Field identification of morphological and physiological traits in two special mutants with strong tolerance and high sensitivity to drought stress in upland rice(Oryza sativa L.)

The two mutants idr1-1 and 297-28, which were obtained from the radiation mutation of HD297 and IAPAR9, were used as experimental materials in this study for a 2-year(2012 and 2013) experiment about field drought resistance identification in Beijing, China. Key agronomic traits and water-related physiological indexes were observed and measured, including the leaf anti-dead level(LADL), days to heading, plant height, setting percentage, aboveground biomass, leaf water potential(LWP), net photosynthetic rate(Pn) and transpiration rate. The results showed that the mutant idr1-1 that was under drought stress(DS) conditions for 2 years had the highest LADL grades(1.3 and 2.0) among all the materials, and they were 2–3 grades stronger than the wild-type IAPAR9 with an average that was 21.4% higher for the setting percentage than the wild type. Compared with the IAPAR9 for the 2-year average delay in the days to heading and the reduction rates in the plant height, setting percentage, and aboveground biomass under DS compared with the well-watered(WW) treatment, idr1-1 showed 3.2% less delay and 19.1, 16.4, and 6.1% less reduction, respectively. The idr1-1 in the LWP always exhibited the highest performance among all the materials. The Pn of idr1-1 under severe and mild DS comparing with that under WW was slightly decreased and even slightly increased, respectively, leading to an average reduction rate of only 0.92%, which was 26.93% less than that of IAPAR9. Under the severe DS, idr1-1 still showed the highest value of 16.88 μmol CO2 m–2 s–1 among all the materials and was significantly higher than that of IAPAR9(11.66 μmol CO2 m–2 s–1). Furthermore, only idr1-1 had the increased and the highest transpiration rate values(7.6 and 6.04 mmol H2 O m–2 s–1) under both mild and severe DS compared with the values under WW, when the transpiration rate of all the other materials significantly decreased. By contrast, the 297-28 in terms of the LADL grade under DS was the lowest(7.0), and it was four grades weaker than its wildtype HD297 and even one grade weaker than the drought-sensitive paddy rice SN265. For the 2-year average reduction rates in aboveground biomass and plant heights under DS compared with those under the WW, 297-28 was 31.6 and 31.8% higher than HD297, respectively. Meanwhile, 297-28 showed the worst performance for the LWP, Pn, and transpiration rate. These results suggest that idr1-1 might be a superior drought tolerant mutant of upland rice found in China. It has a strong ability to maintain and even enhance leaf transpiration while maintaining a high plant water potential under DS, thus supporting a high Pn and alleviating the delay in agronomic trait development and yield loss effectively. 297-28 is a much more highly drought-sensitive mutant that is even more sensitive than paddy rice varieties. The two mutants could be used as drought tolerance controls for rice germplasm identification and the drought resistant mechanism studies in the future. idr1-1 is also suitable for breeding drought-tolerant and lodging-resistant high-yield rice varieties.     

GsMAPK4, a positive regulator of soybean tolerance to salinity stress

Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signaling pathways and the signaling molecules for salinity tolerance regulation have not been clarified. In this study, a proteomic technology based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins responsible for salinity tolerance in soybean plant. Real-time quantitative PCR (qRT-PCR) and Western blotting (WB) were used to verify the results of 2-DE/MS. Based on the results of 2-DE and MS, we selected glucosyltransferase (GsGT4), 4-coumarate, coenzyme A ligase (Gs4CL1), mitogen-activated protein kinase 4 (GsMAPK4), dehydration responsive element binding protein (GsDREB1), and soybean cold-regulated gene (GsSRC1) in the salinity tolerant soybean variety, and GsMAPK4 for subsequent research. We transformed soybean plants with mitogen-activated-protein kinase 4 (GsMAPK4) and screened the resulting transgenics soybean plants using PCR and WB, which confirmed the expression of GsMAPK4 in transgenic soybean. GsMAPK4-overexpressed transgenic plants showed significantly increased tolerance to salt stress, suggesting that GsMAPK4 played a pivotal role in salinity tolerance. Our research will provide new insights for better understanding the salinity tolerance regulation at molecular level.     

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)

Laccase(EC 1.10.3.2)is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2(PxLac2),from the third instar larvae using polymerase chain reaction(PCR)and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR(qPCR).The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant(K_m)of 0.97 mmol L~(-1),maximal reaction speed(V_m)of 56.82 U mL~(-1),and activation energy(E_a)of 17.36 kJ mol~(-1) to oxidize2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt).Type II copper enhanced laccase activity below 0.8mmol L~(-1) and reduced enzyme activity above 0.8 mmol L~(-1) with an IC_(50) concentration of 1.26 mmol L~(-1).This study provides insights into the biological function of laccase.     

Effects of the severity and timing of basal leaf removal on the amino acids profiles of Sauvignon Blanc grapes and wines

The effects of the severity and timing of leaf removal(LR) on the amino acids of Sauvignon Blanc grapes and wines were studied during the 2017 growing season. High-performance liquid chromatography(HPLC) was used to analyze the amino acids profiles of grape berries and wines. The basal leaves were removed at three time points(40, 56 and 72 days after flowering, named LR40, LR56 and LR72, respectively) at two severity levels(one at which the first, third, and fifth basal leaves of each shoot were removed(50% level); and another at which the first six basal leaves were removed(100% level)). The results showed that leaf removal had little impact on total soluble solids(°Brix), titratable acidity, pH or berry weight. The LR72-50% treated grapes had higher berry weight, titratable acidity and °Brix than those of the other treatments. The highest concentrations of total amino acids and of total amino acids except proline were detected in LR72-50% treated grapes(2 952.58 and 2 764.36 mg L~(-1), respectively); the lowest were detected in LR72-100% treated grapes(2 172.82 and 2 038.71 mg L~(-1), respectively). LR72-50% treatment significantly promoted the synthesis of aspartic acid, serine, arginine, alanine, aminobutyric acid and proline at both severity levels for grapes, the concentrations of all of these amino acids were increased relative to the control concentrations. The LR72-50%, LR40-100% and LR72-100% treated wines had higher total amino acids concentrations and higher concentrations of some individual amino acids, such as arginine, alanine and serine, than did the control wines. Of all the amino acids studied, glycine, tyrosine, cysteine, methionine and lysine were not significantly influenced by the timing or severity basal defoliation in grapes and wines. The present study reveals the effects of the timing and severity of leaf removal on the amino acids profiles of grapes and wines.     

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.: MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.). Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp, encoding 424 amino acids. Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri. The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain. qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species, and was highly expressed in the root, while the expression of MdPUB29 was significantly inhibited by exogenous NaCl. Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions. Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29. The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions, suggesting that MdPUB29 may positively regulate salt tolerance.     

Molecular,serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China

Apple stem pitting virus (ASPV) is an important causal agent of pear diseases. Nowadays, the infection status and molecular characteristics of the virus in old pear trees have never been investigated. In this study, we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar ‘Chili’ specifically grown at Laiyang area in China. ASPV-LYC possesses a chimeric genome consisting of 9 273 nucleotides excluding a poly(A) tail at its 3’ end and harboring a recombination region in its open reading frame (ORF1) with Aurora-1 and KL9 identified as the major and minor parents. Western blot analysis with antisera against recombinant coat proteins (CPs) of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates, but with differential activities. Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates. The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.     

Estimates on nitrogen uptake in the subsequent wheat by above-ground and root residue and rhizodeposition of using peanut labeled with 15N isotope on the North China Plain

Leguminous crops play a vital role in enhancing crop yield and improving soil fertility. Therefore, it can be used as an organic N source for improving soil fertility. The purpose of this study was to (i) quantify the amounts of N derived from rhizodeposition, root and above-ground biomass of peanut residue in comparison with wheat and (ii) estimate the effect of the residual N on the wheat-growing season in the subsequent year. The plants of peanut and wheat were stem fed with ¹⁵N urea using the cotton-wick method at the Wuqiao Station of China Agricultural University in 2014. The experiment consisted of four residue-returning strategies in a randomized complete-block design: (i) no return of crop residue (CR0); (ii) return of above-ground biomass of peanut crop (CR1); (iii) return of peanut root biomass (CR2); and (iv) return of all residue of the whole peanut plant (CR3). The 31.5 and 21% of the labeled ¹⁵N isotope were accumulated in the above-ground tissues (leaves and stems) of peanuts and wheat, respectively. N rhizodeposition of peanuts and wheat accounted for 14.91 and 3.61% of the BG¹⁵N, respectively. The ¹⁵N from the below-ground ¹⁵N-labeled of peanuts were supplied 11.3, 5.9, 13.5, and 6.1% of in the CR0, CR1, CR2, and CR3 treatments, respectively. Peanut straw contributes a significant proportion of N to the soil through the decomposition of plant residues and N rhizodeposition. With the current production level on the NCP, it is estimated that peanut straw can potentially replace 104 500 tons of synthetic N fertilizer per year. The inclusion of peanut in rotation with cereal can significantly reduce the use of N fertilizer and enhance the system sustainability.     

A rapid,simple, and sensitive immunoagglutination assay with silica nanoparticles for serotype identification of Pseudomonas aeruginosa

An agglutination test based on colored silica nanoparticles (colored SiNps) was established to detect serotypes of Pseudomonas aeruginosa. Monodisperse colored SiNps were used as agglutination test carriers. The colored SiNps were prepared through reverse microemulsion with reactive dyes, sensitized with 11 kinds of mono-specific antibodies against P. aeruginosa, and denoted as IgG-colored SiNps. Eleven kinds of IgG-colored SiNps were individually mixed with P. aeruginosa on a glass slide. Different serotypes of P. aeruginosa could be identified by agglutination test with evident agglutination. The P. aeruginosa could be detected in a range from 3.6 × 10⁵ to 3.6 × 10¹² cfu mL^?1. This new agglutination test was confirmed to be a specific, sensitive, fast, easy-to-perform, and cost-efficient tool for the routine diagnosis of P. aeruginosa.     

Evaluating the efficacy of an attenuated Streptococcus equi ssp. zooepidemicus vaccine produced by multi-gene deletion in pathogenicity island SeseCisland_4

Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL~(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL~(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL~(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL~(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.     

台湾鱼木对干旱胁迫的生理响应

通过盆栽控水试验,测定台湾鱼木在不同干旱条件下生长、生理指标的变化,研究台湾鱼木对干旱胁迫的生理响应。结果表明:在干旱初期,丙二醛(MDA)浓度上升,植物细胞膜受到一定程度的损伤,随之可溶性糖(SS)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性增加,阻止或减少羟基自由基的形成,抵御活性氧伤害膜保护系统。在干旱中期,渗透调节物质可溶性糖(SS)含量增加,过氧化物酶(POD)活性增强,表明植物细胞启动渗透调节和抗氧化酶系统避免干旱胁迫对植物细胞产生损伤。在干旱后期,丙二醛(MDA)呈现逐渐递增的趋势,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性由峰值下降;叶片相对含水量逐渐降低。相对叶绿素含量(SPAD)初期增加,随后呈现下降趋势,表明抗氧化酶系统超过了调节极限,植物细胞受到了严重损伤。结论:台湾鱼木通过渗透调节和启动抗氧化酶系统,对干旱胁迫表现出一定耐受性,在土壤相对含水量为27.78%时,达到耐受极限。台湾鱼木作为乡土树种之一,可以在我国热带亚热带地区的城市园林中应用推广。     

Comparison of phenolic profiles and antioxidant activities in skins and pulps of eleven grape cultivars(Vitis vinifera L.)

Eleven grape cultivars were analysed to explore the variety differences of fresh grape phenolic profiles. The results showed that free phenolics were predominant in grape skins and pulps, and showed the higher antioxidant activities than bound. In 11 cultivars, Muscat Kyoho extracts had the highest total phenolic content in skins(10.525 mg GAE g~(–1) FW) and pulps(1.134 mg GAE g~(–1) FW), and exhibited the highest DPPH radical scavening capacity(EC_(50)=11.7 μg mL~(–1)) and oxygen radical absorbance capacity(ORAC) value(190.57 μmol TE g~(–1) FW) of free phenolic in skin. In addition, the most abundant phenolics in grape skins were found to be flavonoids such as kaempferol in Kyoho skin(541.2 μg g~(–1) FW), rutin, catechin and epicatechin in Muscat Kyoho skin(262.3, 86.3 and 70.0 μg g~(–1) FW, respectively). Furthermore, the principal component analysis showed a strong difference of phenolic profiles with the cultivars, existing forms and distributions. Pearson correlation coefficient analysis showed a significant linear correlation between total phenolic content and antioxidant activity(P0.05). Therefore, both skins and pulps were rich sources of bioactive phenolic compounds, and Muscat Kyoho was the ideal source among all samples.     

Population genetic structure of Chinese Puccinia triticina races based on multi-locus sequences

Puccinia triticina, the causal agent of wheat leaf rust, is one of the most devastating rust fungi attacking wheat worldwide. Seventy-six isolates of the wheat leaf rust pathogen from Yunnan, Sichuan, Gansu and Henan provinces, China, were tested on wheat leaf rust differentials and the population structure was analyzed using four presumably neutral partial sequence markers such as elongation factor-1α(EF-1α), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), β-tubulin(TUB) and the second largest RNA polymerase subunit(RPB2). The phenotypic diversity of Yunnan and Sichuan populations was higher than that of Gansu and Henan populations. The four populations were separated into two clusters based on the pathogenic data. A total of 12 single nucleotide polymorphisms(SNPs) and 32 haplotypes were identified among the four sequences. The 32 haplotypes were divided into two clusters in a neighbor-joining tree. Bayesian analyses also identified two clusters. Pairwise FST between populations in different regions were significantly different(P0.05). Analysis of molecular variance(AMOVA) indicated that 68% of the total genetic variation was within populations.     

Characterization of TaCOMT genes associated with stem lignin content in common wheat and development of a gene-specific marker

Stem lignin content(SLC) in common wheat(Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase(COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT genes and development of gene-specific markers could enable marker-assisted selection in wheat breeding. In the present study, the full-length genomic DNA(gDNA) sequences of TaCOMT genes located on chromosomes 3 A, 3 B, and 3 D were cloned by homologous cloning. Two allelic variants, TaCOMT-3 Ba and TaCOMT-3 Bb, were identified and differed by a 222-bp insertion/deletion(InDel) in the 3′-untranslated region(3′-UTR). A co-dominant gene-specific marker based on this InDel was developed and designated as Ta COMT-3 BM. A total of 157 wheat cultivars and advanced lines grown in four environments were used to validate the associations between allelic patterns and SLC. The SLC of cultivars with TaCOMT-3 Ba was significantly(P0.01) higher than that of those with TaCOMT-3 Bb, and the marker TaCOMT-3 BM could be effectively used in wheat breeding.     

Dynamic changes of root proteome reveal diverse responsive proteins in maize subjected to cadmium stress

Toxic symptoms and tolerance mechanisms of heavy metal in maize are well documented. However, limited information is available regarding the changes in the proteome of maize seedling roots in response to cadmium(Cd) stress. Here, we employed an i TRAQ-based quantitative proteomic approach to characterize the dynamic alterations in the root proteome during early developmental in maize seedling. We conducted our proteomic experiments in three-day seedling subjected to Cd stress, using roots in four time points. We identified a total of 733, 307, 499, and 576 differentially abundant proteins after 12, 24, 48, or 72 h of treatment, respectively. These proteins displayed different functions, such as ribosomal synthesis, reactive oxygen species homeostasis, cell wall organization, cellular metabolism, and carbohydrate and energy metabolism. Of the 166 and 177 proteins with higher and lower abundance identified in at least two time points, 14 were common for three time points. We selected nine proteins to verify their expression using quantitative real-time PCR. Proteins involved in the ribosome pathway were especially responsive to Cd stress. Functional characterization of the proteins and the pathways identified in this study could help our understanding of the complicated molecular mechanism involved in Cd stress responses and create a list of candidate gene responsible for Cd tolerance in maize seeding roots.     

The synergistic advantage of combining chloropicrin or dazomet with fosthiazate nematicide to control root-knot nematode in cucumber production

The highly-damaging root-knot nematode(Meloidogyne spp., RKN) cannot be reliably controlled using only a nematicide such as fosthiazate because of increasing pest resistance. In laboratory and greenhouse trials, we showed that chloropicrin(CP) or dazomet(DZ) synergized the efficacy of fosthiazate against RKN. The combination significantly extended the degradation half-life of fosthiazate by an average of about 1.25 times. CP or DZ with fosthiazate reduced the time for fosthiazate to penetrate the RKN cuticle compared to fosthiazate alone. CP or DZ combined with low or medium rate of fosthiazate increased the total cucumber yield, compared to the use of each product alone. A low-dose fosthiazate with DZ improved total yield more than a low dose fosthiazate with CP. Extending the half-life of fosthiazate and reducing the time for fosthiazate or fumigant to penetrate the RKN cuticle were the two features that gave the fumigant-fosthiazate combination its synergistic advantage over these products used singularly. This synergy provides the opportunity for farmers to use a low dose of fosthiazate which lowers the risk of RKN resistance. Farmers could combine DZ at 30 g m~(-2) with fosthiazate at a low rate of 0.375 g m~(-2) to control RKN and adequately control two major soil-borne diseases in cucumber greenhouses.     

Residue management induced changes in soil organic carbon and total nitrogen under different tillage practices in the North China Plain

Crop residue retention has been considered a practicable strategy to improve soil organic carbon(SOC) and total nitrogen(TN), but the effectiveness of residue retention might be different under varied tillage practices. To evaluate the effects of residue management on the distribution and stocks of SOC and TN under different tillage practices, a bifactorial experiment with three levels for tillage practices(no-tillage, rotary tillage, and conventional tillage) and two levels for residue managements(residue retention and residue removal) was conducted in the North China Plain(NCP). Results showed that after a short experimental duration(3–4 years), concentrations of SOC and TN in the 0–10 cm layer were higher under no-tillage than under conventional tillage, no matter whether crop residues were retained or not. Residue retention increased SOC and TN concentrations in the upper layers of soil to some degree for all tillage practices, as compared with residue removal, with the greatest increment of SOC concentration occurred in the 0–10 cm layer under rotary tillage, but in the 10–30 cm layer under conventional tillage. The stocks of SOC in the 0–50 cm depth increased from 49.89 Mg ha–1 with residue removal to 53.03 Mg ha–1 with residue retention. However, no-tillage did not increase SOC stock to a depth of 50 cm relative to conventional tillage, and increased only by 5.35% as compared with rotary tillage. Thus, residue retention may contribute more towards SOC sequestration than no-tillage. Furthermore, the combination between residue retention and no-tillage has the greatest advantage in enhancing SOC and TN in the NCP region.     

Phenotypic characterization and genetic mapping of the dwarf mutant m34 in maize

Plant height is one of the most important agronomic traits associated with yield in maize.In this study,a gibberellins(GA)-insensitive dwarf mutant,m34,was screened from inbred line Ye478 by treatment with the chemical mutagen ethylmethanesulfonate(EMS).Compared to Ye478,m34 showed a dwarf phenotype with shorter internodes,and smaller leaf length and width,but with similar leaf number.Furthermore,m34 exhibited smaller guard cells in internodes than Ye478,suggesting that smaller cells might contribute to its dwarf phenotype.Genetic analysis indicated that the m34 dwarf phenotype was controlled by a recessive nuclear gene.An F2 population derived from a cross between m34 and B73 was used for mutational gene cloning and this gene was mapped to a chromosome region between umc2189 and umc1553 in chromosome 1 bin1.10,which harbored a previously identified dwarf gene Zm VP8.Sequencing analysis showed a nucleotide substitution(G1606 to A1606)in the sixth exon of ZmVP8,which resulted in an amino acid change(E531 to K531)from Ye478 to m34.This amino acid change resulted in anα-helix changing to aβ-sheet in the secondary protein structure and the‘SPEC’domain changed to a‘BOT1NT’domain in the tertiary protein structure.Taken together,these results suggested that m34 is a novel allelic mutant originally derived from Ye478 that is useful for further ZmVP8 functional analysis in maize.