Notes & Tips

A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues

Sulfur dioxide (SO₂) is the most frequently used preservative for table grapes, yet the preservation mechanism remains unclear. To ascertain the specific genes and pathways involved in SO₂ preservation, RNA-seq technology was employed to characterize the transcriptome profile of grapes during postharvest storage. A total of 22,288 genes were identified, of which 377 genes were differentially expressed (≥ 2-fold change) between SO₂ and control groups, mainly enriched in secondary metabolism, plant-pathogen interactions, plant hormone signaling, etc. Numerous genes encoding pathogenesis-related (PR) proteins exhibited higher expression levels in SO₂ group, while the activities of disease-resistant enzymes, such as β-1,3-glucanase (PR2) and chitinase (PR3) significantly increased by 28.9 % and 29.3 % (P < 0.05), indicating SO₂-induced plant resistance. Exposure to SO₂ also enhanced the expression of genes encoding essential enzymes of secondary metabolism, and significantly increased both the activities of critical enzymes for the biosynthesis of secondary metabolites, such as phenylalanine ammonia-lyase (PAL) and 4-coumarate-CoA ligase (4CL), and the contents of secondary metabolites such as total phenol, flavonoid, anthocyanin, and lignin, demonstrating an enhanced chemical and physical barriers in SO₂-fumigated grapes. Meanwhile, the genes associated with ethylene signaling and cell wall degradation were down-regulated by SO₂ preservative, and some ethylene-responsive elements were identified in the promoter regions of cell wall hydrolase genes, suggesting that SO₂-inhibited ethylene signaling might contribute to maintaining cell wall integrity. Altogether, gene expression patterns and cellular physiological processes were altered in grapes exposed to SO₂, which helped maintain fruit quality and prolong postharvest life by regulating the defense responses and fruit ageing.

Agropyron cristatum (2n = 4x = 28, PPPP), which harbours many high-yield and disease-resistance genes, is a promising donor for wheat improvement. Narrow genetic diversity and the trade-off between grain weight and grain number have become bottlenecks for increasing grain yield in wheat. In this study, a novel translocation line, WAT650l, was derived from the chromosome 6P addition line 4844–12, which can simultaneously increase both grain number per spike (GNS) and thousand-grain weight (TGW). Cytological analysis and molecular marker analysis revealed that WAT650l was a 5BL·5BS-6PL (bin 12–17) translocation line. Assessment of agronomic traits and analysis of the BC₄F₂ and BC₅F₂ populations suggested that the 6PL terminal chromosome segment in WAT650l resulted in increased grain number per spike (average increased by 14.07 grains), thousand-grain weight (average increased by 4.31 g), flag leaf length, plant height, spikelet number per spike and kernel number per spikelet during the two growing seasons of 2020–2021 and 2021–2022. Additionally, the increased GNS locus and high-TGW locus of WAT650l were mapped to the bins 16–17 and 12–13, respectively, on chromosome 6PL by genetic population analysis of three translocation lines. In summary, we provide a valuable germplasm resource for broadening the genetic base of wheat and overcoming the negative relationship between GNS and TGW in wheat breeding.

The potential of dietary miRNAs as functional molecules affecting the expression of animal and human genes have led many researches. Nevertheless, there is a lack of consensus and the evaluation of their presence in cells or living organisms samples have yielded contradicting results derived from non-reliable miRNA analysis.

This work reviews the difficulties in the analysis of dietary miRNA and compares the techniques and methods used for their extraction, detection, sequencing, and quantitation. Special attention will be pointed out to the potential of new techniques such as mass spectrometry and, also, to the performance of dietary miRNA as functional molecules.

Key findings and conclusions: Difficulties in the analysis of dietary miRNA are observed in all steps of the analytical process. Sample preparation is hindered due to the low concentration and degradability of dietary miRNA and to the sample complexity and presence of interfering molecules. Moreover, the short sequence lengths of miRNA, the different number of copies per cell, and the high sequence similarity within miRNAs can affect the detection and quantitation of miRNA. A suitable experimental design and an extraction of miRNA and the use of accurate, sensitive, selective, reproducible, and high-throughput techniques is key to overcome these problems. The use of mass spectrometry, ion mobility and approaches based on nanomaterials are promising but still not applied to the analysis of dietary miRNA. This review urges for techniques and methods guaranteeing the correct analysis of dietary miRNA in living organisms to confirm their role in the prevalence of important diseases.

Steam explosion coupling high-temperature short-time sterilization (SE-HTST) was exploited to modify cellulosic biomass medium properties and promote high-solid fermentation (HSF). Biomass characterization analysis showed that SE-HTST enlarged microstructural pores and cavities in solid media, providing more effective space for microbial growth. Meanwhile, SE-HTST helped to release glucose from the cellulose with 35.8 ± 4.5, 20.0 ± 2.3, and 12.3 ± 5.7 mg glucose/g dry medium at 24, 48, and 72 h of fermentation, which were 3.1, 2.3, and 1.5 times higher than that in medium from conventional thermal sterilization (CTS), respectively. SE-HTST increased the viable cell and spore number of Bacillus subtilis by 1.8 and 1.6 times at 72 h of fermentation compared to CTS. Moreover, the expressions of master transcriptional gene spo0A and the early sigma factors of sigF and sigE genes gradually increased in the SE-HTST medium, showing enhanced sporulation in HSF. Therefore, SE-HTST is an effective strategy for facilitating cellulose degradation, improving glucose nutrients in biomass medium, and promoting sporulation-regulatory gene expression during high-solid fermentation, which enhances the production of microbial ecological agents using B. subtilis significantly.

Trehalose-6-phosphate synthase (TPS) performs the first step in the biosynthetic pathway of trehalose-6-phosphate and trehalose. These two molecules play key roles in the control of carbon allocation and of stress responses in plants. We investigated the organization of the TPS gene family and its developmental and environmental expression regulation in grapevine, a major horticultural crop. We identified three novel genes in the family, and assessed the expression of the 11 family members in tissues and developmental phases. Two potentially biosynthetic TPS isoforms belonging to Class I were preferentially expressed in leaf (VvTPS1_A) and in fruit (VvTPS1_B) respectively. Sucrose treatment induced expression of VvTPS1_B, but not of VvTPS1_A, and a progressive decrease of sucrose concentration. Expression of a few Class II genes was affected by sucrose treatment. Application of osmotic stress by withdrawing irrigation also induced a decrease in sucrose and an increase of glucose content, and down-regulation of the VvTPS1_A gene. We discuss the possible role of these potential biosynthetic TPS genes. Subgroups of TPS genes, including both Class I and ClassII isoforms, followed a co-expression pattern in different conditions, suggesting that Class II TPS proteins may directly or indirectly interact with TPS biosynthetic genes. Our results pave the way for clarification of the role of TPS isoforms in grapevine responses to environmental stress.

The effect of a postharvest treatment with sulfur dioxide (SO₂) under commercial conditions on table grapes (Vitis vinifera L. ‘Muscat Hamburg’) quality and the possible action mechanisms were investigated. The results indicated that SO₂ treatment resulted in an improved control of infections in grapes during storage. The fresh fruit rate of SO₂ treatment group was significantly higher than that of the control group. Furthermore, SO₂ markedly enhanced activity of phenylalanine ammonia lyase (PAL), promoted the accumulation of phenolic compounds including total phenol, flavonoid and lignin, and improved the polyphenol oxidase (PPO) activity. Moreover, SO₂ significantly elevated transcription levels of pathogenesis-related (PR) proteins genes chitinase3 (CHI3), CHI1b and β-1,3-glucanase (PR2). Consistently, the activities of chitinase and β-1,3-glucanase remarkably increased in SO₂ treatment group. Our founding indicates that SO₂ activates defense responses associated with secondary metabolism and PR proteins for enhanced disease resistance and thereby thus extending the storage life of table grapes.