Analysis of PAT1 subfamily members in the GRAS family of upland cotton and functional characterization of GhSCL13-2A in Verticillium dahliae resistance.

KEY MESSAGE: GhSCL13-2A, a member of the PAT1 subfamily in the GRAS family, positively regulates cotton resistance to Verticillium dahliae by mediating the jasmonic acid and salicylic acid signaling pathways and accumulation of reactive oxygen species. Verticillium wilt (VW) is a devastating disease of upland cotton (Gossypium hirsutum) that is primarily caused by the soil-borne fungus Verticillium dahliae. Scarecrow-like (SCL) proteins are known to be involved in plant abiotic and biotic stress responses, but their roles in cotton defense responses are still unclear. In this study, a total of 25 GhPAT1 subfamily members in the GRAS family were identified in upland cotton. Gene organization and protein domain analysis showed that GhPAT1 members were highly conserved. GhPAT1 genes were widely expressed in various tissues and at multiple developmental stages, and they were responsive to jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) signals. Furthermore, GhSCL13-2A was induced by V. dahliae infection. V. dahliae resistance was enhanced in Arabidopsis thaliana by ectopic overexpression of GhSCL13-2A, whereas cotton GhSCL13-2A knockdowns showed increased susceptibility. Levels of reactive oxygen species (ROS) and JA were also increased and SA content was decreased in GhSCL13-2A knockdowns. At the gene expression level, PR genes and SA signaling marker genes were down-regulated and JA signaling marker genes were upregulated in GhSCL13-2A knockdowns. GhSCL13-2A was shown to be localized to the cell membrane and the nucleus. Yeast two-hybrid and luciferase complementation assays indicated that GhSCL13-2A interacted with GhERF5. In Arabidopsis, V. dahliae resistance was enhanced by GhERF5 overexpression; in cotton, resistance was reduced in GhERF5 knockdowns. This study revealed a positive role of GhSCL13-2A in V. dahliae resistance, establishing it as a strong candidate gene for future breeding of V. dahliae-resistant cotton cultivars.

Analysis of the PRA1 Genes in Cotton Identifies the Role of GhPRA1.B1-1A in Verticillium dahliae Resistance.

Verticillium wilt in cotton (Gossypium hirsutum) is primarily caused by Verticillium dahliae. Previous data suggest that prenylated RAB acceptors (PRAs) play essential roles in environmental plant adaptation, although the potential roles of PRA1 in cotton are unclear. Therefore, in this study, PRA1 family members were identified in G. hirsutum, and their roles in biotic and abiotic stresses were analyzed. Thirty-seven GhPRA1 family members were identified in upland cotton, which were divided into eight groups. Gene structure and domain analyses revealed that the sequences of GhPRA1 members in each group were highly conserved. Many environmental stress-related and hormone-response cis-acting elements were identified in the GhPRA1 promoter regions, indicating that they may respond to biotic and abiotic stresses. Expression analysis revealed that GhPRA1 members were widely expressed in upland cotton. The GhPRA1 genes responded to abiotic stress: drought, cold, salt, and heat stress. GhPRA1.B1-1A expression increased after V. dahliae infection. Furthermore, the functional role of GhPRA1.B1-1A was confirmed by overexpression in Arabidopsis thaliana, which enhanced the resistance to V. dahliae. In contrast, V. dahliae resistance was significantly weakened via virus-induced gene silencing of GhPRA1.B1-1A in upland cotton. Simultaneously, reactive oxygen species accumulation; the H2O2, salicylic acid, and jasmonic acid contents; and callose deposition were significantly decreased in cotton plants with GhPRA1.B1-1A silencing. These findings contribute to a better understanding of the biological roles of GhPRA1 proteins and provide candidate genes for cotton breeders for breeding V. dahliae-resistant cultivars.

Identification and expression analysis of arabinogalactan protein genes in cotton reveal the function of GhAGP15 in Verticillium dahliae resistance.

Verticillium wilt, primarily caused by the fungal pathogen Verticillium dahliae, is a serious disease in cotton. Arabinogalactan proteins (AGPs), a class of hydroxyproline-rich glycoproteins, have been widely implicated in plant growth and environmental adaptation. The purpose of this study is to identify and characterize AGP members in cotton plants and explore their roles in responding to environmental stressors. In total, 65 GhAGP members were identified in upland cotton (Gossypium hirsutum), along with 43, 35, and 37 AGP members that were also identified in G. barbadense, G. arboreum, and G. raimondii, respectively. According to gene structure and protein domains analysis, GhAGP genes in upland cotton are highly conserved. Meanwhile, tandem duplication events have occurred frequently throughout cotton's evolutionary history. Expression analysis showed that GhAGP genes were widely expressed during growth and development and in response to abiotic stressors. Many cis-elements related to hormonal responses and environmental stressors were detected in GhAGP promoter regions. GhAGP genes participate in responding to cold, drought, and salt stress, and were sensitive to ET signaling. Furthermore, the expression level of GhAGP15 was elevated during V. dahliae infection and resistance against V. dahliae in upland cotton was significantly weakened by silencing GhAGP15 using a virus-induced gene silencing (VIGS) approach. Our results further suggest that the function of GhAGP15 in V. dahliae resistance might be involved in regulation of the JA, SA, and reactive oxygen species (ROS) pathways. The comprehensive analysis of AGP genes in cotton performed in this study provides a basic framework for further functional research of these genes.

Evolution and Characterization of Acetyl Coenzyme A: Diacylglycerol Acyltransferase Genes in Cotton Identify the Roles of GhDGAT3D in Oil Biosynthesis and Fatty Acid Composition.

Cottonseed oil is rich in unsaturated fatty acids (UFAs) and serves as an edible oil in human nutrition. Reports suggest that acyl-coenzyme A: diacylglycerol acyltransferases (DGAT) and wax ester synthase/DGAT (WSD1) genes encode a key group of enzymes that catalyze the final step for triacylglycerol biosynthesis and enable an important rate-limiting process. However, their roles in oil biosynthesis and the fatty acid profile of cotton seed are poorly understood. Therefore, the aim of this study was to identify and characterize DGAT and WSD1 genes in cotton plants and examine their roles in oil biosynthesis, the fatty acid profile of cotton seeds, and abiotic stress responses. In this study, 36 GhDGAT and GhWSD1 genes were identified in upland cotton (G. hirsutum) and found to be clustered into four groups: GhDGAT1, GhDGAT2, GhDGAT3, and GhWSD1. Gene structure and domain analyses showed that the GhDGAT and GhWSD1 genes in each group are highly conserved. Gene synteny analysis indicated that segmental and tandem duplication events occurred frequently during cotton evolution. Expression analysis revealed that GhDGAT and GhWSD1 genes function widely in cotton development and stress responses; moreover, several environmental stress and hormone response-related cis-elements were detected in the GhDGAT and GhWSD1 promoter regions. The predicted target transcription factors and miRNAs imply an extensive role of GhDGAT and GhWSD1 genes in stress responses. Increases in GhDGAT3 gene expression with increases in cottonseed oil accumulation were observed. Transformation study results showed that there was an increase in C18:1 content and a decrease in C18:2 and C18:3 contents in seeds of Arabidopsis transgenic plants overexpressing GhDGAT3D compared with that of control plants. Overall, these findings contributed to the understanding of the functions of GhDGAT and GhWSD1 genes in upland cotton, providing basic information for further research.

Evolutionary and Characteristic Analysis of RING-DUF1117 E3 Ubiquitin Ligase Genes in Gossypium Discerning the Role of GhRDUF4D in Verticillium dahliae Resistance.

Verticillium wilt, primarily induced by the soil-borne fungus Verticillium dahliae, is a serious threat to cotton fiber production. There are a large number of really interesting new gene (RING) domain-containing E3 ubiquitin ligases in Arabidopsis, of which three (At2g39720 (AtRHC2A), At3g46620 (AtRDUF1), and At5g59550 (AtRDUF2)) have a domain of unknown function (DUF) 1117 domain in their C-terminal regions. This study aimed to detect and characterize the RDUF members in cotton, to gain an insight into their roles in cotton's adaptation to environmental stressors. In this study, a total of 6, 7, 14, and 14 RDUF (RING-DUF1117) genes were detected in Gossypium arboretum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These RDUF genes were classified into three groups. The genes in each group were highly conserved based on gene structure and domain analysis. Gene duplication analysis revealed that segmental duplication occurred during cotton evolution. Expression analysis revealed that the GhRDUF genes were widely expressed during cotton growth and under abiotic stresses. Many cis-elements related to hormone response and environment stressors were identified in GhRDUF promoters. The predicted target miRNAs and transcription factors implied that GhRDUFs might be regulated by gra-miR482c, as well as by transcription factors, including MYB, C2H2, and Dof. The GhRDUF genes responded to cold, drought, and salt stress and were sensitive to jasmonic acid, salicylic acid, and ethylene signals. Meanwhile, GhRDUF4D expression levels were enhanced after V. dahliae infection. Subsequently, GhRDUF4D was verified by overexpression in Arabidopsis and virus-induced gene silencing treatment in upland cotton. We observed that V. dahliae resistance was significantly enhanced in transgenic Arabidopsis, and weakened in GhRDUF4D silenced plants. This study conducted a comprehensive analysis of the RDUF genes in Gossypium, hereby providing basic information for further functional studies.

Lip morphology estimation models based on three-dimensional images in a modern adult population from China.

Lips are the main part of the lower facial soft tissue and are vital to forensic facial approximation (FFA). Facial soft tissue thickness (FSTT) and linear measurements in three dimensions are used in the quantitative analysis of lip morphology. With most FSTT analysis methods, the surface of soft tissue is unexplicit. Our study aimed to determine FSTT and explore the relationship between the hard and soft tissues of lips in different skeletal occlusions based on cone-beam CT (CBCT) and 3dMD images in a Chinese population. The FSTT of 11 landmarks in CBCT and 29 lip measurements in CBCT and 3dMD of 180 healthy Chinese individuals (90 males, 90 females) between 18 and 30 years were analyzed. The subjects were randomly divided into two groups with different skeletal occlusions distributed equally: 156 subjects in the experimental group to establish the prediction regression formulae of lip morphology and 24 subjects in the test group to assess the accuracy of the formulae. The results indicated that FSTT in the lower lip region varied among different skeletal occlusions. Furthermore, sex discrepancy was noted in the FSTT in midline landmarks and linear measurements. Measurements showing the highest correlation between soft and hard tissues were between total upper lip height and Ns-Pr (0.563 in males, 0.651 in females). The stepwise multiple regression equations were verified to be reliable with an average error of 1.246 mm. The method of combining CBCT with 3dMD provides a new perspective in predicting lip morphology and expands the database for FFA.

Three-dimensional superimposition of digital models for individual identification.

Dentition is an individualizing structure in humans that may be potentially utilized in individual identification. However, research on the use of three-dimensional (3D) digital models for personal identification is rare. This study aimed to develop a method for individual identification based on a 3D image registration algorithm and assess its feasibility in practice. Twenty-eight college students were recruited; for each subject, a dental cast and an intraoral scan were taken at different time points, and digital models were acquired. The digital models of the dental casts and intraoral scans were assumed as antemortem and postmortem dentition, respectively. Additional 72 dental casts were extracted from a hospital database as a suspect pool together with 28 antemortem models. The dentition images of all of the models were extracted. Correntropy was introduced into the traditional iterative closest point algorithm to compare each postmortem 3D dentition with 3D dentitions in the suspect pool. Point-to-point root mean square (RMS) distances were calculated, and then 28 matches and 2772 mismatches were obtained. Statistical analysis was performed using the Mann-Whitney U test, which showed significant differences in RMS between matches (0.18±0.03mm) and mismatches (1.04±0.67mm) (P<0.05). All of the RMS values of the matched models were below 0.27mm. The percentage of accurate identification reached 100% in the present study. These results indicate that this method for individual identification based on 3D superimposition of digital models is effective in personal identification.

Inhibitory effects of peripheral administration of exendin-4 on food intake are attenuated by lesions of the central nucleus of amygdala.

The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 suppresses food intake and decreases body weight. However, the mediating site(s) of action of the anorectic effects induced by peripheral administration of GLP-1R agonists are not well known. The present study investigated the effects of bilateral lesions of the central nucleus of the amygdala (CeA) on the suppression of chow and high-sucrose food intake by exendin-4 in rats. Adult male Sprague-Dawley rats with bilateral sham or electrolytic lesions (400 µA; 25 s) of the CeA were used for this experiment. No significant difference was found in the daily chow intake and high-sucrose food intake in rats with CeA lesion compared to sham-operated rats. Bilateral lesions of the CeA significantly attenuated the suppression of high-sucrose food intake by i.p. exendin-4, but not the suppression of chow intake. These results suggest that a mediating role of the CeA on the peripheral effects of exendin-4.

The effects of mepiquat chloride on the lateral root initiation of cotton seedlings are associated with auxin and auxin-conjugate homeostasis.

BACKGROUND: Mepiquat chloride (MC) is a plant growth regulator widely used in cotton (Gossypium hirsutum L.) production to suppress excessive vegetative growth, increase root growth and avoid yield losses. To increase root growth, cotton seeds were treated with MC to increase the number of lateral root (LRs) and improve drought resistance. An increased indole-3-acetic acid (IAA) pool appeared to correlate with LR growth, and the principal source of IAA in germinating seeds is IAA conjugates. Here, the role of IAA homeostasis and signaling was investigated in cotton seedlings treated with MC. RESULTS: In the present research, MC significantly increased endogenous IAA levels in the roots, which promoted lateral root initiation (LRI) by upregulating GhARF7/19 and GhLBD18s and subsequently increasing LR quantity and elongation. The levels of IAA-amide conjugates significantly decreased in MC-treated seedlings compared with untreated control seedlings. Sixteen members of the cotton IAA amidohydrolase (IAH) gene family were identified, of which GhIAR3a, GhIAR3b, GhILR1, GhILL3 and GhILL6 were expressed during cotton seed germination. Compared with those in untreated control seedlings, the expression levels of GhIAR3a, GhIAR3b, GhILR1 and GhILL6 in the MC-treated seedlings were markedly elevated. The GhIAR3a/b and GhILR1 genes were cloned and expressed in Escherichia coli; these recombinant proteins exhibited hydrolytic activity that could cleave IAA-phenyalanine (Phe), IAA-methionine (Met), IAA-glycine (Gly) and IAA-leucine (Leu) in vitro, while only GhIAR3a hydrolyzed IAA-alanine (Ala) efficiently. The content of GhIAR3a, as detected via an established sandwich enzyme-linked immunosorbent assay (ELISA), increased in the MC-treated seedlings compared with the untreated control seedlings. In addition, the Arabidopsis iar3 mutant was less responsive to MC-induced LR growth than was wild type. CONCLUSIONS: These findings suggested that MC application could mediate IAA homeostasis via increased IAA levels from IAA-amide conjugate hydrolysis by accelerating IAH gene expression, which might promote LRI and increase the LR quantity and elongation.

[Effect of estrogen on genioglossus and hypoglossal nucleus of female rats].

PUEPOSE: To study the hypoglossal nucleus spontaneous discharge and genioglossus function of SD rats with different estrogen levels, and explore the possible central pathways of estrogen protecting upper airway stability. METHODS: Thirty SD rats were randomly divided into 3 groups: sham operation group (Sham), ovariectomized group (OVX) and ovariectomized + estrogen covering group (OVX+E2). The hypoglossal nucleus spontaneous discharge frequency and maximum amplitude of spontaneous discharge were measured with extracellular recording technique；BL-420 biological was used to detect the genioglossus EMG and contraction. The measurements included the average frequency, the maximum frequency, the integral amplitude, the maximum amplitude of the genioglossus, the amplitude of the evoked EMG, the critical fusion frequency, the 50% tonic contraction, and the intensity of the stimulus. SPSS 13.0 software package was used to analyze the data with One-way ANOVA. RESULTS: The hypoglossal nucleus spontaneous discharge frequency and maximum amplitude of spontaneous discharge in OVX group were significantly lower than the Sham group and OVX+E2 group (P<0.05). The genioglossus EMG maximum frequency, average frequency, amplitude and integral maximum discharge magnitude, 50% tonic contraction drive of genioglossus in OVX group were significantly lower than the Sham and OVX+E2 group (P<0.05); the threshold stimulation strength and critical fusion frequency in OVX group were significantly higher than the Sham and OVX + E2 group (P<0.05); there was no significant difference between OVX+ E2 group and the Sham group (P>0.05); there was no significant difference among the three groups (P>0.05). CONCLUSIONS: Estrogen changes excitability of hypoglossal nucleus and affect contraction of genioglossus. Estrogen levels can affect genioglossus contraction function and the ability of resistance to fatigue.

[Presurgical nasoalveolar molding in infants with cleft lip and palate: analysis of 29 cases].

PURPOSE: The objective of this study was to treat the cleft lip and alveolus, nasal deformity with presurgical nasoalveolar molding (PNAM), to elucidate the problems and treatment methods, which may be helpful for the use of PNAM in clinic. METHODS: Twenty nine infants with cleft lip and palate (CLP) were treated with PNAM in our center. There were 19 unilateral and 10 bilateral CLP patients. The initial visit time was 3 to 150 days after birth. Treatment time ranged from 2.5 to 3 months. The appliance was modified at 2-week interval. RESULTS: According to the evaluation standards, 17 infants were treated successfully with the closure of cleft lip and alveolar processes, reposition of the deformed nasal cartilages, and increased length of columella. The lip and nasal deformities of 9 infants were corrected partly, which were helpful for surgery. There were 3 infants giving up PNAM. CONCLUSIONS: There were five important facts for the successful treatment, including initial visit time, impression of the intraoral cleft defect, modification of the plate and the nasal stent, and use of nasal splints. Orthodontics and plastic surgeons should have the same views for PNAM in infants, which will advance the treatment level for cleft lip and palate.

17beta-Estradiol accentuates contractility of rat genioglossal muscle via regulation of estrogen receptor alpha.

OBJECTIVE: This study in rat genioglossus muscle (GG) was designed to test the hypothesis that the effects of estrogen are at least in part, meditated directly by the estrogen receptors (ERs) of muscle. DESIGN: Eighty-eight-week-old female Sprague-Dawley rats were randomly assigned to five groups: (1) normal animals (Normal); (2) sham operation animals (Sham); (3) ovariectomized animals without estrogen replacement (OVX); (4) ovariectomized animals with olive oil replacement (OVX+O); (5) ovariectomized animals with 17beta-estradiol replacement (OVX+E2). Six weeks later, GG was assessed in vivo for contractile properties and further analysis for ERs expression was carried out including real-time quantitative RT-PCR, immunohistochemistry and Western blotting. RESULTS: The maximal twitch tension, 70%-decay time and fatigue index of GG decreased significantly in OVX group when compared with Normal group (P<0.05, P<0.05, P<0.05). However, all the three parameters reversed in OVX+E2 group especially fatigue index. Further analysis showed a clear expression of ERalpha and ERbeta in rat GG. The expression of both ERalpha protein and ERalpha mRNA was both significantly decreased in OVX group (P<0.05) and recovered back to previous level after receiving 17beta-estradiol replacement (P<0.05). But neither ERbeta protein nor ERbeta mRNA was regulated by estrogen deprivation and replacement. CONCLUSION: The results demonstrated that the contractility of GG was accentuated by estrogen. Moreover, these effects were at least in part, meditated directly via regulation of the expression of ERalpha. It might contribute to the protective effects of estrogen on the patency of upper airway and the pathogenesis of obstructive sleep apnea hypopnoea syndrome.

Effects of ovariectomy on rat genioglossal muscle contractile properties and fiber-type distribution.

OBJECTIVE: To test the hypothesis that ovariectomy has no effects on contractile, histochemical, or biochemical properties of the rat genioglossus (GG). MATERIALS AND METHODS: Eight-week-old female Sprague-Dawley rats were randomly assigned into three groups: normal group (Normal), sham-operated group (Sham), and ovariectomized group (OVX). Four weeks later, genioglossal electromyography activity (EMGgg) and contractile properties were measured, including relative integrated EMG (iEMG), maximal twitch tension, 70%-decay time, and fatigue index (FI). Then rats were sacrificed and paired GG were removed for further analysis. Adenosine-triphosphatase (ATPase) staining was performed to determine the percent fiber-type distribution and to identify cross-sectional area (CSA) of muscle fibers. Myosin heavy chain (MHC) phenotypes were determined by gel electrophoresis. RESULTS: Ovariectomy reduced EMG activity and contractile properties of the GG. Following ovariectomy, the CSA of type IIA and the proportion of MHCIIA decreased significantly. The MHC isoform composition of GG transferred from relative slow-twitch to fast-twitch isoform, following the order MHCIIB --> MHCIIX --> MHCIIA. Sham operation had no effect on any of the parameters. CONCLUSIONS: The hypothesis is rejected. The contractile properties of the GG are sensitive to ovariectomy. These changes were, at least in part, associated with changes in the amount and type of contractile protein expressed.

Effects of sex hormones on genioglossal muscle contractility and SR Ca2+-ATPase activity in aged rat.

This investigation was designed to examine whether short-term administration of sex hormones could produce changes in contractile properties and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) function in genioglossal muscle (GG) of aged male rats. Twenty-four aged male rats were randomly divided into three groups to receive an intramuscular injection of either 0.1mg/kg oestrogen (group A), 2.5 mg/kg testosterone (group B), or 0.2 ml sterile peanut oil (group C, control), twice a week, during 4 weeks. After hormone treatment, in vivo isometric contractile properties were determined using surgically prepared GG muscles with platinum electrodes for stimulation of the medial branch of the hypoglossus nerve. Sarcoplasmic reticulum Ca(2+)-ATPase activity was measured in muscle homogenates by detecting the amount of inorganic phosphorus ion released in a standard coupled enzyme assay. SERCA1 mRNA level was observed using a real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). It was found that, compared with group C, testosterone treatment decreased the fatigue resistance in GG muscles (p<.05), whereas no change was observed in the isometric twitch (P(t)) and tetanic tension (P(0)) (p>.05). By contrast, in oestrogen treated GG muscles, no significant modification was found either in the contractile or in endurance properties (p>.05). The change in GG fatigue resistance of group B was associated with a marked decrease in SR Ca(2+)-ATPase activity when compared with that of the control group (p<.01). Furthermore, SERCA1 mRNA level was also down regulated in group B (p<.05). No prominent differences in SR Ca(2+)-ATPase activity and SERCA1 mRNA expression existed between group A and group C (p>.05). The present results show that exogenous testosterone produces more pronounced changes in GG muscle fatigue resistance than oestrogen does by acting at SR Ca(2+)-ATPase activity and SERCA gene expression.

[Effects of ovariectomy on electromyography and phenotype in rat genioglossus].

OBJECTIVE: To investigate the effects of ovariectomy on genioglossus (GG) electromyography and myofibre phenotype in female rats and to explore the proposal mechanisms underling ovarian hormone protecting to the patency of upper airway (UA). METHODS: 30 female Sprague-Dawley rats were randomly assigned to three groups: normal group (NORMAL), sham group (SHAM) and ovariectomized group (OVX). Six weeks later, electromyography activity of the genioglossus (EMGgg) was obtained from inserted bipolar electrodes. The integrated EMG (iEMG) was analyed. Genioglossus was stained with ATPase staining. The fibre phenotype composition and cross sectional area (CSA) of type II A and II B/ II X fibres were measured. RESULTS: EMGgg had no significant variety between the normal and sham group (P >0.05). Compared to normal group, iEMG was significantly decreased in OVX group (P < 0.05). GG myofibres were composed of II phenotye, including II A (40.27 +/- 1.01)% and II B/ II X (59.73 +/- 1.01)%, fibres composition and CSA of types II A and II B/II X had no significant variety between the normal and sham group. Compared to normal group, fibres composition had no significant variety in OVX group (P > 0.05), but the CSA of type II A was significantly decreased (P < 0.05). CONCLUSION: The decreased EMGgg of ovariectomized rat maybe relates to the decrease of CSA in type II A fibre. Ovarian hormone may protect the patency of upper airway through affecting muscle function of UA dilator.