Experimental Study on the Localized Deformation and Damage Behavior of Polymer-Bonded Explosive Simulant under Cyclic Compression.

Uniaxial cyclic compression tests were performed to investigate the compression deformation and damage of polymer-bonded explosive (PBX) simulant, particularly shear localization. The macroscopic mechanical behavior and mesoscale failure mechanisms of the PBX simulant were analyzed by optical observation and SEM scanning methods. After each cyclic compression, the specimen was scanned by X-ray computed tomography (CT), and the internal 3D deformation of the specimen was calculated using the digital volume correlation (DVC) method. The results show that the stress-strain curve of the PBX simulant exhibits five stages and coincides with the morphological changes on the surface of the specimen. The mesoscale failure mechanism is dominated by particle interface debonding and binder tearing, accompanied by a small amount of particle breakage. There are three bifurcation points (T1, T2, and T3) in the curves of the normal and shear strain components with compression strain. It was found that these bifurcation points can reflect the full progression of the specimen from inconspicuous damage to uniformly distributed damage, shear localization, and eventual macroscopic fracture. The strain invariant I1 can quantitatively and completely characterize the deformation and damage processes of the PBX simulant under cyclic compression.

The tRNA thiolation-mediated translational control is essential for plant immunity.

Plants have evolved sophisticated mechanisms to regulate gene expression to activate immune responses against pathogen infections. However, how the translation system contributes to plant immunity is largely unknown. The evolutionarily conserved thiolation modification of transfer RNA (tRNA) ensures efficient decoding during translation. Here, we show that tRNA thiolation is required for plant immunity in Arabidopsis. We identify a cgb mutant that is hyper-susceptible to the pathogen Pseudomonas syringae. CGB encodes ROL5, a homolog of yeast NCS6 required for tRNA thiolation. ROL5 physically interacts with CTU2, a homolog of yeast NCS2. Mutations in either ROL5 or CTU2 result in loss of tRNA thiolation. Further analyses reveal that both transcriptome and proteome reprogramming during immune responses are compromised in cgb. Notably, the translation of salicylic acid receptor NPR1 is reduced in cgb, resulting in compromised salicylic acid signaling. Our study not only reveals a regulatory mechanism for plant immunity but also uncovers an additional biological function of tRNA thiolation.

Characterization and Potential Function Analysis of the SRS Gene Family in Brassica napus.

SRS (SHI-related sequence) transcription factors play a crucial role in plant growth, development, and abiotic stress response. Although Brassica napus (B. napus) is one of the most important oil crops in the world, the role of SRS genes in B. napus (BnSRS) has not been well investigated. Therefore, we employed a bioinformatics approach to identify BnSRS genes from genomic data and investigated their characteristics, functions, and expression patterns, to gain a better understanding of how this gene family is involved in plant development and growth. The results revealed that there were 34 BnSRS gene family members in the genomic sequence of B. napus, unevenly distributed throughout the sequence. Based on the phylogenetic analysis, these BnSRS genes could be divided into four subgroups, with each group sharing comparable conserved motifs and gene structure. Analysis of the upstream promoter region showed that BnSRS genes may regulate hormone responses, biotic and abiotic stress response, growth, and development in B. napus. The protein-protein interaction analysis revealed the involvement of BnSRS genes in various biological processes and metabolic pathways. Our analysis of BnSRS gene expression showed that 23 BnSRS genes in the callus tissue exhibited a dominant expression pattern, suggesting their critical involvement in cell dedifferentiation, cell division, and tissue development. In addition, association analysis between genotype and agronomic traits revealed that BnSRS genes may be linked to some important agronomic traits in B. napus, suggesting that BnSRS genes were widely involved in the regulation of important agronomic traits (including C16.0, C18.0, C18.1, C18.2 C18.3, C20.1, C22.1, GLU, protein, TSW, and FFT). In this study, we predicted the evolutionary relationships and potential functions of BnSRS gene family members, providing a basis for the development of BnSRS gene functions which could facilitate targeted functional studies and genetic improvement for elite breeding in B. napus.

Protein kinase ATR inhibits E3 ubiquitin ligase CRL4PRL1 to stabilize ribonucleotide reductase in response to replication stress.

The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4PRL1, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4PRL1 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.

The ATR-WEE1 kinase module promotes SUPPRESSOR OF GAMMA RESPONSE 1 translation to activate replication stress responses.

DNA replication stress threatens genome stability and is a hallmark of cancer in humans. The evolutionarily conserved kinases ATR (ATM and RAD3-related) and WEE1 are essential for the activation of replication stress responses. Translational control is an important mechanism that regulates gene expression, but its role in replication stress responses is largely unknown. Here we show that ATR-WEE1 control the translation of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a master transcription factor required for replication stress responses in Arabidopsis thaliana. Through genetic screening, we found that the loss of GENERAL CONTROL NONDEREPRESSIBLE 20 (GCN20) or GCN1, which function together to inhibit protein translation, suppressed the hypersensitivity of the atr or wee1 mutant to replication stress. Biochemically, WEE1 inhibits GCN20 by phosphorylating it; phosphorylated GCN20 is subsequently polyubiquitinated and degraded. Ribosome profiling experiments revealed that that loss of GCN20 enhanced the translation efficiency of SOG1, while overexpressing GCN20 had the opposite effect. The loss of SOG1 reduced the resistance of wee1 gcn20 to replication stress, whereas overexpressing SOG1 enhanced the resistance to atr or wee1 to replication stress. These results suggest that ATR-WEE1 inhibits GCN20-GCN1 activity to promote the translation of SOG1 during replication stress. These findings link translational control to replication stress responses in Arabidopsis.

Functional and evolutionary study of MLO gene family in the regulation of Sclerotinia stem rot resistance in Brassica napus L.

BACKGROUND: Oilseed rape (Brassica napus L.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungus Sclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance in B. napus is quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding in B. napus. RESULTS: Here, we performed a genome-wide association study (GWAS) using a natural population of B. napus consisting of 222 accessions to identify BnaA08g25340D (BnMLO2_2) as a candidate gene that regulates the SSR resistance. BnMLO2_2 was a member of seven homolog genes of Arabidopsis Mildew Locus O 2 (MLO2) and the significantly SNPs were mainly distributed in the promoter of BnMLO2_2, suggesting a role of BnMLO2_2 expression level in the regulation of SSR resistance. We expressed BnMLO2_2 in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues of B. napus revealed that BnMLO2_2 had the most expression level in leaf and silique tissues among all the 7 BnMLO2 members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis, mlo2 plants displayed reduced resistance to SSR, whereas overexpression of MLO2 conferred plants an enhanced SSR resistance. Moreover, a higher expression level of MLO2 showed a stronger SSR resistance in the transgenic plants. The regulation of MLO2 in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion of MLO family in Brassica crops. CONCLUSION: Our study revealed an important role of BnMLO2 in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance in B. napus and also new insights into understanding of MLO family evolution in Brassica crops.

A simple and highly efficient strategy to induce both paternal and maternal haploids through temperature manipulation.

Haploid production by outcrossing with inducers is one of the key technologies to revolutionize breeding. A promising approach for developing haploid inducers is by manipulating centromere-specific histone H3 (CENH3/CENPA)1. GFP-tailswap, a CENH3-based inducer, induces paternal haploids at around 30% and maternal haploids at around 5% (ref. 2). However, male sterility of GFP-tailswap makes high-demand maternal haploid induction more challenging. Our study describes a simple and highly effective method for improving both directions of haploid production. Lower temperatures dramatically enhance pollen vigour but reduce haploid induction efficiency, while higher temperatures act oppositely. Importantly, the effects of temperatures on pollen vigour and on haploid induction efficiency are independent. These features enable us to easily induce maternal haploids at around 24.8% by using pollen of inducers grown at lower temperatures to pollinate target plants, followed by switching to high temperatures for haploid induction. Moreover, paternal haploid induction can be simplified and enhanced by growing the inducer at higher temperatures pre- and post-pollination. Our findings provide new clues for developing and using CENH3-based haploid inducers in crops.

The multi-BRCT domain protein DDRM2 promotes the recruitment of RAD51 to DNA damage sites to facilitate homologous recombination.

DNA double-strand breaks (DSBs) are the most toxic form of DNA damage in cells. Homologous recombination (HR) is an error-free repair mechanism for DSBs as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. Through genetic screens for DNA damage response mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including bryophytes, the earliest land plant lineage. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. In support, genetic analysis suggests that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Mechanistically, DDRM2 interacts with the core HR protein RAD51 and is required for the recruitment of RAD51 to DSB sites. Our study reveals that SOG1-DDRM2-RAD51 is a novel module for HR, providing a potential target for improving the efficiency of gene targeting.

The SMC5/6 complex recruits the PAF1 complex to facilitate DNA double-strand break repair in Arabidopsis.

DNA double-strand breaks (DSBs) are one of the most toxic forms of DNA damage, which threatens genome stability. Homologous recombination is an error-free DSB repair pathway, in which the evolutionarily conserved SMC5/6 complex (SMC5/6) plays essential roles. The PAF1 complex (PAF1C) is well known to regulate transcription. Here we show that SMC5/6 recruits PAF1C to facilitate DSB repair in plants. In a genetic screen for DNA damage response mutants (DDRMs), we found that the Arabidopsis ddrm4 mutant is hypersensitive to DSB-inducing agents and is defective in homologous recombination. DDRM4 encodes PAF1, a core subunit of PAF1C. Further biochemical and genetic studies reveal that SMC5/6 recruits PAF1C to DSB sites, where PAF1C further recruits the E2 ubiquitin-conjugating enzymes UBC1/2, which interact with the E3 ubiquitin ligases HUB1/2 to mediate the monoubiquitination of histone H2B at DSBs. These results implicate SMC5/6-PAF1C-UBC1/2-HUB1/2 as a new axis for DSB repair through homologous recombination, revealing a new mechanism of SMC5/6 and uncovering a novel function of PAF1C.

APC/CCDC20 targets SCFFBL17 to activate replication stress responses in Arabidopsis.

DNA replication stress threatens genome stability and affects plant growth and development. How plants resolve replication stress is poorly understood. The protein kinase WEE1-mediated cell cycle arrest is required for replication stress responses. The E3 ubiquitin ligases anaphase-promoting complex/cyclosome (APC/C) and Skp1/Cullin 1/F-box (SCF) are essential regulators of the cell cycle. Here, we show that APC/CCDC20 mediates the degradation of SCFFBL17 during replication stress responses in Arabidopsis thaliana. Biochemically, WEE1 interacts with and phosphorylates the APC/C co-activator APC10, which enhances the interaction between F-BOX-LIKE17 (FBL17) and CELL DIVISION CYCLE 20 (CDC20), an activator of APC/C. Both APC10 and CDC20 are required for the polyubiquitination and degradation of FBL17. Genetically, silencing CDC20 or APC10 confers plant hypersensitivity to replication stress, which is suppressed by loss of FBL17. Collectively, our study suggests that WEE1 activates APC/C to inhibit FBL17, providing insight into replication stress responses in plants.

Salicylic acid inhibits gibberellin signaling through receptor interactions.

It is well known that plants activate defense responses at the cost of growth. However, the underlying molecular mechanisms are not well understood. The phytohormones salicylic acid (SA) and gibberellin (GA) promote defense response and growth, respectively. Here we show that SA inhibits GA signaling to repress plant growth. We found that the SA receptor NPR1 interacts with the GA receptor GID1. Further biochemical studies revealed that NPR1 functions as an adaptor of ubiquitin E3 ligase to promote the polyubiquitination and degradation of GID1, which enhances the stability of DELLA proteins, the negative regulators of GA signaling. Genetic analysis suggested that NPR1, GID1, and DELLA proteins are all required for the SA-mediated growth inhibition. Collectively, our study not only uncovers a novel regulatory mechanism of growth-defense trade-off but also reveals the interaction of hormone receptors as a new mode of hormonal crosstalk.

Recent advances in functional conservation and divergence of recombinase RAD51 and DMC1.

Meiosis is a specialized cell division that occurs in reproductive cells during sexual reproduction. It contains once DNA replication following nucleus division twice, thus producing haploid gametes. Fusion of male and female gametes restores genome to the diploid level, which not only ensures the genome stability between generations during sexual reproduction, but also leads to genetic diversity among offspring. Meiosis homologous recombination (HR) is one of the crucial events during meiotic prophase I, and it not only ensures the subsequently faithful segregation of homologous chromosomes (homologs), but also exchanges genetic information between homologs with greatly increasing the genetic diversity of progeny. RAD51 (RADiation sensitive 51) and DMC1 (disruption Meiotic cDNA 1) are essential recombinases for the HR process, and have certain commonalities and differences. In this review, we summarize and compare the conserved and differentiated features of RAD51 and DMC1 in terms of origin, evolution, structure, and function, we also provide an outlook on future research directions to further understand and study the molecular mechanisms in regulation of meiotic recombination.

A plant-specific module for homologous recombination repair.

Homologous recombination repair (HR) is an error-free DNA damage repair pathway to maintain genome stability and a basis of gene targeting using genome-editing tools. However, the mechanisms of HR in plants are still poorly understood. Through genetic screens for DNA damage response mutants (DDRM) in Arabidopsis, we find that a plant-specific ubiquitin E3 ligase DDRM1 is required for HR. DDRM1 contains an N-terminal BRCT (BRCA1 C-terminal) domain and a C-terminal RING (really interesting new gene) domain and is highly conserved in plants including mosses. The ddrm1 mutant is defective in HR and thus is hypersensitive to DNA-damaging reagents. Biochemical studies reveal that DDRM1 interacts with and ubiquitinates the transcription factor SOG1, a plant-specific master regulator of DNA damage responses. Interestingly, DDRM1-mediated ubiquitination promotes the stability of SOG1. Consistently, genetic data support that SOG1 functions downstream of DDRM1. Our study reveals that DDRM1-SOG1 is a plant-specific module for HR and highlights the importance of ubiquitination in HR.

The shikimate pathway regulates programmed cell death.

Programmed cell death (PCD) is essential for both plant development and stress responses including immunity. However, how plants control PCD is not well-understood. The shikimate pathway is one of the most important metabolic pathways in plants, but its relationship to PCD is unknown. Here, we show that the shikimate pathway promotes PCD in Arabidopsis. We identify a photoperiod-dependent lesion-mimic mutant named Lesion in short-day (lis), which forms spontaneous lesions in short-day conditions. Map-based cloning and whole-genome resequencing reveal that LIS encodes MEE32, a bifunctional enzyme in the shikimate pathway. Metabolic analysis shows that the level of shikimate is dramatically increased in lis. Through genetic screenings, three suppressors of lis (slis) are identified and the causal genes are cloned. SLISes encode proteins upstream of MEE32 in the shikimate pathway. Furthermore, exogenous shikimate treatment causes PCD. Our study uncovers a link between the shikimate pathway and PCD, and suggests that the accumulation of shikimate is an alternative explanation for the action of glyphosate, the most successful herbicide.

A Transcription Factor Regulates Gene Expression in Chloroplasts.

The chloroplast is a semi-autonomous organelle with its own genome. The expression of chloroplast genes depends on both chloroplasts and the nucleus. Although many nucleus-encoded proteins have been shown to localize in chloroplasts and are essential for chloroplast gene expression, it is not clear whether transcription factors can regulate gene expression in chloroplasts. Here we report that the transcription factor NAC102 localizes in both chloroplasts and nucleus in Arabidopsis. Specifically, NAC102 localizes in chloroplast nucleoids. Yeast two-hybrid assay and co-immunoprecipitation assay suggested that NAC102 interacts with chloroplast RNA polymerases. Furthermore, overexpression of NAC102 in chloroplasts leads to reduced chloroplast gene expression and chlorophyll content, indicating that NAC102 functions as a repressor in chloroplasts. Our study not only revealed that transcription factors are new regulators of chloroplast gene expression, but also discovered that transcription factors can function in chloroplasts in addition to the canonical organelle nucleus.

Loss of an ABC transporter in Arabidopsis thaliana confers hypersensitivity to the anti-cancer drug bleomycin.

Bleomycin (BLM) is used as an anti-cancer drug clinically. However, some cancer cells are resistant to BLM, which limits the usage of BLM in chemotherapy. But the underlying mechanism of such resistance is poorly understood. Here we show that the ATP binding cassette (ABC) transporter ABCC3 is required for the BLM-resistance in Arabidopsis. In a genetic screen for ddrm (DNA damage response mutants), we found that loss of ABCC3 confers the hypersensitivity to BLM. In contrast, overexpression of ABCC3 enhances the resistance to BLM. We further found that the expression of ABCC3 is induced by BLM, which is dependent on the protein kinase ATM and the transcription factor SOG1, two master regulators of DNA damage response. Our study revealed that the ABC transporter contributes to BLM-resistance, indicating that the combination of ABC transporter inhibitors and BLM may enhance the efficacy of BLM in cancer therapy.

RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis.

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination.

A novel WEE1 pathway for replication stress responses.

DNA replication stress poses a severe threat to genome stability and is a hallmark of cancer as well as a target for cancer therapy. It is well known that the evolutionarily conserved protein kinase WEE1 regulates replication stress responses by directly phosphorylating and inhibiting the major cell cycle driver CDKs in many organisms. Here, we report a novel WEE1 pathway. We found that Arabidopsis WEE1 directly interacts with and phosphorylates the E3 ubiquitin ligase FBL17 that promotes the degradation of CDK inhibitors. The phosphorylated FBL17 is further polyubiquitinated and degraded, thereby leading to the accumulation of CDK inhibitors and the inhibition of CDKs. In strong support for this model, either loss of function of FBL17 or overexpression of CDK inhibitors suppresses the hypersensitivity of the wee1 mutant to replication stress. Intriguingly, human WEE1 also phosphorylates and destabilizes the FBL17 equivalent protein SKP2, indicating that this is a conserved mechanism. This study reveals that the WEE1-FBL17/SKP2-CKIs-CDKs axis is a molecular framework for replication stress responses, which may have clinical implications because the WEE1 inhibitor AZD1775 is currently in phase II clinical trial as an anticancer drug.

The ATR-WEE1 kinase module inhibits the MAC complex to regulate replication stress response.

DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.