Functional Characterization Research Articles

Whey protein and maltodextrin conjugated foam-mat dried honey powder: Functional, physicochemical, structural, rheological and thermal characterization

Honey in its natural form possesses considerable challenges in mass production and transportation owing to its viscosity and stickiness, making it prone to crystallization over time. Dried honey powder makes it convenient for handling and processing. In this study, foam mat dried powder (FMDP) was obtained by foam mat drying technique in which whey protein isolate (WPI) and maltodextrin (MD) were used as foaming agent and foam stabilizer, respectively. The obtained FMDP was characterized in terms of physical, chemical, functional, phytochemical, rheological and structural properties to study the impact of foaming agents. WPI was found to improve the functionality, stability and glass transition temperature and on the other hand, MD was found to enhance the stability, crystallinity and the color of the FMDP. FTIR helped in understanding the changes in the functional groups attributed to the foaming agents. The crystallinity and the microstructural changes in the FMDP were observed from X-ray diffractograms and SEM micrographs, respectively. The rheological characterization confirmed the viscous nature of honey. The study confirmed the interaction between proteins and polysaccharides, which subsequently improved the heat stability, functional properties, solubility, emulsifying capacity, antioxidant properties, and solubility. The dried honey samples demonstrated minimal caking, low cohesiveness (Hausner ratio: 1.16 ± 0.027–1.29 ± 0.041), good and fair flowability (ranging between 14.25 ± 2.02–22.52 ± 2.25). Therefore, it can be concluded that during this study, protein and polysaccharide conjugate FMDP was obtained with better quality attributes, which can be further incorporated during various product development in food industries.

Functional characterization of a novel Chlamydomonas reinhardtii hydrolase involved in biotransformation of chloramphenicol

Microalgae-based biotechnology is one of the most promising alternatives to conventional methods for the removal of antibiotic contaminants from diverse water matrices. However, current knowledge regarding the biochemical mechanisms and catabolic enzymes involved in microalgal biodegradation of antibiotics is scant, which limits the development of enhancement strategies to increase their engineering feasibility. In this study, we investigated the removal dynamics of amphenicols (chloramphenicol, thiamphenicol, and florfenicol), which are widely used in aquaculture, by Chlamydomonas reinhardtii under different growth modes (autotrophy, heterotrophy, and mixotrophy). We found C. reinhardtii removed >92 % chloramphenicol (CLP) in mixotrophic conditions. Intriguingly, gamma-glutamyl hydrolase (GGH) in C. reinhardtii was most significantly upregulated according to the comparative proteomics, and we demonstrated that GGH can directly bind to CLP at the Pro77 site to induce acetylation of the hydroxyl group at C3 position, which generated CLP 3-acetate. This identified role of microalgal GGH is mechanistically distinct from that of animal counterparts. Our results provide a valuable enzyme toolbox for biocatalysis and reveal a new enzymatic function of microalgal GGH. As proof of concept, we also analyzed the occurrence of these three amphenicols and their degradation intermediate worldwide, which showed a frequent distribution of the investigated chemicals at a global scale. This study describes a novel catalytic enzyme to improve the engineering feasibility of microalgae-based biotechnologies. It also raises issues regarding the different microalgal enzymatic transformations of emerging contaminants because these enzymes might function differently from their counterparts in animals.

Characterization of switchgrass (Panicum virgatum L.) PvKSL1 as a levopimaradiene/abietadiene-type diterpene synthase.

The diverse class of plant diterpenoid metabolites serves important functions in mediating growth, chemical defence, and ecological adaptation. In major monocot crops, such as maize (Zea mays), rice (Oryza sativa), and barley (Hordeum vulgare), diterpenoids function as core components of biotic and abiotic stress resilience. Switchgrass (Panicum virgatum) is a perennial grass valued as a stress-resilient biofuel model crop. Previously we identified an unusually large diterpene synthase family that produces both common and species-specific diterpenoids, several of which accumulate in response to abiotic stress. Here, we report discovery and functional characterization of a previously unrecognized monofunctional class I diterpene synthase (PvKSL1) via in vivo co-expression assays with different copalyl pyrophosphate (CPP) isomers, structural and mutagenesis studies, as well as genomic and transcriptomic analyses. In particular, PvKSL1 converts ent-CPP into ent-abietadiene, ent-palustradiene, ent-levopimaradiene, and ent-neoabietadiene via a 13-hydroxy-8(14)-ent-abietene intermediate. Notably, although featuring a distinct ent-stereochemistry, this product profile is near-identical to bifunctional (+)-levopimaradiene/abietadiene synthases occurring in conifer trees. PvKSL1 has three of four active site residues previously shown to control (+)-levopimaradiene/abietadiene synthase catalytic specificity. However, mutagenesis studies suggest a distinct catalytic mechanism in PvKSL1. Genome localization of PvKSL1 distant from other diterpene synthases, and its phylogenetic distinctiveness from known abietane-forming diterpene synthases, support an independent evolution of PvKSL1 activity. Albeit at low levels, PvKSL1 gene expression predominantly in roots suggests a role of diterpenoid formation in belowground tissue. Together, these findings expand the known chemical and functional space of diterpenoid metabolism in monocot crops.

Cloning, recombinant expression, and characterization of a Rhipicephalus microplus carboxylesterase.

Background The Rhipicephalus microplus carboxylesterase (CBE) is involved in synthetic pyrethroid (SP) hydrolysis and historic evidence suggests that a non-synonymous mutation (Asp374Asn) in CBE is associated with increased resistance towards SP-based acaricides. Functional expression and characterization of the wild-type and mutant CBE is required to understand the impact of the mutation on SP-based resistance. Methods The R. microplus CBE gene was cloned and functionally expressed in Pichia pastoris following the removal of the native signal peptide. Site directed mutagenesis was used to introduce the Asp374Asn substitution. Results Functional expression, characterization, and purification of both wild-type and mutant R. microplus CBE proteins was achieved using affinity chromatography under native conditions. Conclusions This report provides the necessary information for the tick research community to produce recombinant tick derived CBE proteins and to characterize the recombinant proteins towards substrates of interest.

A biallelic mutation in CACNA2D2 associated with developmental and epileptic encephalopathy affects calcium channel-dependent as well as synaptic functions of α2δ-2.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α2δ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α2δ-2. Our data show that the corresponding p.R596P mutation in mouse α2δ-2 drastically decreases membrane expression and synaptic targeting of α2δ-2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies.

Biocompatible melanin from the marine black yeast Hortaea werneckii R23 with antioxidant and photoprotection property.

Pigments from diverse sources have a great deal of interest due to its multifaceted applications. Hence, this study reports the physicochemical and functional characterization of the black pigment melanin from the marine black yeast Hortaea werneckii R23. In the present study, Hortaea werneckii R23, produced a black pigment in the yeast biomass. The pigment was extracted from the harvested yeast biomass and followed by pigment purification, characterization and identification was done. Physicochemical characterization of the pigment showed acid precipitation, alkali solubilization, insolubility in most organic solvents and water. The black pigment was confirmed as melanin based on ultraviolet-visible spectroscopy, Fourier-transform infrared, and Nuclear magnetic resonance spectroscopy analyses. Furthermore, the analyses of the elemental composition indicated that the pigment possessed a moderately high percentage of nitrogen and also detectable proportion of sulfur. All these Physicochemical properties indicated that H. werneckii melanin (HwM) mostly consisted of eumelanin. HwM exhibited strong antioxidant potential as reactive oxygen species (ROS) scavenger by in vitro DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS (2,2-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid) radical scavenging assay, and lipid peroxidation assay. The photoprotectant role of HwM on UV-irradiated human epithelial cells (HEp-2) revealed its potential effect in photoprotection. In addition, cytotoxicity study by XTT and SRB assay confirmed its biocompatibility with HEp-2 cells. From these findings, it is evident that the HwM from the marine black yeast possesses strong antioxidant and photoprotectant activity, moreover, it is biocompatible to human epithelial cells. So HwM could be used as a protective agent against oxidative stress associated disorders in an environment-friendly perspective.

Decisional components of motor responses are not related to online response control: Evidence from lexical decision and speed-accuracy tradeoff manipulations.

Evidence suggests that decision processes can propagate to motor-response execution. However, the functional characterization of motor decisional components is not yet fully understood. By combining a classic lexical decision experiment with manipulations of speed-accuracy tradeoff (SAT), the present experiment assessed the hypothesis that decisional effects on chronometric measures of motor-response execution are related to online response control. The electromyographic (EMG) signal associated with manual button-press responses was used to dissociate the premotor component (from stimulus onset until the onset of the EMG activity) from the motor component (from EMG onset until the button-press), thus enabling the assessment of decision-related effects in terms of motor-response duration within single-trial reaction times. Other than replicating all the previously reported SAT effects, the experiment revealed hindered control processes when the instructions emphasized speed over accuracy, as indicated by measures of response control such as partial errors, fast errors, and correction likelihood. Nonetheless, the lexicality effect on motor responses, consisting of slower motor times for pseudowords compared to words, was impervious to any SAT modulation. The results suggest that SAT-induced variations in decision and response control policies may not be the prominent determinant of decision-related effects on motor times, highlighting the multiple "cognitive" components that affect peripheral response execution.

Functional characterization and structural basis of an efficient ochratoxin A-degrading amidohydrolase

Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an appealing strategy, and the search for more efficient enzymes is a prerequisite for achieving this goal. Here, a novel amidohydrolase, termed PwADH, was demonstrated to exhibit 7.3-fold higher activity than that of the most efficient OTA-degrading ADH3 previously reported. Cryo-electron microscopy structure analysis indicated that additional hydrogen-bond interactions among OTA and the adjacent residue H163, the more compact substrate-binding pocket, and the wider entry to the substrate-access cavity might account for the more efficient OTA-degrading activity of PwADH compared with that of ADH3. We conducted a structure-guided rational design of PwADH and obtained an upgraded variant, G88D, whose OTA-degrading activity was elevated by 1.2-fold. In addition, PwADH and the upgraded G88D were successfully expressed in the industrial yeast Pichia pastoris, and their catalytic activities were compared to those of their counterparts produced in E. coli, revealing the feasibility of producing PwADH and its variants in industrial yeast strains. These results illustrate the structural basis of a novel, efficient OTA-degrading amidohydrolase and will be beneficial for the development of high-efficiency OTA-degrading approaches.

On generalized notions of consistency and reinstatement and their preservation in formal argumentation

We present a conceptualization providing an original domain-independent perspective on two crucial properties in reasoning: consistency and reinstatement. They emerge as a pair of dual characteristics, representing complementary requirements on the outcomes of reasoning processes. Central to our formalization are two underlying parametric relations: incompatibility and reinstatement violation. Different instances of these relations give rise to a spectrum of consistency and reinstatement scenarios. As a demonstration of versatility and expressive power of our approach we provide a characterization of various abstract argumentation semantics which are expressed as combinations of distinct consistency and reinstatement constraints. Moreover, we conduct an investigation into preserving these essential properties across different reasoning stages. Specifically, we delve into scenarios where a labelling is derived from other labellings through a synthesis function, using the synthesis of argument justification as an illustrative instance. We achieve a general characterization of consistency preservation synthesis functions, while we unveil an impossibility result concerning reinstatement preservation, leading us to explore an alternative notion to ensure feasibility. Our exploration reveals a weakness in the traditional definition of argument justification, for which we propose a refined version overcoming this limitation.

Genome-wide analysis of the HD-Zip IV gene family in Camellia sinensis and functional characterization of CsHDZIV3 under trichome development

The HD-Zip IV transcription factors, which belong to the homeodomain leucine zipper (HD-ZIP) family, are essential for the development of epidermal cells in plants. However, there is limited information about HD-Zip IV in tea plant (Camellia sinensis L.). Here we identified 10 HD-Zip IV members (CsHDZIV1∼10) in tea plant, which are distributed in 8 chromosomes with two segmentally duplicated gene pairs. The exon number of CsHDZIVs ranges from 9 to 11, with most genes having two conserved motifs in the 3’UTR. CsHDZIV1∼9 have almost identical motifs and tertiary structures. The promoter region of CsHDZIVs was predicted to harbor light, phytohormone, stress, growth and developmental elements. 9 CsHDZIVs are divided into 5 subfamilies and located in the nucleus. They exhibited expression trends across 12 tea plant tissues and responded to PEG, cold, NaCl and shading treatments. CsHDZIV3 overexpression partially and completely restored trichome development defects of gl2 leaves and stems, respectively. CsHDZIV3 showed histochemical staining in Arabidopsis trichomes and protein interaction with itself. It might regulate trichome development through forming homodimers. These findings contribute to functional study of tea plant HD-Zip IV and provide guiding significance for the investigation of trichome development and the improvement of trichome traits.

Functional characterization of the SDR42E1 reveals its role in vitamin D biosynthesis

Vitamin D deficiency poses a widespread health challenge, shaped by environmental and genetic determinants. A recent discovery identified a genetic regulator, rs11542462, in the SDR42E1 gene, though its biological implications remain largely unexplored. Our bioinformatic assessments revealed pronounced SDR42E1 expression in skin keratinocytes and the analogous HaCaT human keratinocyte cell lines, prompting us to select the latter as an experimental model. Employing CRISPR/Cas9 gene-editing technology and multi-omics approach, we discovered that depleting SDR42E1 showed a 1.6-fold disruption in steroid biosynthesis pathway (P-value = 0.03), considerably affecting crucial vitamin D biosynthesis regulators. Notably, SERPINB2 (P-value = 2.17 × 10−103), EBP (P-value = 2.46 × 10−13), and DHCR7 (P-value = 8.03 × 10−09) elevated by ∼2–3 fold, while ALPP (P-value <2.2 × 10−308), SLC7A5 (P-value = 1.96 × 10−215), and CYP26A1 (P-value = 1.06 × 10−08) downregulated by ∼1.5–3 fold. These alterations resulted in accumulation of 7-dehydrocholesterol precursor and reduction of vitamin D3 production, as evidenced by the drug enrichment (P-value = 4.39 × 10−06) and total vitamin D quantification (R2 = 0.935, P-value = 0.0016) analyses. Our investigation unveils SDR42E1's significance in vitamin D homeostasis, emphasizing the potential of precision medicine in addressing vitamin D deficiency through understanding its genetic basis.

Structure and mechanism of the osmoregulated choline transporter BetT.

The choline-glycine betaine pathway plays an important role in bacterial survival in hyperosmotic environments. Osmotic activation of the choline transporter BetT promotes the uptake of external choline for synthesizing the osmoprotective glycine betaine. Here, we report the cryo-electron microscopy structures of Pseudomonas syringae BetT in the apo and choline-bound states. Our structure shows that BetT forms a domain-swapped trimer with the C-terminal domain (CTD) of one protomer interacting with the transmembrane domain (TMD) of a neighboring protomer. The substrate choline is bound within a tryptophan prism at the central part of TMD. Together with functional characterization, our results suggest that in Pseudomonas species, including the plant pathogen P. syringae and the human pathogen Pseudomonas aeruginosa, BetT is locked at a low-activity state through CTD-mediated autoinhibition in the absence of osmotic stress, and its hyperosmotic activation involves the release of this autoinhibition.

Facile preparation of a novel core-shell fiber S-scheme C/CoS2@BiOBr heterojunction photocatalyst for efficient degradation of Congo red under visible light

Generally, it is of great scientific significance to improve the utilization of solar energy resources. However, it is still a huge challenge at present. For this reason, the construction of photocatalysts with heterostructures is an effective way. In this paper, a novel core-shell fiber-like C/CoS2@BiOBr S-scheme heterojunction with visible light response was rationally designed by in-situ reduction of MOF through anion exchange strategy. Density functional theory (DFT) calculation and characterization analysis show that the internal electric field is formed between C/CoS2 and BiOBr after contact. This is beneficial to charge transfer and separation, and the S-sheme transfer path endows the separated electrons and holes with large redox potential to participate in the reaction directly. Moreover, the addition of BiOBr significantly enhanced the visible light absorption of the composites and improved the photochemical properties. It is worth noting that C/CoS2@BiOBr has excellent catalytic ability, and the optimal value of photodegradation of Congo red (CR) reaches more than 99 % within 15 min of visible light irradiation. Consequently, the active species were •O2- and h+. •O2- play a dominant role in the photocatalytic process. The space charge transfer and possible separation mechanisms of the new heterostructures are further proposed. This study provides some new ideas for the preparation of efficient water purification photocatalysts.

Germplasm Resources and Genetic Breeding of Huang-Qi (Astragali Radix): A Systematic Review.

Huang-Qi (Astragali radix) is one of the most widely used herbs in traditional Chinese medicine, derived from the dried roots of Astragalus membranaceus or Astragalus membranaceus var. mongholicus. To date, more than 200 compounds have been reported to be isolated and identified in Huang-Qi. However, information pertaining to Huang-Qi breeding is considerably fragmented, with fundamental gaps in knowledge, creating a bottleneck in effective breeding strategies. This review systematically introduces Huang-Qi germplasm resources, genetic diversity, and genetic breeding, including wild species and cultivars, and summarizes the breeding strategy for cultivars and the results thereof as well as recent progress in the functional characterization of the structural and regulatory genes related to horticultural traits. Perspectives about the resource protection and utilization, breeding, and industrialization of Huang-Qi in the future are also briefly discussed.

High-performance cellulose/thermoplastic polyurethane composites enabled by interaction-modulated cellulose regeneration

Strong interfacial adhesion between cellulose and other polymers is critical to achieve the properties required for specific applications in composite materials. Here, we developed a method for the simultaneous homogeneous dissolution of cellulose and thermoplastic polyurethane (TPU) in 1,8-diazabicyclo (5.4.0) undec-7-ene levulinate/dimethyl sulfoxide ([DBUH]Lev/DMSO) solvent. This process is essential for preparing cellulose/TPU composite films and fibers through interaction-modulated cellulose regeneration. Both cellulose and TPU can be easily dissolved together in [DBUH]Lev/DMSO solvent under mild conditions. The resulting cellulose/TPU solutions exhibited strong temperature sensitivity, shear-thinning behavior and viscoelasticity, making them suitable for cast films and continuous spinning. More importantly, research findings, including density functional theory calculations and experimental characterization, confirmed the high compatibility and interaction modulability of cellulose and TPU in the composite films. The representative C90T10 sample (cellulose/TPU, 90/10) showed high transparency (90 % at 800 nm) and excellent mechanical properties (tensile strength: 176 MPa; elongation at break: 8.1 %). Additionally, the maximum tensile strength and elongation at the break of the composite fiber from C90T10 were 214 MPa and 48.1 %, respectively. This method may provide a feasible approach to design and produce homogeneous environmentally friendly composites of cellulose and other polymers at the molecular level.

Functional characterization of variants in the regulatory regions of PCSK9 gene

Functional characterization of variants in the regulatory regions of PCSK9 gene

A gene cassette Vd276-280 in Verticillium dahliae contains two genes that affect melanized microsclerotium formation and virulence.

Verticillium dahliae is a soilborne phytopathogenic fungus causing Verticillium wilt on hundreds of plant species. Several sequenced genomes of V. dahliae are available, but functional characterization of most genes has just begun. Based on our previous comparison of the transcriptome from the wild-type and ΔVdCf2 strains, a significant upregulation of the gene cassette, Vd276-280, in the ΔVdCf2 strain was observed. In this study, the functional characterization of the Vd276-280 gene cassette was performed. Agrobacterium-mediated knockout of this gene cassette in V. dahliae significantly inhibited conidiation, melanized microsclerotium formation in the mutant strains, and their virulence towards cotton. Furthermore, deletion of individual genes in the Vd276-280 gene cassette identified that the disruption of VDAG_07276 and VDAG_07280 delayed microsclerotium formation, inhibited conidiation, and reduced virulence towards cotton. Our data suggest that VDAG_07276 and VDAG_07280 in the Vd276-280 gene cassette mainly act as positive regulators of development and virulence in V. dahliae.

Functional characterization of uncertain significance variants (VUS) in patients with familial hypercholesterolemia (FH) through the application of flow cytometry

Functional characterization of uncertain significance variants (VUS) in patients with familial hypercholesterolemia (FH) through the application of flow cytometry

Functional characterization of TrGSTF15, a glutathione S-transferase gene family member, on the transport and accumulation of anthocyanins and proanthocyanidins in Trifolium repens

Anthocyanins and proanthocyanidins (PAs) are important secondary metabolites in plants, high contents of which are an important goal for quality breeding of white clover (Trifolium repens). However, the involvement of glutathione S-transferase (GST) in the transport of anthocyanins and PAs remains unexplored in white clover. This study identified 153 different TrGSTs in white clover. At the transcriptional level, compared to other TrGSTFs, TrGSTF10 and TrGSTF15 are highly expressed in the ‘Purple’ white clover, and they may work with the anthocyanin biosynthesis structural genes CHS and CHI to contribute to pigment buildup in white clover. Subcellular localization confirmed that TrGSTF10 and TrGSTF15 are located in the cytoplasm. Additionally, molecular docking experiments showed that TrGSTF10 and TrGSTF15 have similar binding affinity with two flavonoid monomers. Overexpression of TrGSTF15 complemented the deficiency of anthocyanin coloring and PA accumulation in the Arabidopsis tt19 mutant. The initial findings of this research indicate that TrGSTF15 encodes an important transporter of anthocyanin and PA in white clover, thus providing a new perspective for the further exploration of related transport and regulatory mechanisms.

Insights into ACO genes across Rosaceae: evolution, expression, and regulatory networks in fruit development.

ACO (1-aminocyclopropane-1-carboxylic acid) serves as a pivotal enzyme within the plant ethylene synthesis pathway, exerting influence over critical facets of plant biology such as flowering, fruit ripening, and seed development. This study aims to identify ACO genes from representative Rosaceae genomes, reconstruct their phylogenetic relationships by integrating synteny information, and investigate their expression patterns and networks during fruit development. we utilize a specialized Hidden Markov Model (HMM), crafted on the sequence attributes of ACO gene-encoded proteins, to systematically identify and analyze ACO gene family members across 12 representative species within the Rosaceae botanical family. Through transcriptome analysis, we delineate the expression patterns of ACO genes in six distinct Rosaceae fruits. Our investigation reveals the presence of 62 ACO genes distributed among the surveyed Rosaceae species, characterized by hydrophilic proteins predominantly expressed within the cytoplasm. Phylogenetic analysis categorizes these ACO genes into three discernible classes, namely Class I, Class II, and Class III. Further scrutiny via collinearity assessment indicates a lack of collinearity relationships among these classes, highlighting variations in conserved motifs and promoter types within each class. Transcriptome analysis unveils significant disparities in both expression levels and trends of ACO genes in fruits exhibiting respiratory bursts compared to those that do not. Employing Weighted Gene Co-Expression Network Analysis (WGCNA), we discern that the co-expression correlation of ACO genes within loquat fruit notably differs from that observed in apples. Our findings, derived from Gene Ontology (GO) enrichment results, signify the involvement of ACO genes and their co-expressed counterparts in biological processes linked to terpenoid metabolism and carbohydrate synthesis in loquat. Moreover, our exploration of gene regulatory networks (GRN) highlights the potential pivotal role of the GNAT transcription factor (Ejapchr1G00010380) in governing the overexpression of the ACO gene (Ejapchr10G00001110) within loquat fruits. The constructed HMM of ACO proteins offers a precise and systematic method for identifying plant ACO proteins, facilitating phylogenetic reconstruction. ACO genes from representative Rosaceae fruits exhibit diverse expression and regulative patterns, warranting further function characterizations.