Salt Treatment Research Articles

Comparative Transcriptome Analysis Provides Insights into the Effect of Epicuticular Wax Accumulation on Salt Stress in Coconuts.

The coconut is an important tropical economical crop and exhibits high tolerance to various types of salinity stress. However, little is known about the molecular mechanism underlying its salt tolerance. In this study, RNA-Seq was applied to examine the different genes expressed in four coconut varieties when exposed to a salt environment, resulting in the generation of data for 48 transcriptomes. Comparative transcriptome analysis showed that some genes involved in cutin and wax biosynthesis were significantly upregulated in salt treatment compared to the control, including CYP86A4, HTH, CER1, CER2, CER3, DCR, GPAT4, LTP3, LTP4, and LTP5. In particular, the expression of CER2 was induced more than sixfold, with an RPKM value of up to 205 ten days after salt treatment in Hainan Tall coconut, demonstrating superior capacity in salt tolerance compared to dwarf coconut varieties. However, for yellow dwarf and red dwarf coconut varieties, the expression level of the CER2 gene was low at four different time points after exposure to salt treatment, suggesting that this gene may contribute to the divergence in salt tolerance between tall and dwarf coconut varieties. Cytological evidence showed a higher abundance of cuticle accumulation in tall coconut and severe damage to cuticular wax in dwarf coconut.

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

BackgroundPopulus simonii × P. nigra is an ideal material for studying the molecular mechanisms of woody plants. In recent years, research on Populus simonii × P. nigra has increasingly focused on the application of transgenic technology to improve salt tolerance. However, the rapid characterization of gene functions has been hampered by the long growth cycle and exceedingly poor transformation efficiency. Protoplasts are an important tool for plant gene engineering, which can assist with challenging genetic transformation and the protracted growth cycle of Populus simonii × P. nigra. This study established an optimized system for the preparation and transformation of protoplasts from Populus simonii × P. nigra leaves, making genetic research on Populus simonii × P. nigra faster and more convenient. Major Latex Protein (MLP) family genes play a crucial role in plant salt stress response. In the previous study, we discovered that PsnMLP328 can be induced by salt treatment, which suggested that this gene may be involved in response to salt stress. Protein localization is a suggestion for its function. Therefore, we conducted subcellular localization analysis using protoplasts of Populus simonii × P. nigra to study the function of the PsnMLP328 gene preliminarily.ResultsThis study established an optimized system for the preparation and transformation of Populus simonii × P. nigra protoplasts. The research results indicate that the optimal separation scheme for the protoplasts of Populus simonii × P. nigra leaves included 2.5% cellulase R-10, 0.6% macerozyme R-10, 0.3% pectolyase Y-23, and 0.8 M mannitol. After enzymatic digestion for 5 h, the yield of obtained protoplasts could reach up to 2 × 107 protoplasts/gFW, with a high viability of 98%. We carried out the subcellular localization analysis based on the optimized transient transformation system, and the results indicated that the MLP328 protein is localized in the nucleus and cytoplasm; thereby proving the effectiveness of the transformation system.ConclusionIn summary, this study successfully established an efficient system for preparing and transforming leaf protoplasts of Populus simonii × P. nigra, laying the foundation for future research on gene function and expression of Populus simonii × P. nigra.

GhVOZ1-AVP1 module positively regulates salt tolerance in upland cotton (Gossypium hirsutum L.)

Vascular Plant One‑zinc Finger (VOZ) transcription factor can respond to a variety of abiotic stresses, however its function in cotton and the molecular mechanisms of response to salt tolerance remained unclear. In this study, we found that GhVOZ1 is highly expressed in stamen and stem of cotton under normal conditions. The expression of GhVOZ1 increased significantly after 3 h of salt treatment in three-leaf staged upland cotton. Overexpressed transgenic lines of GhVOZ1 in Arabidopsis and upland cotton were treated with salt stress and we found that GhVOZ1 could respond positively to salt stress. GhVOZ1 can regulate Arabidopsis Vacuolar Proton Pump Pyrophosphatase (H+-PPase) gene (AVP1) expression through specific binding to GCGTCTAAAGTACGC site on GhAVP1 promoter, which was examined through Dual-luciferase assay and Electrophoretic mobility shift assay (EMSA). AVP1 expression was significantly increased in Arabidopsis with GhVOZ1 overexpression, while GhAVP1 expression was decreased in virus induced gene silenced (VIGS) cotton plants of GhVOZ1. Knockdown of GhAVP1 expression in cotton plants by VIGS showed decreased superoxide dismutase (SOD) and peroxidase (POD) activities, whereas an increased malondialdehyde (MDA) content and ultimately decreased salt tolerance. The GhVOZ1-AVP1 module could maintain sodium ion homeostasis through cell ion transport and positively regulate the salt tolerance in cotton, providing new ideas and insights for the study of salt tolerance.

Identification and expression analysis of calcium-dependent protein kinase family in oat (Avena sativa L.) and their functions in response to saline-alkali stresses.

Calcium-dependent protein kinases (CDPKs) serve as calcium ion sensors and play crucial roles in all aspects of plant life cycle. While CDPK gene family has been extensively studied in various plants, there is limited information available for CDPK members in oat, an important cereal crop worldwide. Totally, 60 AsCDPK genes were identified in oat genome and were classified into four subfamilies based on their phylogenetic relationship. The members within each subfamily shared similar gene structure and conserved motifs. Collinearity analysis revealed that AsCDPK gene amplification was attributed to segmental duplication events and underwent strong purifying selection. AsCDPK promoters were predicted to contain cis-acting elements associated with hormones, biotic and abiotic stresses. AsCDPK gene expressions were induced by different salt stresses, exhibiting stress-specific under different salt treatments. Moreover, overexpression of AsCDPK26 gene enhanced salt resistance in C. reinhardtii, a single-cell photoautotrophic model plants. Further analysis revealed a significant correlation between AsCDPK26 and Na+/H+ antiporter 1 (p<0.05), suggesting that AsCDPK26 may interact with ion transporter to modulate salt resistance. These results not only provide valuable insights into AsCDPK genes in response to different salt stresses, but also lay the foundation to mine novel candidates for improving salt tolerance in oat and other crops.

Genetic transformation of GmFBX322 gene and salt tolerance physiology in soybean.

Soybean is one of the most important food crops, breeding salt-tolerant soybean varieties is of great significance to alleviate soybean shortage. In this study, the F-box protein family homologous gene GmFBX322 was cloned from the soybean variety Williams 82 and overexpressed in the Shennong 9 soybean variety to further study and explore the physiological mechanism of soybean salt tolerance. GmFBX322 was constructed on the vector pTF101:35S, and integrated into the genome of Shennong 9 soybean variety by Agrobacterium EHA101-mediated cotyledonary node transformation technology, and 4 overexpressed transgenic lines were obtained, molecular assays were performed on the transformed plants. The expression of GmFBX322 was detected by qRT-PCR and it was found that the leaves of the 4 transgenic lines increased by 2.49, 2.46, 2.77, 2.95 times compared with the wild type; after salt treatment for 12 hours, it was found that the expression of wild type Shennong 9 Inducible expression of GmFBX322. After 72 hours of salt treatment, the leaves of wild-type Shennong 9 soybean plants showed obvious wilting and chlorosis, while the leaves of GmFBX322 plants overexpressing GmFBX322 showed no obvious changes. The leaves were taken at 0, 6, 12, 24, and 48 hours of salt stress to determine the antioxidant activity. Ability and osmotic adjustment level, etc. The results showed that the catalase activity in the leaves of the transgenic lines 2265, 2267, 2269, and 2271 was 2.47, 2.53, 3.59, 2.96 times that of the wild-type plant after 48 hours of salt treatment; the soluble sugar content was 1.22, 1.14, and 1.22 of the wild-type plant. 1.14, 1.57 times; the proline content is 2.20, 1.83, 1.65, 1.84 times of the wild type. After comparing the physiological indicators determined by the experiment, the transgenic lines performed better than the control group, indicating that overexpression of GmFBX322 can enhance the salt tolerance of soybean plants. To verify the function of GmFBX322 gene related to stress resistance, add it to the candidate gene of stress resistance, and provide scientific basis for the selection and breeding of salt-tolerant varieties.

Evaluation of Genetic variability and viability association of eight Mungbean (Vigna radiata L.) varieties under different level of salinity stress

An investigation was carried to study the mean performance, variability and the associations between characters in mungbean. For this study eight mungbean genotypes viz., ADT 2, BM-2002-1, ML 5, AKM 4, Vishal, Vamban, TARM 1 and Utkarsh were used. The experiment was conducted in the Seed Science and Technology (sst) Laboratory and at Pot culture yard, Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalai nagar, Tamil nadu from September 2016 to December 2017 with four salt treatments (0, 2, 4 and 6 DSM-1) in ramdomized block design with three replications. Observation were on seven seedling characters such as germination percentage, seedling shoot length, seedling root length, seedling shoot fresh weight, seedling root fresh weight, seedling shoot dry weight, seedling root dry weight and seven quantitative characters such as days to first flowering, plant height, number of branches/plant, number of pods/plant, number of seeds/pod, 100 seed weight and seed yield/plant. Four elite genotypes were found to be superior based on the mean performance namely Utkarsh, TARM 1, Vamban and Vishal among the various levels of salinity.

Arbuscular mycorrhizal fungi communities and promoting the growth of alfalfa in saline ecosystems of northern China.

Arbuscular mycorrhizal fungi (AMF) are universally distributed in soils, including saline soils, and can form mycorrhizal symbiosis with the vast majority of higher plants. This symbiosis can reduce soil salinity and influence plant growth and development by improving nutrient uptake, increasing plant antioxidant enzyme activity, and regulating hormone levels. In this study, rhizosphere soil from eight plants in the Songnen saline-alkaline grassland was used to isolate, characterize, and screen the indigenous advantageous AMF. The promoting effect of AMF on alfalfa (Medicago sativa L.) under salt treatment was also investigated. The findings showed that 40 species of AMF in six genera were identified by high-throughput sequencing. Glomus mosseae (G.m) and Glomus etunicatum (G.e) are the dominant species in saline ecosystems of northern China. Alfalfa inoculated with Glomus mosseae and Glomus etunicatum under different salt concentrations could be infested and form a symbiotic system. The mycorrhizal colonization rate and mycorrhizal dependence of G.m inoculation were significantly higher than those of G.e inoculation. With increasing salt concentration, inoculation increased alfalfa plant height, fresh weight, chlorophyll content, proline (Pro), soluble sugar (SS), soluble protein (SP), peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activity while decreasing the malondialdehyde (MDA) content and superoxide anion production rate. The results highlight that inoculation with G.m and G.e effectively alleviated salinity stress, with G.m inoculation having a significant influence on salt resistance in alfalfa. AMF might play a key role in alfalfa growth and survival under harsh salt conditions.

Synthesis of phosphate stabilised iodanes and their application in intramolecular aryl migrations.

A diverse set of hydroxy-benzo[e]iodadioxaphosphinine oxides and derived diaryl iodonium salts are prepared and two examples are characterized by X-ray crystallography, featuring an out-of-plane geometry of the hypervalent bond for both compound classes. Treatment of the phosphate-stabilized diaryliodonium salts with Ca(OH)2 results in an efficient base-induced intramolecular aryl migration under aqueous conditions, yielding iodo-substituted diaryl ethers with yields up to 94%. Our findings highlight the synthetic potential of this previously underexplored compound class in organic transformations.

Integrated transcriptome and metabolome analysis of salinity tolerance in response to foliar application of choline chloride in rice (Oryza sativa L.).

Salt stress is a major abiotic stress that affects crop growth and productivity. Choline Chloride (CC) has been shown to enhance salt tolerance in various crops, but the underlying molecular mechanisms in rice remain unclear. To investigate the regulatory mechanism of CC-mediated salt tolerance in rice, we conducted morpho-physiological, metabolomic, and transcriptomic analyses on two rice varieties (WSY, salt-tolerant, and HHZ, salt-sensitive) treated with 500 mg·L-1 CC under 0.3% NaCl stress. Our results showed that foliar application of CC improved morpho-physiological parameters such as root traits, seedling height, seedling strength index, seedling fullness, leaf area, photosynthetic parameters, photosynthetic pigments, starch, and fructose content under salt stress, while decreasing soluble sugar, sucrose, and sucrose phosphate synthase levels. Transcriptomic analysis revealed that CC regulation combined with salt treatment induced changes in the expression of genes related to starch and sucrose metabolism, the citric acid cycle, carbon sequestration in photosynthetic organs, carbon metabolism, and photosynthetic antenna proteins in both rice varieties. Metabolomic analysis further supported these findings, indicating that photosynthesis, carbon metabolism, and carbon fixation pathways were crucial in CC-mediated salt tolerance. The combined transcriptomic and metabolomic data suggest that CC treatment enhances rice salt tolerance by activating distinct transcriptional cascades and phytohormone signaling, along with multiple antioxidants and unique metabolic pathways. These findings provide a basis for further understanding the mechanisms of metabolite synthesis and gene regulation induced by CC in rice in response to salt stress, and may inform strategies for improving crop resilience to salt stress.

Low-temperature enhancement in the extraction of phosphorus from metallurgical-grade silicon by simultaneously re-constructing phosphorus-rich phases of Ca3P2 and CaAl2Si2-P

A novel method of combined Si-Al solvent refining with CaCl2-CaF2-CaO molten salt treatment for phosphorus removal from metallurgical-grade silicon was investigated. With the mass ratio of salt (45 wt.%CaCl2–45 wt.%CaF2–10 wt.%CaO) to the alloy (phosphorus-doped Al-Si) being 1:1, phosphorus-rich phases of Ca3P2 and CaAl2Si2-P were successfully reconstructed at 1250 °C for 1 h. After acid leaching, phosphorus content decreased from 3 wt.% to 0.24 wt.%, corresponding to a phosphorus removal rate of 91.95%.

The efficacy of salt treatment for Dactylogyrus extensus (Monogenea) infection in Carp (Cyprinus carpio)

Salt is widely recommended as a cost-effective and readily available compound against freshwater fish parasites in aquaculture; however, a limited number of studies provide scientific evidence regarding the efficacy of salt use despite its frequent use as an anti-parasitic in fish culture. Dactylogyrus is a severe gill parasite, causing considerable losses in freshwater aquaculture. The current study aimed to evaluate the anti-parasitic efficacy of salt against Dactylogyrus extensus in Cyprinus carpio. In vitro, mortality of D. extensus showed time- and concentration-dependent patterns. In vivo, the anti-parasitic effectiveness of salt to D. extensus was assessed at 23.56% after exposure to salt at a concentration of 1.25 g/L for 10 min. Anti-parasitic efficacy of salt in short-term application in carp can be categorised between slight and mild against monogenean, D. extensus.

The synthesis of 3D organic-inorganic hybrid heterojunctions of g-C3N4 nanoneedles@Ti3C2 MXene with superior photocatalytic H2 evolution.

3D organic-inorganic hybrid heterojunctions of 1D carbon nitride (g-C3N4) nanoneedles anchoring on 2D Ti3C2 MXene nanosheets have been constructed via a green molten salt treatment. The Ti3C2 MXene nanosheets and molten salt treatment guide the formation of ordered nanoneedle-like g-C3N4. Both the formed heterojunctions and the nanoneedle-like structures contribute charge separation and transfer for superior photocatalytic H2 evolution.

Gamma Irradiation Promotes the Growth Rate of Thai Pigmented Rice As Well As Inducing the Accumulation of Bioactive Compounds and Carbohydrate Hydrolyzing Enzymes Inhibitors (α-Glucosidase and α-Amylase) under Salt Conditions.

Rice contains many bioactive compounds that perform various biological activities. Some of these compounds have been identified as α-glucosidase and α-amylase inhibitors, including guaiacol, vanillin, methyl vanillate, vanillic acid, syringic acid, and 2-pentyl furan. In this study, we assessed the growth rate, photosynthetic pigment content, phenolic content, and flavonoid content of gamma-irradiated Thai pigmented rice. Bioactive components of gamma-irradiated rice that had been subjected to salt treatment were also investigated. The findings showed that production of photosynthetic pigments, which are associated with plant growth, was induced by low gamma exposure. Phenolic and flavonoid content of rice was increased after gamma irradiation at 5 to 1,000 Gy. Both gamma irradiation and the salt conditions changed the quantity of vanillin, methyl vanillate, and vanillic acid in the rice. However, at a salt concentration of 40 mM, the salt stress had more of an effect than the gamma dosage. However, the high concentrations of methyl vanillate and vanillic acid detected in the rice under salt conditions were ameliorated by gamma irradiation. Guaiacol served as the substrate of guaiacol peroxidase for catalyzed reactive oxygen species, as evidenced by the observation that the guaiacol content of rice decreased between increased gamma dosages. A gamma dose of 40 to 1,000 Gy resulted in the production of syringic acid. Under salt stress, syringic acid buildup was also seen to be ameliorated by gamma irradiation. In comparison to salt conditions, particularly for 20 mM salt, gamma irradiation had less of an impact on the 2-pentyl furan in rice.

Tuz Stresine Maruz Bırakılan Aspir (Carthamus tinctorius L.) Bitkisinin Morfolojik, Fizyolojik ve Biyokimyasal Tepkileri

The morphological, physiological (biomass, water content-WC) and biochemical (proline, membrane damage-malondialdehyde-MDA, H2O2 content) responses of safflower to NaCI salt stress in different concentrations (0, 50, 75, 150, and 300 mM) were investigated for the first time in in vitro conditions in this study. At the end of the 3-week period, it was determined that NaCl had a negative effect on germination percentages. The percentage of germination was 100% in the control group, while it decreased to 30% in 150 mM NaCl and 5% in 300 mM. In general, morphological development of seedlings was significantly slowed down and seedling growth was not observed at 300 mM concentration. It was determined that the WC, fresh weights, shoot and root length decreased in all NaCl concentrations but there was no significant decrease in dry weights. MDA, proline and H2O2 contents increased in safflower seedlings in parallel with the intensity of salt treatments. While the highest MDA and proline content was found in 150 mM NaCl treatment, the highest H2O2 content was found in 75 mM NaCl treatment. In the light of these data, it has been proven that the Balcı safflower variety is sensitive by showing a negative effect on the applied salt concentrations.

The overlooked ion: Unraveling the effects of magnesium-specific toxicity on willows under sulphate salinity

Among all the current stressors on global agricultural systems, soil salinity is one of the most significant and continues to escalate. Traditionally, sodium chloride is the salt employed for studying salt stress in plants, but for many regions, sulphates are the predominant salts that cause soil degradation and curtail agricultural production. Willow trees (Salix spp.) have been identified as potential candidates for multipurpose establishment on salinity-affected marginal agriculture fields. To compare the impacts of sodium and magnesium sulphate salts, 12 native and hybrid willow genotypes were subjected to four sodium and magnesium sulphate salt treatments for 12 weeks. Magnesium sulphate treated willows showed more significant growth impacts, and displayed treatment-specific inhibition of photosynthesis, increased disruption of mineral nutrient balance, and greater inhibition of sulphur assimilation, compared to the trees treated with same level of sodium and same treatment osmolality. In addition, it was apparent that sulphate treatments encouraged sulphur uptake and translocation to the leaves, where it was partitioned to organic sulphur, but did not pool in small organic sulphur-containing compounds. The advantages and disadvantages of two different salt tolerance strategies were compared through a case study with two native willows, a sodium excluder and a sodium accumulator.

The effect of maltodextrin on properties of sated egg yolk

Salted eggs are mainly produced through salting treatments that taste salty. However, high sodium intake is part of the pathophysiology of hypertension. Thus, this study aimed to develop low-sodium egg yolks with salting treatment using sodium chloride (NaCl) and maltodextrin to reduce salt content in the salting process. Salted egg yolks were produced using methods in the salting process using maltodextrin (10% and 20% supplement) and were compared to salted eggs brined with 20 and 26% sodium chloride (NaCl) solutions. The moisture content, salt content, color, and texture properties of salted egg yolks during salting for up to 35 days were determined. Nevertheless, salted egg yolks produced with the salting solution using maltodextrin supplement had a significant salt content (0.21-3.30 mg/g) that was lower than the amount discovered in commercial salted yolks (2.84-4.15 mg/g). The results revealed that the maltodextrin substitution affected the salted yolks' properties. The salt contents of all salted egg yolk samples gradually increased during the salting process, along with slight decreases in moisture content as salting time and salt solution concentration increased. The lightness (L*) and the yellowness (b*) decreased while the redness (a*) increased. The hardness, adhesiveness, cohesiveness, gumminess, and chewiness of salted egg yolks increased rapidly over the time of salting, while springiness decreased during the initial stages of salting and reached almost constant levels at the end of salting. As the salting time increased to 35 days, the salted yolk gradually became dark reddish. The maximum denaturation temperature (Tmax) and denaturation enthalpy (DH) of egg proteins increased with increasing salting time. These effects were most pronounced due to the high maltodextrin content of the salting solution. This study suggests that this approach with maltodextrin substitution using the shell egg salting protocol can produce low-sodium salted eggs.

Genome-wide identification and expression analysis of the chlorophyll a/b binding protein gene family in oilseed (Brassica napus L.) under salt stress conditions

To elucidate the functions and molecular mechanisms of BnLhc gene family members in Brassica napus under different exogenous hormones and abiotic stress, we identified 57 BnLhc gene family members from the entire genome of B. napus variety ZS11 using Arabidopsis Lhc protein as a seed sequence combined with phyll A-B binding protein PF00504. We performed comprehensive analyses including physicochemical property analysis, structural analysis, phylogenetic relationship analysis, homology relationship analysis, protein interaction network analysis, microRNA-target gene interaction analysis and GO enrichment analysis. Additionally, based on transcriptome data, we investigated response patterns of BnLhc genes to various exogenous stress conditions. Comparative evaluation between non-salt (NS:0) and salt-treated (TS: 1) rapeseed plants revealed significant inhibition in leaf chlorophyll content, biomass (W), leaf area (LA), root surface area (RA), as well as chloroplasts and roots under salt treatment. Furthermore, qPCR was employed to assess the relative expression levels of BnLhc genes associated with salt tolerance. Our findings indicate that while BnLhcs were upregulated in leaves upon exposure to salt signals suggesting their potential involvement in enhancing rape's resistance to salt stress through ion redistribution processes; they were downregulated in roots. This study provides valuable insights into the functionality and molecular genetic mechanisms underlying the role BnLhc gene family

Genome-Wide Identification, Expression and Interaction Analyses of PP2C Family Genes in Chenopodium quinoa.

Plant protein phosphatase 2Cs (PP2Cs) function as inhibitors in protein kinase cascades involved in various processes and are crucial participants in both plant development and signaling pathways activated by abiotic stress. In this study, a genome-wide study was conducted on the CqPP2C gene family. A total of putative 117 CqPP2C genes were identified. Comprehensive analyses of physicochemical properties, chromosome localization and subcellular localization were conducted. According to phylogenetic analysis, CqPP2Cs were divided into 13 subfamilies. CqPP2Cs in the same subfamily had similar gene structures, and conserved motifs and all the CqPP2C proteins had the type 2C phosphatase domains. The expansion of CqPP2Cs through gene duplication was primarily driven by segmental duplication, and all duplicated CqPP2Cs underwent evolutionary changes guided by purifying selection. The expression of CqPP2Cs in various tissues under different abiotic stresses was analyzed using RNA-seq data. The findings indicated that CqPP2C genes played a role in regulating both the developmental processes and stress responses of quinoa. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis of six CqPP2C genes in subfamily A revealed that they were up-regulated or down-regulated under salt and drought treatments. Furthermore, the results of yeast two-hybrid assays revealed that subfamily A CqPP2Cs interacted not only with subclass III CqSnRK2s but also with subclass II CqSnRK2s. Subfamily A CqPP2Cs could interact with CqSnRK2s in different combinations and intensities in a variety of biological processes and biological threats. Overall, our results will be useful for understanding the functions of CqPP2C in regulating ABA signals and responding to abiotic stress.

Selection and validation of reference genes for normalization of gene expression in Floccularia luteovirens

Floccularia luteovirens is one of the rare edible fungi with high nutritional value found on the Qinghai-Tibet Plateau. However, research at the molecular level on this species is currently constrained due to the lack of reliable reference genes for this species. Thirteen potential reference genes (ACT, GAPDH, EF-Tu, SAMDC, UBI, CLN1, β-TUB, γ-TUB, GTP, H3, UBC, UBC-E2, and GTPBP1) were chosen for the present study, and their expression under various abiotic conditions was investigated. Stability of gene expression was tested using GeNorm, NormFinder, BestKeeper, Delta–Ct, and RefFinder. The results showed that the most suitable reference genes for salt treatment were ACT and EF-Tu. Under drought stress, γ-TUB and UBC-E2 would be suitable for normalization. Under oxidative stress, the reference genes H3 and GAPDH worked well. Under heat stress, the reference genes EF-Tu and γ-TUB were suggested. Under extreme pH stress, UBC-E2 and H3 were appropriate reference genes. Under cadmium stress, the reference genes ACT and UBC-E2 functioned well. In different tissues, H3 and GTPBP1 were appropriate reference genes. The optimal internal reference genes when analyzing all samples were H3 and SAMDC. The expression level of HSP90 was studied to further validate the applicability of the genes identified in this study.

The Genome-Wide Profiling of Alternative Splicing in Willow under Salt Stress

Alternative splicing (AS) is an important post-transcriptional regulatory model that can change the normal transcript expression level and possibly result in protein diversity. In this study, we conducted the full-length transcript sequencing of Salix matsudana Koidz 9901 leaves under salt treatment using the PromethION platform. A total of 4786 AS genes (9307 AS events) were determined, accounting for 7.45% of all the transcribed genes. Of them, intron retention (IR) events accounted for the most AS events (46.05%), followed by alternative 3′ splice sites (A3SS). During salt stress, the percentage of IR events decreased, and the percentage of the others increased. Statistical results showed that 5′GG was the most common motif at the 5′ end of the intron in the AS events, and GG3′ was the most common motif at the 3′ end. 5′GG-AG3′ was the most common splice mode in the AS events. The occurrence of AS events was significantly related to the exon number, exon length, intron length, GC content, and expression abundance of the genes. During salt stress, the number of AS genes gradually increased, and they mainly participated in purine and chlorophyll metabolism, RNA transport, and autophagy. Meanwhile, the AS sites of the gene increased during salt treatment, indicating the complexity of the AS events by salt stress. A comparison of differentially expressed genes (DEGs) and differentially alternative splicing (DAS) genes during salt stress revealed that they had a different mechanism of gene expression regulation when subjected to salt stress. These results expand our knowledge of AS events and shed light on and improve our understanding of plant resistance to salt tolerance in willow.