Abstract Alkaline salt stress is a key environmental factor restricting the sustainable development of the apple industry, significantly affecting the yield and quality of apple. In recent years, strigolactone (SLs) has been proven to play a central regulatory role in plant stress responses. However, its role and mechanism under alkaline salt stress remain unknown. Based on this, we found that exogenous application of the SL analog GR245DS can significantly enhance the adaptability of apple to alkaline salt stress. To elucidate the underlying molecular mechanisms, RNA sequencing (RNA-seq) analysis identified the key transcription factor MdbHLH1, whose expression was strongly induced by alkaline salt stress. Overexpression of MdbHLH1 conferred a salt-alkali tolerant phenotype. Further investigation demonstrated that MdbHLH1 directly binds to and activates the promoter of MdAT1 (Alkali Tolerance 1), a crucial alkali-tolerance gene. The MdbHLH1-MdAT1 module enhances alkaline salt stress resistance by promoting hydrogen peroxide (H2O2) efflux and alleviating oxidative damage. More in-depth studies revealed that MdbHLH1 interacts with MdD53 (MdDWARF53), a repressor in the SL signaling pathway. SL signaling induces ubiquitination and degradation of MdD53, thereby releasing MdbHLH1 to activate MdAT1 expression and ultimately improving alkaline stress tolerance in apple. This study elucidates a key SL-MdD53-MdbHLH1-MdAT1 regulatory pathway that enhances saline-alkali tolerance in apple by mitigating oxidative stress, thereby providing mechanistic insights into apple’s adaptation to saline-alkali environments.

Abstract Leaf chlorosis and senescence are key indicators of prolonged drought stress. In this study, we found that suppressing the chlorophyll b reductase gene (LpNOL) delayed drought-induced leaf chlorosis in perennial ryegrass (Lolium perenne). Through a yeast one-hybrid (Y1H) library screen, we identified a NAC transcription factor, designated Chlorophyll b Degradation Regulator 1 (LpCbDR1), as a direct activator of LpNOL. Subcellular localization analysis confirmed that LpCbDR1 localizes to the nucleus, and its direct binding to the LpNOL promoter was validated by electrophoretic mobility shift assay (EMSA) and CUT&Tag-qPCR assays. Overexpression of LpCbDR1 accelerated leaf senescence, whereas knockdown of LpCbDR1 delayed leaf senescence. Notably, LpCbDR1’s expression was not only upregulated during leaf senescence but also induced by osmotic stress, promoting further investigation into its role and underlying mechanisms in regulating drought tolerance. Phenotypic analysis showed that LpCbDR1-overexpressing lines exhibited significantly higher drought tolerance compared to wild-type (WT) plants, while LpCbDR1-RNAi lines were drought-sensitive than WT. Integrated RNA-seq and CUT&Tag analysis identified LpPLA7 and LpERF1B as downstream targets of LpCbDR1. Directly binding of LpCbDR1 to the promoter of LpPLA7 and LpERF1B was confirmed by Y1H, EMSA, and CUT&Tag-qPCR assays. Both LpPLA7 and LpERF1B were drought-inducible, and functional validation revealed that overexpression of either gene enhanced osmotic stress tolerance in both WT and LpCbDR1-RNAi backgrounds. Collectively, this study demonstrates that LpCbDR1 regulates natural, dark-, and drought-induced leaf senescence by activating LpNOL, and improves drought tolerance at least partially through direct activation of LpPLA7 and LpERF1B in perennial ryegrass.

Abstract Volatile terpenes constitute a predominant class of floral scent emitted by Paeonia lactiflora. Despite their ecological and economical significance, the genetic blueprint of the underlying biosynthetic pathway remains poorly elucidated. Although a few terpene synthase (TPS) genes have been reported, the broader network of genes orchestrating terpene production in P. lactiflora is still largely unresolved. In this study, we attempted to address this gap by exploring the terpene biosynthetic pathway genes in P. lactiflora ‘Zifengyu’. β-caryophyllene, geraniol, citronellol and 1, 8-cineole were identified as the dominant floral terpenes, and catalytic functions of key proteins- terpene synthase (PlTPS), Nudix hydrolase (PlNUDX) and prenyltransferase (PlPT) were comprehensively characterized. Briefly, biochemical analyses revealed that six of the nine identified PlTPS proteins utilized diverse prenyl diphosphates to generate both monoterpenes and sesquiterpenes, while their products specificity were determined by plastidic or cytosolic localizations in planta. In particular, PlTPS4, PlTPS5 and PlTPS9 catalyzed the production of β-caryophyllene, 1, 8-cineole and geraniol, respectively. Besides, two amino acid residues were found to drive catalytic activity and product profiles in PlTPS4 and PlTPS5. Markedly, PlNUDX hydrolyzed GPP and NPP to yield geraniol and nerol thereby providing a plastid-independent pathway for monoterpene biosynthesis, and prenyltransferases were further functionally characterized to clarify the supply of prenyl diphosphates feeding into volatile terpenes. Collectively, these findings not only provide a mechanistic framework for understanding floral terpene biosynthesis in P. lactiflora but also reveal alternative metabolic routes that enrich its volatile profiles which could be utilized in scent improvement of ornamental plants.

Abstract Global warming is increasing the frequency of heat stress, a major abiotic constraint on crop growth and productivity. Hydrogen sulfide (H2S), a novel gasotransmitter, has been reported to enhance crops’ heat tolerance, yet its underlying mechanism remains poorly understood. Here, we provide genetic evidence confirming that L-cysteine desulfhydrase (SlLCD1, Solyc01g068160) was the enzymatic source of endogenous H2S in tomato heat adaptation. Dual activation of H2S signaling through both SlLCD1 overexpression and exogenous application enhanced tomato heat tolerance. Conversely, CRISPR/Cas9-generated SlLCD1 mutants (cr-sllcd1), deficient in heat-induced H2S production, displayed heightened heat sensitivity with accelerated wilting and increased oxidative damage, which was rescued by exogenous H2S application. Compared to wild-type plants, the mutants showed a compromised heat-induced increase in antioxidant enzyme activities and levels. This defect, along with the concomitant ROS accumulation and oxidative damage, was reversed by H2S pretreatment, underscoring the critical role of the SlLCD1-H2S module in maintaining ROS homeostasis during heat adaptation. Additionally, cr-sllcd1 mutants exhibited attenuated heat-induced stomatal closure and increased stomatal density. H2S pretreatment rescued both of these defects, thereby optimizing the trade-off among transpirational cooling, water conservation, and photosynthetic efficiency. Overall, the SlLCD1-H2S module confers heat tolerance by a dual mechanism, coordinately enhancing antioxidant capacity and fine-tuning stomatal dynamics. Our study elucidates an important component of the H2S signaling pathway in plant heat tolerance and offers a promising tractable target for developing heat-tolerant tomato cultivars.

Abstract Rapeseed (Brassica napus L.) is one of the most important oil crops worldwide. In our previous work, we generated a high-throughput CRISPR library whereby a knockout collection was established for rapeseed breeding and functional genomics. However, the collection remains small and several promising candidate genes still await functional validation. Here, we report an update of this collection by constructing a small-scale CRISPR mutant library based on the elite commercial cultivar Zhongshuang 11 (ZS11). We first generated 326 independent T0 lines using an optimized protocol for ZS11 transformation and regeneration with a high positive rate of 94.2%. Analysis of the editing outcomes revealed a mutagenesis frequency of 68.4%. We then phenotyped this new collection and unraveled possible key genes underlying the variations in seed oil content (SOC) and plant height. Finally, we functionally validated BnFAB1B and BnEDA32, two candidate genes identified from our knockout collection. The results confirmed that loss-of-function of BnFAB1B significantly increases SOC, indicating its great agronomic potential, whereas knockout of the nuclear-localized BnEDA32 severely disrupts seed oil accumulation. This study provides a valuable knockout collection of the elite cultivar ZS11 and new genes for creating superior rapeseed germplasm.

Abstract Astragalus membranaceus var. mongholicus (AMM) is the principal botanical source of Huangqi, a traditional medicinal herb whose therapeutic value primarily stems from the accumulation of isoflavones and other bioactive compounds in the roots. In this study, field surveys across major AMM production regions revealed pronounced natural variation in stem coloration. Chemical analysis showed that the roots of the red-stemmed type contained significantly higher levels of four bioactive isoflavones and volatile organic compounds than those in green-stemmed plants. Metabolomic profiling further revealed a specific enrichment of cyanidin-based anthocyanins in the red stems, establishing the metabolic basis of the red stem phenotype. Both transcriptomic and metabolomic analyses indicated an overall upregulation of the flavonoid and phenylpropanoid biosynthetic pathways in the stem and root tissues of red-stemmed AMM. Weighted gene co-expression network analysis (WGCNA) identified six key genes (AmC4H, AmCHS, AmCHI, AmF3H, AmF3′H, and AmBZ1) that were strongly associated with the red stem phenotype, all of which were specifically highly expressed in red stems. Functional assays confirmed their roles in anthocyanin biosynthesis. Molecular modeling provided further insights into the substrate specificity of AmBZ1. This study proposes stem color as a visible phenotypic reference for early-stage germplasm selection in AMM, and characterizes the molecular basis underlying red stem formation, providing a foundation for elite germplasm development and molecular breeding.

Abstract Plasmopara viticola, the causal agent of grapevine downy mildew, exhibits substantial intraspecific variation in pathogenicity and genetic diversity, yet the genomic features underlying this variation remain incompletely characterized. Here, we sequenced and assembled two P. viticola isolates, PvH (from Vitis vinifera) and PvS (from V. amurensis), using PacBio HiFi sequencing, and performed comparative genomic analysis. Two complete genome assemblies (17 chromosomes) of P. viticola (PvH: 115.3 Mb; PvS: 113.0 Mb) were generated and revealed that nearly 90% of the putative effectors exist as local duplicated gene clusters. Comparative genomics uncovered distinct intraspecific expansion, deletion and diversification of putative effectors driven by local segmental, tandem, and proximal duplication events in P. viticola. Specifically, PvH exhibited a ~1.4-fold increase in CRNs (PvH: 237; PvS: 183; PV221: 169) and harbored 35 strain-specific CRNs. These differential effectors were predominantly clustered in complex structural variation hotspots (SVs, duplication and inversion). Notably, 104 putative effectors—including 21 RxLRs, 59 CRNs, and 24 CAZymes—located within inversion regions. Together, our results highlight a highly dynamic genome architecture in P. viticola, in which SV and local gene duplication are closely associated with effector diversification. This study provides a genome-resolved comparative framework for understanding intraspecific genomic diversity in P. viticola and establishes a foundation for future population-level and functional investigations.

Abstract Tomato (Solanum lycopersicum) is a nutrient-rich and flavorful vegetable, ranking among the most consumed globally. In recent years, consumers have increasingly demanded high-quality tomatoes, prompting the extensive research into the key factors and relevant molecular mechanisms regulating the formation of fruit quality. Coloration is a crucial aspect determining the appearance quality of tomato fruits and directly affecting their commercial value. This coloration is intrinsically linked to the composition and abundance of special chemical compounds in fruits, particularly chlorophyll and carotenoids. Chlorophyll is the predominant pigment accumulated in the early stages of fruit development and plays a vital role in photosynthesis. As the fruit ripens, chlorophyll undergoes gradual degradation, while carotenoids are abundantly synthesized, resulting in a striking color transition from green to red. Chlorophyll and carotenoids are essential natural pigments and antioxidants that are indispensable for both coloration and nutritional value of tomato fruits. This review presents a comprehensive overview of the metabolic pathways and regulatory mechanisms of these metabolites, aiming to provide novel insights and strategies for improving tomato quality to meet the growing consumer demand for fruits with appealing coloration and enhanced nutrients.

Abstract Safflower is featured with time-honored medical and economic values and developing into diverse phenotypic and genetic variations. In this study, to explore the critical genes associated with color phenotypes and flavonoid derivatives biosynthesis of safflower, BSA-seq, conflated with transcriptomic and metabolic methods were performed in two extreme colors (yellow and white) in the population of “ZHH0119” and “XHH007”. After crossing two parent plants reciprocally, the F4 generation of two accessions were used to construct near-isogenic gene pools for the two extreme traits of yellow and white safflower. BSA-seq results located five QTLs regions on chromosomes 2, 8, 9, 10 and 12 including six CtPALs, three CtC4Hs, two Ct4CLs, one CtCHS, thirty-two CtUGTs and seventy CtCYPs which tied to the yellow color phenotype of safflower. Through transcriptome analysis of two accessions and at different flowering stages, 1 CtPAL, 5 CtC4Hs, 4 CtCHSs, 3 CtCHIs, 3 CtFLSs, 48 CtUGTs, 51 CtCYPs and 75 transcription factors were revealed as significantly upregulated in the yellow accession compared to the white. Integrated analysis identified eight CtUGTs (CtUGT50-57) which exhibited significant positive correlations with chalcone glycosides of yellow safflower. Based on functional characterization, CtUGT52 was found to boost Hydroxysafflor yellow A (HSYA) content in yellow safflower which possessing substrate promiscuity (chalones, flavonols and flavonoids) and catalytic promiscuity (flavonols and flavonoids), revealing its vital role in the HSYA biosynthesis through transgenic overexpression. Combining catalytic mechanism verification of CtUGT52 towards phloretin, kaempferol and luteolin, our study to some extent, elucidated the modification function of CtUGTs for flavonoid aglycones in the flavonoid biosynthesis pathway of safflower.