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1.  Improved Nutritive Quality and Salt Resistance in Transgenic Maize by Simultaneously Overexpression of a Natural Lysine-Rich Protein Gene, SBgLR, and an ERF Transcription Factor Gene, TSRF1 
Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9–10 and 19–11, ZmMYC1 in line 19–11 and ZmSDR in line 19–11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt treatment, the expressions of the ten stress-related genes were significantly induced in both wild-type (WT) and transgenic lines. However, compared to WT, the increases of ZmP5CS1 in all these three transgenic lines and ZmP5CS2 in line 9–10 were less than WT plants. This study provides an effective approach of maize genetic engineering for improved nutritive quality and salt tolerance.
doi:10.3390/ijms14059459
PMCID: PMC3676793  PMID: 23629675
Zea may L.; high lysine; high protein; salt tolerance; marker-free
2.  Characterization of miRNAs in Response to Short-Term Waterlogging in Three Inbred Lines of Zea mays 
PLoS ONE  2012;7(6):e39786.
Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the microRNA-mediated gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic stress responses. To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and their 92 predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17). Based on our studies, miR159, miR164, miR167, miR393, miR408 and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress and development related cis-regulatory elements on the promoters of these miRNAs; and a probable miRNA-mediated gene regulatory network in response to short-term waterlogging stress was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a gene regulatory network; and crosstalk occurs between different biochemical pathways.
doi:10.1371/journal.pone.0039786
PMCID: PMC3387268  PMID: 22768123
3.  Distribution and Expression of the ZmpA Metalloprotease in the Burkholderia cepacia Complex 
Journal of Bacteriology  2005;187(24):8247-8255.
The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extracellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.
doi:10.1128/JB.187.24.8247-8255.2005
PMCID: PMC1316997  PMID: 16321929
4.  Subtractive hybridization-mediated analysis of genes and in silico prediction of associated microRNAs under waterlogged conditions in sugarcane (Saccharum spp.) 
FEBS Open Bio  2014;4:533-541.
Highlights
•Waterlogging adversely affects sugarcane productivity and quality.•A subtractive cDNA library was prepared from sugarcane leaf tissue and sequenced to generate ESTs.•EST sequences were used to identify transcripts induced by waterlogging.•The sequenced clones were classified by predicted functions and stress-related genes formed the largest class.•EST sequences were also used to identify putative novel microRNAs and their targets.
Sugarcane is an important tropical cash crop meeting 75% of world sugar demand and it is fast becoming an energy crop for the production of bio-fuel ethanol. A considerable area under sugarcane is prone to waterlogging which adversely affects both cane productivity and quality. In an effort to elucidate the genes underlying plant responses to waterlogging, a subtractive cDNA library was prepared from leaf tissue. cDNA clones were sequenced and annotated for their putative functions. Major groups of ESTs were related to stress (15%), catalytic activity (13%), cell growth (10%) and transport related proteins (6%). A few stress-related genes were identified, including senescence-associated protein, dehydration-responsive family protein, and heat shock cognate 70 kDa protein. A bioinformatics search was carried out to discover novel microRNAs (miRNAs) that can be regulated in sugarcane plants subjected to waterlogging stress. Taking advantage of the presence of miRNA precursors in the related sorghum genome, seven candidate mature miRNAs were identified in sugarcane. The application of subtraction technology allowed the identification of differentially expressed sequences and novel miRNAs in sugarcane under waterlogging stress. The comparative global transcript profiling in sugarcane plants undertaken in this study suggests that proteins associated with stress response, signal transduction, metabolic activity and ion transport play important role in conferring waterlogging tolerance in sugarcane.
doi:10.1016/j.fob.2014.05.007
PMCID: PMC4087145  PMID: 25009768
ABA, abscisic acid; ADF, actin depolymerizing factor; ESTs, expressed sequence tags; Hsps, heat shock proteins; PTGS, post-transcriptional gene silencing; REMs, remorins; SSA, snow pea pod senescence-associated; SUCEST, sugarcane EST database; TGS, transcriptional gene silencing; EST; Novel miRNAs; Sugarcane; Suppression subtractive hybridization; Waterlogging stress
5.  Identification of transcriptome induced in roots of maize seedlings at the late stage of waterlogging 
BMC Plant Biology  2010;10:189.
Background
Plants respond to low oxygen stress, particularly that caused by waterlogging, by altering transcription and translation. Previous studies have mostly focused on revealing the mechanism of the response at the early stage, and there is limited information about the transcriptional profile of genes in maize roots at the late stage of waterlogging. The genetic basis of waterlogging tolerance is largely unknown. In this study, the transcriptome at the late stage of waterlogging was assayed in root cells of the tolerant inbred line HZ32, using suppression subtractive hybridization (SSH). A forward SSH library using RNA populations from four time points (12 h, 16 h, 20 h and 24 h) after waterlogging treatment was constructed to reveal up-regulated genes, and transcriptional and linkage data was integrated to identify candidate genes for waterlogging tolerance.
Results
Reverse Northern analysis of a set of 768 cDNA clones from the SSH library revealed a large number of genes were up-regulated by waterlogging. A total of 465 ESTs were assembled into 296 unigenes. Bioinformatic analysis revealed that the genes were involved in complex pathways, such as signal transduction, protein degradation, ion transport, carbon and amino acid metabolism, and transcriptional and translational regulation, and might play important roles at the late stage of the response to waterlogging. A significant number of unigenes were of unknown function. Approximately 67% of the unigenes could be aligned on the maize genome and 63 of them were co-located within reported QTLs.
Conclusion
The late response to waterlogging in maize roots involves a broad spectrum of genes, which are mainly associated with two response processes: defense at the early stage and adaption at the late stage. Signal transduction plays a key role in activating genes related to the tolerance mechanism for survival during prolonged waterlogging. The crosstalk between carbon and amino acid metabolism reveals that amino acid metabolism performs two main roles at the late stage: the regulation of cytoplasmic pH and energy supply through breakdown of the carbon skeleton.
doi:10.1186/1471-2229-10-189
PMCID: PMC2956539  PMID: 20738849
6.  Dynamic QTL Analysis and Candidate Gene Mapping for Waterlogging Tolerance at Maize Seedling Stage 
PLoS ONE  2013;8(11):e79305.
Soil waterlogging is one of the major abiotic stresses adversely affecting maize growth and yield. To identify dynamic expression of genes or quantitative trait loci (QTL), QTL associated with plant height, root length, root dry weight, shoot dry weight and total dry weight were identified via conditional analysis in a mixed linear model and inclusive composite interval mapping method at three respective periods under waterlogging and control conditions. A total of 13, 19 and 23 QTL were detected at stages 3D|0D (the period during 0–3 d of waterlogging), 6D|3D and 9D|6D, respectively. The effects of each QTL were moderate and distributed over nine chromosomes, singly explaining 4.14–18.88% of the phenotypic variation. Six QTL (ph6-1, rl1-2, sdw4-1, sdw7-1, tdw4-1 and tdw7-1) were identified at two consistent stages of seedling development, which could reflect a continuous expression of genes; the remaining QTL were detected at only one stage. Thus, expression of most QTL was influenced by the developmental status. In order to provide additional evidence regarding the role of corresponding genes in waterlogging tolerance, mapping of Expressed Sequence Tags markers and microRNAs were conducted. Seven candidate genes were observed to co-localize with the identified QTL on chromosomes 1, 4, 6, 7 and 9, and may be important candidate genes for waterlogging tolerance. These results are a good starting point for understanding the genetic basis for selectively expressing of QTL in different stress periods and the common genetic control mechanism of the co-localized traits.
doi:10.1371/journal.pone.0079305
PMCID: PMC3828346  PMID: 24244474
7.  The Transcriptome of Brassica napus L. Roots under Waterlogging at the Seedling Stage 
Although rapeseed (Brassica napus L.) is known to be affected by waterlogging, the genetic basis of waterlogging tolerance by rapeseed is largely unknown. In this study, the transcriptome under 0 h and 12 h of waterlogging was assayed in the roots of ZS9, a tolerant variety, using digital gene expression (DGE). A total of 4432 differentially expressed genes were identified, indicating that the response to waterlogging in rapeseed is complicated. The assignments of the annotated genes based on GO (Gene Ontology) revealed there were more genes induced under waterlogging in “oxidation reduction”, “secondary metabolism”, “transcription regulation”, and “translation regulation”; suggesting these four pathways are enhanced under waterlogging. Analysis of the 200 most highly expressed genes illustrated that 144 under normal conditions were down-regulated by waterlogging, while up to 191 under waterlogging were those induced in response to stress. The expression of genes involved under waterlogging is mediated by multiple levels of transcriptional, post-transcriptional, translational and post-translational regulation, including phosphorylation and protein degradation; in particular, protein degradation might be involved in the negative regulation in response to this stress. Our results provide new insight into the response to waterlogging and will help to identify important candidate genes.
doi:10.3390/ijms14022637
PMCID: PMC3588007  PMID: 23358252
rapeseed (Brassica napus L.); waterlogging; DGE (digital gene expression); roots; transcriptome
8.  De novo sequencing, assembly, and analysis of the Taxodium ‘Zhongshansa’ roots and shoots transcriptome in response to short-term waterlogging 
BMC Plant Biology  2014;14:201.
Background
Taxodium is renowned for its strong tolerance to waterlogging stress, thus it has great ecological and economic potential. However, the scant genomic resources in genus Taxodium have greatly hindered further exploration of its underlying flood-tolerance mechanism. Taxodium ‘Zhongshansa’ is an interspecies hybrid of T. distichum and T. mucronatum, and has been widely planted in southeastern China. To understand the genetic basis of its flood tolerance, we analyzed the transcriptomes of Taxodium ‘Zhongshansa’ roots and shoots in response to short-term waterlogging.
Results
RNA-seq was used to analyze genome-wide transcriptome changes of Taxodium ‘Zhongshansa 406’ clone root and shoot treated with 1 h of soil-waterlogging stress. After de novo assembly, 108,692 unigenes were achieved, and 70,260 (64.64%) of them were annotated. There were 2090 differentially expressed genes (DEGs) found in roots and 394 in shoots, with 174 shared by both of them, indicating that the aerial parts were also affected. Under waterlogging stress, the primary reaction of hypoxic-treated root was to activate the antioxidative defense system to prevent cells experiencing reactive oxygen species (ROS) poisoning. As respiration was inhibited and ATP decreased, another quick coping mechanism was repressing the energy-consuming biosynthetic processes through the whole plant. The glycolysis and fermentation pathway was activated to maintain ATP production in the hypoxic root. Constantly, the demand for carbohydrates increased, and carbohydrate metabolism were accumulated in the root as well as the shoot, possibly indicating that systemic communications between waterlogged and non-waterlogged tissues facilated survival. Amino acid metabolism was also greatly influenced, with down-regulation of genes involvedin serine degradation and up-regulation of aspartic acid degradation. Additionally, a non-symbiotic hemoglobin class 1 gene was up-regulated, which may also help the ATP production. Moreover, the gene expression pattern of 5 unigenes involving in the glycolysis pathway revealed by qRT-PCR confirmed the RNA-Seq data.
Conclusions
We conclude that ROS detoxification and energy maintenance were the primary coping mechanisms of ‘Zhongshansa’ in surviving oxygen deficiency, which may be responsible for its remarkable waterlogging tolerance. Our study not only provided the first large-scale assessment of genomic resources of Taxodium but also guidelines for probing the molecular mechanism underlying ‘Zhongshansa’ waterlogging tolerance.
doi:10.1186/s12870-014-0201-y
PMCID: PMC4222663  PMID: 25055883
Taxodium; Waterlogging; Stress; Transcriptome; qRT-PCR
9.  Mapping of QTL Associated with Waterlogging Tolerance during the Seedling Stage in Maize 
Annals of Botany  2007;99(6):1067-1081.
Background and Aims
Soil waterlogging is a major environmental stress that suppresses maize (Zea mays) growth and yield. To identify quantitative trait loci (QTL) associated with waterlogging tolerance at the maize seedling stage, a F2 population consisting of 288 F2:3 lines was created from a cross between two maize genotypes, ‘HZ32’ (waterlogging-tolerant) and ‘K12’ (waterlogging-sensitive).
Methods
The F2 population was genotyped and a base-map of 1710·5 cM length was constructed with an average marker space of 11·5 cM based on 177 SSR (simple sequence repeat) markers. QTL associated with root length, root dry weight, plant height, shoot dry weight, total dry weight and waterlogging tolerance coefficient were identified via composite interval mapping (CIM) under waterlogging and control conditions in 2004 (EXP.1) and 2005 (EXP.2), respectively.
Key Results and Conclusions
Twenty-five and thirty-four QTL were detected in EXP.1 and EXP.2, respectively. The effects of each QTL were moderate, ranging from 3·9 to 37·3 %. Several major QTL determining shoot dry weight, root dry weight, total dry weight, plant height and their waterlogging tolerance coefficient each mapped on chromosomes 4 and 9. These QTL were detected consistently in both experiments. Secondary QTL influencing tolerance were also identified and located on chromosomes 1, 2, 3, 6, 7 and 10. These QTL were specific to particular traits or environments. Although the detected regions need to be mapped more precisely, the findings and QTL found in this study may provide useful information for marker-assisted selection (MAS) and further genetic studies on maize waterlogging tolerance.
doi:10.1093/aob/mcm055
PMCID: PMC3244372  PMID: 17470902
Maize (Zea mays); waterlogging tolerance; genome mapping; SSR marker; QTL; epistasis effect
10.  Elucidation of the molecular responses to waterlogging in Jatropha roots by transcriptome profiling 
Jatropha (Jatropha curcas) is a promising oil-seed crop for biodiesel production. However, the species is highly sensitive to waterlogging, which can result in stunted growth and yield loss. To date, the molecular mechanisms underlying the responses to waterlogging in Jatropha remain elusive. Here, the transcriptome adjustment of Jatropha roots to waterlogging was examined by high-throughput RNA-sequencing (RNA-seq). The results indicated that 24 h of waterlogging caused significant changes in mRNA abundance of 1968 genes. Comprehensive gene ontology and functional enrichment analysis of root transcriptome revealed that waterlogging promoted responses to hypoxia and anaerobic respiration. On the other hand, the stress inhibited carbohydrate synthesis, cell wall biogenesis, and growth. The results also highlighted the roles of ethylene, nitrate, and nitric oxide in waterlogging acclimation. In addition, transcriptome profiling identified 85 waterlogging-induced transcription factors including members of AP2/ERF, MYB, and WRKY families implying that reprogramming of gene expression is a vital mechanism for waterlogging acclimation. Comparative analysis of differentially regulated transcripts in response to waterlogging among Arabidopsis, gray poplar, Jatropha, and rice further revealed not only conserved but species-specific regulation. Our findings unraveled the molecular responses to waterlogging in Jatropha and provided new perspectives for developing a waterlogging tolerant cultivar in the future.
doi:10.3389/fpls.2014.00658
PMCID: PMC4251292  PMID: 25520726
Jatropha curcas; genomics; low oxygen; ERFs; transcriptional control; RNA-seq
11.  A Comparison of Screening Methods to Identify Waterlogging Tolerance in the Field in Brassica napus L. during Plant Ontogeny 
PLoS ONE  2014;9(3):e89731.
Waterlogging tolerance is typically evaluated at a specific development stage, with an implicit assumption that differences in waterlogging tolerance expressed in these systems will result in improved yield performance in fields. It is necessary to examine these criteria in fields.
In the present study, three experiments were conducted to screen waterlogging tolerance in 25 rapeseed (Brassica napus L.) varieties at different developmental stages, such as seedling establishment stage and seedling stage at controlled environment, and maturity stage in the fields. The assessments for physiological parameters at three growth stages suggest that there were difference of waterlogging tolerance at all the development stages, providing an important basis for further development of breeding more tolerant materials. The results indicated that flash waterlogging restricts plant growth and growth is still restored after removal of the stress. Correlation analysis between waterlogging tolerance coefficient (WTC) of yield and other traits revealed that there was consistency in waterlogging tolerance of the genotypes until maturity, and good tolerance at seedling establishment stage and seedling stage can guarantee tolerance in later stages. The waterlogging-tolerant plants could be selected using some specific traits at any stage, and selections would be more effective at the seedling establishment stage.
Thus, our study provides a method for screening waterlogging tolerance, which would enable the suitable basis for initial selection of a large number of germplasm or breeding populations for waterlogging tolerance and help for verifying their potential utility in crop-improvement.
doi:10.1371/journal.pone.0089731
PMCID: PMC3940661  PMID: 24594687
12.  Identification of QTLs for yield and yield components of barley under different growth conditions*  
Waterlogging is a major abiotic stress limiting barley (Hordeum vulgare L.) yield and its stability in areas with excessive rainfall. Identification of genomic regions influencing the response of yield and its components to waterlogging stress will enhance our understanding of the genetics of waterlogging tolerance and the development of more tolerant barley cultivars. Quantitative trait loci (QTLs) for grain yield and its components were identified using 156 doubled haploid (DH) lines derived from a cross between the cultivars Yerong (waterlogging-tolerant) and Franklin (waterlogging-sensitive) grown under different conditions (waterlogged and well drained). A total of 31 QTLs were identified for the measured characters from two experiments with two growth environments. The phenotypic variation explained by individual QTLs ranged from 4.74% to 55.34%. Several major QTLs determining kernel weight (KW), grains per spike (GS), spikes per plant (SP), spike length (SL) and grain yield (GY) were detected on the same region of chromosome 2H, indicating close linkage or pleiotropy of the gene(s) controlling these traits. Some different QTLs were identified under waterlogging conditions, and thus different markers may have to be used in selecting cultivars suitable for high rainfall areas.
doi:10.1631/jzus.B0900332
PMCID: PMC2833401  PMID: 20205303
Barley (Hordeum vulgare L.); Waterlogging tolerance; Yield; Quantitative trait locus (QTL)
13.  A Genome-Wide Characterization of MicroRNA Genes in Maize 
PLoS Genetics  2009;5(11):e1000716.
MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR–RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with ∼35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.
Author Summary
MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally and play roles in diverse pathways including those acting on development and responses to stress. Here, we describe a genome-wide computational prediction of maize miRNA genes and their characterization with respect to expression, putative targets, evolution following whole genome duplication, and allelic diversity. The structures of unprocessed primary miRNA transcripts were determined by 5′ RACE and 3′ RACE. Expression profiles were surveyed in five tissue types by deep-sequencing of small RNA libraries. We predicted miRNA targets computationally based on the most recent maize protein annotations. Analysis of the predicted functions of target genes, on the basis of gene ontology, supported their roles in regulatory processes. We identified putative orthologs in Sorghum based on an analysis of synteny and found that maize-homoeologous miRNA genes were retained more frequently than expected. We also explored miRNA nucleotide diversity among many maize inbred lines and partially inbred teosinte lines. The results indicated that mature miRNA genes were highly conserved during their evolution. This preliminary characterization based on our findings provides a framework for future analysis of miRNA genes and their roles in key traits of maize as feed, fodder, and biofuel.
doi:10.1371/journal.pgen.1000716
PMCID: PMC2773440  PMID: 19936050
14.  Genome-Wide Expression of Transcriptomes and Their Co-Expression Pattern in Subtropical Maize (Zea mays L.) under Waterlogging Stress 
PLoS ONE  2013;8(8):e70433.
Waterlogging causes extensive damage to maize crops in tropical and subtropical regions. The identification of tolerance genes and their interactions at the molecular level will be helpful to engineer tolerant genotypes. A whole-genome transcriptome assay revealed the specific role of genes in response to waterlogging stress in susceptible and tolerant genotypes. Genes involved in the synthesis of ethylene and auxin, cell wall metabolism, activation of G-proteins and formation of aerenchyma and adventitious roots, were upregulated in the tolerant genotype. Many transcription factors, particularly ERFs, MYB, HSPs, MAPK, and LOB-domain protein were involved in regulation of these traits. Genes responsible for scavenging of ROS generated under stress were expressed along with those involved in carbohydrate metabolism. The physical locations of 21 genes expressed in the tolerant genotype were found to correspond with the marker intervals of known QTLs responsible for development of adaptive traits. Among the candidate genes, most showed synteny with genes of sorghum and foxtail millet. Co-expression analysis of 528 microarray samples including 16 samples from the present study generated seven functional modules each in the two genotypes, with differing characteristics. In the tolerant genotype, stress genes were co-expressed along with peroxidase and fermentation pathway genes.
doi:10.1371/journal.pone.0070433
PMCID: PMC3735631  PMID: 23936429
15.  Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions 
PLoS ONE  2014;9(4):e93724.
Background
Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied.
Methodology/Principal Findings
In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments.
Conclusions/Significance
These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.
doi:10.1371/journal.pone.0093724
PMCID: PMC3974806  PMID: 24699822
16.  Burkholderia cenocepacia ZmpB Is a Broad-Specificity Zinc Metalloprotease Involved in Virulence  
Infection and Immunity  2006;74(7):4083-4093.
In previous studies we characterized the Burkholderia cenocepacia ZmpA zinc metalloprotease. In this study, we determined that B. cenocepacia has an additional metalloprotease, which we designated ZmpB. The zmpB gene is present in the same species as zmpA and was detected in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria, and B. pyrrocinia but was absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The zmpB gene was expressed, and ZmpB was purified from Escherichia coli by using the pPROEXHTa His6 Tag expression system. ZmpB has a predicted preproenzyme structure typical of thermolysin-like proteases and is distantly related to Bacillus cereus bacillolysin. ZmpB was expressed as a 63-kDa preproenzyme precursor that was autocatalytically cleaved into mature ZmpB (35 kDa) and a 27-kDa prepropeptide. EDTA, 1,10-phenanthroline, and Zn2+ cations inhibited ZmpB enzyme activity, indicating that it is a metalloprotease. ZmpB had proteolytic activity against α-1 proteinase inhibitor, α2-macrogobulin, type IV collagen, fibronectin, lactoferrin, transferrin, and immunoglobulins. B. cenocepacia zmpB and zmpA zmpB mutants had no proteolytic activity against casein and were less virulent in a rat agar bead chronic infection model, indicating that zmpB is involved in B. cenocepacia virulence. Expression of zmpB was regulated by both the CepIR and CciIR quorum-sensing systems.
doi:10.1128/IAI.00297-06
PMCID: PMC1489746  PMID: 16790782
17.  A Single Locus Is Responsible for Salinity Tolerance in a Chinese Landrace Barley (Hordeum vulgare L.) 
PLoS ONE  2012;7(8):e43079.
Introduction
Salinity and waterlogging are two major abiotic stresses severely limiting barley production. The lack of a reliable screening method makes it very hard to improve the tolerance through breeding programs.
Methods
This work used 188 DH lines from a cross between a Chinese landrace variety, TX9425 (waterlogging and salinity tolerant), and a Japanese malting barley, Naso Nijo (waterlogging and salinity sensitive), to identify QTLs associated with the tolerance.
Results
Four QTLs were found for waterlogging tolerance. The salinity tolerance was evaluated with both a hydroponic system and in potting mixture. In the trial with potting mixture, only one major QTL was identified to associate with salinity tolerance. This QTL explained nearly 50% of the phenotypic variation, which makes it possible for further fine mapping and cloning of the gene. This QTL was also identified in the hydroponic experiment for different salt-related traits. The position of this QTL was located at a similar position to one of the major QTLs for waterlogging tolerance, indicating the possibility of similar mechanisms controlling both waterlogging and salinity tolerance.
Conclusion
The markers associated with the QTL provided a unique opportunity in breeding programs for selection of salinity and waterlogging tolerance.
doi:10.1371/journal.pone.0043079
PMCID: PMC3423432  PMID: 22916210
18.  Occurrence and Evolution of the Paralogous Zinc Metalloproteases IgA1 Protease, ZmpB, ZmpC, and ZmpD in Streptococcus pneumoniae and Related Commensal Species 
mBio  2012;3(5):e00303-12.
ABSTRACT
The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host.
IMPORTANCE
The paralogous zinc metalloproteases IgA1 protease, ZmpB, ZmpC, and ZmpD have been identified as crucial for virulence of the human pathogen Streptococcus pneumoniae. This study maps the presence of the corresponding genes and enzyme activities in S. pneumoniae and in related commensal species of the genera Streptococcus, Gemella, and Granulicatella. The distribution, genome location, and sequence diversification indicate that zmpB is the ancestral gene predating the evolution of today’s humanoid species. The ZmpB protease may play an important but yet unidentified role in the association of streptococci of the Mitis and Salivarius groups with their human host, as it is ubiquitous in these two groups, except for a fragmented gene in Streptococcus thermophilus, the only species not associated with humans. The relative sequence diversification of the IgA1 protease, ZmpB, and ZmpD is striking evidence of differences in selection for diversification of these surface-exposed proteins in the pathogen S. pneumoniae compared to the closely related commensal streptococci.
doi:10.1128/mBio.00303-12
PMCID: PMC3518915  PMID: 23033471
19.  Waterlogging-induced changes in root architecture of germplasm accessions of the tropical forage grass Brachiaria humidicola 
AoB Plants  2014;6:plu017.
Brachiaria humidicola, a tropical forage grass, develops aerenchyma in nodal roots to adapt to waterlogging. A large body of work has focused on the functional role of aerenchyma in nodal roots under waterlogged soil conditions. On the other hand, quantification of responses of lateral roots to waterlogging has been often overlooked in past work. Our data indicated that although waterlogging reduced the overall proportion of lateral roots, its proportion significantly increased in the top 10 cm of the soil. This suggests that soil flooding increases lateral root proliferation of B. humidicola in upper soil layers. This may compensate the reduction of root surface area brought by the restriction of root growth at depths below 30 cm into waterlogged soil.
Waterlogging is one of the major factors limiting the productivity of pastures in the humid tropics. Brachiaria humidicola is a forage grass commonly used in zones prone to temporary waterlogging. Brachiaria humidicola accessions adapt to waterlogging by increasing aerenchyma in nodal roots above constitutive levels to improve oxygenation of root tissues. In some accessions, waterlogging reduces the number of lateral roots developed from main root axes. Waterlogging-induced reduction of lateral roots could be of adaptive value as lateral roots consume oxygen supplied from above ground via their parent root. However, a reduction in lateral root development could also be detrimental by decreasing the surface area for nutrient and water absorption. To examine the impact of waterlogging on lateral root development, an outdoor study was conducted to test differences in vertical root distribution (in terms of dry mass and length) and the proportion of lateral roots to the total root system (sum of nodal and lateral roots) down the soil profile under drained or waterlogged soil conditions. Plant material consisted of 12 B. humidicola accessions from the gene bank of the International Center for Tropical Agriculture, Colombia. Rooting depth was restricted by 21 days of waterlogging and confined to the first 30 cm below the soil surface. Although waterlogging reduced the overall proportion of lateral roots, its proportion significantly increased in the top 10 cm of the soil. This suggests that soil flooding increases lateral root proliferation of B. humidicola in the upper soil layers. This may compensate for the reduction of root surface area brought about by the restriction of root growth at depths below 30 cm. Further work is needed to test the relative efficiency of nodal and lateral roots for nutrient and water uptake under waterlogged soil conditions.
doi:10.1093/aobpla/plu017
PMCID: PMC4038435  PMID: 24876299
Lateral root proportion; oxygen deficiency; rooting depth; root length; soil flooding; vertical root distribution.
20.  Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress 
Amino Acids  2010;39(4):1043-1053.
Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.
doi:10.1007/s00726-010-0596-1
PMCID: PMC2945468  PMID: 20414691
Glycine max; Soybean; Alanine aminotransferase; Hypoxic stress; Waterlogging; Nitrogen fertilisation
21.  Review of wheat improvement for waterlogging tolerance in Australia and India: the importance of anaerobiosis and element toxicities associated with different soils 
Annals of Botany  2008;103(2):221-235.
Background and Aims
The lack of knowledge about key traits in field environments is a major constraint to germplasm improvement and crop management because waterlogging-prone environments are highly diverse and complex, and the mechanisms of tolerance to waterlogging include a large range of traits. A model is proposed that waterlogging tolerance is a product of tolerance to anaerobiosis and high microelement concentrations. This is further evaluated with the aim of prioritizing traits required for waterlogging tolerance of wheat in the field.
Methods
Waterlogging tolerance mechanisms of wheat are evaluated in a range of diverse environments through a review of past research in Australia and India; this includes selected soils and plant data, including plant growth under waterlogged and drained conditions in different environments. Measurements focus on changes in redox potential and concentrations of diverse elements in soils and plants during waterlogging.
Key Results
(a) Waterlogging tolerance of wheat in one location often does not relate to another, and (b) element toxicities are often a major constraint in waterlogged environments. Important element toxicities in different soils during waterlogging include Mn, Fe, Na, Al and B. This is the first time that Al and B toxicities have been indicated for wheat in waterlogged soils in India. These results support and extend the well-known interactions of salinity/Na and waterlogging/hypoxia tolerance.
Conclusions
Diverse element toxicities (or deficiencies) that are exacerbated during waterlogging are proposed as a major reason why waterlogging tolerance at one site is often not replicated at another. Recommendations for germplasm improvement for waterlogging tolerance include use of inductively coupled plasma analyses of soils and plants.
doi:10.1093/aob/mcn137
PMCID: PMC2707304  PMID: 18708642
Waterlogging; microelements; toxicity; redox potential; wheat; anaerobiosis
22.  Comparative mapping of quantitative trait loci associated with waterlogging tolerance in barley (Hordeum vulgare L.) 
BMC Genomics  2008;9:401.
Background
Resistance to soil waterlogging stress is an important plant breeding objective in high rainfall or poorly drained areas across many countries in the world. The present study was conducted to identify quantitative trait loci (QTLs) associated with waterlogging tolerance (e.g. leaf chlorosis, plant survival and biomass reduction) in barley and compare the QTLs identified across two seasons and in two different populations using a composite map constructed with SSRs, RFLP and Diversity Array Technology (DArT) markers.
Results
Twenty QTLs for waterlogging tolerance related traits were found in the two barley double haploid (DH) populations. Several of these QTLs were validated through replication of experiments across seasons or by co-location across populations. Some of these QTLs affected multiple waterlogging tolerance related traits, for example, QTL Qwt4-1 contributed not only to reducing barley leaf chlorosis, but also increasing plant biomass under waterlogging stress, whereas other QTLs controlled both leaf chlorosis and plant survival.
Conclusion
Improving waterlogging tolerance in barley is still at an early stage compared with other traits. QTLs identified in this study have made it possible to use marker assisted selection (MAS) in combination with traditional field selection to significantly enhance barley breeding for waterlogging tolerance. There may be some degree of homoeologous relationship between QTLs controlling barley waterlogging tolerance and that in other crops as discussed in this study.
doi:10.1186/1471-2164-9-401
PMCID: PMC2533678  PMID: 18752688
23.  Functional Analysis of the Burkholderia cenocepacia ZmpA Metalloprotease 
Journal of Bacteriology  2005;187(13):4421-4429.
Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His6 tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His6-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H465 with G465 or A465, E377 with A377 or D377, or H380 with P380 or A380. Mutagenesis of H465, E377, or H380 resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H380 was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil α-1 proteinase inhibitor, α2-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.
doi:10.1128/JB.187.13.4421-4429.2005
PMCID: PMC1151788  PMID: 15968051
24.  Barley responses to combined waterlogging and salinity stress: separating effects of oxygen deprivation and elemental toxicity 
Salinity and waterlogging are two major factors affecting crop production around the world and often occur together (e.g., salt brought to the surface by rising water tables). While the physiological and molecular mechanisms of plant responses to each of these environmental constraints are studied in detail, the mechanisms underlying plant tolerance to their combined stress are much less understood. In this study, whole-plant physiological responses to individual/combined salinity and waterlogging stresses were studied using two barley varieties grown in either vermiculite (semi-hydroponics) or sandy loam. Two weeks of combined salinity and waterlogging treatment significantly decreased plant biomass, chlorophyll content, maximal quantum efficiency of PSII and water content (WC) in both varieties, while the percentage of chlorotic and necrotic leaves and leaf sap osmolality increased. The adverse effects of the combined stresses were much stronger in the waterlogging-sensitive variety Naso Nijo. Compared with salinity stress alone, the combined stress resulted in a 2-fold increase in leaf Na+, but a 40% decrease in leaf K+ content. Importantly, the effects of the combined stress were more pronounced in sandy loam compared with vermiculite and correlated with changes in the soil redox potential and accumulation of Mn and Fe in the waterlogged soils. It is concluded that hypoxia alone is not a major factor determining differential plant growth under adverse stress conditions, and that elemental toxicities resulting from changes in soil redox potential have a major impact on genotypic differences in plant physiological and agronomical responses. These results are further discussed in the context of plant breeding for waterlogging stress tolerance.
doi:10.3389/fpls.2013.00313
PMCID: PMC3743405  PMID: 23967003
salinity; waterlogging; microelement toxicity; barley; breeding; manganese; potassium; sodium
25.  Waterlogging Tolerance of Crops: Breeding, Mechanism of Tolerance, Molecular Approaches, and Future Prospects 
BioMed Research International  2012;2013:963525.
Submergence or flood is one of the major harmful abiotic stresses in the low-lying countries and crop losses due to waterlogging are considerably high. Plant breeding techniques, conventional or genetic engineering, might be an effective and economic way of developing crops to grow successfully in waterlogged condition. Marker assisted selection (MAS) is a new and more effective approach which can identify genomic regions of crops under stress, which could not be done previously. The discovery of comprehensive molecular linkage maps enables us to do the pyramiding of desirable traits to improve in submergence tolerance through MAS. However, because of genetic and environmental interaction, too many genes encoding a trait, and using undesirable populations the mapping of QTL was hampered to ensure proper growth and yield under waterlogged conditions Steady advances in the field of genomics and proteomics over the years will be helpful to increase the breeding programs which will help to accomplish a significant progress in the field crop variety development and also improvement in near future. Waterlogging response of soybean and major cereal crops, as rice, wheat, barley, and maize and discovery of QTL related with tolerance of waterlogging, development of resistant variety, and, in addition, future prospects have also been discussed.
doi:10.1155/2013/963525
PMCID: PMC3591200  PMID: 23484164

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