Characterization of salt tolerant wheat genotypes by using morpho-physiological, biochemical, and molecular analysis.

Food security is facing a major threat from salinity and there is a need to develop salt tolerant crop varieties to ensure that the demand for food from the world's increasing population is met. Salinity mostly occurs in arid and semi-arid regions. It may cause many adverse physiological effects on plants, i.e., toxic ion accumulation, disturbed osmotic potential, and decreased crop yield. The present study aimed to investigate the morphological, physiological, biochemical, and genetic parameters of wheat genotypes under salt stress. Six wheat genotypes were screened for salt tolerance at the seedling and maturity stage. Seeds were sown at 0 and 150 mM of salinity level. Biochemical traits, i.e., shoot/root fresh and dry weight, chlorophyll a/b and total chlorophyll contents, shoot nitrogen, shoot phosphorus, proline, and carbohydrates were measured. Wheat genotypes showed a significant increase in free amino acids, shoot nitrogen, and total soluble proteins under saline conditions. Higher Na+/K+ ratio and free amino acids were estimated under 150 mM NaCl treatment in Pasban-90 and found to be the most salt-tolerant genotype. By contrast, reduced proline, total chlorophyll, and Na+/K+ ratio were found in Kohistan-97 marking it to be sensitive to stress. Expression analysis of HKTs genes was performed to validate the results of two contrasting genotypes. The differential expression of HKT2; 1 and HKT2; 3 explained the tissue and genotype specific epigenetic variations. Our findings indicated that these selected genotypes can be further used for molecular studies to find out QTLs/genes related to salinity. This suggests that, in contrasting wheat genotypes, there is a differentially induced defense response to salt stress, indicating a functional correlation between salt stress tolerance and differential expression pattern in wheat.

Mitogen-activated protein kinase TaMPK3 suppresses ABA response by destabilising TaPYL4 receptor in wheat.

Abscisic acid (ABA) receptors are considered as the targeted manipulation of ABA sensitivity and water productivity in plants. Regulation of their stability or activity will directly affect ABA signalling. Mitogen-activated protein kinase (MAPK) cascades link multiple environmental and plant developmental cues. However, the molecular mechanism of ABA signalling and MAPK cascade interaction remains largely elusive. TaMPK3 overexpression decreases drought tolerance and wheat sensitivity to ABA, significantly weakening ABA's inhibitory effects on growth. Under drought stress, overexpression lines show lower survival rates, shoot fresh weight and proline content, but higher malondialdehyde levels at seedling stage, as well as decreased grain width and 1000 grain weight in both glasshouse and field conditions at the adult stage. TaMPK3-RNAi increases drought tolerance. TaMPK3 interaction with TaPYL4 leads to decreased TaPYL4 levels by promoting its ubiquitin-mediated degradation, whereas ABA treatment diminishes TaMPK3-TaPYL interactions. In addition, the expression of ABA signalling proteins is impaired in TaMPK3-overexpressing wheat plants under ABA treatment. The MPK3-PYL interaction module was found to be conserved across monocots and dicots. Our results suggest that the MPK3-PYL module could serve as a negative regulatory mechanism for balancing appropriate drought stress response with normal plant growth signalling in wheat.

Identification of a Pm4 Allele as a Powdery Mildew Resistance Gene in Wheat Line Xiaomaomai.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive foliar diseases of wheat. In this study, we combined the bulked segregant RNA sequencing (BSR-seq) and comparative genomics analysis to localize the powdery mildew resistance gene in Chinese landrace Xiaomaomai. Genetic analysis of F1 plants from a crossing of Xiaomaomai × Lumai23 and the derived F2 population suggests that a single recessive gene, designated as pmXMM, confers the resistance in this germplasm. A genetic linkage map was constructed using the newly developed SNP markers and pmXMM was mapped to the distal end of chromosome 2AL. The two flanking markers 2AL15 and 2AL34 were closely linked to pmXMM at the genetic distance of 3.9 cM and 1.4 cM, respectively. Using the diagnostic primers of Pm4, we confirmed that Xiaomaomai carries a Pm4 allele and the gene function was further validated by the virus-induced gene silencing (VIGS). In addition, we systematically analyzed pmXMM in comparison with the other Pm4 alleles. The results suggest that pmXMM is identical to Pm4d and Pm4e at sequence level. Pm4b is also not different from Pm4c according to their genome/amino acid sequences. Only a few nucleotide variances were detected between pmXMM and Pm4a/b, which indicate the haplotype variation of the Pm4 gene.

Transcriptomic profiling of wheat stem during meiosis in response to freezing stress.

Low temperature injury in spring has seriously destabilized the production and grain quality of common wheat. However, the molecular mechanisms underlying spring frost tolerance remain elusive. In this study, we investigated the response of a frost-tolerant wheat variety Zhongmai8444 to freezing stress at the meiotic stage. Transcriptome profiles over a time course were subsequently generated by high-throughput sequencing. Our results revealed that the prolonged freezing temperature led to the significant reductions in plant height and seed setting rate. Cell wall thickening in the vascular tissue was also observed in the stems. RNA-seq analyses demonstrated the identification of 1010 up-regulated and 230 down-regulated genes shared by all time points of freezing treatment. Enrichment analysis revealed that gene activity related to hormone signal transduction and cell wall biosynthesis was significantly modulated under freezing. In addition, among the identified differentially expressed genes, 111 transcription factors belonging to multiple gene families exhibited dynamic expression pattern. This study provided valuable gene resources beneficial for the breeding of wheat varieties with improved spring frost tolerance.

Genome-Wide Analysis of the Soybean Calmodulin-Binding Protein 60 Family and Identification of GmCBP60A-1 Responses to Drought and Salt Stresses.

Calmodulin-binding protein 60 (CBP60) members constitute a plant-specific protein family that plays an important role in plant growth and development. In the soybean genome, nineteen CBP60 members were identified and analyzed for their corresponding sequences and structures to explore their functions. Among GmCBP60A-1, which primarily locates in the cytomembrane, was significantly induced by drought and salt stresses. The overexpression of GmCBP60A-1 enhanced drought and salt tolerance in Arabidopsis, which showed better state in the germination of seeds and the root growth of seedlings. In the soybean hairy roots experiment, the overexpression of GmCBP60A-1 increased proline content, lowered water loss rate and malondialdehyde (MDA) content, all of which likely enhanced the drought and salt tolerance of soybean seedlings. Under stress conditions, drought and salt response-related genes showed significant differences in expression in hairy root soybean plants of GmCBP60A-1-overexpressing and hairy root soybean plants of RNAi. The present study identified GmCBP60A-1 as an important gene in response to salt and drought stresses based on the functional analysis of this gene and its potential underlying mechanisms in soybean stress-tolerance.

New Solution-Processed Surface Treatment to Improve the Photovoltaic Properties of Electrodeposited Cu(In,Ga)Se2 (CIGSe) Solar Cells.

The surface Ga content for a CIGSe absorber was closely related to variation in the open-circuit voltage (VOC), while it was generally low on a CIGSe surface fabricated by two-step selenization. In this work, a solution-processed surface treatment based on spin-coating GaCl3 solution onto a CIGSe surface was applied to increase the Ga content on the surface. XPS, XRD, Raman spectroscopy, and band gap extraction based on the external quantum efficiency response demonstrated that GaCl3 post deposition treatment (GaCl3-PDT) can be used to enhance the Ga content on the surface of a CIGSe absorber. Meanwhile, a solution-processed surface treatment with KSCN (KSCN-PDT) was employed to form a transmission barrier for holes by moving the valence band maximum downward and decreasing the interface recombination between the CdS and CIGSe layers. Admittance spectroscopy results revealed that deep defects were passivated by GaCl3-PDT or KSCN-PDT. By applying the combination of GaCl3-PDT and KSCN-PDT, a champion device was realized that exhibited an efficiency of 13.5% with an improved VOC of 610 mV. Comparing the efficiency of the untreated CIGSe solar cells (11.7%), the CIGSe device efficiency with GaCl3-PDT and KSCN-PDT exhibited 15% enhancement.

Molecular Characterization of All-Stage and Adult-Plant Resistance Loci Against Powdery Mildew in Winter Wheat Cultivar Liangxing 99 Using BSR-Seq Technology.

Winter wheat cultivar Liangxing 99, which carries gene Pm52, is resistant to powdery mildew at both seedling and adult-plant stages. An F2:6 recombinant inbred line population from cross Liangxing 99 × Zhongzuo 9504 was phenotyped with Blumeria graminis f. sp. tritici isolate Bgt27 at the adult-plant stage in four field tests and the seedling stage in a greenhouse test. The analysis of bulk segregant RNA sequencing (BSR-Seq) identified a single-nucleotide polymorphism-enriched locus, Qaprpm.caas.2B, on chromosome 2BL in the same genomic interval of Pm52 associated with the all-stage resistance (ASR) and Qaprpm.caas.7A on chromosome 7AL associated with the adult-plant resistance (APR) against the disease. Qaprpm.caas.2B was detected in a 1.3 cM genetic interval between markers Xicscl726 and XicsK128 in which Pm52 was placed with a range of logarithm of odds (LOD) values from 28.1 to 34.6, and the phenotype variations explained in terms of maximum disease severity (MDS) ranged from 45 to 52%. The LOD peak of Qaprpm.caas.7A was localized in a 4.6 cM interval between markers XicsK7A8 and XicsK7A26 and explained the phenotypic variation of MDS ranging from 13 to 16%. The results of this study confirmed Pm52 for ASR and identified Qaprpm.caas.7A for APR to powdery mildew in Liangxing 99.

Molecular mapping and identification of a candidate gene for new locus Hg2 conferring hairy glume in wheat.

Glume hairiness or pubescence that occurs in hexaploid common wheat and its relatives at different ploidy levels is a distinct morphological marker. Current knowledge about the genetic control of wheat glume hairiness is based on study of Hg1 (formerly Hg) on chromosome 1AS. Here, we report characterization of a new locus for hairy glume Hg2 in synthetic hexaploid wheat line CIGM86.944. Hg2 was inherited a dominant allele. Bulked segregant analysis and RNA-sequencing (BSR-Seq) was performed on an F2:3 population from cross CIGM86.944 × Shannong 29 (glabrous glume), which localized Hg2 in a 2.02 cM genetic interval corresponding to ∼1.08 Mb (754,001,564-755,082,433 Mb) on chromosome 2BL in the Chinese Spring reference genome. Gene annotation and expression identified TraesCS2B02 G562300.1 encoding diacylglycerol kinase 5 protein and TraesCS2B02 G561400.1 encoding a wound-responsive family protein as possible candidate genes regulating development of glume hairiness. The identification of Hg2 provides new insights into the genetic control of glume hairiness in wheat.

Genome-Wide Analysis of the Catharanthus roseus RLK1-Like in Soybean and GmCrRLK1L20 Responds to Drought and Salt Stresses.

Abiotic stresses, such as drought and salinity, severely affects the growth, development and productivity of the plants. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase family is involved in several processes in the plant life cycle. However, there have been few studies addressing the functions of CrRLK1L proteins in soybean. In this study, 38 CrRLK1L genes were identified in the soybean genome (Glycine max Wm82.a2.v1). Phylogenetic analysis demonstrated that soybean CrRLK1L genes were grouped into clusters, cluster I, II, III. The chromosomal mapping demonstrated that 38 CrRLK1L genes were located in 14 of 20 soybean chromosomes. None were discovered on chromosomes 1, 4, 6, 7, 11, and 14. Gene structure analysis indicated that 73.6% soybean CrRLK1L genes were characterized by a lack of introns.15.7% soybean CrRLK1L genes only had one intron and 10.5% soybean CrRLK1L genes had more than one intron. Five genes were obtained from soybean drought- and salt-induced transcriptome databases and were found to be highly up-regulated. GmCrRLK1L20 was notably up-regulated under drought and salinity stresses, and was therefore studied further. Subcellular localization analysis revealed that the GmCrRLK1L20 protein was located in the cell membrane. The overexpression of the GmCrRLK1L20 gene in soybean hairy roots improved both drought tolerance and salt stresses and enhanced the expression of the stress-responsive genes GmMYB84, GmWRKY40, GmDREB-like, GmGST15, GmNAC29, and GmbZIP78. These results indicated that GmCrRLK1L20 could play a vital role in defending against drought and salinity stresses in soybean.

Genomic-Wide Analysis of the PLC Family and Detection of GmPI-PLC7 Responses to Drought and Salt Stresses in Soybean.

Phospholipase C (PLC) performs significant functions in a variety of biological processes, including plant growth and development. The PLC family of enzymes principally catalyze the hydrolysis of phospholipids in organisms. This exhaustive exploration of soybean GmPLC members using genome databases resulted in the identification of 15 phosphatidylinositol-specific PLC (GmPI-PLC) and 9 phosphatidylcholine-hydrolyzing PLC (GmNPC) genes. Chromosomal location analysis indicated that GmPLC genes mapped to 10 of the 20 soybean chromosomes. Phylogenetic relationship analysis revealed that GmPLC genes distributed into two groups in soybean, the PI-PLC and NPC groups. The expression patterns and tissue expression analysis showed that GmPLCs were differentially expressed in response to abiotic stresses. GmPI-PLC7 was selected to further explore the role of PLC in soybean response to drought and salt stresses by a series of experiments. Compared with the transgenic empty vector (EV) control lines, over-expression of GmPI-PLC7 (OE) conferred higher drought and salt tolerance in soybean, while the GmPI-PLC7-RNAi (RNAi) lines exhibited the opposite phenotypes. Plant tissue staining and physiological parameters observed from drought- and salt-stressed plants showed that stress increased the contents of chlorophyll, oxygen free radical (O2 -), hydrogen peroxide (H2O2) and NADH oxidase (NOX) to amounts higher than those observed in non-stressed plants. This study provides new insights in the functional analysis of GmPLC genes in response to abiotic stresses.

A prognostic nomogram incorporating red cell distribution width for patients with intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is the second most common subtype of stroke with higher mortality and morbidity, and it lacks effective prognostic markers. The aim of this research is to construct newly valuable prognostic nomogram incorporating red blood cell distribution width (RDW) for ICH patients.We retrospectively analyzed 953 adult patients with ICH. The impacts of RDW on short-term mortality and functional prognosis were calculated using Akaike information criterion (AIC), Bayesian information criteria (BIC) and the area under the curve (AUC) respectively, which could be used to compare with Glasgow coma scale (GCS) and ICH score. The independent factors of prognosis were identified by univariate and multivariate logistic regression analysis. A nomogram based on RDW for nerve functional prognosis was further constructed and validated. Its clinical value was subsequently explored utilizing decision curve analysis.Cumulative clinical results were retrieved for 235 inpatients from Jan 2012 to June 2017. In 30-day mortality sets, GCS and ICH score had better prognostic performance than RDW (AUC: 0.929 and 0.917 vs 0.764; AIC: 124.101 and 134.188 vs 221.372; BIC: 131.021 and 141.107 vs 228.291). In 30-day functional prognosis sets, the consequences of evaluation systems were inconsistent. GCS was the best parameter for predicting outcome using AIC (262.350 vs 276.392 and 264.756) and BIC (269.269 vs 283.311 and 271.675). However, RDW was higher than GCS and ICH score considering AUC (0.784 vs 0.759 and 0.722). Age, GCS, RDW, platelet distribution width, and surgery were independent prognostic factors by multivariate logistic regression analysis, and those coefficients were used to formulate a nomogram. This nomogram can provide accurate prediction with the concordance index of 0.880 (95% CI, 0.837-0.922) higher than Harrell's concordance index of GCS system 0.759 (95% CI, 0.698-0.819) and RDW 0.784 (95% CI, 0.721-0.847). The calibration plots showed optimal consistency between bootstrap-predicted and the actual observed values of 30-day unfavorable prognosis. Decision curve analysis showed an increased net benefit for utilizing the nomogram.High RDW values are associated with an unfavorable outcome after ICH. The established nomogram incorporating RDW should be considered for a 30-day functional prognosis.

A Novel Metal Precursor Structure for Electrodepositing Ultrathin CIGSe Thin-Film Solar Cell with High Efficiency.

The two-step process of electrodeposition and selenization is one of the most effective methods for producing CIGSe and CZTSe solar cells at a low cost. However, it is difficult to prepare the ultrathin CIGSe absorber by electrodeposition due to the nonuniform deposition of Cu on the Mo substrate. In this study, Cu was deposited on a Sb film instead of a Mo film, and the 3D growth mode of Cu was changed. Uniform and smooth ultrathin Cu films were fabricated on the Sb film using a pulse frequency over a range from 1000 to 10,000 Hz and a pulse current density ranging from 31.25 to 62.5 mA/cm2. Owing to the improved uniformity of Cu/In/Ga films, the thickness of the CIGSe absorber was reduced from 2 to 0.36 µm with Sb incorporation. In addition, the effects of Sb-doping on the CIGSe absorbers and the device performance were investigated. The crystallinity of the CIGSe films was improved, and the interface recombination of the solar cells was reduced by Sb incorporation. Ultimately, CIGSe thin-film solar cells with efficiencies of 5.25 and 11.27% were obtained with CIGSe absorber thicknesses of 0.36 and 1.2 µm, respectively.

Formation of Cl-Doped ZnO Thin Films by a Cathodic Electrodeposition for Use as a Window Layer in CIGS Solar Cells.

Zinc oxide films that are prepared by radio frequency (RF) sputtering are widely used as window layers in copper indium gallium diselenide (CIGS) solar cells. To reduce their production cost, the electrodeposition method for preparing Cl-doped zinc oxide (ZnO:Cl), rather than sputtering, was studied. The electrodeposition parameters of injected current density and the pH of the electrolyte solution were studied. A moderate current density was used to yield high quality zinc oxides. The pH of the electrolyte greatly affected the formation of ZnO films. The pH value of the electrolyte that ensured that zinc oxides of high quality are obtained was close to seven. Electrodeposited ZnO:Cl films had higher transmittance than ZnO:Al films in the near-infrared region and so they can be used to improve the performance of solar cells. Our experiments revealed that the CIGS solar cells with electrodeposited ZnO:Cl films as a window layer were slightly more efficient than those with sputtered ZnO:Al films.

Modified Back Contact Interface of CZTSe Thin Film Solar Cells: Elimination of Double Layer Distribution in Absorber Layer.

Double layer distribution exists in Cu2SnZnSe4 (CZTSe) thin films prepared by selenizing the metallic precursors, which will degrade the back contact of Mo substrate to absorber layer and thus suppressing the performance of solar cell. In this work, the double-layer distribution of CZTSe film is eliminated entirely and the formation of MoSe2 interfacial layer is inhibited successfully. CZTSe film is prepared by selenizing the precursor deposited by electrodeposition method under Se and SnSe x mixed atmosphere. It is found that the insufficient reaction between ZnSe and Cu-Sn-Se phases in the bottom of the film is the reason why the double layer distribution of CZTSe film is formed. By increasing Sn content in the metallic precursor, thus making up the loss of Sn because of the decomposition of CZTSe and facilitate the diffusion of liquid Cu2Se, the double layer distribution is eliminated entirely. The crystallization of the formed thin film is dense and the grains go through the entire film without voids. And there is no obvious MoSe2 layer formed between CZTSe and Mo. As a consequence, the series resistance of the solar cell reduces significantly to 0.14 Ω cm2 and a CZTSe solar cell with efficiency of 7.2% is fabricated.

Controllable Growth of Ga Film Electrodeposited from Aqueous Solution and Cu(In,Ga)Se2 Solar Cells.

Electrodepositon of Ga film is very challenging due to the high standard reduction potential (-0.53 V vs SHE for Ga3+). In this study, Ga film with compact structure was successfully deposited on the Mo/Cu/In substrate by the pulse current electrodeposition (PCE) method using GaCl3 aqueous solution. A high deposition rate of Ga3+ and H+ can be achieved by applying a large overpotential induced by high pulse current. In the meanwhile, the concentration polarization induced by cation depletion can be minimized by changing the pulse frequency and duty cycle. Uniform and smooth Ga film was fabricated at high deposition rate with pulse current density 125 mA/cm2, pulse frequency 5 Hz, and duty cycle 0.25. Ga film was then selenized together with electrodeposited Cu and In films to make a CIGSe absorber film for solar cells. The solar cell based on the Ga film presents conversion efficiency of 11.04%, fill factor of 63.40%, and Voc of 505 mV, which is much better than those based on the inhomogeneous and rough Ga film prepared by the DCE method, indicating the pulse current electrodeposition process is promising for the fabrication of CIGSe solar cell.

Effect of Sn Content in a CuSnZn Metal Precursor on Formation of MoSe2 Film during Selenization in Se+SnSe Vapor.

The preparation of Cu2ZnSnSe4 (CZTSe) thin films by the selenization of an electrodeposited copper-tin-zinc (CuSnZn) precursor with various Sn contents in low-pressure Se+SnSex vapor was studied. Scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS) measurements revealed that the Sn content of the precursor that is used in selenization in a low-pressure Se+SnSex vapor atmosphere only slightly affects the elemental composition of the formed CZTSe films. However, the Sn content of the precursor significantly affects the grain size and surface morphology of CZTSe films. A metal precursor with a very Sn-poor composition produces CZTSe films with large grains and a rough surface, while a metal precursor with a very Sn-rich composition procures CZTSe films with small grains and a compact surface. X-ray diffraction (XRD) and SEM revealed that the metal precursor with a Sn-rich composition can grow a thicker MoSe2 thin film at CZTSe/Mo interface than one with a Sn-poor composition, possibly because excess Sn in the precursor may catalyze the formation of MoSe2 thin film. A CZTSe solar cell with an efficiency of 7.94%was realized by using an electrodeposited metal precursor with a Sn/Cu ratio of 0.5 in selenization in a low-pressure Se+SnSex vapor.

[A simple error-prone PCR method through dATP reduction].

UNLABELLED: The error-prone PCR is one of the main methods for in vitro gene mutagenesis, usually through adding Mn2+ increasing Mg2+ and dCTP/dTTP concentration. OBJECTIVE AND METHODS: In this study, both the antifungal protein gene Ace-AMP1 from Allium cepa and the Bt toxin gene cry1A(c) from Bacillus thuringiensis were subjected to PCR mutagenesis through reducing the dATP concentration, but without adding Mn2+ or adjusting other PCR components. RESULTS: The result showed that the rates of base mutation and sequence variation were increased along with the decrease of dATP concentrations. When dTTP/dCTP/dGTP: dATP equaled 20:1-40:1, the rate of base mutation was between 1.4% and 1.8%, and the rate of sequence variation was between 77.8% and 100%. DISCUSSION AND CONCLUSION: This method is simple and practical, and enables the process optimization of several mutagenic factors in conventional error-prone PCR. Moreover, as the resulting base mutations were mainly A x T-->G x C transition, the present method provides a new way to improve the GC content of gene by in vitro mutagenesis. The mutagenesis method of simply reducing single dNTP concentration could improve AT or GC content of the target gene, it is an expansion of error-prone PCR.

CZTSe solar cells prepared by electrodeposition of Cu/Sn/Zn stack layer followed by selenization at low Se pressure.

Cu2ZnSnSe4 (CZTSe) thin films are prepared by the electrodeposition of stack copper/tin/zinc (Cu/Sn/Zn) precursors, followed by selenization with a tin source at a substrate temperature of 530°C. Three selenization processes were performed herein to study the effects of the source of tin on the quality of CZTSe thin films that are formed at low Se pressure. Much elemental Sn is lost from CZTSe thin films during selenization without a source of tin. The loss of Sn from CZTSe thin films in selenization was suppressed herein using a tin source at 400°C (A2) or 530°C (A3). A copper-poor and zinc-rich CZTSe absorber layer with Cu/Sn, Zn/Sn, Cu/(Zn + Sn), and Zn/(Cu + Zn + Sn) with metallic element ratios of 1.86, 1.24, 0.83, and 0.3, respectively, was obtained in a selenization with a tin source at 530°C. The crystallized CZTSe thin film exhibited an increasingly (112)-preferred orientation at higher tin selenide (SnSe x ) partial pressure. The lack of any obvious Mo-Se phase-related diffraction peaks in the X-ray diffraction (XRD) diffraction patterns may have arisen from the low Se pressure in the selenization processes. The scanning electron microscope (SEM) images reveal a compact surface morphology and a moderate grain size. CZTSe solar cells with an efficiency of 4.81% were produced by the low-cost fabrication process that is elucidated herein.