Phase separation underlies signaling activation of oncogenic NTRK fusions.

NTRK (neurotrophic tyrosine receptor kinase) gene fusions that encode chimeric proteins exhibiting constitutive activity of tropomyosin receptor kinases (TRK), are oncogenic drivers in multiple cancer types. However, the underlying mechanisms in oncogenesis that involve various N-terminal fusion partners of NTRK fusions remain elusive. Here, we show that NTRK fusion proteins form liquid-like condensates driven by their N-terminal fusion partners. The kinase reactions are accelerated in these condensates where the complexes for downstream signaling activation are also concentrated. Our work demonstrates that the phase separation driven by NTRK fusions is not only critical for TRK activation, but the condensates formed through phase separation serve as organizational hubs for oncogenic signaling.

Machine Learning for Myocardial Infarction Compared With Guideline-Recommended Diagnostic Pathways.

BACKGROUND: Collaboration for the Diagnosis and Evaluation of Acute Coronary Syndrome (CoDE-ACS) is a validated clinical decision support tool that uses machine learning with or without serial cardiac troponin measurements at a flexible time point to calculate the probability of myocardial infarction (MI). How CoDE-ACS performs at different time points for serial measurement and compares with guideline-recommended diagnostic pathways that rely on fixed thresholds and time points is uncertain. METHODS: Patients with possible MI without ST-segment-elevation were enrolled at 12 sites in 5 countries and underwent serial high-sensitivity cardiac troponin I concentration measurement at 0, 1, and 2 hours. Diagnostic performance of the CoDE-ACS model at each time point was determined for index type 1 MI and the effectiveness of previously validated low- and high-probability scores compared with guideline-recommended European Society of Cardiology (ESC) 0/1-hour, ESC 0/2-hour, and High-STEACS (High-Sensitivity Troponin in the Evaluation of Patients With Suspected Acute Coronary Syndrome) pathways. RESULTS: In total, 4105 patients (mean age, 61 years [interquartile range, 50-74]; 32% women) were included, among whom 575 (14%) had type 1 MI. At presentation, CoDE-ACS identified 56% of patients as low probability, with a negative predictive value and sensitivity of 99.7% (95% CI, 99.5%-99.9%) and 99.0% (98.6%-99.2%), ruling out more patients than the ESC 0-hour and High-STEACS (25% and 35%) pathways. Incorporating a second cardiac troponin measurement, CoDE-ACS identified 65% or 68% of patients as low probability at 1 or 2 hours, for an identical negative predictive value of 99.7% (99.5%-99.9%); 19% or 18% as high probability, with a positive predictive value of 64.9% (63.5%-66.4%) and 68.8% (67.3%-70.1%); and 16% or 14% as intermediate probability. In comparison, after serial measurements, the ESC 0/1-hour, ESC 0/2-hour, and High-STEACS pathways identified 49%, 53%, and 71% of patients as low risk, with a negative predictive value of 100% (99.9%-100%), 100% (99.9%-100%), and 99.7% (99.5%-99.8%); and 20%, 19%, or 29% as high risk, with a positive predictive value of 61.5% (60.0%-63.0%), 65.8% (64.3%-67.2%), and 48.3% (46.8%-49.8%), resulting in 31%, 28%, or 0%, who require further observation in the emergency department, respectively. CONCLUSIONS: CoDE-ACS performs consistently irrespective of the timing of serial cardiac troponin measurement, identifying more patients as low probability with comparable performance to guideline-recommended pathways for MI. Whether care guided by probabilities can improve the early diagnosis of MI requires prospective evaluation. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00470587.

Multiparameter Assessment of Foam Cell Formation Progression Using a Dual-Color Switchable Fluorescence Probe.

The assessment of atherosclerosis (AS) progression has emerged as a prominent area of research. Monitoring various pathological features of foam cell (FC) formation is imperative to comprehensively assess AS progression. Herein, a simple benzospiropyran-julolidine-based probe, BSJD, with switchable dual-color imaging ability was developed. This probe can dynamically and reversibly adjust its molecular structure and fluorescent properties in different polar and pH environments. Such a polarity and pH dual-responsive characteristic makes it superior to single-responsive probes in dual-color imaging of lipid droplets (LDs) and lysosomes as well as monitoring their interaction. By simultaneously tracking various pathological features, including LD accumulation and size changes, lysosome dysfunction, and dynamically regulated lipophagy, more comprehensive information can be obtained for multiparameter assessment of FC formation progression. Using BSJD, not only the activation of lipophagy in the early stages and inhibition in the later phases during FC formation are clearly observed but also the important roles of lipophagy in regulating lipid metabolism and alleviating FC formation are demonstrated. Furthermore, BSJD is demonstrated to be capable of rapidly imaging FC plaque sites in AS mice with fast pharmacokinetics. Altogether, BSJD holds great promise as a dual-color organelle-imaging tool for investigating disease-related LD and lysosome changes and their interactions.

Receptor-like kinases and their signaling cascades for plant male fertility: loyal messengers.

Receptor-like kinases (RLKs) are evolved for plant cell-cell communications. The typical RLK protein contains an extracellular and hypervariable N-terminus to perceive various signals, a transmembrane domain to anchor into plasma membrane, and a cytoplasmic, highly conserved kinase domain to phosphorylate target proteins. To date, RLKs have manifested their significance in a myriad of biological processes during plant reproductive growth, especially in male fertility. This review first summarizes a recent update on RLKs and their interacting protein partners controlling anther and pollen development, pollen release from dehisced anther, and pollen function during pollination and fertilization. Then, regulatory networks of RLK signaling pathways are proposed. In addition, we predict RLKs in maize and rice genome, obtain homologs of well-studied RLKs from phylogeny of three subfamilies and then analyze their expression patterns in developing anthers of maize and rice to excavate potential RLKs regulating male fertility in crops. Finally, current challenges and future prospects regarding RLKs are discussed. This review will contribute to a better understanding of plant male fertility control by RLKs, creating potential male sterile lines, and inspiring innovative crop breeding methods.

Targeting androgen receptor phase separation to overcome antiandrogen resistance.

Patients with castration-resistant prostate cancer inevitably acquire resistance to antiandrogen therapies in part because of androgen receptor (AR) mutations or splice variants enabling restored AR signaling. Here we show that ligand-activated AR can form transcriptionally active condensates. Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role. AR liquid-liquid phase separation behaviors faithfully report transcriptional activity and antiandrogen efficacy. Antiandrogens can promote phase separation and transcriptional activity of AR-resistant mutants in a ligand-independent manner. We conducted a phase-separation-based phenotypic screen and identified ET516 that specifically disrupts AR condensates, effectively suppresses AR transcriptional activity and inhibits the proliferation and tumor growth of prostate cancer cells expressing AR-resistant mutants. Our results demonstrate liquid-liquid phase separation as an emerging mechanism underlying drug resistance and show that targeting phase separation may provide a feasible approach for drug discovery.

A Multimodal Omics Framework to Empower Target Discovery for Cardiovascular Regeneration.

Ischaemic heart disease is a global healthcare challenge with high morbidity and mortality. Early revascularisation in acute myocardial infarction has improved survival; however, limited regenerative capacity and microvascular dysfunction often lead to impaired function and the development of heart failure. New mechanistic insights are required to identify robust targets for the development of novel strategies to promote regeneration. Single-cell RNA sequencing (scRNA-seq) has enabled profiling and analysis of the transcriptomes of individual cells at high resolution. Applications of scRNA-seq have generated single-cell atlases for multiple species, revealed distinct cellular compositions for different regions of the heart, and defined multiple mechanisms involved in myocardial injury-induced regeneration. In this review, we summarise findings from studies of healthy and injured hearts in multiple species and spanning different developmental stages. Based on this transformative technology, we propose a multi-species, multi-omics, meta-analysis framework to drive the discovery of new targets to promote cardiovascular regeneration.

LncRNA CTBP1-DT-encoded microprotein DDUP sustains DNA damage response signalling to trigger dual DNA repair mechanisms.

Sustaining DNA damage response (DDR) signalling via retention of DDR factors at damaged sites is important for transmitting damage-sensing and repair signals. Herein, we found that DNA damage provoked the association of ribosomes with IRES region in lncRNA CTBP1-DT, which overcame the negative effect of upstream open reading frames (uORFs), and elicited the novel microprotein DNA damage-upregulated protein (DDUP) translation via a cap-independent translation mechanism. Activated ATR kinase-mediated phosphorylation of DDUP induced a drastic 'dense-to-loose' conformational change, which sustained the RAD18/RAD51C and RAD18/PCNA complex at damaged sites and initiated RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair mechanisms. Importantly, treatment with ATR inhibitor abolished the effect of DDUP on chromatin retention of RAD51C and PCNA, thereby leading to hypersensitivity of cancer cells to DNA-damaging chemotherapeutics. Taken together, our results uncover a plausible mechanism underlying the DDR sustaining and might represent an attractive therapeutic strategy in improvement of DNA damage-based anticancer therapies.

The role of the diencephalon in the guidance of thalamocortical axons in mice.

Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.

Thermally activated delayed fluorescence of a Ir(III) complex: absorption and emission properties, nonradiative rates, and mechanism.

One recent experimental study reported a Ir(III) complex with thermally activated delayed fluorescence (TADF) phenomenon in solution, but its luminescent mechanism is elusive. In this work, we combined density functional theory (DFT), time-dependent DFT (TDDFT) and multi-state complete active space second-order perturbation theory (MS-CASPT2) methods to investigate excited-state properties, photophysics, and emission mechanism of this Ir(III) complex. Two main absorption bands observed in experiments can be attributed to the electronic transition from the S0 state to the S1 and S2 states; while, the fluorescence and phosphorescence are generated from the S1 and T1 states, respectively. Both the S1 and T1 states have clear metal-to-ligand charge transfer (MLCT) character. The present computational results reveal a three-state model including the S0, S1 and T1 states to rationalize the TADF behavior. The small energy gap between the S1 and T1 states benefits the forward and reverse intersystem crossing (ISC and rISC) processes. At 300 K, the rISC rate is five orders of magnitude larger than the phosphorescence rate therefore enabling TADF. At 77 K, the rISC rate is sharply decreased but remains close to the phosphorescence rate; therefore, in addition to the phosphorescence, the delayed fluorescence could also contribute to the experimental emission. The estimated TADF lifetime agrees well with experiments, 9.80 vs. 6.67 µs, which further verifies this three-state model.

Molecular regulation of ZmMs7 required for maize male fertility and development of a dominant male-sterility system in multiple species.

Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and female fertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops.

A Systematic Investigation of Lipid Transfer Proteins Involved in Male Fertility and Other Biological Processes in Maize.

Plant lipid transfer proteins (LTPs) play essential roles in various biological processes, including anther and pollen development, vegetative organ development, seed development and germination, and stress response, but the research progress varies greatly among Arabidopsis, rice and maize. Here, we presented a preliminary introduction and characterization of the whole 65 LTP genes in maize, and performed a phylogenetic tree and gene ontology analysis of the LTP family members in maize. We compared the research progresses of the reported LTP genes involved in male fertility and other biological processes in Arabidopsis and rice, and thus provided some implications for their maize orthologs, which will provide useful clues for the investigation of LTP transporters in maize. We predicted the functions of LTP genes based on bioinformatic analyses of their spatiotemporal expression patterns by using RNA-seq and qRT-PCR assays. Finally, we discussed the advances and challenges in substrate identification of plant LTPs, and presented the future research directions of LTPs in plants. This study provides a basic framework for functional research and the potential application of LTPs in multiple plants, especially for male sterility research and application in maize.

The OsEIL1-OsERF115-target gene regulatory module controls grain size and weight in rice.

Grain size is one of the essential determinants of rice yield. Our previous studies revealed that ethylene plays an important role in grain-size control; however, the precise mechanism remains to be determined. Here, we report that the ethylene response factor OsERF115 functions as a key downstream regulator for ethylene-mediated grain development. OsERF115 encodes an AP2/ERF-type transcriptional factor that is specifically expressed in young spikelets and developing caryopses. Overexpression of OsERF115 significantly increases grain length, width, thickness and weight by promoting longitudinal elongation and transverse division of spikelet hull cells, as well as enhancing grain-filling activity, whereas its knockout mutations lead to the opposite effects, suggesting that OsERF115 positively regulates grain size and weight. OsERF115 transcription is strongly induced by ethylene, and OsEIL1 directly binds to the promoter to activate its expression. OsERF115 acts as a transcriptional repressor to directly or indirectly modulate a set of grain-size genes during spikelet growth and endosperm development. Importantly, haplotype analysis reveals that the SNP variations in the EIN3-binding sites of OsERF115 promoter are significantly associated with the OsERF115 expression levels and grain weight, suggesting that natural variations in the OsERF115 promoter contribute to grain-size diversity. In addition, the OsERF115 orthologues are identified only in grass species, implying a conserved and unique role in the grain development of cereal crops. Our results provide insights into the molecular mechanism of ethylene-mediated grain-size control and a potential strategy based on the OsEIL1-OsERF115-target gene regulatory module for genetic improvement of rice yield.

Thermally Activated Delayed Fluorescence Mechanism of a Bicyclic "Carbene-Metal-Amide" Copper Compound: DFT/MRCI Studies and Roles of Excited-State Structure Relaxation.

Herein we investigated the luminescence mechanism of one "carbene-metal-amide" copper compound with thermally activated delayed fluorescence (TADF) using density functional theory (DFT)/multireference configuration interaction, DFT, and time-dependent DFT methods with the polarizable continuum model. The experimentally observed low-energy absorption and emission peaks are assigned to the S1 state, which exhibits clear interligand and partial ligand-to-metal charge-transfer character. Moreover, it was found that a three-state (S0, S1, and T1) model is sufficient to describe the TADF mechanism, and the T2 state should play a negligible role. The calculated S1-T1 energy gap of 0.10 eV and proper spin-orbit couplings facilitate the reverse intersystem crossing (rISC) from T1 to S1. At 298 K, the rISC rate of T1 → S1 (∼106 s-1) is more than 3 orders of magnitude larger than the T1 phosphorescence rate (∼103 s-1), thereby enabling TADF. However, it disappears at 77 K because of a very slow rISC rate (∼101 s-1). The calculated TADF rate, lifetime, and quantum yield agree very well with the experimental data. Methodologically, the present work shows that only considering excited-state information at the Franck-Condon point is insufficient for certain emitting systems and including excited-state structure relaxation is important.

Combined QM (MS-CASPT2)/MM studies on photocyclization and photoisomerization of a fulgide derivative in toluene solution.

Photocyclization and photoisomerization of fulgides have been extensively studied experimentally and computationally due to their significant potential applications for example as photoswitches in memory devices. However, the reported excited-state decay mechanisms of fulgides do not include the effects of solvation explicitly to date. Herein, calculations using the high-level MS-CASPT2//CASSCF method were conducted to explore the photoinduced excited-state decay processes of the Eα conformer of a fulgide derivative in toluene with solvent effects treated by implicit PCM and explicit QM/MM models, respectively. Several minima and conical intersections were optimized successfully in and between the S0 and S1 states; then, two nonadiabatic excited-state decay channels that could efficiently drive the system to the ground state were proposed based on the excited-state ring-closure and isomerization paths. In addition, we also found that in the ring-closure path, the potential energy surface is essentially barrierless before approaching the conical intersection, while it needs to overcome a small energy barrier along the E → Z photoisomerization path for the nonadiabatic S1 → S0 internal conversion process. The present computational results could provide useful mechanistic insights into the photoinduced cyclization and isomerization reactions of fulgide and its derivatives.

Prediction of microvascular obstruction by coronary artery angiography score after acute ST-segment elevation myocardial infarction: a single-center retrospective observational study.

BACKGROUND: Some coronary artery angiography (CAG) scores are associated with the no-reflow phenomenon after percutaneous coronary intervention (PCI) in patients with acute ST-segment elevation myocardial infarction (STEMI). However, quality evidence regarding the association between the CAG scores and microvascular injury is still needed. Our study aimed to validate the ability of the CAG scores in predicting microvascular obstruction (MVO) detected by cardiac magnetic resonance (CMR) imaging. METHODS: From October 2020 to October 2021, 141 consecutive patients with acute STEMI who underwent primary PCI and CMR were retrospectively reviewed. CMR imaging was performed between 3 and 7 days after PCI. The patients were divided into MVO and non-MVO group based on the CMR results. Three CAG scores (SYNTAX score, SYNTAX II score and Gensini score) were used to assess the severity of coronary artery atherosclerotic burden. RESULTS: A total of 122 patients were included (mean age 60.6 ± 12.8 years). MVO occurred in 51 patients (41.8%). Patients with MVO had higher SYNTAX scores, SYNTAX II scores and Gensini scores than those without MVO (all p < 0.001). The Gensini score (r = 0.567, p < 0.001) showed the strongest correlation with infarction size than SYNTAX score (r = 0.521, p < 0.001) and SYNTAX II score (r = 0.509, p < 0.001). The areas under the receiver operator characteristic curves of SYNTAX score, SYNTAX II score and Gensini score for predicting MVO patients were 0.726, 0.774 and 0.807. In multivariable regression analysis, peak troponin I (odd ratio [OR] = 1.236, p = 0.001) and SYNTAX II score (OR = 11.636, p = 0.010) were identified as independent predictors of MVO. CONCLUSIONS: In patients with acute STEMI undergoing primary PCI treatment, the peak troponin I and SYNTAX II score may be an independent predictor of MVO.

Transposable elements drive rapid phenotypic variation in Capsella rubella.

Rapid phenotypic changes in traits of adaptive significance are crucial for organisms to thrive in changing environments. How such phenotypic variation is achieved rapidly, despite limited genetic variation in species that experience a genetic bottleneck is unknown. Capsella rubella, an annual and inbreeding forb (Brassicaceae), is a great system for studying this basic question. Its distribution is wider than those of its congeneric species, despite an extreme genetic bottleneck event that severely diminished its genetic variation. Here, we demonstrate that transposable elements (TEs) are an important source of genetic variation that could account for its high phenotypic diversity. TEs are (i) highly enriched in C. rubella compared with its outcrossing sister species Capsella grandiflora, and (ii) 4.2% of polymorphic TEs in C. rubella are associated with variation in the expression levels of their adjacent genes. Furthermore, we show that frequent TE insertions at FLOWERING LOCUS C (FLC) in natural populations of C. rubella could explain 12.5% of the natural variation in flowering time, a key life history trait correlated with fitness and adaptation. In particular, we show that a recent TE insertion at the 3' UTR of FLC affects mRNA stability, which results in reducing its steady-state expression levels, to promote the onset of flowering. Our results highlight that TE insertions can drive rapid phenotypic variation, which could potentially help with adaptation to changing environments in a species with limited standing genetic variation.

ATP-Binding Cassette G Transporters and Their Multiple Roles Especially for Male Fertility in Arabidopsis, Rice and Maize.

ATP-binding cassette subfamily G (ABCG) transporters are extensive in plants and play essential roles in various processes influencing plant fitness, but the research progress varies greatly among Arabidopsis, rice and maize. In this review, we present a consolidated nomenclature and characterization of the whole 51 ABCG transporters in maize, perform a phylogenetic analysis and classification of the ABCG subfamily members in maize, and summarize the latest research advances in ABCG transporters for these three plant species. ABCG transporters are involved in diverse processes in Arabidopsis and rice, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport, which provide useful clues for the functional investigation of ABCG transporters in maize. Finally, we discuss the current challenges and future perspectives for the identification and mechanism analysis of substrates for plant ABCG transporters. This review provides a basic framework for functional research and the potential application of ABCG transporters in multiple plants, including maize.

Circular RNA circIKBKB promotes breast cancer bone metastasis through sustaining NF-κB/bone remodeling factors signaling.

BACKGROUND: Breast cancer (BC) has a marked tendency to spread to the bone, resulting in significant skeletal complications and mortality. Recently, circular RNAs (circRNAs) have been reported to contribute to cancer initiation and progression. However, the function and mechanism of circRNAs in BC bone metastasis (BC-BM) remain largely unknown. METHODS: Bone-metastatic circRNAs were screened using circRNAs deep sequencing and validated using in situ hybridization in BC tissues with or without bone metastasis. The role of circIKBKB in inducing bone pre-metastatic niche formation and bone metastasis was determined using osteoclastogenesis, immunofluorescence and bone resorption pit assays. The mechanism underlying circIKBKB-mediated activation of NF-κB/bone remodeling factors signaling and EIF4A3-induced circIKBKB were investigated using RNA pull-down, luciferase reporter, chromatin isolation by RNA purification and enzyme-linked immunosorbent assays. RESULTS: We identified that a novel circRNA, circIKBKB, was upregulated significantly in bone-metastatic BC tissues. Overexpressing circIKBKB enhanced the capability of BC cells to induce formation of bone pre-metastatic niche dramatically by promoting osteoclastogenesis in vivo and in vitro. Mechanically, circIKBKB activated NF-κB pathway via promoting IKKß-mediated IκBα phosphorylation, inhibiting IκBα feedback loop and facilitating NF-κB to the promoters of multiple bone remodeling factors. Moreover, EIF4A3, acted acting as a pre-mRNA splicing factor, promoted cyclization of circIKBKB by directly binding to the circIKBKB flanking region. Importantly, treatment with inhibitor eIF4A3-IN-2 reduced circIKBKB expression and inhibited breast cancer bone metastasis effectively. CONCLUSION: We revealed a plausible mechanism for circIKBKB-mediated NF-κB hyperactivation in bone-metastatic BC, which might represent a potential strategy to treat breast cancer bone metastasis.

CRISPR/Cas9-based discovery of maize transcription factors regulating male sterility and their functional conservation in plants.

Identifying genic male-sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with Arabidopsis and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9-mediated gene mutagenesis, and then, 12 genes in eight types, including ZmbHLH51, ZmbHLH122, ZmTGA9-1/-2/-3, ZmTGA10, ZmMYB84, ZmMYB33-1/-2, ZmPHD11 and ZmLBD10/27, were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably, ZmTGA9-1/-2/-3 triple-gene mutants and ZmMYB33-1/-2 double-gene mutants displayed complete male sterility, but their double- or single-gene mutants showed male fertility. Similarly, ZmLBD10/27 double-gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition, ZmbHLH51 was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co-segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14-type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and Arabidopsis. These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male-sterility lines in maize.

ZmMs25 encoding a plastid-localized fatty acyl reductase is critical for anther and pollen development in maize.

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.