Cell cycle-dependent kinase inhibitor GhKRP6, a direct target of GhBES1.4, participates in BR regulation of cell expansion in cotton.

The steroidal hormone brassinosteroid (BR) has been shown to positively regulate cell expansion in plants. However, the specific mechanism by which BR controls this process has not been fully understood. In this study, RNA-seq and DAP-seq analysis of GhBES1.4 (a core transcription factor in BR signaling) were used to identify a cotton cell cycle-dependent kinase inhibitor called GhKRP6. The study found that GhKRP6 was significantly induced by the BR hormone and that GhBES1.4 directly promoted the expression of GhKRP6 by binding to the CACGTG motif in its promoter region. GhKRP6-silenced cotton plants had smaller leaves with more cells and reduced cell size. Furthermore, endoreduplication was inhibited, which affected cell expansion and ultimately decreased fiber length and seed size in GhKRP6-silenced plants compared with the control. The KEGG enrichment results of control and VIGS-GhKRP6 plants revealed differential expression of genes related to cell wall biosynthesis, MAPK, and plant hormone transduction pathways - all of which are related to cell expansion. Additionally, some cyclin-dependent kinase (CDK) genes were upregulated in the plants with silenced GhKRP6. Our study also found that GhKRP6 could interact directly with a cell cycle-dependent kinase called GhCDKG. Taken together, these results suggest that BR signaling influences cell expansion by directly modulating the expression of cell cycle-dependent kinase inhibitor GhKRP6 via GhBES1.4.

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 Small Auxin-Up RNA (SAUR) Genes Regulating Root Growth Angle (RGA) in Apple.

Small auxin upregulated RNAs (SAURs) are primary auxin response genes; the function of regulating root growth angle (RGA) is unclear in the apple rootstock. We firstly identified 96 MdSAUR genes families from new apple genome GDDH13 using the resequence database of 'Baleng Crab (BC)' and 'M9'. A total of 25 MdSAUR genes, regulating the formation of RGA, were screened for the expression profiles in stems and roots and the allelic variants of quantitative trait loci (QTL). Finally, through the joint analysis of network and protein-protein interaction, MdSAUR2, MdSAUR29, MdSAUR60, MdSAUR62, MdSAUR69, MdSAUR71, and MdSAUR84 were screened as the main candidate genes for regulating RGA. This study provides a new insight for further revealing the regulatory mechanism of RGA in apple dwarf rootstocks.

[Effect of moxibustion on quality of life in tumor patients: network Meta-analysis].

OBJECTIVE: To rank the effectiveness of various moxibustion methods on the quality of life in tumor patients, and explore the best treatment plan of moxibustion for improving the quality of life in tumor patients from the perspective of evidence-based medicine. METHODS: The Chinese and English literature of randomized controlled trial (RCT) of the effect of moxibustion on the quality of life in tumor patients were searched in PubMed, EMbase, Cochrane Library, CNKI, SinoMed, Wanfang and VIP. The retrieval time was from the establishment of the databases to October 31, 2020. The R3.6.2 and Stata15.0 software were used for network Meta-analysis based on Bayesian model. RESULTS: A total of 30 Chinese RCTs were included, including 2 169 patients, involving 16 interventions. In terms of the effectiveness of improving quality of life, the top three treatments were special moxibustion plus other therapies 1 (either of tendon acupuncture, acupoint pressing, acupoint injection, etc.), wheat-grain moxibustion and mild moxibustion. The special moxibustion methods were the combination of fire-dragon moxibustion, thunder-fire moxibustion, fuyang fire moxibustion and moxa salt-bag moxibustion. The number of literature of these four moxibustion methods was small. Considering the clinical application of moxibustion, it was concluded that wheat-grain moxibustion ranked first. CONCLUSION: The adjuvant treatment of wheat-grain moxibustion is more effective than other moxibustion methods on improving the quality of life in tumor patients, but the results needed to be further verified because the bias risk of RCT included in this study is high and the sample size is small.

Direct contact moxibustion promotes apoptosis of gastric cancer cells in rats by regulating intestinal flora.

OBJECTIVE: To examine whether direct contact moxibustion (DCM) can prevent and treat gastric cancer (GC) by regulating intestinal flora in rats. METHODS: Male Wistar rats were randomly divided into normal group, normal + DCM control group, model group, and model + DCM group. Gastric cancer rats were induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG, 20 mg/mL) by gavage. At the same time, the model rats and normal rats were given DCM at Zusanli (ST36), Weishu (BL21), and Zhongwan (CV12) for 16 weeks. After treatment, gastric tissues were collected to analyze the pathological changes and the apoptosis of gastric mucosa cells. In addition, the cecal stool was taken and analyzed by 16s rRNA sequencing. RESULTS: Gastric cancer-like pathological changes and different abundance of the intestinal flora were found in the model group. DCM promoted mucosa tissue apoptosis and regulated the abnormal changes of the intestinal microflora caused by MNNG; DCM also inhibited the growth of Ruminococcaceae and Prevotellaceae flora and promoted the growth of probiotic Akkermansia. Furthermore, DCM made the composition and abundance of intestinal microflora in the GC rats tending to the normal rats. CONCLUSION: DCM stimulating Zusanli (ST36), Weishu (BL21), and Zhongwan (CV12) promoted the apoptosis of gastric mucosa and delayed the progression of gastric cancer, possibly by decreasing Ruminococcaceae and Prevotellaceae bacteria (bacteria that produce short-chain fatty acids in the intestine) and promoting the growth of probiotic Akkermansia.

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.).

Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum.

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

Identification and Characterization of the ERF Subfamily B3 Group Revealed GhERF13.12 Improves Salt Tolerance in Upland Cotton.

The APETALA2 (AP2)/ethylene response factor plays vital functions in response to environmental stimulus. The ethylene response factor (ERF) subfamily B3 group belongs to the AP2/ERF superfamily and contains a single AP2/ERF domain. Phylogenetic analysis of the ERF subfamily B3 group genes from Arabdiposis thaliana, Gossypium arboreum, Gossypium hirsutum, and Gossypium raimondii made it possible to divide them into three groups and showed that the ERF subfamily B3 group genes are conserved in cotton. Collinearity analysis identified172 orthologous/paralogous gene pairs between G. arboreum and G. hirsutum; 178 between G. hirsutum and G. raimondii; and 1,392 in G. hirsutum. The GhERF subfamily B3 group gene family experienced massive gene family expansion through either segmental or whole genome duplication events, with most genes showing signature compatible with the action of purifying selection during evolution. Most G. hirsutum ERF subfamily B3 group genes are responsive to salt stress. GhERF13.12 transgenic Arabidopsis showed enhanced salt stress tolerance and exhibited regulation of related biochemical parameters and enhanced expression of genes participating in ABA signaling, proline biosynthesis, and ROS scavenging. In addition, the silencing of the GhERF13.12 gene leads to increased sensitivity to salt stress in cotton. These results indicate that the ERF subfamily B3 group had remained conserved during evolution and that GhERF13.12 induces salt stress tolerance in Arabidopsis and cotton.

Comparative phosphoproteomic analysis of BR-defective mutant reveals a key role of GhSK13 in regulating cotton fiber development.

Brassinosteroid (BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1 (pag1), and its corresponding wild-type (ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage (10 days after pollination (DPA)) and secondary cell wall synthesis stage (20 DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs (GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor Gh_DNF_YB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.

Start-up and operational performance of the partial nitrification process in a sequencing batch reactor (SBR) coupled with a micro-aeration system.

Partial nitrification (PN) of ammonia to nitrite is investigated in a lab-scale sequencing batch reactor (SBR) coupled with both a microporous aeration system and a mechanical agitation system at a moderate temperature of (27 ±â€¯1 °C). The SBR has a high actual oxygen transfer efficiency (AOTE) of 2.0% and dynamical efficiency (DE) of 20.0%. Alkalinity consumption declined with the decreasing ratios of HCO3- to NH4+-N in the influent from 2.57, 1.96, 1.91 to 1.66, while the pH of the effluent is constantly maintained at 7.5 ±â€¯0.1. The SBR is successfully operated for 195 days at a nitrogen loading rate (NLR) of up to 2.82 kg·m-3.d-1, achieving a nitrite accumulation rate (NAR) of over 90%. The high-throughput sequencing shows that the ratio of Nitrosomonas, the dominant species, is up to 29.83%.

Identification of Histone H3 (HH3) Genes in Gossypium hirsutum Revealed Diverse Expression During Ovule Development and Stress Responses.

Histone acts as the core for nucleosomes and is a key protein component of chromatin. Among different histone variants, histone H3 (HH3) variants have been reported to play vital roles in plant development. However, biological information and evolutionary relationships of HH3 genes in cotton remain to be elucidated. The current study identified 34 HH3 genes in Gossypium hirsutum. Phylogenetic analysis classified HH3 genes of 19 plant species into eight distinct clades. Sequence logos analysis among Arabidopsis, rice, and G. hirsutum amino acid residues showed higher conservation in amino acids. Using collinearity analysis, we identified 81 orthologous/paralogous gene pairs among the four genomes (A, D, At, and Dt) of cotton. Further, orthologous/paralogous and the Ka/Ks ratio demonstrated that cotton HH3 genes experienced strong purifying selection pressure with restricted functional divergence resulting from segmental and whole genome duplication. Expression pattern analysis indicated that GhHH3 genes were preferentially expressed in cotton ovule tissues. Additionally, GhHH3 gene expression can be regulated by abiotic stresses (cold, heat, sodium chloride (NaCl), and polyethylene glycol (PEG)) and phytohormonal (brassinolide (BL), gibberellic acid (GA), indole-3-acetic acid (IAA), salicylic acid (SA), and methyl jasmonate (MeJA)) treatments, suggesting that GhHH3 genes might play roles in abiotic and hormone stress resistance. Taken together, this work provides important information to decipher complete molecular and physiological functions of HH3 genes in cotton.

Genome-Wide Identification and Characterization of the PERK Gene Family in Gossypium hirsutum Reveals Gene Duplication and Functional Divergence.

Proline-rich extensin-like receptor kinases (PERKs) are an important class of receptor kinases in plants. Receptor kinases comprise large gene families in many plant species, including the 15 PERK genes in Arabidopsis. At present, there is no comprehensive published study of PERK genes in G. hirsutum. Our study identified 33 PERK genes in G. hirsutum. Phylogenetic analysis of conserved PERK protein sequences from 15 plant species grouped them into four well defined clades. The GhPERK gene family is an evolutionarily advanced gene family that lost its introns over time. Several cis-elements were identified in the promoter regions of the GhPERK genes that are important in regulating growth, development, light responses and the response to several stresses. In addition, we found evidence for gene loss or addition through segmental or whole genome duplication in cotton. Gene duplication and synteny analysis identified 149 orthologous/paralogous gene pairs. Ka/Ks values show that most GhPERK genes experienced strong purifying selection during the rapid evolution of the gene family. GhPERK genes showed high expression levels in leaves and during ovule development. Furthermore, the expression of GhPERK genes can be regulated by abiotic stresses and phytohormone treatments. Additionally, PERK genes could be involved in several molecular, biological and physiological processes that might be the result of functional divergence.

Transcriptome profiling of Gossypium arboreum during fiber initiation and the genome-wide identification of trihelix transcription factors.

Cotton fiber initiation is the first step in fiber development, and it determines the yield. Here, genome-wide transcriptome profiling of Gossypium arboreum was performed to determine the molecular basis of cotton fiber initiation. A comparison of the transcriptomes of fiber-bearing ovules at -0.5, 0, 0.5, 1, 1.5, 2, 2.5 and 3 d post-anthesis detected 12,049 differentially expressed genes that mainly participated in ribosome, carbon metabolism and amino acid biosynthesis pathways. Genes encoding alcohol dehydrogenase 1 and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, involving in fatty acid degradation and flavonoid biosynthesis, were enriched. Furthermore, 1049 differentially expressed transcription factors were identified. Among these, 17 were trihelix family transcription factors, which play important roles in plant development and responses to biotic and abiotic stresses. In total, 52 full-length trihelix genes, named as GaGTs, were identified in G. arboreum and located in 12 of the 13 cotton chromosomes. Transcriptomic data and a quantitative real-time PCR analysis indicated that several GaGTs were significantly induced during fiber initiation in G. arboreum. Thus, the genome-wide comprehensive analysis of gene expression in G. arboreum fiber initiation will serve as a useful resource for unraveling the functions of specific genes. The phylogenetic relationships and expression analyses of the G. arboreum trihelix genes established a solid foundation for future comprehensive functional analyses of the GaGTs.

Genome-wide analysis of BES1 genes in Gossypium revealed their evolutionary conserved roles in brassinosteroid signaling.

Brassinosteroids (BRs), which are essential phytohormones for plant growth and development, are important for cotton fiber development. Additionally, BES1 transcription factors are critical for BR signal transduction. However, cotton BES1 family genes have not been comprehensively characterized. In this study, we identified 11 BES1 genes in G. arboreum, 11 in G. raimondii, 16 in G. barbadense, and 22 in G. hirsutum. The BES1 sequences were significantly conserved in the Arabidopsis thaliana, rice, and upland cotton genomes. A total of 94 BES1 genes from 10 different plant species were divided into three clades according to the neighbor-joining and minimum-evolution methods. Moreover, the exon/intron patterns and motif distributions were highly conserved among the A. thaliana and cotton BES1 genes. The collinearity among the orthologs from the At and Dt subgenomes was estimated. Segmental duplications in the At and Dt subgenomes were primarily responsible for the expansion of the cotton BES1 gene family. Of the GhBES1 genes, GhBES1.4_At/Dt exhibited BL-induced expression and was predominantly expressed in fibers. Furthermore, Col-0/mGhBES1.4_At plants produced curled leaves with long and bent petioles. These transgenic plants also exhibited decreased hypocotyl sensitivity to brassinazole and constitutive BR induced/repressed gene expression patterns. The constitutive BR responses of the plants overexpressing mGhBES1.4_At were similar to those of the bes1-D mutant.

Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses.

Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells.

A lower pH value benefits regeneration of Trichosanthes kirilowii by somatic embryogenesis, involving rhizoid tubers (RTBs), a novel structure.

A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0-9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids.