The aggravation of soil salinization has become one of the major factors that threaten crop growth and yield. Rhizobia, as an important biological nitrogen-fixing microorganism, can establish symbiotic relationships with legumes to improve their nitrogen-fixing ability and stress tolerance. Trehalose, a non-reducing disaccharide that is widely found in bacteria, fungi, and plants, can protect cellular structures and maintain the viability of cells under stress conditions. However, it remains to be determined whether the endogenous trehalose level in rhizobia could affect its stress tolerance and nitrogen-fixing capabilities. In this study, we constructed four engineered rhizobial strains to examine the effects of the overexpression and knockout of the trehalose synthesis genes otsA/otsB in the rhizobium strain CCBAU25338 on its salt tolerance and nitrogen-fixing capacity. The results indicated that the overexpression of otsA, rather than the otsB gene, significantly enhanced both the stress tolerance and nitrogen-fixing abilities of the strains. Furthermore, the inoculation of otsA-overexpressing recombinant cells leads to greater agronomic traits in the host plant’s peanuts under salinity conditions. We hope our findings may serve as valuable references for the future development of efficient and superior engineered rhizobial strains for peanut cultivation.

Neocinnamomum caudatum (Nees) Merr. is an underutilized woody oil plant with seeds rich in long-chain fatty acids and polyunsaturated fatty acids. Reliable quantitative real-time PCR (RT-qPCR) analysis is essential for investigating the molecular mechanisms underlying seed oil biosynthesis, but suitable reference genes have not yet been validated in this species. Here, seven candidate reference genes, namely EF-1α, ACT2, ACT11, UBQ11, TUA, F-BOX, and GAPDH, were selected from transcriptomic data and evaluated in leaves, flowers, and developing seeds of N. caudatum. Their expression stability was assessed using geNorm, NormFinder, and BestKeeper, followed by comprehensive ranking with RankAggreg. Among all tested samples (leaves, flowers and developing seeds combined), GAPDH was identified as the most stable reference gene, whereas EF-1α was the least stable. For developing seeds alone, TUA showed the highest stability, while EF-1α exhibited poor stability. In leaf and flower samples, ACT11 was the most stable gene, whereas TUA was unsuitable for normalization. The expression patterns of NcFAD2 and NcFatB, two genes involved in fatty acid biosynthesis, were used to validate the selected reference genes. Stable reference genes and the optimized multi-gene combination generated consistent expression profiles, while unstable reference genes caused evident distortion. This study provides the first systematic evaluation of reference genes for qRT-PCR analysis in N. caudatum and offers a practical foundation for future functional studies of lipid metabolism in this woody oil plant.

Plastid-localized glycerol‑3‑phosphate acyltransferase (ATS1) catalyzes the initial acylation step in the prokaryotic branch of glycerolipid biosynthesis and is pivotal for membrane lipid remodeling. In this study, we investigated the role of Arabidopsis thaliana ATS1 (AtATS1) in salt tolerance using transgenic Brassica napus lines overexpressing AtATS1 and wild-type plants. Under 150 mM salt stress, AtATS1-overexpressing lines exhibited significantly compromised tolerance, characterized by severe growth inhibition and cotyledon abscission compared to wild-type plants. Physiological assays further revealed that despite a hyper-induction of antioxidant enzymes (SOD and POD), the transgenic lines suffered from excessive reactive oxygen species (ROS) accumulation and elevated malondialdehyde (MDA) levels, indicating a failure to maintain redox homeostasis. Transcriptomic analysis demonstrated that AtATS1 overexpression disrupts the stress response by misallocating metabolic resources toward glucosinolate biosynthesis and constitutively repressing the MAPK signaling cascade (including WRKY42, MPK15, and MEKK1). In conclusion, these findings identify AtATS1 as a negative regulator of salt tolerance in B. napus. This study provides new insights into the link between lipid metabolism and abiotic stress responses and highlights the need for careful modulation of glycerolipid biosynthesis when engineering salt-resilient rapeseed varieties.

Perilla frutescens is a specialty oilseed crop rich in polyunsaturated fatty acids, particularly α-linolenic acid (ALA), and the molecular regulation of seed lipid metabolism is an active area of research. Reverse-transcription quantitative PCR (RT-qPCR) is essential for investigating the molecular mechanisms underlying seed oil biosynthesis; however, systematically validated reference genes are lacking for this species. Based on P. frutescens transcriptome data, seven commonly used housekeeping genes—PfActin, Pf18S (18S pre-ribosomal assembly protein GAR2-like), PfEF-1α, PfCYP, PfTUA, PfTUB, and PfGAPDH—were initially selected. After PfGAPDH was excluded because of its extremely low abundance and unstable expression, the remaining six candidates were evaluated in vegetative organs (leaves, stems, and roots), seeds at different developmental stages, and leaves subjected to PEG 6000 treatment. Expression stability was independently assessed using geNorm, NormFinder, and BestKeeper, and the resulting stability values were integrated with RefFinder. Pf18S showed the highest overall stability in the complete sample set and developing seeds. Under osmotic stress, PfTUB was the most stable single reference gene, and geNorm identified PfTUB + Pf18S as the optimal two-gene combination. In vegetative organs, PfTUB ranked first and PfTUA second, whereas PfEF-1α was among the least stable genes in most sample sets. The recommended two-gene combinations were PfTUA + PfTUB for vegetative organs, Pf18S + PfTUA for developing seeds, and PfTUB + Pf18S for osmotic stress. Biological validation showed that normalization to Pf18S reproduced spatiotemporal expression patterns of six key lipid-biosynthesis genes that were consistent with the dynamic accumulation of total seed oil and ALA. These results support a tiered normalization strategy using Pf18S for preliminary screening and condition-specific reference-gene pairs for precise quantification.