Genome-wide characterization of DNA methyltransferase family genes implies GhDMT6 improving tolerance of salt and drought on cotton.

BACKGROUND: DNA methylation is an important epigenetic mode of genomic DNA modification and plays a vital role in maintaining epigenetic content and regulating gene expression. Cytosine-5 DNA methyltransferase (C5-MTase) are the key enzymes in the process of DNA methylation. However, there is no systematic analysis of the C5-MTase in cotton so far, and the function of DNMT2 genes has not been studied. METHODS: In this study, the whole genome of cotton C5-MTase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome, and the function of GhDMT6 was further validated by VIGS experiments and subcellular localization analysis. RESULTS: 33 C5-MTases were identified from three cotton genomes, and were divided into four subfamilies by systematic evolutionary analysis. After the protein domain alignment of C5-MTases in cotton, 6 highly conserved motifs were found in the C-terminus of 33 proteins involved in methylation modification, which indicated that C5-MTases had a basic catalytic methylation function. These proteins were divided into four classes based on the N-terminal difference, of which DNMT2 lacks the N-terminal regulatory domain. The expression of C5-MTases in different parts of cotton was different under different stress treatments, which indicated the functional diversity of cotton C5-MTase gene family. Among the C5-MTases, the GhDMT6 had a obvious up-regulated expression. After silencing GhDMT6 with VIGS, the phenotype of cotton seedlings under different stress treatments showed a significant difference. Compared with cotton seedlings that did not silence GhDMT6, cotton seedlings silencing GhDMT6 showed significant stress resistance. CONCLUSION: The results show that C5-MTases plays an important role in cotton stress response, which is beneficial to further explore the function of DNMT2 subfamily genes.

Systematic analysis and expression of Gossypium ATG8 family reveals the roles of GhATG8f responding to salt stress in cotton.

KEY MESSAGE: Comprehensive analysis of Gossypium ATG8 family indicates that GhATG8f could improve salt tolerance of cotton by increasing SOD, POD and CAT activity and proline accumulation. In plants, autophagy is regulated by several genes that play important roles in initiating and controlling the process. ATG8, functioning as a protein similar to ubiquitin, is involved in crucial tasks throughout the autophagosome formation process. In this research, we conducted an extensive and all-encompassing investigation of 64 ATG8 genes across four varieties of cotton. According to the subcellular localization prediction results, 49 genes were found in the cytoplasm, 6 genes in the chloroplast, 1 gene in the peroxisome, 5 genes in the nucleus, and 3 genes in the extracellular region. Phylogenetic analysis categorized a total of 5 subfamilies containing sixty-four ATG8 genes. The expression of the majority of GhATG8 genes was induced by salt, drought, cold, and heat stresses, as revealed by RNA-seq and real-time PCR. Analysis of cis-elements in the promoters of GhATG8 genes revealed the predominant presence of responsive elements for plant hormones and abiotic stress, suggesting that GhATG8 genes might have significant functions in abiotic stress response. Furthermore, we additionally performed a gene interaction network analysis for the GhATG8 proteins. The salt stress resistance of cotton was reduced due to the downregulation of GhATG8f expression, resulting in decreased activity of CAT, SOD, and POD enzymes, as well as decreased fresh weight and proline accumulation. In summary, our research is the initial exploration of ATG8 gene components in cotton, providing a basis for future investigations into the regulatory mechanisms of ATG8 genes in autophagy and their response to abiotic stress.

GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton.

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-ß (lycopene ß-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas. Synopsis: Lycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

Enhancing stimulation of cyaniding, GhLDOX3 activates reactive oxygen species to regulate tolerance of alkalinity negatively in cotton.

Anthocyanins belong to flavonoid secondary metabolites that act as plant pigments to give flowers and fruits different colors and as "scavengers" of reactive oxygen species (ROS) to protect plants from abiotic and biotic stresses. Few studies linked anthocyanins to alkaline resistance so far. In this study, anthocyanin synthesis-related gene leucoanthocyanidin dioxygenase (LDOX) was screened as a candidate gene to explore its relationship with alkali stress. The results found that pYL156: GhLDOX3 lines treated with 50 mM Na2CO3 (pH 11.11) for 24 h showed a significant increase in peroxidase (POD) activity, a decrease in total anthocyanin content and an increase in cyanidin content and a decrease in ROS accumulation compared to pYL156. The overexpressed (OE) lines, ldox mutant and wild-type (WT) lines in Arabidopsis were treated with 50 mM Na2CO3, 100 mM Na2CO3 and 150 mM Na2CO3 for 8 d, respectively. The wilted degree of the OE lines was more severe than WT lines, and less severe in the mutant lines in the 150 mM Na2CO3 treatment. After treatment, the expression levels of AtCAT and AtGSH genes related to antioxidant system in OE lines were significantly lower than in WT, and the expression levels of AtCAT and AtGSH in mutant lines were significantly higher than in WT. In conclusion, the above results suggest GhLDOX3 played a negative regulatory role in the mechanism of resisting Na2CO3 stress. Therefore, it can be considered in cotton breeding to improve the alkali tolerance of cotton by regulating the expression of related genes.

Evolutionary dynamics in financial markets with heterogeneities in investment strategies and reference points.

In nature and human societies, the effects of homogeneous and heterogeneous characteristics on the evolution of collective behaviors are quite different from each other. By incorporating pair pattern strategies and reference point strategies into an agent-based model, we have investigated the effects of homogeneous and heterogeneous investment strategies and reference points on price movement. In the market flooded with the investors with homogeneous investment strategies or homogeneous reference points, large price fluctuations occur. In the market flooded with the investors with heterogeneous investment strategies or heterogeneous reference points, moderate price fluctuations occur. The coexistence of different kinds of investment strategies can not only refrain from the occurrence of large price fluctuations but also the occurrence of no-trading states. The present model reveals that the coexistence of heterogeneous populations, whether they are the individuals with heterogeneous investment strategies or heterogeneous reference points of stock prices, is an important factor for the stability of the stock market.

Raffinose degradation-related gene GhAGAL3 was screened out responding to salinity stress through expression patterns of GhAGALs family genes.

A-galactosidases (AGALs), the oligosaccharide (RFO) catabolic genes of the raffinose family, play crucial roles in plant growth and development and in adversity stress. They can break down the non-reducing terminal galactose residues of glycolipids and sugar chains. In this study, the whole genome of AGALs was analyzed. Bioinformatics analysis was conducted to analyze members of the AGAL family in Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, and Gossypium raimondii. Meanwhile, RT-qPCR was carried out to analyze the expression patterns of AGAL family members in different tissues of terrestrial cotton. It was found that a series of environmental factors stimulated the expression of the GhAGAL3 gene. The function of GhAGAL3 was verified through virus-induced gene silencing (VIGS). As a result, GhAGAL3 gene silencing resulted in milder wilting of seedlings than the controls, and a significant increase in the raffinose content in cotton, indicating that GhAGAL3 responded to NaCl stress. The increase in raffinose content improved the tolerance of cotton. Findings in this study lay an important foundation for further research on the role of the GhAGAL3 gene family in the molecular mechanism of abiotic stress resistance in cotton.

Genome-wide identification of GAD family genes suggests GhGAD6 functionally respond to Cd2+ stress in cotton.

Glutamate decarboxylase (GAD) mainly regulated the biosynthesis of γ-aminobutyric acid (GABA) and played an important role in plant growth and stress resistance. To explore the potential function of GAD in cotton growth, the genome-wide identification, structure, and expression analysis of GAD genes were performed in this study. There were 10, 9, 5, and 5 GAD genes identified in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. GAD was divided into four clades according to the protein motif composition, gene structure, and phylogenetic relationship. The segmental duplication was the main way of the GAD gene family evolution. Most GhGADs respond to abiotic stress. Clade Ⅲ GAD was induced by Cd2+ stress, especially GhGAD6, and silencing GhGAD6 would lead to more serious Cd2+ poisoning in cotton. The oxidative damage caused by Cd2+ stress was relieved by increasing the GABA content. It was speculated that the decreased expression of GhGAD6 reduced the content of GABA in vivo and caused the accumulation of ROS. This study will further expand our understanding of the relationship between the evolution and function of the GhGAD gene family and provide new genetic resources for cotton breeding under environmental stress and phytoremediation.

Transformation and functional verification of Cry5Aa in cotton.

Most of the cotton bollworm-resistant genes applied in cotton are more than 20 years and they all belong to Cry1Ab/c family, but the insect-resistant effects of Cry5Aa on cotton were rarely reported. The possible risk of resistance is increasing. The study synthesized a novel bollworm-resistant gene Cry5Aa artificially based on preferences of cotton codon. The new gene was transferred to cotton through the method of pollen tube pathway. The transgenic strains were identified by kanamycin test in field and laboratory PCR analysis. Meanwhile, an insect resistance test was conducted by artificial bollworm feeding with transgenic leaves and GK19 was used as a control in this study. Results showed that rate of positive transgenic strains with kanamycin resistance in the first generation (T1), the second generation (T2) and the third generation (T3) respectively were 7.76%, 73.1% and 95.5%. However, PCR analysis showed that the positive strain rate in T1, T2 and T3 were 2.35%, 55.8% and 94.5%, respectively. The resistant assay of cotton bollworm showed that the mortality rate of the second, third and fourth instar larva feed by the transgenic cotton leaves, were 85.42%, 73.35% and 62.79%, respectively. There was a significant difference between transgenic plant of Cry5Aa and GK19 in insect resistance. Finally, we also conducted the further analysis of gene expression patterns, gene flow and the effect on non-target pest in the study. The results showed that Cry5Aa gene had less environmental impact, and Cry5Aa has been transferred successfully and expressed stably in cotton. Therefore, the novel bollworm resistance gene can partially replace the current insect-resistance gene of Lepidoptera insects.

Analysis of potential strategies for cadmium stress tolerance revealed by transcriptome analysis of upland cotton.

In recent years, heavy metal pollution has become a more serious global problem, and all countries are actively engaged in finding methods to remediate heavy metal-contaminated soil. We conducted transcriptome sequencing of the roots of cotton grown under three different cadmium concentrations, and analysed the potential strategies for coping with cadmium stress. Through Gene Ontology analysis, we found that most of the genes differentially regulated under cadmium stress were associated with catalytic activity and binding action, especially metal iron binding, and specific metabolic and cellular processes. The genes responsive to cadmium stress were mainly related to membrane and response to stimulus. The KEGG pathways enriched differentially expressed genes were associated with secondary metabolite production, Starch and sucrose metabolism, flavonoid biosynthesis, phenylalanina metalism and biosynthesis, in order to improve the activity of antioxidant system, repair systems and transport system and reduction of cadmium toxicity. There are three main mechanisms by which cotton responds to cadmium stress: thickening of physical barriers, oxidation resistance and detoxification complexation. Meanwhile, identified a potential cotton-specific stress response pathway involving brassinolide, and ethylene signaling pathways. Further investigation is needed to define the specific molecular mechanisms underlying cotton tolerance to cadmium stress. In this study potential coping strategies of cotton root under cadmium stress were revealed. Our findings can guide the selection of cotton breeds that absorb high levels of cadmium.

Improving medication adherence with adjuvant aromatase inhibitor in women with breast cancer: study protocol of a randomised controlled trial to evaluate the effect of short message service (SMS) reminder.

BACKGROUND: Medication adherence refers to whether a patient takes medication according to the frequency prescribed, or continues to take a prescribed medication. Inadequate adherence to medication may cause alterations in risk-benefit ratios, resulting in reduced benefits, increased risks or both, and is significantly associated with adverse clinical outcomes and higher healthcare costs. We aim to examine the effect of a computer generated short message service (SMS) reminder in improving medication adherence, and inhibiting the aromatisation process amongst breast cancer women receiving oral aromatase inhibitor therapy. METHODS/DESIGN: In this randomised controlled trial, eligible patients will be equally allocated to receive either SMS reminder or standard care. The former receives weekly SMS reminder to take medication while the latter does not receive any. The primary endpoint of medication adherence at 1-year is assessed using the Simplified Medication Adherence Questionnaire, and compared using the χ2 test. Adjustment for baseline covariate and potential confounders will be made using the logistic regression. Secondary outcomes involving estrone and androstenedione levels will be compared using the analysis of covariance, whereas the estradiol levels (< 18.4 pmol/L versus ≥18.4 pmol/L) will be compared using the χ2 test, and the logistic regression. Further, the assessment of knowledge, attitude, behaviour, and barriers and facilitating factors of medication adherence will be made via logistic regression. DISCUSSION: This will be the first study to evaluate short-term clinical outcomes from SMS reminder for breast cancer patients on aromatase inhibitor therapy. Random allocation to SMS reminder or control arm ensures that patients in both arms will be comparable with respect to demographic and clinical characteristics, and any difference in outcomes can be attributed to the intervention. Participants are not blinded to the assignment of intervention, thus there may be potential for bias in outcome assessments. TRIAL REGISTRATION: NCT02524548 . Retrospectively registered on 17 August 2015.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield.

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls.