Whole genome-wide transcript profiling to identify differentially expressed genes associated with seed field emergence in two soybean low phytate mutants.

BACKGROUND: Seed germination is important to soybean (Glycine max) growth and development, ultimately affecting soybean yield. A lower seed field emergence has been the main hindrance for breeding soybeans low in phytate. Although this reduction could be overcome by additional breeding and selection, the mechanisms of seed germination in different low phytate mutants remain unknown. In this study, we performed a comparative transcript analysis of two low phytate soybean mutants (TW-1 and TW-1-M), which have the same mutation, a 2 bp deletion in GmMIPS1, but show a significant difference in seed field emergence, TW-1-M was higher than that of TW-1 . RESULTS: Numerous genes analyzed by RNA-Seq showed markedly different expression levels between TW-1-M and TW-1 mutants. Approximately 30,000-35,000 read-mapped genes and ~21000-25000 expressed genes were identified for each library. There were ~3900-9200 differentially expressed genes (DEGs) in each contrast library, the number of up-regulated genes was similar with down-regulated genes in the mutant TW-1and TW-1-M. Gene ontology functional categories of DEGs indicated that the ethylene-mediated signaling pathway, the abscisic acid-mediated signaling pathway, response to hormone, ethylene biosynthetic process, ethylene metabolic process, regulation of hormone levels, and oxidation-reduction process, regulation of flavonoid biosynthetic process and regulation of abscisic acid-activated signaling pathway had high correlations with seed germination. In total, 2457 DEGs involved in the above functional categories were identified. Twenty-two genes with 20 biological functions were the most highly up/down- regulated (absolute value Log2FC >5) in the high field emergence mutant TW-1-M and were related to metabolic or signaling pathways. Fifty-seven genes with 36 biological functions had the greatest expression abundance (FRPM >100) in germination-related pathways. CONCLUSIONS: Seed germination in the soybean low phytate mutants is a very complex process, which involves a series of physiological, morphological and transcriptional changes. Compared with TW-1, TW-1-M had a very different gene expression profile, which included genes related to plant hormones, antioxidation, anti-stress and energy metabolism processes. Our research provides a molecular basis for understanding germination mechanisms, and is also an important resource for the genetic analysis of germination in low phytate crops. Plant hormone- and antioxidation-related genes might strongly contribute to the high germination rate in the TW-1-M mutant.

Natural variation underlies alterations in Nramp aluminum transporter (NRAT1) expression and function that play a key role in rice aluminum tolerance.

Aluminum (Al) toxicity is a major constraint for crop production on acid soils which compose ∼ 40% of arable land in the tropics and subtropics. Rice is the most Al-tolerant cereal crop and offers a good model for identifying Al tolerance genes and mechanisms. Here we investigated natural variation in the rice Nramp aluminum transporter (NRAT1) gene encoding a root plasma membrane Al uptake transporter previously hypothesized to underlie a unique Al tolerance mechanism. DNA sequence variation in the NRAT1 coding and regulatory regions was associated with changes in NRAT1 expression and NRAT1 Al transport properties. These sequence changes resulted in significant differences in Al tolerance that were found to be associated with changes in the Al content of root cell wall and cell sap in 24 representative rice lines from a rice association panel. Expression of the tolerant OsNRAT1 allele in yeast resulted in higher Al uptake than did the sensitive allele and conferred greater Al tolerance when expressed in transgenic Arabidopsis. These findings indicate that NRAT1 plays an important role in rice Al tolerance by reducing the level of toxic Al in the root cell wall and transporting Al into the root cell, where it is ultimately sequestered in the vacuole. Given its ability to enhance Al tolerance in rice and Arabidopsis, this work suggests that the NRAT1 gene or its orthologs may be useful tools for enhancing Al tolerance in a wide range of plant species.

How a "Smart regulatory" platform can improve the property fee adjustment dilemma.

In the current process of social and economic development, the cost of living and property management is continuously increasing. In order to maintain the normal operation of property management and provide high-quality services, it is necessary to adjust the property fees reasonably. If the property fees cannot be adjusted for a long time, it will have a detrimental impact on the development of the community. However, the traditional regulatory model has failed to accurately define the quality of property services, leading to information asymmetry and a lack of trust between the two parties, resulting in a deadlock in property fee adjustments. With the rise of intelligent management, a new direction for property fee adjustments has been provided. This article first analyzes the pricing dilemma under traditional regulation through game theory. Then, based on the theory of "Smart regulatory", it proposes the idea of constructing a "Smart regulatory" platform and explores the feasibility of this model using game theory and Matlab simulations. The study found that the "Smart regulatory" platform can resolve information asymmetry and encourage both parties to cooperate in the game. After the establishment of the platform, property fee adjustments are influenced by government supervision, government penalties, and the magnitude of property fee increase.

The reduction of reactive oxygen species formation by mitochondrial alternative respiration in tomato basal defense against TMV infection.

The role of mitochondrial alternative oxidase (AOX) and the relationship between systemic AOX induction, ROS formation, and systemic plant basal defense to Tobacco mosaic virus (TMV) were investigated in tomato plants. The results showed that TMV inoculation significantly increased the level of AOX gene transcripts, ubiquinone reduction levels, pyruvate content, and cyanide-resistant respiration (CN-resistant R) in upper, un-inoculated leaves. Pretreatment with potassium cyanide (KCN, a cytochrome pathway inhibitor) greatly increased CN-resistant R and reduced reactive oxygen species (ROS) formation, while application of salicylhydroxamic acid (SHAM, an AOX inhibitor) blocked the AOX activity and enhanced the production of ROS in the plants. Furthermore, TMV systemic infection was enhanced by SHAM and reduced by KCN pretreatment, as compared with the un-pretreated TMV counterpart. In addition, KCN application significantly diminished TMV-induced increase in antioxidant enzyme activities and dehydroascorbate/total ascorbate pool, while an opposite change was observed with SHAM-pretreated plants. These results suggest that the systemic induction of the mitochondrial AOX pathway plays a critical role in the reduction of ROS to enhance basal defenses. Additional antioxidant systems were also coordinately regulated in the maintenance of the cellular redox homeostasis.

Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

Decades of Genetic Research on Soybean mosaic virus Resistance in Soybean.

This review summarizes the history and current state of the known genetic basis for soybean resistance to Soybean mosaic virus (SMV), and examines how the integration of molecular markers has been utilized in breeding for crop improvement. SVM causes yield loss and seed quality reduction in soybean based on the SMV strain and the host genotype. Understanding the molecular underpinnings of SMV-soybean interactions and the genes conferring resistance to SMV has been a focus of intense research interest for decades. Soybean reactions are classified into three main responses: resistant, necrotic, or susceptible. Significant progress has been achieved that has greatly increased the understanding of soybean germplasm diversity, differential reactions to SMV strains, genotype-strain interactions, genes/alleles conferring specific reactions, and interactions among resistance genes and alleles. Many studies that aimed to uncover the physical position of resistance genes have been published in recent decades, collectively proposing different candidate genes. The studies on SMV resistance loci revealed that the resistance genes are mainly distributed on three chromosomes. Resistance has been pyramided in various combinations for durable resistance to SMV strains. The causative genes are still elusive despite early successes in identifying resistance alleles in soybean; however, a gene at the Rsv4 locus has been well validated.

Multiplex single nucleotide polymorphism (SNP) assay for detection of soybean mosaic virus resistance genes in soybean.

Soybean mosaic virus (SMV) is one of the most destructive viral diseases in soybean (Glycine max). Three independent loci for SMV resistance have been identified in soybean germplasm. The use of genetic resistance is the most effective method of controlling this disease. Marker assisted selection (MAS) has become very important and useful in the effort of selecting genes for SMV resistance. Single nucleotide polymorphism (SNP), because of its abundance and high-throughput potential, is a powerful tool in genome mapping, association studies, diversity analysis, and tagging of important genes in plant genomics. In this study, a 10 SNPs plus one insert/deletion (InDel) multiplex assay was developed for SMV resistance: two SNPs were developed from the candidate gene 3gG2 at Rsv1 locus, two SNPs selected from the clone N11PF linked to Rsv1, one 'BARC' SNP screened from soybean chromosome 13 [linkage group (LG) F] near Rsv1, two 'BARC' SNPs from probe A519 linked to Rsv3, one 'BARC' SNP from chromosome 14 (LG B2) near Rsv3, and two 'BARC' SNPs from chromosome 2 (LG D1b) near Rsv4, plus one InDel marker from expressed sequence tag (EST) AW307114 linked to Rsv4. This 11 SNP/InDel multiplex assay showed polymorphism among 47 diverse soybean germplasm, indicating this assay can be used to investigate the mode of inheritance in a SMV resistant soybean line carrying Rsv1, Rsv3, and/or Rsv4 through a segregating population with phenotypic data, and to select a specific gene or pyramid two or three genes for SMV resistance through MAS in soybean breeding program. The presence of two SMV resistance genes (Rsv1 and Rsv3) in J05 soybean was confirmed by the SNP assay.

Systemic induction and role of mitochondrial alternative oxidase and nitric oxide in a compatible tomato-Tobacco mosaic virus interaction.

The role of mitochondrial alternative oxidase (AOX) and the relationship between AOX and nitric oxide (NO) in virus-induced systemic defense to Tobacco mosaic virus (TMV) were investigated in susceptible tomato (Solanum lycopersicum) plants. TMV inoculation to the lower leaves induced a rapid NO synthesis and AOX activation in upper uninoculated leaves as early as 0.5 day postinoculation. Application of exogenous potassium cyanide (KCN, a cytochrome pathway inhibitor) at nonlethal concentrations and NO donor diethylamine NONOate (DEA/NO) to the upper uninoculated leaves greatly induced accumulation of AOX transcript, reduced TMV viral RNA accumulation, and increased the leaf photochemical quantum yield at photosystem II. Pretreatment with NO scavenger almost completely blocked TMV-induced AOX induction and substantially increased TMV susceptibility. Salicylhydroxamic acid (SHAM, an AOX inhibitor) pretreatment reduced the DEA/NO-induced cyanide-resistant respiration and partially compromised induced resistance to TMV. Conversely, KCN and SHAM pretreatment had very little effect on generation of NO, and pretreatment with NO scavenger did not affect KCN-induced AOX induction and TMV resistance. These results suggest that TMV-induced NO generation acts upstream and mediates AOX induction which, in turn, induces mitochondrial alternative electron transport and triggers systemic basal defense against the viral pathogen.

Decreased energy synthesis is partially compensated by a switch to sucrose synthase pathway of sucrose degradation in restricted root of tomato plants.

Tomato (Solanum lycopersium L.) plants were grown hydroponically to investigate the changes of energy metabolism and adaptive mechanism in response to root restriction. Root restriction resulted in a significant increase in root lipid peroxidation and reduction in leaf net CO(2) assimilation rate, which was accompanied by increase of alcohol dehydrogenase (ADH; EC 1.1.1.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activities. Total, cytochrome pathway, and alternative pathway respirations were all decreased in the roots after 15 days of root restriction treatment. Accompanied with the decrease of ATP content, ratio of invertase/sucrose synthase activity was increased in the restricted roots together with a decrease in glucose content and an increase in fructose content. We concluded that the decreased energy synthesis under root restriction condition was partially compensated by the energy-conserving sucrose synthase pathway of sucrose metabolism.

Salt-Responsive Transcriptome Profiling of Suaeda glauca via RNA Sequencing.

BACKGROUND: Suaeda glauca, a succulent halophyte of the Chenopodiaceae family, is widely distributed in coastal areas of China. Suaeda glauca is highly resistant to salt and alkali stresses. In the present study, the salt-responsive transcriptome of Suaeda glauca was analyzed to identify genes involved in salt tolerance and study halophilic mechanisms in this halophyte. RESULTS: Illumina HiSeq 2500 was used to sequence cDNA libraries from salt-treated and control samples with three replicates each treatment. De novo assembly of the six transcriptomes identified 75,445 unigenes. A total of 23,901 (31.68%) unigenes were annotated. Compared with transcriptomes from the three salt-treated and three salt-free samples, 231 differentially expressed genes (DEGs) were detected (including 130 up-regulated genes and 101 down-regulated genes), and 195 unigenes were functionally annotated. Based on the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) classifications of the DEGs, more attention should be paid to transcripts associated with signal transduction, transporters, the cell wall and growth, defense metabolism and transcription factors involved in salt tolerance. CONCLUSIONS: This report provides a genome-wide transcriptional analysis of a halophyte, Suaeda glauca, under salt stress. Further studies of the genetic basis of salt tolerance in halophytes are warranted.

[Drug susceptibility and UL97 gene mutation analysis of cytomegalovirus in recipients of hematopoietic stem cell transplantation].

OBJECTIVE: To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods. METHODS: HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing. RESULTS: HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance. CONCLUSIONS: Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Effects of two low phytic acid mutations on seed quality and nutritional traits in soybean (Glycine max L. Merr).

Reduction of phytic acid in soybean seeds has the potential to improve the nutritional value of soybean meal and lessen phosphorus pollution in large scale animal farming. The objective of this study was to assess the effect of two new low phytic acid (LPA) mutations on seed quality and nutritional traits. Multilocation/season comparative analyses showed that the two mutations did not affect the concentration of crude protein, any of the individual amino acids, crude oil, and individual saturated fatty acids. Among other traits, Gm-lpa-TW75-1 had consistently higher sucrose contents (+47.4-86.1%) and lower raffinose contents (-74.2 to -84.3%) than those of wild type (WT) parent Taiwan 75; Gm-lpa-ZC-2 had higher total isoflavone contents (3038.8-4305.4 microg/g) than its parent Zhechun # 3 (1583.6-2644.9 microg/g) in all environments. Further tests of homozygous F(3) progenies of the cross Gm-lpa-ZC-2 x Wuxing # 4 (WT variety) showed that LPA lines had a mean content of total isoflavone significantly higher than WT lines. This study demonstrated that two LPA mutant genes have no negative effects on seed quality and nutritional traits; they instead have the potential to improve a few other properties. Therefore, these two mutant genes are valuable genetic resources for breeding high quality soybean varieties.

Putrescine enhancement of tolerance to root-zone hypoxia in Cucumis sativus: a role for increased nitrate reduction.

Cucumber (Cucumis sativus L.) plants were subjected to hypoxic stress with or without a pretreatment of putrescine (Put) to investigate whether nitrate reduction is involved in the enhancement effects of Put on tolerance to root-zone hypoxia. Both hypoxic stress and exogenous Put application significantly increased the contents of endogenous Put, spermidine and spermine. Plants grown under hypoxic conditions exhibited reductions in plant growth rate, NAD+/NADH ratio, ATP concentration, and consequent lowered cell viability in roots. The detrimental effects, however, were significantly alleviated by the addition of Put into the nutrient solution 24 h before the administration of hypoxia. Transcript levels of NR (nitrate reductase) and its cofactor binding domain genes FAD (FAD binding) and CYP51G1 (Heme binding), the activity of nitrate reductase (NR, EC 1.6.6.1) and the nitrate reduction process were each greatly enhanced by Put application, particularly in roots exposed to hypoxia. Lactate dehydrogenase (EC 1.1.1.27) activity was independent of aeration condition and Put application, whereas alcohol dehydrogenase (EC 1.1.1.1) activity was significantly increased after exposure to hypoxia, but did not increase after Put application. Put failed to alleviate the hypoxia injury of root electrolyte leakage when NR was inhibited by tungstate in the nutrient solution. These results suggest that Put enhances tolerance to hypoxia by increasing the transcript levels of NR and its cofactor binding domain genes, thereby stimulating the activities of NR and nitrate reduction to maintain the redox and energy status.