution to the developments of HCC. In this study, the expressions of BAIAP2-AS1 and SOX4 were distinctly upregulated in HCC cells and tissues, and high BAIAP2-AS1 may be a novel biomarker for HCC. E2F1 activated BAIAP2-AS1 expression. The silence of BAIAP2-AS1 inhibited the proliferation and metastasis of HepG2 and PLC5 cells. Assays for relationship verification showed that BAIAP2-AS1 regulated the expression of SOX4 and miR-361-3p. Rescue experiments further confirmed the positive interaction between miR-361-3p and BAIAP2-AS1 as well as between miR-361-3p and SOX4. Overall, BAIAP2-AS1 modulated the miR-361-3p/SOX4 axis to promote the development of HCC. Thus, our study offers a potential therapeutic target for treating HCC.

Towards breeding of rapeseed (Brassica napus) with alien cytoplasm and powdery mildew resistance from Ethiopian mustard (Brassica carinata).

Powdery mildew (PM), caused by Erysiphe cruciferarum, is an epidemic of oil rapeseed (Brassica napus L.) growing worldwide, but PM resistant germplasm is rare in this species. We screened 102 accessions of B. napus and other cruciferous species and found an Ethiopian mustard (Brassica carinata) cultivar 'White flower' immune to PM in both the field and greenhouse. Outcrossing in the female parent 'White flower' was promoted by using a chemical gametocide tribenuron-methyl, to obtain hybrid seeds of distant hybridization with an elite B. napus cultivar 'Zhongshuang11'. Three true F1 hybrids with B. carinata cytoplasm were obtained without using embryo rescue, which showed complete male sterility and light yellow petals. The hybrid plants and the progenies derived from backcrossing were validated using morphological traits, seed quality, and molecular markers. Five lines in the BC1F3 generation, named 'W7-1', 'W7-4', 'W7-6', 'W8-1', and 'W8-3', and one BC2F2 line 'W3PS-1', whose young leaf was yellow green, were identified to be resistant or moderately resistant to PM. The seed quality and some morphological traits of these lines resembled the parent 'Zhongshuang11', indicating that the resistance gene(s) has been preliminarily introduced into B. napus.

Sublethal application of various sulfonylurea and imidazolinone herbicides favors outcrossing and hybrid seed production in oilseed rape.

BACKGROUND: Acetolactate synthase (ALS)-inhibiting herbicides from the chemical families of sulfonylureas and imidazolinones are used worldwide. However, drift or sprayer contamination from some sulfonylurea herbicides causes a high level of male sterility in cruciferous species, especially oilseed rape (OSR). In this paper, we evaluated the gametocidal effects of 27 ALS-inhibiting herbicides that were sprayed on OSR plants at the bolting stage. RESULTS: OSR anther development was very sensitive to sublethal exposure to most ALS-inhibiting herbicides. The application of 18 out of the 20 tested sulfonylureas (except ethametsulfuron and ethoxysulfuron), two imidazolinones (imazethapyr and imazamox), and one sulfonylamino-carbonyltriazolinone (flucarbazone-sodium) at suitable rates could induce male sterility. Eight of the herbicides, including chlorsulfuron (at application rates of 60-120 mg/ha), halosulfuron-methyl (300-600 mg/ha), sulfosulfuron (400-600 mg/ha), triflusulfuron-methyl (500-750 mg/ha), pyrazosulfuron-ethyl (150-225 mg/ha), nicosulfuron (200-300 mg/ha), imazethapyr (750-1125 mg/ha), and imazamox (400-800 mg/ha), could induce over 90% male sterility and over 60% relative outcrossed seed set in six cultivars with different origins. These eight chemicals could be used as new gametocides for hybrid seed production. This study also examined the possibility of external application of these gametocides on several unstable Polima cytoplasmic male sterile and thermosensitive genic male sterile lines. Although the outcrossed seed set of the treated lines was slightly reduced, the gametocide application significantly increased the seed purity of the resulting hybrid. CONCLUSION: The finding of the gametocidal effects of most sulfonylureas and imidazolinones are of great importance for developing new functions for ALS-inhibiting herbicides. The application of gametocides will also greatly promote the safe utilization of environment-sensitive male sterility in hybrid seed production. Unexpectedly, the application of three triazolopyrimidines (florasulam, flumetsulam, and penoxsulam) and one pyrimidinylthiobenzoate (bispyribac-sodium) did not cause male sterility, although these herbicides obviously inhibited the activity of ALS and plant growth. This result suggests that inhibition of ALS activity does not always lead to male sterility in plants, and these gametocides may also inhibit other biological functions vital for microspore development.

How exposure to ALS-inhibiting gametocide tribenuron-methyl induces male sterility in rapeseed.

BACKGROUND: Acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl (TBM) is an efficient gametocide that can cause rapeseed (Brassica napus L.) to become male sterile and outcrossing. To find the reason the TBM treatment leads to male sterility, an integrated study using cytological, physiological, and transcriptomic methods was conducted. RESULTS: Some temporary symptoms, including the discoloration of young leaves and a short halt of raceme elongation, were observed in the rapeseed plants exposed to TBM at an application rate of 1 µg per plant. Both chloroplasts in young leaves and plastids in anthers were deformed. TBM also reduced the leaf photosynthetic rate and the contents of chlorophyll, soluble sugar and pyruvate. Both the tapetal cells and uni-nucleate microspores in the treated plants showed large autophagic vacuoles, and the tissue degenerated quickly. A transcriptomic comparison with the control identified 200 upregulated and 163 downregulated differential expression genes in the small flower buds of the TBM treatment. The genes encoding functionally important proteins, including glucan endo-1,3-beta-glucosidase A6, QUARTET3 (QRT3), ARABIDOPSIS ANTHER 7 (ATA7), non-specific lipid-transfer protein LTP11 and LTP12, histone-lysine N-methyltransferase ATXR6, spermidine coumaroyl-CoA acyltransferase (SCT), and photosystem II reaction centre protein psbB, were downregulated by TBM exposure. Some important genes encoding autophagy-related protein ATG8a and metabolic detoxification related proteins, including DTX1, DTX6, DTX35, cytosolic sulfotransferase SOT12, and six members of glutathione S-transferase, were upregulated. In addition, several genes related to hormone stimulus, such as 1-aminocyclopropane-1-carboxylate synthase 8 (ACS8), ethylene-responsive factor ERF1A, ERF1, ERF71, CRF6, and RAP2-3, were also upregulated. The transcriptional regulation is in accordance with the functional abnormalities of pollen wall formation, lipid metabolism, chloroplast structure, ethylene generation, cell cycle, and tissue autophagy. CONCLUSION: The results suggested that except for ALS, the metabolic pathways related to lipid metabolism, pollen exine formation, photosynthesis and hormone response are associated with male sterility induced by TBM. The results provide new insight into the molecular mechanisms of inducing male sterility by sulfonylurea.

Acetolactate Synthase-Inhibiting Gametocide Amidosulfuron Causes Chloroplast Destruction, Tissue Autophagy, and Elevation of Ethylene Release in Rapeseed.

Background: Acetolactate synthase (ALS)-inhibiting herbicides amidosulfuron (Hoestar) is an efficient gametocide that can induce male sterility in rapeseed (Brassica napus L.). We conducted an integrated study of cytological, transcriptomic, and physiological analysis to decipher the gametocidal effect of amidosulfuron. Results: In the first several days after exposure to amidosulfuron at a gametocidal dose of ca. 1 µg per plant, the plants showed the earliest symptoms including short retard of raceme elongation, slight chlorosis on leaf, and decrease of photosynthesis rate. Chloroplasts in leaf and anther epidermis, and tapetal plastids were deformed. Both tapetal cell and uni-nucleate microspore showed autophagic vacuoles and degenerated quickly. The amidosulfuron treatment caused reduction of photosynthetic rate and the contents of leaf chlorophyll, soluble sugar and pyruvate, as well as content alteration of several free amino acids in the treated plants. A comparison of transcriptomic profiling data of the young flower buds of the treated plants with the control identified 142 up-regulated and 201 down-regulated differential expression transcripts with functional annotations. Down-regulation of several interesting genes encoding PAIR1, SDS, PPD2, HFM1, CSTF77, A6, ALA6, UGE1, FLA20, A9, bHLH91, and putative cell wall protein LOC106368794, and up-regulation of autophagy-related protein ATG8A indicated functional abnormalities about cell cycle, cell wall formation, chloroplast structure, and tissue autophagy. Ethylene-responsive transcription factor RAP2-11-like was up-regulated in the flower buds and ethylene release rate was also elevated. The transcriptional regulation in the amidosulfuron-treated plants was in line with the cytological and physiological changes. Conclusions: The results suggested that metabolic decrease related to photosynthesis and energy supply are associated with male sterility induced by amidosulfuron. The results provide insights into the molecular mechanisms of gametocide-induced male sterility and expand the knowledge on the transcriptomic complexity of the plants exposure to sulfonylurea herbicide.

TGMS in Rapeseed (Brassica napus) Resulted in Aberrant Transcriptional Regulation, Asynchronous Microsporocyte Meiosis, Defective Tapetum, and Fused Sexine.

The thermo-sensitive genic male sterility (TGMS) line SP2S is a spontaneous rapeseed mutation with several traits that are favorable for the production of two-line hybrids. To uncover the key cellular events and genetic regulation associated with TGMS expression, a combined study using cytological observation, transcriptome profiling, and gene expression analysis was conducted for SP2S and its near-isogenic line SP2F grown under warm conditions. Asynchronous microsporocyte meiosis and abnormal tapetal plastids and elaioplasts were demonstrated in the anther of SP2S. The tetrad microspore did not undergo mitosis before the cytoplasm degenerated. Delayed degradation of the tetrad wall, which led to tetrad microspore aggregation, resulted in postponement of sexine (outer layer of pollen exine) formation and sexine fusion in the tetrad. The nexine (foot layer of exine) was also absent. The delay of tetrad wall degradation and abnormality of the exine structure suggested that the defective tapetum lost important functions. Based on transcriptomic comparisons between young flower buds of SP2S and SP2F plants, a total of 465 differentially expressed transcripts (DETs) were identified, including 303 up-regulated DETs and 162 down-regulated DETs in SP2S. Several genes encoding small RNA degrading nuclease 2, small RNA 2'-O-methyltransferase, thioredoxin reductase 2, regulatory subunit A alpha isoform of serine/threonine-protein phosphatase 2A, glycine rich protein 1A, transcription factor bHLH25, leucine-rich repeat receptor kinase At3g14840 like, and fasciclin-like arabinogalactan proteins FLA19 and FLA20 were greatly depressed in SP2S. Interestingly, a POLLENLESS3-LIKE 2 gene encoding the Arabidopsis MS5 homologous protein, which is necessary for microsporocyte meiosis, was down-regulated in SP2S. Other genes that were up-regulated in SP2S encoded glucanase A6, ethylene-responsive transcription factor 1A-like, pollen-specific SF3, stress-associated endoplasmic reticulum protein 2, WRKY transcription factors and pentatricopeptide repeat (PPR) protein At1g07590. The tapetum-development-related genes, including BnEMS1, BnDYT1, and BnAMS, were slightly up-regulated in 3-mm-long flower buds or their anthers, and their downstream genes, BnMS1 and BnMYB80, which affect callose dissolution and exine formation, were greatly up-regulated in SP2S. This aberrant genetic regulation corresponded well with the cytological abnormalities. The results suggested that expression of TGMS associates with complex transcriptional regulation.

Exposure to trace amounts of sulfonylurea herbicide tribenuron-methyl causes male sterility in 17 species or subspecies of cruciferous plants.

BACKGROUND: For most cruciferous plants, which are known as important crops and a number of weeds, hybrid breeding is hampered by the unavailability of a pollination control system. Male sterility induced by a gametocide can be useful for the utilization of plant heterosis. RESULTS: The gametocidal effect of sulfonylurea herbicide tribenuron-methyl was tested across seventeen cruciferous species or subspecies including Brassica juncea, B. carinata, B. oleracea ssp. capitata, B. oleracea ssp. acephala, B. rapa ssp. pekinensis, B. rapa ssp. chinensis, B. rapa ssp. parachinensis, B. nigra, Orychophragmus violaceus, Matthiola incana, Raphanus sativa, Sisymbrium altissimum, Eruca sativa, Sinapis alba, Sinapis arvensis, Capsella bursa-pastoris and Camelina sativa. The plants of 23 cultivars in these species or subspecies were foliar sprayed with 10 ml of 0.2 or 0.4 mg/L of tribenuron-methyl before the vacuolated microspore formed in the largest flower buds; the application was repeated ten to twelve days afterwards. Tribenuron-methyl exposure significantly changed the flowering phenology and reproductive function. The treated plants demonstrated a one to four day delay in flowering time and a shortened duration of flowering, as well as other slight phytotoxic effects including a reduction in plant height and floral organ size. Approximately 80% to 100% male sterility, which was estimated by both pollen staining and selfing seed-set rate, was induced in the plants. As a result, plants were rendered functionally able to out-cross, with an average 87% and 54% manually pollinated seed-set rate compared to the corresponding controls at the 0.2 mg/L and 0.4 mg/L doses, respectively. CONCLUSIONS: The results suggested that male reproductive function was much more sensitive to tribenuron-methyl exposure than female function. This sulfonylurea herbicide has a promising use as the gametocide for hybrid production in cruciferous plants.

De novo transcriptome reconstruction of a thermo-sensitive male sterility mutant in rapeseed (Brassica napus; Brassicaceae).

PREMISE OF THE STUDY: SP2S is a spontaneous thermo-sensitive genic male sterility (TGMS) mutation that facilitates two-line hybrid breeding in Brassica napus (Brassicaceae). De novo assembly of the floral bud transcriptome of SP2S can provide a foundation for deciphering the transcriptional regulation of SP2S in response to temperature change. METHODS: mRNAs of the young floral buds of SP2S and its near-isogenic line SP2F grown under cool (16°C)/warm (22°C) conditions were sequenced on an Illumina Solexa platform, producing 239.7 million short reads with a total length of 19.95 Gbp. RESULTS: The reads were assembled de novo using the Trinity program, resulting in 135,702 transcripts with an average length of 784 bp, an N50 value of 1221 bp, and a total length of 107 Mbp. We identified 24,157 cDNA-derived simple sequence repeats in the assembly. We found 137 and 195 single-nucleotide polymorphisms and 49 and 51 differentially regulated KEGG orthology groups when comparing sample SP2S at 22°C vs. SP2S at 16°C and sample SP2S at 22°C vs. SP2F at 22°C, respectively. DISCUSSION: The numerous differentially expressed genes and the derived single-nucleotide polymorphisms show abnormal transcriptional regulation in the TGMS system. These results outline an intricate transcriptional regulation that occurred in the rapeseed TGMS SP2S when the temperature changed.

[Roles of proto-oncogene c-erbB2 during the initiation growth of rat primordial follicles].

OBJECTIVE: To study the expression and possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles. METHODS: Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth culture system. In-situ hybridization, RT-PCR and immunohistochemistry were performed to assess the expressions of c-erbB2 mRNA and protein during the initiation growth of primordial follicles and after the effect of EGF. Western blot was used to observe the PCNA, p-ERK1/2 contents and correlation analysis was used to study the correlation relationship between contents of p-ERK1/2 and expressions of c-erbB2 mRNA at the same time of the primordial follicles growth. RESULTS: PCNA protein levels appeared to be more intense during the initiation growth of primordial follicles, EGF could promote the proliferation and differentiation of the primordial follicles. c-erbB2 mRNA existed in the oocytes endochylema and ErbB2 existed in the oocytes membrane, the expressions of c-erbB2 mRNA and ErbB2 appeared to be more intense when primordial follicles were cultured for 8 d than cultured for 0 d in the Waymouth culture system and were further increased with 50 ng/ml EGF for 4 d and 8 d. The same results were observed by RT-PCR, too. p-ERK1/2 protein levels were consistent with the changes of c-erbB2 mRNA and protein. Furthermore, Spearman rank correlation analysis showed there was a significant positive correlation relationship between the changes of p-ERK1/2 and the changes of c-erbB2 mRNA during the primordial follicles growth and after the effect of EGF (rs = 0.900, P < 0.05). CONCLUSION: It was suggested that proto-oncogene c-erbB2 may be play an important role during the initiation growth of primordial follicles with EGF, and it is indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.

[The expression of proto-oncogene c-erbB2 and its role in the initiation of primordial follicle growth in rat ovary].

Little is known about the factors that control the initiation of growth of primordial follicles. The objective of the present study was to investigate the effect of c-erbB2 on the onset of primordial follicle development, and whether c-erbB2 mediates the effect of epidermal growth factor (EGF) in this process. We synthesized three pairs of siRNAs targeting the c-erbB2 mRNA and transferred them into the newborn rat ovary cultured in vitro with Metafectene. After siRNAs transfection, the efficiency of siRNAs was tested by examining c-erbB2 mRNA and protein levels. The level of c-erbB2 mRNA was reduced by 49.6%, 46.7% and 82.6% respectively after transfecting siRNA1, siRNA2 and siRNA3, and the level of ErbB2 protein also reduced remarkably after siRNA3 transfection. c-erbB2 siRNA3 significantly inhibited the primordial follicle initiation and development; EGF augmented primordial follicles formation, but the effect was abolished by c-erbB2 siRNA3. All of these results suggest that c-erbB2 plays an important role in primordial follicle development and folliculogenesis initiation, and mediates the effect of EGF on primordial follicle development.

[Role of MAPK and PKC signaling pathways in the regulation of c-erbB2 in the primordial follicles onset].

Our previous studies showed that the proto-oncogene c-erbB2 played an important role in primordial follicles growth. The present study was conducted to investigate the role of MAPK and PKC signaling pathways in the primordial follicle onset in neonatal rats, and the relationship between c-erbB2 and MAPK/PKC signaling pathways. Ovaries collected from 2-day-old Sprague-Dawley rats were cultured in the Waymouth culture system in vitro. Ovaries were transfected with c-erbB2 siRNA, or treated with PD98059 (50 mumol/L) or Calphostin (0.5 mumol/L) in the culture medium. RT-PCR was performed to measure the expression of c-erbB2 mRNA, and Western blot analysis was performed to measure the expression of ErbB2, MAPK and PKC protein after the neonatal rat ovaries were cultured for 8 d. The quantities of every-stage follicles of ovaries cultured for 8 d were obtained in histological section stained with hematoxylin eosin. The results showed that c-erbB2 siRNA reduced the levels of c-erbB2 mRNA (P<0.01) and the levels of ErbB2, MAPK and PKC protein (P<0.01) significantly. But the levels of c-erbB2 mRNA and ErbB2 protein exhibited no change (P>0.05) in the ovaries cultured with PD98059 or Calphostin. After the ovaries were transfected with c-erbB2 siRNA or cultured with PD98059 or Calphostin for 8 d, the quantities of primary follicles and second follicles were lower than those in the control group (P<0.05 or P<0.01), but the quantity of the primordial follicles was higher than that in the control group (P<0.01). These results suggest that proto-oncogene c-erbB2 promotes the initiation of primordial follicle growth through the MAPK and PKC signal transduction, and c-erbB2 is possibly the upstream of PKC and MAPK signaling pathway in the regulation of primordial follicle onset.

[Attempting to enhance the efficiency of T-DNA insertional mutant application in rice].

T-DNA tagging method is a high throughput system for identifying and cloning novel genes from T-DNA-inserted mutant population created via genetic transformation by Agrobacterium tumefaciens. However, the efficiency of using T-DNA-inserted mutant population to clone genes in rice was much lower than in Arabidopsis. In this study, a rice tagged line with two copies of T-DNA segments inserted independently to each other was screened out via a series of verification tests, including the co-segregated analysis between the mutated character and the sequence of T-DNA or the genomic sequence flanking inserted T-DNA. From this tagged line, two inserted incidents were separated from the progeny population of a plant heterozygous in two tagged sites, and some plants with the target trait and one of the inserted incidents were obtained, which were important basic materials for the subsequently co-segregated analysis between the mutated character and the sequence of inserted T-DNA, and for cloning the mutant gene in future. Based on this study, we have some thoughts about the gene cloning from the T-DNA tagged lines with more than one inserted sequence independently and put forward to discuss with colleagues.

[Antioxidative activity of water-soluble extracts from Fructus Polygoni Orientalis].

OBJECTIVE: To study the antioxidative activity of the water-soluble extracts from Fructus Polygoni Orientalis. METHODS: Malondialdehyde and superoxide dismutase were measured by thiobarbituric acid assay, nitrite method, respectively. The activity of glutathione peroxidase was determined by 5,5'-dithionbis (2-nitrobenzoic acid) photometric method. Lipofuscin was determined by Sohal method in the brain of senile model mice induced by D-galactose. The extracts of 0.8, 1.6 and 3.2 g.kg(-1).d(-1) were administered to the mice separately through oral gavage daily for 7 weeks. RESULTS: Malondialdehyde content increased, activities of superoxide dismutase and glutathione peroxidase in the heart, liver and kidney decreased. Levels of lipofuscin in the brain of senile model mice induced by D-galactose increased. The three doses of the extracts could inhibit the levels of lipofuscin in the brain, malondialdehyde in the heart, liver and kidney. These results showed that there was a negative dose-effect relationship. The extracts also up-regulated the levels of superoxide dismutase and glutathione peroxidase in the heart, liver and kidney with a positive dose-effect relationship. CONCLUSION: The water-soluble extracts exert its antioxidative activity by eliminating free hydroxyl radicals, increasing activities of superoxide dismutase and glutathione peroxidase.