Introduction: Miscanthus sinensis ‘Zebrinus’ is a landscape plant with high ornamental value, whose core ornamental feature is determined by the irregularly distributed yellow variegation on its leaves, supporting its extensive application in landscape design and configuration. M. sinensis ‘Zebrinus’, as a typical variegated-leaf gramineous plant, possesses a key phenotypic trait of leaf variegation that distinguishes it from ordinary Miscanthus species. However, up to the present moment, we know little about the molecular regulatory mechanism underlying this unique variegation, with relevant research carried out in the exploratory stage. Methods: This study was performed with the use of two leaf phenotypes [Yellow area of variegated leaves (YS) and Green area of variegated leaves (GS)] of M. sinensis ‘Zebrinus’. Differential metabolites between GS and YS leaf samples was conducted using the metabolomic analysis, with a focus on identifying key metabolites associated with leaf variegation. Furthermore, gene expression profiles of GS and YS leaves were acquired through transcriptome sequencing. With the screening of differentially expressed genes (DEGs), this study also carried out functional annotation and pathway enrichment analysis. Moreover, the expression levels of candidate genes in GS and YS leaves were measured via quantitative real-time polymerase chain reaction (qRT-PCR). In addition, a “gene-metabolite” regulatory network was constructed by integrating the metabolomic and transcriptomic data to screen out the key metabolites and core genes responsible for regulating leaf variegation in M. sinensis ‘Zebrinus’. Results: Metabolomic analysis identified 4,036 common metabolites in GS and YS samples, with major enrichment in the flavonoid biosynthesis pathway. Secondary classification of this pathway indicated that flavonoids had the highest content. Further comparison of the expression levels of key metabolites revealed that the accumulation patterns of neohesperidin, taxifolin, naringenin, and xanthohumol in YS were all higher than those in GS, with naringenin showing the most significant difference, suggesting that it might be the core metabolite regulating leaf spot formation. According to subsequent transcriptome sequencing, 5,252 DEGs were screened out from the YS and GS samples, which were mainly enriched in flavonoid biosynthesis phenylpropanoid biosynthesis and other pathways. qRT-PCR presented the highest expression level in chalcone synthase ( CHS ). Integration of metabolome and transcriptome demonstrated significant enrichment of differential metabolites and DEGs in the flavonoid biosynthesis pathway. Additionally, correlation network graph analysis suggested the highest correlation of naringenin with CHS . Discussion: This study identifies the core intrinsic regulatory mechanism underlying leaf variegation in M. sinensis ‘Zebrinus’ through integrated metabolomic and transcriptomic analysis. CHS has a strong correlation with naringenin, suggesting that the transcriptional regulation of the CHS gene may directly affect the biosynthesis of naringenin. The synergistic effect of the two may be one of the key molecular mechanisms underlying the formation of yellow leaf variegation.

Leaf architecture critically impacts crop yield. The Auxin Response Factor (ARF) family is a key regulator of leaf development, yet remains uncharacterized in the important legume crop chickpea (Cicer arietinum L.), which bears pinnate compound leaves. Here, we performed a genome-wide identification and analysis of ARF genes in chickpea. We identified 33 CaARF genes and resolved their phylogenetic structure through comparison with six other key dicot species. The analysis revealed a deeply conserved core set of ARF proteins across species, all sharing the N-terminal DNA-binding domain (DBD), with most the C-terminal PB1 domain, connected by a middle region (MR). We also uncovered instances of lineage-specific expansion, e.g., a chickpea-specific ARF clade, which is characterized by the absence of the C-terminal PB1 domain. Expression profiling using public transcriptome data and qRT-PCR revealed distinct spatiotemporal expression patterns for CaARF genes across tissues and during compound leaf development. Detailed in situ hybridization analysis for selected candidates, chosen based on phylogenetic proximity to known leaf-development-related ARFs in other species, localized their transcripts to specific regions within compound leaf primordia. Focusing on CaARF5, the closest ortholog of Arabidopsis MONOPTEROS/ARF5, we confirmed its nuclear localization and dynamic expression during chickpea leaf development. Functional complementation assays demonstrated that CaARF5 could restore developmental defects in the Arabidopsis mp mutant. Our study establishes an evolutionary and molecular framework for the chickpea ARF family, highlighting conserved features and species-specific innovations, and provides essential resources for future research on auxin-mediated leaf development and ARF-targeted legume breeding.

Improving fruit quality is a core challenge in apple production in Southwest China, where long-term clean tillage has driven red soil acidification and fertility decline. However, how different cover crop species influence fruit quality through soil fungal pathways in this region remains unclear. Here, we compared five cover crop treatments—natural grass, alfalfa, perennial ryegrass, white clover, and orchardgrass—against clean tillage in a field experiment, integrating ITS sequencing with multivariate statistical analyses to evaluate their effects on soil properties, fungal communities, and apple fruit quality. Orchardgrass (DG) showed the most favorable performance, increasing sugar–acid ratio (+28.74%) and vitamin C content (+42.16%), followed by alfalfa and natural grass, while ryegrass had the weakest effects. Soil fertility improvement followed the same ranking, and soil pH, organic matter, and nitrogen were identified as key factors associated with fungal community shifts. Fungal community composition and diversity were closely linked to soil properties and fruit quality traits, with sugar–acid ratio co-regulated by nitrogen, pH, and fungal diversity, while vitamin C content was directly associated with cover crop treatment. These findings suggest that orchardgrass may be a promising cover crop for enhancing fruit flavor and nutritional quality under the subtropical red-soil conditions tested in this study, offering practical implications for cover crop selection, though the long-term stability of these effects requires further validation through multi-year trials.

Alfalfa (Medicago sativa L.) is a major forage legume worldwide. Developing multifoliolate germplasm has been explored as a strategy to improve forage nutritive value and support more efficient forage livestock production. Here, we developed a multifoliolate population, SJ-ML, using CRISPR/Cas9-generated palmate-like pentafoliate mutants as donor parents. Molecular and phenotypic analyses indicated a stable inheritance of the edited alleles and the multifoliolate trait in SJ-ML. SJ-ML was evaluated under solar greenhouse and field conditions, with the recipient cultivar ‘Aohan’ as the greenhouse control and the commercial cultivars ‘Galaxie-Max’ and ‘GN5’ as field controls. SJ-ML showed a greater leaf area and a higher leaf-to-stem ratio, without reductions in yield or plant height. Nutritive analyses indicated that SJ-ML had a higher crude protein content, relative feed value, digestible dry matter, and dry matter intake, while acid detergent fiber, neutral detergent fiber, and lignin were lower than those of the controls. Across regrowth stages, SJ-ML generally maintained a higher nutritive value than controls. These results support SJ-ML as a multifoliolate germplasm resource for improving nutritive value without a trade-off in agronomic traits, with potential relevance for sustainable agriculture through enhanced forage protein value and a reduced reliance on supplemental protein in some ration contexts.