Cloning and functional verification of the male sterile gene BrQRT3 in Chinese cabbage.

Chinese cabbage is a cross-pollinated crop with significant heterosis, and male sterile lines are an important way to produce hybrid seeds. In this study, a male sterile mutant msm0795 was identified in an EMS-mutagenized population of Chinese cabbage. Cytological observations revealed that the microspores failed to separate after the tetrad stage, and thus developed into abnormal pollen grains, resulting in anther abortion. MutMap combined with Kompetitive Allele Specific PCR genotyping showed that BraA01g011280.3.5 C was identified as the candidate gene, which encodes polygalacturonase QRT3 and plays a direct role in the degradation of pollen mother cell wall during microspore development, named BrQRT3. Subcellular localization and expression analyses demonstrated that BrQRT3 was localized in the cell membrane and was ubiquitously expressed in roots, stems, leaves, flower buds, and flowers, but the expression of BrQRT3 was gradually suppressed with the anther development. Ectopic expression confirmed that over-expression of BrQRT3 in qrt3 background Arabidopsis mutant can rescue the pollen defects caused by loss of AtQRT3 function. It is the first time to achieve a male sterile mutant caused by the mutation of BrQRT3 in Chinese cabbage. These findings contribute to elucidate the mechanism of BrQRT3 in regulating stamen development of Chinese cabbage.

Comparative metabolic profiling of different pakchoi cultivars reveals nutritional diversity via widely targeted metabolomics.

Pakchoi (Brassica rapa ssp. chinensis) is cultivated for its high nutritional value; however, the nutritional diversity of different pakchoi cultivars is rarely investigated. Herein, we performed widely targeted metabolic profiling analyses of five popular pakchois. A total of 670 metabolites were detected, which could be divided into 13 categories. The accumulation patterns of main nutritional metabolites among the five pakchois were significantly different and complementary. Moreover, the pakchoi cultivar 'QYC' showed quite different metabolomic profiles compared with other pakchois. The Venn diagram showed that the 75 differential metabolites were shared among the comparison groups ('QYC' vs. 'MET'/ 'NBC'/ 'PPQ'/ 'XQC'), of which 52 metabolites were upregulated in 'QYC'. The phenolic acids had the largest variations between 'QYC' and the other pakchoi cultivars. These findings expand metabolomic information on different pakchoi cultivars and further provide new insights into the selection and breeding of excellent pakchoi cultivars.

BrKCS6 mutation conferred a bright glossy phenotype to Chinese cabbage.

KEY MESSAGE: BrKCS6 encoding 3-ketoacyl-CoA synthases was cloned through MutMap and KASP analysis, and its function was verified via allelic mutants in Chinese cabbage. Bright and glossy green appearance is an attractive commodity character for leafy vegetables and is mainly caused by the absence of epicuticular wax crystals. In this study, two allelic epicuticular wax crystal deficiency mutants, wdm9 and wdm10, were obtained from an EMS mutagenesis population of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Genetic analysis revealed that the mutant phenotype was controlled by a recessive nuclear gene. BrKCS6 encoding 3-ketoacyl-CoA synthases was identified as the candidate gene by MutMap and KASP analysis. A SNP (G to A) on BrKCS6 in wdm9 led to the amino acid substitution from serine (S) to phenylalanine (F), and another SNP (G to A) in wdm10 resulted in the amino acid substitution from serine (S) to leucine (L). Both SNPs are located in the ACP_syn_III_C conserved domain, corresponding to two highly conserved sites among BrKCS6 and its homologs. These two amino acid substitutions changed the secondary structure and the three-dimensional structure of BrKCS6 protein. qRT-PCR results showed that the relative expression of BrKCS6 significantly decreased in the flower, stem, and leaves in mutant, and the relative expressions of the downstream key genes of BrKCS6 were down-regulated in mutant. We were the first to clone the precious glossy bright gene BrKCS6 which has a great potential for commodity quality breeding in Chinese cabbage.

BrACOS5 mutations induced male sterility via impeding pollen exine formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: BrACOS5 mutations led to male sterility of Chinese cabbage verified in three allelic male-sterile mutants. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the major vegetable crops in East Asia, and the utilization of male-sterile line is an important measure for its hybrid seed production. Herein, we isolated three allelic male-sterile mutants, msm1-1, msm1-2 and msm1-3, from an ethyl methane sulfonate (EMS) mutagenized population of Chinese cabbage double-haploid (DH) line 'FT', whose microspores were completely aborted with severely absent exine, and tapetums were abnormally developed. Genetic analyses indicated that the three male-sterile mutants belonged to allelic mutation and were triggered by the same recessive nuclear gene. MutMap-based gene mapping and kompetitive allele-specific PCR (KASP) analysis demonstrated that three different single-nucleotide polymorphisms (SNPs) of BraA09g012710.3C were responsible for the male sterility of msm1-1/2/3, respectively. BraA09g012710.3C is orthologous of Arabidopsis thaliana ACOS5 (AT1G62940), encoding an acyl-CoA synthetase in sporopollenin biosynthesis, and specifically expressed in anther, so we named BraA09g012710.3C as BrACOS5. BrACOS5 localizes to the endoplasmic reticulum (ER). Mutations of BrACOS5 resulted in decreased enzyme activities and altered fatty acid contents in msm1 anthers. As well as the transcript accumulations of putative orthologs involved in sporopollenin biosynthesis were significantly down-regulated excluding BrPKSA. These results provide strong evidence for the integral role of BrACOS5 in conserved sporopollenin biosynthesis pathway and also contribute to uncovering exine development pattern and underlying male sterility mechanism in Chinese cabbage.

Suberoylanilide hydroxamic acid induced microspore embryogenesis and promoted plantlet regeneration in ornamental kale (Brassica oleracea var. acephala).

Isolated Microspore Culture (IMC) is an efficient method to obtain the homozygous strain; however, it is difficult to apply in ornamental kale due to its low rate of microspore embryogenesis. Histone acetylation is an important epigenetic mechanism and may affect the changes of the microspore development pathway, promoting microspore embryogenesis. Here, microspores from three cut-flower ornamental kales, namely Crane Feather Queen (CFQ), Crane Pink (CP), and Crane Bicolor (CB), were treated with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) to induce embryogenesis. The haploid 'CFQ' microspore plantlets were doubled with colchicine. The results for 'CFQ' revealed that, the appropriate concentration of SAHA was 0.03 µM and obtained 17.27 embryos per bud. For 'CP,' the appropriate concentration of SAHA was 0.045 µM and obtained 11.19 embryos per bud. For 'CB,' the appropriate concentration of SAHA was 0.045 µM and obtained 6.10 embryos per bud. Haploid 'CFQ' microspore plantlets were treated with 75 mg/L colchicine for 7 d and the doubling rate was 41.7%. Haploid 'CFQ' plantlets were treated with 1000 mg/L colchicine by root-soaking for 4 h and the doubling rate was 64.3%. SAHA could promote microspore embryogenesis, and colchicine root soaking was more effective than adding colchicine to the medium for haploid plantlet doubling in cut-flower ornamental kale.

Defect in BrMS1, a PHD-finger transcription factor, induces male sterility in ethyl methane sulfonate-mutagenized Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Male sterility is an ideal character for the female parent in commercial hybrid seed production in Chinese cabbages. We identified three allele male sterile mutants msm2-1/2/3 in progenies of ethyl methane sulfonate mutagenized Chinese cabbage. It was proved that their male sterilities were controlled by a same recessive nuclear gene. Cytological observation showed that the delayed tapetal programmed cell death (PCD) as well as the abnormal pollen exine and intine led to pollen abortion in these mutants. MutMap combined with KASP analyses showed that BraA10g019050.3C, a homologous gene of AtMS1 encoding a PHD-finger transcription factor and regulated pollen development, was the causal gene. A single-nucleotide mutation from G to A occurred at the 2443th base of BrMS1 in msm2-1 which results in premature termination of the PHD-finger protein translation; a single-nucleotide mutation from G to A existed at 1372th base in msm2-2 that makes for frameshift mutation; a single-nucleotide mutation from G to A distributed at 1887th base in msm2-3 which issues in the amino acid changed from Asp to Asn. The three allelic mutations in BrMS1 all led to the male sterile phenotype, which revealed its function in stamen development. Quantitative reverse transcription polymerase chain reaction analysis indicated that BrMS1 specially expressed in the anther at the early stage of pollen development and its expression level was higher in msm2-1/2/3 than that in the wild-type "FT." BrMS1 was located at the nucleus and a length of 12 amino acid residues at the C-terminus had transcriptional activation activity. RNA-seq indicated that the mutation in BrMS1 affected the transcript level of genes related to the tapetum PCD and pollen wall formation, which brought out the pollen abortion. These male sterile mutants we developed provided a novel gene resource for hybrid breeding in Chinese cabbage.

Mutation in BrGGL7 gene encoding a GDSL esterase / lipase causes male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: MutMap and KASP analyses revealed that the BrGGL7 gene is responsible for the male-sterile trait of ftms1 in Chinese cabbage, with functional verification in Arabidopsis. The application of a male-sterile line is an ideal approach of hybrid seed production in Chinese cabbage. In this study, we obtained a male-sterile mutant (ftms1) from the double haploid line 'FT' using ethyl methane sulfonate (EMS) mutagenesis. The mutant was completely sterile due to abnormal enlargement and vacuolization of the tapetum cells. A single recessive nuclear gene was found to control male sterility in the mutant, while MutMap and KASP analyses identified BraA05g022470.3C (BrGGL7), which encodes a GDSL esterase / lipase, as the candidate mutant gene. A single nucleotide substitution from C to T occurred within the domain of BrGGL7 in ftms1, resulting in premature translation termination in the fourth exon. Meanwhile, qRT-PCR analysis indicated that BrGGL7 was prominently expressed in the anthers, and expression was greater in the wild-type 'FT' than ftms1. Genetic complementation of the orthologous Arabidopsis ggl7 mutant further confirmed the role of BrGGL7 in pollen development. These findings suggest that BrGGL7 plays a fundamental role in pollen formation, providing important insight into the molecular mechanisms underlying male sterility in Chinese cabbage.

Prognostic Value of lncRNA DRAIC and miR-3940-3p in Lung Adenocarcinoma and Their Effect on Lung Adenocarcinoma Cell Progression.

PURPOSE: Lung adenocarcinoma (LUAD) is a most common malignant tumor, even worse for diseases with relatively poor prognosis. Non-coding RNAs have the potential to be biomarkers for the prognosis of various cancers. LncRNA DRAIC and miR-3940-3p have been screened as dysregulated RNAs in LUAD. The clinical significance and biological function of lncRNA DRAIC and miR-3940-3p in LUAD were assessed in this study. PATIENTS AND METHODS: A total of 122 cases of LUAD patients with complete clinical information were enrolled. The expression levels of lncRNA DRAIC and miR-3940-3p were determined via RT-qPCR in LUAD tissues and cells. The relationship between lncRNA DRAIC or miR-3940-3p expression and the clinicopathological features of patients was analyzed based on the Pearson Chi-square test. For the prognostic value, the Kaplan-Meier plot and multi-variate Cox proportional regression analysis were introduced. Finally, the effect of lnc DRAIC and miR-3940-3p on the LUAD cellular function was investigated by CCK-8 and Transwell assay. RESULTS: lnc DRAIC was upregulated in LUAD tissues and cells, but miR-3940-3p was downregulated. Both of them showed significant associations with and TNM stage, lymph node metastasis, and a poor prognosis. Lnc-DRAIC and miR-3940-3p have the potential as independent prognostic factors for LUAD. Furthermore, the inhibition of lnc DRAIC can inhibit cell proliferation, migration, and invasion of LUAD partly as a ceRNA of miR-3940-3p. CONCLUSION: lncRNA DRAIC/miR-3940-3p axis may be involved in the progression of LUAD and can be developed to promising prognostic factors, which may provide new insights into the treatment of LUAD.

The head fixation based on skull cap: An improved protocol used in single unit recording in the vestibular system.

Single unit recording has an important application in neuroscience, especially in the vestibular system such as visual stabilization, posture maintenance, spatial orientation and cognition. However, single unit recording conducted in living animals is a demanding technique and non-ideal mechanical stability between the recording location of nerve tissues and the tip of microelectrode always results in failure to obtain successful recordings in the vestibular system. In order to improve the mechanical stability during single unit recording, we constructed a novel head fixation method based on skull cap. This article describes in detail how to construct this novel head fixation. Following the step-by-step procedure mentioned in this article will provide a high-quality mechanical stability for single unit recording in the vestibular system, allowing us to successfully record the nonlinear neural dynamic response over a big magnitude motion stimulation. This improvement of head fixation contributes to the in-depth understanding of the vestibular system.

The real identity and sensory overlap mechanism of special vestibular afferent neurons that sense both rotation and linear force.

AIMS: Although the vestibular system has been widely investigated over the past 50 years, there is still an unsolved mystery. Some special vestibular afferent (SVA) neurons responding to both rotation and linear force were found through neurophysiological techniques, however, the sensory overlap mechanism of SVA neurons is still unclear, which may be closely related to vestibular-related diseases. MATERIALS AND METHODS: To address the above-mentioned problem, a cupula buoyancy theory was established in the present study, where SVA neurons were considered semicircular canal afferent (SCCA) neurons. Then labyrinth anatomy and neural response dynamics of vestibular afferent neurons in chinchilla were investigated through vestibular labyrinth reconstruction and single unit recording technique, respectively. KEY FINDINGS: We analyzed the deflections of cupulae under multiple conditions with the help of Amira Software and predicted the neural response law of SCCA neurons to linear force based on the cupula buoyancy theory. Data analysis confirmed that the basic response characteristic of SVA neurons had no significant difference to those of SCCA neurons, but were significantly different from those of otolith afferent neurons. Further, the actual responses of SVA neurons to linear force are completely consistent with our predictions. These results strongly suggest that SVA neurons actually are SCCA neurons, and the cupula buoyancy theory is the key to the sensory overlap mechanism of SCCA neurons. SIGNIFICANCE: Our study revealed the real identity of SVA neurons and provided a reasonable mechanism for sensory overlap of rotation and linear force, which improved our understanding about the vestibular system.

Investigation of genes associated with petal variations between diploid and autotetraploid in Chinese cabbage (Brassica rapa L. ssp. pekinensis) by RNA-seq and sRNA-seq.

Polyploidy promotes morphological, physiological, and reproductive diversity in plants. The imminent effect of chromosome doubling in plants is the enlargement of organs such as flowers and fruits, which increases the commercial value of crops. Flowering plays a vital role in the growth and development of angiosperms. Here, we prepared an isolated microspore culture of 'FT', a doubled haploid (DH) line of Chinese cabbage (Brassica rapa L. ssp. pekinensis), and obtained diploid and autotetraploid plants with the same genetic background. Compared with diploids, the autotetraploids were characterized by large floral organs, dark petals, delayed flowering, and reduced fertility. The indole-3-acetic acid (IAA) and jasmonic acid (JA) levels in autotetraploid petals were significantly higher and the abscisic acid (ABA) level was significantly lower than those in the diploid petals. The lutein level in autotetraploid petals was nearly two times higher than that in the diploid petals. A comparative transcriptome analysis revealed 14,412 differentially expressed genes (DEGs) between the diploids and autotetraploids, and they were enriched in 117 Gene Ontology terms and 110 Kyoto Encyclopedia of Genes and Genomes pathways. We detected 231 DEGs related to phytohormone signal transduction and 29 DEGs involved in carotenoid biosynthesis. An miRNA-target mRNA analysis showed that 32 DEGs regulated by 16 DEMs were associated with flowering timing (BraA03000336, BraA09004319, and BraA09000515), petal development (BraA05002408, BraA01004006, BraA09004069, and BraA04000966), flower opening (BraA07000350), and pollen development (BraA01000720, BraA09005727, and BraA01000253). This study provides information to help elucidate the molecular mechanisms underlying phenotypic variations induced by autopolyploidy in Chinese cabbage.

Improvement of Mechanical Stability for Single Unit Recording Based on Skull Cap in Living Chinchilla.

Three-point head fixation was constructed to provide mechanical stability for single unit recording (SUR) on vestibular sensory system in living chinchilla previously. However, it is no more qualified to this work when the stimulation intensity becomes large because of frequent unit losing and neuron damage, which strongly implies that the mechanical stability has been broken during the stimulation. Here, we constructed a novel head fixation (skull cap assistant head fixation) provided by skull cap on the basis of three-point head fixation in order to improve the mechanical stability for SUR under the stimulation with large magnitude. The large area bone connection is the feature and advantage of this improved method, which directly fixes the tested local nervous tissue and microelectrode in an intact stable system through skull cap except two ear bars and a tube face mask. Our data exhibited that skull cap assistant head fixation could significantly improve the success rate of neural response activity recording in the population of semicircular canal neurons under the stimulation with large intensity (amplitude ≥100 deg/s). Based on the analysis of neural response activity and noise base-line during stimulation, our data further indicated that this method could significantly improve the mechanical stability for SUR during high-speed motion stimulation on vestibular system in living chinchilla. Skull cap assistant head fixation extends the application of SUR on vestibular neuron in linear response range and provides a solid foundation for electrophysiological research on vestibular sensory system in further studies.

The reliability of nonlinear least-squares algorithm for data analysis of neural response activity during sinusoidal rotational stimulation in semicircular canal neurons.

Although many mathematical methods were used to analyze the neural activity under sinusoidal stimulation within linear response range in vestibular system, the reliabilities of these methods are still not reported, especially in nonlinear response range. Here we chose nonlinear least-squares algorithm (NLSA) with sinusoidal model to analyze the neural response of semicircular canal neurons (SCNs) during sinusoidal rotational stimulation (SRS) over a nonlinear response range. Our aim was to acquire a reliable mathematical method for data analysis under SRS in vestibular system. Our data indicated that the reliability of this method in an entire SCNs population was quite satisfactory. However, the reliability was strongly negatively depended on the neural discharge regularity. In addition, stimulation parameters were the vital impact factors influencing the reliability. The frequency had a significant negative effect but the amplitude had a conspicuous positive effect on the reliability. Thus, NLSA with sinusoidal model resulted a reliable mathematical tool for data analysis of neural response activity under SRS in vestibular system and more suitable for those under the stimulation with low frequency but high amplitude, suggesting that this method can be used in nonlinear response range. This method broke out of the restriction of neural activity analysis under nonlinear response range and provided a solid foundation for future study in nonlinear response range in vestibular system.