Transcriptomic Profiling Highlights the ABA Response Role of BnSIP1-1 in Brassica napus.

BnSIP1-1 is the first identified SIP1 (6b Interacting Protein1) subfamily gene of the trihelix transcription factor family from Brassica napus (B. napus). We previously used a reverse genetic method to reveal its abiotic stress response function in endowing plants resistance to drought and salinity, as well as ABA (Abscisic acid). However, the molecular mechanisms of BnSIP1-1 are unclear. In this study, the global transcriptome files of BnSIP1-1-overexpressing transgenic and wildtype B. napus seedlings under ABA treatment were constructed using RNA-seq. A total of 1823 and 5512 DEGs (Differentially Expressed Genes) were identified in OE vs. WT and OE_ABA vs. WT_ABA comparison groups, which included 751 and 2567 up-regulated DEGs, and 1072 and 2945 down-regulated DEGs, separately. The impact of overexpressed BnSIP1-1 on plants was amplified by ABA, indicating BnSIP1-1 was an ABA-conditioned responsive gene. More interestingly, we found the reasons for BnSIP1-1 increasing plants' insensitivity to ABA were not by regulating ABA synthesis and catabolism, but by manipulating ABA transportation, ABA signal perception and transduction, inositol phosphate metabolism, as well as endomembrane trafficking, indirectly suggesting this gene may play roles upstream of the core ABA response pathway. Our results provided new insights into improving the knowledge about the function of BnSIP1-1 and the ABA signaling mechanism in B. napus.

Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line.

The genome-wide long hairpin RNA interference (lhRNAi) library is an important resource for plant gene function research. Molecularly characterizing lhRNAi mutant lines is crucial for identifying candidate genes associated with corresponding phenotypes. In this study, a dwarf and sterile line named P198 was screened from the Brassica napus (B. napus) RNAi library. Three different methods confirmed that eight copies of T-DNA are present in the P198 genome. However, only four insertion positions were identified in three chromosomes using fusion primer and nested integrated polymerase chain reaction. Therefore, the T-DNA insertion sites and copy number were further investigated using Oxford Nanopore Technologies (ONT) sequencing, and it was found that at least seven copies of T-DNA were inserted into three insertion sites. Based on the obtained T-DNA insertion sites and hairpin RNA (hpRNA) cassette sequences, three candidate genes related to the P198 phenotype were identified. Furthermore, the potential differentially expressed genes and pathways involved in the dwarfism and sterility phenotype of P198 were investigated by RNA-seq. These results demonstrate the advantage of applying ONT sequencing to investigate the molecular characteristics of transgenic lines and expand our understanding of the complex molecular mechanism of dwarfism and male sterility in B. napus.

Evolution of the Brassicaceae-specific MS5-Like family and neofunctionalization of the novel MALE STERILITY 5 gene essential for male fertility in Brassica napus.

New genes (or lineage-specific genes) can facilitate functional innovations. MALE STERILITY 5 (MS5) in Brassica napus is a fertility-related new gene, which has two wild-type alleles (BnMS5a and BnMS5c ) and two mutant alleles (BnMS5b and BnMS5d ) that could induce male sterility. Here, we studied the history and functional evolution of MS5 homologs in plants by phylogenetic analysis and molecular genetic experiments. We identified 727 MS5 homologs and found that they define a Brassicaceae-specific gene family that has expanded partly via multiple tandem gene duplications and also probably transpositions. The MS5 in B. napus is inherited from a basic diploid ancestor of B. rapa. Molecular genetic experiments indicate that BnMS5a and BnMS5c are functionally distinct in B. napus and that BnMS5d can inhibit BnMS5a in B. napus in a dosage-dependent manner. The BnMS5a protein can move in coordination with meiotic telomeres and interact with the nuclear envelope protein SUN1, with a possible crucial role in meiotic chromosome behavior. In summary, BnMS5 belongs to a Brassicaceae-specific new gene family, and has gained a novel function that is essential for male fertility in B. napus through neofunctionalization that has likely occurred since the origin of B. rapa.

Rolling Circle Amplification (RCA)-Mediated Genome-Wide ihpRNAi Mutant Library Construction in Brassica napus.

With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.

Fine mapping of a dominant thermo-sensitive genic male sterility gene (BntsMs) in rapeseed (Brassica napus) with AFLP- and Brassica rapa-derived PCR markers.

KEY MESSAGE: A new thermo-sensitive dominant genic male sterility (TSDGMS) line of Brassica napus was found and mapped in this paper. Our result will greatly accelerate the map-based cloning of the BntsMs gene. TE5A is a thermo-sensitive dominant genic male sterility line originating from spontaneous mutation of the inbred line TE5 in Brassica napus and provides a promising system for the development of hybrid cultivars. Genetic analysis has revealed that the BntsMs mutant is controlled by a single, dominant gene. Here, we describe the fine mapping of BntsMs using amplified fragment length polymorphism (AFLP) and intron polymorphism (IP) methodologies. We screened 1,024 primer combinations and then identified five AFLP markers linked to the BntsMs gene, two of which were successfully converted into sequence-characterised amplified region (SCAR) markers. The linkage of the markers was identified by analysing a large BC2 population of 700 recessive-fertility individuals. Two SCAR markers were found in the flanking region of the BntsMs gene at distance of 3.5 and 4.8 cm. Based on sequence information from the previously screened AFLP markers and on genome organisation comparisons of the A genome of Brassica rapa and Arabidopsis, seven IP markers linked to the BntsMs gene were developed. By analysing the 700 recessive-fertility individuals, two IP markers, IP004 and IP470, were localised to the flanking region of the BntsMs gene at a distance of 0.3 and 0.2 cm, respectively. A comparison of the B. rapa and Arabidopsis genomes revealed 27 genes of B. rapa in the flanking region of these two IP markers. It is likely that the molecular markers developed from these investigations will greatly accelerate the positional cloning of the BntsMs gene.

[Plant Trihelix transcription factors family].

The Trihelix transcription factor family has raised great concerns only in recent years. It was named after its conserved DNA binding domain containing three tandem helix (helix-loop-helix-loop-helix), which could bind specifically with GT element, a light-responsive DNA element. So, this family is also known as GT factors. At the early stage of study, the knowledge of this family was only confined to their functions in regulation of light-responsive genes. However, recent researches indicated that Trihelix family also plays important roles in different growth and development processes involving flowers, stomata, trichomes, embryos, and seeds, as well as roles in response to abiotic and biotic stresses. This review mainly focused on the structural characteristics, classification, and the latest functional research progresses on the Trihelix family.

Time series prediction of COVID-19 transmission in America using LSTM and XGBoost algorithms.

In this paper, we establish daily confirmed infected cases prediction models for the time series data of America by applying both the long short-term memory (LSTM) and extreme gradient boosting (XGBoost) algorithms, and employ four performance parameters as MAE, MSE, RMSE, and MAPE to evaluate the effect of model fitting. LSTM is applied to reliably estimate accuracy due to the long-term attribute and diversity of COVID-19 epidemic data. Using XGBoost model, we conduct a sensitivity analysis to determine the robustness of predictive model to parameter features. Our results reveal that achieving a reduction in the contact rate between susceptible and infected individuals by isolated the uninfected individuals, can effectively reduce the number of daily confirmed cases. By combining the restrictive social distancing and contact tracing, the elimination of ongoing COVID-19 pandemic is possible. Our predictions are based on real time series data with reasonable assumptions, whereas the accurate course of epidemic heavily depends on how and when quarantine, isolation and precautionary measures are enforced.

SDG711 Is Involved in Rice Seed Development through Regulation of Starch Metabolism Gene Expression in Coordination with Other Histone Modifications.

SDG711 is a histone H3K27me2/3 transmethylase in rice, a homolog of CLF in Arabidopsis, and plays key roles in regulating flowering time and panicle development. In this work, we investigated the role of SDG711 in rice seed development. Overexpression and downregulation of SDG711 lead to a decrease and increase in the expression level of genes related to starch accumulation, resulting in smaller seeds or even seed abortion. ChIP assay showed that SDG711-mediated H3K27me3 changed significantly in genes related to endosperm development, and SDG711 can directly bind to the gene body region of several starch synthesis genes and amylase genes. In addition, H3K4me3 and H3K9ac modifications also cooperate with H3K27me3 to regulate the development of the endosperm. Our results suggest that the crosstalk between SDG711-mediated H3K27me3 and H3K4me3, and H3K9ac are involved in starch accumulation to control normal seed development.

Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice.

High temperature (HT) stress is a major environmental stress that limits agricultural production worldwide. Discovery and application of genes promoting high temperature tolerance is essential to enhance crop tolerance to heat stress. Proteins associated with chaperone and protein folding plays an important role in the high temperature stress response of plants. MTH1745 (MtPDI), a disulfide isomerase-like protein (PDI) with a chaperone function and disulfide isomerase activity from Methanothermobacter thermautotrophicus delta H, was selected for studying the heat stress tolerance using an ectopic expression method in rice. Through molecular identification via quantitative real-time PCR and western blot, we demonstrated that the MtPDI gene was expressed stably in transgenic rice. Heat stress tolerance and survival ratio were significantly improved in seedling transgenic rice. At the same time, proline content, superoxide dismutase (SOD) and peroxidase (POD) activities were increased in MtPDI transgenic rice with a reduced malondialdehyde (MDA) content. Moreover, increased content of thiols group was discovered in transgenic plants. These results indicate that heterologous expression of MtPDI from extremophiles could confer heat stress tolerance of transgenic rice through the accumulation of proline content, the synergistic increase of the antioxidant enzymes activity and elevated production of more thiols group, which finally ameliorated the oxidative damage.

Nuclear Envelope-Associated Chromosome Dynamics during Meiotic Prophase I.

Chromosome dynamics during meiotic prophase I are associated with a series of major events such as chromosomal reorganization and condensation, pairing/synapsis and recombination of the homologs, and chromosome movements at the nuclear envelope (NE). The NE is the barrier separating the nucleus from the cytoplasm and thus plays a central role in NE-associated chromosomal movements during meiosis. Previous studies have shown in various species that NE-linked chromosome dynamics are actually driven by the cytoskeleton. The linker of nucleoskeleton and cytoskeleton (LINC) complexes are important constituents of the NE that facilitate in the transfer of cytoskeletal forces across the NE to individual chromosomes. The LINCs consist of the inner and outer NE proteins Sad1/UNC-84 (SUN), and Klarsicht/Anc-1/Syne (KASH) domain proteins. Meiosis-specific adaptations of the LINC components and unique modifications of the NE are required during chromosomal movements. Nonetheless, the actual role of the NE in chromosomic dynamic movements in plants remains elusive. This review summarizes the findings of recent studies on meiosis-specific constituents and modifications of the NE and corresponding nucleoplasmic/cytoplasmic adaptors being involved in NE-associated movement of meiotic chromosomes, as well as describes the potential molecular network of transferring cytoplasm-derived forces into meiotic chromosomes in model organisms. It helps to gain a better understanding of the NE-associated meiotic chromosomal movements in plants.

BnSIP1-1, a Trihelix Family Gene, Mediates Abiotic Stress Tolerance and ABA Signaling in Brassica napus.

The trihelix family genes have important functions in light-relevant and other developmental processes, but their roles in response to adverse environment are largely unclear. In this study, we identified a new gene, BnSIP1-1, which fell in the SIP1 (6b INTERACTING PROTEIN1) clade of the trihelix family with two trihelix DNA binding domains and a fourth amphipathic α-helix. BnSIP1-1 protein specifically targeted to the nucleus, and its expression can be induced by abscisic acid (ABA) and different stresses. Overexpression of BnSIP1-1 improved seed germination under osmotic pressure, salt, and ABA treatments. Moreover, BnSIP1-1 decreased the susceptibility of transgenic seedlings to osmotic pressure and ABA treatments, whereas there was no difference under salt stress between the transgenic and wild-type seedlings. ABA level in the transgenic seedlings leaves was higher than those in the control plants under normal condition. Under exogenous ABA treatment and mannitol stress, the accumulation of ABA in the transgenic plants was higher than that in the control plants; while under salt stress, the difference of ABA content before treatment was gradually smaller with the prolongation of salt treatment time, then after 24 h of treatment the ABA level was similar in transgenic and wild-type plants. The transcription levels of several general stress marker genes (BnRD29A, BnERD15, and BnLEA1) were higher in the transgenic plants than the wild-type plants, whereas salt-responsive genes (BnSOS1, BnNHX1, and BnHKT) were not significantly different or even reduced compared with the wild-type plants, which indicated that BnSIP1-1 specifically exerted different regulatory mechanisms on the osmotic- and salt-response pathways in seedling period. Overall, these findings suggested that BnSIP1-1 played roles in ABA synthesis and signaling, salt and osmotic stress response. To date, information about the involvement of the Brassica napus trihelix gene in abiotic response is scarce. Here, we firstly reported abiotic stress response and possible function mechanisms of a new trihelix gene in B. napus.

Aberrant Meiotic Prophase I Leads to Genic Male Sterility in the Novel TE5A Mutant of Brassica napus.

Genic male sterility (GMS) has already been extensively utilized for hybrid rapeseed production. TE5A is a novel thermo-sensitive dominant GMS line in Brassica napus, however, its mechanisms of GMS remain largely unclear. Histological and Transmission electron microscopy (TEM) analyses of anthers showed that the male gamete development of TE5A was arrested at meiosis prophase I. EdU uptake of S-phase meiocytes revealed that the TE5A mutant could accomplish DNA replication, however, chromosomal and fluorescence in situ hybridization (FISH) analyses of TE5A showed that homologous chromosomes could not pair, synapse, condense and form bivalents. We then analyzed the transcriptome differences between young floral buds of sterile plants and its near-isogenic fertile plants through RNA-Seq. A total of 3,841 differentially expressed genes (DEGs) were obtained, some of which were associated with homologous chromosome behavior and cell cycle control during meiosis. Dynamic expression changes of selected candidate DEGs were then analyzed at different anther developmental stages. The present study not only demonstrated that the TE5A mutant had defects in meiotic prophase I via detailed cytological analysis, but also provided a global insight into GMS-associated DEGs and elucidated the mechanisms of GMS in TE5A through RNA-Seq.

Identification and Analysis of MS5d: A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes.

Here, we report the identification of the Brassica-specific gene MS5d, which is responsible for male sterility in Brassica napus. The MS5d gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5d gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5d, encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5d likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus.

Elucidation of Cross-Talk and Specificity of Early Response Mechanisms to Salt and PEG-Simulated Drought Stresses in Brassica napus Using Comparative Proteomic Analysis.

To understand the cross-talk and specificity of the early responses of plants to salt and drought, we performed physiological and proteome analyses of Brassica napus seedlings pretreated with 245 mM NaCl or 25% polyethylene glycol (PEG) 6000 under identical osmotic pressure (-1.0 MPa). Significant decreases in water content and photosynthetic rate and excessive accumulation of compatible osmolytes and oxidative damage were observed in response to both stresses. Unexpectedly, the drought response was more severe than the salt response. We further identified 45 common differentially expressed proteins (DEPs), 143 salt-specific DEPs and 160 drought-specific DEPs by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. The proteome quantitative data were then confirmed by multiple reaction monitoring (MRM). The differences in the proteomic profiles between drought-treated and salt-treated seedlings exceeded the similarities in the early stress responses. Signal perception and transduction, transport and membrane trafficking, and photosynthesis-related proteins were enriched as part of the molecular cross-talk and specificity mechanism in the early responses to the two abiotic stresses. The Ca2+ signaling, G protein-related signaling, 14-3-3 signaling pathway and phosphorylation cascades were the common signal transduction pathways shared by both salt and drought stress responses; however, the proteins with executive functions varied. These results indicate functional specialization of family proteins in response to different stresses, i.e., CDPK21, TPR, and CTR1 specific to phosphorylation cascades under early salt stress, whereas STN7 and BSL were specific to phosphorylation cascades under early drought stress. Only the calcium-binding EF-hand family protein and ZKT were clearly identified as signaling proteins that acted as cross-talk nodes for salt and drought signaling pathways. Our study provides new clues and insights for developing strategies to improve the tolerance of crops to complex, multiple environmental stresses.

Enhanced seed oil content by overexpressing genes related to triacylglyceride synthesis.

Oilseed rape (Brassica napus) is one of the most important oilseed crops globally. To meet increasing demand for oil-based products, the ability to enhance desirable oil content in the seed is required. This study assessed the capability of five genes in the triacylglyceride (TAG) synthesis pathway to enhance oil content. The genes BnGPDH, BnGPAT, BnDGAT, ScGPDH and ScLPAAT were overexpressed separately in a tobacco (Nicotiana benthamiana) model system, and simultaneously by pyramiding in B. napus, under the control of a seed specific Napin promoter. ScLPAAT transgenic plants showed a significant increase of 6.84% to 8.55% in oil content in tobacco seeds, while a ~4% increase was noted for BnGPDH and BnGPAT transgenic seeds. Seed-specific overexpression of all four genes in B. napus resulted in as high a 12.57% to 14.46% increased in seed oil content when compared to WT, equaling close to the sum of the single-gene overexpression increases in tobacco. Taken together, our study demonstrates that BnGPDH, BnGPAT and ScLPAAT may effectively increase seed oil content, and that simultaneous overexpression of these in transgenic B. napus may further enhance the desirable oil content relative to single-gene overexpressors.

Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF.

The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.