AtSBT6.1 is the primary physiological convertase for AtRALF1, while AtSBT5.3 contributes under ectopic AtRALF1 overexpression. The 5-kDa rapid alkalinization factor (RALF) peptide is widespread in the plant kingdom. In Arabidopsis, the RALF family comprises 37 RALF-like peptides. AtRALF1 is a 49-amino-acid peptide derived from a 120-amino-acid precursor, but the protease responsible for its maturation has remained unresolved. We used in silico expression profiling to select subtilases (AtSBTs) potentially involved in AtRALF1 precursor processing. T-DNA insertion AtSBT mutants, and F3 homozygous lines derived from crosses between AtSBT mutants, and an AtRALF1-overexpressing line were analyzed for primary root length (PRL) and lateral root primordium (LRP) density. PRL and LRP density were higher in atsbt6.1 than in wild-type plants, whereas atsbt5.3 was indistinguishable from the wild-type. Ectopic expression of AtRALF1 driven by the 35S promoter in atsbt6.1 and atsbt5.3 backgrounds indicated that both AtSBT6.1 and AtSBT5.3 contribute to AtRALF1 precursor processing under overexpression conditions. In contrast, the phenotypes of single mutants indicate that AtSBT6.1 plays the predominant role in AtRALF1 precursor processing when AtRALF1 is expressed from its endogenous promoter. Transient expression assays further showed that AtRALF1 precursor processing occurs in the Golgi complex. These findings identify AtSBT6.1 as the main subtilase for AtRALF1 maturation in planta and reveal context-dependent participation of AtSBT5.3 under ectopic AtRALF1 expression.
Standardized in vitro phenotypes provide genetically tractable traits that integrate GWAS, QTL mapping, and multi-omics, enabling mechanistic insight and genetics-informed improvement of plant regeneration. Plant tissue culture (PTC) has traditionally been employed as a technical tool for propagation, transformation, and germplasm conservation. This review reframes in vitro culture systems as quantitative phenotyping platforms that enable rigorous genetic dissection of complex traits, such as regeneration competence, stress tolerance, and developmental plasticity. By operating under highly standardized and reproducible conditions, in vitro systems reduce environmental noise and expose genotype-dependent variation that is often obscured in field or greenhouse experiments. When formalized as quantitative traits, culture responses, including callus induction, organogenesis, somatic embryogenesis, and stress survival, become amenable to genome-wide association studies (GWAS) and quantitative trait locus (QTL) mapping. Synthesizing evidence across diverse plant species, this review demonstrates that in vitro traits are genetically structured, polygenic, and biologically meaningful, reflecting coordinated regulation of hormonal signaling, chromatin accessibility, stress response pathways, and developmental gene networks. Numerous GWAS and QTL studies have identified loci and candidate genes underlying regeneration efficiency, embryogenic competence, and stress-responsive culture performance, confirming that tissue culture responsiveness is rooted in genomic architecture rather than protocol effects alone. The interpretive power of genetic mapping is substantially enhanced through integration with bioinformatic and multi-omics layers, including transcriptomics, epigenomics, proteomics, metabolomics, and network-based analyses, thereby refining candidate prioritization and facilitating mechanistic inference. This review further examines the dual role of somaclonal variation as both a constraint on clonal fidelity and a source of genetic and epigenetic diversity that can reveal cryptic genotype–phenotype relationships. Emphasis is placed on study design principles, phenotyping challenges, statistical modeling strategies, and integrative pipelines that connect genotype to in vitro phenotype and ultimately to breeding and biotechnology applications. This synthesis highlights in vitro culture systems as powerful, genetics-informed platforms for precision breeding, gene validation, and supporting efforts to more predictably and rationally manipulate complex traits, particularly in horticultural and clonally propagated crops.
A targeted Pseudomonas-based synthetic microbial community reshaped rhizosphere bacterial communities and altered potassium partitioning under stress conditions, suggesting a potential role in mitigating antibiotic-induced dysbiosis. Sustainable agriculture primarily relies on biological practices to improve crop yield and resilience. Synthetic bacterial communities (SynComs) are a promising approach to promote plant growth; however, their effectiveness in situ is influenced by interactions with native microbial ecosystems and by environmental stressors, such as low pH and antibiotic use. In this context, beneficial microorganisms like Pseudomonas may enhance nutrient availability and mitigate antibiotic-induced dysbiosis by supporting microbial community reassembly and functional stability, thereby improving plant health. This study evaluated the effects of a Pseudomonas-based SynCom on nutrient accumulation and rhizosphere microbial dynamics in citrus plants subjected to streptomycin-induced dysbiosis and low-pH conditions. Selected Pseudomonas strains, chosen for their compatibility and adaptability to low-pH conditions, and through whole-genome sequencing, were applied at equal concentrations (OD600 = 0.1). SynCom treatment increased Chloroflexota abundance in the rhizosphere while reducing Actinomycetota and Pseudomonadota after 24 days. By Day 45, the abundance of Actinomycetota decreased, with slight increases in the other two phyla. At the genus level, SynCom application reduced the abundance of Pseudomonas and Agrobacterium, while enriching the Klebsiella community. Plants treated with SynCom under stress exhibited a 25
Phylogenomics of 921 WHIRLY genes across 330 plants reveals stepwise subfamily origins, purifying selection, and a novel AtWHY1/2/3 hetero-oligomeric stress complex. Whirly (WHY) transcription factors are a plant-specific, highly conserved family with central roles in organellar genome maintenance, retrograde signaling, and abiotic stress response, but their deep evolutionary trajectories across the full green plant lineage have remained largely unresolved. In the present study, 921 WHY genes were identified across 330 different species from algae to land plants, which were classified into three subfamilies (WHY I, WHY II, and WHY III) in this study. Structural characteristics of different subfamilies showed significant differences in the number of exons and protein motifs, which may have arisen due to functional differentiation. Collinearity was primarily observed among members of the same subfamily, and network clustering analysis further divided all WHY genes into three clusters, with genes within each cluster exhibiting significant collinearity. All three subfamilies are under intense long-term purifying selection, with average Ka/Ks ratios far below 1, indicating stringent evolutionary constraints on their core conserved single-stranded DNA binding domain. Using yeast two-hybrid assays, protein molecular docking and site-specific interaction validation, we further uncovered a previously unreported stable hetero-oligomeric complex formed by AtWHY1, AtWHY2, and AtWHY3, assembled via defined hydrogen bond networks and salt bridges at the conserved interface residues, which acts as a combinatorial regulatory switch to coordinate distinct subcellular stress responses. This work establishes a unified evolutionary framework for the WHY family and provides new mechanistic insights into how their hetero-oligomerization drives specialized stress adaptation during plant territorialization.
