Genome-wide identification and comparative gene family analyses have been commonly performed to investigate species-specific evolution linked to various traits and molecular pathways. However, most previous studies were limited to gene screening in a single reference genome, failing to account for the gene presence/absence variations (gPAVs) in a species. Here, we propose an innovative pangenome-based approach of gene family analyses based on orthologous gene groups (OGGs). Using the basic helix-loop-helix (bHLH) transcription factor family in barley as an example, we identified 161 ∼ 176 bHLHs in 20 barley genomes, which could be classified into 201 OGGs. These 201 OGGs were further classified into 140 core, 12 soft-core, 29 shell, and 20 line-specific/cloud bHLHs, revealing a complete profile of bHLH in barley. Using a genome-scan approach, we overcome the genome annotation bias and identified on average 1.5 un-annotated core bHLHs per barley genome. We found that all core bHLHs belong to whole genome/segmental duplicates whilst dispensable bHLHs were more likely to result from small scale duplication events. Interestingly, we noticed that the dispensable bHLHs tended to enrich in specific subfamilies SF13, SF27, and SF28, implying the potential biased expansion of specific bHLHs in barley. We found that 50% of the bHLHs contain at least one intact transposon element within the 2kb upstream-to-downstream region. bHLHs with CNV have 1.48 TEs on average, significantly higher than 1.36 for core bHLH without CNV, supporting TEs' potential role in bHLH expansion. Selection pressure analyses showed that dispensable bHLHs had experienced clear relaxed selection compared to core bHLHs, consistent with their conservation patterns. We further integrate pangenome with recently available barley pantranscriptome data in 5 tissues and discovered apparent transcriptional divergence within and across bHLH subfamilies. We conclude that pangenome-based gene family analyses can better describe the genuine evolution status of bHLHs untapped before and provided novel insights into bHLH evolution in barley. We expect this study will inspire similar analyses in many other gene families and species.

The unfolded protein response (UPR) is a vital cellular pathway that maintains endoplasmic reticulum (ER) homeostasis under conditions of ER stress, which is associated with the degradation of misfolded proteins. However, the role of ER-associated degradation in plant-microbe interactions has yet to be explored. In this study, we identified a novel viral protein βV1, encoded by tomato yellow leaf curl betasatellite (TYLCCNB), is an ER-localized protein that triggers ER aggregation. Transient expression of βV1 in Nicotiana benthamiana induces robust ER stress and activates the bZIP17/28 branch of the UPR signaling pathway. The induction of bZIP17/28 by βV1 is crucial for successful virus infection. Furthermore, we demonstrate that βV1 is unstable in N. benthamiana mesophyll cells, as it is targeted for autophagic degradation. The autophagy-related protein ATG18a, a key component of autophagosomes, participates in the degradation of βV1, thereby exerting an anti-viral role. Taken together, our results unveil a novel function of the βV1 protein and provide the first evidence of the involvement of bZIP17/28 and ATG18a in ER-associated autophagic degradation during geminivirus infection. These findings significantly expand our comprehension of the arms race dynamics between plants and viruses.

Heterosis is extensively utilized in the two-line hybrid breeding system. Photo-/thermo-sensitive genic male sterile (P/TGMS) lines are key components of two-line hybrid rice. TGMS lines containing tms5 have significantly advanced two-line hybrid rice breeding. We cloned the TMS5 gene and found that TMS5 is a tRNA cyclic phosphatase that can remove 2',3'-cyclic phosphate (cP) from cP-ΔCCA-tRNAs for efficient repair to ensure maintenance of mature tRNA levels. tms5 mutation causes increased cP-ΔCCA-tRNAs and reduced mature tRNAs, leading to male sterility at the restrictive temperatures. However, the regulatory network of tms5-mediated TGMS remains to be elucidated. Here, we identified that an E3 ligase OsHel2 cooperates with TMS5 to regulate TGMS at the restrictive temperatures. Consistently, both the accumulation of 2',3'-cP-ΔCCA-tRNAs and insufficiency of mature tRNAs in tms5 mutant were largely recovered in the tms5 oshel2-1 mutant. A lesion in OsHel2 results in partial readthrough of the stalled sequences, thereby evading ribosome-associated protein quality control (RQC) surveillance. Our findings reveal a mechanism by which the OsHel2 impede readthrough of stalled mRNA sequences to regulate male fertility in TGMS rice, thus providing a paradigm for investigating how disorders in the components of the RQC pathway impair cellular functions and lead to diseases or defects in other organisms.

The introduction of Reduced height (Rht) genes into wheat varieties results in semidwarf plant architecture with largely improved lodging resistance and harvest indices. Therefore, the exploration of new Rht gene resources to breed semidwarf wheat cultivars has been a major strategy for guaranteeing high and stable grain yields of wheat since the 1960s. In this study, we report the map-based cloning of TaERF-A1, which encodes an AP2/ERF transcription factor and acts as a positive regulator of wheat stem elongation, as a new gene for regulating plant height and spike length. The natural variant TaERF-A1, characterized by a substitution from Phe (derived from Nongda3338) to Ser (derived from Jingdong6) at position 178, significantly weakened the stability of the TaERF-A1 protein. As a result, this substitution led to partly attenuated transcriptional activation of TaERF-A1-targeted downstream genes, including TaPIF4, resulting in the restriction of stem and spike elongation. Importantly, introgression of the semidwarfing-related allele TaERF-A1 in wheat materials significantly enhanced lodging resistance, especially in dense cropping systems. Therefore, our study reveals TaERF-A1 as a new Rht gene resource for breeding semidwarf wheat varieties with increased yield stability.

Plant diseases, caused by a wide range of pathogens, severely reduce crop yield, quality and pose a threat to global food security. Developing broad-spectrum resistance (BSR) in crops is a key strategy to control crop diseases and safeguard crop production. Cloning of disease-resistance (R) genes and understanding their underlying molecular mechanisms provides new genetic resources and strategies for crop breeding. Novel genetic engineering and genome editing tools have accelerated the study of BSR genes and engineering of BSR in crops, and this area represents the primary focus of this review. We first summarize recent advances in the understanding of the plant immune system. We then examine progress in understanding molecular mechanisms underlying BSR in crops. Finally, we highlight diverse strategies employed to achieve BSR, such as gene stacking to combine multiple R genes, multiplexed genome editing of susceptibility (S) genes and promoters of executor R genes, editing cis-regulatory elements for fine-tuning gene expression, RNA interference, saturation mutagenesis, and precise genomic insertions. Genetic studies and engineering of BSR accelerate breeding of disease-resistant cultivars and crop improvement, which will act to safeguard global food security.

Current methods used in genome-wide association studies frequently lack power due to their inability to detect heterogeneous associations and rare and multiallelic variants. To address these issues, quantile regression was integrated for the first time with a compressed variance component multi-locus random-SNP-effect mixed linear model (3VmrMLM) to propose q3VmrMLM for detecting heterogeneous quantitative trait nucleotides (QTNs) and QTN-by-environment interactions (QEIs), while q3VmrMLM-Hap was designed to identify multiallelic haplotypes and rare variants. In Monte Carlo simulation studies, q3VmrMLM had higher power than 3VmrMLM, SKAT, and iQRAT. In the re-analysis of 10 traits in 1439 rice hybrids, 261 known genes were identified only by q3VmrMLM and q3VmrMLM-Hap, while 175 known genes were detected commonly by the new and existing methods. Of all the significant QTNs with known genes, q3VmrMLM (179: 140 variance heterogeneity and 157 quantile effect heterogeneity) found more heterogeneous QTNs than 3VmrMLM (123), SKAT (27) and iQRAT (29), q3VmrMLM-Hap (121) mapped more low-frequency (<0.05) QTNs than q3VmrMLM (51), 3VmrMLM (43), SKAT (11) and iQRAT (12), and q3VmrMLM-Hap (12), q3VmrMLM (16) and 3VmrMLM (12) had similar power in identifying gene-by-environment interactions. All significant and suggested QTNs achieved the highest predictive accuracy (r=0.9045). In conclusion, this study provides a new and complementary approach to mining genes and unrevealing the genetic architecture of complex traits in crops.

Brassinosteroids (BRs) are steroidal phytohormones indispensable for plant growth, development, and responses to environmental stresses. The export of bioactive BRs to the apoplast is essential for BR signalling initiation, which requires binding of BR molecule to the extracellular domains of the plasma membrane-localized receptor complex. We have previously shown that the Arabidopsis thaliana ATP-binding cassette (ABC) transporter, ABCB19, functions as a BR exporter, and together with its close homologue, ABCB1, positively regulate BR signalling. Here, we demonstrate that ABCB1 is another BR transporter. The ATP hydrolysis activity of ABCB1 was stimulated by bioactive BRs, and its transport activity was confirmed in proteoliposomes and protoplasts. Structures of ABCB1 in substrate-unbound (apo), brassinolide (BL)-bound, and ATP plus BL-bound states were determined. In the BL-bound structure, BL was bound to the hydrophobic cavity formed by the transmembrane domain, and triggered local conformational changes. Together, our data provide additional insights into the ABC transporter-mediated BR export.

Hybrid breeding is widely acknowledged as the most effective method for increasing crop yield, particularly in maize and rice. However, a major challenge in hybrid breeding is selecting desirable combinations from a vast pool of potential crosses. Genomic selection (GS) has emerged as a powerful tool to tackle this challenge, but its success in practical breeding depends on prediction accuracy. Several strategies have been explored to enhance the prediction accuracy for complex traits, such as incorporating functional markers and multi-omics data. Metabolome-wide association studies (MWAS) help identify metabolites closely linked to phenotype, known as metabolic markers. However, the use of preselected metabolic markers from parental lines to predict hybrid performance has not yet been explored. In this study, we developed a novel approach called metabolic marker-assisted genomic prediction (MM_GP) that incorporates significant metabolites identified from MWAS into GS models to improve the accuracy of genomic hybrid prediction. In maize and rice hybrid populations, MM_GP outperformed GP for all traits, regardless of the methods used (GBLUP or XGBoost). On average, MM_GP yielded 4.6% and 13.6% higher predictive abilities compared to GP in maize and rice, respectively. Additionally, MM_GP could match or even surpass the predictive ability of M_GP (integrated genomic-metabolomic prediction) for most traits. Notably, integrating only six metabolic markers significantly related to multiple traits resulted in a 5.0% and 3.1% higher average predictive ability than GP and M_GP in maize, respectively. With the advancement of high-throughput metabolomics technologies and prediction models, this approach holds great promise to revolutionize genomic hybrid breeding by enhancing its accuracy and efficiency.

For optimum photosynthetic productivity it is crucial for plants to swiftly transition between light harvesting and photoprotective states as light conditions change in the field. The PsbS protein plays a pivotal role in this process by switching the light harvesting antenna, LHCII, into the photoprotective state, qE, to avoid photoinhibition in high light environment. However, the molecular mechanism of PsbS action upon LHCII have remained unclear. In our study, we identified its specific aminoacid domains that are essential for the function. Using the aminoacid point-mutagenesis of PsbS in vivo we found that the activation of photoprotection involves dynamic changes in the oligomeric state and conformation of PsbS, with two residues, E67 and E173, playing a key role in this process. Further, the replacement of hydrophobic phenylalanine residues in transmembrane helixes II (F83, F84, F87) and IV (F191, F193, F194) with tyrosine revealed that phenylalanine localised in helix IV could play a significant role in hydrophobic interactions of PsbS with LHCII. The removal of the 3 helix (H3) aminoacids I74, Y75, E76 did not affect the amplitude but resulted in a strongly delayed recovery of qE in darkness. These findings provide new insights into the molecular architecture of PsbS that are essential for regulating light harvesting in higher plants. Moreover, the combination of experimental mutagenesis with AI-assisted protein folding evolutionary scale model approach (ESMFold) opens new avenues for intelligently manipulating protein functions in silico to streamline and evaluate the experimental point mutagenesis strategies.

The dynamic shuttling of proteins between the nucleus and cytoplasm orchestrates vital functions in eukaryotes. Here, we unveil multifaceted functions of Arabidopsis Sin3-associated protein 18 kDa (SAP18) in regulating development and heat stress tolerance. Proteomic analysis demonstrated that SAP18 is a core component of the nuclear Apoptosis- and Splicing-Associated Protein (ASAP) complex in Arabidopsis, contributing to the precise splicing of genes associated with leaf development. Genetic analysis further confirmed SAP18's critical role in different developmental processes as part of the ASAP complex, including leaf morphogenesis and flowering time. Interestingly, upon heat shock SAP18 translocates from the nucleus to cytoplasmic stress granules and processing bodies. The heat-sensitive phenotype of SAP18 loss-of-function mutant revealed its novel role in plant thermoprotection. Our findings significantly expand our understanding of SAP18 relevance for plant growth, linking nuclear splicing with cytoplasmic stress responses, and providing new perspectives for future exploration of plant thermotolerance mechanisms.

Grain size, which encompasses grain length, width, and thickness, is a critical determinant of both grain weight and quality in rice. Despite the extensive regulatory networks known to determine grain length and width, the pathway(s) that regulate grain thickness remain to be clarified. Here, we present the map-based cloning and characterization of qGT3, a major quantitative trait locus for grain thickness in rice that encodes the MADS-domain transcription factor OsMADS1. Our findings demonstrate that OsMADS1 regulates grain thickness by affecting sugar delivery during grain filling, and we show that OsMADS1 modulates expression of the downstream monosaccharide transporter gene MST4. A natural variant leads to alternative splicing and thus to a truncated OsMADS1 protein with attenuated transcriptional repressor activity. The truncated OsMADS1 protein results in increased expression of MST4, leading to enhanced loading of monosaccharides into the developing endosperm and thereby increasing grain thickness and improving grain quality. In addition, our results reveal that NF-YB1 and NF-YC12 interact directly with OsMADS1, acting as cofactors to enhance its transcriptional activity toward MST4. Collectively, these findings reveal a novel molecular mechanism underlying grain thickness regulation that is controlled by the OsMADS1-NF-YB1-YC12 complex and has great potential for synergistic improvement of grain yield and quality in rice.

Increasing soil salinization has led to severe losses of plant yield and quality. Thus, it is urgent to investigate the molecular mechanism of the salt stress response. In this study, we took systematically analyzed cotton root response to salt stress by single-cell transcriptomics technology; 56,281 high-quality cells were totally obtained from 5-days-old lateral root tips of Gossypium arboreum under natural growth and different salt-treatment conditions. Ten cell types with an array of novel marker genes were synthetically identified and confirmed with in situ RNA hybridization, and some specific-type cells of pesudotime analysis also pointed out their potential differentiation trajectory. The prominent changes of cell numbers responding to salt stress were observed on outer epidermal and inner endodermic cells, which were significantly enriched in response to stress, amide biosynthetic process, glutathione metabolism, and glycolysis/gluconeogenesis. Other functional aggregations were concentrated on plant-type primary cell wall biogenesis, defense response, phenylpropanoid biosynthesis and metabolic pathways by analyzing the abundant differentially expressed genes (DEGs) identified from multiple comparisons. Some candidate DEGs related with transcription factors and plant hormones responding to salt stress were also identified, of which the function of Ga03G2153, an annotated auxin-responsive GH3.6, was confirmed by using virus-induced gene silencing (VIGS). The GaGH3.6-silenced plants presented severe stress-susceptive phenotype, and suffered more serious oxidative damages by detecting some physiological and biochemical indexes, indicating that GaGH3.6 might participate in salt tolerance in cotton through regulating oxidation-reduction process. For the first time, a transcriptional atlas of cotton roots under salt stress were characterized at a single-cell resolution, which explored the cellular heterogeneityand differentiation trajectory, providing valuable insights into the molecular mechanism underlying stress tolerance in plants.