Genome-Wide Identification and Salt Stress Response Analysis of the bZIP Transcription Factor Family in Sugar Beet.

As one of the largest transcription factor families in plants, bZIP transcription factors play important regulatory roles in different biological processes, especially in the process of stress response. Salt stress inhibits the growth and yield of sugar beet. However, bZIP-related studies in sugar beet (Beta vulgaris L.) have not been reported. This study aimed to identify the bZIP transcription factors in sugar beet and analyze their biological functions and response patterns to salt stress. Using bioinformatics, 48 BvbZIP genes were identified in the genome of sugar beet, encoding 77 proteins with large structural differences. Collinearity analysis showed that three pairs of BvbZIP genes were fragment replication genes. The BvbZIP genes were grouped according to the phylogenetic tree topology and conserved structures, and the results are consistent with those reported in Arabidopsis. Under salt stress, the expression levels of most BvbZIP genes were decreased, and only eight genes were up-regulated. GO analysis showed that the BvbZIP genes were mainly negatively regulated in stress response. Protein interaction prediction showed that the BvbZIP genes were mainly involved in light signaling and ABA signal transduction, and also played a certain role in stress responses. In this study, the structures and biological functions of the BvbZIP genes were analyzed to provide foundational data for further mechanistic studies and for facilitating the efforts toward the molecular breeding of stress-resilient sugar beet.

Quantitative redox proteomics revealed molecular mechanisms of salt tolerance in the roots of sugar beet monomeric addition line M14.

BACKGROUND: Salt stress is often associated with excessive production of reactive oxygen species (ROS). Oxidative stress caused by the accumulation of ROS is a major factor that negatively affects crop growth and yield. Root is the primary organ that senses and transmits the salt stress signal to the whole plant. How oxidative stress affect redox sensitive proteins in the roots is not known. RESULTS: In this study, the redox proteome of sugar beet M14 roots under salt stress was investigated. Using iTRAQ reporters, we determined that salt stress caused significant changes in the abundance of many proteins (2305 at 20 min salt stress and 2663 at 10 min salt stress). Using iodoTMT reporters, a total of 95 redox proteins were determined to be responsive to salt stress after normalizing again total protein level changes. Notably, most of the differential redox proteins were involved in metabolism, ROS homeostasis, and stress and defense, while a small number play a role in transport, biosynthesis, signal transduction, transcription and photosynthesis. Transcription levels of 14 genes encoding the identified redox proteins were analyzed using qRT-PCR. All the genes were induced by salt stress at the transcriptional level. CONCLUSIONS: Based on the redox proteomics results, we construct a map of the regulatory network of M14 root redox proteins in response to salt stress. This study further refines the molecular mechanism of salt resistance at the level of protein redox regulation.

Functional Characterization of Sugar Beet M14 Antioxidant Enzymes in Plant Salt Stress Tolerance.

Salt stress can cause cellular dehydration, which induces oxidative stress by increasing the production of reactive oxygen species (ROS) in plants. They may play signaling roles and cause structural damages to the cells. To overcome the negative impacts, the plant ROS scavenging system plays a vital role in maintaining the cellular redox homeostasis. The special sugar beet apomictic monosomic additional M14 line (BvM14) showed strong salt stress tolerance. Comparative proteomics revealed that six antioxidant enzymes (glycolate oxidase (GOX), peroxiredoxin (PrxR), thioredoxin (Trx), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase3 (DHAR3)) in BvM14 were responsive to salt stress. In this work, the full-length cDNAs of genes encoding these enzymes in the redox system were cloned from the BvM14. Ectopic expression of the six genes reduced the oxidative damage of transgenic plants by regulating the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), ascorbic acid (AsA), and glutathione (GSH), and thus enhanced the tolerance of transgenic plants to salt stress. This work has charecterized the roles that the antioxidant enzymes play in the BvM14 response to salt stress and provided useful genetic resources for engineering and marker-based breeding of crops that are sensitive to salt stress.

Genome-wide identification and characterization of the soybean SOD family during alkaline stress.

BACKGROUND: Superoxide dismutase (SOD) proteins, as one kind of the antioxidant enzymes, play critical roles in plant response to various environment stresses. Even though its functions in the oxidative stress were very well characterized, the roles of SOD family genes in regulating alkaline stress response are not fully reported. METHODS: We identified the potential family members by using Hidden Markov model and soybean genome database. The neighbor-joining phylogenetic tree and exon-intron structures were generated by using software MEGA 5.0 and GSDS online server, respectively. Furthermore, the conserved motifs were analyzed by MEME online server. The syntenic analysis was conducted using Circos-0.69. Additionally, the expression levels of soybean SOD genes under alkaline stress were identified by qRT-PCR. RESULTS: In this study, we identified 13 potential SOD genes in soybean genome. Phylogenetic analysis suggested that SOD genes could be classified into three subfamilies, including MnSODs (GmMSD1-2), FeSODs (GmFSD1-5) and Cu/ZnSODs (GmCSD1-6). We further investigated the gene structure, chromosomal locations and gene-duplication, conserved domains and promoter cis-elements of the soybean SOD genes. We also explored the expression profiles of soybean SOD genes in different tissues and alkaline, salt and cold stresses, based on the transcriptome data. In addition, we detected their expression patterns in roots and leaves by qRT-PCR under alkaline stress, and found that different SOD subfamily genes may play different roles in response to alkaline stress. These results also confirmed the hypothesis that the great evolutionary divergence may contribute to the potential functional diversity in soybean SOD genes. Taken together, we established a foundation for further functional characterization of soybean SOD genes in response to alkaline stress in the future.

Genome-Wide Analysis of Glycine soja Response Regulator GsRR Genes Under Alkali and Salt Stresses.

Soil salt-alkalization is a dramatic challenging factor for plant growth. Wild soybean (Glycine soja) exhibits a favorable trait of superior tolerance to salt-alkali stress, and recent discoveries show that response regulator family genes are involved in diverse abiotic stresses. Genomic and transcriptomic analyses of all response regulator genes in wild soybean will provide insight into their function in plant stress response. In this study, we identified and characterized a total of 56 Glycine soja response regulator (GsRR) genes. Phylogenetic analysis suggested that GsRR genes could be classified into five subclasses (A1, A2, B1, B2, and C). We further investigated the chromosome locations, gene duplications and conserved domains of the GsRRs. Furthermore, the clustering analysis of GsRR transcript profiles revealed five different expression patterns under alkali stress. The A1 and A2 subclasses display significantly higher transcriptional levels than the B subclass. In addition, quantitative real-time PCR results verified that the GsRR genes were also significantly influenced by salt stress. Notably, GsRR2a in the A1 subclass showed opposite expression patterns under salt stress comparing with alkali stress. Moreover, overexpression of GsRR2a in Arabidopsis significantly improved the tolerance to alkali stress, but not salt stress. These results suggest the important roles of GsRR genes in response to salt and alkaline stresses, and also provide valuable clues for further functional characterization of GsRR family genes.

A potential efflux boron transporter gene GsBOR2, positively regulates Arabidopsis bicarbonate tolerance.

Soil alkalization severely restricts agricultural production and economic development worldwide, this problem is far more serious in Songnen Plain, the largest commodity grain base of China. However, little research has been done concerning the mechanisms of plant responses to alkaline stress to date. In this study, we isolated an alkali inducible gene GsBOR2 from Glycine soja on the basis of RNA seq data. GsBOR2 sh high protein sequence similarity with the known boron transporters in other species. The expression of GsBOR2 was highly up-regulated by 50 mM NaHCO3 treatment and displayed tissue specificity. We then generated the transgenic Arabidopsis overexpressing GsBOR2 and found that the transgenic lines exhibited enhanced alkaline tolerance compared to wild type plants, as illustrated by longer roots and greater shoot biomass. Moreover, GsBOR2 overexpression was also capable of increasing plant resistance to KHCO3 treatment but not to high-pH stress. Functional complementation of Scbor1 mutant yeasts suggested that GsBOR2 could likely mediate the efflux of boron from cells. Taken together, the alkali responsive gene GsBOR2 is a positive regulator of plant bicarbonate tolerance.

Genome-wide analysis and expression profiling of PP2C clade D under saline and alkali stresses in wild soybean and Arabidopsis.

Protein phosphatase 2Cs (PP2Cs) belong to the largest protein phosphatase family in plants. Some members have been described as being negative modulators of plant growth and development, as well as responses to hormones and environmental stimuli. However, little is known about the members of PP2C clade D, which may be involved in the regulation of signaling pathways, especially in response to saline and alkali stresses. Here, we identified 13 PP2C orthologs from the wild soybean (Glycine soja) genome. We examined the sequence characteristics, chromosome locations and duplications, gene structures, and promoter cis-elements of the PP2C clade D genes in Arabidopsis and wild soybean. Our results showed that GsPP2C clade D (GsAPD) genes exhibit more gene duplications than AtPP2C clade D genes. Plant hormone and abiotic stress-responsive elements were identified in the promoter regions of most PP2C genes. Moreover, we investigated their expression patterns in roots, stems, and leaves. Quantitative real-time PCR analyses revealed that the expression levels of representative GsPP2C and AtPP2C clade D genes were significantly influenced by alkali and salt stresses, suggesting that these genes might be associated with or directly involved in the relevant stress signaling pathways. Our results established a foundation for further functional characterization of PP2C clade D genes in the future.

GsSLAH3, a Glycine soja slow type anion channel homolog, positively modulates plant bicarbonate stress tolerance.

Alkaline stress is a major form of abiotic stress that severely inhibits plant growth and development, thus restricting crop productivity. However, little is known about how plants respond to alkali. In this study, a slow-type anion channel homolog 3 gene, GsSLAH3, was isolated and functionally characterized. Bioinformatics analysis showed that the GsSLAH3 protein contains 10 transmembrane helices. Consistently, GsSLAH3 was found to locate on plasma membrane by transient expression in onion epidermal cells. In wild soybeans, GsSLAH3 expression was induced by NaHCO3 treatment, suggesting its involvement in plant response to alkaline stress. Ectopic expression of GsSLAH3 in yeast increased sensitivity to alkali treatment. Dramatically, overexpression of GsSLAH3 in Arabidopsis thaliana enhanced alkaline tolerance during the germination, seedling and adult stages. More interestingly, we found that transgenic lines also improved plant tolerance to KHCO3 rather than high pH treatment. A nitrate content analysis of Arabidopsis shoots showed that GsSLAH3 overexpressing lines accumulated more NO3- than wild-type. In summary, our data suggest that GsSLAH3 is a positive alkali responsive gene that increases bicarbonate resistance specifically.

GsCHX19.3, a member of cation/H+ exchanger superfamily from wild soybean contributes to high salinity and carbonate alkaline tolerance.

Cation/H+ exchangers (CHX) are characterized to be involved in plant growth, development and stress responses. Although soybean genome sequencing has been completed, the CHX family hasn't yet been systematically analyzed, especially in wild soybean. Here, through Hidden Markov Model search against Glycine soja proteome, 34 GsCHXs were identified and phylogenetically clustered into five groups. Members within each group showed high conservation in motif architecture. Interestingly, according to our previous RNA-seq data, only Group IVa members exhibited highly induced expression under carbonate alkaline stress. Among them, GsCHX19.3 displayed the greatest up-regulation in response to carbonate alkaline stress, which was further confirmed by quantitative real-time PCR analysis. We also observed the ubiquitous expression of GsCHX19.3 in different tissues and its localization on plasma membrane. Moreover, we found that GsCHX19.3 expression in AXT4K, a yeast mutant lacking four ion transporters conferred resistance to low K+ at alkali pH, as well as carbonate stress. Consistently, in Arabidopsis, GsCHX19.3 overexpression increased plant tolerance both to high salt and carbonate alkaline stresses. Furthermore, we also confirmed that GsCHX19.3 transgenic lines showed lower Na+ concentration but higher K+/Na+ values under salt-alkaline stress. Taken together, our findings indicated that GsCHX19.3 contributed to high salinity and carbonate alkaline tolerance.

A novel Glycine soja homeodomain-leucine zipper (HD-Zip) I gene, Gshdz4, positively regulates bicarbonate tolerance and responds to osmotic stress in Arabidopsis.

BACKGROUND: Wild soybean (Glycine soja) is a highly adaptive plant species which can grow well in saline-alkaline soils. In soybean genome, there exist about 140 HD-Zip (Homeodomain-leucine Zipper) genes. HD-Zip transcription factor family is one of the largest plant specific superfamilies and plays important roles in response to abiotic stresses. Although HD-Zip transcription factors have been broadly reported to be involved in plant resistance to abiotic stresses like salt and drought, their roles in response to bicarbonate stress is largely unknown. RESULTS: From our previous transcriptome profile analysis of wild soybean treated by 50 mM NaHCO3, we identified an HD-Zip gene (Gshdz4) which showed high response to the alkaline stress. Our result of qRT-PCR showed that the expression of Gshdz4 was induced by alkaline stress (NaHCO3) in both leaves and roots of wild soybean. Overexpression of Gshdz4 in Arabidopsis resulted in enhanced tolerance to NaHCO3 and KHCO3 during the process of plant growth and development. However, the growths of transgenic and WT plants were not significantly different on the medium with high pH adjusted by KOH, implicating Gshdz4 is only responsible for resisting HCO3 (-) but not high pH. The transgenic plants had less MDA contents but higher POD activities and chlorophyll contents than the WT plants. Moreover, the transcript levels of stress-related genes, such as NADP-ME, H (+) -Ppase, RD29B and KIN1 were increased with greater extent in the transgenic plants than the wild plants. On the contrary, Gshdz4 overexpression lines were much sensitive to osmotic stress at seed germination and stocking stages compared to the wild plants. CONCLUSIONS: We revealed that the important and special roles of Gshdz4 in enhancing bicarbonate tolerance and responding to osmotic stress. It is the first time to elucidate these novel functions of HD-ZIP transcription factors. All the evidences broaden our understanding of functions of HD-Zip family and provide clues for uncovering the mechanisms of high tolerance of wild soybean to saline-alkaline stresses.

GsERF6, an ethylene-responsive factor from Glycine soja, mediates the regulation of plant bicarbonate tolerance in Arabidopsis.

MAIN CONCLUSION: This is an original study focus on ERF gene response to alkaline stress. GsERF6 functions as transcription factor and significantly enhanced plant tolerance to bicarbonate (HCO 3 (-) ) in transgenic Arabidopsis . Alkaline stress is one of the most harmful, but little studied environmental factors, which negatively affects plant growth, development and yield. The cause of alkaline stress is mainly due to the damaging consequence of high concentration of the bicarbonate ion, high-pH, and osmotic shock to plants. The AP2/ERF family genes encode plant-specific transcription factors involved in diverse environmental stresses. However, little is known about their physiological functions, especially in alkaline stress responses. In this study, we functionally characterized a novel ERF subfamily gene, GsERF6 from alkaline-tolerant wild soybean (Glycine soja). In wild soybean, GsERF6 was rapidly induced by NaHCO3 treatment, and its overexpression in Arabidopsis enhanced transgenic plant tolerance to NaHCO3 challenge. Interestingly, GsERF6 transgenic lines also displayed increased tolerance to KHCO3 treatment, but not to high pH stress, implicating that GsERF6 may participate specifically in bicarbonate stress responses. We also found that GsERF6 overexpression up-regulated the transcription levels of bicarbonate-stress-inducible genes such as NADP-ME, H (+)-Ppase and H (+)-ATPase, as well as downstream stress-tolerant genes such as RD29A, COR47 and KINI. GsERF6 overexpression and NaHCO3 stress also altered the expression patterns of plant hormone synthesis and hormone-responsive genes. Conjointly, our results suggested that GsERF6 is a positive regulator of plant alkaline stress by increasing bicarbonate ionic resistance specifically, providing a new insight into the regulation of gene expression under alkaline conditions.

Functional characterization of a Glycine soja Ca(2+)ATPase in salt-alkaline stress responses.

It is widely accepted that Ca(2+)ATPase family proteins play important roles in plant environmental stress responses. However, up to now, most researches are limited in the reference plants Arabidopsis and rice. The function of Ca(2+)ATPases from non-reference plants was rarely reported, especially its regulatory role in carbonate alkaline stress responses. Hence, in this study, we identified the P-type II Ca(2+)ATPase family genes in soybean genome, determined their chromosomal location and gene architecture, and analyzed their amino acid sequence and evolutionary relationship. Based on above results, we pointed out the existence of gene duplication for soybean Ca(2+)ATPases. Then, we investigated the expression profiles of the ACA subfamily genes in wild soybean (Glycine soja) under carbonate alkaline stress, and functionally characterized one representative gene GsACA1 by using transgenic alfalfa. Our results suggested that GsACA1 overexpression in alfalfa obviously increased plant tolerance to both carbonate alkaline and neutral salt stresses, as evidenced by lower levels of membrane permeability and MDA content, but higher levels of SOD activity, proline concentration and chlorophyll content under stress conditions. Taken together, for the first time, we reported a P-type II Ca(2+)ATPase from wild soybean, GsACA1, which could positively regulate plant tolerance to both carbonate alkaline and neutral salt stresses.

Overexpression of GsGSTU13 and SCMRP in Medicago sativa confers increased salt-alkaline tolerance and methionine content.

Tau-class glutathione S-transferases (GSTUs) are ubiquitous proteins encoded by a large gene family in plants, which play important roles in combating different environmental stresses. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three GSTUs (GsGSTU13, GsGSTU14 and GsGSTU19) as potential salt-alkaline stress-responsive genes. Two of them, GsGSTU14 and GsGSTU19, have been shown to positively regulate plant salt-alkaline tolerance. In this study, we further demonstrated the positive function of GsGSTU13 in plant salt-alkaline stress responses by overexpressing it in Medicago sativa. Stress tolerance tests suggested that GsGSTU13 transgenic lines showed better growth and physiological indicators than wild alfalfa (cv. Zhaodong) under alkaline stress. Considering the shortage of methionine in alfalfa, we then co-transformed GsGSTU13 into two main alfalfa cultivars in Heilongjiang Province (cv. Zhaodong and cv. Nongjing No. 1) together with SCMRP, a synthesized gene that could improve the methionine content. We found that GsGSTU13/SCMRP transgenic alfalfa displayed not only higher methionine content but also higher tolerance to alkaline and salt stresses, respectively. Taken together, our results demonstrate that GsGSTU13 acts as a positive regulator in plant responses to salt and alkaline stresses, and can be used as a good candidate for generation of salt-alkaline tolerant crops.

GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja, Plays Differential Roles in Plant Responses to Bicarbonate, Salt and Osmotic Stresses.

Calcium, as the most widely accepted messenger, plays an important role in plant stress responses through calcium-dependent signaling pathways. The calmodulin-like family genes (CMLs) encode Ca2+ sensors and function in signaling transduction in response to environmental stimuli. However, until now, the function of plant CML proteins, especially soybean CMLs, is largely unknown. Here, we isolated a Glycine soja CML protein GsCML27, with four conserved EF-hands domains, and identified it as a calcium-binding protein through far-UV CD spectroscopy. We further found that expression of GsCML27 was induced by bicarbonate, salt and osmotic stresses. Interestingly, ectopic expression of GsCML27 in Arabidopsis enhanced plant tolerance to bicarbonate stress, but decreased the salt and osmotic tolerance during the seed germination and early growth stages. Furthermore, we found that ectopic expression of GsCML27 decreases salt tolerance through modifying both the cellular ionic (Na+, K+) content and the osmotic stress regulation. GsCML27 ectopic expression also decreased the expression levels of osmotic stress-responsive genes. Moreover, we also showed that GsCML27 localized in the whole cell, including cytoplasm, plasma membrane and nucleus in Arabidopsis protoplasts and onion epidermal cells, and displayed high expression in roots and embryos. Together, these data present evidence that GsCML27 as a Ca2+-binding EF-hand protein plays a role in plant responses to bicarbonate, salt and osmotic stresses.

Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress.

Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H+-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants.

Wild soybean roots depend on specific transcription factors and oxidation reduction related genesin response to alkaline stress.

Soil alkalinity is an important environmental problem limiting agricultural productivity. Wild soybean (Glycine soja) shows strong alkaline stress tolerance, so it is an ideal plant candidate for studying the molecular mechanisms of alkaline tolerance and identifying alkaline stress-responsive genes. However, limited information is available about G. soja responses to alkaline stress on a genomic scale. Therefore, in the present study, we used RNA sequencing to compare transcript profiles of G. soja root responses to sodium bicarbonate (NaHCO3) at six time points, and a total of 68,138,478 pairs of clean reads were obtained using the Illumina GAIIX. Expression patterns of 46,404 G. soja genes were profiled in all six samples based on RNA-seq data using Cufflinks software. Then, t12 transcription factors from MYB, WRKY, NAC, bZIP, C2H2, HB, and TIFY families and 12 oxidation reduction related genes were chosen and verified to be induced in response to alkaline stress by using quantitative real-time polymerase chain reaction (qRT-PCR). The GO functional annotation analysis showed that besides "transcriptional regulation" and "oxidation reduction," these genes were involved in a variety of processes, such as "binding" and "response to stress." This is the first comprehensive transcriptome profiling analysis of wild soybean root under alkaline stress by RNA sequencing. Our results highlight changes in the gene expression patterns and identify a set of genes induced by NaHCO3 stress. These findings provide a base for the global analyses of G. soja alkaline stress tolerance mechanisms.

GsSKP21, a Glycine soja S-phase kinase-associated protein, mediates the regulation of plant alkaline tolerance and ABA sensitivity.

Plant SKP1-like family proteins, components of the SCF complex E3 ligases, are involved in the regulation of plant development and stress responses. Little is known about the precise function of SKP genes in plant responses to environmental stresses. GsSKP21 was initially identified as a potential stress-responsive gene based on the transcriptome sequencing of Glycine soja. In this study, we found that GsSKP21 protein contains highly conserved SKP domains in its N terminus and an extra unidentified domain in its C terminus. The transcript abundance of GsSKP21, detected by quantitative real-time PCR, was induced under the treatment of alkali and salt stresses. Overexpression of GsSKP21 in Arabidopsis dramatically increased plant tolerance to alkali stress. Furthermore, we found that overexpression of GsSKP21 resulted in decreased ABA sensitivity during both the seed germination and early seedling growth stages. GsSKP21 mediated ABA signaling by altering the expression levels of the ABA signaling-related and ABA-induced genes. We also investigated the tissue expression specificity and subcellular localization of GsSKP21. These results suggest that GsSKP21 is important for plant tolerance to alkali stress and plays a critical regulatory role in the ABA-mediated stress response.

Ectopic expression of GsPPCK3 and SCMRP in Medicago sativa enhances plant alkaline stress tolerance and methionine content.

So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), H(+)-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.

CNVD: text mining-based copy number variation in disease database.

Copy number variation (CNV) is a kind of chromosomal structural reorganization that has been detected, in this decade, mainly by high-throughput biological technology. Researchers have found that CNVs are ubiquitous in many species and accumulating evidence indicates that CNVs are closely related with complex diseases. The investigation of chromosomal structural alterations has begun to reveal some important clues to the pathologic causes of diseases and to the disease process. However, many of the published studies have focused on a single disease and, so far, the experimental results have not been systematically collected or organized. Manual text mining from 6301 published papers was used to build the Copy Number Variation in Disease database (CNVD). CNVD contains CNV information for 792 diseases in 22 species from diverse types of experiments, thus, ensuring high confidence and comprehensive representation of the relationship between the CNVs and the diseases. In addition, multiple query modes and visualized results are provided in the CNVD database. With its user-friendly interface and the integrated CNV information for different diseases, CNVD will offer a truly comprehensive platform for disease research based on chromosomal structural variations. The CNVD interface is accessible at http://bioinfo.hrbmu.edu.cn/CNVD.

Calculating phenotypic similarity between genes using hierarchical structure data based on semantic similarity.

Phenotypic similarity is correlated with a number of measures of gene function, such as relatedness at the level of direct protein-protein interaction. The phenotypic effect of a deleted or mutated gene, which is one part of gene annotation, has caught broad attention. However, there have been few measures to study phenotypic similarity with the data from Human Phenotype Ontology (HPO) database, therefore more analogous measures should be developed and investigated. We used five semantic similarity-based measures (Jiang and Conrath, Lin, Schlicker, Yu and Wu) to calculate the human phenotypic similarity between genes (PSG) with data from HPO database, and evaluated their accuracy with information of protein-protein interaction, protein complex, protein family, gene function or DNA sequence. Compared with the gene pairs that were random selected, the results of these methods were statistically significant (all P<0.001). Furthermore, we assessed the performance of these five measures by receiver operating characteristic (ROC) curve analysis, and found that most of them performed better than the previous methods. This work had proved that these measures based on semantic similarity for calculation of PSG were effective for hierarchical structure data. Our study contributes to the development and optimization of novel algorithms of PSG calculation and provides more alternative methods to researchers as well as tools and directions for PSG study.