The Contributions of Sub-Communities to the Assembly Process and Ecological Mechanisms of Bacterial Communities along the Cotton Soil-Root Continuum Niche Gradient.

Soil microbes are crucial in shaping the root-associated microbial communities. In this study, we analyzed the effect of the soil-root niche gradient on the diversity, composition, and assembly of the bacterial community and co-occurrence network of two cotton varieties. The results revealed that the bacterial communities in cotton soil-root compartment niches exhibited a skewed species abundance distribution, dominated by abundant taxa showing a strong spatial specificity. The assembly processes of the rhizosphere bacterial communities were mainly driven by stochastic processes, dominated by the enrichment pattern and supplemented by the depletion pattern to recruit bacteria from the bulk soil, resulting in a more stable bacterial community. The assembly processes of the endosphere bacterial communities were determined by processes dominated by the depletion pattern and supplemented by the enrichment pattern to recruit species from the rhizosphere, resulting in a decrease in the stability and complexity of the community co-occurrence network. The compartment niche shaped the diversity of the bacterial communities, and the cotton variety genotype was an important source of diversity in bacterial communities within the compartment niche. We suggest that the moderate taxa contribute to significantly more changes in the diversity of the bacterial community than the rare and abundant taxa during the succession of bacterial communities in the cotton root-soil continuum.

Genome-wide association study identifies GhSAL1 affects cold tolerance at the seedling emergence stage in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Genomic analysis of upland cotton revealed that cold tolerance was associated with ecological distribution. GhSAL1 on chromosome D09 negatively regulated cold tolerance of upland cotton. Cotton can undergo low-temperature stress at the seedling emergence stage, which adversely affects growth and yield; however, the regulatory mechanism underlying cold tolerance remains nebulous. Here, we analyze the phenotypic and physiological parameters in 200 accessions from 5 ecological distributions under constant chilling (CC) and diurnal variation of chilling (DVC) stresses at the seedling emergence stage. All accessions were clustered into four groups, of which Group IV, with most germplasms from the northwest inland region (NIR), had better phenotypes than Groups I-III under the two kinds of chilling stresses. A total of 575 significantly associated single-nucleotide polymorphism (SNP) were identified, and 35 stable genetic quantitative trait loci (QTL) were obtained, of which 5 were associated with traits under CC and DVC stress, respectively, while the remaining 25 were co-associated. The accumulation of dry weight (DW) of seedling was associated with the flavonoid biosynthesis process regulated by Gh_A10G0500. The emergence rate (ER), DW, and total length of seedling (TL) under CC stress were associated with the SNPs variation of Gh_D09G0189 (GhSAL1). GhSAL1HapB was the elite haplotype, which increased ER, DW, and TL by 19.04%, 11.26%, and 7.69%, respectively, compared with that of GhSAL1HapA. The results of virus-induced gene silencing (VIGS) experiment and determination of metabolic substrate content preliminarily illustrated that GhSAL1 negatively regulated cotton cold tolerance through IP3-Ca2+ signaling pathway. The elite haplotypes and candidate genes identified in this study could be used to improve cold tolerance at the seedling emergence stage in future upland cotton breeding.

GhMYB102 promotes drought resistance by regulating drought-responsive genes and ABA biosynthesis in cotton (Gossypium hirsutum L.).

The MYB transcription factor (TF) family is among the largest TF families and plays an important role in plant growth and stress response. However, few studies have investigated the role of the MYB gene in drought resistance in cotton. In this study, we analysed the drought transcriptomic data of cotton and identified that the GhMYB102 gene was significantly upregulated in upland cotton during the early stages of drought stress. Bioinformatics analysis showed that the amino acid sequence encoded by GhMYB102 contained two highly conserved MYB binding domains belonging to R2R3-MYB TFs. GhMYB102 was most closely related to AtMYB102. GhMYB102 is mainly expressed in roots and is induced by abiotic stresses and abscisic acid (ABA); it is localised in the nucleus and has transcriptional activation activity. Silencing of GhMYB102 decreased plant drought resistance. In addition, dual-luciferase assays and yeast single hybridisation analysis showed that GhMYB102 could directly bind the MYB motif elements in the promoter regions of GhNCED1 and GhZAT10. These results indicate that GhMYB102 plays a positive role in drought tolerance by regulating the expression of GhNCED1 and GhZAT10. Thus, GhMYB102 enhances drought resistance by participating in ABA biosynthesis or regulating the expression of drought-responsive genes.

Genome-wide association study reveals that GhTRL1 and GhPIN8 affect cotton root development.

KEY MESSAGE: Two regions located at chromosome A05 and D04 were found to be significantly associated with 0-0.5 mm and 0.5-2 mm diameter roots, respectively, and two candidate genes related to root development were identified. Roots absorb water and nutrients, and play an important role in plant growth. However, there are few genetic developmental studies on cotton root structural traits. In this study, we used 200 upland cotton (Gossypium hirsutum L.) varieties to analyze the phenotypic variation of 43 traits. A total of 2001 related single-nucleotide polymorphism (SNP) sites located within or near 1046 genes were detected through a genome-wide association study (GWAS). The 32 root traits were linked to SNPs that corresponded to 317 nonrepetitive genes. For SNPs associated with root length and 0-0.5 mm diameter root traits, a significant peak appeared on chromosome A05 (between 21.91 and 22.24 Mb). For SNPs associated with root surface area, root volume and 0.5-2 mm diameter root traits, a significant peak appeared on chromosome D04 (between 7.35 and 7.70 Mb). Within these two key regions, SNPs were detected in the promoter and coding regions of two candidate genes, GhTRL1-A05 and GhPIN8-D04. The expression levels of these two genes also changed significantly according to transcriptome sequencing and quantitative real-time PCR (qRT-PCR). After silencing the GhTRL1 and GhPIN8 genes via virus-induced gene silencing (VIGS), we found that the plants expressing TRV2::GhTRL1 and TRV2::GhPIN8 had a reduced root length, surface area. Moreover, the contents of cis-12-oxo-phytodienoic acid (cis-OPDA), isopentenyl adenosine (iPR) and cis-zeatin (cZ) in the roots of the plants expressing TRV2::GhTRL1 decreased. This study contributes to the cultivation and improvement of cotton varieties.

Development of pyrazolo[3,4-d]pyrimidin-4-one scaffold as novel CDK2 inhibitors: Design, synthesis, and biological evaluation.

A series of pyrazolo[3,4-d]pyrimidin-4-one scaffold were designed and synthesized as novel CDK2 inhibitors. By analyzing the common motifs of various known inhibitors, the designed compounds 1 were virtually screen for their inhibitory activity by docking into the active pocket of CDK2. The influence of different substitutes on the docking results was investigated. A total of 15 pyrazolo[3,4-d]pyrimidin-4-ones 1 were synthesized by Paal-Knorr reaction, pyrimidine ring closure, bromination, Suzuki coupling reaction, amide formation and Knoevenagel condensation. The Cell Counting Kit-8 (CCK-8) was used to evaluate the inhibitory activity of pyrazolo[3,4-d]pyrimidin-4-ones 1 in the breast cancer cell line MCF-7 in vitro using Etoposide as a reference control substance. The screening results demonstrated that the designed compounds have significant antiproliferative activity, and compounds 1e and 1j were the most active compounds with IC50 values of 10.79 µM and 10.88 µM, respectively, being better than that of Etoposide (IC50 = 18.75 µM). The enzyme inhibition assay was carried out against CDK2, the results indicated that the compounds 1e and 1j significantly inhibited CDK2 with IC50 values of 1.71 µM and 1.60 µM.

Genome-wide association study identifies variants of GhSAD1 conferring cold tolerance in cotton.

Cold stress is a major environmental factor affecting plant growth and development. Although some plants have developed resistance to cold stress, the molecular mechanisms underlying this process are poorly understood. Using genome-wide association mapping with 200 cotton accessions collected from different regions, we identified variations in the short chain alcohol dehydrogenase gene, GhSAD1, that responds to cold stress. Virus-induced gene silencing and overexpression in Arabidopsis revealed that GhSAD1 fulfils important roles in cold stress responses. Ectopic expression of a haploid genotype of GhSAD1 (GhSAD1HapB) in Arabidopsis increased cold tolerance. Silencing of GhSAD1HapB resulted in a decrease in abscisic acid (ABA) content. Conversely, overexpression of GhSAD1HapB increased ABA content. GhSAD1HapB regulates cold stress responses in cotton through modulation of C-repeat binding factor activity, which regulates ABA signalling. GhSAD1HapB induces the expression of COLD-REGULATED (COR) genes and increases the amount of metabolites associated with cold stress tolerance. Overexpression of GhSAD1HapB partially complements the phenotype of the Arabidopsis ABA2 mutant, aba2-1. Collectively, these findings increase our understanding of the mechanisms underlying GhSAD1-mediated cold stress responses in cotton.

Ectopic expression of GhIQD14 (cotton IQ67 domain-containing protein 14) causes twisted organ and modulates secondary wall formation in Arabidopsis.

In plants, although KNOX genes are known to regulate secondary cell wall (SCW) formation, their protein-regulating mechanisms remain largely unknown. Here, we showed that GhKNL1, which regulates SCW formation and fiber development in cotton, could interact with an IQ67 domain containing protein (GhIQD14) in yeast. Confocal observation showed that GhIQD14 was localized to the microtubules. In Arabidopsis, ectopic expression of GhIQD14 caused hypocotyls to be sensitive to microtubule depolymerization agent, organ twisting of seedlings, trichomes, rosette leaves, and capsules, as well as severely irregular xylem vessels and thicker interfascicular fiber cell walls in the inflorescence stem. Furthermore, we found that GhIQD14 interacted with AtKNAT7 in vivo, and instantaneous co-expression of GhIQD14 and AtKNAT7 in tobacco showed that GhIQD14 weakened the distribution of AtKNAT7 in the nucleus, bringing it into the microtubules, thus affecting the SCW formation related genes expression. Our results suggested that GhIQD14 might be involved in the morphological development and SCW formation in cotton.

Transcriptomic analyses show that 24-epibrassinolide (EBR) promotes cold tolerance in cotton seedlings.

In plants, brassinosteroids (BRs) are a class of steroidal hormones that are involved in numerous physiological responses. However, the function of BRs in cold tolerance in cotton has not been explored. In this study, cotton seedlings were treated with five concentrations (0, 0.05, 0.1, 0.2, 0.5 and 1.0 mg/L) of 24-Epibrassinolide (EBR) at 4°C. We measured the electrolyte leakage, malondialdehyde (MDA) content, proline content, and net photosynthesis rate (Pn) of the seedlings, which showed that EBR treatment increased cold tolerance in cotton in a dose-dependent manner, and that 0.2 mg/L is an optimum concentration for enhancing cold tolerance. The function of EBR in cotton cotyledons was investigated in the control 0 mg/L (Cold+water) and 0.2 mg/L (Cold+EBR) treatments using RNA-Seq. A total of 4,001 differentially expressed genes (DEGs), including 2,591 up-regulated genes and 1,409 down-regulated genes were identified. Gene Ontology (GO) and biochemical pathway enrichment analyses showed that EBR is involved in the genetic information process, secondary metabolism, and also inhibits abscisic acid (ABA) and ethylene (ETH) signal transduction. In this study, physiological experiments showed that EBR can increase cold tolerance in cotton seedlings, and the comprehensive RNA-seq data shed light on the mechanisms through which EBR increases cold tolerance in cotton seedlings.

Comparative Transcriptome Analysis Provides Insights into the Seed Germination in Cotton in Response to Chilling Stress.

Gossypium hirsutum L., is a widely cultivated cotton species around the world, but its production is seriously threatened by its susceptibility to chilling stress. Low temperature affects its germination, and the underlying molecular mechanisms are rarely known, particularly from a transcriptional perspective. In this study, transcriptomic profiles were analyzed and compared between two cotton varieties, the cold-tolerant variety KN27-3 and susceptible variety XLZ38. A total of 7535 differentially expressed genes (DEGs) were identified. Among them, the transcripts involved in energy metabolism were significantly enriched during germination based on analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as glycolysis/gluconeogenesis, tricarboxylic acid cycle (TCA cycle), and glyoxylate cycle (GAC). Results from further GO enrichment analysis show the earlier appearance of DNA integration, meristem growth, cotyledon morphogenesis, and other biological processes in KN27-3 compared with XLZ38 under chilling conditions. The synthesis of asparagine, GDP-mannose, and trehalose and the catabolic process of raffinose were activated. DEGs encoding antioxidants (spermidine) and antioxidase (CAT1, GPX4, DHAR2, and APX1) were much more up-regulated in embryos of KN27-3. The content of auxin (IAA), cis-zeatin riboside (cZR), and trans-zeatin riboside (tZR) in KN27-3 are higher than that in XLZ38 at five stages (from 12 h to 54 h). GA3 was expressed at a higher level in KN27-3 from 18 h to 54 h post imbibition compared to that in XLZ38. And abscisic acid (ABA) content of KN27-3 is lower than that in XLZ38 at five stages. Results from hormone content measurements and the related gene expression analysis indicated that IAA, CTK, and GA3 may promote germination of the cold-tolerant variety, while ABA inhibits it. These results expand the understanding of cottonseed germination and physiological regulations under chilling conditions by multiple pathways.

Transcriptome analysis and identification of genes associated with fruiting branch internode elongation in upland cotton.

BACKGROUND: Appropriate plant architecture can improve the amount of cotton boll opening and allow increased planting density, thus increasing the level of cotton mechanical harvesting and cotton yields. The internodes of cotton fruiting branches are an important part of cotton plant architecture. Thus, studying the molecular mechanism of internode elongation in cotton fruiting branches is highly important. RESULTS: In this study, we selected internodes of cotton fruiting branches at three different stages from two cultivars whose internode lengths differed significantly. A total of 76,331 genes were detected by transcriptome sequencing. By KEGG pathway analysis, we found that DEGs were significantly enriched in the plant hormone signal transduction pathway. The transcriptional data and qRT-PCR results showed that members of the GH3 gene family, which are involved in auxin signal transduction, and CKX enzymes, which can reduce the level of CKs, were highly expressed in the cultivar XLZ77, which has relatively short internodes. Genes related to ethylene synthase (ACS), EIN2/3 and ERF in the ethylene signal transduction pathway and genes related to JAR1, COI1 and MYC2 in the JA signal transduction pathway were also highly expressed in XLZ77. Plant hormone determination results showed that the IAA and CK contents significantly decreased in cultivar XLZ77 compared with those in cultivar L28, while the ACC (the precursor of ethylene) and JA contents significantly increased. GO enrichment analysis revealed that the GO categories associated with promoting cell elongation, such as cell division, the cell cycle process and cell wall organization, were significantly enriched, and related genes were highly expressed in L28. However, genes related to the sphingolipid metabolic process and lignin biosynthetic process, whose expression can affect cell elongation, were highly expressed in XLZ77. In addition, 2067 TFs were differentially expressed. The WRKY, ERF and bHLH TF families were the top three largest families whose members were active in the two varieties, and the expression levels of most of the genes encoding these TFs were upregulated in XLZ77. CONCLUSIONS: Auxin and CK are positive regulators of internode elongation in cotton branches. In contrast, ethylene and JA may act as negative regulators of internode elongation in cotton branches. Furthermore, the WRKY, ERF and bHLH TFs were identified as important inhibitors of internode elongation in cotton. In XLZ77(a short-internode variety), the mass synthesis of ethylene and amino acid conjugation of auxin led to the inhibition of plant cell elongation, while an increase in JA content and degradation of CKs led to a slow rate of cell division, which eventually resulted in a phenotype that presented relatively short internodes on the fruiting branches. The results of this study not only provide gene resources for the genetic improvement of cotton plant architecture but also lay a foundation for improved understanding of the molecular mechanism of the internode elongation of cotton branches.

Analysis of Drought Tolerance and Associated Traits in Upland Cotton at the Seedling Stage.

(1) Background: Upland cotton (Gossypium hirsutum L.) is the most important natural fiber worldwide, and it is extensively planted and plentifully used in the textile industry. Major cotton planting regions are frequently affected by abiotic stress, especially drought stress. Drought resistance is a complex, quantitative trait. A genome-wide association study (GWAS) constitutes an efficient method for dissecting the genetic architecture of complex traits. In this study, the drought resistance of a population of 316 upland cotton accessions was studied via GWAS. (2) Methods: GWAS methodology was employed to identify relationships between molecular markers or candidate genes and phenotypes of interest. (3) Results: A total of 8, 3, and 6 SNPs were associated with the euphylla wilting score (EWS), cotyledon wilting score (CWS), and leaf temperature (LT), respectively, based on a general linear model and a factored spectrally transformed linear mixed model. For these traits, 7 QTLs were found, of which 2 each were located on chromosomes A05, A11, and D03, and of which 1 was located on chromosome A01. Importantly, in the candidate regions WRKY70, GhCIPK6, SnRK2.6, and NET1A, which are involved in the response to abscisic acid (ABA), the mitogen-activated protein kinase (MAPK) signaling pathway and the calcium transduction pathway were identified in upland cotton at the seedling stage under drought stress according to annotation information and linkage disequilibrium (LD) block analysis. Moreover, RNA sequencing analysis showed that WRKY70, GhCIPK6, SnRK2.6, and NET1A were induced by drought stress, and the expression of these genes was significantly different between normal and drought stress conditions. (4) Conclusions: The present study should provide some genomic resources for drought resistance in upland cotton. Moreover, the germplasm of the different phenotypes, the detected SNPs and, the potential candidate genes will be helpful for molecular marker-assisted breeding studies about increased drought resistance in upland cotton.

The compensation effects of physiology and yield in cotton after drought stress.

The objective of this study was to investigate the root growth compensatory effects and cotton yield under drought stress. The results indicate that the root dry weight, boll weight, and cotton yield increased in both the drought-resistant cultivar (CCRI-45) and the drought-sensitive cultivar (CCRI-60). Compensation effects were exhibited under the three-day drought stress treatment at a soil relative water content (SRWC) of 60% and 45% during the seedling stage, and flowering and boll-forming stage over two years. The yield of the drought-resistant cultivar (CCRI-45) was higher than the control, however, following the six-day 45% SRWC drought treatments, the yield of the drought-sensitive cultivar (CCRI-60) was lower than the control. The soluble sugar content, proline content, superoxide dismutase (SOD) activity, and peroxidase (POD) activity of the roots increased under drought stress and then decreased after re-watering, although the values remained higher than those of the controls for a short period. These physiological measures may represent stress reactions and thus may not indicate factors that result in compensation effects. However, catalase (CAT) activity and gibberellic acid (GA) content of the roots decreased under drought stress. After re-watering, the CAT activity and the GA content increased and were significantly higher than those of the controls under the six-day 60% SRWC and 45% SRWC drought treatments. The abscisic acid (ABA) content of the roots increased under drought stress. After re-watering, the ABA content decreased to a lower level under the three and six-day 60% SRWC and 45% SRWC drought treatments than in the controls. According to an analysis of various indicators, the interaction between ABA and GA signals may play an important role in root growth compensatory effects. In summary, the results demonstrate that moderate drought stress is beneficial to root growth and yield. This conclusion is of great significance to improving our understanding of the maximum utilization of limited water resources.

Single-point Mutation of an Histidine-aspartic Domain-containing Gene involving in Chloroplast Ribosome Biogenesis Leads to White Fine Stripe Leaf in Rice.

Plant leaves are a crucial organ associated closely with chloroplast development, photosynthesis rate and crop productivity. In this study, a white fine stripe leaf 1 (wfsl1) mutant was isolated and characterized from the japonica rice Zhonghua11 (ZH11) after ethyl methanesulfonate mutagenesis. The wfsl1 displayed white fine stripe leaves since tillering stage and abnormal chloroplast structure. Map-based cloning and Bioinformatic analysis indicated that WFSL1 on chromosome 1 contains an "A" to "T" substitution in protein coding region, and encodes a putative metal-dependent phosphohydrolase with HD domain at the N-terminus. WFSL1 was targeted to the chloroplasts and had higher expression in mature leaves and sheaths. RNA-seq analysis revealed that chloroplast development and photosynthesis genes were significantly affected in wfsl1 plants. Levels of WFSL1 and chloroplast encoded proteins were decreased in wfsl1 mutants via western blot analysis. Compared with WT, wfsl1 exhibits lower Chl content and defective in biogenesis of chloroplast ribosomes, which resulted in reduced grain yield. Taken together, our results show that WFSL1 is critical for chloroplast development, ribosome biogenesis, and light energy utilization, finally affects grain yield.

Genomic and phenotypic analyses of Pseudomonas psychrotolerans PRS08-11306 reveal a turnerbactin biosynthesis gene cluster that contributes to nitrogen fixation.

Plant-microbe interactions can provide agronomic benefits, such as enhancing nutrient uptake and providing fixed nitrogen. The Pseudomonas psychrotolerans strain PRS08-11306 was isolated from rice seeds and can enhance plant growth. Here, we analyzed the P. psychrotolerans genome, which is ∼5Mb, with 4389 coding sequences, 77 tRNAs, and 7 rRNAs. Genome analysis identified a cluster of turnerbactin biosynthetic genes, which are responsible for the production of a catecholate siderophore and contribute to nitrogen fixation for the host. Analysis of the transcription factor mutant ΔrpoS, which does not express this gene cluster, confirmed the relationship between the gene cluster and siderophore production. The nitrogen fixation characteristics of the cluster were confirmed in a plant growth-promoting experiment. The annotated full genome sequence of this strain sheds light on the role of P. psychrotolerans PRS08-11306 as a plant beneficial bacterium.

White stripe leaf 12 (WSL12), encoding a nucleoside diphosphate kinase 2 (OsNDPK2), regulates chloroplast development and abiotic stress response in rice (Oryza sativa L.).

Chloroplast is a crucial organelle for plant photosynthesis and maintaining normal life activities in higher plants. Although some genes related to chloroplast development and pigment synthesis have been identified or cloned in rice, little is known about the relationship between these genes and abiotic stress response. In this study, we identified a novel mutant white stripe leaf 12 (wsl12) affecting pigment synthesis, chloroplast development and abiotic stress response in rice. The mutant phenotype was obvious at seeding and tillering stages and in response to the temperature change. Genetic analysis of reciprocal crosses between wsl12 and wild-type plants showed that wsl12 was a recessive mutant in a single nuclear locus. Map-based cloning revealed that the WSL12 locus encoded OsNDPK2, one of the three nucleoside diphosphate kinases (OsNDPKs). WSL12 expressed in all tested tissues, while it highly expressed in leaves and young tissues. The WSL12 protein localized to the chloroplast. The wsl12 mutant showed higher superoxide anion level and enhanced sensitivity to abscisic acid (ABA) and salinity. The transcription pattern of many genes involved in chlorophyll biosynthesis, ABA synthesis, light signaling pathway, reactive oxygen species-scavenging pathway and the other two OsNDPKs was altered in the wsl12 mutant. These results indicate that the OsNDPK2 encoded by WSL12 plays an important role in chloroplast development and chlorophyll biosynthesis by regulating the expression levels of related genes. In addition, WSL12 also affects the response to abiotic stress, such as ABA and salinity in rice, and is beneficial to molecular breeding of stress tolerance.

[Low dose all-trans retinoic acid and androgen therapy for patients with myelodysplastic syndrome].

To explore therapeutic efficacy of androgens and low dose all-trans retinoic acid (ATRA) for myelodysplastic syndrome (MDS) patients, 55 patients of MDS were observed, including 41 cases of refractory anemia (RA), 11 cases of refractory anemia with excess of blasts (RAEB), 2 cases of refractory anemia with excess of blasts in transformation (RAEB-t) and 1 case of chronic myeloic-monocytic leukemia (CMML). These patients received danazol (600 mg/day) or stanazol (6 mg/day) and ATRA (10 mg/day) for at least 3 months. The results showed that according to MDS international working group response criteria, at the end of three months,complete remission (CR) was seen in 1 patient, partial remission (PR) was found in 2 patients. Hematologic improvement: major response (MaR) were seen in 15 patients, minor response (MiR) were seen in 4 patients. The total response rate was 35.8%. In conclusion, danazol or stanazol in combination with low dose ATRA are partialy effective in therapy for patients with low-risk myelodysplastic syndrome.