Dan Yao

The soybean GmPEAMT1/GmPLMT double-knockout mutant (Dko) was constructed to investigate how the disruption of phospholipid-related genes affects reproductive traits, seed nutrient composition, and associated molecular responses under experimental conditions. Dko plants showed coordinated changes in photosynthetic parameters and reproductive structures: the number of pollen grains and ovules was reduced, whereas pollen and ovule size-related traits were increased, while mature seed number and 100-seed weight were maintained. Developing Dko seeds showed higher protein content and increased levels of 12 out of 18 measured amino acids, particularly alanine and cysteine. Transcriptomic and qRT-PCR analyses further indicated coordinated changes in amino acid, pantothenate, coenzyme A, glycerolipid, and glycerophospholipid-related pathways. Lipidomics provided complementary evidence of changes in phospholipid-associated and other lipid molecular features, whereas total seed lipid content showed no significant difference from the control. Together, these results suggest that GmPEAMT1/GmPLMT disruption was associated with variations in reproductive traits at the developmental stages examined and seed nutrient allocation, with lipid-feature changes representing one component of a broader multi-omic response. The proposed lipid and amino acid metabolic remodeling in GmPEAMT1/GmPLMT double-knockout soybean seeds should be regarded as a working hypothesis because direct metabolic flux, protein abundance, and enzyme activity are not measured.

Two MYB transcription factors, GmMYB73 and GmMYB85, were identified as positive regulators of soybean oil biosynthesis. They enhance seed oil content and fatty acid composition through distinct protein interaction partners. This study establishes a novel 'ncRNA-TF-oil trait' regulatory framework in soybean. MYB transcription factors (MYBs), which function as trans-acting regulatory proteins containing the conserved MYB DNA-binding domain, represent the largest family of transcription factors in plants. In this study, we investigated the regulatory role of soybean MYB transcription factors in lipid biosynthesis. By integrating existing transcriptome data from soybean pods at defined developmental stages (R3-R5 stages, days after flowering, DAF), we systematically identified two MYB transcription factors (GmMYB73 and GmMYB85). To investigate the biological functions of GmMYB73/GmMYB85, we constructed plant overexpression vectors and generated transgenic Arabidopsis thaliana lines. The transgenic seeds showed significantly increased thousand-grain weight (by 10.94 and 10.53% for GmMYB73 and GmMYB85, respectively) and elevated triglyceride content (by 11.34 and 9.07%, respectively). Conversely, we observed substantial reductions in soluble sugars (decreased by 34.44 and 16.67%) and starch content (decreased by 2.08 and 43.75%). Analysis of fatty acid composition revealed significant increases in three major unsaturated fatty acids: oleic acid (up by 127.82 and 205.95%), linoleic acid (increased by 138.92 and 187.98%), and α-linolenic acid (elevated by 125.97 and 172.64%). Through screening in the soybean young pod transcriptome database, upstream genes of GmMYB73 and GmMYB85 were identified as lncRNA44155 and lncRNA22084. Quantitative PCR confirmed their interaction in transgenic hairy roots, with lncRNA44155 and lncRNA22084 activating GmMYB73 and GmMYB85 expression respectively. Protein interaction analysis demonstrated differential regulatory mechanisms: GmMYB73 physically associates with the fatty acid biosynthetic enzyme GmFAB2, whereas GmMYB85 directly binds to GmLEC1b, the master controller of seed lipid accumulation. This study provides the first evidence that MYB transcription factors regulate soybean oil metabolism through a noncoding RNA-mediated multilevel mechanism, establishing a novel 'ncRNA-TF-oil trait' regulatory framework.

Soybean (Glycine max) is an important model crop for studying plant functional genes, such as the NAC transcription factor (TF) gene family. The NAC transcription factor (TF) family is one of the largest plant-specific TF families and plays critical roles in plant growth, development, and stress responses. In this study, we performed a pan-genome-wide analysis of NAC genes using 29 soybean genomes. A total of 5051 NAC genes were identified and clustered into 245 orthologous gene groups (OGGs), including 58 core, 88 soft-core, 32 shell, and 67 cloud groups. Based on phylogenetic relationships, the representative NAC OGGs were assigned to 18 subfamilies, 17 of which contained soybean NAC genes. Gene duplication analysis indicated that whole-genome duplication (WGD)/segmental duplication was the predominant driver of NAC family expansion, accounting for 90.88% of duplication events. Approximately 39.30% of NAC genes carried at least one intact transposable element (TE) within 2 kb upstream or downstream regions. NAC genes with copy number variation (CNV) harbored more nearby TEs than non-CNV genes (1.54 vs. 1.31 TEs per gene), and dispensable NAC genes contained more nearby TEs than core NAC genes (1.59 vs. 1.33 TEs per gene). These results indicate a significant association between local TE abundance and NAC gene CNV or dispensability. Selection pressure analysis showed that dispensable NAC genes had higher Ka, Ks, and Ka/Ks values than core genes, suggesting relatively relaxed evolutionary constraints. Expression profiling across six tissues revealed distinct transcriptional patterns among NAC subfamilies. Structurally conserved subfamilies generally showed broader expression, whereas structurally divergent subfamilies displayed greater expression variability. Regulatory network and Gene Ontology (GO) enrichment analyses suggested that conserved subfamilies were mainly associated with stress responses, while divergent subfamilies were related to cell wall regulation, signal transduction, and ion homeostasis. Further analysis of Wm82 drought RNA-seq data prioritized several putative drought-responsive NAC candidates, including Glyma.16G043200, Glyma.06G248900, Glyma.07G050600, Glyma.12G206900, and Glyma.18G261300. Overall, these findings elucidate the mechanisms of expansion and the functional divergence of the NAC gene family at the soybean pan-genome level, providing a theoretical basis for understanding NAC gene evolution and facilitating future crop improvement.

Seabuckthorn (Hippophae rhamnoides Linn.) is a commonly utilized medicinal crop with various applications in the treatment of different diseases. Two particularly noteworthy nutrients in seabuckthorn fruit are seabuckthorn oil and flavonoids. In recent years, seabuckthorn oil has attracted considerable attention due to its perceived benefits for beauty and healthcare. Consequently, there is a clear need for further research into seabuckthorn oil. While numerous studies have been conducted on the regulation of oil by the FAD2 gene family, there is a paucity of literature examining the molecular mechanism of FAD2 gene involvement in seabuckthorn oil regulation. Accordingly, two FAD2 genes have been identified in seabuckthorn, which are classified differently and perform distinct functions. Both genes are located in the endoplasmic reticulum. Following transient expression in seabuckthorn fruits, it was observed that HrFAD2-1 and HrFAD2-3 were capable of influencing the synthesis of α-linolenic acid, with HrFAD2-1 additionally demonstrated to facilitate the synthesis of lysophosphatidic acid. All of the aforementioned genes have been observed to promote jasmonic acid (JA) synthesis. The heterologous transformation of Linum usitatissimum demonstrates that both HrFAD2-1 and HrFAD2-3 are capable of promoting plant growth. The HrFAD2-1 gene was observed to significantly increase the content of major fatty acids in Linum usitatissimum Linn seeds, whereas the HrFAD2-3 gene appeared to be primarily involved in the regulation of plant growth and development. In conclusion, a preliminary investigation into the functions of the HrFAD2-1 and HrFAD2-3 genes in fatty acid synthesis was conducted. This revealed that HrFAD2-1 is closely associated with oleic acid synthesis and acts as a negative regulator. Furthermore, our findings will provide a foundation for subsequent investigations into the fatty acid synthesis pathway in Hippophae rhamnoides oil, offering a theoretical basis for subsequent studies at the molecular level.

Soybean is an important grain, oil and feed crop, which plays an important role in ensuring national food security. However, soil salinization hinders and destroys the normal physiological metabolism of soybean, resulting in the abnormal growth or death of soybean. The XTH gene can modify the plant cell wall and participate in the response and adaptation of plants to negative stress. To elucidate the role of the overexpressed GmXTH1 gene under NaCl-induced stress in soybean, we determined the germination rate, the germination potential, the germination index, seedling SOD activity, POD activity, the MDA content and the MDA content during the germination stage of the overexpressed lines of the GmXTH1 gene, the OEAs (OEA1, OEA2 and OEA3), the interference expression line IEA2, the control mutant M18, the CAT content and the chlorophyll content. The relative expression of the GmXTH1 gene in the material OEA1 and the contents of Na⁺ and K⁺ in the roots after stress were also determined. The results showed that OEAs exhibited enhanced germination indices, including the germination rate and germination potential, and were less sensitive to stress compared with the mutant M18. In contrast, the inhibitory effect of NaCl was more pronounced in the line with a disturbed expression of GmXTH1 (IEA2). The OEAs exhibited more enzyme activities and a lower MDA content, indicating reduced oxidative stress, and maintained higher chlorophyll levels, suggesting improved photosynthetic capacity. Relative expression analysis showed that the GmXTH1 gene was rapidly up-regulated in response to NaCl, peaking at 4 h after treatment, and subsequently declining. This temporal expression pattern correlated with the enhanced salt tolerance observed in OEA1. Notably, OEA1 accumulated more Na⁺ and maintained higher K⁺ levels, indicating effective ionic homeostasis under stress. Collectively, these results suggest that the overexpression of the GmXTH1 gene may positively regulate plant responses to salt stress by modulating the antioxidant defense and ion transport mechanisms.

With the rapid advances in next-generation sequencing technology, numerous non-protein-coding transcripts have been identified, including long noncoding RNAs (lncRNAs), which are functional RNAs comprising more than 200 nucleotides. Although lncRNA-mediated regulatory processes have been extensively investigated in animals, there has been considerably less research on plant lncRNAs. Nevertheless, multiple studies on major crops showed lncRNAs are involved in crucial processes, including growth and development, reproduction, and stress responses. This review summarizes the progress in the research on lncRNA roles in several major crops, presents key strategies for exploring lncRNAs in crops, and discusses current challenges and future prospects. The insights provided in this review will enhance our comprehension of lncRNA functions in crops, with potential implications for improving crop genetics and breeding.

Plant roots play crucial roles in their response to drought conditions. However, the molecular responses in soybean roots to drought stress remain unclear. We investigated the alterations in the protein expression in the roots of a drought-resistant soybean cultivar ‘Jiyu 47’ during the seedling phase based on tandem mass tag (TMT) proteomics analysis. The results revealed significant variations in the expression of the proteins involved in several metabolic pathways in soybean roots, including sucrose metabolism, abscisic acid (ABA) metabolism, and the ATP-binding cassette (ABC) transporter pathway. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed a coordinated expression pattern of the proteins involved in the various cellular pathways responding to drought stress in soybean. The increased production of sucrose and betaine enhanced the inhibition of the damage caused by reactive oxygen species (ROS) and the tolerance of drought stress. The results of the physiological variations showed that sucrose metabolism, ABA metabolic mechanism, and the ABC transporter pathways played an important role in the antioxidant defense system in response to drought stress in soybean roots. The results of quantitative real-time PCR revealed the up-regulated expression of three genes (i.e., GmPYR1, GmHO-1, and GmSOD) involved in ABA biosynthesis and the signaling pathway. This study provides novel insights into the comprehension of the molecular pathways regulating the soybean root response to drought stress.

Soybean [Glycine max (Linn.)Merr.]is an important oil crop. Long noncoding RNAs (lncRNAs) play a varietyof functions in plants. However, their function in the soybean oilsynthesis pathway is yet to be uncovered. Here, the lncRNA43234 gene related to soybean oil synthesis was screened, and the full-lengthcDNA sequence of the lncRNA was obtained using rapid amplificationof cDNA ends. Overexpression of lncRNA43234 increasedthe content of crude protein in seeds, decreased the content of oleicacid, and affected the content of alanine and arginine in free aminoacids. RNA interference of the lncRNA43234 gene decreasedthe crude protein content in seeds. Quantitative real-time polymerasechain reaction analysis revealed that lncRNA43234 influenced the expression of XM_014775786.1 associatedwith phosphatidylinositol metabolism by acting as a decoy for miRNA10420, thereby affecting the content of soybean oil.Our results provide insights into how lncRNA-mediated competing endogenousRNA regulatory networks are involved in soybean oil synthesis.

Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

Δ¹²-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family—GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C—and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T₁ generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.