Characterization and interaction analysis of Eg14-3-3s involved in oil biosynthesis for MAS in Elaeis guineensis

Abstract

Members of the 14-3-3 protein family regulate lipid biosynthesis through interactions with transcription factors such as WRINKLED1 (WRI1); however, the roles of the Eg14-3-3 isoforms in oil palm remain poorly understood. In this study, we investigated whether Eg14-3-3 proteins play a role in oil biosynthesis and evaluated their interaction with EgWRI1. Subcellular localization predictions indicated that Eg14-3-3 proteins are present in both the cytoplasm and the nucleus. Protein-protein interaction analyses revealed that several Eg14-3-3 isoforms interact with EgWRI1, with the interaction of the Eg14-3-3-like protein D isoform X2 being the strongest. Functional analysis revealed that transient expression of the Eg14-3-3-like protein D isoform X2 significantly increased oil accumulation. Furthermore, high-resolution melting (HRM) markers targeting the Eg14-3-3-like protein D isoform X2 and EgWRI1 could effectively distinguish between low- and high-yielding cultivars. Overall, the integration of functional genomics and HRM-based marker development represents an effective strategy for accelerating oil palm breeding.

Keywords:
14-3-3 proteins; oil palm; Elaeis guineensis; protein-protein interaction; HRM-based marker

INTRODUCTION

The oil palm (Elaeis guineensis Jacq.) is among the most important oil-producing crops worldwide and has a wide range of applications, including in the food industry and in biodiesel production (Murphy et al. 2021). Maximizing the yield-density relationship through improved cultivars can help mitigate future tropical deforestation and biodiversity loss by reducing the land area required to meet global oil demand. Therefore, increasing oil yield through genetic improvement has become a major focus of oil palm research (Babu et al. 2021, Chaves et al. 2021, Murphy et al. 2021). Marker-based breeding approaches that integrate genomic, functional, and transcriptomic information can facilitate the development of molecular markers associated with high-yield traits, thereby enhancing the efficiency of breeding programs (Pott et al. 2021, Pacheco et al. 2023, Martin et al. 2025).

In oil palm, oil predominantly accumulates in the mesocarp storage tissue, where genes involved in lipid biosynthesis are highly expressed, including key genes responsible for fatty acid modification and triacylglycerol (TAG) assembly (Martin et al. 2025, Yuan et al. 2025). Several transcriptional regulators play crucial roles in oil accumulation and biosynthesis by controlling carbon partitioning at the metabolic level, ultimately leading to increased oil yield. Among these regulators, WRINKLED1 (WRI1) is a central transcription factor that regulates glycolysis and fatty acid biosynthesis, contributing to increased seed oil content (Yang et al. 2022, Zhang et al. 2024, Song et al. 2025). In oil palm, the expression of EgWRI1 is greatest in the mesocarp, the primary oil-accumulating tissue, and its expression increases during fruit development, particularly at the ripening stage. EgWRI1 is a divergent ortholog of Arabidopsis thaliana WRI1 (AtWRI1) and is characterized by a 93-amino acid deletion at the C-terminus compared with AtWRI1. Despite this divergence, EgWRI1 is highly expressed in the mesocarp and is believed to regulate plastidial pyruvate supply and fatty acid synthesis, thereby promoting lipid accumulation (Bourgis et al. 2011, Singh et al. 2013, Zhang et al. 2019).

In plants, 14-3-3 proteins have been extensively studied in Arabidopsis and Oryza sativa, in which they are known to play key roles in flowering regulation, metabolic processes, and the accumulation of seed storage reserves, including starch and lipids. 14-3-3 proteins constitute a family of conserved regulatory molecules that modulate multiple signaling pathways by functioning as phosphopeptide-binding proteins and by facilitating protein trafficking through the formation of protein-protein interaction complexes (Obsilova and Obsil 2022, Strini et al. 2022, Tang et al. 2025). Plant 14-3-3 isoforms are encoded by multigene families with slightly divergent sequences and are involved in diverse biosynthetic and signaling processes during plant growth and development, including interactions with transcription factors and receptor proteins (Nakkaew et al. 2013, Ma et al. 2016, Camoni et al. 2018, Obsilova and Obsil 2022, Yang et al. 2022, Fan et al. 2024). Recent studies have reported that AtWRI1 interacts with At14-3-3 proteins and that the coexpression of these proteins leads to increased oil accumulation in transgenic plants by increasing transcriptional activity and stabilizing WRI1 protein levels (Ma et al. 2016). Our previous work revealed that the Eg14-3-3 ω protein plays important roles in glycolysis, fatty acid biosynthesis, and oil accumulation in plant cells (Nakkaew et al. 2013). However, the other Eg14-3-3 isoforms have not been systematically studied, and comparative analyses of their interactions with EgWRI1 remain unexplored.

Therefore, the aim of this study was to perform comprehensive molecular characterization and in silico sequence analysis of the Eg14-3-3 gene family, including phylogenetic relationship analysis. In vivo protein-protein interactions between the Eg14-3-3 isoforms and EgWRI1 were investigated using yeast two-hybrid (Y2H) assays. Functional analyses were conducted through transient overexpression of selected genes in tobacco leaves to evaluate their effects on oil accumulation. Finally, the Eg14-3-3-like protein D isoform X2 and EgWRI1 were identified as promising target genes for marker-assisted selection. A high-resolution melting (HRM) approach based on the 5′ UTRs of the Eg14-3-3-like protein D isoform X2 and EgWRI1 was developed to discriminate between oil palm cultivars, providing a practical molecular tool for breeding programs aimed at improving oil yield.

MATERIAL AND METHODS

Identification and phylogenetic analysis of the Eg14-3-3 gene family

Based on our previous research, five Eg14-3-3 genes were cloned (Phongdara et al. 2012), whereas seven other Eg14-3-3 genes were identified in the genome database of oil palm (Elaeis guineensis var. tenera taxid:51953) (Singh et al. 2013). The identities of twelve Eg14-3-3 candidate genes were analyzed using the BLASTN and BLAST genome programs via Web BLAST. They were subsequently mapped to oil palm chromosomes on the basis of the E. guineensis genome database (NCBI), and chromosomal positions and a distribution map were generated using MapInspect software. In addition, the exon‒intron structures of the Eg14-3-3 genes were generated using the Gene Structure Display Server version 2.0 (GSDS; http://gsds.cbi.pku.edu.cn/). In silico physicochemical characterization was performed using the ExPASy server (https://www.expasy.org/). Subcellular localizations were predicted using pLoc-mPlant (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/).

Phylogenetic analysis of the Eg14-3-3 protein family

The sequences of the twelve Eg14-3-3 protein family members, along with all orthologs of 14-3-3 proteins, were obtained from NCBI. Multiple sequence alignment analysis was performed using ClustalX2.1 and was visualized using GENEDOC software. The phylogenetic tree analysis and evolutionary history were inferred using the neighbor-joining method with 1,000 replicates in MEGA 11.0 software. Subsequently, the phylogenetic tree was visualized and renamed using iTOL (https://itol.embl.de/).

In vivo analysis of protein‒protein interactions between Eg14-3-3s and EgWRI1

In vivo analysis of protein‒protein interactions between Eg14-3-3s and EgWRI1 was performed using a yeast two-hybrid assay and quantitative β-galactosidase assays. First, specific primer pairs were designed based on the CDSs of the twelve Eg14-3-3 genes. cDNA synthesis was performed using SuperScript™ III Reverse Transcriptase (Invitrogen, United States) according to the manufacturer’s instructions. Full-length amplification was performed using 5x FIREPol® Master Mix (Solis BioDyne, Estonia) according to the manufacturer’s instructions, allowing the full sequences of the Eg14-3-3 genes to be cloned and sequenced. The Eg14-3-3 genes were subsequently cloned, inserted into the pGBKT7 vector, and transformed into the Saccharomyces cerevisiae strain Y2HGold. EgWRI1 was cloned, inserted into the pGADT7 vector, and transformed into S. cerevisiae strain Y187 using the Yeastmaker™ Yeast Transformation System 2 according to the manufacturer’s protocol (Clontech, United States). Colony PCR using specific primers was then performed on selected colonies (Table S1). Comparative analyses of their interactions were conducted using drop dilution assays on TDO (SD/-Trp/-Leu/-His) with X-α-gal. To determine the relative strength of the protein-protein interactions, a pellet X-gal assay (PXG assay) was performed using the Möckli and Auerbach procedure (Möckli and Auerbach 2004). Yeast cultures were grown to an OD₆₀₀ of 0.8, pelleted, resuspended in 20 µL of PBS, lysed using two freeze-thaw cycles (3 min cycle^-1), and incubated for 26 h. β-Galactosidase activity was subsequently calculated using the equation: 1,000 × Corrvalue/[t × V × OD₆₀₀], following the method of Trimborn et al. (2022). The assay was performed in three biological and two technical replicates (Trimborn et al. 2022). Statistical comparisons between groups were conducted using ANOVA with post hoc tests in SPSS software version 26.

Transient expression of Eg14-3-3 and fatty acid analysis in tobacco

For the transient experimental analyses, tobacco (Nicotiana tabacum) was grown in an enclosed field at the Prince of Songkla University, and two-month-old tobacco plants were selected for transient expression analysis. Constructs encoding Eg14-3-3-like protein and Eg14-3-3-like protein D isoform X2 were generated in pCAMBIA1303 (Cambia, Australia) under the control of the 35SCaMV promoter, and the sequence was verified for orientation before being transformed into Agrobacterium tumefaciens LBA4404 (Invitrogen, United States). The transformants were cultured, activated using 200 μM acetosyringone (AS; Invitrogen, United States), and infiltrated into N. tabacum leaves using a syringe. After 5 days, the leaves were harvested, and total lipids were extracted using the Folch procedure with minor modifications (Bourgis et al. 2011). In brief, frozen samples (300 mg; n = 4 independent leaves per group) were ground into powder and homogenized with a homogenizer pestle in 30 mL of chloroform/methanol (2:1, v/v) (RCI Labscan, Thailand). The mixture was centrifuged at 4,000 rpm for 10 min, after which the supernatant was collected. The debris was re-extracted in 15 mL of chloroform/methanol (2:1, v/v), mixed with 10 ml of 0.88% potassium chloride in water, and centrifuged at 4,000 rpm for 10 min. The chloroform phase (bottom layer) was collected, evaporated to dryness, and reconstituted with 1 ml of hexane (RCI Labscan, Thailand). Finally, the extracted lipids were analyzed. Total fatty acids were converted to fatty acid methyl esters (FAMEs) by the Office of Scientific Instrument and Testing of Prince of Songkla University using an Agilent 7890A gas chromatograph and an Agilent CP9080 column (Agilent Technologies, United States). The percentage composition of each fatty acid was then quantified and compared in duplicate.

DNA extraction, real-time PCR, and high-resolution melting (HRM) analysis

Genomic DNA from E. guineensis leaves was extracted from fifteen-year-old plants representing low- and high-yield variants of tenera oil palm cultivars (n = 8 per group). The samples were provided by Prof. Dr. Theera Eksomtramage from the Agricultural Research Station, Klong Hoi Khong, Songkhla, Thailand. Oil yield data for these samples were recorded over five years; high-yield variants produced 26.32-43.68 kg of oil per tree per year, whereas low-yield variants produced 1.98-12.43 kg per tree per year (Nakkaew et al. 2008). The collected samples were immediately frozen in liquid nitrogen and stored at −80 °C until genomic DNA extraction was performed using a Plant Genomic DNA Mini Kit (Geneaid Biotech, Taiwan) following the manufacturer’s instructions. The DNA samples (50 ng) were subjected to real-time PCR with 5′-UTR-targeting primers and iQ™ SYBR^® Green Supermix (Bio-Rad, United States) according to the manufacturer’s instructions (Table S1). The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products were denatured at 95 °C for 10 s, and the melting temperature was increased from 65 °C to 95 °C in 0.5 °C increments via a Bio-Rad qPCR CFX96 Real-Time System (Bio-Rad, United States). Melting curves were subsequently analyzed using CFX Manager™ software, and HRM analysis was performed using Precision Melt Analysis™ software with default parameters. Each sample was analyzed in two independent runs (Nakkaew et al. 2024).

RESULTS AND DISCUSSION

Localization in chromosomes and gene structure of the Eg14-3-3 gene family

Based on recent research, five Eg14-3-3 genes of Elaeis guineensis were cloned (Phongdara et al. 2012), and an additional seven Eg14-3-3 genes were identified in the oil palm genome in the NCBI database. Sequence analysis of these twelve putative Eg14-3-3 family genes revealed gene lengths ranging from 670 to 1,618 bp in length (Table S2). Their chromosomal localization was analyzed and mapped based on the EG5 oil palm genome assembly (GCF_000442705.1). Ten of the twelve Eg14-3-3 genes were mapped to six of sixteen chromosomes (chromosomes 1, 3, 6, 7, 13 and 15) (Figure 1A). The remaining two genes, encoding Eg14-3-3-like protein GF14 kappa and Eg14-3-3-like protein 16R, are located on the unplaced genomic scaffolds EG5 p5_sc00068 and EG5 p5_sc26766, respectively (Table S2). In addition, gene structure analysis revealed that most Eg14-3-3 genes contain between 4 and 7 exons, including untranslated regions (UTRs) with upstream/downstream regions and introns of variable lengths. In contrast, the genes encoding Eg14-3-3-like protein 16R and Eg14-3-3-like protein GF14 omega had only two exons and one exon, respectively (Figure 1B, Table S2). These analyses reveal substantial structural diversity within the Eg14-3-3 gene family in oil palm, similar to the variation observed among 14-3-3s in Arabidopsis thaliana and Oryza sativa (Yao et al. 2007, Fan et al. 2024, Zhang et al. 2024). Subcellular localization predictions indicated that all the Eg14-3-3 proteins are typically localized in the nucleus, while four members also exhibited potential cytoplasmic localization (Table S2). These predicted localization results suggest that Eg14-3-3 proteins have functional flexibility and may play a role in various biological processes, which is consistent with Arabidopsis 14-3-3 proteins that interact with WRINKLED1 (WRI1) to regulate transcription during oil biosynthesis to increase seed oil content (Ma et al. 2016). Multiple sequence alignment revealed that all the Eg14-3-3 proteins contained conserved 14-3-3 protein signature motifs, including signatures I and II, with the exception of Eg14-3-3 protein 6 and Eg14-3-3-like protein 16R (Figure 2A). Phylogenetic analysis was performed to classify the Eg14-3-3 proteins into two distinct subgroups: five epsilon and seven non-epsilon isoforms. These groups clustered closely with the corresponding epsilon and non-epsilon 14-3-3 proteins from A. thaliana and O. sativa (Figure 2B) (Yao et al. 2007, Zhang et al. 2024). This close evolutionary relationship suggests that these genes have conserved biological functions, including roles in metabolism, signal transduction, and lipid-related regulatory processes during plant development (Ma et al. 2016, Camoni et al. 2018, Gao et al. 2021, Yang et al. 2022). However, experimental validation of the subcellular localization of the Eg14-3-3 proteins and the functional relationship between the Eg14-3-3 proteins and EgWRI1 in oil palm is lacking and requires further investigation.

Figure 1
Structural analysis of oil palm Eg14-3-3 genes. A. MapInspect was used to plot the chromosomal location of each Eg14-3-3 gene based on the distribution analysis (scale: one million bases (Mb)). B. The gene structures of Eg14-3-3 were analyzed using the GSDS database. The ε-group genes are shown in blue, whereas the non-ε-group genes are shown in red.

Figure 2
Multiple amino acid sequence alignments and phylogenetic analysis of the Eg14-3-3 protein family. A. The aligned protein sequences of the Eg14-3-3 protein family were visualized using GeneDoc; 14-3-3 protein signature motifs are shown in red boxes. B. Phylogenetic analysis of the Eg14-3-3 protein family using the neighbor-joining method with 1,000 bootstrap replicates.

In vivo interactions between the Eg14-3-3 protein and EgWRI1

The full-length coding sequences of the Eg14-3-3 gene family members and EgWRI1 were used in the interaction analysis (Figure S1). However, only five Eg14-3-3 genes were successfully cloned: those encoding Eg14-3-3-like protein D, Eg14-3-3-like protein, Eg14-3-3-like protein D isoform X2, Eg14-3-3-like protein GF14 iota, and Eg14-3-3-like protein 16R. Protein‒protein interaction analysis using TDO with an X-α-gal assay revealed that the interactions of Eg14-3-3-like protein D isoform X2 and Eg14-3-3-like protein GF14 iota with EgWRI1 were the strongest (Figure 3A). Quantitative X-gal activity analysis further revealed that compared with Eg14-3-3-like protein and Eg14-3-3-like protein GF14 iota, the interaction of Eg14-3-3-like protein D isoform X2 with EgWRI1 was significantly stronger (p < 0.05; Student’s t test) (Figure 3B). These results suggest that the Eg14-3-3-like protein D isoform X2 may play a key regulatory role. This is similar to Arabidopsis 14-3-3 proteins, which have been shown to strongly interact with AtWRI1 in yeast two-hybrid assays (Ma et al. 2016), and it represents a promising candidate for functional studies on its regulatory role in the regulation of oil biosynthesis.

Figure 3
A. Yeast two-hybrid and B relative quantitative X-gal activity results showing interactions between the Eg14-3-3 proteins and EgWRI1. The relative X-gal activity represents three biological and two technical replicates and is normalized using the negative control value. The error bars indicate the standard deviation, whereas the asterisks indicate a statistically significant difference between Eg14-3-3-like protein D isoform X2 and pGBKT7 (p < 0.05 based on ANOVA with post hoc tests). Significant differences between groups are marked with different letters.

Functional analysis of Eg14-3-3s via transient expression in tobacco leaves

The transient expression of the Eg14-3-3-like protein and the Eg14-3-3-like protein D isoform X2 in tobacco leaves resulted in notable changes in fatty acid composition. Total fatty acids were extracted, converted to fatty acid methyl esters (FAMEs), and analyzed by gas chromatography (GC) using 1 µg of C:17 as an internal standard. Compared with the control (pCAMBIA1303), both transgenic constructs led to increased unsaturated oil production (Figure 4A-C). Fatty acid profiling revealed significant increases in saturated fatty acids, which were quantified (Figure S3). The results revealed that palmitic acid (C16:0) concentrations reached 48.03 ± 1.59 and 43.53 ± 2.87 µg, whereas stearic acid (C18:0) concentrations reached 30.05 ± 0.72 and 28.49 ± 1.88 µg in leaves expressing the Eg14-3-3-like protein and the Eg14-3-3-like protein D isoform X2, respectively. In contrast, the control (pCAMBIA1303) presented lower levels of palmitic acid (38.73 ± 8.46 µg) and stearic acid (27.76 ± 6.06 µg). In addition, increased levels of unsaturated fatty acids were observed, including linoleic acid (C18:2) with values of 18.85 ± 0.91 and 12.26 ± 0.81 µg, and linolenic acid (C18:3) with values of 48.84 ± 2.36 and 45.01 ± 2.96 µg. By comparison, the control (pCAMBIA1303) resulted in lower levels of linoleic acid (5.25 ± 1.17 µg) and linolenic acid (11.38 ± 2.49 µg). Notably, oleic acid (C18:1) was detected exclusively in leaves transiently expressing both Eg14-3-3 constructs. These findings suggest that the expression of Eg14-3-3-like proteins and the expression of the Eg14-3-3-like protein D isoform X2 increase oil accumulation. These findings are consistent with those of previous studies reporting that 14-3-3 proteins act synergistically with WRI1 to increase triacylglycerol (TAG) accumulation and stabilize WRI1 in Nicotiana benthamiana leaves (Ma et al. 2016). Therefore, the observed differences in fatty acid accumulation suggest that these two Eg14-3-3 proteins may participate in lipid metabolism. The observed differences may be associated with differential gene expression potentially influenced by variants in the 5′ untranslated region (5′ UTR).

Figure 4
Fatty acid profiling based on gas chromatography analysis of tobacco leaves transiently expressing Eg14-3-3-like proteins. A. and Eg14-3-3-like protein D isoform X2. B. compared with the control group, C and C:17 as an internal standard.

High-resolution melting (HRM) analysis for genotyping of oil palm cultivars

Previous results revealed a strong interaction between the Eg14-3-3-like protein D isoform X2 and EgWRI1 and its positive effect on fatty acid accumulation. Therefore, the 5′ untranslated region (5′ UTR) of the Eg14-3-3-like protein D isoform X2 was analyzed for regulatory elements. Similar to those reported in the EgWRI1 promoter, functional cis-elements, including ethylene-responsive transcription factor motifs and a CCAAT box binding domain, were identified (Zhang et al. 2019). Specific primer pairs targeting the 5′ UTRs of the Eg14-3-3-like protein D isoform X2 and EgWRI1 based on the E. guineensis var. tenera genome (taxid: 51953) were designed (Table S1), and fragments of the expected HRM amplicons (~880 bp and ~460 bp, respectively) were amplified and directly sequenced (Figures S2 and S3). HRM analysis using these primers could successfully distinguish between low- and high-yield tenera cultivars. Normalized temperature-shifted melting curves clearly separated low-yield (red lines) and high-yield (green lines) genotypes (Figure 5A-D). These results revealed that the 5′ UTRs of the Eg14-3-3-like protein D isoform X2 and EgWRI1 can serve as reliable molecular markers for distinguishing oil palm cultivars with different oil yields. HRM-based marker-assisted selection provides an efficient and rapid tool for breeders to identify high-yielding genotypes, thereby accelerating breeding programs and improving the sustainability of oil palm production (Li et al. 2018, Pacheco et al. 2023, Nakkaew et al. 2024).

Figure 5
High-resolution melting analysis using the 5′ UTR of the Eg14-3-3-like protein D isoform X2 (5A and 5B) and the 5′ UTR of EgWRI1 (5C and 5D). Red and green lines indicate low- and high-yielding variants of tenera oil palm cultivars, respectively (n = 8).

CONCLUSIONS

Oil palm (Elaeis guineensis) is among the most important oil-producing crops worldwide. However, its long breeding cycle presents major challenges for improving yield and sustainable production. Integrating genomics, functional analysis, and marker-assisted selection offers an effective strategy for accelerating breeding programs. Understanding molecular mechanisms, including transcriptional regulators associated with oil accumulation, is therefore critical for oil crop improvement (Pott et al. 2021, Chen et al. 2024, Martin et al. 2025, Song et al. 2025). In this study, an analysis of the interaction of Eg14-3-3s with EgWRI1 in oil palm was conducted. Twelve putative Eg14-3-3s were identified and characterized, revealing diverse gene structures and conserved evolutionary relationships with 14-3-3 proteins from Arabidopsis and Oryza. Subcellular localization predictions suggested that these proteins are predominantly localized in the nucleus, which is consistent with their roles in transcriptional regulation. However, only five Eg14-3-3 genes were successfully cloned; the remaining seven genes could not be cloned, possibly because of their low expression levels and highly variable expression patterns across different developmental stages. Protein-protein interaction analysis revealed that the Eg14-3-3-like protein D isoform X2 interacts most strongly with EgWRI1, and transient expression and fatty acid profiling confirmed that it significantly increases fatty acid accumulation, which is consistent with the synergistic regulation of oil biosynthesis by 14-3-3 proteins and WRI1 in Arabidopsis (Ma et al. 2016, Yang et al. 2022). Moreover, HRM-based genotyping using the 5′ UTRs of the Eg14-3-3-like protein D isoform X2 and EgWRI1 could successfully distinguish between low- and high-yielding E. guineensis tenera cultivars. These findings revealed the Eg14-3-3-like protein D isoform X2 and EgWRI1 as possible markers for further validation in large-scale studies and provide a useful approach for marker-assisted selection in oil palm breeding. However, HRM-based genotyping markers should be modified by labeling specific primers with fluorescent dyes to increase the accuracy and specificity of HRM genotyping analysis. The application of such markers has the potential to enable early identification of high-yielding cultivars, shorten breeding cycles, and support the development of more efficient and sustainable oil palm production.

ACKNOWLEDGEMENTS

The authors wish to thank The Center of Genomics and Bioinformatics Research Molecular Biotechnology and Bioinformatics, Faculty of Science , for instrument support. This research and innovation activity is funded by National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Grant No SCI6505048S and SCI6505049S) and the Faculty of Science Research Fund, Prince of Songkla University, Contract no. 1-2563-02-008.

Data Availability Statement

The supplementary file and datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

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Edited by

• SCIENTIFIC EDITOR:
  Luiz Antônio dos Santos Dias

Publication Dates

• Publication in this collection
  28 Sept 2026
• Date of issue
  2026

History

• Received
  29 Sept 2025
• Accepted
  19 July 2026
• Published
  07 Aug 2026