Identification of cotton PIP5K genes and role of GhPIP5K9a in primary root development.

Phosphatidylinositol 4 phosphate 5-kinase (PIP5K) is crucial for the phosphatidylinositol (PI) signaling pathway. It plays a significant role in plant growth and development, as well as stress response. However, its effects on cotton are unknown. This study identified PIP5K genes from four cotton species and conducted bioinformatic analyses, with a particular emphasis on the functions of GhPIP5K9a in primary roots. The results showed that cotton PIP5Ks were classified into four subgroups. Analysis of gene structure and motif composition showed obvious conservation within each subgroup. Synteny analysis suggested that the PIP5K gene family experienced significant expansion due to both whole-genome duplication (WGD) and segmental duplication. Transcriptomic data analysis revealed that the majority of GhPIP5K genes had the either low or undetectable levels of expression. Moreover, GhPIP5K9a is highly expressed in the root and was located in plasmalemma. Suppression of GhPIP5K9a transcripts resulted in longer primary roots, longer primary root cells and increased auxin polar transport-related genes expression, and decreased abscisic acid (ABA) content, indicating that GhPIP5K9a negatively regulates cotton primary root growth. This study lays the foundation for further exploration of the role of the PIP5K genes in cotton.

GhTKPR1_8 functions to inhibit anther dehiscence and reduce pollen viability in cotton.

Sporopollenin, as the main component of the pollen exine, is a highly resistant polymer that provides structural integrity under unfavourable environmental conditions. Tetraketone α-pyrone reductase 1 (TKPR1) is essential for sporopollenin formation, catalyzing the reduction of tetraketone carbonyl to hydroxylated α-pyrone. The functional role of TKPR1 in male sterility has been reported in flowering plants such as maize, rice, and Arabidopsis. However, the molecular cloning and functional characterization of TKPR1 in cotton remain unaddressed. In this study, we identified 68 TKPR1s from four cotton species, categorized into three clades. Transcriptomics and RT-qPCR demonstrated that GhTKPR1_8 exhibited typical expression patterns in the tetrad stage of the anther. GhTKPR1_8 was localized to the endoplasmic reticulum. Moreover, ABORTED MICROSPORES (GhAMS) transcriptionally activated GhTKPR1_8 as indicated by luciferase complementation tests. GhTKPR1_8-knockdown inhibited anther dehiscence and reduced pollen viability in cotton. Additionally, overexpression of GhTKPR1_8 in the attkpr1 mutant restored its male sterile phenotype. This study offers novel insights into the investigation of TKPR1 in cotton while providing genetic resources for studying male sterility.

Exploring the role of GhN/AINV23: implications for plant growth, development, and drought tolerance.

BACKGROUND: Neutral/alkaline invertases (N/AINVs) play a crucial role in plant growth, development, and stress response, by irreversibly hydrolyzing sucrose into glucose and fructose. However, research on cotton in this area is limited. This study aims to investigate GhN/AINV23, a neutral/alkaline invertase in cotton, including its characteristics and biological functions. RESULTS: In our study, we analyzed the sequence information, three-dimensional (3D) model, phylogenetic tree, and cis-elements of GhN/AINV23. The localization of GhN/AINV23 was determined to be in the cytoplasm and cell membrane. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that GhN/AINV23 expression was induced by abscisic acid (ABA), exogenous sucrose and low exogenous glucose, and inhibited by high exogenous glucose. In Arabidopsis, overexpression of GhN/AINV23 promoted vegetative phase change, root development, and drought tolerance. Additionally, the virus-induced gene silencing (VIGS) assay indicated that the inhibition of GhN/AINV23 expression made cotton more susceptible to drought stress, suggesting that GhN/AINV23 positively regulates plant drought tolerance. CONCLUSION: Our research indicates that GhN/AINV23 plays a significant role in plant vegetative phase change, root development, and drought response. These findings provide a valuable foundation for utilizing GhN/AINV23 to improve cotton yield.

GhSTP18, a member of sugar transport proteins family, negatively regulates salt stress in cotton.

The sugar transporter protein (STP) family has been shown to play important roles in plant growth, development, and stress response. However, it has not been studied in cotton compared to other major crops. In this study, we identified 90 STP genes from four cotton species, performed bioinformatic analysis, and focused on the role of GhSTP18 in salt stress. According to our results, cotton STP proteins were divided into four subgroups according to the phylogenetic tree. A synteny analysis suggested that whole-genome duplication (WGD) and segmental duplication were key drivers in the expansion of the STP gene family. The transcriptomic data analysis showed that 29 GhSTP genes exhibited sink-specific expression. Quantitative real time-polymerase chain reaction (qRT-PCR) analyses revealed that expression of GhSTP18 was induced by salt treatment, heat treatment, cold treatment, and drought treatment, and continuously increased during a salt stress time course. Notably, GhSTP18 encodes a plasma membrane-localized galactose transporter. Suppression of GhSTP18 transcription by a virus-induced gene silencing (VIGS) assay reduced sensitivity to salt stress in cotton, indicating that GhSTP18 negatively regulates plant salt tolerance. These results provide an important reference and resource for further studying and deploying STP genes for cotton improvement.

Cotton proteomics: Dissecting the stress response mechanisms in cotton.

The natural environment of plants comprises a complex set of biotic and abiotic stresses, and plant responses to these stresses are complex as well. Plant proteomics approaches have significantly revealed dynamic changes in plant proteome responses to stress and developmental processes. Thus, we reviewed the recent advances in cotton proteomics research under changing environmental conditions, considering the progress and challenging factors. Finally, we highlight how single-cell proteomics is revolutionizing plant research at the proteomics level. We envision that future cotton proteomics research at the single-cell level will provide a more complete understanding of cotton's response to stresses.

GhBOP1 as a Key Factor of Ribosomal Biogenesis: Development of Wrinkled Leaves in Upland Cotton.

Block of proliferation 1 (BOP1) is a key protein that helps in the maturation of ribosomes and promotes the progression of the cell cycle. However, its role in the leaf morphogenesis of cotton remains unknown. Herein, we report and study the function of GhBOP1 isolated from Gossypium hirsutum. The sequence alignment revealed that BOP1 protein was highly conserved among different species. The yeast two-hybrid experiments, bimolecular fluorescence complementation, and luciferase complementation techniques revealed that GhBOP1 interact with GhPES and GhWDR12. Subcellular localization experiments revealed that GhBOP1, GhPES and GhWDR12 were localized at the nucleolus. Suppression of GhBOP1 transcripts resulted in the uneven bending of leaf margins and the presence of young wrinkled leaves by virus-induced gene silencing assay. Abnormal palisade arrangements and the presence of large upper epidermal cells were observed in the paraffin sections of the wrinkled leaves. Meanwhile, a jasmonic acid-related gene, GhOPR3, expression was increased. In addition, a negative effect was exerted on the cell cycle and the downregulation of the auxin-related genes was also observed. These results suggest that GhBOP1 plays a critical role in the development of wrinkled cotton leaves, and the process is potentially modulated through phytohormone signaling.

Functional divergence of GhAP1.1 and GhFUL2 associated with flowering regulation in upland cotton (Gossypium hirsutum L.).

The AP1/FUL transcription factors are important for floral development, but the underlying molecular mechanisms remain unclear. In this study, we cloned and identified two AP1/FUL-like genes, GhAP1.1 and GhFUL2, in upland cotton, which is a commonly cultivated economically valuable crop. Sequence alignment and phylogenetic analysis indicated that GhAP1.1 and GhFUL2, which are encoded by genes in the AP1/FUL clade, have conserved N-terminal regions but diverse C-terminal domains. Quantitative real-time PCR analysis revealed that GhAP1.1 and GhFUL2 were expressed in the flower and root, and showed opposite expression patterns during shoot apical meristem development. The upregulated expression of GhAP1.1 in Arabidopsis did not result in significant changes to the flowering time or floral organ development, and the transcript levels of the florigen FT increased and those of LFY decreased. Overexpression of GhFUL2 in Arabidopsis delayed flowering and promoted bolting by decreasing FT and LFY transcript levels. Silencing GhFUL2 in cotton dramatically increased the expression of GhFT and GhAP1.3 and promoted flowering. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that GhAP1.1 could interact with the SVP homolog GhSVP2.2, whereas GhFUL2 formed heterodimers with GhSEP3/GhSEP4 homologs and GhSVP2.2. The present results demonstrated that the functional divergence of GhAP1.1 and GhFUL2, which involved changes in sequences and expression patterns, influenced the regulation of cotton flower development.

Improvement of plant tolerance to drought stress by cotton tubby-like protein 30 through stomatal movement regulation.

INTRODUCTION: Cotton is a vital industrial crop that is gradually shifting to planting in arid areas. However, tubby-like proteins (TULPs) involved in plant response to various stresses are rarely reported in cotton. The present study exhibited that GhTULP30 transcription in cotton was induced by drought stress. OBJECTIVE: The present study demonstrated the improvement of plant tolerance to drought stress by GhTULP30 through regulation of stomatal movement. METHODS: GhTULP30 response to drought and salt stress was preliminarily confirmed by qRT-PCR and yeast stress experiments. Ectopic expression in Arabidopsis and endogenous gene silencing in cotton were used to determine stomatal movement. Yeast two-hybrid and spilt-luciferase were used to screen the interacting proteins. RESULTS: Ectopic expression of GhTULP30 in yeast markedly improved yeast cell tolerance to salt and drought. Overexpression of GhTULP30 made Arabidopsis seeds more resistant to drought and salt stress during seed germination and increased the stomata closing speed of the plant under drought stress conditions. Silencing of GhTULP30 in cotton by virus-induced gene silencing (VIGS) technology slowed down the closure speed of stomata under drought stress and decreased the length and width of the stomata. The trypan blue and diaminobenzidine staining exhibited the severity of leaf cell necrosis of GhTULP30-silenced plants. Additionally, the contents of proline, malondialdehyde, and catalase of GhTULP30-silenced plants exhibited significant variations, with obvious leaf wilting. Protein interaction experiments exhibited the interaction of GhTULP30 with GhSKP1B and GhXERICO. CONCLUSION: GhTULP30 participates in plant response to drought stress. The present study provides a reference and direction for further exploration of TULP functions in cotton plants.

Postpartum women's views on human milk banking in a city in Southeast China: a cross-sectional survey.

BACKGROUND: Donor human milk is the best alternative for preterm infants when their mother's own milk is insufficient or unavailable. The development of human milk banks in China started late, and in most of these banks, the amount of donor human milk is insufficient for clinical demand. Moreover, many mothers are reluctant to use donor human milk due to safety concerns. It is important to understand the potential supply and demand of donor human milk before establishing a new human milk bank. This study aimed to understand women's acceptance of human milk banking in Wenzhou, southeastern China. METHODS: A cross-sectional study was conducted in three community health centers in Wenzhou, southeast China, in December 2020. Data were collected from 305 postpartum women selected through convenience sampling. Sociodemographic, perinatal and breastfeeding characteristics, awareness and knowledge of human milk banking and willingness to donate human milk, and to accept donor human milk were assessed. Multivariable logistic regression analysis was used to explore independent predictors of willingness to donate human milk and to accept donor human milk. RESULTS: Only 17% (52/305) of our participants had heard of human milk banking prior to this survey. The prevalence of willingness to donate human milk and use donor human milk in our study was 73.4% (224/305) and 44.6% (136/305), respectively. Employment (adjusted odds ratio [AOR] 2.30; 95% confidence interval [CI] 1.17, 4.50) and human milk banking knowledge (AOR 1.23; 95% CI 1.12, 1.35) were independent predictors of willingness to donate human milk. Monthly household income in the previous year (AOR 2.18; 95% CI 1.17, 4.06), awareness of human milk banking (AOR 2.41; 95% CI 1.24, 4.67) and knowledge of human milk banking (AOR 1.22; 95% CI 1.11, 1.35) were significantly associated with willingness to accept donor human milk. CONCLUSIONS: In our study, awareness of human milk banks among women in the first year postpartum was low. More mothers were willing to donate human milk than to use donor human milk to feed their children. In our study, knowledge of human milk banking was a predictor of both willingness to donate human milk and willingness to use donor human milk. Programs with detailed information on human milk banking are needed to help mothers improve their knowledge and increase acceptance of human milk banking.

Evolutionary Relationships and Divergence of KNOTTED1-Like Family Genes Involved in Salt Tolerance and Development in Cotton (Gossypium hirsutum L.).

The KNOX (KNOTTED1-like homeobox) transcription factors play an important role in leaf, shoot apical meristem and seed development and respond to biotic and abiotic stresses. In this study, we analyzed the diversity and evolutionary history of the KNOX gene family in the genome of tetraploid cotton (Gossypium hirsutum). Forty-four putative KNOX genes were identified. All KNOX genes from seven higher plant species were classified into KNOXI, KNOXII, and KNATM clades based on a phylogenetic analysis. Chromosomal localization and collinearity analysis suggested that whole-genome duplication and a polyploidization event contributed to the expansion of the cotton KNOX gene family. Analyses of expression profiles revealed that the GhKNOX genes likely responded to diverse stresses and were involved in cotton growth developmental processes. Silencing of GhKNOX2 enhanced the salt tolerance of cotton seedlings, whereas silencing of GhKNOX10 and GhKNOX14 reduced seedling tolerance to salt stress. Silencing of GhSTM3 influenced the cotton flowering time and plant development. These findings clarify the evolution of the cotton KNOX gene family and provide a foundation for future functional studies of KNOX proteins in cotton growth and development and response to abiotic stresses.

Genome-Wide Analysis of the Phospholipase D Family in Five Cotton Species, and Potential Role of GhPLD2 in Fiber Development and Anther Dehiscence.

Phospholipase D (PLD) and its hydrolysis product phosphatidic acid play an important role in the regulation of several cellular processes, including root growth, pollen tube elongation, and microtubule reorganization. Here, we systematically identified and analyzed the membership, characterization, and evolutionary relationship of PLDs in five species of cotton. The results of the transcriptomic analysis suggested that the evaluated PLD genes showed high expression levels in anther tissue and during the fiber initiation and elongation periods. Quantitative real-time polymerase chain reaction showed differential expression of GhPLD genes in the anthers of photoperiod sensitive male sterility mutant 5 (psm5). Previous research on multiple stable quantitative trait loci also suggests the role of PLD genes in the fiber development. Further analyses showed that GhPLD2 protein is localized to the plasma membrane. The virus-induced gene silencing of GhPLD2 in cotton seedlings repressed its expression by 40-70%, which led to a reduction in reactive oxygen species (ROS) levels, 22% anther indehiscence, and disrupted fiber initiation and elongation. Thus, we inferred that GhPLD2 may promote ROS production, which, in turn, may regulate anther dehiscence and fiber development.

Identification and characterization of the AINV genes in five Gossypium species with potential functions of GhAINVs under abiotic stress.

Acid invertase (AINV) is a kind of sucrose hydrolase with an important role in plants. Currently, the AINV genes have not been systematically studied in cotton. In this study, a total of 92 AINV genes were identified in five cotton species. The phylogenetic analysis revealed that the AINV proteins were divided into two subgroups in cotton: vacuolar invertase (VINV) and cell wall invertase (CWINV). The analysis of gene structures, conserved motifs, and three-dimensional protein structures suggested that GhAINVs were significantly conserved. The synteny analysis showed that whole-genome duplication was the main force promoting the expansion of the AINV gene family. The cis-element, transcriptome, and quantitative real time-polymerase chain reaction (qRT-PCR) showed that some GhAINVs were possibly associated with stress response. GhCWINV4, highly expressed in PEG treatment, was cloned, and subsequent virus-induced gene silencing assay confirmed that this gene was involved in the drought stress response. Overall, this study might be helpful for further analyzing the biological function of AINVs and provide clues for improving the resistance of cotton to stress.

Genome-wide analysis of JMJ-C histone demethylase family involved in salt-tolerance in Gossypium hirsutum L.

The jumonji C (JMJ-C) domain-containing protein is a histone demethylase and is involved in plant stress. However, the function of the JMJ-C gene family in cotton is still not confirmed. Herein, 25, 26, 52, and 53 members belonging to the JMJ-C gene family were identified in Gossypium raimondii, Gossypium arboreum, Gossypium hirsutum, and Gossypium barbadense, respectively. Based on phylogenetic relationships and conserved domains, the JMJ-C genes were categorized into five subfamilies, KDM3, KDM4, KDM5, JMJC, and JMJD6. The chromosomal location, gene structure, motif compositions, and cis-elements have been displayed. The collinear investigation showed that whole-genome duplication event is the mainly power to drive JMJ-C gene family expansion. Transcriptome and qRT-PCR analysis revealed that eight GhJMJs were induced by salt and PEG treatment. Further assays confirmed that GhJMJ34/40 greatly improved salt and osmotic tolerance in Saccharomyces cerevisiae. These results help clarify JMJ-C protein functions in preparation for further study.

GhN/AINV13 positively regulates cotton stress tolerance by interacting with the 14-3-3 protein.

Neutral/alkaline invertases (N/AINVs) are sucrose hydrolases with important roles in plants. In this study, 15, 15, 15, 29, and 30 N/AINVs were identified in the Gossypium species, G. raimondii, G. herbaceum, G. arboreum, G. hirsutum, and G. barbadense, respectively. Along with two previously discovered branches, α and ß, a new clade γ was first discovered in our study. Investigation of gene collinearity showed that whole-genome duplication (WGD) and polyploidization were responsible for the expansion of the N/AINV gene family in allopolyploid Gossypium. Moreover, expression patterns revealed that GhN/AINV3/13/17/23/24/28 from the ß clade is highly expressed during the period of fiber initiation. The invertase activity of GhN/AINV13 and GhN/AINV23 were confirmed by restoring defects of invertase-deficient yeast mutant SEY2102. Treatments of abiotic stress showed that most GhN/AINVs were induced in response to polyethylene glycol (PEG) or salt stress. A virus-induced gene-silencing (VIGS) experiment and yeast two-hybrid assay demonstrated that GhN/AINV13 may interact with their positive regulators Gh14-3-3 proteins and participate in the fiber initiation or stress tolerance of cotton. Our results provided fundamental information regarding N/AINVs and highlight their potential functions in cotton stress tolerance.

GhTULP34, a member of tubby-like proteins, interacts with GhSKP1A to negatively regulate plant osmotic stress.

Tubby-like protein genes (TULPs), present in the form of large multigene families, play important roles in environmental stress. However, little is known regarding the TULP family genes in cotton. In this study, we systematically identified and analyzed the membership, characterization, and evolutionary relationship of TULPs in four species of cotton. Transcriptome analysis indicated that GhTULPs participate in environmental stress and cotton tissue development. At the same time, we also predicted and analyzed the potential molecular regulatory mechanisms and functions of TULPs. GhTULP34, as a candidate gene, significantly reduced the germination rate of transgenic Arabidopsis plants under salt stress, and inhibited root development and stomatal closure under mannitol stress. The yeast two-hybrid and luciferase (LUC) systems showed that GhTULP34 can interact with GhSKP1A, a subunit of the SCF-type (Skp1-Cullin-1-F-box) complex. This study will provide a basis and reference for future research on their roles in stress tolerance.

Comprehensive Genome-Wide Analysis of Thaumatin-Like Gene Family in Four Cotton Species and Functional Identification of GhTLP19 Involved in Regulating Tolerance to Verticillium dahlia and Drought.

Thaumatin-like proteins (TLPs) present in the form of large multigene families play important roles in biotic stress and abiotic stress. However, there has been no systematic analysis of the TLPs in cotton. In this study, comprehensive identification and evolutionary analysis of TLPs in four species of cotton were conducted. In total, 50, 48, 91, and 90 homologous sequences were identified in Gossypium raimondii, G. arboreum, G. barbadense, and G. hirsutum, respectively. Gene structure, protein motifs, and gene expression were further investigated. Transcriptome and quantitative real-time PCR analysis indicated that GhTLPs participate in abiotic, biotic stress and cotton fiber development. GhTLP19 on chromosome At05 was selected as a candidate gene for further study. When GhTLP19 was silenced by virus-induced gene silencing (VIGS) in cotton, with the increase of malondialdehyde (MDA) content and the decrease of catalase (CAT) content, and as the increase of disease index (DI) and hyphae accumulation, the plants were more sensitive to drought and Verticillium dahliae. Furthermore, the GhTLP19 overexpressing Arabidopsis transgenic lines exhibited higher proline content, thicker and longer trichomes and more tolerance to drought when compared to wild type. This study will provide a basis and reference for future research on their roles in stress tolerance and fiber development.

The effectiveness of massage on peri-operative anxiety in adults: A meta-analysis of randomized controlled trials and controlled clinical trials.

BACKGROUND AND PURPOSE: and purpose: Massage has gained increasing attention for reducing peri-operative anxiety. We aimed to investigate the effectiveness of massage for peri-operative anxiety in adults. METHODS: Six English electronic databases were comprehensively searched from their inception to February 2020. Subgroup analysis, quality assessment, sensitivity analysis, meta-regression and publication bias assessment were performed. RESULTS: Twenty-five controlled trials comprising 2494 participants were included. The meta-analysis indicated that massage could significantly reduce peri-operative anxiety for most types of surgical patients. Specifically, it was effective for pre-, intra- and post-operative anxiety. Acupoint or specific body reflex area massage showed a larger effect than general massage did. Massage delivered by professionals and non-professionals were both effective. Massage lasting 10-20 min per session was the most worthy of recommendation. Massage was concomitant with the improvement of peri-operative vital signs and post-operative pain. CONCLUSION: Massage is a promising complementary therapy for ameliorating peri-operative anxiety in adults.

EMBRYONIC FLOWER2B, coming from a stable QTL, represses the floral transition in cotton.

The EMBRYONIC FLOWER 2 (EMF2) gene encodes a VEFS (VRN2-EMF2-FIS2-Su(z)12) domain protein involved in plant growth and development. Herein, genome-wide characterization of the VEFS-box gene family in Gossypium raimondii, G. arboreum, G. barbadense, and G. hirsutum was performed with a total of 3, 3, 6, and 6 homologous sequences respectively identified in the four species. The gene structure, protein motifs, and gene expression were further investigated. Based on our previous research on multiple stable quantitative trait loci for early maturity, GhEMF2B on chromosome D03 was selected as a candidate gene for further study. Quantitative real-time PCR analysis indicated that GhEMF2B was upregulated in the apical buds of late-maturing cultivars at the fourth and fifth true-leaf stages compared to that of early-maturing cultivars. Virus-induced gene silencing of GhEMF2B in cotton seedlings repressed expression by 50%-70%, which led to earlier floral bud development, young curled leaves, and abnormal petal formation. Further analysis demonstrated that the silencing of GhEMF2B enhanced the expression levels of the positive floral regulators AGAMOUS-LIKE 6 (GhAGL6), FLOWERING LOCUS T (GhFT), and APETALA 1 (GhAP1). Thus, it can be inferred that GhEMF2B plays important roles in the floral transition and development of cotton.

Progress of Research on the Regulatory Pathway of the Plant Shade-Avoidance Syndrome.

When subject to vegetational shading, shade-avoiding plants detect neighbors by perceiving reduced light quantity and altered light quality. The former includes decreases in the ratio of red to far-red wavelengths (low R:FR) and low blue light ratio (LBL) predominantly detected by phytochromes and cryptochromes, respectively. By integrating multiple signals, plants generate a suite of responses, such as elongation of a variety of organs, accelerated flowering, and reduced branching, which are collectively termed the shade-avoidance syndrome (SAS). To trigger the SAS, interactions between photoreceptors and phytochrome-interacting factors are the general switch for activation of downstream signaling pathways. A number of transcription factor families and phytohormones, especially auxin, gibberellins, ethylene, and brassinosteroids, are involved in the SAS processes. In this review, shade signals, the major photoreceptors involved, and the phenotypic characteristics of the shade-intolerant plant Arabidopsis thaliana are described in detail. In addition, integration of the signaling mechanisms that link photoreceptors with multiple hormone signaling pathways is presented and future research directions are discussed.